Kinetic characterization of the catalytic mechanism of methionine sulfoxide reductase B from Neisseria meningitidis

Methionine sulfoxide reductases catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The methionine sulfoxide reductases family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the sulfoxide function. Both enzymes are monomeric and share a similar three-step chemical mechanism. First, in the reductase step, a sulfenic acid intermediate is formed with a concomitant release of 1 mol of methionine per mol of enzyme. Then, an intradisulfide bond is formed. Finally, Msrs return back to reduced forms via reduction by thioredoxin. In the present study, it is shown for the Neisseria meningitidis MsrB that (1) the reductase step is rate-determining in the process leading to formation of the disulfide bond and (2) the thioredoxin-recycling process is rate-limiting. Moreover, the data suggest that within the thioredoxin-recycling process, the rate-limiting step takes place after the two-electron chemical exchange and thus is associated with the release of oxidized thioredoxin.     

Trait and phylogenetic diversity provide insights into community assembly of reef‐associated shrimps (Palaemonidae) at different spatial scales across the Chagos Archipelago

Coral reefs are the most biodiverse marine ecosystem and one of the most threatened by global climate change impacts. The vast majority of diversity on reefs is comprised of small invertebrates that live within the reef structure, termed the cryptofauna. This component of biodiversity is hugely understudied, and many species remain undescribed. This study represents a rare analysis of assembly processes structuring a distinct group of cryptofauna, the Palaemonidae, in the Chagos Archipelago, a reef ecosystem under minimal direct human impacts in the central Indian Ocean. The Palaemonidae are a diverse group of Caridae (infraorder of shrimps) that inhabit many different niches on coral reefs and are of particular interest because of their varied habitat associations. Phylogenetic and trait diversity and phylogenetic signal were used to infer likely drivers of community structure. The mechanisms driving palaemonid community assembly and maintenance in the Chagos Archipelago showed distinct spatial patterns. At local scales, among coral colonies and among reefs fringing individual atolls, significant trait, and phylogenetic clustering patterns suggest environmental filtering may be a dominant ecological process driving Palaemonidae community structure, although local competition through equalizing mechanisms may also play a role in shaping the local community structure. Importantly, we also tested the robustness of phylogenetic diversity to changes in evolutionary information as multi‐gene phylogenies are resource intensive and for large families, such as the Palaemonidae, are often incomplete. These tests demonstrated a very modest impact on phylogenetic community structure, with only one of the four genes (PEPCK gene) in the phylogeny affecting phylogenetic diversity patterns, which provides useful information for future studies on large families with incomplete phylogenies. These findings contribute to our limited knowledge of this component of biodiversity in a marine locality as close to undisturbed by humans as can be found. It also provides a rare evaluation of phylogenetic diversity methods.     

When History Repeats Itself: Exploring the Genetic Architecture of Host-Plant Adaptation in Two Closely Related Lepidopteran Species

The genus Ostrinia includes two allopatric maize pests across Eurasia, namely the European corn borer (ECB, O. nubilalis) and the Asian corn borer (ACB, O. furnacalis). A third species, the Adzuki bean borer (ABB, O. scapulalis), occurs in sympatry with both the ECB and the ACB. The ABB mostly feeds on native dicots, which probably correspond to the ancestral host plant type for the genus Ostrinia. This situation offers the opportunity to characterize the two presumably independent adaptations or preadaptations to maize that occurred in the ECB and ACB. In the present study, we aimed at deciphering the genetic architecture of these two adaptations to maize, a monocot host plant recently introduced into Eurasia. To this end, we performed a genome scan analysis based on 684 AFLP markers in 12 populations of ECB, ACB and ABB. We detected 2 outlier AFLP loci when comparing French populations of the ECB and ABB, and 9 outliers when comparing Chinese populations of the ACB and ABB. These outliers were different in both countries, and we found no evidence of linkage disequilibrium between any two of them. These results suggest that adaptation or preadaptation to maize relies on a different genetic architecture in the ECB and ACB. However, this conclusion must be considered in light of the constraints inherent to genome scan approaches and of the intricate evolution of adaptation and reproductive isolation in the Ostrinia spp. complex.     

Sex‐specific density‐dependent secretion of glucocorticoids in lizards: insights from laboratory and field experiments

Negative density feedbacks have been extensively described in animal species and involve both consumptive (i.e. trophic interactions) and non‐consumptive (i.e. social interactions) mechanisms. Glucocorticoids are a major component of the physiological stress response and homeostasis, and therefore make a good candidate for proximate determinants of negative density feedbacks. Here, we combined laboratory and field experiments with enclosed populations to investigate the relationship between density, social stress and plasma corticosterone levels in the common lizard Zootoca vivipara. This species exhibits strong negative density feedbacks that affect females more than males, and its life history is sensitive to experimentally‐induced chronic elevation of corticosterone plasma levels. We found that prolonged crowding in the laboratory can trigger a chronic secretion of corticosterone independent from food restriction. In the field experiments, corticosterone levels of females were not affected by population density. Corticosterone levels of males increased with population density but only during the late activity season in a first field experiment where we manipulated density. They also increased with density during the mating season but only in populations with a female‐biased sex ratio in a second field experiment where we crossed manipulated density and adult sex ratio. Altogether, our results provide limited evidence for a role of basal corticosterone secretion in density feedbacks in this species. Context and density‐dependent effects in males may arise from changes in behavior caused by competition for resources, male–male competition, and mating.     

Metagenomic exploration of antibiotic resistance in soil

The ongoing development of metagenomic approaches is providing the means to explore antibiotic resistance in nature and address questions that could not be answered previously with conventional culture-based strategies. The number of available environmental metagenomic sequence datasets is rapidly expanding and henceforth offer the ability to gain a more comprehensive understanding of antibiotic resistance at the global scale. Although there is now evidence that the environment constitutes a vast reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin, a better understanding of their diversity, prevalence and ecological significance may help predict the emergence and spreading of newly acquired resistances. Recent applications of metagenomic approaches to the study of ARGDs in natural environments such as soil should help overcome challenges concerning expanding antibiotic resistances.     

Compositional biases and polyalanine runs in humans

Human proteins containing polyalanine tracts tend to have runs of other amino acids and their open reading frames (ORFs) display a biased codon usage. Their alanine, glycine, proline, and histidine content strongly correlates with the GC content of the third codon base, suggesting that the compositional specificity of these proteins is dictated to a great extent by the evolution of their ORFs.     

Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo . Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.     

Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).     

Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD) 2 in Host Defense during Pneumococcal Pneumonia

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2 ^-/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2 ^-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2 ^-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2 ^-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2 ^-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2 ^-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.