Vismodegib Suppresses TRAIL-mediated Liver Injury in a Mouse Model of Nonalcoholic Steatohepatitis

Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.     

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O₂ and H₂O₂. SOD also reacts with H₂O₂, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO₂) and H₂O₂, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper.     

Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.     

Microbial Communities in Chesapeake & Ohio Canal National Historical Park and Their Function as Indicators of Water Quality

Abstract

The objective of this study was to establish meaningful relationships between prokaryotic community profiles and water quality parameters in different water bodies (spring, stream, cave, and mine) in the middle reach of the Chesapeake & Ohio Canal National Historic Park (C&O), Maryland. The microbial profiles in the water samples were determined using metagenomic analysis. The relationships between microbial phylogenetic profiles and water quality parameters were investigated using principal component analysis (PCA) and redundancy analysis (RDA). The most abundant phyla identified in most samples were Proteobacteria (55.4%), Bacteroidetes (12.3%), Actinobacteria (10.6%), Firmicutes (2.4%), Planktomycetes (1.8%), Verrucomicrobia (1.5%), Chloroflexi (1.5%), and Acidobacteria (1.3%), which are major bacterial and archaeal groups typically observed in natural freshwater environments. PCA showed that water chemistry was determined primarily by the geology of the site and the type of water source (i.e., spring, stream, cave, or mine). Most samples located in carbonate formations correlated with high alkalinity, inorganic carbon, and calcium, representing the typical karstic geochemistry. RDA shows that pH, electrical conductivity, temperature and nutrients including nitrate, phosphate, and sulfate, were significant determinants of the microbial ecology.     

Cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance

Abstract

Entomopathogenic fungi are among the most successful biocontrol agents for preventing economic loss from insects. The identification of virulent species or isolates, the development of formulation technology and the improvement of efficiency are avenues being pursuing by researchers in diverse scientific disciplines. A successful entomopathogenic fungus deploys a combination of mechanical and biochemical processes to overcome the first defensive barrier in insects, the integument. A precise understanding of the mechanisms underlying fungal pathogenicity, particularly the roles of enzymes such as proteases, is essential in order to highlight the potential of entomopathogenic fungi and increase their virulence via genetic modifications. Cuticle-degrading proteases are divided into subtilisin-like (Pr1) and trypsin-like (Pr2) proteases, which are secreted in the initial stages of penetration. The biochemical structure contains the catalytic triad Asp39, His69 and Ser224 in addition to Ca²⁺ binding sites. Studies have shown a molecular weight of almost 19–47?kDa, an optimal pH of 7–12 and an optimal temperature of 35–45?°C. Different species or isolates of entomopathogenic fungi exhibit differences in the secretion and activity of cuticle-degrading proteases, which may indicate their virulence capacity. Genetic engineering techniques have been developed to create isolates with protease overexpression. Such isolates have significantly higher virulence against the host because they not only ensure fungal penetration but also exhibit direct toxicity to insects.