Cytolethal distending toxin B inoculation leads to distinct gut microtypes and IBS-D-like microRNA-mediated gene expression changes in a rodent model.

Diarrhea-predominant irritable bowel syndrome (IBS-D), associated with increased intestinal permeability, inflammation, and small intestinal bacterial overgrowth, can be triggered by acute gastroenteritis. Cytolethal distending toxin B (CdtB) is produced by gastroenteritis-causing pathogens and may underlie IBS-D development, through molecular mimicry with vinculin. Here, we examine the effects of exposure to CdtB alone on gut microbiome composition, host intestinal gene expression, and IBS-D-like phenotypes in a rat model. CdtB-inoculated rats exhibited increased anti-CdtB levels, which correlated with increased stool wet weights, pro-inflammatory cytokines (TNFα, IL2) and predicted microbial metabolic pathways including inflammatory responses, TNF responses, and diarrhea. Three distinct ileal microbiome profiles (microtypes) were identified in CdtB-inoculated rats. The first microtype (most like controls) had altered relative abundance (RA) of genera Bifidobacterium, Lactococcus, and Rothia. The second had lower microbial diversity, higher Escherichia-Shigella RA, higher absolute E. coli abundance, and altered host ileal tissue expression of immune-response and TNF-response genes compared to controls. The third microtype had higher microbial diversity, higher RA of hydrogen sulfide (H2S)-producer Desulfovibrio, and increased expression of H2S-associated pain/serotonin response genes. All CdtB-inoculated rats exhibited decreased ileal expression of cell junction component mRNAs, including vinculin-associated proteins. Significantly, cluster-specific microRNA-mRNA interactions controlling intestinal permeability, visceral hypersensitivity/pain, and gastrointestinal motility genes, including several previously associated with IBS were seen. These findings demonstrate that exposure to CdtB toxin alone results in IBS-like phenotypes including inflammation and diarrhea-like stool, decreased expression of intestinal barrier components, and altered ileal microtypes that influenced changes in microRNA-modulated gene expression and predicted metabolic pathways consistent with specific IBS-D symptoms.

Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis.

Introduction: Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown. Methods: Here, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays. Results and discussion: We identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages-inhibiting translation and protein turnover and inducing apoptosis-whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies.

Asporin, an extracellular matrix protein, is a beneficial regulator of cardiac remodeling.

Heart failure is accompanied by adverse cardiac remodeling involving extracellular matrix (ECM). Cardiac ECM acts as a major reservoir for many proteins including growth factors, cytokines, collagens, and proteoglycans. Activated fibroblasts during cardiac injury can alter the composition and activity of these ECM proteins. Through unbiased analysis of a microarray dataset of human heart tissue comparing normal hearts (n = 135) to hearts with ischemic cardiomyopathy (n = 94), we identified Asporin (ASPN) as the top differentially regulated gene (DEG) in ischemic cardiomyopathy; its gene-ontology terms relate closely to fibrosis and cell death. ASPN is a Class I small leucine repeat protein member implicated in cancer, osteoarthritis, and periodontal ligament mineralization. However, its role in cardiac remodeling is still unknown. Here, we initially confirmed our big dataset analysis through cells, mice, and clinical atrial biopsy samples to demonstrate increased Aspn expression after pressure overload or cardiac ischemia/reperfusion injury. We tested the hypothesis that Aspn, being a TGFß1 inhibitor, can attenuate fibrosis in mouse models of cardiac injury. We found that Aspn is released by cardiac fibroblasts and attenuates TGFß signaling. Moreover, Aspn-/- mice displayed increased fibrosis and decreased cardiac function after pressure overload by transverse aortic constriction (TAC) in mice. In addition, Aspn protected cardiomyocytes from hypoxia/reoxygenation-induced cell death and regulated mitochondrial bioenergetics in cardiomyocytes. Increased infarct size after ischemia/reperfusion injury in Aspn-/- mice confirmed Aspn's contribution to cardiomyocyte viability. Echocardiography revealed greater reduction in left ventricular systolic function post-I/R in the Aspn-/- animals compared to wild type. Furthermore, we developed an ASPN-mimic peptide using molecular modeling and docking which when administered to mice prevented TAC-induced fibrosis and preserved heart function. The peptide also reduced infarct size after I/R in mice, demonstrating the translational potential of ASPN-based therapy. Thus, we establish the role of ASPN as a critical ECM molecule that regulates cardiac remodeling to preserve heart function.

Proteomics of Mouse Heart Ventricles Reveals Mitochondria and Metabolism as Major Targets of a Post-Infarction Short-Acting GLP1Ra-Therapy.

Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein-protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867.

Hypothermia promotes mitochondrial elongation In cardiac cells via inhibition of Drp1.

Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in surgery, stroke, cardiac arrest and organ preservation. Protection is afforded principally by a reduction of metabolism, manifesting as reduced rates of oxygen uptake, preservation of ATP levels, and a curtailing of ischemic calcium overload. The effects of non-ischemic hypothermic stress are relatively unknown. We sought to investigate the effects of clinically mild-to-severe hypothermia on mitochondrial morphology, oxygen consumption and protein expression in normoxic hearts and cardiac cells. Normoxic perfusion of rat hearts at 28-32 °C was associated with inhibition of mitochondrial fission, evidenced by a reduced abundance of the active phosphorylated form of the fission receptor Drp1 (pDrp1S616). Abundance of the same residue was reduced in H9c2 cells subjected to hypothermic culture (25-32 °C), in addition to a reduced abundance of the Drp1 receptor MFF. Hypothermia-treated H9c2 cardiomyocytes exhibited elongated mitochondria and depressed rates of mitochondrial-associated oxygen consumption, which persisted upon rewarming. Hypothermia also promoted a reduction in mRNA expression of the capsaicin receptor TRPV1 in H9c2 cells. When normothermic H9c2 cells were transfected with TRPV1 siRNA we observed reduced pDrp1S616 and MFF abundance, elongated mitochondria, and reduced rates of mitochondrial-associated oxygen consumption, mimicking the effects of hypothermic culture. In conclusion hypothermia promoted elongation of cardiac mitochondria via reduced pDrp1S616 abundance which was also associated with suppression of cellular oxygen consumption. Silencing of TRPV1 in H9c2 cardiomyocytes reproduced the morphological and respirometric phenotype of hypothermia. This report demonstrates a novel mechanism of cold-induced inhibition of mitochondrial fission.

At the heart of mitochondrial quality control: many roads to the top.

Mitochondrial quality control depends upon selective elimination of damaged mitochondria, replacement by mitochondrial biogenesis, redistribution of mitochondrial components across the network by fusion, and segregation of damaged mitochondria by fission prior to mitophagy. In this review, we focus on mitochondrial dynamics (fusion/fission), mitophagy, and other mechanisms supporting mitochondrial quality control including maintenance of mtDNA and the mitochondrial unfolded protein response, particularly in the context of the heart.

Attenuation of Adverse Postinfarction Left Ventricular Remodeling with Empagliflozin Enhances Mitochondria-Linked Cellular Energetics and Mitochondrial Biogenesis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin are known to reduce the risk of hospitalizations related to heart failure irrespective of diabetic state. Meanwhile, adverse cardiac remodeling remains the leading cause of heart failure and death in the USA. Thus, understanding the mechanisms that are responsible for the beneficial effects of SGLT2 inhibitors is of the utmost relevance and importance. Our previous work illustrated a connection between adverse cardiac remodeling and the regulation of mitochondrial turnover and cellular energetics using a short-acting glucagon-like peptide-1 receptor agonist (GLP1Ra). Here, we sought to determine if the mechanism of the SGLT2 inhibitor empagliflozin (EMPA) in ameliorating adverse remodeling was similar and/or to identify what differences exist, if any. To this end, we administered permanent coronary artery ligation to induce adverse remodeling in wild-type and Parkin knockout mice and examined the progression of adverse cardiac remodeling with or without EMPA treatment over time. Like GLP1Ra, we found that EMPA affords a robust attenuation of PCAL-induced adverse remodeling. Interestingly, unlike the GLP1Ra, EMPA does not require Parkin to improve/maintain mitochondria-related cellular energetics and afford its benefits against developing adverse remodeling. These findings suggests that further investigation of EMPA is warranted as a potential path for developing therapy against adverse cardiac remodeling for patients that may have Parkin and/or mitophagy-related deficiencies.

Percepção de oportunidades de aprendizado de funcionários técnico-administrativos no âmbito universitário / Perception learning opportunities of technical-administrative staff in the university scope / Percepción del personal técnico y administrativo sobre las oportunidades de aprendizaje en las universidades

Resumo Este estudo tem por objetivo identificar a percepção de oportunidades de aprendizagem e sua relação com o estilo de liderança exercido pelos funcionários técnico-administrativos no âmbito universitário, a intenção de rotatividade e capital psicológico no trabalho. Foi realizado um estudo transversal, com 102 funcionários técnico-administrativos, por meio de um questionário de autopreenchimento contendo dados referentes a idade, escolaridade, tempo de serviço na instituição e se o respondente exercia um cargo de liderança. Foram aplicadas escalas relacionadas à percepção de oportunidades de aprendizagem, avaliação do estilo gerencial, intenção de rotatividade e capital psicológico no trabalho. Os resultados revelam que os funcionários identificam oportunidades de aprendizado e a presença de três estilos gerenciais no âmbito universitário. Também se observou que os funcionários se identificam como capitais psicológicos no trabalho e possuem baixa intenção de rotatividade. Assim, este trabalho buscou ampliar a literatura ainda escassa envolvendo funcionários técnico-administrativos em âmbito universitário. (AU)

