Determination of the DNA methylation level of the marbled crayfish: an increase in sample throughput by an optimised sample preparation

Using a previously described capillary electrophoretic method with laser-induced fluorescence detection the genomic methylation level can be determined exactly. We present a sample preparation that eliminates the surplus of fluorescence marker used for coupling resulting in an increase of sample throughput from 75 to 250 analyses per week. The sensitivity of the method was also increased, which allows the determination of methylation levels under 1%. With these changes in sample preparation a methylation level of 1.64+/-0.03% in hepatopancreas DNA of the recently discovered marbled crayfish could be determined.     

Morphological variation of the spermatheca in the garden snail Cantareus aspersus

Spermathecal morphology is known to play an important role in postcopulatory sexual selection of many invertebrates. In helicid land snails, the spermatheca is subdivided into tubules, whose number is sometimes subject to a strong inter-individual variation. Significance of this variation for postcopulatory sexual selection is unknown, but it might be related to cryptic female choice. In the present work, we have investigated the fine multi-tubular structure of the sperm storage organ in Cantareus aspersus. We found between 3 and 13 tubules per individual in a single population, which represents a degree of variation rarely observed in helicid land snails.     

Recognizing protein–protein interfaces with empirical potentials and reduced amino acid alphabets

Background  

In structural genomics, an important goal is the detection and classification of protein–protein interactions, given the structures of the interacting partners. We have developed empirical energy functions to identify native structures of protein–protein complexes among sets of decoy structures. To understand the role of amino acid diversity, we parameterized a series of functions, using a hierarchy of amino acid alphabets of increasing complexity, with 2, 3, 4, 6, and 20 amino acid groups. Compared to previous work, we used the simplest possible functional form, with residue–residue interactions and a stepwise distance-dependence. We used increased computational ressources, however, constructing 290,000 decoys for 219 protein–protein complexes, with a realistic docking protocol where the protein partners are flexible and interact through a molecular mechanics energy function. The energy parameters were optimized to correctly assign as many native complexes as possible. To resolve the multiple minimum problem in parameter space, over 64000 starting parameter guesses were tried for each energy function. The optimized functions were tested by cross validation on subsets of our native and decoy structures, by blind tests on series of native and decoy structures available on the Web, and on models for 13 complexes submitted to the CAPRI structure prediction experiment.     

Effects of litter removal and mowing on germination and establishment of two fen-grassland species along a productivity gradient

To study the factors that limit the occurrence of species in fen grasslands, the effects of mowing and litter removal on germination, growth and establishment of two common species (Silene flos-cuculi andLotus pedunculatus) were analyzed along a productivity gradient at seven sites in Northern Germany. The sites differed in nutrient availability, vegetation composition and standing crop. In autumn 2002 a field experiment was set up at each site, including factorial combinations of two treatments (litter removal, mowing). Seeds and juvenile individuals of both species were transferred within the treatment combinations. The number of germinated and transplanted individuals was recorded during the 2003 and 2004 seasons and biomass of survived plants weighed in late summer 2004. Mowing and litter removal had positive effects on the number of germinated seeds of both species. The effect of litter removal was mostly significant at sites with a comparatively low nutrient availability and standing crop (350 g/m²), while the effect of mowing was more important at the site with the highest standing crop (1000 g/m²). In some cases the presence of litter had a positive effect on the survival of individuals, which may be caused by extremely dry weather conditions in 2003. The experiment showed that plant species reaction on mowing and litter removal differed both according to their life stage and the site conditions. These differences could be interpreted in context with Shifting Limitation Hypothesis because the abolition of seed limitation by sowing and creation of additional gaps for germination was most efficient at low productive sites, while the high standing crop reduced first of all the establishment probability, which could be increased by mowing.     

On the origin of lipid asymmetry: the flip side of ion transport

Membrane lipid asymmetry influences a multitude of cellular functions, including membrane vesiculation, cell division, and lifespan. Most cells retain the bulk of aminophospholipids to the cytosolic membrane leaflet by means of ATP-fuelled flippases or translocases. Converging lines of evidence indicate that members of the P(4) subfamily of P-type ATPases catalyze aminophospholipid transport and create lipid asymmetry in late secretory and endocytic compartments. Yet P-type ATPases usually pump small cations or metal ions. Atomic structures revealed important aspects of the transport mechanism, and sequence homology indicates that this mechanism is conserved throughout the family. Consequently, understanding how P(4) ATPases acquired the ability to translocate phospholipids instead of simple ions has become a major focus of interest.     

Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection

Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4⁺ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4⁺ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4⁺ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4⁺ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.     

A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host’s inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.