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INTRODUCTION: Gut microbial communities are critical players in the pathogenesis of obesity. Pregnancy is associated with increased bacterial load and changes in gut bacterial diversity. Sparse data exist regarding composition of gut microbial communities in obesity combined with pregnancy. MATERIAL AND METHODS: Banked tissues were collected under sterile conditions during necropsy, from three non-obese (nOb) and four obese (Ob) near-term pregnant baboons. Sequences were assigned taxonomy using the Ribosomal Database Project classifier. Microbiome abundance and its difference between distinct groups were assessed by a nonparametric test. RESULTS: Three families predominated in both the nOb and Ob colonic microbiome: Prevotellaceae (25.98% and 32.71% respectively), Ruminococcaceae (12.96% and 7.48%), and Lachnospiraceae (8.78% and 11.74%). Seven families of the colon microbiome displayed differences between Ob and nOb groups. CONCLUSION: Changes in gut microbiome in pregnant obese animals open the venue for dietary manipulation in pregnancy.
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Bactérias/isolamento & purificação , Microbioma Gastrointestinal , Doenças dos Macacos/microbiologia , Obesidade/microbiologia , Papio/microbiologia , Animais , Bactérias/classificação , Feminino , GravidezRESUMO
The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.
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Evolução Biológica , Pandemias , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Exotoxinas/genética , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Saúde Global , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neutrófilos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose , Filogenia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Transcriptoma , VirulênciaRESUMO
ERK (extracellular-signal-regulated kinase) activation leads to disruption of tight junctions in some epithelial monolayers, whereas it prevents disruption of tight junctions in other epithelia. The factors responsible for such contrasting influences of ERK on tight junction integrity are unknown. The present study investigated the effect of the state of cell differentiation on ERK-mediated regulation of tight junctions in Caco-2 cell monolayers. EGF (epidermal growth factor) potentiated H2O2-induced tight junction disruption in under-differentiated cell monolayers, which was attenuated by the MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitor U0126. In contrast, EGF prevented H2O2-induced disruption of tight junctions in differentiated cell monolayers, which was also attenuated by U0126. Knockdown of ERK1/2 enhanced tight junction integrity and accelerated assembly of tight junctions in under-differentiated cell monolayers, whereas it had the opposite effect in differentiated cell monolayers. Regulated expression of wild-type and constitutively active MEK1 disrupted tight junctions, and the expression of dominant-negative MEK1 enhanced tight junction integrity in under-differentiated cells, whereas contrasting responses were recorded in differentiated cells. EGF prevented both H2O2-induced association of PP2A (protein phosphatase 2A), and loss of association of PKCζ (protein kinase Cζ), with occludin by an ERK-dependent mechanism in differentiated cell monolayers, but not in under-differentiated cell monolayers. Active ERK was distributed in the intracellular compartment in under-differentiated cell monolayers, whereas it was localized mainly in the perijunctional region in differentiated cell monolayers. Thus ERK may exhibit its contrasting influences on tight junction integrity in under-differentiated and differentiated epithelial cells by virtue of differences in its subcellular distribution and ability to regulate the association of PKCζ and PP2A with tight junction proteins.
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Diferenciação Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Junções Íntimas , Células CACO-2 , Células Epiteliais/citologia , Células Epiteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/análise , Humanos , MAP Quinase Quinase 1/fisiologia , Ligação Proteica , Proteína Quinase C/metabolismo , Proteína Fosfatase 2/metabolismo , Distribuição TecidualRESUMO
Recently published exploratory studies based on exposure to outdoor fine particulates, defined as particles with a nominal mean diameter less than or equal to 2.5 µm (PM2.5) indicate that the pollutant may play a role in mental health conditions, such as major depressive disorder. This paper details a model that can estimate the United States (US) major depressive disorder burden attributable to indoor PM2.5 exposure, locally modifiable through input parameter calibrations. By utilizing concentration values in an exposure-response function, along with relative risk values derived from epidemiological studies, the model estimated the prevalence of expected cases of major depressive disorder in multiple scenarios. Model results show that exposure to indoor PM2.5 might contribute to 476,000 cases of major depressive disorder in the US (95% confidence interval 11,000-1,100,000), approximately 2.7% of the total number of cases reported annually. Increasing heating, ventilation, and air conditioning (HVAC) filter efficiency in a residential dwelling results in minor reductions in depressive disorders in rural or urban locations in the US. Nevertheless, a minimum efficiency reporting value (MERV) 13 filter does have a benefit/cost ratio at or near one when smoking occurs indoors; during wildfires; or in locations with elevated outdoor PM2.5 concentrations. The approach undertaken herein could provide a transparent strategy for investment into the built environment to improve the mental health of the occupants.
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Striking individual differences in severity of group A streptococcal (GAS) sepsis have been noted, even among patients infected with the same bacterial strain. We had provided evidence that HLA class II allelic variation contributes significantly to differences in systemic disease severity by modulating host responses to streptococcal superantigens. Inasmuch as the bacteria produce additional virulence factors that participate in the pathogenesis of this complex disease, we sought to identify additional gene networks modulating GAS sepsis. Accordingly, we applied a systems genetics approach using a panel of advanced recombinant inbred mice. By analyzing disease phenotypes in the context of mice genotypes we identified a highly significant quantitative trait locus (QTL) on Chromosome 2 between 22 and 34 Mb that strongly predicts disease severity, accounting for 25%-30% of variance. This QTL harbors several polymorphic genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, cocitation networks, and biological relevance, and identified interleukin1 alpha and prostaglandin E synthases pathways as key networks involved in modulating GAS sepsis severity. The association of GAS sepsis with multiple pathways underscores the complexity of traits modulating GAS sepsis and provides a powerful approach for analyzing interactive traits affecting outcomes of other infectious diseases.
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Mapeamento Cromossômico/métodos , Predisposição Genética para Doença , Sepse/genética , Infecções Estreptocócicas/genética , Streptococcus pyogenes/patogenicidade , Animais , Bacteriemia , Feminino , Regulação Bacteriana da Expressão Gênica , Genômica , Genótipo , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Locos de Características Quantitativas , Recombinação Genética , Sepse/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/fisiologiaRESUMO
Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for â¼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of â¼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets--by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.
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Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Variação Genética , Animais , Densidade Óssea/genética , Caenorhabditis elegans , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Biblioteca Gênica , Estudo de Associação Genômica Ampla , Genômica , Humanos , Camundongos , Camundongos Endogâmicos DBA , Locos de Características QuantitativasRESUMO
In the Xenopus laevis oocyte there is a million fold more transcription factor IIIA (TFIIIA) and its corresponding mRNA than in a somatic cell. These high levels of TFIIIA gene expression are achieved primarily by transcriptional regulation. The TATA box along with three positive cis-elements in the control region of the TFIIIA gene located at positions -269 to -264 (E1), -235 to -220 (E2), and -669 to -636 (E3) are required for this high level of expression in oocytes. The proteins that bind E1 and E3 of the TFIIIA gene have been identified as Xenopus USF (Xl-USF) and B3 (homolog of Vg1 RBP/VERA). In this study the B2 protein was found to bind E2 in a zinc-dependent fashion and anti-human Sp1 (but not Sp2, Sp3, nor Sp4) supershifted the B2:element 2 complex. The E2 binding protein was purified by DNA affinity chromatography. Based on supershift analysis, molecular weight estimation experiments, and purified human Sp1 DNA binding affinity tests the data strongly support the idea that the B2 protein is the Xenopus ortholog of Sp1, but not Sp2, Sp3, nor Sp4. Xl-USF binds to element 1 of the TFIIIA gene which is immediately adjacent to element 2. Coimmunoprecipitation experiments using crude whole oocyte extracts revealed that Xenopus Sp1 and USF or closely related factors are present together in a high-affinity complex. This structure contributes positively to the initiation of TFIIIA gene transcription in Xenopus oocytes.
Assuntos
Proteínas de Ligação a DNA , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição TFIIA/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Humanos , Immunoblotting , Peso Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Elementos de Resposta/genética , Fator de Transcrição Sp1/imunologia , Fator de Transcrição TFIIA/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores Estimuladores Upstream , Xenopus laevis/genética , Dedos de Zinco/genéticaRESUMO
The selective expression of the Xenopus TFIIIA gene in immature oocytes is principally regulated by a single 5'-flanking DNA sequence element, termed element 3 (i.e. E3). We describe the isolation and characterization of a cDNA for a protein present in immature Xenopus ooctyes, termed B3.65, which appears to bind to and activate E3-mediated expression. The approximate molecular weight of the E3 binding protein(s) was determined by ultraviolet light cross-linking analysis. B3.65, a protein of the appropriate molecular weight, was purified biochemically from immature Xenopus ooctye extracts by affinity chromatography. Antiserum to purified B3.65 super-shifted the E3 activator complex. In addition, B3.65 mRNA was found to be highly enriched in immature oocytes. All of these data are consistent with B3.65 either being the E3 activator, or antigenically related to the specific activator required for XenopusTFIIIA gene transcription. B3.65 is a member of the K-homologous (KH) domain family of proteins, with almost absolute identity to Xenopus Vg1 RBP/VERA (97%) and significant similarity to human koc (82%). The koc mRNA is over-expressed in human pancreatic cancer tissues, and B3.65 mRNA was detected in Xenopus pancreas and kidney. Interestingly, KH proteins, like Vg1RBP/VERA, are most commonly associated with RNA metabolism, in their capacity to regulate RNA localization, stability, and translation. Our results suggest that B3.65 is a key regulator of both RNA- and DNA metabolism.
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Fator de Transcrição TFIIIA/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Northern Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Peso Molecular , Oócitos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
The cellular heterogeneity of brain poses a particularly thorny issue in genome-wide gene expression studies. Because laser capture microdissection (LCM) enables the precise extraction of a small area of tissue, we combined LCM with neuronal track tracing to collect nucleus accumbens shell neurons that project to ventral pallidum, which are of particular interest in the study of reward and addiction. Four independent biological samples of accumbens projection neurons were obtained. Approximately 500 pg of total RNA from each sample was then amplified linearly and subjected to Affymetrix microarray and Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD) transcriptome sequencing (RNA-seq). A total of 375 million 50-bp reads were obtained from RNA-seq. Approximately 57% of these reads were mapped to the rat reference genome (Baylor 3.4/rn4). Approximately 11,000 unique RefSeq genes and 100,000 unique exons were identified from each sample. Of the unmapped reads, the quality scores were 4.74 ± 0.42 lower than the mapped reads. When RNA-seq and microarray data from the same samples were compared, Pearson correlations were between 0.764 and 0.798. The variances in data obtained for the four samples by microarray and RNA-seq were similar for medium to high abundance genes, but less among low abundance genes detected by microarray. Analysis of 34 genes by real-time polymerase chain reaction showed higher correlation with RNA-seq (0.66) than with microarray (0.46). Further analysis showed 20-30 million 50-bp reads are sufficient to provide estimates of gene expression levels comparable to those produced by microarray. In summary, this study showed that picogram quantities of total RNA obtained by LCM of â¼700 individual neurons is sufficient to take advantage of the benefits provided by the transcriptome sequencing technology, such as low background noise, high dynamic range, and high precision.
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The onset of infection and the switch from primary to secondary niches are dramatic environmental changes that not only alter bacterial transcriptional programs, but also perturb their sociomicrobiology, often driving minor subpopulations with mutant phenotypes to prevail in specific niches. Having previously reported that M1T1 Streptococcus pyogenes become hypervirulent in mice due to selection of mutants in the covRS regulatory genes, we set out to dissect the impact of these mutations in vitro and in vivo from the impact of other adaptive events. Using a murine subcutaneous chamber model to sample the bacteria prior to selection or expansion of mutants, we compared gene expression dynamics of wild type (WT) and previously isolated animal-passaged (AP) covS mutant bacteria both in vitro and in vivo, and we found extensive transcriptional alterations of pathoadaptive and metabolic gene sets associated with invasion, immune evasion, tissue-dissemination, and metabolic reprogramming. In contrast to the virulence-associated differences between WT and AP bacteria, Phenotype Microarray analysis showed minor in vitro phenotypic differences between the two isogenic variants. Additionally, our results reflect that WT bacteria's rapid host-adaptive transcriptional reprogramming was not sufficient for their survival, and they were outnumbered by hypervirulent covS mutants with SpeB(-)/Sda(high) phenotype, which survived up to 14 days in mice chambers. Our findings demonstrate the engagement of unique regulatory modules in niche adaptation, implicate a critical role for bacterial genetic heterogeneity that surpasses transcriptional in vivo adaptation, and portray the dynamics underlying the selection of hypervirulent covS mutants over their parental WT cells.
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Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Seleção Genética , Streptococcus pyogenes/genética , Animais , Evolução Biológica , Interações Hospedeiro-Patógeno/genética , Camundongos , Streptococcus pyogenes/patogenicidade , Virulência/genéticaRESUMO
Common sequence variants within a gene often generate important differences in expression of corresponding mRNAs. This high level of local (allelic) control-or cis modulation-rivals that produced by gene targeting, but expression is titrated finely over a range of levels. We are interested in exploiting this allelic variation to study gene function and downstream consequences of differences in expression dosage. We have used several bioinformatics and molecular approaches to estimate error rates in the discovery of cis modulation and to analyze some of the biological and technical confounds that contribute to the variation in gene expression profiling. Our analysis of SNPs and alternative transcripts, combined with eQTL maps and selective gene resequencing, revealed that between 17 and 25% of apparent cis modulation is caused by SNPs that overlap probes rather than by genuine quantitative differences in mRNA levels. This estimate climbs to 40-50% when qualitative differences between isoform variants are included. We have developed an analytical approach to filter differences in expression and improve the yield of genuine cis-modulated transcripts to approximately 80%. This improvement is important because the resulting variation can be successfully used to study downstream consequences of altered expression on higher-order phenotypes. Using a systems genetics approach we show that two validated cis-modulated genes, Stk25 and Rasd2, are likely to control expression of downstream targets and affect disease susceptibility.
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Alelos , Biologia Computacional , Perfilação da Expressão Gênica , Expressão Gênica/genética , Regiões 3' não Traduzidas/genética , Processamento Alternativo , Animais , Bases de Dados Genéticas , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Group A Streptococcus (GAS) causes rare but life-threatening syndromes of necrotizing fasciitis and toxic shock-like syndrome in humans. The GAS serotype M1T1 clone has globally disseminated, and mutations in the control of virulence regulatory sensor kinase (covRS) operon correlate with severe invasive disease. Here, a cohort of non-M1 GAS was screened to determine whether mutation in covRS triggers systemic dissemination in divergent M serotypes. A GAS disease model defining parameters governing invasive propensity of differing M types is proposed. The vast majority of GAS infection is benign. Nonetheless, many divergent M types possess limited capacity to cause invasive infection. M1T1 GAS readily switch to a covRS mutant form that is neutrophil resistant and frequently associated with systemic infection. Whilst non-M1 GAS are shown in this study to less frequently accumulate covRS mutations in vivo, such mutants are isolated from invasive infections and exhibit neutrophil resistance and enhanced virulence. The reduced capacity of non-M1 GAS to switch to the hypervirulent covRS mutant form provides an explanation for the comparatively less frequent isolation of non-M1 serotypes from invasive human infections.