Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Tot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔV_Tot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Tot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.     

Oxidative and Reductive Routes of Glycerol and Glucose Fermentation by Escherichia coli Batch Cultures and Their Regulation by Oxidizing and Reducing Reagents at Different pHs

Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E _h ). During glycerol utilization at pH 7.5 ?pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E _h dropped down to negative values (?150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H₂ was evolved at the middle log phase while during glucose fermentation H₂ was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H₂ formation. Reducing reagents dl-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H₂ production. The findings point out the importance of reductive conditions for glycerol fermentation and H₂ production by E. coli.     

A quantitative trait locus on chromosome 4 affects cycling of hematopoietic stem and progenitor cells through regulation of TGF-beta 2 responsiveness

The hematopoietic stem and progenitor cell (HSPC) compartment is subject to extensive quantitative genetic variation. We have previously shown that TGF-beta2 at low concentrations enhances flt3 ligand-induced growth of HSPCs, while it is potently antiproliferative at higher concentrations. This in vitro enhancing effect was subject to quantitative genetic variation, for which a quantitative trait locus (QTL) was tentatively mapped to chromosome 4 (chr.4). Tgfb2(+/-) mice have a smaller and more slowly cycling HSPC compartment, which has a decreased serial repopulation capacity, and are less susceptible to the lethal effect of high doses of 5-fluorouracil. To unequivocally demonstrate that these phenotypes can be attributed to the enhancing effect of TGF-beta2 on HSPC proliferation observed in vitro and are therefore subject to mouse strain-dependent variation as well, we generated congenic mice where the telomeric region of chr.4 was introgressed from DBA/2 into C57BL/6 mice. In these mice, the enhancing effect of TGF-beta2 on flt3 signaling, but not the generic antiproliferative effect of high concentrations of TGF-beta2, was abrogated, confirming the location of this QTL, which we named tb2r1, on chr.4. These mice shared a smaller and more slowly cycling HSPC compartment and increased 5-fluorouracil resistance but not a decreased serial repopulation capacity with Tgfb2(+/-) mice. The concordance of phenotypes between Tgfb2(+/-) and congenic mice indicates that HSPC frequency and cycling are regulated by tb2r1, while an additional QTL in the telomeric region of chr.4 may regulate the serial repopulation capacity of hematopoietic stem cells.     

New indices in morphometry of nuclear structure in the NIH 3T3 cell line

We analyzed the connection of changes in nucleus ploidy with changes in nucleolar apparatus of NIH 3T3 cells. The quantity of nucleoli does not depend on the quantity of nucleolar DNA, but instead depends on euploidy: the majority of euploid cells have 1-3 nucleoli. The quantity of DNA in the nucleolus is correlated with the quantity of nucleolar DNA, and does not depend on ploidy changes. The nucleolar area has a tendency to increase in line with an increase in their numbers in the nucleus. The relationship of the quantity of DNA in the nucleolus with that of the nucleus is stable. During the process of increase in the number of nucleoli in a nucleus, there is a corresponding decrease in the quantity of DNA in each nucleolus, and there is likewise no increase in the sum of nucleolar DNA. The ratio of sums of the nucleolar perimeters to nuclear perimeter is a significant factor, which increases linearly along with an increase in the number of nucleoli in a nucleus.     

A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages

The coincidence between infections with parasitic worms and the reduced prevalence of allergic disease in humans and in animal models has prompted the search for helminth molecules with antiallergic and antiinflammatory potential. We report herein that filarial cystatin, a secreted protease inhibitor of filarial nematodes, suppresses Th2-related inflammation and the ensuing asthmatic disease in a murine model of OVA-induced allergic airway responsiveness. Treatment with recombinant filarial cystatin inhibited eosinophil recruitment, reduced levels of OVA-specific and total IgE, down-regulated IL-4 production, and suppressed allergic airway hyperreactivity when applied during or after sensitization and before challenge with the allergen. Depletion of macrophages by clodronate-containing liposomes prevented the curative effects and restored the levels of infiltrating cells, IgE, and allergic airway reactivity. Blocking of IL-10 by application of anti-IL-10 receptor Abs restored the reduced number of infiltrating cells and the levels of OVA-specific IgE. In contrast, depletion of regulatory T cells by anti-CD25 Abs had only limited effects. Cystatin also modulated macrophage-mediated inflammation in a murine model of dextran sulfate sodium-induced colitis, leading to reduction of inflammatory infiltrations and epithelial damage. Our data demonstrate that treatment with a single helminth protein can exert the antiallergic effects of helminth infections.     

