Human epigenetic and transcriptional T cell differentiation atlas for identifying functional T cell-specific enhancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

Regulation network analysis of testicular seminoma at various stages of progression.

Testicular seminoma has become the most common solid malignancy in young men, especially in the 20s group. We obtained the gene expression profile of human testicular seminoma cells from NCBI, identified the differentially expressed genes of testicular seminoma cells of different stages, and constructed the regulation networks of different stages of testicular seminoma using bioinformatics methodology. Forty differentially expressed genes of testicular seminoma cells of different stages were identified. These genes and pathways are apparently involved in the progression of testicular seminoma.

Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet.

The aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat and high-fructose corn syrup (ALIOS diet). Specifically, we examined whether treatment with liraglutide could reduce hepatic insulin resistance and steatosis as well as improve cardiac function. Male C57BL/6J mice were pair fed or fed ad libitum either standard chow or the ALIOS diet. After 8 wk the mice were further subdivided and received daily injections of either liraglutide or saline for 4 wk. Hyperinsulinemic-euglycemic clamp studies were performed after 6 wk, revealing hepatic insulin resistance. Glucose tolerance and insulin resistance tests were performed at 8 and 12 wk prior to and following liraglutide treatment. Liver pathology, cardiac measurements, blood chemistry, and RNA and protein analyses were performed. Clamp studies revealed hepatic insulin resistance after 6 wk of ALIOS diet. Liraglutide reduced visceral adiposity and liver weight (P < 0.001). As expected, liraglutide improved glucose and insulin tolerance. Liraglutide improved hypertension (P < 0.05) and reduced cardiac hypertrophy. Surprisingly, liver from liraglutide-treated mice had significantly higher levels of fatty acid binding protein, acyl-CoA oxidase II, very long-chain acyl-CoA dehydrogenase, and microsomal triglyceride transfer protein. We conclude that liraglutide reduces the harmful effects of an ALIOS diet by improving insulin sensitivity and by reducing lipid accumulation in liver through multiple mechanisms including, transport, and increase ß-oxidation.

Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against hepatic fibrosis.

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad-/-) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3-Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad-/- mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad-/-mice than in wild-type mice. Adiponectin also promotes SOCS-3-Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)-MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.

Adiponectin modulates C-jun N-terminal kinase and mammalian target of rapamycin and inhibits hepatocellular carcinoma.

BACKGROUND & AIMS: Epidemiological studies have shown that obesity is a risk factor for hepatocellular carcinoma (HCC). Lower adiponectin levels are associated with poor prognosis in obese HCC patients, hence it is plausible that adiponectin acts as a negative regulator of HCC. We investigated the effects of adiponectin on HCC development and its molecular mechanisms. METHODS: Assays with Huh7 and HepG2 HCC cells were used to examine the signal transduction pathways involved in the protective functions of adiponectin in HCC. These studies were followed by in vivo approaches using HCC xenografts and tumor analysis. Results from in vitro and in vivo findings were corroborated using human HCC tissue microarray and analysis of clinicopathological characteristics. RESULTS: Adiponectin increased apoptosis of HCC cells through activation of caspase-3. Adiponectin increased phosphorylation of c-Jun-N-terminal kinase (JNK) and inhibition of c-Jun-N-terminal kinase-phosphorylation inhibited adiponectin-induced apoptosis and caspase-3 activation. Adiponectin increased phosphorylation of 5'-adenosine monophosphate-activated protein kinase and tumor suppressor tuberous sclerosis complex 2 and inhibited mammalian target of rapamycin phosphorylation. Inhibition of 5'-adenosine monophosphate-activated protein kinase phosphorylation not only inhibited adiponectin-induced c-Jun-N-terminal kinase phosphorylation, but also blocked biological effects of adiponectin. Adiponectin substantially reduced liver tumorigenesis in nude mice. Importantly, analysis of adiponectin expression levels in tissue microarray of human HCC patients revealed an inverse correlation of adiponectin expression with tumor size. CONCLUSIONS: Adiponectin protects against liver tumorigenesis; its reduced expression is associated with poor prognosis in obese patients with HCC.

Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis.

