RAPD PCR detects co-colonisation of multiple Group B Streptococcus genotypes: a practical molecular technique for screening multiple colonies

Group B Streptococcus (GBS) is a leading cause of neonatal meningitis, pneumonia, and sepsis. The biggest contributing factor of neonatal infections is due to vertical transmission from maternal colonisation of GBS in the genitourinary tract. Multiple serotype colonisation is often not investigated in epidemiological studies, but it is an important consideration for serotype-based vaccine development and implementation to ensure less abundant serotypes are not under-represented. In this study, we show that RAPD PCR is a quick tool useful in screening the presence of genetically different strains using multiple colony picks from a single patient swab. We observed a maximum of five different GBS strains colonising a single patient at a specific time

MUC1-C drives myeloid leukaemogenesis and resistance to treatment by a survivin-mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy-resistant disease. The MUC1-C oncoprotein governs critical pathways of tumorigenesis, including self-renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1-C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1-C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1-C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1-C with silencing or pharmacologic inhibition with GO-203 led to a decrease in active β-catenin levels and, in-turn, down-regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1-C was also associated with increased sensitivity of AML cells to Cytarabine (Ara-C) treatment by a survivin-dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1-C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO-203 with Ara-C for the treatment of patients with AML

Sex-Dependent Changes in miRNA Expression in the Bed Nucleus of the Stria Terminalis Following Stress

Anxiety disorders disproportionately affect women compared to men, which may arise from sex differences in stress responses. MiRNAs are small non-coding RNAs known to regulate gene expression through actions on mRNAs. MiRNAs are regulated, in part, by factors such as stress and gonadal sex, and they have been implicated in the pathophysiology of multiple psychiatric disorders. Here, we assessed putative sex differences in miRNA expression in the bed nucleus of the stria terminalis (BNST) – a sexually dimorphic brain region implicated in anxiety – of adult male and female rats that had been exposed to social isolation (SI) stress throughout adolescence. To assess the translational utility of our results, we assessed if childhood trauma in humans resulted in changes in blood miRNA expression that are similar to those observed in rats. Male and female Sprague-Dawley rats underwent SI during adolescence or remained group housed (GH) and were tested for anxiety-like behavior in the elevated plus maze as adults. Small RNA sequencing was performed on tissue extracted from the BNST. Furthermore, we re-analyzed an already available small RNA sequencing data set from the Grady Trauma Project (GTP) from men and women to identify circulating miRNAs that are associated with childhood trauma exposure. Our results indicated that there were greater anxiogenic-like effects and changes in BNST miRNA expression in SI versus GH females compared to SI versus GH males. In addition, we found nine miRNAs that were regulated in both the BNST from SI compared to GH rats and in blood samples from humans exposed to childhood trauma. These studies emphasize the utility of rodent models in studying neurobiological mechanisms underlying psychiatric disorders and suggest that rodent models could be used to identify novel sex-specific pharmacotherapies for anxiety disorders

Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells

High Resolution Methylome Map of Rat Indicates Role of Intragenic DNA Methylation in Identification of Coding Region

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability

Ephrin-A5 Suppresses Neurotrophin Evoked Neuronal Motility, ERK Activation and Gene Expression

During brain development, growth cones respond to attractive and repulsive axon guidance cues. How growth cones integrate guidance instructions is poorly understood. Here, we demonstrate a link between BDNF (brain derived neurotrophic factor), promoting axonal branching and ephrin-A5, mediating axonal repulsion via Eph receptor tyrosine kinase activation. BDNF enhanced growth cone filopodial dynamics and neurite branching of primary neurons. We show that ephrin-A5 antagonized this BDNF-evoked neuronal motility. BDNF increased ERK phosphorylation (P-ERK) and nuclear ERK entry. Ephrin-A5 suppressed BDNF-induced ERK activity and might sequester P-ERK in the cytoplasm. Neurotrophins are well established stimulators of a neuronal immediate early gene (IEG) response. This is confirmed in this study by e.g. c-fos, Egr1 and Arc upregulation upon BDNF application. This BDNF-evoked IEG response required the transcription factor SRF (serum response factor). Notably, ephrin-A5 suppressed a BDNF-evoked neuronal IEG response, suggesting a role of Eph receptors in modulating gene expression. In opposite to IEGs, long-term ephrin-A5 application induced cytoskeletal gene expression of tropomyosin and actinin. To uncover specific Eph receptors mediating ephrin-As impact on neurotrophin signaling, EphA7 deficient mice were analyzed. In EphA7 deficient neurons alterations in growth cone morphology were observed. However, ephrin-A5 still counteracted neurotrophin signaling suggesting that EphA7 is not required for ephrin and BDNF crosstalk. In sum, our data suggest an interaction of ephrin-As and neurotrophin signaling pathways converging at ERK signaling and nuclear gene activity. As ephrins are involved in development and function of many organs, such modulation of receptor tyrosine kinase signaling and gene expression by Ephs might not be limited to the nervous system

The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies