The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

Circular RNAs are temporospatially regulated throughout development and ageing in the rat.

Circular RNAs (circRNAs) are covalently closed structural isoforms of linear mRNA which have been observed across a broad range of species and tissues. Here, we provide a comprehensive circRNAs expression catalogue for the rat including 8 organs of both sexes during 4 developmental stages using a public RNAseq dataset. These analyses revealed thousands of circular RNA species, many expressed in an organ-specific manner along with their host genes which were enriched with tissue-specific biological functions. A large number of circRNAs also displayed a developmental-dependent expression pattern and are accumulated during ageing. CircRNAs also displayed some sexually dimorphic expression, with gender associated differences observed in various tissues and developmental stages. These observations suggest that circRNAs are dynamically expressed in a spatial-, temporal- and gender-specific manner in mammals, and may have important biological function in differentiation, development and aging.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

MiR-137: an important player in neural development and neoplastic transformation.

MicroRNAs (miRNAs) represent an important class of small regulatory RNAs that control gene expression posttranscriptionally by targeting mRNAs for degradation or translation inhibition. Early studies have revealed a complex role for miRNAs in major biological processes such as development, differentiation, growth and metabolism. MiR-137 in particular, has been of great interest due to its critical role in brain function and putative involvement in the etiology of both neuropsychiatric disorders and cancer. Several lines of evidence suggest that development, differentiation and maturation of the nervous system is strongly linked to the expression of miR-137 and its regulation of a large number of downstream target genes in various pathways. Dysregulation of this molecule has also been implicated in major mental illnesses through its position in a variant allele highly associated with schizophrenia in the largest mega genome-wide association studies. Interestingly, miR-137 has also been shown to act as a tumor suppressor, with numerous studies finding reduced expression in neoplasia including brain tumor. Restoration of miR-137 expression has also been shown to inhibit cell proliferation, migration and metastasis, and induce cell cycle arrest, differentiation and apoptosis. These properties of miR-137 propose its potential for prognosis, diagnosis and as a therapeutic target for treatment of several human neurological and neoplastic disorders. In this review, we provide details on the discovery, targets, function, regulation and disease involvement of miR-137 with a broad look at recent discovery in this area.

Correction: Safety of Seasonal Malaria Chemoprevention (SMC) with Sulfadoxine-Pyrimethamine plus Amodiaquine when Delivered to Children under 10 Years of Age by District Health Services in Senegal: Results from a Stepped-Wedge Cluster Randomized Trial.

[This corrects the article DOI: 10.1371/journal.pone.0162563.].

Safety of Seasonal Malaria Chemoprevention (SMC) with Sulfadoxine-Pyrimethamine plus Amodiaquine when Delivered to Children under 10 Years of Age by District Health Services in Senegal: Results from a Stepped-Wedge Cluster Randomized Trial.

BACKGROUND: It is recommended that children aged 3 months to five years of age living in areas of seasonal transmission in the sub-Sahel should receive Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine (SPAQ) during the malaria transmission season. The purpose of this study was to evaluate the safety of SMC with SPAQ in children when delivered by community health workers in three districts in Senegal where SMC was introduced over three years, in children from 3 months of age to five years of age in the first year, then in children up to 10 years of age. METHODS: A surveillance system was established to record all deaths and all malaria cases diagnosed at health facilities and a pharmacovigilance system was established to detect adverse drug reactions. Health posts were randomized to introduce SMC in a stepped wedge design. SMC with SPAQ was administered once per month from September to November, by nine health-posts in 2008, by 27 in 2009 and by 45 in 2010. RESULTS: After three years, 780,000 documented courses of SMC had been administered. High coverage was achieved. No serious adverse events attributable to the intervention were detected, despite a high level of surveillance. CONCLUSIONS: SMC is being implemented in countries of the sub-Sahel for children under 5 years of age, but in some areas the age distribution of cases of malaria may justify extending this age limit, as has been done in Senegal. Our results show that SMC is well tolerated in children under five and in older children. However, pharmacovigilance should be maintained where SMC is implemented and provision for strengthening national pharmacovigilance systems should be included in plans for SMC implementation. TRIAL REGISTRATION: ClinicalTrials.gov NCT 00712374.

Developmental suppression of schizophrenia-associated miR-137 alters sensorimotor function in zebrafish.