UNLABELLED: Obesity is rapidly becoming a pandemic and is associated with increased carcinogenesis. Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related carcinogenesis. Recently, we reported the oncogenic role of leptin including its potential to increase tumor invasiveness and migration of hepatocellular carcinoma (HCC) cells. In the present study we investigated whether adiponectin could antagonize the oncogenic actions of leptin in HCC. We employed HCC cell lines HepG2 and Huh7, the nude mice-xenograft model of HCC, and immunohistochemistry data from tissue-microarray to demonstrate the antagonistic role of adiponectin on the oncogenic actions of leptin. Adiponectin treatment inhibited leptin-induced cell proliferation of HCC cells. Using scratch-migration and electric cell-substrate impedance-sensing-based migration assays, we found that adiponectin inhibited leptin-induced migration of HCC cells. Adiponectin treatment effectively blocked leptin-induced invasion of HCC cells in Matrigel invasion assays. Although leptin inhibited apoptosis in HCC cells, we found that adiponectin treatment induced apoptosis even in the presence of leptin. Analysis of the underlying molecular mechanisms revealed that adiponectin treatment reduced leptin-induced Stat3 and Akt phosphorylation. Adiponectin also increased suppressor of cytokine signaling (SOCS3), a physiologic negative regulator of leptin signal transduction. Importantly, adiponectin significantly reduced leptin-induced tumor burden in nude mice. In HCC samples, leptin expression significantly correlated with HCC proliferation as evaluated by Ki-67, whereas adiponectin expression correlated significantly with increased disease-free survival and inversely with tumor size and local recurrence. CONCLUSION: Collectively, these data demonstrate that adiponectin has the molecular potential to inhibit the oncogenic actions of leptin by blocking downstream effector molecules.

Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart.

Increased coronary blood vessel development could potentially benefit patients with ischemic heart disease. In a model of stress-induced myocardial ischemia, intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor-5 (FGF-5) resulted in messenger RNA and protein expression of the transferred gene. Two weeks after gene transfer, regional abnormalities in stress-induced function and blood flow were improved, effects that persisted for 12 weeks. Improved blood flow and function were associated with evidence of angiogenesis. This report documents, for the first time, successful amelioration of abnormalities in myocardial blood flow and function following in vivo gene transfer.

Safety effects of street lighting on roadway segments: Development of a crash modification function.

Objective: Nighttime crashes are overrepresented on the U.S. highway system. Roadway lighting, which provides additional visibility by supplementing vehicle headlights, has been identified as an effective countermeasure to improve nighttime safety. However, the existing literature does not provide a thorough understanding of the effects of street lighting photometric characteristics on nighttime crash occurrence on roadway segments. This study aimed to investigate the relationship between lighting photometric measures and nighttime crash risk on roadway segments and develop a crash modification function/factor (CMF). Methods: The research team collected horizontal illuminance data on 440 roadway segments between 2 successive signalized intersections in Florida for 2012-2014 and matched 4 years of nighttime and daylight crash data (2011-2014). Random parameter negative binomial models were estimated for both nighttime and daylight crash frequencies. The expected night-to-day crash odds ratio, as an equivalent of CMF, was derived from the fitted models with the correction of estimation variances. The confidence intervals (CIs) of the developed CMF were estimated using the Cox method. Results: The coefficient of the mean of horizontal illuminance is significantly negative in the nighttime model. The coefficients of the standard deviation of horizontal illuminance are significantly positive and normally distributed in both the nighttime and daylight models. The significance of the standard deviation in the daylight model captures the confounding effects-a high standard deviation correlates with high traffic exposures, poor safety design standards, and low maintenance quality. The CMF based on the expected daylight-to-day odds ratio was developed as an exponential function of the increments and the increment squares of the mean and the standard deviation of horizontal illuminance. Its 95% CIs indicate that the CMF is almost significant over the whole range. Other significant variables contributing to nighttime crash risk include annual average daily traffic, truck percentage, segment length, access density, undivided roads, and urban/city limits. Conclusions: Horizontal illuminance characteristics have a significant impact on nighttime crash risk on roadway segments. An increase in the mean of horizontal illuminance, indicating an improvement in average lighting level, tends to decrease nighttime crash risk; an increase in the standard deviation, representing a poor uniformity of lighting pattern on a roadway segment, is more likely to raise nighttime crash risk. Because the 2 measures are strongly correlated in a low mean range (<0.44 fc), the 2 photometric measures need to be considered together to interpret the safety effects of lighting patterns. The standard deviation shows better performance in measuring lighting uniformity on a roadway segment than the traditional ratios (max-to-min and mean-to-min). However, a new photometric measure is needed to capture the true lighting pattern influencing driver vision at night.

Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi alpha 2) and beta-adrenergic receptors in a porcine model of chronic episodic myocardial ischemia.

