GDP release preferentially occurs on the phosphate side in heterotrimeric G-proteins

After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of G(α).     

RAD51 and Breast Cancer Susceptibility: No Evidence for Rare Variant Association in the Breast Cancer Family Registry Study

Background

Although inherited breast cancer has been associated with germline mutations in genes that are functionally involved in the DNA homologous recombination repair (HRR) pathway, including BRCA1, BRCA2, TP53, ATM, BRIP1, CHEK2 and PALB2, about 70% of breast cancer heritability remains unexplained. Because of their critical functions in maintaining genome integrity and already well-established associations with breast cancer susceptibility, it is likely that additional genes involved in the HRR pathway harbor sequence variants associated with increased risk of breast cancer. RAD51 plays a central biological function in DNA repair and despite the fact that rare, likely dysfunctional variants in three of its five paralogs, RAD51C, RAD51D, and XRCC2, have been associated with breast and/or ovarian cancer risk, no population-based case-control mutation screening data are available for the RAD51 gene. We thus postulated that RAD51 could harbor rare germline mutations that confer increased risk of breast cancer.

Methodology/Principal Findings

We screened the coding exons and proximal splice junction regions of the gene for germline sequence variation in 1,330 early-onset breast cancer cases and 1,123 controls from the Breast Cancer Family Registry, using the same population-based sampling and analytical strategy that we developed for assessment of rare sequence variants in ATM and CHEK2. In total, 12 distinct very rare or private variants were characterized in RAD51, with 10 cases (0.75%) and 9 controls (0.80%) carrying such a variant. Variants were either likely neutral missense substitutions (3), silent substitutions (4) or non-coding substitutions (5) that were predicted to have little effect on efficiency of the splicing machinery.

Conclusion

Altogether, our data suggest that RAD51 tolerates so little dysfunctional sequence variation that rare variants in the gene contribute little, if anything, to breast cancer susceptibility.     

A complete collection of single‐gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.     

A microarray system for Y chromosomal and mitochondrial single nucleotide polymorphism analysis in chimpanzee populations

Chimpanzee populations are diminishing as a consequence of human activities, and as a result this species is now endangered. In the context of conservation programmes, genetic data can add vital information, for instance on the genetic diversity and structure of threatened populations. Single nucleotide polymorphisms (SNP) are biallelic markers that are widely used in human molecular studies and can be implemented in efficient microarray systems. This technology offers the potential of robust, multiplexed SNP genotyping at low reagent cost in other organisms than humans, but it is not commonly used yet in wild population studies. Here, we describe the characterization of new SNPs in Y-chromosomal intronic regions in chimpanzees and also identify SNPs from mitochondrial genes, with the aim of developing a microarray system that permits the simultaneous study of both paternal and maternal lineages. Our system consists of 42 SNPs for the Y chromosome and 45 SNPs for the mitochondrial genome. We demonstrate the applicability of this microarray in a captive population where genotypes accurately reflected its large pedigree. Two wild-living populations were also analysed and the results show that the microarray will be a useful tool alongside microsatellite markers, since it supplies complementary information about population structure and ecology. SNP genotyping using microarray technology, therefore, is a promising approach and may become an essential tool in conservation genetics to help in the management and study of captive and wild-living populations. Moreover, microarrays that combine SNPs from different genomic regions could replace microsatellite typing in the future.     

Effects of transfer to sea water on standard and routine metabolic rates in smolting Atlantic salmon at different stages of seawater adaptability

Juvenile Atlantic salmon were transferred to sea water at three different times corresponding to different levels of seawater tolerance, i.e. before (A) and at two times (B and C) during the completion of parr-smolt transformation. Changes in their standard and routine metabolic rates were evaluated by measuring oxygen consumption. The time at which these transfers had been carried out and, consequently, the fish adaptability to sea water affected their post-transfer metabolism. Indeed, transfer A induced a decrease in mean metabolic rate; under standard conditions, this could be ascribed to a lowered protein turnover. The depression in routine oxygen consumption, which occurred at daylight, was interpreted as an adaptative response to a possible limitation in oxygen supply to the tissues. This limitation would result from a decrease both in the gas diffusing capacity of gills and in oxygen affinity of haemoglobin assumed from changes in plasma ions. Transfers B and C increased standard metabolism; this was probably induced by a stimulation of protein turnover. No outstanding change was noticed in routine oxygen consumption of fish after transfer B. On the other hand, the re-occurrence of a circadian rhythm of routine metabolism in sea water after transfer C is interpreted as the disappearance of physiological disturbances following a prolonged stay of fish in fresh water.     

Metabarcoding for the parallel identification of several hundred predators and their prey: Application to bat species diet analysis

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two‐step PCR protocol enabling the “all at once” taxonomic identification of bats and their arthropod prey for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods, and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity and amplification biases. Our parallel identification strategy of predators and prey reduces the risk of mis‐assigning prey to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost‐effective screening tool for addressing evolutionary ecological issues or developing “chirosurveillance” and conservation strategies.     

Cardboard nesting cavities may promote the development of Osmia cornuta and reduce infestation of kleptoparasitic mites

Mason bees (Osmia spp.) are receiving increasing attention as they are very effective pollinators of multiple crops. However, different bee-associated kleptoparasites are known which can be detrimental to the development of the bees’ offspring. To date, clear evidence-based guidelines to control these kleptoparasites are lacking for growers. This study investigates whether different types of artificial nesting materials—cardboard tubes and wooden grooved boards—influence the development of the European orchard bee (Osmia cornuta) as well as the infestation rate of kleptoparasitic mites in the bees’ nest. This preliminary study provides insight that cardboard tubes reduce the infestation rate of mites by 81.8% and as such promote successful development of the European orchard bee by 15.5%. Using cardboard tubes over wooden grooved boards should be advised to growers as an effective management strategy to avoid infestation of kleptoparasitic mites and to enhance successful bee development.