Chronic ethanol exposure produces time- and brain region-dependent changes in gene coexpression networks.

Repeated ethanol exposure and withdrawal in mice increases voluntary drinking and represents an animal model of physical dependence. We examined time- and brain region-dependent changes in gene coexpression networks in amygdala (AMY), nucleus accumbens (NAC), prefrontal cortex (PFC), and liver after four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure in C57BL/6J mice. Microarrays were used to compare gene expression profiles at 0-, 8-, and 120-hours following the last ethanol exposure. Each brain region exhibited a large number of differentially expressed genes (2,000-3,000) at the 0- and 8-hour time points, but fewer changes were detected at the 120-hour time point (400-600). Within each region, there was little gene overlap across time (~20%). All brain regions were significantly enriched with differentially expressed immune-related genes at the 8-hour time point. Weighted gene correlation network analysis identified modules that were highly enriched with differentially expressed genes at the 0- and 8-hour time points with virtually no enrichment at 120 hours. Modules enriched for both ethanol-responsive and cell-specific genes were identified in each brain region. These results indicate that chronic alcohol exposure causes global 'rewiring' of coexpression systems involving glial and immune signaling as well as neuronal genes.

Comparative whole genome sequencing of community-associated methicillin-resistant Staphylococcus aureus sequence type 8 from primary care clinics in a Texas community.

STUDY OBJECTIVE: Our understanding of the molecular dynamics driving the community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic at the whole genome level is limited. We sought to assess the use of whole genome sequencing (WGS) to evaluate the genomic diversity and genotypic prediction of antimicrobial resistance of CA-MRSA isolates from patients in South Texas. DESIGN: Comparative whole genome sequencing. ISOLATES: Thirteen clinical CA-MRSA isolates recovered from patients presenting with skin and soft tissue infections to nine primary care clinics in the South Texas Ambulatory Research Network between 2010 and 2013. MEASUREMENTS AND MAIN RESULTS: Comparative WGS was performed on the 13 MRSA sequence type 8 clinical isolates. We compared the resistome of genes encoding for antibiotic resistance with a phenotypically derived antibiogram using standard antimicrobial susceptibility testing. The strains differed by an average of 72 single nucleotide polymorphisms (SNPs) per isolate in the core genome compared with FPR3757 (USA300, reference strain). There were a total of 623 unique SNPs in the core genome (range 47-88 SNPs per isolate). We identified 19 nonsynonymous SNPs in genes encoding proven or putative S. aureus virulence determinants in the core genome. There was complete concordance between genotypic evidence for antimicrobial resistance and the phenotypically derived antibiogram. CONCLUSION: Overall, although these CA-MRSA isolates were similar on the level of clonal type, clinical syndrome, and geographic area, the strains were diverse at the genome level. Furthermore, our findings provide important proof-of-concept information for using WGS as a potential front-end screening tool for antimicrobial resistance predictions.

Integration of miRNA and protein profiling reveals coordinated neuroadaptations in the alcohol-dependent mouse brain.

The molecular mechanisms underlying alcohol dependence involve different neurochemical systems and are brain region-dependent. Chronic Intermittent Ethanol (CIE) procedure, combined with a Two-Bottle Choice voluntary drinking paradigm, represents one of the best available animal models for alcohol dependence and relapse drinking. MicroRNAs, master regulators of the cellular transcriptome and proteome, can regulate their targets in a cooperative, combinatorial fashion, ensuring fine tuning and control over a large number of cellular functions. We analyzed cortex and midbrain microRNA expression levels using an integrative approach to combine and relate data to previous protein profiling from the same CIE-subjected samples, and examined the significance of the data in terms of relative contribution to alcohol consumption and dependence. MicroRNA levels were significantly altered in CIE-exposed dependent mice compared with their non-dependent controls. More importantly, our integrative analysis identified modules of coexpressed microRNAs that were highly correlated with CIE effects and predicted target genes encoding differentially expressed proteins. Coexpressed CIE-relevant proteins, in turn, were often negatively correlated with specific microRNA modules. Our results provide evidence that microRNA-orchestrated translational imbalances are driving the behavioral transition from alcohol consumption to dependence. This study represents the first attempt to combine ex vivo microRNA and protein expression on a global scale from the same mammalian brain samples. The integrative systems approach used here will improve our understanding of brain adaptive changes in response to drug abuse and suggests the potential therapeutic use of microRNAs as tools to prevent or compensate multiple neuroadaptations underlying addictive behavior.

Positively correlated miRNA-mRNA regulatory networks in mouse frontal cortex during early stages of alcohol dependence.

BACKGROUND: Although the study of gene regulation via the action of specific microRNAs (miRNAs) has experienced a boom in recent years, the analysis of genome-wide interaction networks among miRNAs and respective targeted mRNAs has lagged behind. MicroRNAs simultaneously target many transcripts and fine-tune the expression of genes through cooperative/combinatorial targeting. Therefore, they have a large regulatory potential that could widely impact development and progression of diseases, as well as contribute unpredicted collateral effects due to their natural, pathophysiological, or treatment-induced modulation. We support the viewpoint that whole mirnome-transcriptome interaction analysis is required to better understand the mechanisms and potential consequences of miRNA regulation and/or deregulation in relevant biological models. In this study, we tested the hypotheses that ethanol consumption induces changes in miRNA-mRNA interaction networks in the mouse frontal cortex and that some of the changes observed in the mouse are equivalent to changes in similar brain regions from human alcoholics. RESULTS: miRNA-mRNA interaction networks responding to ethanol insult were identified by differential expression analysis and weighted gene coexpression network analysis (WGCNA). Important pathways (coexpressed modular networks detected by WGCNA) and hub genes central to the neuronal response to ethanol are highlighted, as well as key miRNAs that regulate these processes and therefore represent potential therapeutic targets for treating alcohol addiction. Importantly, we discovered a conserved signature of changing miRNAs between ethanol-treated mice and human alcoholics, which provides a valuable tool for future biomarker/diagnostic studies in humans. We report positively correlated miRNA-mRNA expression networks that suggest an adaptive, targeted miRNA response due to binge ethanol drinking. CONCLUSIONS: This study provides new evidence for the role of miRNA regulation in brain homeostasis and sheds new light on current understanding of the development of alcohol dependence. To our knowledge this is the first report that activated expression of miRNAs correlates with activated expression of mRNAs rather than with mRNA downregulation in an in vivo model. We speculate that early activation of miRNAs designed to limit the effects of alcohol-induced genes may be an essential adaptive response during disease progression.

Understanding Alcoholism Through microRNA Signatures in Brains of Human Alcoholics.

Advances in the fields of genomics and genetics in the last decade have identified a large number of genes that can potentially influence alcohol-drinking behavior in humans as well as animal models. Consequently, the task of identifying efficient molecular targets that could be used to develop effective therapeutics against the disease has become increasingly daunting. One of the reasons for this is the fact that each of the many alcohol-responsive genes only contributes a small effect to the overall mechanism and disease phenotype, as is characteristic of complex traits. Current research trends are hence shifting toward the analysis of gene networks rather than emphasizing individual genes. The discovery of microRNAs and their mechanisms of action on regulation of transcript level and protein translation have made evident the utility of these small non-coding RNA molecules that act as central coordinators of multiple cross-communicating cellular pathways. Cells exploit the fact that a single microRNA can target hundreds of mRNA transcripts and that a single mRNA transcript can be simultaneously targeted by distinct microRNAs, to ensure fine-tuned and/or redundant control over a large number of cellular functions. By the same token, we can use these properties of microRNAs to develop novel, targeted strategies to combat complex disorders. In this review, we will focus on recent discoveries of microRNA signatures in brain of human alcoholics supporting the hypothesis that changes in gene expression and regulation by microRNAs are responsible for long-term neuroadaptations occurring during development of alcoholism. We also discuss insights into the potential modulation of epigenetic regulators by a subset of microRNAs. Taken together, microRNA activity may be controlling many of the cellular mechanisms already known to be involved in the development of alcoholism, and suggests potential targets for the development of novel therapeutic interventions.

Up-regulation of microRNAs in brain of human alcoholics.

