Time and treatment-dependent changes in genes essential for a) intra-tissue biosynthesis of androgens , b) establishment of endometrial decidualization  and c) endometrial receptivity  were measured. Decidualization of ESC resulted in significant time-dependent changes in expression of AKR1C3 and SRD5A1 and secretion of T/DHT. Addition of flutamide significantly reduced secretion of IGFBP1 and prolactin and altered the expression of endometrial receptivity markers. Intracrine biosynthesis of endometrial androgens during decidualization may play a key role in endometrial receptivity and offer a novel target for fertility treatment.The endometrium is a complex, steroid-dependent tissue that undergoes dynamic cyclical remodelling. Transformation of stromal fibroblasts (ESC) into specialised secretory cells  is fundamental to the establishment of a receptive endometrial microenvironment which can support and maintain pregnancy. Androgen receptors (AR) are present in ESC; in other tissues local metabolism of ovarian and adrenal-derived androgens regulate AR-dependent gene expression. We hypothesised that altered expression/activity of androgen biosynthetic enzymes would regulate tissue availability of bioactive androgens and the process of decidualization. Primary human ESC were treated  In fertile women the endometrium ‘prepares’ for implantation of a blastocyst by a period of post-ovulatory (progesterone-dependent) tissue remodelling. This process is controlled by endometrial stromal cells (ESC) that undergo decidualization, a process of differentiation, that results in transformation from proliferating fibroblasts to specialised secretory cells capable of producing factors that promote endometrial receptivity and regulate multiple cell types including those of the immune and vascular systemsCYP19A1)SRD5A1). In the 1970s Rose et al. detected conversion to DHT in human endometrial homogenates co-incubated with radiolabelled testosterone9The human endometrium is an androgen target tissue and androgen receptors (AR) are expressed in the endometrium throughout the menstrual cycle. Our detailed analysis of full thickness sections of human endometrium has revealed intense regional and stage-dependent immunoexpression in ESCin vitro decidualization phenocopies the post-ovulatory differentiation of ESC during a fertile cycle with characteristic changes in cellular morphologyin vitro studies have used the synthetic progestin medroxyprogesterone acetate (MPA) in place of progesterone. As MPA is reported to activate the AR, in addition to its well-known role as a progesterone receptor (PR) agonist1516in silico and knockdown approaches have reported that DHT and AR-dependent signaling can regulate distinct gene networks in decidualized human ESC with evidence for a role in cell survival, cell cycle regulation and cytoskeletal organisation4Ar have altered neuroendocrine signalling and ovarian function and are subfertileAr plays a key role in uterine developmentPrimary stromal cells isolated from human endometrium (ESC) can be stimulated to undergo a robust decidualization response by the addition of progesterone and the second messenger molecule cyclic adenosine monophosphate (cAMP)In androgen target tissues local activation of circulating precursors regulate AR-dependent gene expression but whether local metabolism and activation of androgens occurs within the endometrium is not known. In the current study we tested the hypothesis that changes in intra-uterine bioavailability of androgens would have a direct impact on endometrial tissue function. We demonstrate that decidualization of ESC is associated with local biosynthesis of androgens. Our novel data demonstrate that intracrine androgens can complement the effects of progesterone by acting to enhance the decidualization response and support an optimal environment for successful establishment of pregnancy.AKR1C3 mRNA were increased in ESC treated with decidualization media (DM) for 1, 2 and 4 days with a significant peak in expression detected after 2 and 4 days of treatment  ESC exhibited classic rounded, ‘epithelioid’ morphology . ConsistSPP1, MAOA and EDNRB: changes induced by addition of flutamide were consistent with a role for androgens in regulation of these 3 genes. Osteopontin/Secreted Phosphoprotein 1 (SPP1) is reported to be an essential mediator of implantation and receptivity in women24SPP1 mRNA was significantly increased in ESC treated to decidualize (DEC) for 4 (SPP1 mRNA were lower in cells treated with flutamide and this was significant at the 8-day time point (Candidate receptivity genes were selected by comparing androgen-regulated genes22me point  and undeMAOA) mRNA concentrations after 1  binds members of the endothelin family of proteins that are reported to regulate endometrial blood flowrol at 4 .Endometrial receptivity is a time-sensitive process underpinned by dynamic changes in the transformation of the stromal compartment and coordinated stromal-epithelial cross-talk. To our knowledge we present the first data that demonstrate endometrial stromal cells promote formation of a pro-androgenic microenvironment during decidualization. Critically, we found that changes in androgen metabolism and biosynthesis are time-dependent, suggesting androgens will be most abundant within areas of the tissue at early stages of decidualization. We found that incubation with the selective AR antagonist flutamide had a significant effect on gene expression within primary human ESC over the time course of decidualization. Notably in this study we did not add exogenous T or DHT therefore the impact of flutamide on gene transcription and protein biosynthesis provides evidence of intracrine androgen activity. We noted that blocking intracrine androgens both inhibited and delayed expression of proteins implicated as markers of the decidualization response and endometrial receptivity  response to decidualization induced by estradiol (E2) and progesterone was significantly reduced, however this was only in a small subset of the patients that were analysedInsufficient androgen signalling may be detrimental to the regulation of decidualization however exposure to excess androgens may also adversely affect endometrial function. Elevated AR expression has been reported in endometrium of women with polycystic ovarian syndrome (PCOS)32in vitro fertilisation or intracytoplasmic sperm injectionThe widely reported age-related decline in fertility is often considered as an exclusively ovarian phenomenon but there is emerging evidence that reduced androgen bioavailability may result in endometrial ‘senescence’ a phenomenon that has received little attention to date. Concentrations of circulating androgens decline precipitously with age(CYP19A1) by 8 days decidualizationIn the current study we provide further novel insights into the regulation of decidualization by profiling discrete responses of ESC across a time-course up to 8 days whereas many other studies only consider the response of cells after 6 or more days incubation15early remodelling events by affecting endometrial transcription and altering the expression of decidualization and receptivity markers. These data suggest intra-uterine androgens may be critical for decidualization and thus impact on establishment and maintenance of pregnancy.The results of this study suggest a new paradigm for understanding control of endometrial remodelling in which local androgens modulate decidualization and promote coordinated development of the endometrial microenvironment. Critically, we describe a novel role for androgens that is both distinct from and complementary to the long-established role of progesterone in promoting post-ovulatory tissue remodelling in women. We demonstrate that androgens regulate Human endometrial tissue  was obtained from women undergoing surgery for non-malignant gynaecological conditions. None of the women were receiving hormonal therapy or suffering from endometriosis. Cycle phase was determined as previously reportedCYC as internal control. Primers/probes are given in Supplemental Table 1.Isolation of mRNAs, preparation of cDNAs, and analysis by qRTPCR was performed according to standard protocols (11); samples were quantified by standard curve method or by the comparative ΔΔCt method with Western blotting was performed using 50 μg/lane total cell lysates; membranes were probed with rabbit or goat anti-SRD5A1 ; rabbit anti-AKR1C3 ; rabbit anti-Monoamine Oxidase A  or rabbit anti-Endothelin B Receptor (Abcam ab129102). SRD5A1 was not detected at the predicted molecular weight (29 kDa) but consistently detected at around 100 kDa at all reducing conditions tested . Identity of the protein was confirmed using two different specific antibodies; no bands were detected if the primary antibody was incubated with specific blocking peptide (SRD5A1 (A-18) peptide sc-20396 P) or in controls lacking the primary antibodies. In all cases loading control was goat anti-actin . Membranes were incubated with species-specific fluorescent-conjugated secondary antibodies and visualised using the Licor Odyssey system (Licor). Protein bands detected by Western blot were quantified by performing densitometry analysis using ImageJ (NIH.gov). Data were normalised to Actin and expressed as fold-change relative to control treatment.Immunofluorescence was carried out on ESC grown on chamber slides (BD Biosciences) incubated with control media or DM to identify the localisation of the proteins within the cell and as numbers of primary cells were limited only one time point was selected (4 days). In line with expectations, both AKR1C3 and SRD5A1 were localised to the cytoplasm of the primary ESC a finding consistent with expression within the endoplasmic reticulum. Primary antibodies; rabbit anti-SRD5A1  and rabbit anti-AKR1C3  were incubated overnight at 4 °C. Antigen detection was performed using a Tyramide signal amplification (Perkin Elmer) system followed by SYTOXGreen nuclear counterstain (Life technologies). Images were captured using a LSM 710 Confocal microscope (Zeiss). Cell morphology was assessed by phase-contract imaging using Axiovert 200 inverted microscope (Zeiss) with 5× objective.Testosterone (T), dihydrotestosterone (DHT) and Insulin-like Growth Factor Binding Protein 1 (IGFBP1) were measured in culture supernatants. Media were assayed in duplicate. Assay specificities are described in Supplemental Tables 2–4.Statistical analysis was performed using Graphpad prism. Student’s t test or One-way ANOVA were used to determine significance between treatments in data that were normally distributed. Non-parametric testing was utilised where sample sizes were insufficient to confirm normality of data distribution; Mann-Whitney test or Kruskal-Wallis test was used to assess differences between treatments. Where data were analysed as fold change significance was tested using one sample t test and a theoretical mean of 1. Criterion for significance was p < 0.05. All data are presented as mean ± SEM.How to cite this article: Gibson, D. A. et al. Intracrine Androgens Enhance Decidualization and Modulate Expression of Human Endometrial Receptivity Genes. Sci. Rep. 6, 19970; doi: 10.1038/srep19970 (2016).


Pluripotent embryonic stem cells grown under standard conditions (ESC) have a markedly glycolytic profile, which is shared with many different types of cancer cells. Thus, some therapeutic strategies suggest that pharmacologically shifting cancer cells towards an oxidative phenotype, using glycolysis inhibitors, may reduce cancer aggressiveness. Given the metabolic parallels between cancer and stemness would chemotherapeutical agents have an effect on pluripotency, and could a strategy involving these agents be envisioned to modulate stem cell fate in an accessible manner? In this manuscript we attempted to determine the effects of 3-bromopyruvate (3BrP) in pluripotency. Although it has other intracellular targets, this compound is a potent inhibitor of glycolysis enzymes thought to be important to maintain a glycolytic profile. The goal was also to determine if we could contribute towards a pharmacologically accessible metabolic strategy to influence cell differentiation.Mouse embryonic stem cells (mESC) grown under standard pluripotency conditions (in the presence of Leukemia Inducing Factor- LIF) were treated with 3BrP. As a positive control for differentiation other mESCs were grown without LIF. Overall our results demonstrate that 3BrP negatively affects pluripotency, forcing cells to become less glycolytic and with more active mitochondria. These changes in metabolism are correlated with increased differentiation, even under pluripotency conditions (i.e. in the presence of LIF). However, 3BrP also significantly impaired cell function, and may have other roles besides affecting the metabolic profile of mESCs.Treatment of mESCs with 3BrP triggered a metabolic switch and loss of pluripotency, even in the presence of LIF. Interestingly, the positive control for differentiation allowed for a distinction between 3BrP effects and changes associated with spontaneous differentiation/loss of pluripotency in the absence of LIF. Additionally, there was a slight differentiation bias towards mesoderm in the presence of 3BrP. However, the side effects on cellular function suggest that the use of this drug is probably not adequate to efficiently push cells towards specific differentiation fates. Furthhibition , 8. Morehibition , and thehibition .Importantly, the metabolic characteristics of pluripotent stem cells (PSCs) are common to proliferative cells in general, and thus similar to some types of cancer cells. Common metabolic strategies between cancer and stemness include high levels of hexokinase II (HKII) linked to the outer mitochondrial membrane and a pyruvate dehydrogenase (PDH) cycle promoting the conversion of pyruvate to lactate rather than to acetyl-CoA . HexokinTaken together these data suggest that pharmacological strategies linked to the targeting of metabolic characteristics that define active cancer cells may also be useful in modulating pluripotent stem cell fate. Although it may also have other targets, 3-brompyruvate (3BrP) is a chemical pyruvate analog that functions as a potent inhibitor of glycolytic enzymes, most notably, but not exclusively, HKII , 22. ImpGiven that ESCs are also highly proliferative, and share some common features with tumor cells, we wondered if 3BrP would also affect ESC pluripotency and could, in addition, be used as a practical and inexpensive tool to modulate stem cell fate by promoting differentiation by inducing a metabolic glycolysis-OXPHOS shift. The goal of the present work was not to determine the importance of specific glycolysis enzymes towards the maintenance of pluripotency, for which a totally different experimental approach would be necessary, but rather to establish if the specific compound 3BrP could be a viable alternative for cell culture metabolic modulation in order to obtain specific cell lineages.2 and 5% CO2. E14Tg2a mESC were passaged when the right confluence was achieved, usually two or three days after platting. Briefly, 0.1% gelatin (Sigma-Aldrich) was added to plates and allowed to coat for ten minutes at 37°C. Afterwards, the excess was removed and supplemented Knockout-DMEM media was added. Cells were plated at a final density of 5000cells/cm for all experimental conditions: ESCs in control conditions (with LIF), in the absence of LIF (differentiation control) and in the presence of two different 3BrP (Sigma-Aldrich) concentrations (25 and 50 μM) plus LIF. 3BrP was freshly prepared and added every 24hours and experiments were conducted after 60h of incubation with 3BrP+LIF or in the absence of LIF. It is important to stress that all 3BrP experiments were carried out under strict pluripotency conditions (i.e. in the presence of LIF) in order to determine if this glycolytic inhibitor pushed the cells towards differentiation even under pluripotency conditions. Thus 3BrP experiments were compared to ESCs cultured with (pluripotency control) or without (differentiation control) LIF. The exception was the embryoid body experiments, specific for differentiation, as noted below.Mouse embryonic stem cell line E14Tg2a was kindly provided by Miguel Ramalho-Santos  and characterized elsewhere , 27. Cel2 and 5% CO2. Viability was assessed counting 100 cells per condition; green fluorescent cells without red fluorescence were counted as live cells and a cell with both stains as dead cells using a fluorescent microscope.In order to monitor cell viability the LIVE/DEAD Kit (Invitrogen) was used according to manufacturers’ instructions. The Kit consists of two DNA binding fluorescent dyes: SYBR 14 which is membrane permeable staining the nucleus green for all cells, and PI (propidium iodide). PI only enters cells with compromised membrane integrity, thus staining the nucleus of dead cells red. Briefly, cells were collected at the 60h time point by enzymatic dissociation with accutase (Gibco LIFe Technologies), and centrifuged for 5 min at 1200rpm. The pellet was ressuspended in D-PBS and 6μM of SYBR 14 and 0.48mM of PI were added to the cell suspension that was then incubated for 20 min at 37°C, 20%O2 and 5% CO2 and were solubilized with 300 μl of isopropanol with 0.04M HCl. Intensity was measured colorimetrically at 570 nm. Raw data was normalized to total cell number for each condition and then metabolic activity was normalized to the control.MTT was reconstituted according to the manufacturers’ instructions (Sigma-Aldrich) and was used at a final concentration of 0.5mg/ml. This assay is routinely to monitor cell proliferation/metabolic activity. This is due to the fact that MTT is reduced by cellular dehydrogenases (using both NADPH and NADH) present in the cells, and this will produce violet formazan crystals that are soluble in acidified isopropanol . Cells wIt is well accepted that stem cells that are self-renewing and pluripotent present high levels of AP . The AlkAll dyes noted below were analyzed by flow cytometry  and 20000 gated cells were acquired/analyzed per condition with the Cell Quest Pro Acquisition software (BD Biosciences).2 and 5% CO2 in the dark in 1ml of D-PBS. Afterwards cells were centrifuged to remove excess TMRM and pellets were ressuspended with 500μl of PBS, kept on ice and analyzed. In order to define the proper gates for an accurate analysis we used cells without TMRM as a blank control and TMRM labeled cells incubated with 250 μM of CCCP, a potent mitochondrial uncoupler, as a negative control.Mitochondrial membrane potential (MMP) was monitored using tetramethylrhodamine methyl (TMRM-Invitrogen) –32 a lipTo evaluate intracellular amounts of superoxide anion we used MitoSOX Red (Molecular Probes) that emits fluorescence after selectively reacting with superoxide in mitochondria. MitoSOX Red was prepared according to the manufacturers’ instructions and cells were incubated for 30 min at 37°C in the dark with a final concentration of 3μM of the probe. To properly define the analysis gates we used cells without the probe as a negative control and cells that were incubated with Antimycin A (100μM) as a positive control, given that this potent mitochondrial complex III inhibitor acts as a ROS inducer –35.To infer effects on cell proliferation we examined the expression of the proliferating cell nuclear antigen (PCNA), given its role in DNA replication and repair, and that it is highly expressed in rapidly proliferating cells . Cells wFor all flow cytometry experiments representative data is also available in The protocol for adenine nucleotide content analysis was as previously described . Sampleshttp://pga.mgh.harvard.edu/primerbank/) and ordered from Integrated DNA Technologies (IDT). SsoFast EvaGreen Supermix (Bio-Rad) was used to perform RT-PCR analysis according with Bio-Rad instructions. Samples were run in CFX96 Touch Real-Time PCR Detection System and mRNA fold change was calculated using the -ΔΔCt method.RNA isolation and DNA cleanup was performed as described previously . RNA conIn order to obtain protein extracts for Western blot analysis mESC were lysed with 100μl of RIPA buffer (Sigma-Aldrich) supplemented with 2mM of phenylmethylsulphonyl fluoride-PMSF (Sigma-Aldrich) and 2x Halt phosphatase inhibitor cocktail  as described elsewhere . ProteinProtein samples for Western blot were prepared by diluting 30μg of protein in Laemmli sample buffer (Bio-Rad) and water, given that the volume of sample buffer had to contain 30μg of protein (water was used to adjust the volume). A total volume of 30μl per sample was prepared and denatured at 95°C in a dry bath. After this step samples were loaded into 12% Acrilamide Tris-HCl gel and electrophoresis was performed in a Mini protean