Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.     

IL17 Mediates Pelvic Pain in Experimental Autoimmune Prostatitis (EAP)

Chronic pelvic pain syndrome (CPPS) is the most common form of prostatitis, accounting for 90–95% of all diagnoses. It is a complex multi-symptom syndrome with unknown etiology and limited effective treatments. Previous investigations highlight roles for inflammatory mediators in disease progression by correlating levels of cytokines and chemokines with patient reported symptom scores. It is hypothesized that alteration of adaptive immune mechanisms results in autoimmunity and subsequent development of pain. Mouse models of CPPS have been developed to delineate these immune mechanisms driving pain in humans. Using the experimental autoimmune prostatitis (EAP) in C57BL/6 mice model of CPPS we examined the role of CD4+T-cell subsets in the development and maintenance of prostate pain, by tactile allodynia behavioral testing and flow cytometry. In tandem with increased CD4+IL17A+ T-cells upon EAP induction, prophylactic treatment with an anti-IL17 antibody one-day prior to EAP induction prevented the onset of pelvic pain. Therapeutic blockade of IL17 did not reverse pain symptoms indicating that IL17 is essential for development but not maintenance of chronic pain in EAP. Furthermore we identified a cytokine, IL7, to be associated with increased symptom severity in CPPS patients and is increased in patient prostatic secretions and the prostates of EAP mice. IL7 is fundamental to development of IL17 producing cells and plays a role in maturation of auto-reactive T-cells, it is also associated with autoimmune disorders including multiple sclerosis and type-1 diabetes. More recently a growing body of research has pointed to IL17’s role in development of neuropathic and chronic pain. This report presents novel data on the role of CD4+IL17+ T-cells in development and maintenance of pain in EAP and CPPS.     

High temperature pre-digestion of corn stover biomass for improved product yields

Introduction

The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation.

Results

We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions.

Conclusion

Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.

     

A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals

Diseases are an emerging threat to ocean ecosystems. Coral reefs, in particular, are experiencing a worldwide decline because of disease and bleaching, which have been exacerbated by rising seawater temperatures. Yet, the ecological mechanisms behind most coral diseases remain unidentified. Here, we demonstrate that a coral pathogen, Vibrio coralliilyticus, uses chemotaxis and chemokinesis to target the mucus of its coral host, Pocillopora damicornis. A primary driver of this response is the host metabolite dimethylsulfoniopropionate (DMSP), a key element in the global sulfur cycle and a potent foraging cue throughout the marine food web. Coral mucus is rich in DMSP, and we found that DMSP alone elicits chemotactic responses of comparable intensity to whole mucus. Furthermore, in heat-stressed coral fragments, DMSP concentrations increased fivefold and the pathogen''s chemotactic response was correspondingly enhanced. Intriguingly, despite being a rich source of carbon and sulfur, DMSP is not metabolized by the pathogen, suggesting that it is used purely as an infochemical for host location. These results reveal a new role for DMSP in coral disease, demonstrate the importance of chemical signaling and swimming behavior in the recruitment of pathogens to corals and highlight the impact of increased seawater temperatures on disease pathways.     

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.     

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.     

Metabolite profiling of polar steroid constituents in the Far Eastern starfish Aphelasterias japonica using LC–ESI MS/MS

Numerous polar steroids are characteristic metabolites of starfish which determine physiological activities of their extracts. The Far Eastern starfish Aphelasterias japonica is a rich source of different steroid glycosides and polyhydroxysteroids. For detailed analysis of complicated mixture of steroids from this species, isolated by solid-phase extraction, a liquid chromatography–electrospray tandem mass spectrometry (LC–ESI MS/MS) approach was selected and applied. The characteristic fragmentations in ESI MS/MS spectra of steroid glycosides allowed determining types of aglycones, presence of sulfate groups, sugar sequences as well as branching. In addition, main structural features of polyhydroxylated polar steroids including position of hydroxylation and level of sulfation were also established. Totally, 68 metabolites, comprising of 33 asterosaponins, 28 sulfated polyhydroxysteroid mono- and biosides and 7 sulfated polyhydroxysteroid compounds were found by this method. In addition to 15 previously isolated compounds from A. japonica, many new steroid glycosides including asterosaponins with unusual carbohydrate chains were discovered and characterized by their ESI product ion mass spectra. Some details of biosynthesis of polyhydroxylated steroids and their conjugated forms in the species studied such as a role of sulfation and order of introduction of hydroxyl group in A. japonica were proposed.