BACKGROUND: MicroRNAs (miRNAs) are small, noncoding oligonucleotides with an important role in posttranscriptional regulation of gene expression at the level of translation and mRNA degradation. Recent studies have revealed that miRNAs play important roles in a variety of biological processes, such as cell proliferation, neuronal differentiation, developmental timing, synapse function, and neurogenesis. A single miRNA can target hundreds of mRNA transcripts for either translation repression or degradation, but the function of many human miRNAs is not known. METHODS: miRNA array analysis was performed on the prefrontal cortex of 27 individual human cases (14 alcoholics and 13 matched controls). Target genes for differentially expressed miRNAs were predicted using multiple target prediction algorithms and a consensus approach, and predicted targets were matched against differentially expressed mRNAs from the same samples. Over- and under-representation analysis was performed using hypergeometric probability and z-score tests. RESULTS: Approximately 35 miRNAs were significantly up-regulated in the alcoholic group compared with controls. Target prediction showed a large degree of overlap with our published cDNA microarray data. Functional classification of the predicted target genes of the regulated miRNAs includes apoptosis, cell cycle, cell adhesion, nervous system development, and cell-cell signaling. CONCLUSIONS: These data suggest that the reduced expression of genes in human alcoholic cases may be because of the up-regulated miRNAs. Cellular processes fundamental to neuronal plasticity appear to represent major targets of the suggested miRNA regulation.

Temperature-sensitive mutant of the Caenorhabditis elegans neurotoxic MEC-4(d) DEG/ENaC channel identifies a site required for trafficking or surface maintenance.

DEG/ENaC channel subunits are two transmembrane domain proteins that assemble into heteromeric complexes to perform diverse biological functions that include sensory perception, electrolyte balance, and synaptic plasticity. Hyperactivation of neuronally expressed DEG/ENaCs that conduct both Na+ and Ca2+, however, can potently induce necrotic neuronal death in vivo. For example, Caenorhabditis elegans DEG/ENaC MEC-4 comprises the core subunit of a touch-transducing ion channel critical for mechanosensation that when hyperactivated by a mec-4(d) mutation induces necrosis of the sensory neurons in which it is expressed. Thus, studies of the MEC-4 channel have provided insight into both normal channel biology and neurotoxicity mechanisms. Here we report on intragenic mec-4 mutations identified in a screen for suppressors of mec-4(d)-induced necrosis, with a focus on detailed characterization of allele bz2 that has the distinctive phenotype of inducing dramatic neuronal swelling without being fully penetrant for toxicity. The bz2 mutation encodes substitution A745T, which is situated in the intracellular C-terminal domain of MEC-4. We show that this substitution renders both MEC-4 and MEC-4(d) activity strongly temperature sensitive. In addition, we show that both in Xenopus oocytes and in vivo, substitution A745T disrupts channel trafficking or maintenance of the MEC-4 subunit at the cell surface. This is the first demonstration of a C-terminal domain that affects trafficking of a neuronally expressed DEG/ENaC. Moreover, this study reveals that neuronal swelling occurs prior to commitment to necrotic death and defines a powerful new tool for inducible necrosis initiation.

Demostración, mediante enzymeba, del sobrediagnóstico de amebiasis intestinal asociado al examen microscópico de heces.Reporte de un estudio en Cienfuegos,Cuba / Demonstration, using Enzymeba test, of the overdiafnosis of instestinal amebiasis associated to the microscopical examination of faeces.Report of a study in Cienfuegos,Cuba

El sobrediagnóstico microscópico de amebiasis intestinal ha sido ampliamente reportado pero pocas veces objetivamente demostrado. En el presente trabajo, empleando Enzymeba, un procedimiento diagnóstico de amebiasis intestinal desarrollado en el Instituto de Medicina Tropical "Pedro Kourí", Ciudad de La Habana, abordamos este problema. Duranteseis meses se colectaron en los servicios de parasitologia de la casi totalidad de los policlínicos(13 de 14), y de los dos mayores hospitales(2 de 3) de la provincia de Cienfuegos, todas las muestras de heces en las que el personal técnico correspondiente había ing/formado la detección por observación microscópica de uno o más estadíos de Entamoeba histolytica. En sólo 61(14,4 por cento) de 424 muestras colectadas Enzymeba confirmó el hallazgo microscópico previo. No encontramos diferencia estadísticamente significativa(p>0.05) entre la eficiencia del diagnóstico microscópico en los policlínicos de aquella provincia.