tetra cell Bio-Rad apparatus. After protein size separation in the gel, proteins were blotted into a PDVF membrane (Bio-Rad) and blocked in a solution of 5% powder milk (Bio-Rad) in Tris-Buffered Saline with Tween (TBST). Afterwards, membranes were incubated overnight at 4°C with the primary antibodies: mouse anti-Nanog , rabbit anti-Oct4, mouse anti-P53, rabbit anti-PDH, rabbit anti-GAPDH , mouse anti-Hexokinase I, mouse anti-Hexokinase II, mouse anti-Caspase3  and mouse anti-beta-Actin . Membranes were washed and then incubated with the correct HRP-conjugated secondary antibody. Proteins were detected using the Clarity Western ECL Substrate (Bio-Rad) and membranes were developed using the VersaDoc Imaging system (Bio-Rad). Protein quantification was performed using Quantity One software and results were normalized to beta-Actin levels for each condition.To promote and monitor ESC differentiation the EB assay was used. mESCs were platted in a 60mm non-adherent petri dish at a cellular density equal to one million cells and were left for three days in a 37°C, 5% CO2 incubator with KODMEM without LIF supplementation in order to form EB. Cell media was changed during this period by gentle aspiration of the non-adherent dishes into falcons and allowing the cells to sediment. At day 3 KODMEM media was supplemented with 10% FBS and EB were transferred into new normal gelatin coated 100mm dishes. EBs were incubated overnight in a 37°C, 5% CO2 incubator to allow them to attach to the dish. From that day on EB were grown with basic DMEM media with no serum supplementation and media changed every other day. At day 14 EBs were collected for protein isolation. In this case 3BrP experiments were carried out in the absence of LIF, as the goals was to see if the drug created a differentiation bias. Therefore there is only one control in this case (EB formation without 3BrP), unlike what took place in the pluripotency experiments mentioned earlier.SPSS Statistics 21.0 (SPSS Inc.) was used to perform statistical analysis. All the raw data was verified for normality and homoscedasticity and accordingly the appropriate parametric tests for paired samples were applied. Given that all the experimental conditions were collected/analyzed at the same time a repeated measure ANOVA was used. Sphericity was assessed and, when violated, the Greenhouse-Geisser correction was applied before performing any POST-HOC tests. When data violated the normal distribution assumption a Friedman ANOVA was performed. All data is expressed as mean ± standard error of mean (SEM) reflecting the number of experiments. Statistical significance was determined at p ≤ 0.05.Given that 3BrP has been described as lowering cell viability by increasing ROS production and activating the mitochondrial apoptosis pathway in glycolytic cells , 41, we Does 3BrP affect pluripotency in the presence of LIF? To address this issue pluripotency was first monitored by analyzing gene expression and protein levels for two of the major regulators of the pluripotency network: Oct4 and Nanog. Both Western Blot  and RT-PGiven that 3BrP has been described to cause mitochondrial depolarization  and thatMitochondrial ROS production, in the form of mitochondria-produced superoxide anion, was also analyzed as another indicator of mitochondrial activity and possible target for 3BrP induced changes in ESC status. Superoxide anion production was quantified by measuring MitoSOX Red fluorescence by flow cytometry, and showed a similar trend as noted for TMRM; all the experimental conditions presented a slight increase in superoxide anion production when compared to the control, although this increase was statistically significant for 50 μM 3BrP . This isTaking into account the significant effect of 3BrP in mitochondrial function we wondered if this might impact cell energy status. To address this issue, adenine nucleotides were quantified by High-performance liquid chromatography HPLC; . In accoConsidering that 3BrP is a known apoptosis inducer, and even though we did not see significant differences in terms of cell viability, we performed AnnexinV/PI analysis by flow cytometry . Notably3BrP has been described to inhibit Hexokinase II (HKII) and, more recently, Gapdh . Thus weTo determine if 3BrP could influence ESC differentiation we used the embryoid body assay, where cells are left to differentiate spontaneously by removing LIF, in this case in the presence or absence of 3BrP. The first thing to point out is the fact that control cells presented markers for the three germ layers upon differentiation, clearly demonstrating that ESCs were indeed pluripotent . Given tEmbryonic stem cells (ESC) in general rely more on glycolysis and a shift to a predominantly oxidative metabolism (OXPHOS) is though to be needed for differentiation to take place . Due to Considering the metabolic parallelism between ESC and cancer cells , we wondWe show that 3BrP has a negative impact on colony morphology, cell number, viability and cell proliferation, although not as negative as growing ESCs without LIF. Overall, in the presence of 3BrP colonies were smaller and borders started to lose definition. These observations are paralleled by significant differences in total cell number, although with no detrimental effects on viability, suggesting that 3BrP affects cell proliferation rates. The effects of 3BrP on cell viability are well described, as this compound causes both apoptosis and necrosis by depleting ATP due to impairment of mitochondrial function , 48. TheAlthough 3BrP is possibly causing toxic effects we could not rule out an additional impact in the cell cycle. Both pluripotent stem cells and certain cancer cells proliferate extensively, and thus require bioenergetic and biosynthetic blocks for cell growth and division . Given tAs discussed previously, 3BrP affects mitochondrial function in cancer cells, so we investigated mitochondrial-related changes in ESC in the presence of this compound. Overall mitochondria become more active, reflected by the significant increase in MMP following 3BrP treatment, accompanied by an increase in the production of superoxide anion. For the higher concentration of 3BrP energy charge decreased, a reflection from the significant increase in AMP level and the tendency for a decrease in ATP levels, which could mean that the increment in MMP and ROS production is starting to be detrimental to mitochondrial function. MTT results also suggest a slower metabolic state of cells exposed to 3BrP, and given that MTT measures dehydrogenase activity treated cells could have a lower capability to manage oxidative stress as well. This outcome is characteristic of 3BrP. On the other hand, as with PCNA results, cells grown without LIF were slightly different, with higher concentrations of ATP and AMP, and a significant increase in MTT, thus suggesting that these cells are more active and capable of managing oxidative stress.Besides a detrimental effect on mitochondrial function and apoptosis effect 3BrP could affect metabolic enzymes promoting a metabolic shift that would result in the loss of pluripotency. In order to address this question we quantified protein and mRNA levels for Hexokinase I and II, as well as other downstream glycolytic enzymes. Although results were not linear, they suggest a decrease in both Hexokinase isoforms and Gapdh (both purported targets for 3BrP inhibition ), at theOverall our results suggest that by using 3BrP we are promoting pluripotency loss even under pluripotency culture conditions, and possibly a shift in metabolism, considering that mitochondria are more active. However, the effects of 3BrP in the presence of LIF do not always mirror what takes place under normal differentiation conditions (absence of LIF), suggesting that 3BrP is not simply shifting ESCs towards differentiation, but also causing other effects, and thus that the parallelism between stemness and cancer must be treated with caution. In addition, differentiation seems to be accompanied by a decrease in Gapdh and Hif 1α levels, which is very interesting given that Hif-1α participates in the maintenance of stem cell identity by interacting with core pluripotency transcriptional factors, and also increases the expression of glycolytic enzymes , 8, 16.Although embryonic stem cells (ESC) rely more on glycolysis it is important to note that there may be a metabolically bivalent metabolic state early in cell commitment . RegardlS1 Fig(A) intracellular amounts of superoxide using MitoSOX Red (Molecular Probes); (B) evaluation of the expression of the proliferating cell nuclear antigen (PCNA) and (C) mitochondrial membrane potential using TMRM (Invitrogen). For MitoSOX Red and PCNA experimental conditions were: Control (upper left panel); Control without LIF (upper right); 3BrP 25 μM+LIF (lower left) and 3BrP 50 μM + LIF (lower right). For TMRM analysis the Dot-Plots are: Control (upper left panel); Control without LIF ; Control without LIF for 5 days (upper right panel); 3BrP 25 μM+ LIF (lower left panel) and 3BrP 50 μM + LIF (lower right panel). The histograms show representative experiments in an overlay display in order to better represent the data.ESCs were maintained in control conditions (with LIF), in the absence of LIF (differentiation control) and in the presence of two different 3BrP concentrations (25 and 50 μM) plus LIF. Dot-Plots and overlay of Histograms for analysis of: (TIF)Click here for additional data file.S1 TablePrimers were used for the genes listed as described in the text.(DOCX)Click here for additional data file.


