Genomic Diversity among Drug Sensitive and Multidrug Resistant Isolates of Mycobacterium tuberculosis with Identical DNA Fingerprints

complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients.We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations. isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs

Evidence That Ca2+ within the Microdomain of the L-Type Voltage Gated Ca2+ Channel Activates ERK in MIN6 Cells in Response to Glucagon-Like Peptide-1

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca2+ through L-type voltage gated Ca2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation

Spontaneous DNA damage to the nuclear genome promotes senescence, T redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Controlled Chaos of Polymorphic Mucins in a Metazoan Parasite (Schistosoma mansoni) Interacting with Its Invertebrate Host (Biomphalaria glabrata)

Invertebrates were long thought to possess only a simple, effective and hence non-adaptive defence system against microbial and parasitic attacks. However, recent studies have shown that invertebrate immunity also relies on immune receptors that diversify (e.g. in echinoderms, insects and mollusks (Biomphalaria glabrata)). Apparently, individual or population-based polymorphism-generating mechanisms exists that permit the survival of invertebrate species exposed to parasites. Consequently, the generally accepted arms race hypothesis predicts that molecular diversity and polymorphism also exist in parasites of invertebrates. We investigated the diversity and polymorphism of parasite molecules (Schistosoma mansoni Polymorphic Mucins, SmPoMucs) that are key factors for the compatibility of schistosomes interacting with their host, the mollusc Biomphalaria glabrata. We have elucidated the complex cascade of mechanisms acting both at the genomic level and during expression that confer polymorphism to SmPoMuc. We show that SmPoMuc is coded by a multi-gene family whose members frequently recombine. We show that these genes are transcribed in an individual-specific manner, and that for each gene, multiple splice variants exist. Finally, we reveal the impact of this polymorphism on the SmPoMuc glycosylation status. Our data support the view that S. mansoni has evolved a complex hierarchical system that efficiently generates a high degree of polymorphism—a “controlled chaos”—based on a relatively low number of genes. This contrasts with protozoan parasites that generate antigenic variation from large sets of genes such as Trypanosoma cruzi, Trypanosoma brucei and Plasmodium falciparum. Our data support the view that the interaction between parasites and their invertebrate hosts are far more complex than previously thought. While most studies in this matter have focused on invertebrate host diversification, we clearly show that diversifying mechanisms also exist on the parasite side of the interaction. Our findings shed new light on how and why invertebrate immunity develops

Intravesical device-assisted therapies for non-muscle-invasive bladder cancer

Non-muscle-invasive bladder cancer (NMIBC), the most prevalent type of bladder cancer, accounts for ~75% of bladder cancer diagnoses. This disease has a 50% risk of recurrence and 20% risk of progression within 5 years, despite the use of intravesical adjuvant treatments (such as BCG or mitomycin C) that are recommended by clinical guidelines. Intravesical device-assisted therapies, such as radiofrequency-induced thermochemotherapeutic effect (RITE), conductive hyperthermic chemotherapy, and electromotive drug administration (EMDA), have shown promising efficacy. These device-assisted treatments are an attractive alternative to BCG, as issues with supply have been a problem in some countries. RITE might be an effective treatment option for some patients who have experienced BCG failure and are not candidates for radical cystectomy. Data from trials using EMDA suggest that it is effective in high-risk disease but requires further validation, and results of randomized trials are eagerly awaited for conductive hyperthermic chemotherapy. Considerable heterogeneity in patient cohorts, treatment sessions, use of maintenance regimens, and single-arm study design makes it difficult to draw solid conclusions, although randomized controlled trials have been reported for RITE and EMDA