The neurodevelopmentally regulated microRNA miR-137 was strongly implicated as risk locus for schizophrenia in the most recent genome wide association study coordinated by the Psychiatric Genome Consortium (PGC). This molecule is highly conserved in vertebrates enabling the investigation of its function in the developing zebrafish. We utilized this model system to achieve overexpression and suppression of miR-137, both transiently and stably through transgenesis. While miR-137 overexpression was not associated with an observable specific phenotype, downregulation by antisense morpholino and/or transgenic expression of miR-sponge RNA induced significant impairment of both embryonic and larval touch-sensitivity without compromising overall anatomical development. We observed miR-137 expression and activity in sensory neurons including Rohon-Beard neurons and dorsal root ganglia, two neuronal cell types that confer touch-sensitivity in normal zebrafish, suggesting a role of these cell types in the observed phenotype. The lack of obvious anatomical or histological pathology in these cells, however, suggested that subtle axonal network defects or a change in synaptic function and neural connectivity might be responsible for the behavioral phenotype rather than a change in the cellular morphology or neuroanatomy.

Genomic Epidemiology of a Protracted Hospital Outbreak Caused by a Toxin A-Negative Clostridium difficile Sublineage PCR Ribotype 017 Strain in London, England.

Clostridium difficile remains the leading cause of nosocomial diarrhea worldwide, which is largely considered to be due to the production of two potent toxins: TcdA and TcdB. However, PCR ribotype (RT) 017, one of five clonal lineages of human virulent C. difficile, lacks TcdA expression but causes widespread disease. Whole-genome sequencing was applied to 35 isolates from hospitalized patients with C. difficile infection (CDI) and two environmental ward isolates in London, England. The phylogenetic analysis of single nucleotide polymorphisms (SNPs) revealed a clonal cluster of temporally variable isolates from a single hospital ward at University Hospital Lewisham (UHL) that were distinct from other London hospital isolates. De novo assembled genomes revealed a 49-kbp putative conjugative transposon exclusive to this hospital clonal cluster which would not be revealed by current typing methodologies. This study identified three sublineages of C. difficile RT017 that are circulating in London. Similar to the notorious RT027 lineage, which has caused global outbreaks of CDI since 2001, the lineage of toxin-defective RT017 strains appears to be continually evolving. By utilization of WGS technologies to identify SNPs and the evolution of clonal strains, the transmission of outbreaks caused by near-identical isolates can be retraced and identified.

Distinct miRNA expression in dorsal striatal subregions is associated with risk for addiction in rats.

Recently, we published data using an animal model that allowed us to characterize animals into two groups, addiction vulnerable and addiction resilient, where we identified that addiction/relapse vulnerability was associated with deficits in synaptic plasticity-associated gene expression in the dorsal striatum (DS). Notable was the strong reduction in expression for activity-regulated cytoskeleton-associated protein (Arc) considered a master regulator of synaptic plasticity. In the present study, we confirmed that Arc messenger RNA was significantly decreased in the DS, but importantly, we identified that this reduction was restricted to the dorsomedial (DMS) and not dorsolateral striatum (DLS). There is recent evidence of microRNA (miRNA)-associated posttranscriptional suppression of Arc and animal models of addiction have identified a key role for miRNA in the regulation of addiction-relevant genes. In further support of this link, we identified several differentially expressed miRNA with the potential to influence addiction-relevant plasticity genes, including Arc. A key study recently reported that miR-212 expression is protective against compulsive cocaine-seeking. Supporting this hypothesis, we found that miR-212 expression was significantly reduced in the DMS but not DLS of addiction-vulnerable animals. Together, our data provide strong evidence that miRNA promote ongoing plasticity deficits in the DS of addiction-vulnerable animals.

Alteration of imprinted Dlk1-Dio3 miRNA cluster expression in the entorhinal cortex induced by maternal immune activation and adolescent cannabinoid exposure.

