A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes

Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput. 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays. This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved

Factors driving patterns and trends in strandings of small cetaceans

The incidence of cetacean strandings is expected to depend on a combination of factors, including the dis- tribution and abundance of the cetaceans, their prey, and causes of mortality (e.g. natural, fishery bycatch), as well as currents and winds which affect whether carcasses reach the shore. We investigated spatiotemporal patterns and trends in the numbers of strandings of three species of small cetacean in Galicia (NW Spain) and their relationships with meteoro- logical, oceanographic, prey abundance and fishing-related variables, aiming to disentangle the relationship that may exist between these factors, cetacean abundance and mor- tality off the coast. Strandings of 1166 common dolphins (Delphinus delphis), 118 bottlenose dolphins (Tursiops truncatus) and 90 harbour porpoises (Phocoena phocoena) during 2000–2013 were analysed. Generalised additive and generalised additive-mixed model results showed that the variables which best explained the pattern of strandings of the three cetacean species were those related with local ocean meteorology (strength and direction of the North– South component of the winds and the number of days with South-West winds) and the winter North Atlantic Oscil- lation Index. There were no significant relationships with indices of fishing effort or landings. Only bottlenose dolphin showed possible fluctuations in local abundance over the study period. There was no evidence of long-term trends in number of strandings in any of the species and their abun- dances were, therefore, considered to have been relatively stable during the study period.Versión del editor2,01

Model-Derived Dispersal Pathways from Multiple Source Populations Explain Variability of Invertebrate Larval Supply

Background: Predicting the spatial and temporal patterns of marine larval dispersal and supply is a challenging task due to the small size of the larvae and the variability of oceanographic processes. Addressing this problem requires the use of novel approaches capable of capturing the inherent variability in the mechanisms involved. Methodology/Principal Findings: In this study we test whether dispersal and connectivity patterns generated from a biophysical model of larval dispersal of the crab Carcinus maenas, along the west coast of the Iberian Peninsula, can predict the highly variable daily pattern of wind-driven larval supply to an estuary observed during the peak reproductive season (March–June) in 2006 and 2007. Cross-correlations between observed and predicted supply were significant (p,0.05) and strong, ranging from 0.34 to 0.81 at time lags of 26 to+5 d. Importantly, the model correctly predicted observed cross-shelf distributions (Pearson r = 0.82, p,0.001, and r = 0.79, p,0.01, in 2006 and 2007) and indicated that all supply events were comprised of larvae that had been retained within the inner shelf; larvae transported to the outer shelf and beyond never recruited. Estimated average dispersal distances ranged from 57 to 198 km and were only marginally affected by mortality. Conclusions/Significance: The high degree of predicted demographic connectivity over relatively large geographic scales is consistent with the lack of genetic structuring in C. maenas along the Iberian Peninsula. These findings indicate that the dynamic nature of larval dispersal can be captured by mechanistic biophysical models, which can be used to provid

Unusual circulation patterns of the rias baixas induced by minho freshwater intrusion (NW of the Iberian Peninsula)

The Minho River, situated 30 km south of the Rias Baixas, is the most important freshwater source flowing into the Western Galician coast (NW of the Iberian Peninsula). The buoyancy generated by the Minho estuarine plume can reverse the normal circulation pattern inside the Rias Baixas affecting the exchange between the Rias and the ocean, changing the input of nutrients. Nevertheless, this inversion of the circulation patterns is not a well-monitored phenomenon. The only published results based on in situ data related to the presence of the Minho River plume inside the Rias de Vigo and Pontevedra correspond to an event measured on spring 1998. In this case unexpectedly higher inflow surface current velocities were found at the Ria de Pontevedra, located further away from Minho River. Thus, the main aim of this study is to research the main factors inducing this unusual pattern on the circulation of the Rias de Vigo and Pontevedra. A numerical model implementation of MOHID previously developed, calibrated, and validated for this coastal area was used. Several scenarios were performed in order to explain the individual effect of the Minho River, rivers discharging into each Rias, and estuarine morphology changes. According to the model results, the Minho River discharge is a key factor in the establishment of the negative circulation, while small rivers inside the Rias slightly attenuate this circulation. The negative circulation was stronger in Ria de Pontevedra independently of the distance of this coastal system from the Minho River mouth, showing that morphologic estuarine features are the main factor justifying the different local circulation patterns

Response of Cocoa Trees (Theobroma cacao) to a 13-month Dessication Period in Sulawesi, Indonesia

In South-east Asia, ENSO-related droughts represent irregularly occuring hazards for agroforestry systems containing cocoa which are predicted to increase in severity with expected climate warming. To characterize the drought response of mature cocoa tree, we conducted the Sulawesi Throughfall Displacement Experiment in a shaded (Gliricidia sepium) cocoa agroforestry system in Central Sulawesi, Indonesia. Three large sub-canopy roofs were installed to reduce throughfall by about 80% over a 13-month period to test the hypotheses that (i) cocoa trees are sensitive to drought due to their shallow fine root system, and (ii)bean yield is more sensitive to drought than leaf or stem growth. As 83% of fine root (diameter 2mm) was located in the upper 40 cm of the soil, the cocoa tree examined had a very shallow root system. Cocoa and Gliricidia differed in their vertical rooting patterns, thereby reducing competition for water. Despite being exposed for several mnths to soil water contents close to the conventional wilting point, cocoa trees showed no significant decreases in leaf biomass, stem and branch wood production or fine root biomass. Possible causes are active osmotic adjusment in roots, mitigation of drought stress by shading from Gliricidia or other factors. By contrast, production of cocoa bean

Amplification of wildfire area burnt by hydrological drought in the humid tropics

Borneo’s diverse ecosystems, which are typical humid tropical conditions, are deteriorating rapidly, as the area is experiencing recurrent large-scale wildfires, affecting atmospheric composition1, 2, 3, 4 and influencing regional climate processes5, 6. Studies suggest that climate-driven drought regulates wildfires2, 7, 8, 9, but these overlook subsurface processes leading to hydrological drought, an important driver. Here, we show that models which include hydrological processes better predict area burnt than those solely based on climate data. We report that the Borneo landscape10 has experienced a substantial hydrological drying trend since the early twentieth century, leading to progressive tree mortality, more severe than in other tropical regions11. This has caused massive wildfires in lowland Borneo during the past two decades, which we show are clustered in years with large areas of hydrological drought coinciding with strong El Niño events. Statistical modelling evidence shows amplifying wildfires and greater area burnt in response to El Niño/Southern Oscillation (ENSO) strength, when hydrology is considered. These results highlight the importance of considering hydrological drought for wildfire prediction, and we recommend that hydrology should be considered in future studies of the impact of projected ENSO strength, including effects on tropical ecosystems, and biodiversity conservation

Genome-wide association studies of cancer: current insights and future perspectives.

Genome-wide association studies (GWAS) provide an agnostic approach for investigating the genetic basis of complex diseases. In oncology, GWAS of nearly all common malignancies have been performed, and over 450 genetic variants associated with increased risks have been identified. As well as revealing novel pathways important in carcinogenesis, these studies have shown that common genetic variation contributes substantially to the heritable risk of many common cancers. The clinical application of GWAS is starting to provide opportunities for drug discovery and repositioning as well as for cancer prevention. However, deciphering the functional and biological basis of associations is challenging and is in part a barrier to fully unlocking the potential of GWAS