Abstract This study aimed to identify the perception of learning opportunities and its association with the leadership style of the administrative-technical staff within the university scope, the turnover intention, and the psychological capital at work. For that, this cross-sectional study was conducted with data on age, education level, length of service in the institution, and leadership position of 102 administrative and technical employees, collected by means of a self-administered questionnaire. Perceived learning opportunities, management style, turnover intention, and psychological capital at work were evaluated using scales. The results show that the administrative-technical staff identifies the three management styles and learning opportunities within the university scope. Moreover, they identify themselves as psychological capitals at work and possess low turnover intention. This study is expected to expand the (still scarce) literature on administrative and technical staff in the university context. (AU)

Resumen Este estudio tuvo como objetivo identificar las oportunidades de aprendizaje y su relación con el estilo de liderazgo adoptado por el personal técnico y administrativo en las universidades, la intención de rotación y el capital psicológico en el trabajo. Este es un estudio transversal realizado con 102 personales técnicos y administrativos mediante la aplicación de un cuestionario de autoinforme con datos sobre la edad, la educación, el tiempo de servicio en la institución y si ejercían cargo de liderazgo. Se aplicaron las escalas en cuanto a la percepción de las oportunidades de aprendizaje, la evaluación del estilo de gestión, la intención de rotación y el capital psicológico en el trabajo. Los resultados muestran que el personal identifica oportunidades de aprendizaje y la presencia de tres estilos de gestión en la universidad. También se observó que ellos se identifican como capital psicológico en el trabajo y tienen la intención de baja rotación. Por lo tanto, este estudio trató de ampliar la literatura todavía escasa, con la participación del personal técnico y administrativo en el ámbito universitario. (AU)

Ultraviolet A light effectively reduces bacteria and viruses including coronavirus.

Antimicrobial-resistant and novel pathogens continue to emerge, outpacing efforts to contain and treat them. Therefore, there is a crucial need for safe and effective therapies. Ultraviolet-A (UVA) phototherapy is FDA-approved for several dermatological diseases but not for internal applications. We investigated UVA effects on human cells in vitro, mouse colonic tissue in vivo, and UVA efficacy against bacteria, yeast, coxsackievirus group B and coronavirus-229E. Several pathogens and virally transfected human cells were exposed to a series of specific UVA exposure regimens. HeLa, alveolar and primary human tracheal epithelial cell viability was assessed after UVA exposure, and 8-Oxo-2'-deoxyguanosine was measured as an oxidative DNA damage marker. Furthermore, wild-type mice were exposed to intracolonic UVA as an in vivo model to assess safety of internal UVA exposure. Controlled UVA exposure yielded significant reductions in Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Clostridioides difficile, Streptococcus pyogenes, Staphylococcus epidermidis, Proteus mirabilis and Candida albicans. UVA-treated coxsackievirus-transfected HeLa cells exhibited significantly increased cell survival compared to controls. UVA-treated coronavirus-229E-transfected tracheal cells exhibited significant coronavirus spike protein reduction, increased mitochondrial antiviral-signaling protein and decreased coronavirus-229E-induced cell death. Specific controlled UVA exposure had no significant effect on growth or 8-Oxo-2'-deoxyguanosine levels in three types of human cells. Single or repeated in vivo intraluminal UVA exposure produced no discernible endoscopic, histologic or dysplastic changes in mice. These findings suggest that, under specific conditions, UVA reduces various pathogens including coronavirus-229E, and may provide a safe and effective treatment for infectious diseases of internal viscera. Clinical studies are warranted to further elucidate the safety and efficacy of UVA in humans.

Intermittent Use of a Short-Course Glucagon-like Peptide-1 Receptor Agonist Therapy Limits Adverse Cardiac Remodeling via Parkin-dependent Mitochondrial Turnover.

