The 10th Biennial Hatter Cardiovascular Institute workshop: cellular protection—evaluating new directions in the setting of myocardial infarction, ischaemic stroke, and cardio-oncology

Due to its poor capacity for regeneration, the heart is particularly sensitive to the loss of contractile cardiomyocytes. The onslaught of damage caused by ischaemia and reperfusion, occurring during an acute myocardial infarction and the subsequent reperfusion therapy, can wipe out upwards of a billion cardiomyocytes. A similar program of cell death can cause the irreversible loss of neurons in ischaemic stroke. Similar pathways of lethal cell injury can contribute to other pathologies such as left ventricular dysfunction and heart failure caused by cancer therapy. Consequently, strategies designed to protect the heart from lethal cell injury have the potential to be applicable across all three pathologies. The investigators meeting at the 10th Hatter Cardiovascular Institute workshop examined the parallels between ST-segment elevation myocardial infarction (STEMI), ischaemic stroke, and other pathologies that cause the loss of cardiomyocytes including cancer therapeutic cardiotoxicity. They examined the prospects for protection by remote ischaemic conditioning (RIC) in each scenario, and evaluated impasses and novel opportunities for cellular protection, with the future landscape for RIC in the clinical setting to be determined by the outcome of the large ERIC-PPCI/CONDI2 study. It was agreed that the way forward must include measures to improve experimental methodologies, such that they better reflect the clinical scenario and to judiciously select combinations of therapies targeting specific pathways of cellular death and injury

Sensing and Adaptation to Low pH Mediated by Inducible Amino Acid Decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection

Human Genome-Wide RNAi Screen for Host Factors That Modulate Intracellular Salmonella Growth

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity

Evolution of an endofungal Lifestyle: Deductions from the Burkholderia rhizoxinica Genome

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia rhizoxinica </it>is an intracellular symbiont of the phytopathogenic zygomycete <it>Rhizopus microsporus</it>, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, we analysed the whole genome of <it>B. rhizoxinica </it>HKI 0454 - a type strain of endofungal <it>Burkholderia </it>species.</p> <p>Results</p> <p>The genome consists of a structurally conserved chromosome and two plasmids. Compared to free-living <it>Burkholderia </it>species, the genome is smaller in size and harbors less transcriptional regulator genes. Instead, we observed accumulation of transposons over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, we analysed gene clusters coding for nonribosomal peptide synthetases (NRPS). Notably, <it>B. rhizoxinica </it>lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large number of virulence-related factors and putative type III effectors.</p> <p>Conclusions</p> <p><it>B. rhizoxinica </it>is the first endofungal bacterium, whose genome has been sequenced. Here, we present models of evolution, metabolism and tools for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that <it>B. rhizoxinica </it>is in an early phase of adaptation to the intracellular lifestyle (genome in transition). By analysis of tranporters and metabolic pathways we predict how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. <it>In silico </it>analyses of virulence-associated genes, secreted proteins and effectors might inspire future studies on molecular mechanisms underlying bacterial-fungal interaction.</p

Network centrality and organizational aspirations: A behavioral interaction in the context of international strategic alliances

Whereas social network analysis has been associated with organizational aspirations, little is known on how firm's structural positioning, and particularly network centrality, affects organizational aspirations to engage in international strategic alliances (ISA). This study examines the impact of network centrality on firm's internationalization behavior within the ISA domain in response to the performance-aspiration gap. We build on social and behavioral perspectives to predict that network centrality and performance-based aspirations will be associated with the number of ISA the firm engages in. Using a sample of 7760 alliance collaborations from the top 81 global pharmaceutical firms for the period of 1991-2012, we find supporting evidence for most of our arguments

Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors

Intestinal microbiota in human health and disease: the impact of probiotics

The complex communities of microorganisms that colonise the human gastrointestinal tract play an important role in human health. The development of culture-independent molecular techniques has provided new insights in the composition and diversity of the intestinal microbiota. Here, we summarise the present state of the art on the intestinal microbiota with specific attention for the application of high-throughput functional microbiomic approaches to determine the contribution of the intestinal microbiota to human health. Moreover, we review the association between dysbiosis of the microbiota and both intestinal and extra-intestinal diseases. Finally, we discuss the potential of probiotic microorganism to modulate the intestinal microbiota and thereby contribute to health and well-being. The effects of probiotic consumption on the intestinal microbiota are addressed, as well as the development of tailor-made probiotics designed for specific aberrations that are associated with microbial dysbiosis

Multistep enzyme catalyzed reactions for unnatural amino acids.

The use of unnatural amino acids, particularly synthetic α-amino acids, for modern drug discovery research requires the availability of enantiomerically pure isomers. Starting from a racemate, one single enantiomer can be obtained using a deracemization process. The two more common strategies of deracemization are those obtained by stereoinversion and by dynamic kinetic resolution. Both techniques will be here described using as a substrate the D,L-3-(2-naphthyl)-alanine, a non-natural amino acid: the first one employing a multi-enzymatic redox system, the second one combining an hydrolytic enzyme together with a base-catalyzed substrate racemization. In both cases, the final product, L-3-(2-naphthyl)alanine, is recovered with good yield and excellent enantiomeric excess

Salmonella transcriptional signature in Tetrahymena phagosomes and role of acid tolerance in passage through the protist

Salmonella enterica Typhimurium remains undigested in the food vacuoles of the common protist, Tetrahymena. Contrary to its interaction with Acanthamoeba spp., S. Typhimurium is not cytotoxic to Tetrahymena and is egested as viable cells in its fecal pellets. Through microarray gene expression profiling we investigated the factors in S. Typhimurium that are involved in its resistance to digestion by Tetrahymena. The transcriptome of S. Typhimurium in Tetrahymena phagosomes showed that 989 and 1282 genes were altered in expression compared with that in water and in LB culture medium, respectively. A great proportion of the upregulated genes have a role in anaerobic metabolism and the use of alternate electron acceptors. Many genes required for survival and replication within macrophages and human epithelial cells also had increased expression in Tetrahymena, including mgtC, one of the most highly induced genes in all three cells types. A ΔmgtC mutant of S. Typhimurium did not show decreased viability in Tetrahymena, but paradoxically, was egested at a higher cell density than the wild type. The expression of adiA and adiY, which are involved in arginine-dependent acid resistance, also was increased in the protozoan phagosome. A ΔadiAY mutant had lower viability after passage through Tetrahymena, and a higher proportion of S. Typhimurium wild-type cells within pellets remained viable after exposure to pH 3.4 as compared with uningested cells. Our results provide evidence that acid resistance has a role in the resistance of Salmonella to digestion by Tetrahymena and that passage through the protist confers physiological advantages relevant to its contamination cycle