BLAST+: architecture and applications

Background  

Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications.     

Doubly robust tests of exposure effects under high-dimensional confounding

After variable selection, standard inferential procedures for regression parameters may not be uniformly valid; there is no finite-sample size at which a standard test is guaranteed to approximately attain its nominal size. This problem is exacerbated in high-dimensional settings, where variable selection becomes unavoidable. This has prompted a flurry of activity in developing uniformly valid hypothesis tests for a low-dimensional regression parameter (eg, the causal effect of an exposure A on an outcome Y) in high-dimensional models. So far there has been limited focus on model misspecification, although this is inevitable in high-dimensional settings. We propose tests of the null that are uniformly valid under sparsity conditions weaker than those typically invoked in the literature, assuming working models for the exposure and outcome are both correctly specified. When one of the models is misspecified, by amending the procedure for estimating the nuisance parameters, our tests continue to be valid; hence, they are doubly robust. Our proposals are straightforward to implement using existing software for penalized maximum likelihood estimation and do not require sample splitting. We illustrate them in simulations and an analysis of data obtained from the Ghent University intensive care unit.     

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.     

Helminth infection with Litomosoides sigmodontis induces regulatory T cells and inhibits allergic sensitization, airway inflammation, and hyperreactivity in a murine asthma model

Numerous epidemiological studies have shown an inverse correlation between helminth infections and the manifestation of atopic diseases, yet the immunological mechanisms governing this phenomenon are indistinct. We therefore investigated the effects of infection with the filarial parasite Litomosoides sigmodontis on allergen-induced immune reactions and airway disease in a murine model of asthma. Infection with L. sigmodontis suppressed all aspects of the asthmatic phenotype: Ag-specific Ig production, airway reactivity to inhaled methacholine, and pulmonary eosinophilia. Similarly, Ag-specific recall proliferation and overall Th2 cytokine (IL-4, IL-5, and IL-3) production were significantly reduced after L. sigmodontis infection. Analysis of splenic mononuclear cells and mediastinal lymph nodes revealed a significant increase in the numbers of T cells with a regulatory phenotype in infected and sensitized mice compared with sensitized controls. Additionally, surface and intracellular staining for TGF-beta on splenic CD4(+) T cells as well as Ag-specific TGF-beta secretion by splenic mononuclear cells was increased in infected and sensitized animals. Administration of Abs blocking TGF-beta or depleting regulatory T cells in infected animals before allergen sensitization and challenges reversed the suppressive effect with regard to airway hyperreactivity, but did not affect airway inflammation. Despite the dissociate results of the blocking experiments, these data point toward an induction of regulatory T cells and enhanced secretion of the immunomodulatory cytokine TGF-beta as one principle mechanism. In conclusion, our data support the epidemiological evidence and enhance the immunological understanding concerning the impact of helminth infections on atopic diseases thus providing new insights for the development of future studies.     

Selection footprint in the FimH adhesin shows pathoadaptive niche differentiation in Escherichia coli

Spread of biological species from primary into novel habitats leads to within-species adaptive niche differentiation and is commonly driven by acquisition of point mutations in individual genes that increase fitness in the alternative environment. However, finding footprints of adaptive niche differentiation in specific genes remains a challenge. Here we describe a novel method to analyze the footprint of pathogenicity-adaptive, or pathoadaptive, mutations in the Escherichia coli gene encoding FimH-the major, mannose-sensitive adhesin. Analysis of distribution of mutations across the nodes and branches of the FimH phylogenetic network shows (1) zonal separation of evolutionary primary structural variants of FimH and recently derived ones, (2) dramatic differences in the ratio of synonymous and nonsynonymous changes between nodes from different zones, (3) evidence for replacement hot-spots in the FimH protein, (4) differential zonal distribution of FimH variants from commensal and uropathogenic E. coli, and (5) pathoadaptive functional changes in FimH brought by the mutations. The selective footprint in fimH indicates that the pathoadaptive niche differentiation of E. coli is either in its initial stages or undergoing an evolutionary "source/sink" dynamic.