Uncovering the origin of Z-configured double bonds in polyketides: intermediate E-double bond formation during borrelidin biosynthesis

Formation of Z-configured double bonds in reduced polyketides is uncommon and their origins have not been extensively studied. To investigate the origin of the Z-configured double bond in the macrolide borrelidin, the recombinant dehydratase domains BorDH2 and B0rDH3 were assayed with a synthetic analogue of the predicted tetraketide substrate. The configuration of the dehydrated products was determined to be E in both cases by comparison to synthetic standards. Detailed NMR spectroscopic analysis of the biosynthetic intermediate pre-borrelidin confirmed the E,E-configuration of the fulllength polyketide synthase product. In contrast to a previously-proposed hypothesis, our results show that in this case the Z-configured double bond is not formed via dehydration from a 3 L-configured precursor, but rather as the result of a later isomerization process.Marie Curie programme of the European UnionEmmy Noether programme of the Deutsche ForschungsgemeinschaftDAA

Native and bone marrow-derived cell mosaicism in gastric carcinoma in H. pylori-infected p27-deficient mice

OBJECTIVE: Chronic Helicobacter pylori (H. pylori) infection promotes non-cardia gastric cancer. Some mouse models suggest that bone marrow derived cells (BMDC) contribute to Helicobacter-associated gastric carcinogenesis. We determined whether this increased susceptibility to Helicobacter-induced gastric carcinogenesis of p27-deficient mice is dependent upon their p27-null BMDC or their p27-null gastric epithelial cells. DESIGN: Female mice (recipients) were irradiated and transplanted with BMDC from male donors. Wild type (WT) mice in group 1 (control) received BMDC from male GFP-transgenic mice. Female WT and p27 KO mice were engrafted with male p27KO mice BMDC (Group 2) or GFP-transgenic WT BMDC (Group 3). Recipients were infected with H. pylori SS1 for one year. RESULTS: Mice lacking p27 in either the BM pool or gastric epithelium developed significantly more advanced gastric pathology, including high-grade dysplasia. Co-staining of donor BMDC in dysplastic gastric glands was confirmed by immunofluorescence. Gastric expression of IL-1 beta protein was reduced in groups 2 and 3 (p \u3c 0.05 vs control) whereas expression of IFN-gamma and chemokines MIP-1 beta, MIG, IP-10 and RANTES in group 2 were significantly higher than group 3. CONCLUSIONS: Both bone marrow-derived and gastric epithelial cells contribute to the increased gastric cancer susceptibility of p27-deficient H. pylori-infected mice

An Integrated Genomic and Immunoinformatic Approach to \u3cem\u3eH. pylori\u3c/em\u3e Vaccine Design

Background: One useful application of pattern matching algorithms is identification of major histocom-patability complex (MHC) ligands and T-cell epitopes. Peptides that bind to MHC molecules and interact with T cell receptors to stimulate the immune system are critical antigens for protection against infectious pathogens. We describe a genomes-to-vaccine approach to H. pylori vaccine design that takes advantage of immunoinformatics algorithms to rapidly identify T-cell epitope sequences from large genomic datasets. Results: To design a globally relevant vaccine, we used computational methods to identify a core genome comprised of 676 open reading frames (ORFs) from amongst seven genetically and phenotypically diverse H. pylori strains from around the world. Of the 1,241,153 9-mer sequences encoded by these ORFs, 106,791 were identical amongst all seven genomes and 23,654 scored in the top 5% of predicted HLA ligands for at least one of eight archetypal Class II HLA alleles when evaluated by EpiMatrix. To maximize the number of epitopes that can be assessed experimentally, we used a computational algorithm to in-crease epitope density in 20-25 amino acid stretches by assembling potentially immunogenic 9-mers to be identically positioned as they are in the native protein antigen. 1,805 immunogenic consensus sequences (ICS) were generated. 79% of selected ICS epitopes bound to a panel of 6 HLA Class II haplotypes, repre-senting \u3e90% of the global human population. Conclusions: The breadth of H. pylori genome datasets was computationally assessed to rapidly and care-fully determine a core set of genes. Application of immunoinformatics tools to this gene set accurately pre-dicted epitopes with promising properties for T cell-based vaccine development

Human Immune Responses to \u3cem\u3eH. pylori\u3c/em\u3e HLA Class II Epitopes Identified by Immunoinformatic Methods

