Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²⁺) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²⁺ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²⁺ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.     

Hexosamine Biosynthesis Is a Possible Mechanism Underlying Hypoxia’s Effects on Lipid Metabolism in Human Adipocytes

Introduction

Hypoxia regulates adipocyte metabolism. Hexosamine biosynthesis is implicated in murine 3T3L1 adipocyte differentiation and is a possible underlying mechanism for hypoxia’s effects on adipocyte metabolism.

Methods

Lipid metabolism was studied in human visceral and subcutaneous adipocytes in in vitro hypoxic culture with adipophilic staining, glycerol release, and palmitate oxidation assays. Gene expression and hexosamine biosynthesis activation was studied with QRTPCR, immunofluorescence microscopy, and Western blotting.

Results

Hypoxia inhibits lipogenesis and induces basal lipolysis in visceral and subcutaneous human adipocytes. Hypoxia induces fatty acid oxidation in visceral adipocytes but had no effect on fatty acid oxidation in subcutaneous adipocytes. Hypoxia inhibits hexosamine biosynthesis in adipocytes. Inhibition of hexosamine biosynthesis with azaserine attenuates lipogenesis and induces lipolysis in adipocytes in normoxic conditions, while promotion of hexosamine biosynthesis with glucosamine in hypoxic conditions slightly increases lipogenesis.

Conclusions

Hypoxia’s net effect on human adipocyte lipid metabolism would be expected to impair adipocyte buffering capacity and contribute to systemic lipotoxicity. Our data suggest that hypoxia may mediate its effects on lipogenesis and lipolysis through inhibition of hexosamine biosynthesis. Hexosamine biosynthesis represents a target for manipulation of adipocyte metabolism.     

WSX-1 Signalling Inhibits CD4+ T Cell Migration to the Liver during Malaria Infection by Repressing Chemokine-Independent Pathways

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4⁺ T cells in the liver of infected IL27R^−/− (WSX-1^−/−) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4⁺ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4⁺ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1^−/− mice than infected WT mice, and hepatic CD4⁺ T cells from WSX-1^−/− mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4⁺ T cells to the liver of WSX-1^−/− mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4⁺ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4⁺ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.     

Trauma-Related Mortality among Adults in Rural Western Kenya: Characterising Deaths Using Data from a Health and Demographic Surveillance System

Background

Information on trauma-related deaths in low and middle income countries is limited but needed to target public health interventions. Data from a health and demographic surveillance system (HDSS) were examined to characterise such deaths in rural western Kenya.

Methods And Findings

Verbal autopsy data were analysed. Of 11,147 adult deaths between 2003 and 2008, 447 (4%) were attributed to trauma; 71% of these were in males. Trauma contributed 17% of all deaths in males 15 to 24 years; on a population basis mortality rates were greatest in persons over 65 years. Intentional causes accounted for a higher proportion of male than female deaths (RR 2.04, 1.37-3.04) and a higher proportion of deaths of those aged 15 to 65 than older people. Main causes in males were assaults (n=79, 25%) and road traffic injuries (n=47, 15%); and falls for females (n=17, 13%). A significantly greater proportion of deaths from poisoning (RR 5.0, 2.7-9.4) and assault (RR 1.8, 1.2-2.6) occurred among regular consumers of alcohol than among non-regular drinkers. In multivariate analysis, males had a 4-fold higher risk of death from trauma than females (Adjusted Relative Risk; ARR 4.0; 95% CI 1.7-9.4); risk of a trauma death rose with age, with the elderly at 7-fold higher risk (ARR 7.3, 1.1-49.2). Absence of care was the strongest predictor of trauma death (ARR 12.2, 9.4-15.8). Trauma-related deaths were higher among regular alcohol drinkers (ARR 1.5, 1.1-1.9) compared with non-regular drinkers.

Conclusions

While trauma accounts for a small proportion of deaths in this rural area with a high prevalence of HIV, TB and malaria, preventive interventions such as improved road safety, home safety strategies for the elderly, and curbing harmful use of alcohol, are available and could help diminish this burden. Improvements in systems to record underlying causes of death from trauma are required.     

