Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 °C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes

Cost-effectiveness of reducing salt intake in the Pacific Islands: protocol for a before and after intervention study

BackgroundThere is broad consensus that diets high in salt are bad for health and that reducing salt intake is a cost-effective strategy for preventing chronic diseases. The World Health Organization has been supporting the development of salt reduction strategies in the Pacific Islands where salt intakes are thought to be high. However, there are no accurate measures of salt intake in these countries. The aims of this project are to establish baseline levels of salt intake in two Pacific Island countries, implement multi-pronged, cross-sectoral salt reduction programs in both, and determine the effects and cost-effectiveness of the intervention strategies.Methods/DesignIntervention effectiveness will be assessed from cross-sectional surveys before and after population-based salt reduction interventions in Fiji and Samoa. Baseline surveys began in July 2012 and follow-up surveys will be completed by July 2015 after a 2-year intervention period.A three-stage stratified cluster random sampling strategy will be used for the population surveys, building on existing government surveys in each country. Data on salt intake, salt levels in foods and sources of dietary salt measured at baseline will be combined with an in-depth qualitative analysis of stakeholder views to develop and implement targeted interventions to reduce salt intake.DiscussionSalt reduction is a global priority and all Member States of the World Health Organization have agreed on a target to reduce salt intake by 30% by 2025, as part of the global action plan to reduce the burden of non-communicable diseases. The study described by this protocol will be the first to provide a robust assessment of salt intake and the impact of salt reduction interventions in the Pacific Islands. As such, it will inform the development of strategies for other Pacific Island countries and comparable low and middle-income settings around the world.<br /

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies

International collaborative project to compare and track the nutritional composition of fast foods

Background: Chronic diseases are the leading cause of premature death and disability in the world with over-nutrition a primary cause of diet-related ill health. Excess quantities of energy, saturated fat, sugar and salt derived from fast foods contribute importantly to this disease burden. Our objective is to collate and compare nutrient composition data for fast foods as a means of supporting improvements in product formulation. METHODS/DESIGN: Surveys of fast foods will be done in each participating country each year. Information on the nutrient composition for each product will be sought either through direct chemical analysis, from fast food companies, in-store materials or from company websites. Foods will be categorized into major groups for the primary analyses which will compare mean levels of saturated fat, sugar, sodium, energy and serving size at baseline and over time. Countries currently involved include Australia, New Zealand, France, UK, USA, India, Spain, China and Canada, with more anticipated to follow. DISCUSSION: This collaborative approach to the collation and sharing of data will enable low-cost tracking of fast food composition around the world. This project represents a significant step forward in the objective and transparent monitoring of industry and government commitments to improve the quality of fast foods.E Dunford is supported by a Sydney Medical School Foundation scholarship and B Neal by an Australian Research Council Future Fellowship.http://www.biomedcentral.com/1471-2458/12/559am201

High-coverage whole-genome analysis of 1220 cancers reveals hundreds of genes deregulated by rearrangement-mediated cis-regulatory alterations.

The impact of somatic structural variants (SVs) on gene expression in cancer is largely unknown. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data and RNA sequencing from a common set of 1220 cancer cases, we report hundreds of genes for which the presence within 100 kb of an SV breakpoint associates with altered expression. For the majority of these genes, expression increases rather than decreases with corresponding breakpoint events. Up-regulated cancer-associated genes impacted by this phenomenon include TERT, MDM2, CDK4, ERBB2, CD274, PDCD1LG2, and IGF2. TERT-associated breakpoints involve ~3% of cases, most frequently in liver biliary, melanoma, sarcoma, stomach, and kidney cancers. SVs associated with up-regulation of PD1 and PDL1 genes involve ~1% of non-amplified cases. For many genes, SVs are significantly associated with increased numbers or greater proximity of enhancer regulatory elements near the gene. DNA methylation near the promoter is often increased with nearby SV breakpoint, which may involve inactivation of repressor elements

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

The physical, economic and policy drivers of land conversion to forestry in Ireland

peer-reviewedLand use change is fundamentally a product of the interaction of physical land characteristics, economic considerations and agricultural and environmental policies. Researchers are increasingly combining physical and socio-economic spatial data to investigate the drivers of land-use change in relation to policy and economic developments. Focusing on Ireland, this study develops a panel data set of annual afforestation over 2811 small-area boundaries between 1993 and 2007 from vector and raster data sources. Soil type and other physical characteristics are combined with the net returns of converting agricultural land to forestry, based on the micro-simulation of individual farm incomes, to investigate land conversion. A spatial econometric approach is adopted to model the data and a range of physical, economic and policy factors are identified as having a significant effect on afforestation rates. In addition to the financial returns, the availability and quality of land and the implementation of environmental protection policies are identified as important factors in land conversion. The implications of these factors for the goal of forest expansion are discussed in relation to conflicting current and future land use policies.Department of Agriculture, Food and the Marin