H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects

Extended X-ray emission in PKS 1718-649

© ESO 2018. PKS 1718-649 is one of the closest and most comprehensively studied candidates of a young active galactic nucleus (AGN) that is still embedded in its optical host galaxy. The compact radio structure, with a maximal extent of a few parsecs, makes it a member of the group of compact symmetric objects (CSO). Its environment imposes a turnover of the radio synchrotron spectrum towards lower frequencies, also classifying PKS 1718-649 as gigahertz-peaked radio spectrum (GPS) source. Its close proximity has allowed the first detection of extended X-ray emission in a GPS/CSO source with Chandra that is for the most part unrelated to nuclear feedback. However, not much is known about the nature of this emission. By co-adding all archival Chandra data and complementing these datasets with the large effective area of XMM-Newton, we are able to study the detailed physics of the environment of PKS 1718-649. Not only can we confirm that the bulk of the kiloparsec-scale environment emits in the soft X-rays, but we also identify the emitting gas to form a hot, collisionally ionized medium. While the feedback of the central AGN still seems to be constrained to the inner few parsecs, we argue that supernovae are capable of producing the observed large-scale X-ray emission at a rate inferred from its estimated star formation rate

HelicoVax: Epitope-based therapeutic Helicobacter pylori vaccination in a mouse model

Helicobacter pylori is the leading cause of gastritis, peptic ulcer disease and gastric adenocarcinoma and lymphoma in humans. Due to the decreasing efficacy of anti-H. pylori antibiotic therapy in clinical practice, there is renewed interest in the development of anti-H. pylori vaccines. In this study an in silico-based approach was utilized to develop a multi-epitope DNA-prime/peptide-boost immunization strategy using informatics tools. The efficacy of this construct was then assessed as a therapeutic vaccine in a mouse model of gastric cancer induced by chronic H. pylori infection. The multi-epitope vaccine administered intranasally induced a broad immune response as determined by interferon-gamma production in ELISpot assays. This was associated with a significant reduction in H. pylori colonization compared with mice immunized with the same vaccine intramuscularly, given an empty plasmid, or given a whole H. pylori lysate intranasally as the immunogen. Total scores of gastric histological changes were not significantly different among the 4 experimental groups. These results suggest that further development of an epitope-based mucosal vaccine may be beneficial in eradicating H. pylori and reducing the burden of the associated gastric diseases in humans

A CFTR Potentiator in Patients with Cystic Fibrosis and the G551D Mutation

BACKGROUND: Increasing the activity of defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is a potential treatment for cystic fibrosis. METHODS: We conducted a randomized, double-blind, placebo-controlled trial to evaluate ivacaftor (VX-770), a CFTR potentiator, in subjects 12 years of age or older with cystic fibrosis and at least one G551D-CFTR mutation. Subjects were randomly assigned to receive 150 mg of ivacaftor every 12 hours (84 subjects, of whom 83 received at least one dose) or placebo (83, of whom 78 received at least one dose) for 48 weeks. The primary end point was the estimated mean change from baseline through week 24 in the percent of predicted forced expiratory volume in 1 second (FEV(1)). RESULTS: The change from baseline through week 24 in the percent of predicted FEV(1) was greater by 10.6 percentage points in the ivacaftor group than in the placebo group (P<0.001). Effects on pulmonary function were noted by 2 weeks, and a significant treatment effect was maintained through week 48. Subjects receiving ivacaftor were 55% less likely to have a pulmonary exacerbation than were patients receiving placebo, through week 48 (P<0.001). In addition, through week 48, subjects in the ivacaftor group scored 8.6 points higher than did subjects in the placebo group on the respiratory-symptoms domain of the Cystic Fibrosis Questionnaire–revised instrument (a 100-point scale, with higher numbers indicating a lower effect of symptoms on the patient’s quality of life) (P<0.001). By 48 weeks, patients treated with ivacaftor had gained, on average, 2.7 kg more weight than had patients receiving placebo (P<0.001). The change from baseline through week 48 in the concentration of sweat chloride, a measure of CFTR activity, with ivacaftor as compared with placebo was −48.1 mmol per liter (P<0.001). The incidence of adverse events was similar with ivacaftor and placebo, with a lower proportion of serious adverse events with ivacaftor than with placebo (24% vs. 42%). CONCLUSIONS: Ivacaftor was associated with improvements in lung function at 2 weeks that were sustained through 48 weeks. Substantial improvements were also observed in the risk of pulmonary exacerbations, patient-reported respiratory symptoms, weight, and concentration of sweat chloride

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes

Communications Biophysics

Contains reports on nine research projects split into four sections.National Institutes of Health (Grant 5 PO1 NS13126)National Institutes of Health (Grant 5 KO4 NS00113)National Institutes of Health (Training Grant 5 T32 NS07047)National Institutes of Health (Training Grant 1 T32 NS07099)National Science Foundation (Grant BNS77-16861)National Institutes of Health (Grant 5 ROI NS10916)National Institutes of Health (Grant 5 RO1 NS12846)National Science Foundation (Grant BNS77-21751)National Institutes of Health (Grant 1 RO1 NS14092)Edith E. Sturgis FoundationHealth Sciences FundNational Institutes of Health (Grant 2 R01 NS11680)National Institutes of Health (Fellowship 5 F32 NS05327)National Institutes of Health (Grant 2 ROI NS11080)National Institutes of Health (Training Grant 5 T32 GM07301