Predicting the outer membrane proteome of Pasteurella multocida based on consensus prediction enhanced by results integration and manual confirmation

Background Outer membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome. Results In the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane β-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane β-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes. Conclusions The bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria

Hyperosmotic priming of arabidopsis seedlings establishes a long-term somatic memory accompanied by specific changes of the epigenome

<p>Background: In arid and semi-arid environments, drought and soil salinity usually occur at the beginning and end of a plant's life cycle, offering a natural opportunity for the priming of young plants to enhance stress tolerance in mature plants. Chromatin marks, such as histone modifications, provide a potential molecular mechanism for priming plants to environmental stresses, but whether transient exposure of seedlings to hyperosmotic stress leads to chromatin changes that are maintained throughout vegetative growth remains unclear.</p> <p>Results: We have established an effective protocol for hyperosmotic priming in the model plant Arabidopsis, which includes a transient mild salt treatment of seedlings followed by an extensive period of growth in control conditions. Primed plants are identical to non-primed plants in growth and development, yet they display reduced salt uptake and enhanced drought tolerance after a second stress exposure. ChIP-seq analysis of four histone modifications revealed that the priming treatment altered the epigenomic landscape; the changes were small but they were specific for the treated tissue, varied in number and direction depending on the modification, and preferentially targeted transcription factors. Notably, priming leads to shortening and fractionation of H3K27me3 islands. This effect fades over time, but is still apparent after a ten day growth period in control conditions. Several genes with priming-induced differences in H3K27me3 showed altered transcriptional responsiveness to the second stress treatment.</p> <p>Conclusion: Experience of transient hyperosmotic stress by young plants is stored in a long-term somatic memory comprising differences of chromatin status, transcriptional responsiveness and whole plant physiology.</p&gt

Central nervous system involvement in childhood acute lymphoblastic leukemia is linked to upregulation of cholesterol biosynthetic pathways

No abstract available

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself

Distinct Effects of IL-18 on the Engraftment and Function of Human Effector CD8+ T Cells and Regulatory T Cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8+ effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Rα was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies

Automated method for modeling seven-helix transmembrane receptors from experimental data.

A rule-based automated method is presented for modeling the structures of the seven transmembrane helices of G-protein-coupled receptors. The structures are generated by using a simulated annealing Monte Carlo procedure that positions and orients rigid helices to satisfy structural restraints. The restraints are derived from analysis of experimental information from biophysical studies on native and mutant proteins, from analysis of the sequences of related proteins, and from theoretical considerations of protein structure. Calculations are presented for two systems. The method was validated through calculations using appropriate experimental information for bacteriorhodopsin, which produced a model structure with a root mean square (rms) deviation of 1.87 A from the structure determined by electron microscopy. Calculations are also presented using experimental and theoretical information available for bovine rhodopsin to assign the helices to a projection density map and to produce a model of bovine rhodopsin that can be used as a template for modeling other G-protein-coupled receptors

Using experimental information to produce a model of the transmembrane domain of the ion channel phospholamban.

Molecular models of the transmembrane domain of the phospholamban pentamer have been generated by a computational method that uses the experimentally measured effects of systematic single-site mutations as a guiding force in the modeling procedure. This method makes the assumptions that 1) the phospholamban transmembrane domain is a parallel five-helix bundle, and 2) nondisruptive mutation positions are lipid exposed, whereas 3) disruptive or partially disruptive mutations are not. Our procedure requires substantially less computer time than systematic search methods, allowing rapid assessment of the effects of different experimental results on the helix arrangement. The effectiveness of the approach is investigated in test calculations on two helix-dimer systems of known structure. Two independently derived sets of mutagenesis data were used to define the restraints for generating models of phospholamban. Both resulting models are left-handed, highly symmetrical pentamers. Although the overall bundle geometry is very similar in the two models, the orientation of individual helices differs by approximately 50 degrees, resulting in different sets of residues facing the pore. This demonstrates how differences in restraints can have an effect on the model structures generated, and how the violation of these restraints can identify inconsistent experimental data

Alveolar fluid neutrophil elastase activity in the adult respiratory distress syndrome is coinplexed to alpha-2-macroglobulin

We characterized the elastase and antielastase activity of the alveolar fluid of seven patients with the adult respiratory distress syndrome (ARDS) and thirteen normal volunteers. Alpha-l-antitrypsin (AlAT) concentrations were 60-fold higher in ARDS as compared to normal lavage fluid (2,140±498 nM; 36.1±4.2 nM, respectively). ARDS fluid antineutrophil elastase activity was also considerably higher than that of normals (979±204 nM; 31.3±2.9 nM, respectively). Despite the antineutrophil elastase excess, 5 of 7 ARDS lavage samples contained elastase activity (mean, 6.1±2.4 pM) as assayed using low-molecular-mass substrate, while only 1 of 13 normal subjects had detectable elastase activity (0.2 pM) (P < 0.01, compared with ARDS). That this activity was due to alpha-2-macroglobulin (A2MG)-complexed neutrophil elastase was evidenced by (a) the Sephadex G-75 elution profile; (b) the inactivity against insoluble VHielastin; (c) the inhibitory profile with phenylmethylsulfonyl fluoride, methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, ethylene diamine tetraacetic acid, and AlAT; and (d) the immobilization by A2MG antibody bound to polystyrene plates. Furthermore, in agreement with the predicted affinity of AMAT and A2MG for neutrophil elastase, the ratio of A2MG to AMAT in the fluid (0.57%) coincided with the ratio of the A2MG- to AlAT-complexed elastase (0.36%). These findings suggest that the net lung protease-antiprotease balance in ARDS is shifted largely in favor of the antiproteases (chiefly AMAT), and that the antiproteases, AMAT and A2MG, have similar affinities for neutrophil elastase in vivo