Regional myocardial ischemia is associated with increased levels of adenosine and norepinephrine, factors that may alter activation of the beta-adrenergic receptor (beta AR)-G protein-adenylyl cyclase pathway in the heart. We have used the ameroid constrictor model to determine whether alterations in myocardial signal transduction through the beta AR-G protein-adenylyl cyclase pathway occur in the setting of chronic episodes of reversible ischemia. Pigs were instrumented with ameroid occluders placed around the left circumflex coronary artery. 5 wk later, after ameroid closure, flow and function were normal in the ischemic bed, but flow (P = 0.001) and function (P < 0.03) were abnormal when metabolic demands were increased. The ischemic bed showed a reduction in myocardial beta AR number (P < 0.005). Despite regional downregulation of myocardial beta AR number, adenylyl cyclase activity was similar in the ischemic and control beds. Quantitative immunoblotting showed that the cardiac inhibitory GTP-binding protein, Gi alpha 2, was decreased in the ischemic bed (P = 0.02). In contrast, the cardiac stimulatory GTP-binding protein, Gs alpha, was increased in endocardial sections from the ischemic bed (P = < 0.05). Decreased Gi alpha 2 content was associated with decreased inhibition of adenylyl cyclase. Reduced Gi alpha 2 content, in conjunction with increased Gs alpha content in the endocardium, may provide a means by which adrenergic activation is maintained in the setting of chronic episodic myocardial ischemia.

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling associated with chronic reduction in beta-adrenergic receptor agonist activation were found. First, there was coordinate downregulation of Gi alpha 2 and Gs alpha mRNA and protein expression in the left ventricle; reduced G-protein content was also found in the right atrium. Second, in the left ventricle, there was a twofold increase in beta-adrenergic receptor-dependent stimulation of adenylyl cyclase and a persistent high affinity state of the beta-adrenergic receptor. Finally, there was a reduction in left ventricular beta-adrenergic receptor kinase activity, suggesting a previously unrecognized association between the degree of adrenergic activation and myocardial beta-adrenergic receptor kinase expression. The heart appears to adapt in response to chronic beta-adrenergic receptor antagonist administration in a manner that would be expected to offset reduced agonist stimulation. The mechanisms for achieving this extend beyond beta-adrenergic receptor upregulation and include alterations in G-protein expression, beta-adrenergic receptor-Gs interaction, and myocardial beta-adrenergic receptor kinase activity.

Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved.

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n = 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n = 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n = 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n = 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n = 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 microg/kg) on day 1 without ischemia (group VI, n = 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n = 5), the six O/R cycles caused translocation of PKC isoforms epsilon and eta from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the alpha, beta1, beta2, gamma, delta, zeta, iota, lambda, and mu isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n = 5) prevented the translocation of both PKC epsilon and eta induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n = 5) prevented the translocation of PKC eta but not that of epsilon, indicating that the activation of epsilon is necessary for late PC to occur whereas that of eta is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKC-mediated signaling pathway, and implicate epsilon as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.

Functional proteomic analysis of protein kinase C epsilon signaling complexes in the normal heart and during cardioprotection.

Using two-dimensional electrophoresis, mass spectrometry, immunoblotting, and affinity pull-down assays, we found that myocardial protein kinase C epsilon (PKCepsilon) is physically associated with at least 36 known proteins that are organized into structural proteins, signaling molecules, and stress-responsive proteins. Furthermore, we found that the cardioprotection induced by activation of PKCepsilon is coupled with dynamic modulation and recruitment of PKCepsilon-associated proteins. The results suggest heretofore-unrecognized functions of PKCepsilon and provide an integrated framework for the understanding of PKCepsilon-dependent signaling architecture and cardioprotection.

The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation.

Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.

Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy.

To test the hypothesis that activation of the protein kinase C (PKC) epsilon isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCepsilon isoform driven by an alpha-myosin heavy chain promoter. In transgenic mice, the protein level of PKCepsilon in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for beta-myosin heavy chain and alpha-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca(2+) signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca(2+) was increased. No differences were observed in either the levels of cardiac Ca(2+)-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCbeta(2) overexpression, transgenic mice with cardiac-specific overexpression of the active PKCepsilon mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.

Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits.

Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (+119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (+103% versus control) and at 30 minutes (+89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the epsilon isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (+72%) and at 30 minutes (+67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKCepsilon, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKCepsilon but also that of Src and Lck, indicating that Src and Lck are downstream of PKCepsilon in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction.

Protein kinase C epsilon-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling.

