Activation of beta-adrenoceptors mimics preconditioning of rat-isolated atria and ventricles against ischaemic contractile dysfunction

The effects of ischaemia and reoxygenation on cardiac contractile function can be abrogated by ischaemic preconditioning (IPC). We tested whether β-adrenoceptor agonists could mimic IPC and whether IPC was dependent on β-adrenoceptor activation in rat-isolated cardiac tissues. Paced left atria and right ventricular strips were set-up in Krebs solution and isometric developed tension recorded. Ischaemia was simulated by replacing with hypoxic glucose-free Krebs solution for 30 min. IPC and isoprenaline (10−7 M) preconditioning for 10 min were examined. Developed tension post-reoxygenation was expressed as a percentage of the pre-ischaemic baseline. Recovery at 15 min was significantly increased by IPC in atria (47 ± 4.0% vs. 29.3 ± 1.7%, p < 0.05) and ventricles (39.0 ± 5.2% vs. 22.4 ± 2.8%, p < 0.05). At 60 min, isoprenaline-treated atria recovery (75.8 ± 16.6%) was significantly (p < 0.05) greater than controls (47.9 ± 2.3%). Propranolol (10−6 M) abolished both effects. Therefore, both IPC and β-adrenoceptor agonist-induced improvement of contractile recovery was propranolol-sensitive and β-adrenoceptor-mediated

Removal of ecotoxicity of 17α-ethinylestradiol using TAML/peroxide water treatment

17α -ethinylestradiol (EE2), a synthetic oestrogen in oral contraceptives, is one of many pharmaceuticals found in inland waterways worldwide as a result of human consumption and excretion into wastewater treatment systems. At low parts per trillion (ppt), EE2 induces feminisation of male fish, diminishing reproductive success and causing fish population collapse. Intended water quality standards for EE2 set a much needed global precedent. Ozone and activated carbon provide effective wastewater treatments, but their energy intensities and capital/operating costs are formidable barriers to adoption. Here we describe the technical and environmental performance of a fast- developing contender for mitigation of EE2 contamination of wastewater based upon smallmolecule, full-functional peroxidase enzyme replicas called “TAML activators”. From neutral to basic pH, TAML activators with H2O2 efficiently degrade EE2 in pure lab water, municipal effluents and EE2-spiked synthetic urine. TAML/H2O2 treatment curtails estrogenicity in vitro and substantially diminishes fish feminization in vivo. Our results provide a starting point for a future process in which tens of thousands of tonnes of wastewater could be treated per kilogram of catalyst. We suggest TAML/H2O2 is a worthy candidate for exploration as an environmentally compatible, versatile, method for removing EE2 and other pharmaceuticals from municipal wastewaters.Heinz Endowments, the Swiss National Science Foundation, the Steinbrenner Institute for a Steinbrenner Doctoral Fellowship. NMR instrumentation at CMU was partially supported by NSF (CHE-0130903 and CHE-1039870)

Whole Genome Sequence Analysis of Cryptococcus gattii from the Pacific Northwest Reveals Unexpected Diversity

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen

Determination of Beta-Defensin Genomic Copy Number in Different Populations: A Comparison of Three Methods

There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories.In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations.QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number

Fatal Disseminated Cryptococcus gattii Infection in New Mexico

We report a case of fatal disseminated infection with Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico

Division of labour in response to host oxidative burst drives a fatal Cryptococcus gattii outbreak

Cryptococcus gattii is an emerging intracellular pathogen and the cause of the largest primary outbreak of a life-threatening fungal disease in a healthy population. Outbreak strains share a unique mitochondrial gene expression profile and an increased ability to tubularize their mitochondria within host macrophages. However, the underlying mechanism that causes this lineage of C. gattii to be virulent in immunocompetent individuals remains unexplained. Here we show that a subpopulation of intracellular C. gattii adopts a tubular mitochondrial morphology in response to host reactive oxygen species. These fungal cells then facilitate the rapid growth of neighbouring C. gattii cells with non-tubular mitochondria, allowing for effective establishment of the pathogen within a macrophage intracellular niche. Thus, host reactive oxygen species, an essential component of the innate immune response, act as major signalling molecules to trigger a ‘division of labour’ in the intracellular fungal population, leading to increased pathogenesis within this outbreak lineage

Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

Association of LEC and tnpA Helicobacter pylori genes with gastric cancer in a Brazilian population

<p>Abstract</p> <p>Background</p> <p><it>H. pylori </it>seroprevalence in Brazilians varies and is dependent on socioeconomic status, sanitation conditions and ethnicity; furthermore, <it>H. pylori </it>is not always associated with the incidence of gastric cancer, suggesting the role of more virulent strains. The purpose of this study was to analyze the association of more virulent <it>H. pylori </it>strains with gastric cancer.</p> <p>Methods</p> <p>DNA was extracted from gastric biopsies of thirty-four cases of gastric cancer (11 intestinal-type, 23 diffuse-type), and thirty-four of patients with endoscopic gastritis. The presence of <it>cag</it>PAI genes (<it>cagA</it>, <it>cagA </it>promoter, <it>cagE</it>, <it>cagM</it>, <it>tnpB</it>, <it>tnpA</it>, <it>cagT </it>and the left end of the <it>cag</it>II (LEC)) and <it>babA </it>were analyzed by PCR.</p> <p>Results</p> <p>Comparison of <it>H. pylori </it>isolates from gastric cancer and gastritis patients showed significant associations of <it>tnpA </it>and LEC with gastric cancer (73.5% [OR, 6.66; 95% CI, 2.30-19.25] and 58.8% [OR, 10.71; 95% CI, 3.07-37.28] of cases, respectively). Other <it>cag</it>PAI genes were detected in both groups at similar frequencies.</p> <p>Conclusions</p> <p><it>tnpA </it>and LEC of <it>H. pylori cag</it>PAI were associated with gastric cancer; nonetheless, these results were restricted within this group of patients and further studies are needed to confirm these results in a larger sample and determine their role in gastric carcinogenesis.</p

A high-resolution integrated map of copy number polymorphisms within and between breeds of the modern domesticated dog

<p>Abstract</p> <p>Background</p> <p>Structural variation contributes to the rich genetic and phenotypic diversity of the modern domestic dog, <it>Canis lupus familiaris</it>, although compared to other organisms, catalogs of canine copy number variants (CNVs) are poorly defined. To this end, we developed a customized high-density tiling array across the canine genome and used it to discover CNVs in nine genetically diverse dogs and a gray wolf.</p> <p>Results</p> <p>In total, we identified 403 CNVs that overlap 401 genes, which are enriched for defense/immunity, oxidoreductase, protease, receptor, signaling molecule and transporter genes. Furthermore, we performed detailed comparisons between CNVs located within versus outside of segmental duplications (SDs) and find that CNVs in SDs are enriched for gene content and complexity. Finally, we compiled all known dog CNV regions and genotyped them with a custom aCGH chip in 61 dogs from 12 diverse breeds. These data allowed us to perform the first population genetics analysis of canine structural variation and identify CNVs that potentially contribute to breed specific traits.</p> <p>Conclusions</p> <p>Our comprehensive analysis of canine CNVs will be an important resource in genetically dissecting canine phenotypic and behavioral variation.</p