Whole-exome sequencing (WES) arises as a new approach in diagnosing individuals affected by multigenic and complex phenotypes. Herein, we aim to examine whether WES is useful in screening asymptomatic individuals for actionable interventions, which has not yet been established. Twenty-five healthy adults underwent WES, bioinformatics, and manual curation of their exomes. Six participants (24%) harbored significant, management-changing variants in cancer predisposition genes, American College of Medical Genetics, and genomics reportable cardiac diseases and pharmacogenomic biomarkers that have led to clinical recommendations and interventions. Furthermore, more than 80% of the participants (21) carried 1–3 genetic variants with an associated clinical guideline for an altered drug dosing or administration based on the FDA’s table of pharmacogenomics. These results support WES potential not only to answer specific diagnostic questions presented by the relevant personal and/or family history but also to uncover clinically important genetic findings unrelated to the primary indication for sequencing. Next-generation sequencing (NGS) has revolutionized both medical research and diagnostics  published guidelines and recommendations for which are the genes where pathogenic mutations are detected in the course of clinical WES should be reported to the patients even if there is a lack of relevant family history. Currently, it is recommended to include mutations in 56 genes that are associated with highly penetrant, monogenic disorders , all adults (age 25 and above), who expressed interest in sequencing their exome, were recruited from the Integrated Cancer Prevention Center at Tel Aviv Medical Center, Tel Aviv, between 2012 and 2013. None of the study participants had a known inherited disorder prior to testing. All study participants received medical counseling from a certified geneticist (EF) and their primary care physician (NA). All participants signed an informed consent. The local ethics committee and the Ministry of Health in Israel approved the study.Study participants  sample-preparation protocol. DNA libraries were then hybridized to exome-capture probes with NimbleGen SeqCap EZ Human Exome Library, version 2.0 . Exome-enriched libraries were sequenced on the HiSeq 2000 (Illumina) with an average coverage of 80-fold per each sample.http://picard.sourceforge.net). Reads were realigned around detected insertions and deletions (indels) and base qualities recalibrated using GATK. Variant calling was performed using the GATK UnifiedGenotyper tool. Variants with low base or mapping qualities, demonstrating strand bias, aberrant read position distribution or reference versus alternate quality score discrepancy were marked and filtered from subsequent analysis. Variants were annotated using ANNOVAR , NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/) and a personal database of 100 Ashkenazi Jews exomes. Insertions and deletions (Indels) found adjacent to homopolymers longer than 5 bases or repeats were marked and were not considered as true candidates. For monogenic diseases diagnosis, variants with allele frequencies higher than 1% in any of the databases were considered prevalent and were excluded from downstream analysis. For polygenic diseases and pharmacologic-related variants, variants with frequency higher than 10% were excluded. Variant severity was predicted using 10 different prediction tools gathered by dbNSFP , Orphanet (http://www.orpha.net), and the Human Phenotype Ontology , using the Burrows Wheeler Aligner (BWA) . Drug-response associations with level of evidence of 1B or higher  were reported and further crossed with the FDA’s table of pharmacogenomic biomarkers in drug labeling and manually reviewed.Genotype data at loci with reported clinical drug-response predictions were intersected with annotations cataloged in the PharmGKB knowledge base (The multidisciplinary team composed of a genetic counselor (E.F.), an internist and gastroenterologist (N.A.), a pharmacology specialist (M.B.), and a bioinformatics specialist (N.S.) manually reviewed the variants. When needed (Mati Berkovitz), the team sought the input of the appropriate specialist. The team reviewed all variants predicted to be deleterious by combining computational algorithms. This consortium performed literature review using several databases  and classified candidate variants according to variant and gene-level evidence for pathogenicity and description of the clinical phenotype for reporting.APC (OMIM 611731), targeted for I1307K and E1317Q variants. 100% concordance was noted between these and exome analysis. In addition, 1–2 clinically relevant mutations, selected by a multidisciplinary team per each participant were validated via Sanger sequencing with 95% concordance.All participants underwent Sanger sequencing of All participants were Caucasians of Jewish origin, the median age was 52 (35–74) years and 60% were males. Demographics of study participants are presented on Each participant harbored an average of 969,737 coding variants , of which 235 (197–313) were defined as rare and severe, and 23 (1–86) were novel single-nucleotide variants . Two topAn average of 1.4 cancer-related genes with predictable deleterious variants were noted per participant (range 0–3 genes). Three harbored more than one cancer predisposition gene mutation.BRCA1 (OMIM 113705), BRCA2 (OMIM 600185), and PALB2 (OMIM 610355 were present in four patients (16%); Lynch syndrome-related gene sequence alterations – MLH1 (OMIM 120436), MSH2 (OMIM 609309), MSH6 (OMIM 600678), PMS2 (OMIM 600259), and PMS1 (OMIM 600258 were detected in six patients (24%).Breast cancer-related variants – Six patients (24%) harbored deleterious variants in CVS disorders-related genes. Thoracic aortic aneurysms-related variants were detected in five patients (20%) and one patient (5%) had hypertrophic cardiomyopathy-related variant. Variants predicted to be deleterious in cancer, cardiovascular, and pharmacogenomics genes are presented in FigureAfter genetic variants discovery in all participants was concluded and deleterious variants were flagged and validated, a multidisciplinary team assessed all seemingly deleterious variants. The dispersion of variants predicted to harbor deleterious mutations are displayed in FigureCYP2C19  is reportedly associated with reduced efficacy of Clopidogrel treatment. Hence, Clopidogrel dosage was increased.A 55-year-old male with severe ischemic heart disease (three coronary angioplasties) who is treated with Clopidogrel. A nonsynonymous single-nucleotide variation in BRCA2 (rs80359550) was found. At genetic consultation meticulous breast cancer screening by MRI alternating with breast mammograms every 6–12 months as well as risk reduction bilateral salpingo-oophorectomy (RRBSO) were recommended.A 43-year-old female with no family history of breast or ovarian cancer, but a father was diagnosed with pancreatic cancer at age 67 years. A frameshift deleterious mutation – a 19 nucleotide deletion in exon 11 in MSH2 (rs202026056) and MSH6 (rs376220212) were detected. Recommendation to adhere to a Lynch syndrome surveillance scheme was made.A 55-year-old male with two tubulovillous adenomas (7 mm in the transverse colon and 5 mm in the cecum) were resected during routine screening colonoscopy at age 50 years. Two nonsynonymous single-nucleotide variations in MYH11 . The patient was prescribed β-blockers and advised to decrease exertion physical activity.A 54-year-old physically active male with an incidental finding of dilated ascending aorta that was stable for 7 years. He was found to harbor a nonsynonumous single-nucleotide variation in BMPR1A  was detected and led to recommendation to perform gastroscopy and capsule endoscopy with the next colonoscopy procedure.A 48-year-old female without personal or family history of gastrointestinal malignancy. A nonsynonymous single-nucleotide variation in PMS1 (rs142159998) gene was detected and assessed as potentially related to the phenotype and led to the recommendation to continue the appropriate Hereditary nonpolyposis colorectal cancer (HNPCC) surveillance. Her daughter, 42-year old, does not carry this mutation; hence she was switched from an HNPCC surveillance program to normal colorectal cancer (CRC) screening program.A 74-year-old woman who underwent removal of recurrent advanced adenomas on three sequential colonoscopies. A nonsynonymous single-nucleotide variation in Fourteen additional participants were found to carry genetic variants categorized as disease causing in the Human Gene Mutation Database (HGMD) that the medical consulting team had reclassified as reportable variants of uncertain significance.WES is increasingly used in clinical medicine and has the potential not only to answer specific diagnostic questions presented by the relevant personal and/or family history but also to uncover clinically important genetic findings unrelated to the primary indication for sequencing.This study aimed to evaluate the feasibility and yield of applying WES as a screening tool for the general, healthy population. We used an “upside down” approach to genetic-based diagnosis, where subjects were initially screened for pathogenic mutations followed by manual curation and disclosing actionable results to the primary physician and participants with specific recommendations whenever appropriate. Six of the participants (24%) manifested inherited disease-risk variants that are likely pathogenic and have led to significant alterations in the clinical recommendations and treatment. More than 80% of the participants carried 1–3 genetic variants with an associated clinical guideline for an altered drug dosing or administration based on the FDA’s table of pharmacogenomics.Despite major improvements in analyzing and interpreting the genomic data, processing of WES data into actionable decisions is complex. From the clinical geneticists’ perspective the ability to generate a large amount of genetic data for each counseled individual in a single experiment and the need to interpret these data into concrete, clinically relevant recommendations to be disclosed to the participant is a major undertaking. Unlike “classical” genetic testing that focuses on one or a few genes in the context of a specific personal and family history, WES provides a new framework of reference, and emphasizes the need to generate new adoptable guidelines that would ensure that all possible benefits as well as confidentiality are adequately maintained. Manual curation and biological validation remain a crucial step in results accuracy and assigning putative clinical significance. The number of reportable genetic findings from WES is likely to differ with sequencing expertise, variation in pathogenicity classifications and manual validation methodology, as well as the often unknown or unreported family history. Furthermore, the entire spectrum of clinically significant mutation identification is not satisfied by exome sequencing alone at present. Structural variants are not reliably identified and the overall exome coverage varies between genes and exons, so that some genome coding areas are not consistently covered at a read depth that is sufficient for a comprehensive, clinically significant assignment of genetic variants , required a median of 45 min  per genetic variant.One of the major strengths of this study is the comprehensive multidisciplinary follow up of all cases over 10 years in a single tertiary medical center. This enabled thorough documentation of the personal and family phenotype as these evolved, facilitating correlation of the phenotype with the WES generated genotype and allowed to check additional family members according to the genomic findings.However, given the small study sample, of mostly Jewish Ashkenazim from a single medical center in Israel, we have limited ability to generalize the results to other, larger and ethnically diverse populations.Currently, genomic risk information disclosure will need to be conveyed by a trained team of physicians and genetic counselors. Until accepted guidelines and generally accessible and reliable data sources exist for clinical interpretation of genetic variants this approach should be adopted. However, as more evidence accumulates screening asymptomatic individuals, still in its infancy appears as a very promising technology in the near future.In conclusion, this study showed that about a quarter of 25 healthy adult Jewish individuals harbored significant, actionable variants in cancer predisposition genes, ACMG reportable mutations for cardiac diseases and pharmacogenomics biomarkers, variants that have led to clinical recommendations, actions and surveillance.The authors declare that they have no conflict of interest.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.


Early influenza activity during the 2012–13 season  tested positive for influenza virus by rRT-PCR . The proAmong the patients with influenza, 32% had been administered the 2012–13 seasonal influenza vaccine, compared with 50% of the influenza-negative controls . For allAmong the 751 infections with influenza A viruses, 560 (75%) had been subtyped; 546 (98%) of the infections were caused by influenza A (H3N2) viruses . The adjA total of 366 (33%) of the 1,115 cases had infections caused by influenza B viruses . The adjThese updated and age-adjusted VE estimates for the 2012–13 influenza vaccine confirm moderate effectiveness in preventing outpatient medical visits caused by circulating influenza viruses, similar to earlier unadjusted estimates in the United States ,5. OveraConfirmation of the protective benefits of the 2012–13 influenza vaccine among persons aged 6 months–64 years offers further support for the public health benefit of annual seasonal influenza vaccination and supports the expansion of vaccination, particularly among younger age groups. The nonsignificant adjusted VE of 9% against A (H3N2) among persons aged ≥65 years is similar to the estimate in a recent interim report from Europe (One possible explanation for these findings is that some older adults did not mount an effective immune response to the influenza A (H3N2) component of this season’s vaccine. Nonetheless, this finding should not discourage future vaccination by persons aged ≥65 years, who are at greater risk for more severe cases and complications from influenza. Influenza vaccines remain the best preventive tool available, and VE is known to vary by virus type/subtype, age group, season, host immunity, and the outcome measured . This stClinicians should maintain a high index of suspicion for influenza infection among persons with acute respiratory illness while influenza activity is ongoing. Early antiviral treatment can reduce influenza-associated illness severity and complications ; this seThe findings in this report are subject to at least four limitations. First, the observational study design has greater potential for confounding and bias relative to randomized clinical trials. Second, although these midseason VE estimates were adjusted for potential confounders identified in previous studies , additioAlthough imperfect, influenza vaccines remain the best tool currently available for preventing illness from influenza. This report highlights the value of both increasing the use of influenza vaccines, especially among children and young adults, and continuing efforts to develop more effective vaccines and vaccination strategies. Antiviral medications are important for the treatment and control of influenza and should be used as recommended, regardless of patient vaccination status.What is already known on this topic?Annual vaccination is the mainstay of influenza prevention, but overall effectiveness of the influenza vaccine is moderate and varies by year, virus type, and population subgroup. Early unadjusted interim estimates of overall vaccine effectiveness (VE) for the 2012–13 season indicated the vaccine was 62% effective among all ages at preventing medically attended, laboratory-confirmed influenza A and B virus infections.What is added by this report?This report provides updated and adjusted VE estimates for the 2012–13 influenza season based on data from 2,697 children and adults with acute respiratory illness enrolled in the U.S. Influenza Vaccine Effectiveness (Flu VE) Network during December 3, 2012–January 19, 2013. The overall VE  for all ages at preventing medically attended influenza A and B virus infections was 56% . VE was estimated at 47% against influenza A (H3N2) virus infections and 67% against influenza B virus infections. VE against influenza A (H3N2) was lower and not statistically significant among adults aged ≥65 years.What are the implications for public health practice?The 2012–13 seasonal influenza vaccine provides substantial protection for the population overall, which underscores the public health value of vaccination. Nonetheless, some vaccinated persons have become ill with influenza this season, especially among persons aged ≥65 years. Antiviral medications are an important second line of defense against influenza and should be used promptly, as recommended for treatment of suspected influenza in certain patients in high-risk groups, including those aged ≥65 years, regardless of their vaccination status.


FBXW7, an E3-ubiquitin protein ligase in SCFs (SKP1-cullin-F-box) complex, is a major human tumor suppressor gene, and understanding mechanisms by which FBXW7 contributes to tumorigenesis is critical for the treatment of human cancers with FBXW7 deficiency. Long non-coding RNAs (lncRNAs) have emerged as key regulators of various biological processes. Here we have identified a set of lncRNAs that are associated with Fbxw7 deficiency. The correlation network and functional annotation analysis revealed that Fbxw7-associated lncRNAs regulate genes involved in cell cycle, DNA repair, metabolic process, and cell communication and adhesion. The number of coding genes that correlated with individual lncRNAs varied largely. A lncRNA on chromosome 15 (A_30_P01032978), which was upregulated in tumors from Fbxw7 deficient mice was positively correlated with 15 coding genes. High expression of this 15-gene signature was associated with poor prognosis in two independent human breast cancer studies. Our results open possible new avenues to understand mechanisms by which Fbxw7 deficiency increases tumor susceptibility via the alteration of lncRNAs. Long non-coding RNAs (lncRNAs), previously thought of as transcriptional noise, are emerging as key regulators of a multitude of cellular processes by taking part in epigenetic, transcriptional, and post-transcriptional regulation of gene expression ,2. The lFBXW7 is emerging as a major human tumor suppressor that lies at the nexus of several pathways that control cell growth, differentiation and tumorigenesis. Deletion and/or mutations of FBXW7 have been found in cancers from a wide spectrum of human tissues . The oveOur previous studies showed that temporal pharmacological inhibition of the mTOR pathway was sufficient to suppress the tumor development contributed by Fbxw7 loss, suggesting that the Fbxw7-mTOR pathway plays a major role in this radiation-induced carcinogenesis mouse model . We furtFBXW7 is one of the most important tumor suppressor genes in human cancer. However, the mechanisms by which FBXW7 contribute to tumor development still remain largely unclear. To gain insight into the molecular mechanisms, we identified the lncRNAs that are associated with FBXW7 deficiency, since lncRNAs have an emerging role in cancer development and progression. Therefore, we transcriptionally profiled radiation-induced thymic lymphomas from Fbxw7+/− p53+/− mice (n=13) or from p53+/− mice (n=16) using microarrays that contained both mRNAs and lncRNAs. In comparison to transcriptome of thymic lymphomas from p53+/− mice, we found 2205 differentially expressed transcripts in thymic lymphomas from Fbxw7+/− p53+/− mice  based on expression levels in all tumors for 2205 differentially expressed transcripts. Five major subnetworks were observed, four of which contained lncRNAs . Gene onNot surprisingly, we found that the number of coding genes correlated with lncRNAs covered a wide range . While tTwo lncRNAs on chromosome 18 approximately 40 kb apart were found to be correlated with Anxa2 and Cecr2 (lncRNA position: 5119300) and Cecr2, Zeb1 and Zfp438 (lncRNA position: 5162836)  in the lThe interferon enriched subnetwork contained 10 independent lncRNA probes corresponding to three closely spaced genomic loci on chromosome 1 at an interferon-inducible gene family locus . The genTo investigate if FBXW7-associated lncRNAs are relevant to human cancer, we first found human homologs of the coding genes that are significantly correlated with lncRNAs and then examine whether these human homologous genes are correlated with clinical outcome. As shown in In summary, our results open possible new avenues to understand mechanisms by which Fbxw7 deficiency increases tumor susceptibility via the alteration of lncRNAs.All animal experiments were performed at Lawrence Berkeley National Laboratory and the study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was approved by the Animal Welfare and Research Committee of the Lawrence Berkeley National Laboratory. Detailed methods were described previously . BrieflyTotal RNA quality and quantity were determined using Agilent 2100 Bioanalyzer and NanoDrop ND-1000. Agilent SurePrint G3 Mouse GE 8x60K Microarrays were used according to the manufacturer’s protocol . All processes were performed by Ambry Genetics . Microarray data have been deposited at NCBI GEO (accession number: GSE71975).Data normalization was performed using GeneSpring GX12.5 (Agilent Technologies). Signal intensities for each probe were normalized to the 75th percentile without baseline transformation. Genes that were differentially expressed in thymic lymphomas between vehicle and rapamycin treated Fbxw7+/− p53+/− mice and p53+/− mice were identified by the unpaired Student's t-test with a p-value cut-off of less than 0.05 and a fold change criteria of more than 1.3. Correlation analysis was performed in R using Spearman rank order correlation. Transcript correlation networks were generated using Cytoscape using the Expression Correlation Network plugin with a correlation coefficient cut-off of 0.8. Gene enrichment analysis was performed using the Gene Ontology feature of the WEB-based GEne SeT AnaLysis Toolkit ,25.Mouse genes that correlated in expression with lncRNA A_30_P01032978 were converted to human homologs. Gene expression microarray datasets of breast cancer for which disease-free survival was available were downloaded from the NCBI GEO website (GSE1456 and GSE6532). The sum of expression of all genes within the signature was calculated for each breast cancer patient. Kaplan-Meier plots were constructed and a long-rank test was used to determine differences among disease free survival according to the sum of the normalized expression intensities.TableS1TableS2TableS3TableS4TableS5


HIV involvement of the CNS continues to be a significant problem despite successful use of combination antiretroviral therapy (cART). Drugs of abuse can act in concert with HIV proteins to damage glia and neurons, worsening the neurotoxicity caused by HIV alone. Methamphetamine (METH) is a highly addictive psychostimulant drug, abuse of which has reached epidemic proportions and is associated with high-risk sexual behavior, increased HIV transmission, and development of drug resistance. HIV infection and METH dependence can have synergistic pathological effects, with preferential involvement of frontostriatal circuits. At the molecular level, epigenetic alterations have been reported for both HIV-1 infection and drug abuse, but the neuropathological pathways triggered by their combined effects are less known. We investigated epigenetic changes in the brain associated with HIV and METH. We analyzed postmortem frontal cortex tissue from 27 HIV seropositive individuals, 13 of which had a history of METH dependence, in comparison to 14 cases who never used METH. We detected changes in the expression of DNMT1, at mRNA and protein levels, that resulted in the increase of global DNA methylation. Genome-wide profiling of DNA methylation in a subset of cases, showed differential methylation on genes related to neurodegeneration; dopamine metabolism and transport; and oxidative phosphorylation. We provide evidence for the synergy of HIV and METH dependence on the patterns of DNA methylation on the host brain, which results in a distinctive landscape for the comorbid condition. Importantly, we identified new epigenetic targets that might aid in understanding the aggravated neurodegenerative, cognitive, motor and behavioral symptoms observed in persons living with HIV and addictions. Approximately 40 million people worldwide are infected with