Molecular and morphological evidence support four new species in the genus Muscodor from northern Thailand

The genus Muscodor comprises fungal endophytes which produce mixtures of volatile compounds (VOCs) with antimicrobial activities. In the present study, four novel species, Muscodor musae, M. oryzae, M. suthepensis and M. equiseti were isolated from Musa acuminata, Oryza rufipogon, Cinnamomum bejolghota and Equisetum debile, respectively; these are medicinal plants of northern Thailand. The new Muscodor species are distinguished based on morphological and physiological characteristics and on molecular analysis of ITS-rDNA. Volatile compound analysis showed that 2-methylpropanoic acid was the main VOCs produced by M. musae, M. suthepensis and M. equiseti. The mixed volatiles from each fungus showed in vitro antimicrobial activity. Muscodor suthepensis had the highest antifungal activity.     

Halogen bond tunability II: the varying roles of electrostatic and dispersion contributions to attraction in halogen bonds

In a previous study we investigated the effects of aromatic fluorine substitution on the strengths of the halogen bonds in halobenzene…acetone complexes (halo?=?chloro, bromo, and iodo). In this work, we have examined the origins of these halogen bonds (excluding the iodo systems), more specifically, the relative contributions of electrostatic and dispersion forces in these interactions and how these contributions change when halogen σ-holes are modified. These studies have been carried out using density functional symmetry adapted perturbation theory (DFT-SAPT) and through analyses of intermolecular correlation energies and molecular electrostatic potentials. It is found that electrostatic and dispersion contributions to attraction in halogen bonds vary from complex to complex, but are generally quite similar in magnitude. Not surprisingly, increasing the size and positive nature of a halogen’s σ-hole dramatically enhances the strength of the electrostatic component of the halogen bonding interaction. Not so obviously, halogens with larger, more positive σ-holes tend to exhibit weaker dispersion interactions, which is attributable to the lower local polarizabilities of the larger σ-holes.

Nanoporous Polytetrafluoroethylene/Silica Composite Separator as a High‐Performance All‐Vanadium Redox Flow Battery Membrane

A novel low‐cost nanoporous polytetrafluoroethylene (PTFE)/silica composite separator has been prepared and evaluated for its use in an all‐vanadium redox flow battery (VRB). The separator consists of silica particles enmeshed in a PTFE fibril matrix. It possesses unique nanoporous structures with an average pore size of 38 nm and a porosity of 48%. These pores function as the ion transport channels during redox flow battery operation. This separator provides excellent electrochemical performance in the mixed‐acid VRB system. The VRB using this separator delivers impressive energy efficiency, rate capability, and temperature tolerance. In additon, the flow cell using the novel separator also demonstrates an exceptional capacity retention capability over extended cycling, thus offering excellent stability for long‐term operation. The characteristics of low cost, excellent electrochemical performance and proven chemical stability afford the PTFE/silica nanoporous separator great potential as a substitute for the Nafion membrane used in VRB applications.     

Extending the coverage of spectral libraries: A neighbor‐based approach to predicting intensities of peptide fragmentation spectra

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well‐studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor‐based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K‐nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20–60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.     

High genetic diversity within the morphologically conservative dwarf loach, Kichulchoia brevifasciata (Teleostei: Cobitidae), an endangered freshwater fish from South Korea

The dwarf loach, Kichulchoia brevifasciata, is a primary freshwater fish endemic to South Korea (Republic of Korea). Due to its limited geographic range, special habitat requirements, and scarcity, this species has been considered one of the most endangered cobitid loaches in the world. Gene tree and species tree reconstruction derived from mitochondrial and nuclear sequence data supports the exclusivity of K. brevifasciata and the existence of two highly distinct genetic lineages (eastern and western lineages). Intraspecific genetic variation based on the corrected genetic distance ranged from 0.0013 to 0.0017 (cytochrome b) and 0–0.0012 (nuclear loci) within each lineage and 0.0349 (cytochrome b) and 0.0037–0.0104 (nuclear loci) between the lineages. Although morphologically homogeneous, eastern and western lineages were estimated to have diverged roughly 2.79 million years ago (4.25–1.42, 95 % HPD). Future conservation efforts for K. brevifasciata should consider these genetically distinct lineages as separate evolutionary entities and adopt conservation efforts accordingly.