Given that adverse remodeling is the leading cause of heart failure and death in the USA, there is an urgent unmet need to develop new methods in dealing with this devastating disease. Here we evaluated the efficacy of a short-course glucagon-like peptide-1 receptor agonist therapy-specifically 2-quinoxalinamine, 6,7-dichloro-N-(1,1-dimethylethyl)-3-(methylsulfonyl)-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB; aka Compound 2) - in attenuating adverse LV remodeling. We also examined the role, if any, of mitochondrial turnover in this process. Wild-type, Parkin knockout and MitoTimer-expressing mice were subjected to permanent coronary artery ligation, then treated briefly with DMB. LV remodeling and cardiac function were assessed by histology and echocardiography. Autophagy and mitophagy markers were examined by western blot and mitochondrial biogenesis was inferred from MitoTimer protein fluorescence and qPCR. We found that DMB given post-infarction significantly reduced adverse LV remodeling and the decline of cardiac function. This paralleled an increase in autophagy, mitophagy and mitochondrial biogenesis. The salutary effects of the drug were lost in Parkin knockout mice, implicating Parkin-mediated mitophagy as part of its mechanism of action. Our findings suggest that enhancing Parkin-associated mitophagy and mitochondrial biogenesis after infarction is a viable target for therapeutic mitigation of adverse remodeling.

Antiviral Effects of Menthol on Coxsackievirus B.

Coxsackievirus B (CVB) is a common human enterovirus that causes systemic infection but specifically replicates to high titers in the pancreas. It was reported that certain viruses induce mitochondrial fission to support infection. We documented that CVB triggers mitochondrial fission and blocking mitochondrial fission limits infection. The transient receptor potential channels have been implicated in regulating mitochondrial dynamics; namely, the heat and capsaicin receptor transient receptor potential cation channel subfamily V member 1 (TRPV1) contributes to mitochondrial depolarization and fission. When we transiently warmed HeLa cells to 39 °C prior to CVB exposure, infection was heightened, whereas cooling cells to 25 °C reduced infection. Inducing "cold" by stimulating transient receptor potential cation channel subfamily M member 8 (TRPM8) with menthol led to reduced infection and also resulted in lower levels of mitochondrial fission during infection. Additionally, menthol stabilized levels of mitochondrial antiviral signaling (MAVS) which is known to be tied to mitochondrial dynamics. Taken together, this highlights a novel pathway wherein CVB relies on TRPV1 to initiate proviral mitochondrial fission, which may contribute to the disruption of antiviral immunity. TRPM8 has been shown to antagonize TRPV1, and thus we hypothesize that stimulating TRPM8 blocks TRPV1-mediated mitochondrial fragmentation following CVB exposure and attenuates infection.

Dysregulation of microRNAs and target genes networks in human abdominal aortic aneurysm tissues.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a pathological enlargement of infrarenal aorta close to the aortic bifurcation, and it is an important cause of mortality in the elderly. Therefore, the biomarker identification for early diagnosis is of great interest for clinical benefit. It is known that microRNAs (miRNAs) have important roles via target genes regulation in many diseases. This study aimed to identify miRNAs and their target genes involved in the pathogenesis of AAA. METHODS: Tissue samples were obtained from patients who underwent AAA surgery and from organ donors (control group). Quantitative PCR Array was applied to assess 84 genes and 384 miRNAs aiming to identify differentially expressed targets (AAA n = 6, control n = 6), followed by validation in a new cohort (AAA n = 18, control n = 6) by regular qPCR. The functional interaction between validated miRNAs and target genes was performed by the Ingenuity Pathway Analysis (IPA) software. RESULTS: The screening cohort assessed by PCR array identified 10 genes and 59 miRNAs differentially expressed (≥2-fold change, p<0.05). Among these, IPA identified 5 genes and 9 miRNAs with paired interaction. ALOX5, PTGIS, CX3CL1 genes, and miR-193a-3p, 125b-5p, 150-5p maintained a statistical significance in the validation cohort. IPA analysis based on the validated genes and miRNAs revealed that eicosanoid and metalloproteinase/TIMP synthesis are potentially involved in AAA. CONCLUSION: Paired interactions of differentially expressed ALOX5, PTGIS, CX3CL1 genes, and miR-193b-3p, 125b-5p, 150-5p revealed a potentially significant role of the eicosanoid synthesis and metalloproteinase/TIMP pathways in the AAA pathogenesis.

Dysregulation of microRNAs and target genes networks in human abdominal aortic aneurysm tissues

BACKGROUND: Abdominal aortic aneurysm (AAA) is a pathological enlargement of infrarenal aorta close to the aortic bifurcation, and it is an important cause of mortality in the elderly. Therefore, the biomarker identification for early diagnosis is of great interest for clinical benefit. It is known that microRNAs (miRNAs) have important roles via target genes regulation in many diseases. This study aimed to identify miRNAs and their target genes involved in the pathogenesis of AAA. METHODS: Tissue samples were obtained from patients who underwent AAA surgery and from organ donors (control group). Quantitative PCR Array was applied to assess 84 genes and 384 miRNAs aiming to identify differentially expressed targets (AAA n = 6, control n = 6), followed by validation in a new cohort (AAA n = 18, control n = 6) by regular qPCR. The functional interaction between validated miRNAs and target genes was performed by the Ingenuity Pathway Analysis (IPA) software. RESULTS The screening cohort assessed by PCR array identified 10 genes and 59 miRNAs differentially expressed...(AU)

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis.