A significant feature of the cortical neuropathology of schizophrenia is a disturbance in the biogenesis of short non-coding microRNA (miRNA) that regulate translation and stability of mRNA. While the biological origin of this phenomenon has not been defined, it is plausible that it relates to major environmental risk factors associated with the disorder such as exposure to maternal immune activation (MIA) and adolescent cannabis use. To explore this hypothesis, we administered the viral mimic poly I:C to pregnant rats and further exposed some of their maturing offsprings to daily injections of the synthetic cannabinoid HU210 for 14 days starting on postnatal day 35. Whole-genome miRNA expression analysis was then performed on the left and right hemispheres of the entorhinal cortex (EC), a region strongly associated with schizophrenia. Animals exposed to either treatment alone or in combination exhibited significant differences in the expression of miRNA in the left hemisphere, whereas the right hemisphere was less responsive. Hemisphere-associated differences in miRNA expression were greatest in the combined treatment and highly over-represented in a single imprinted locus on chromosome 6q32. This observation was significant as the syntenic 14q32 locus in humans encodes a large proportion of miRNAs differentially expressed in peripheral blood lymphocytes from patients with schizophrenia, suggesting that interaction of early and late environmental insults may affect miRNA expression, in a manner that is relevant to schizophrenia.

The Clostridium difficile PCR ribotype 027 lineage: a pathogen on the move.

Clostridium difficile is a Gram-positive, spore-forming, human and animal pathogen that is the major cause of antibiotic-associated diarrhoea worldwide. The past decade has seen the rapid emergence of the hypervirulent PCR ribotype (RT) 027 complex, which has been associated with increases in the incidence and severity of disease and mortality. In this review, we describe the potential virulence factors that have been reported in strains from the RT 027 complex. We review the emergence, population structure, dissemination and evolution of this lineage.

Genome-wide mRNA and miRNA analysis of peripheral blood mononuclear cells (PBMC) reveals different miRNAs regulating HIV/HCV co-infection.

Co-infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is common due to shared transmission routes. The genomic basis of HIV/HCV co-infection and its regulation by microRNA (miRNA) is unknown. Therefore, our objective was to investigate genome-wide mRNA expression and its regulation by miRNA in primary PBMCs derived from 27 patients (5 HCV - mono-infected, 5 HIV-mono-infected, 12 HCV/HIV co-infected, and 5 healthy controls). This revealed 27 miRNAs and 476 mRNAs as differentially expressed (DE) in HCV/HIV co-infection when compared to controls (adj p<0.05). Our study shows the first evidence of miRNAs specific for co-infection, several of which are correlated with key gene targets demonstrating functional relationships to pathways in cancer, immune-function, and metabolism. Notable was the up regulation of HCV-specific miR-122 in co-infection (FC>50, p=4.02E-06), which may have clinical/biological implications.

The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing.

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

Decreased cortical muscarinic M1 receptors in schizophrenia are associated with changes in gene promoter methylation, mRNA and gene targeting microRNA.

Many studies have shown decreased cortical muscarinic M1 receptors (CHRM1) in schizophrenia (Sz), with one study showing Sz can be separated into two populations based on a marked loss of CHRM1 (-75%) in -25% of people (Def-Sz) with the disorder. To better understand the mechanism contributing to the loss of CHRM1 in Def-Sz, we measured specific markers of gene expression in the cortex of people with Sz as a whole, people differentiated into Def-Sz and people with Sz that do not have a deficit in cortical CHRM1 (Non-Def-Sz) and health controls. We now report that cortical CHRM1 gene promoter methylation and CHRM1 mRNA are decrease in Sz, Def-Sz and Non-Def-Sz but levels of the micro RNA (miR)-107, a CHRM1 targeting miR, are increased only in Def-Sz. We also report in vitro data strongly supporting the notion that miR-107 levels regulate CHRM1 expression. These data suggest there is a reversal of the expected inverse relationship between gene promoter methylation and CHRM1 mRNA in people with Sz and that a breakdown in gene promoter methylation control of CHRM1 expression is contributing to the global pathophysiology of the syndrome. In addition, our data argues that increased levels of at least one miR, miR-107, is contributing to the marked loss of cortical CHRM1 in Def-Sz and this may be a differentiating pathophysiology. These latter data continue to support the hypothesis that microRNAs (miRNA) have a role in the underlying neurobiology of Sz but argue they are differentially affected in subsets of people within that syndrome.

Increased inflammatory markers identified in the dorsolateral prefrontal cortex of individuals with schizophrenia.