the Human Immunodeficiency Virus (HIV). HIV traffics into the central nervous system (CNS) early after infection and could be associated with a spectrum of neurobehavioral conditions ranging from minor neurocognitive disorder (MND) to HIV-associated dementia (HAD) HIV involvement of the CNS continues to be a significant problem despite successful use of combination antiretroviral therapy (cART), which has decreased the incidence of HAD but has not greatly affected the prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) Multiple behavioral risk factors contribute to the transmission of HIV, including injection drug use that represents the second most risky behavior in the United States, accounting for one-third of the AIDS cases HIV infection and METH dependence are frequently comorbid and may have synergistic pathological effects, with preferential involvement of frontostriatal circuits Epigenetic mechanisms play an important role during the infection with retroviruses, including HIV-1, as they mediate the integration of the virus into the host genome, a crucial step in the viral life cycle c-fos and fos-b genes in the nucleus accumbens, which is associated with increased acetylation of histone 4 residues in their promoters Drug abuse alone is known to induce epigenetic changes in the brain which have been related to addictive behaviors Although the epigenetic mechanisms induced by either HIV-1 infection or drug abuse are somewhat defined, the molecular consequences of their co-occurrence are much less explored. The combination of HIV and METH, for example, results in greater interneuron loss and higher activation of reverse transcriptase in macrophages In the present report we provide evidence that concurrent HIV-1 infection and METH use alters the methylome of the host brain, inducing epigenetic and transcriptional changes that appear to be specific for the comorbid condition. We detected increased levels of DNMT1 on the brains of HIV-seropositive cases who used METH, which resulted in increased global DNA methylation. Analysis of the individual loci the showed differential methylation showed enrichment for neurodegenerative diseases; dopamine metabolism; and oxidative phosphorylation, pathways associated with neuronal damage and previously reported to be affected during HIV infection and METH use, and which might be associated with the aggravated neurodegenerative, cognitive, motor and behavioral symptoms observed on HIV seropositive individuals who use METH.We evaluated 27 HIV seropositive cases (HIV+) from the National NeuroAIDS Tissue Consortium (NNTC) autopsy cohort . FourteeWe obtained postmortem brain tissue banked at the University of California, San Diego HIV Neurobehavioral Research Program (HNRP) under the California NeuroAIDS Tissue Network (CNTN) study. The parent study was reviewed and approved by the University of California, San Diego Human Research Protections Program. All subjects provided written informed consent prior to participating in the study and donating tissue.DNA and RNA were extracted from frozen postmortem human frontal cortex samples using the DNeasy Blood and Tissue Mini kit and the RNeasy Lipid Tissue Mini kit (Qiagen), respectively. Quantification of viral DNA and unspliced cellular HIV RNA in the brain samples was performed by the HNRP Neurovirology Core (UCSD) as previously described Genomic DNA was extracted from 25 mg of frozen brain tissue  with DNeasy Blood & Tissue Mini kit (Qiagen). DNA methylation was measured in 200 ng of genomic DNA with the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek). Each sample was run in triplicate.2 First Strand kit (Qiagen) from 1 µg of total RNA. Real-time PCR analysis was performed using specific probes for DNMT1 ; DNMT3B ; TGFBR3 ; and NET1  using Taqman Fast Advanced Master Mix (Life Technologies) on the StepOnePlus real-time PCR system (Applied Biosystems) according to the manufacturer instructions. PCR reactions were performed in duplicate. Relative quantification of gene expression was calculated using β-actin  as an internal control and expressed as the inverse ratio to threshold cycle (1/dCt).Transcript abundance was determined by quantitative real-time PCR (qPCR) using Taqman technology (Life Technologies). Briefly, total RNA was extracted from frozen cortical samples using the RNeasy Lipid Tissue Mini kit (Qiagen) and was reversed-transcribed using RTDNMT1 protein levels were quantified by western blot and immnuhistochemistry as described earlier i+Agej+ARTk+ εijkl where Yijkl represents the lth observation on the ith METH jth Age kth ART µ is the common effect for the whole experiment. εijkl represents the random error present in the lth observation on the ith METH jth Age kth ART. The errors εijkl were assumed to be normally and independently distributed with mean 0 and standard deviation δ for all measurements.Briefly, 500 ng of genomic DNA were converted by bisulfite treatment using the EZ DNA Methylation kit (Zymo) according to the manufacturer’s instructions. Genome-wide methylation profiling was performed on 200 ng of bisulfite-treated DNA using the Illumina Infinium HumanMethylation 450 k BeadChip (Illumina) at the Case Western Reserve University Department of Genetics and Genome Sciences Genomics Core, following standardized protocols at the facility and according to the manufacturer’s instructions. Beadchips were scanned on an Illumina HiScan SQ. The methylation status of a specific CpG site was expressed as β values, calculated as the ratio of the fluorescence intensity signals of the methylated (M) and unmethylated (U) alleles, β = Max/[Max+Max+100]. β values range between 0 (non-methylated) and 1 (completely methylated). The Illumina GenomeStudio Software (version 2011.1) was used to assess quality and extract the DNA methylation signals from scanned arrays. Methylation data was extracted as raw signals with no background subtraction and data was normalized to control probes present on the array. Sample methylation profiles including average β and intensity signals for methylated (M) and unmethylated (UM) probes obtained from the whole array are available as For the validation of microarray discovery we performed Methylation-Sensitive High Resolution Melting (MS-HRM) analysis on a group of genes showing differential methylation in the HIV seropositive (HIV+)/METH+ group, including NET1; TTL7; TGFBR3; SCN1A; UNC5D and APBA1. Primers were designed for bisulfite-converted DNA using Methyl Primer Express software (Applied Biosystems) to produce an amplicon <300 bp that overlapped the Illumina probe-set that showed differential methylation and also included neighbor CpG sites to increase the magnitude of the delta of their melting temperatures (Tm). Presence of CpGs in the primer sequence was avoided to prevent bias due to preferential amplification of the unmethylated target DNA t test  with a significance of p<0.05 or Mann Whitney test, as indicated. Correlation between viral RNA and global DNA methylation was calculated by Spearman’s rho. Linear regression was used to analyze the relation between loci-associated methylation levels and transcript abundance (Prism Graph Pad Software). Fold change of methylation was determined by analysis of variance multivariable analysis using Partek Genomics Suite (Partek) and computing for false discovery rate (FRD) at q<0.05 as described earlier.Statistical analysis was performed using Student’s In the present study we analyzed a total of 27 HIV seropositive cases (HIV+), whose clinical characteristics are presented in t test, unpaired; two tailed at p<0.05 significance).Antemortem neurocognitive diagnosis identified a high rate of cognitive impairment in HIV+METH− cases (13 out of 14 cases) with mostly milder disease (mild neurocognitive disorder (MND) or asymptomatic neurocognitive impairment (ANI)). None had HIV-associated dementia (HAD). In the HIV+METH+ group, 62% individuals had cognitive impairment, with 5 cases of MND and 1 case of HAD. The relatively high rate of cognitive impairment in the non-METH cases may reflect brain injury that occurred before cART became available to these subjects, whose exposure to ARV on average was significantly shorter than the METH group  and for the 13 cases of the HIV+METH+ group (100%). Not all the subjects abused of all the substances .We first quantified the levels of HIV DNA and RNA content on the frontal cortex of the HIV seropositive individuals included in the study, comparing cases with and without METH use . AlthougAs HIV infection and METH use have been reported to alter epigenetic regulation, we next investigated the levels of global DNA methylation in the brain. The HIV+METH+ group had increased global methylation in the frontal cortex , which cThe observed changes in global methylation might have important consequences in the host brain. We therefore explored the epigenetic changes induced by HIV disease in concert with METH dependence by profiling genome-wide DNA methylation in the frontal cortex of a subgroup of samples from our initial cohort, including 6 HIV+METH+ and 6 HIV+METH− cases. Methylome analysis was performed using the Infinium Human 450 K beadchip and GenomeStudio. For the analysis of differential methylation, we applied the Illumina Custom model after normalization to control probes present in the array and using the HIV+METH− group as the reference. We selected probes showing absolute Delta β-values>|0.2| at p<0.01  q <0.001 to control for multiple comparisons) as differentially methylated (DM), a threshold previously suggested to improve detection of DM probes in this array platform with 99% confidence Detection levels were similar among groups, with an average of 485,381 CpG detected at p<0.01. A locus was called as detected at p<0.01 level if the mean signal intensity from multiple probes for that CpG locus was significantly higher at the level of p<0.01 than the negative control on the same chip.www.pantherdb.org) We first compared the methylation profiles between HIV+METH+ cases with HIV+METH− subjects. We detected 235 CpG with differential methylation, with 54% of DM loci showing decreased methylation and the remaining 46% showing increased methylation in the HIV+METH+ . DistribIn order to incorporate in the analysis other biological and clinical factors other than drug use that might modify DNA methylation, we performed a second analysis by exporting normalized β values from Genome Studio into PARTEK Genomic Suite Differential methylation analysis reporting loci with absolute fold change >1.2 at a FDR<0.05 identified a total of 446 gene-associated CGs with differential methylation in the brains of HIV+METH+ subjects, 441 corresponding to autosomal loci; with 204 presenting increased methylation  and 237 We partially verified the array findings by Methylation-Sensitive High Resolution Melting (MS-HRM) analysis, a novel form of real time PCR that uses thermal denaturation differences in bisulfite-converted DNA to create a methylation profile against known methylation standards. We tested a group of genes showing differential methylation in the HIV+METH+ cluster, including UNC5D; TGFBR3 and NET1 that were hypermethylated; and TTL7; SCN1A and APBA1 that were hypomethylated . We obseTGFBR3 transcripts in the HIV+METH+ group Click here for additional data file.Figure S2Pathway analysis showing enrichment for Neurological disease.(PDF)Click here for additional data file.Table S1Complete list of differentially methylated genes.(DOCX)Click here for additional data file.File S1Individual sample methylation profile for all interrogated probes on the array for HIV+METH− cases.(ZIP)Click here for additional data file.File S2Individual sample methylation profile for all interrogated probes on the array for HIV+METH− cases.(ZIP)Click here for additional data file.File S3Individual sample methylation profile for all interrogated probes on the array for HIV+METH+ cases.(ZIP)Click here for additional data file.File S4Individual sample methylation profile for all interrogated probes on the array for HIV+METH+ cases.(ZIP)Click here for additional data file.