BACKGROUND: During pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence. METHODS: We examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis. RESULTS: Pancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin. CONCLUSION: Our findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

Coxsackievirus B infection induces the extracellular release of miR-590-5p, a proviral microRNA.

Coxsackievirus B is a significant human pathogen and is a leading cause of myocarditis. We and others have observed that certain enteroviruses including coxsackievirus B cause infected cells to shed extracellular vesicles containing infectious virus. Recent reports have shown that vesicle-bound virus can infect more efficiently than free virus. Though microRNAs are differentially regulated in cells following infection, few have been associated with the vesicles shed from infected cells. Here we report exclusive trafficking of specific microRNAs into viral vesicles compared to vesicles from non-infected cells. We found that the most highly-expressed unique microRNA in viral vesicles was miR-590-5p, which facilitates prolonged viral replication by blocking apoptotic factors. Cells over-expressing this miR were significantly more susceptible to infection. This may be a mechanism by which coxsackievirus B boosts subsequent rounds of infection by co-packaging virus and a select set of pro-viral microRNAs in extracellular vesicles.

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.

Analysis of Circulating miR-1, miR-23a, and miR-26a in Atrial Fibrillation Patients Undergoing Coronary Bypass Artery Grafting Surgery.

Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. From a pathophysiological point of view, a myriad of factors such as trauma, atrial dilation, ischemia, mechanical myopericarditis, autonomic imbalance, loss of connexins, AF nest remodeling, inflammation, sutures, and dysfunction caused by postextracorporeal circulation can contribute to postoperative atrial fibrillation (POAF) resulting in a longer hospital stay and consequently higher cost. Recent studies showed that short fragments of RNA, called microRNA (miRNA), can contribute to the development of several cardiovascular diseases, including AF. The aim of this study was to evaluate the levels of circulating miRNAs (miR-1, -23a, and -26a) that can be involved in POAF. Patients submitted to coronary artery bypass graft surgery were grouped in POAF (24 patients) and without POAF (24 patients). Results showed older age, longer clamp-time, and more days in the intensive care unit as well as a longer total hospital stay in the POAF group. Preoperative levels of circulating miRNAs were similar. Analysis of miRNAs revealed significantly lower circulating levels of miRNA-23a (P = 0.02) and -26a (P = 0.01) in the POAF group during the postoperative period. Receiver operating characteristic (ROC) analysis showed the area under the ROC curve of miR-23a and miR-26a for predicting FA was 0.63 (95% confidence interval [CI]: 0.51-0.74; P = 0.02) and 0.66 (95% CI: 0.55-0.77; P = 0.01), respectively. Our data suggests that circulating miRNA-23a and -26a may be involved in the underlying biology of postoperative AF development.

ABCC3 Polymorphisms and mRNA Expression Influence the Concentration of a Carboxylic Acid Metabolite in Patients on Clopidogrel and Aspirin Therapy.

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.

nalysis of circulating miR-1, miR-23a, and miR-26a in atrial fibrillation patients undergoing coronary bypass artery grafting surgery

Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. From a pathophysiological point of view, a myriad of factors such as trauma, atrial dilation, ischemia, mechanical myopericarditis, autonomic imbalance, loss of connexins, AF nest remodeling, inflammation, sutures, and dysfunction caused by postextracorporeal circulation can contribute to postoperative atrial fibrillation (POAF) resulting in a longer hospital stay and consequently higher cost. Recent studies showed that short fragments of RNA, called microRNA (miRNA), can contribute to the development of several cardiovascular diseases, including AF. The aim of this study was to evaluate the levels of circulating miRNAs (miR-1, -23a, and -26a) that can be involved in POAF. Patients submitted to coronary artery bypass graft surgery were grouped in POAF (24 patients) and without POAF (24 patients). Results showed older age, longer clamp-time, and more days in the intensive care unit as well as a longer total hospital stay in the POAF group. Preoperative levels of circulating miRNAs were similar...

ABCC3 Polymorphisms and mRNA Expression Influence the / Concentration of a Carboxylic Acid Metabolite in Patients on / Clopidogrel and Aspirin Therapy

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.