Upregulation of the immune response may be involved in the pathogenesis of schizophrenia with changes occurring in both peripheral blood and brain tissue. To date, microarray technology has provided a limited view of specific inflammatory transcripts in brain perhaps due to sensitivity issues. Here we used SOLiD Next Generation Sequencing to quantify neuroimmune mRNA expression levels in the dorsolateral prefrontal cortex of 20 individuals with schizophrenia and their matched controls. We detected 798 differentially regulated transcripts present in people with schizophrenia compared with controls. Ingenuity pathway analysis identified the inflammatory response as a key change. Using quantitative real-time PCR we confirmed the changes in candidate cytokines and immune modulators, including interleukin (IL)-6, IL-8, IL-1ß and SERPINA3. The density of major histocompatibility complex-II-positive cells morphologically resembling microglia was significantly increased in schizophrenia and correlated with IL-1ß expression. A group of individuals, most of whom had schizophrenia, were found to have increased inflammatory mRNA expression. In summary, we have demonstrated changes in an inflammatory response pathway that are present in ∼40% of people diagnosed with schizophrenia. This suggests that therapies aimed at immune system attenuation in schizophrenia may be of direct benefit in the brain.

Genome-wide supported variant MIR137 and severe negative symptoms predict membership of an impaired cognitive subtype of schizophrenia.

Progress in determining the aetiology of schizophrenia (Sz) has arguably been limited by a poorly defined phenotype. We sought to delineate empirically derived cognitive subtypes of Sz to investigate the association of a genetic variant identified in a recent genome-wide association study with specific phenotypic characteristics of Sz. We applied Grade of Membership (GoM) analyses to 617 patients meeting ICD-10 criteria for Sz (n=526) or schizoaffective disorder (n=91), using cognitive performance indicators collected within the Australian Schizophrenia Research Bank. Cognitive variables included subscales from the Repeatable Battery for the Assessment of Neuropsychological Status, the Controlled Oral Word Association Test and the Letter Number Sequencing Test, and standardised estimates of premorbid and current intelligence quotient. The most parsimonious GoM solution yielded two subtypes of clinical cases reflecting those with cognitive deficits (CDs; N=294), comprising 47.6% of the sample who were impaired across all cognitive measures, and a cognitively spared group (CS; N=323) made up of the remaining 52.4% who performed relatively well on all cognitive tests. The CD subgroup were more likely to be unemployed, had an earlier illness onset, and greater severity of functional disability and negative symptoms than the CS group. Risk alleles on the MIR137 single-nucleotide polymorphism (SNP) predicted membership of CD subtype only in combination with higher severity of negative symptoms. These findings provide the first evidence for association of the MIR137 SNP with a specific Sz phenotype characterised by severe CDs and negative symptoms, consistent with the emerging role of microRNAs in the regulation of proteins responsible for neural development and function.

Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England.

BACKGROUND: Marked increases in Clostridium difficile infection (CDI) incidence, driven by epidemic strain spread, is a global phenomenon. METHODS: The Clostridium difficile Ribotyping Network (CDRN) was established in 2007 as part of enhanced CDI surveillance in England, to facilitate the recognition and control of epidemic strains. We report on changes in CDI epidemiology in England in the first 3 years of CDRN. RESULTS: CDRN received 12,603 fecal specimens, comprising significantly (P < .05) increasing numbers and proportions of national CDI cases in 2007-2008 (n = 2109, 3.8%), 2008-2009 (n = 4774, 13.2%), and 2009-2010 (n = 5720, 22.3%). The C. difficile recovery rate was 90%, yielding 11,294 isolates for ribotyping. Rates of 9 of the 10 most common ribotypes changed significantly (P < .05) during 2007-2010. Clostridium difficile ribotype 027 predominated, but decreased markedly from 55% to 36% and 21% in 2007-2008, 2008-2009, and 2009-2010, respectively. The largest regional variations in prevalence occurred for ribotypes 027, 002, 015, and 078. Cephalosporin and fluoroquinolone use in CDI cases was reported significantly (P < .05) less frequently during 2007-2010. Mortality data were subject to potential reporting bias, but there was a significant decrease in CDI-associated deaths during 2007-2010, which may have been due to multiple factors, including reduced prevalence of ribotype 027. CONCLUSIONS: Access to C. difficile ribotyping was associated with significant changes in the prevalence of epidemic strains, especially ribotype 027. These changes coincided with markedly reduced CDI incidence and related mortality in England. CDI control programs should include prospective access to C. difficile typing and analysis of risk factors for CDI and outcomes.