Phenotypic diversity,population structure and stress protein-based capacitoring in populations of Xeropicta derbentina,a heat-tolerant land snail species

The shell colour of many pulmonate land snail species is highly diverse. Besides a genetic basis, environmentally triggered epigenetic mechanisms including stress proteins as evolutionary capacitors are thought to influence such phenotypic diversity. In this study, we investigated the relationship of stress protein (Hsp70) levels with temperature stress tolerance, population structure and phenotypic diversity within and among different populations of a xerophilic Mediterranean snail species (Xeropicta derbentina). Hsp70 levels varied considerably among populations, and were significantly associated with shell colour diversity: individuals in populations exhibiting low diversity expressed higher Hsp70 levels both constitutively and under heat stress than those of phenotypically diverse populations. In contrast, population structure (cytochrome c oxidase subunit I gene) did not correlate with phenotypic diversity. However, genetic parameters (both within and among population differences) were able to explain variation in Hsp70 induction at elevated but non-pathologic temperatures. Our observation that (1) population structure had a high explanatory potential for Hsp70 induction and that (2) Hsp70 levels, in turn, correlated with phenotypic diversity while (3) population structure and phenotypic diversity failed to correlate provides empirical evidence for Hsp70 to act as a mediator between genotypic variation and phenotype and thus for chaperone-driven evolutionary capacitance in natural populations.     

Food allergy enhances allergic asthma in mice

Background

Atopic march refers to the typical transition from a food allergy in early childhood to allergic asthma in older children and adults. However the precise interplay of events involving gut, skin and pulmonary inflammation in this process is not completely understood.

Objectives

To develop a mouse model of mixed food and respiratory allergy mimicking the atopic march and better understand the impact of food allergies on asthma.

Methods

Food allergy to ovalbumin (OVA) was induced through intra-peritoneal sensitization and intra-gastric challenge, and/or a respiratory allergy to house dust mite (HDM) was obtained through percutaneous sensitization and intra-nasal challenges with dermatophagoides farinae (Der f) extract. Digestive, respiratory and systemic parameters were analyzed.

Results

OVA-mediated gut allergy was associated with an increase in jejunum permeability, and a worsening of Der f-induced asthma with stronger airway hyperresponsiveness and pulmonary cell infiltration, notably eosinophils. There was overproduction of the pro-eosinophil chemokine RANTES in broncho-alveolar lavages associated with an enhanced Th2 cytokine secretion and increased total and Der f-specific IgE when the two allergies were present. Both AHR and lung inflammation increased after a second pulmonary challenge.

Conclusion

Gut sensitization to OVA amplifies Der f-induced asthma in mice.     

Label‐free characterization of carbonic anhydrase—novel inhibitor interactions using surface plasmon resonance,isothermal titration calorimetry and fluorescence‐based thermal shift assays

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. K_D values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of K_D were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the K_D value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity‐based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (k_a, k_d, and K_D), leads us to choose this methodology for the study of new potential inhibitors. Copyright © 2013 John Wiley & Sons, Ltd.     

An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue

Background

Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue.

Results

Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study.

Conclusions

Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

     

Ocean acidification boosts reproduction in fish via indirect effects

Ocean acidification affects species populations and biodiversity through direct negative effects on physiology and behaviour. The indirect effects of elevated CO₂ are less well known and can sometimes be counterintuitive. Reproduction lies at the crux of species population replenishment, but we do not know how ocean acidification affects reproduction in the wild. Here, we use natural CO₂ vents at a temperate rocky reef and show that even though ocean acidification acts as a direct stressor, it can indirectly increase energy budgets of fish to stimulate reproduction at no cost to physiological homeostasis. Female fish maintained energy levels by compensation: They reduced activity (foraging and aggression) to increase reproduction. In male fish, increased reproductive investment was linked to increased energy intake as mediated by intensified foraging on more abundant prey. Greater biomass of prey at the vents was linked to greater biomass of algae, as mediated by a fertilisation effect of elevated CO₂ on primary production. Additionally, the abundance and aggression of paternal carers were elevated at the CO₂ vents, which may further boost reproductive success. These positive indirect effects of elevated CO₂ were only observed for the species of fish that was generalistic and competitively dominant, but not for 3 species of subordinate and more specialised fishes. Hence, species that capitalise on future resource enrichment can accelerate their reproduction and increase their populations, thereby altering species communities in a future ocean.

Ocean acidification affects species populations and diversity through direct negative effects on physiology and behavior, but the indirect effects are less clear. Using volcanic carbon dioxide vents as natural analogues of future ocean acidification, this study shows that elevated CO2 can stimulate fish reproduction in the wild through increased food abundance, leading to increased energy budgets at no cost to physiological homeostasis.     

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.