Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.     

The Day-Hospital of the University Hospital,Bobo Dioulasso: An Example of Optimized HIV Management in Southern Burkina Faso

ObjectivesTo evaluate the epidemiological evolution of patients with HIV (PtHIV), between 2002 and 2012, in a day-hospital that became an HIV reference centre for south-west Burkina Faso.ResultsA total of 7320 patients have been treated at the centre since 2002; the active file of patients increased from 147 in 2002 to 3684 patients in 2012. Mean age was stable at 38.4 years and the majority were female (71%). The delay to initiation of antiretroviral (ARV) treatment after HIV diagnosis decreased from 12.9 months in 2002 to 7.2 months in 2012. The percentage of PtHIV lost to follow-up, untreated for HIV and deaths all decreased after 2005. Voluntary anonymous screening and/or an evocative clinical picture were the main reasons for HIV diagnosis, usually at a late stage (41.1% at WHO stage 3). Virological success increased due to a decrease in time to initiation of ARV treatment and an increase in percentage of patients treated (90.5% in 2012, mainly with 1^st line drugs). However, there was also a slight increase in the rate of therapeutic failures and the percentage of patients who progressed to 2^nd or 3^rd line-ARVs.ConclusionOur day-hospital is a good example of the implementation of a specialist centre for the management of PtHIV in a resource-limited country (Burkina Faso).     

Influence of Glycosylation Inhibition on the Binding of KIR3DL1 to HLA-B*57:01

Viral infections can affect the glycosylation pattern of glycoproteins involved in antiviral immunity. Given the importance of protein glycosylation for immune function, we investigated the effect that modulation of the highly conserved HLA class I N-glycan has on KIR:HLA interactions and NK cell function. We focused on HLA-B*57:01 and its interaction with KIR3DL1, which has been shown to play a critical role in determining the progression of a number of human diseases, including human immunodeficiency virus-1 infection. 721.221 cells stably expressing HLA-B*57:01 were treated with a panel of glycosylation enzyme inhibitors, and HLA class I expression and KIR3DL1 binding was quantified. In addition, the functional outcomes of HLA-B*57:01 N-glycan disruption/modulation on KIR3DL1ζ⁺ Jurkat reporter cells and primary human KIR3DL1⁺ NK cells was assessed. Different glycosylation enzyme inhibitors had varying effects on HLA-B*57:01 expression and KIR3DL1-Fc binding. The most remarkable effect was that of tunicamycin, an inhibitor of the first step of N-glycosylation, which resulted in significantly reduced KIR3DL1-Fc binding despite sustained expression of HLA-B*57:01 on 721.221 cells. This effect was paralleled by decreased activation of KIR3DL1ζ⁺ Jurkat reporter cells, as well as increased degranulation of primary human KIR3DL1⁺ NK cell clones when encountering HLA-B*57:01-expressing 721.221 cells that were pre-treated with tunicamycin. Overall, these results demonstrate that N-glycosylation of HLA class I is important for KIR:HLA binding and has an impact on NK cell function.     

Developmental Reorganization of the Cognitive Network in Pediatric Epilepsy

Traditional approaches to understanding cognition in children with epilepsy (CWE) involve cross-sectional or prospective examination of diverse test measures, an approach that does not inform the interrelationship between different abilities or how interrelationships evolve prospectively. Here we utilize graph theory techniques to interrogate the development of cognitive landmarks in CWE and healthy controls (HC) using the two-year percentage change across 20 tests. Additionally, we characterize the development of cognition using traditional analyses, showing that CWE perform worse at baseline, develop in parallel with HC, statically maintaining cognitive differences two years later. Graph analyses, however, showed CWE to exhibit both lower integration and segregation in development of their cognitive networks compared to HC. In conclusion, graph analyses of neuropsychological data capture a dynamic and changing complexity in the interrelationships among diverse cognitive skills, maturation of the cognitive network over time, and the nature of differences between normally developing children and CWE.     

Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and P-selectin inhibition

The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is described. These molecules are difluorinated analogues of CH₂-glycopeptides already reported to act as sLe^x mimics. The α and β anomers of these CF₂-glycopeptides have been prepared, as well as their 1-hydroxy analogues which were present in solution as an equilibrium mixture of α- and β-pyranose and α- and β-furanose forms. These molecules showed inhibitory activities comparable to their CH₂ counterparts with a moderate influence of the pseudo-anomeric center configuration.     

Chips from chips: application to the study of antibody responses to methylated proteins

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.     

Scarlet-Rz1, an EMS-generated hexaploid wheat with tolerance to the soilborne necrotrophic pathogens <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-8 and <Emphasis Type="Italic">R. oryzae</Emphasis>

The necrotrophic root pathogens Rhizoctonia solani AG-8 and R. oryzae cause Rhizoctonia root rot and damping-off, yield-limiting diseases that pose barriers to the adoption of conservation tillage in wheat production systems. Existing control practices are only partially effective, and natural genetic resistance to Rhizoctonia has not been identified in wheat or its close relatives. We report the first genetic resistance/tolerance to R. solani AG-8 and R. oryzae in wheat (Triticum aestivum L. em Thell) germplasm ‘Scarlet-Rz1’. Scarlet-Rz1 was derived from the allohexaploid spring wheat cultivar Scarlet using EMS mutagenesis. Tolerant seedlings displayed substantial root and shoot growth after 14 days in the presence of 100–400 propagules per gram soil of R. solani AG-8 and R. oryzae in greenhouse assays. BC₂F₄ individuals of Scarlet-Rz1 showed a high and consistent degree of tolerance. Seedling tolerance was transmissible and appeared to be dominant or co-dominant. Scarlet-Rz1 is a promising genetic resource for developing Rhizoctonia-tolerant wheat cultivars because the tolerance trait immediately can be deployed into wheat breeding germplasm through cross-hybridization, thereby avoiding difficulties with transfer from secondary or tertiary relatives as well as constraints associated with genetically modified plants. Our findings also demonstrate the utility of chemical mutagenesis for generating tolerance to necrotrophic pathogens in allohexaploid wheat. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. P. A. Okubara and C. M. Steber contributed equally to this work.     

Structural Relaxation During Drying and Rehydration of Food Materials��the Water Effect and the Origin of Hysteresis

The state of water in foodstuffs is a guiding principle in food design, and the equilibrium concept of water activity (Aw) is ubiquitous. It is regarded as a primary variable or “hurdle” in preservation technology, and a key variable influencing chemical reaction during storage. However, the amount of water in any system differs as function of water activity depending whether it is determined by water sorption or desorption. Even though this hysteresis behaviour has already been described in the literature, no physical interpretation of its origin has yet been proposed with respect to detailed molecular organisation. This work shows, for two different food powders, gluten and a milk-based product that the hysteresis disappears when either go through their glass transition. A more complete DSC analysis for gluten during different sorption/desorption cycles demonstrates that the hysteresis is dependent on the ageing of the material, which evolves in the glassy state and is induced by structural relaxation.     

Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle

Carbon monoxide (CO), one of the end products of heme oxygenase activity, inhibits smooth muscle proliferation by decreasing ERK1/2 phosphorylation and cyclin D1 expression, a signaling pathway that is known to be modulated by reactive oxygen species (ROS) in airway smooth muscle cells (ASMCs). Two important sources of ROS involved in cell signaling are the membrane NAD(P)H oxidase and the mitochondrial respiratory chain. Thus, that CO could modulate redox signaling in ASMCs by interacting with the heme moiety of NAD(P)H oxidase and/or the respiratory chain is a plausible hypothesis. Here we show that a recently identified carbon monoxide-releasing molecule, [Ru(CO)3Cl2]2 (or CORM-2) 1) inhibits NAD(P)H oxidase cytochrome b558 activity, 2) increases oxidant production by the mitochondria, and 3) inhibits ASMC proliferation and phosphorylation of the ERK1/2 mitogen-activated protein kinase and expression of cyclin D1, two critical pathways involved in muscle proliferation. No such effects were observed with the negative control (Ru(Me2SO)4Cl2), which does not contain CO groups. Because both diphenylene iodinium or apocynin (inhibitors of NAD(P)H oxidase) and rotenone (a molecule that increases mitochondrial ROS production by blocking the respiratory chain) mimicked the effect of CORM-2 on cyclin D1 expression and ASMC proliferation, the antiproliferative effect of CORM-2 is probably related to inhibition of cytochromes on both NAD(P)H oxidase and the respiratory chain. The involvement of increased mitochondria-derived oxidants is substantiated by the findings showing that the antioxidant N-acetylcysteine partially inhibited the effects of CORM-2. This study provides a new mechanism to explain redox signaling by CO.     

A robust sequencing assay of a thousand amplicons for the high-throughput population monitoring of Alpine ibex immunogenetics

Polymorphism for immune functions can explain significant variation in health and reproductive success within species. Drastic loss in genetic diversity at such loci constitutes an extinction risk and should be monitored in species of conservation concern. However, effective implementations of genome-wide immune polymorphism sets into high-throughput genotyping assays are scarce. Here, we report the design and validation of a microfluidics-based amplicon sequencing assay to comprehensively capture genetic variation in Alpine ibex (Capra ibex). This species represents one of the most successful large mammal restorations recovering from a severely depressed census size and a massive loss in diversity at the major histocompatibility complex (MHC). We analysed 65 whole-genome sequencing sets of the Alpine ibex and related species to select the most representative markers and to prevent primer binding failures. In total, we designed ~1,000 amplicons densely covering the MHC, further immunity-related genes as well as randomly selected genome-wide markers for the assessment of neutral population structure. Our analysis of 158 individuals shows that the genome-wide markers perform equally well at resolving population structure as RAD-sequencing or low-coverage genome sequencing data sets. Immunity-related loci show unexpectedly high degrees of genetic differentiation within the species. Such information can now be used to define highly targeted individual translocations. Our design strategy can be realistically implemented into genetic surveys of a large range of species. In conclusion, leveraging whole-genome sequencing data sets to design targeted amplicon assays allows the simultaneous monitoring of multiple genetic risk factors and can be translated into species conservation recommendations.