An essential role for protein kinase C epsilon (PKCepsilon) has been shown in multiple forms of cardioprotection; however, there is a distinct paucity of information concerning the signaling architecture that is responsible for the manifestation of a protective phenotype. We and others have recently shown that signal transduction may proceed via the formation of signaling complexes (Circ Res. 2001;88:59-62). In order to understand if the assembly of multiprotein complexes is the manner by which signaling is conducted in cardioprotection, we designed a series of experiments to characterize the associations of Src tyrosine kinase with PKCepsilon in a conscious rabbit model of nitric oxide (NO)-induced late preconditioning. Our data demonstrate that PKCepsilon and Src can form functional signaling modules in vitro: PKCepsilon interacts with Src; the association with PKCepsilon activates Src; and adult cardiac cells receiving recombinant adenoviruses encoding PKCepsilon exhibit increased Src activity. Furthermore, our results show that NO-induced late preconditioning involved PKCepsilon-Src module formation and enhanced the enzymatic activity of PKCepsilon-associated Src. Inhibition of PKC blocked cardioprotection, module formation, and PKCepsilon-associated Src activity, providing direct evidence for a functional role of the PKCepsilon-Src module in the orchestration of NO-induced cardioprotection in conscious rabbits.

Downregulation of miR-320a/383-sponge-like long non-coding RNA NLC1-C (narcolepsy candidate-region 1 genes) is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation.

Long non-coding RNAs (lncRNAs), which are extensively transcribed from the genome, have been proposed to be key regulators of diverse biological processes. However, little is known about the role of lncRNAs in regulating spermatogenesis in human males. Here, using microarray technology, we show altered expression of lncRNAs in the testes of infertile men with maturation arrest (MA) or hypospermatogenesis (Hypo), with 757 and 2370 differentially down-regulated and 475 and 163 up-regulated lncRNAs in MA and Hypo, respectively. These findings were confirmed by quantitative real-time PCR (qRT-PCR) assays on select lncRNAs, including HOTTIP, imsrna320, imsrna292 and NLC1-C (narcolepsy candidate-region 1 genes). Interestingly, NLC1-C, also known as long intergenic non-protein-coding RNA162 (LINC00162), was down-regulated in the cytoplasm and accumulated in the nucleus of spermatogonia and primary spermatocytes in the testes of infertile men with mixed patterns of MA compared with normal control. The accumulation of NLC1-C in the nucleus repressed miR-320a and miR-383 transcript and promoted testicular embryonal carcinoma cell proliferation by binding to Nucleolin. Here, we define a novel mechanism by which lncRNAs modulate miRNA expression at the transcriptional level by binding to RNA-binding proteins to regulate human spermatogenesis.

Saturated fat and cholesterol are critical to inducing murine metabolic syndrome with robust nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome (MetS). Up to a third of NAFLD subjects are at risk for developing nonalcoholic steatohepatitis (NASH). Many rodent models fail to replicate both MetS and NASH. The purpose of this study was to develop a reliable mouse model of NASH and MetS using a diet containing cholesterol, saturated fat and carbohydrate that is reflective of Western diets of North Americans. EXPERIMENTAL DESIGN: We used adult male C57BL/6 J 4- to 5-week-old mice and administered a solid diet containing 0.2% cholesterol, 45% of its calories from fat, with 30% of the fat in the form of partially hydrogenated vegetable oil. We also provided carbohydrate largely as high-fructose corn syrup equivalent in water. In a separate cohort, we gave the identical diet in the absence of cholesterol. Glucose and insulin tolerance testing was conducted throughout the feeding period. The feeding was conducted for 16 weeks, and the mice were sacrificed for histological analysis, markers of MetS, liver inflammation, circulating lipids, as well as liver staining for fibrosis and alpha smooth muscle actin (α-SMA). RESULTS: We found that cholesterol significantly increased serum leptin, interleukin-6, liver weight and liver weight/body weight ratio, fibrosis and liver α-SMA. CONCLUSIONS: Mice administered a diet accurately reflecting patterns associated with humans afflicted with MetS can reliably replicate features of MetS, NASH and significant liver fibrosis. The model we describe significantly reduces the time by several months for development of stage 3 hepatic fibrosis.

Merging and scoring molecular interactions utilising existing community standards: tools, use-cases and a case study.

The evidence that two molecules interact in a living cell is often inferred from multiple different experiments. Experimental data is captured in multiple repositories, but there is no simple way to assess the evidence of an interaction occurring in a cellular environment. Merging and scoring of data are commonly required operations after querying for the details of specific molecular interactions, to remove redundancy and assess the strength of accompanying experimental evidence. We have developed both a merging algorithm and a scoring system for molecular interactions based on the proteomics standard initiative-molecular interaction standards. In this manuscript, we introduce these two algorithms and provide community access to the tool suite, describe examples of how these tools are useful to selectively present molecular interaction data and demonstrate a case where the algorithms were successfully used to identify a systematic error in an existing dataset.

Protein kinase C-epsilon mediates phorbol ester-induced phosphorylation of connexin-43.

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKCepsilon) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (> or = 45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKK1 did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKCE significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of > or = 45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKCepsilon.