Batrachochytrium dendrobatidis Shows High Genetic Diversity and Ecological Niche Specificity among Haplotypes in the Maya Mountains of Belize

The amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the globe. Although it has been found in most countries in Central America, its presence has never been assessed in Belize. We set out to determine the range, prevalence, and diversity of Bd using quantitative PCR (qPCR) and sequencing of a portion of the 5.8 s and ITS1-2 regions. Swabs were collected from 524 amphibians of at least 26 species in the protected areas of the Maya Mountains of Belize. We sequenced a subset of 72 samples that had tested positive for Bd by qPCR at least once; 30 samples were verified as Bd. Eight unique Bd haplotypes were identified in the Maya Mountains, five of which were previously undescribed. We identified unique ecological niches for the two most broadly distributed haplotypes. Combined with data showing differing virulence shown in different strains in other studies, the 5.8 s - ITS1-2 region diversity found in this study suggests that there may be substantial differences among populations or haplotypes. Future work should focus on whether specific haplotypes for other genomic regions and possibly pathogenicity can be associated with haplotypes at this locus, as well as the integration of molecular tools with other ecological tools to elucidate the ecology and pathogenicity of Bd

Bronchiolitis: an update on management and prophylaxis.

Bronchiolitis is an acute respiratory illness that is the leading cause of hospitalization in young children less than 2 years of age in the UK. Respiratory syncytial virus is the most common virus associated with bronchiolitis and has the highest disease severity, mortality and cost. Bronchiolitis is generally a self-limiting condition, but can have serious consequences in infants who are very young, premature, or have underlying comorbidities. Management of bronchiolitis in the UK is guided by the National Institute for Health and Care Excellence (2015) guidance. The mainstays of management are largely supportive, consisting of fluid management and respiratory support. Pharmacological interventions including nebulized bronchodilators, steroids and antibiotics generally have limited or no evidence of efficacy and are not advised by National Institute of Health and Care Excellence. Antiviral therapeutics remain in development. As treatments are limited, there have been extensive efforts to develop vaccines, mainly targeting respiratory syncytial virus. At present, the only licensed product is a monoclonal antibody for passive immunisation. Its cost restricts its use to those at highest risk. Vaccines for active immunisation of pregnant women and young infants are also being developed

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer

Molecular approaches to trematode systematics: 'best practice' and implications for future study

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the ‘best molecular practice’ for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient ‘useful’ comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics

A Critical Analysis of Atoh7 (Math5) mRNA Splicing in the Developing Mouse Retina

The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3′RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although ∼10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments

Habitat partitioning and vulnerability of sharks in the Great Barrier Reef Marine Park

Sharks present a critical conservation challenge, but little is known about their spatial distribution and vulnerability, particularly in complex seascapes such as Australia's Great Barrier Reef Marine Park (GBRMP). We review (1) the distribution of shark species among the primary habitats of the GBRMP (coral reefs, inshore/shelf, pelagic and deep-water habitats) (2) the relative exploitation of each species by fisheries, and (3) how current catch rates interact with their vulnerability and trophic index. Excluding rays and chimaeras, we identify a total of 82 shark species in the GBRMP. We find that shark research in the GBRMP has yielded little quantitative information on most species. Reef sharks are largely site-fidelic, but can move large distances and some regularly use non-reef habitats. Inshore and shelf sharks use coastal habitats either exclusively or during specific times in their life cycle (e.g. as nurseries). Virtually nothing is known about the distribution and habitat use of the GBRMP's pelagic and deep-water sharks. At least 46 species (53.5 %) are caught in one or more fisheries, but stock assessments are lacking for most. At least 17 of the sharks caught are considered highly vulnerable to exploitation. We argue that users of shark resources should be responsible for demonstrating that a fishery is sustainable before exploitation is allowed to commence or continue. This fundamental change in management principle will safeguard against stock collapses that have characterised many shark fisheries