Opportunities to treat infection with hepatitis C virus (HCV) are evolving rapidly. From the introduction of interferonα monotherapy in the early 90s' to the approval of telaprevir and boceprevir based triple therapies with pegylated interferonα and ribavirin in 2011, the chances of curing patients infected with HCV genotype 1 have improved dramatically to reach approximately 70%. Significant further improvements, which may cure virtually all hepatitis C patients with an all oral, interferon-free regimen will become available in the very near future. These new direct acting antivirals (DAA) target the viral polymerase with either nucleoside analogues or non nucleosidic inhibitors, the viral protease, and the viral NS5A protein. Several clinical trials have now shown that a combination of sofosbuvir (nucleosidic polymerase inhibitor) with daclatasvir or ledipasvir (NS5A inhibitors), or sofosbuvir with simeprevir (protease inhibitor), or a combination of ABT-450 (proease inhibitor) with ritonavir (ABT-450/r), the nonnucleoside polymerase inhibitor ABT-333 and the NS5A inhibitor ABT-267, can achieve sustained virologic response in up to 95% of naive patients or previously treated patients, even in patients who failed prior treatment with first generation protease inhibitor. As these DAAs are becoming available in early access treatment programs, treatment strategy studies are being performed to optimize treatment regimens with respect to the choice of DAAs and treatment duration, based on viral genotypes, prior treatment exposure, and the presence of liver cirrhosis. The next challenge will be to identify HCV infected patients in the general population and to facilitate access to treatment.


Plasmodium falciparum parasites.Artemisinin resistance is rapidly spreading in Southeast Asia. The efficacy of artemisinin-combination therapy (ACT) continues to be excellent across Africa. We performed parasite transcriptional profiling and genotyping on samples from an antimalarial treatment trial in Uganda. We used qRT-PCR and genotyping to characterize residual circulating parasite populations after treatment with either ACT or ACT-primaquine. Transcripts suggestive of circulating ring stage parasites were present after treatment at a prevalence of >25% until at least 14 days post initiation of treatment. Greater than 98% of all ring stage parasites were cleared within the first 3 days, but subsequently persisted at low concentrations until day 14 after treatment. Genotyping demonstrated a significant decrease in multiplicity of infection within the first 2 days in both ACT and ACT-primaquine arms. However, multiple clone infections persisted until day 14 post treatment. Our data suggest the presence of genetically diverse persisting parasite populations after ACT treatment. Although we did not demonstrate clinical treatment failures after ACT and the viability and transmissibility of persisting ring stage parasites remain to be shown, these findings are of relevance for the interpretation of parasite clearance transmission dynamics and for monitoring drug effects in  P. falciparum malaria, and continued efficacy is crucial for successful elimination and eradication efforts. The high potency of artemisinins results in a rapid initial reduction of the biomass of asexual malaria parasites, while the more slowly eliminated partner drug contributes to parasite clearance and prevents subsequent recrudescent infections468Malaria remains a leading cause of global morbidity and mortality with approximately 584,000 deaths and 198 million clinical cases of malaria in 20132The significance of DNA-based detection of residual parasitemia after ACT treatment is currently unknown. While residual submicroscopic parasitemia has been associated with subsequent recrudescence of infections10Pfs25 QT-NASBA was used for mature gametocyte detection. This analysis demonstrated complete clearance of patent parasitemia at day 3 in both treatment arms, and clearance of mature gametocyte transcripts in 94% of patients by day 14 upon primaquine treatmentP. falciparum parasitesversus mature gametocyte loads upon treatment (PF3D7_1438800 or Pfs25 (from the QT-NASBA data) across patient samples and treatment arms. In contrast the two different gametocyte markers are highly correlated in the AL group  and in the AL-PQ group , and in the subset of persisting parasites in both groups , using a partial correlation test controlling for the time of sampling. Together these data suggest the existence of low-level persistent ring stage parasites upon AL treatment, alone or in combination with primaquine, among African malaria patients from Uganda.In the original analysis of samples analyzed in the AL or AL−PQ treatment arms of the study, total parasitemia and presence of gametocytes were determined by microscopy, and reatment  resultedreatment . We havereatment . This hymsp2 genotyping , however many of them remained >1 , enrollment parasite density  or any other patient or infection characteristics .Residual ring stage parasitemia by day 3 was not significantly associated with patient age . The detection of msp2 genotypes at individual time points is imperfect and influenced by parasite densities, sequestration patterns and differences in amplification efficiency of smaller and larger msp2 allelesIn vitro studies have revealed that parasites may be able to tolerate artemisinin treatment by entering a temporarily growth-arrested state of dormancyThe reason for the persistence of parasite transcripts that is suggestive of ring-stage persistence, after apparently successful treatment is unknown. Low levels of persisting parasites might originate from dormant parasite populations or a sequestered reservoir, might represent remaining parasites during therapy due to therapeutic pharmacokinetic properties, or might be associated with resistance to drugs. We observed no evidence of an association with drug resistance, parasitological and clinical cure rates by microscopy were excellent23in vivo studies are needed to confirm that the persistence of transcripts of ring stage parasites indicate viable parasite populations. These studies should be designed to determine whether the phenomenon of ring stage persistence is a characteristic of ACT treatment and allow for the assessment of late recrudescent infections and/or de novo generation of gametocytes from low levels of persisting parasite populations.In summary, we present preliminary evidence for persisting submicroscopic parasite populations that comprise ring stage parasites and gametocytes after treatment with AL or AL-PQ, treatment combinations that were efficacious in preventing recurrent parasite carriage by microscopy for at least 28 days. While the viability of these parasite populations remains to be established and we observed no evidence that parasite populations have no immediate clinical consequences for the treated patients, our findings are relevant for the interpretation of parasite clearance and transmission dynamics. Future All experiments were performed in accordance with relevant guidelines and regulations. Specifically samples for this study were derived from a previously published randomized, double blind, placebo-controlled trial with four treatment armsP. falciparum mono-infection with a parasite density lower than 500,000 parasites per μL, normal G6PD enzyme function and hemoglobin concentration ≥ 80 g/L were randomized to treatment with AL alone (AL group) or in combination with 0.1, 0.4 or 0.75 mg/kg primaquine (AL-PQ group). For the current analysis, we selected 37 children from the AL arm and 33 children from the AL-PQ arm with a dose of 0.75 mg/kg primaquine, using random tables generated in Stata (version 12.0). Children were followed for 28 days for safety parameters, recrudescence or re-infection. Blood samples were obtained by finger prick on days 0, 3, 7, 10 and 14 after initiation of treatment, and immediately transferred to L6 buffer  and stored at −80 °C until extraction. Nucleic acid material was extracted from 50 μL aliquots using Total Nucleic Acid Isolation Kits–High Performance  and a MagNA Pure LC automated extractor (Roche Applied Science).Children aged 1–10 years with uncomplicated Pfs25 mRNA quantitative nucleic acid sequence based amplificationP. falciparum parasites from patient samples using probe-based qRT-PCR. Reverse transcription was performed from extracted nuclear acid samples using SuperScript III First-Strand Synthesis System . Custom primers  and Taqman probes (Life Technologies) were used at 900 nM and 250 nM final concentrations, respectively, to measure the transcript levels of PF3D7_0501300 , PF3D7_1438800  and PF3D7_1120200  on a Viia7 qRT-PCR machine (Life Technologies).The patient’s parasite burden was quantified using microscopy, gametocyte detection by A linear regression model was developed to estimate the density of circulating ring and mature gametocyte parasites. We used the patient’s patent parasitemia  obtained using Giemsa stained microscopy slide to define the relationship between parasite density and CT values from qRT-PCR  by regression analysis. The regression between microscopy and qRT-PCR was then used to infer parasite density in the remainder of the data points with sub-patent parasite levels . Multiplin vitro conditions with stage-specific populations of P. falciparum strain 3D7  medium for sexual parasite stages30To determine the sensitivity of our ring marker detection by qRT-PCR assay, we performed a cellular dilution experiment under controlled rain 3D7 . Parasitmsp2) using capillary electrophoresis for fragment sizingDNA from all time points was tested for clonal complexity based on genotyping for the polymorphic marker gene merozoite surface protein 2 .


Catastrophic floods generated ~3.2 Ga by rapid groundwater evacuation scoured the Solar System’s most voluminous channels, the southern circum-Chryse outflow channels. Based on Viking Orbiter data analysis, it was hypothesized that these outflows emanated from a global Hesperian cryosphere-confined aquifer that was infused by south polar meltwater infiltration into the planet’s upper crust. In this model, the outflow channels formed along zones of superlithostatic pressure generated by pronounced elevation differences around the Highland-Lowland Dichotomy Boundary. However, the restricted geographic location of the channels indicates that these conditions were not uniform Boundary. Furthermore, some outflow channel sources are too high to have been fed by south polar basal melting. Using more recent mission data, we argue that during the Late Noachian fluvial and glacial sediments were deposited into a clastic wedge within a paleo-basin located in the southern circum-Chryse region, which was then completely submerged under a primordial northern plains ocean. Subsequent Late Hesperian outflow channels were sourced from within these geologic materials and formed by gigantic groundwater outbursts driven by an elevated hydraulic head from the Valles Marineris region. Thus, our findings link the formation of the southern circum-Chryse outflow channels to ancient marine, glacial, and fluvial erosion and sedimentation. At the end of the Noachian Period Mars experienced an abrupt transition into a climate dominated by extremely cold and dry conditions, which resulted in the subsequent confinement of the planet’s hydrosphere in highly pressurized aquifers beneath thick upper crustal permafrost materials111In order to explain the relative localized occurrence47101212The long-held view is that regional groundwater outbursts led to the formation of extensive chaotic terrains  that are the sources of the southern circum-Chryse outflow channels5141516171819121312These chaotic terrains have been mapped in detail by previous investigators41612The southern Margaritifer Terra highlands are extensively dissected by vast canyon systems  and 2a, The terrain contact between the upland and hybrid canyons is defined by elevations ranging between −2050 m and −1900 m , label 12425262725262728We propose the following regional geologic reconstruction leading to the development of the OFCS aquifers: The vast fluvial, or glacial-glaciofluvial, canyon systems of southern Margaritifer Terra42et al.We infer a relatively short-lasting duration for the conditions allowing deep upland erosion connected with the formation of the proposed massive clastic sedimentary wedge based on previous investigations that propose a major spike in erosional and sedimentation rates occurring near the Noachian-Hesperian boundary3435363738394041423435363738394041425 km3 of groundwater and eroded or fluidized sediments were rapidly evacuated from the hypothesized caverns. This value was calculated as the total area of subsided highlands (~4 × 105 km2) times an average negative relief of ~700 m .The rapid groundwater evacuation required to generate the Late Hesperian catastrophic floods would have required groundwater migration via extraordinarily permeable structures, most likely including extensive systems of large interconnected caverns filled with water and/or unconsolidated fluidizable sediment12The distribution of the subsided valleys, here proposed to have been largely controlled by the distribution of the ancient buried troughs, indicates that the hypothesized caverns formed a direct connection between eastern Valles Marineris and a lower-lying equatorial belt of chaotic terrains located at the upstream portions of numerous outflow channels ), where hw is the relief in meters (~3500 m to ~4500 m), g is Martian surface gravity (3.711 m/s2), ρ is water density (1000 kg/m3). These estimates of regional groundwater pressurization approximate or exceed the upper estimate predicted to have caused outflow channel activity in circum-Chryse [10 MPa]2, or contained large volumes of fine-grained sediments. Following the groundwater outbursts, the hydraulic head within the previously confined cavernous water systems would have rapidly diminished due to drawdown of the regional water table, however, continued exsolution of CO2 could have maintained the outflow channel discharges.Elaver Valles , locatedOur proposed scenario implies that the sedimentary deposits that buried the troughs were highly porous and likely included large volumes of hydrated clays and perhaps glacal ice. As Oceanus Borealis receded, these water-saturated deposits would have progressively frozen into extensive areas of ice-rich permafrost .


IRS1 has been implicated in insulin resistance, type 2 diabetes, adverse lipid profile, and cardiovascular risk. To gain further insight into this chromosomal region, we typed four SNPs in a cross-sectional cohort and subjects with type 2 diabetes recruited from the same geographic region. From 16 possible haplotypes, 6 haplotypes with frequencies >0.01 were observed. We identified one haplotype that was protective against insulin resistance (determined by HOMA-IR and fasting plasma insulin levels), type 2 diabetes, an adverse lipid profile, increased C-reactive protein, and asymptomatic atherosclerotic disease (assessed by intima media thickness of the common carotid arteries). BMI and total adipose tissue mass as well as visceral and subcutaneous adipose tissue mass did not differ between the reference and protective haplotypes. In 92 subjects, we observed an association of the protective haplotype with higher skeletal muscle mRNA levels of LOC646736, which is located in the same haplotype block as the informative SNPs and is mainly expressed in skeletal muscle, but only at very low levels in liver or adipose tissues. These data suggest a role for LOC646736 in human insulin resistance and warrant further studies on the functional effects of this locus.The genomic region ~500 kb upstream of  IRS1, was the first type 2 diabetes risk locus identified in a GWAS that was linked to IR and hyperinsulinemia . Furthermore, subjects homozygous for the more common allele of rs2713538 displayed nominal higher expected mean log-transformed IRS1 transcript levels in comparison to carriers of the less common allele .fference . Among tIn silico analyses of LOC646736 transcripts showed that exon 2 has been exapted from a primate-specific Alu short interspersed element (SINE). The exonization of such elements is a mechanism of genomic diversification that appears to be specific for higher mammals [ LOC646736 were observed in greater and lesser hominoids, but not in other mammals. Our in silico analyses identified several open reading frames (ORFs). The largest ORF extending from exon 1 to exon 3 predicts a protein of 124 amino acids with a molecular weight of 14151 Da. Another ORF, present in several transcripts and encoded by exons 5 to 7, predicts a shorter protein of 73 amino acids with a molecular weight of 8705 Da. Clones harboring the respective coding sequences were translated in HeLa cell lysates and gave rise to proteins of the expected sizes. Furthermore, transfections of primary human myoblasts with the two sequences cloned in-frame into enhanced green fluorescent protein (eGFP) expression vectors resulted in a fluorescent signal indicating expression of the respective fusion proteins  that is protective against IR, type 2 diabetes, and preclinical atherosclerosis.The identification of DNA variants underlying diabetes susceptibility and/or its clinical hallmarks should provide a pathophysiological perspective of disease subtypes and therefore may afford the development of more specific therapies , 26. HowIn the original study, rs2943641 was associated with several markers of IR in three populations . HoweverA new finding of our studies is the association of the protective haplotype with reduced CRP levels. Chronic, subclinical inflammation of adipose tissue significantly contributes to IR . While e IRS1 as the causal locus functionally affected by the polymorphisms. The original study showed moderate associations of rs2943641 with IRS1 protein levels in skeletal muscle in the basal state (P = 0.03), but not after in vivo stimulation with insulin. Our IRS1 mRNA results in a smaller population are compatible with the original findings, as subjects homozygous for the less common allele at rs2943641 showed the highest IRS1 transcript levels. Importantly, the original study showed a strong association of IRS1-phosphatidylinositol-3-OH kinase (PI(3)K) activity with rs2943641 genotypes during in vivo insulin infusion (P = 0.001). These data strongly suggest genotype effects on insulin signal transduction in skeletal muscle but do not necessarily argue for the causality of IRS1, as changes in the amino acid sequence of IRS1 or posttranslational modifications would have to be postulated that affect its association with PI(3)K. However, the three known IRS1 alleles resulting in amino acid substitutions  occur with low frequencies (<0.06) [ IRS1 transcript levels in the subcutaneous and visceral adipose tissue in both men and women (N > 500), but the association appeared to be stronger in men [ LOC646736 and upstream of IRS1, with BMI in 152893 men [ IRS1 as the causal locus, it is currently not clear whether insulin signaling in skeletal muscle or adipose tissue biology that may result in insulin resistance is primarily affected by the genomic region upstream of IRS1.Two recent studies have implicated (<0.06) , 29, whi2893 men . Thus, w IRS1 as the causal locus, but they suggest that LOC646376 may also contribute to IR-related phenotypes. First, LOC646736 is localized within the LD block of the informative SNPs; second, the same haplotype was protective against all the interrelated phenotypes studied; third, LOC646736 transcript levels are mainly expressed in skeletal muscle; fourth, the protective haplotype was associated with the greatest abundance of skeletal muscle LOC646736 transcripts; and fifth, skeletal muscle LOC646736 mRNA levels were higher in women than in men.Our results do not exclude LOC646736 transcripts are translated in in vitro expression systems and also after transfection of various cell lines. Clearly, it will be of utmost importance to determine whether the putative proteins are expressed in human tissues. It is also possible that LOC646736 transcripts represent lincRNA, implicated in the coordination of coherent protein responses [ NYAP2, located in a distinct LD block ~400 kb upstream of LOC646736, deserve consideration. NYAP2 is mainly expressed in brain, where it mediates the interaction of PI(3)K with the Wiscott-Aldrich Syndrome Protein Family, member 1 complex, thereby regulating neuronal morphogenesis [ IRS1 and outside of the LOC646736 LD block. Finally, a known copy-number variant (CNV1152.1) is tagged by three SNPs  that are in moderate LD with rs2943634 (r2 ~ 0.44) [To this end, we show that two differentesponses , 31. Henogenesis . miR-570 ~ 0.44) . LOC64736 homologs in rodents may argue against its role in IR. However, additional control mechanisms may have evolved in higher mammalian species. An example for such fine-tuning is TBC1D3, a hominoid-specific gene that promotes insulin signaling by delaying its degradation through suppression of phosphorylation of critical serine residues [As insulin signaling and skeletal muscle metabolism are thought to be similar between humans and rodents, the absence ofresidues . IRS1 that protects against IR and related metabolic abnormalities. Our data are consistent with a role of LOC646736 in IR-related phenotypes. Clearly, functional studies will be required to unravel the complexity associated with this genomic region.In conclusion, our studies have defined a haplotype upstream ofin vitro translation and eGFP constructs, genotypes by SNPs and sex, pairwise linkage disequilibria between SNPs and associations of haplotypes with traits by sex.Supplementary Material contains 17 Supplementary Tables showing additional clinical data of study subjects, sequences of primers used for PCR and generation of