Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.     

Eye size variation reflects habitat and daily activity patterns in colubrid snakes

The functioning of the vertebrate eye depends on its absolute size, which is presumably adapted to specific needs. Eye size variation in lidless and spectacled colubrid snakes was investigated, including 839 specimens belonging to 49 genera, 66 species and subspecies. Variations of adult eye diameters (EDs) in both absolute and relative terms between species were correlated with parameters reflecting behavioral ecology. In absolute terms, eye of arboreal species was larger than in terrestrial and semiaquatic species. For diurnal species, EDs of terrestrial species do not differ from semiaquatic species; for nocturnal species the ED of terrestrial species is larger than fossorial species but not different from semiaquatic species. In relative terms, ED did not differ significantly by habitat for diurnal species. Although the ED of terrestrial species is larger than fossorial species there were no differences for nocturnal species between semiaquatic and fossorial snakes. In contrast to other vertebrates studied to date, colubrid EDs in absolute and relative terms are larger in diurnal than in nocturnal species. These observations suggest that among colubrid snakes, eye size variation reflects adaptation to specific habitats, foraging strategies and daily activities, independently of phylogeny. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Effect of Feed Restriction on Performance and Postprandial Nutrient Metabolism in Pigs Co-Infected with Mycoplasma hyopneumoniae and Swine Influenza Virus

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Microplastics in soil-plant system: effects of nano/microplastics on plant photosynthesis,rhizosphere microbes and soil properties in soil with different residues

Plant and Soil - To investigate the effects of polystyrene microplastics (PS-beads) on the soil properties, photosynthesis of Flowering Chinese cabbage, the rhizosphere microbial community and...     

Microcalorimetric assay of acetylcholinesterase

Comparative assays were made in a spectrophotometer and a microcalorimeter for the reaction between acetylcholinesterase (EC 3.1.1.7) and acetylthiocholine. The rate of light absorbance change and the rate of heat flow were measured from similar and simultaneous reactions in spectrophotometer and microcalorimeter, respectively. At the enzyme activity levels studied, i.e., 0.05–0.15 I.U. in calorimetry and 1–4 I.U. in spectrophotometry, the reaction rates were linear and showed first-order kinetics. A highly significant positive correlation was seen between the two methods (r = 0.997). More importantly, spectrophotometric assay with acetylthiocholine (which utilized a secondary reaction with chromagen, dithiobisnitrobenzoic acid) stood in highly significant positive correlation with calorimetric assays (which did not require a chromagen) either with the same substrate (r = 0.976) or with acetylcholine (r = 0.900). It appears that microcalorimetry can be used in preference to spectrophotometry for enzyme kinetic studies to overcome the complexity of reaction mixture and interference problems and with the advantage of using natural substrates.     

Identification of a rice RNA- and microtubule-binding protein as the multifunctional protein, a peroxisomal enzyme involved in the beta -oxidation of fatty acids.

The control of subcellular mRNA localization and translation is often mediated by protein factors that are directly or indirectly associated with the cytoskeleton. We report the identification and characterization of a rice seed protein that possesses both RNA and microtubule binding activities. In vitro UV cross-linking assays indicated that this protein binds to all mRNA sequences tested, although there was evidence for preferential binding to RNAs that contained A-C nucleotide sequence motifs. The protein was purified to homogeneity using a two-step procedure, and amino acid sequencing identified it as the multifunctional protein (MFP), a peroxisomal enzyme known to possess a number of activities involved in the beta-oxidation of fatty acids. The recombinant version of this rice MFP binds to RNA in UV cross-linking and gel mobility shift experiments, co-sediments specifically with microtubules, and possesses at least two enzymatic activities involved in peroxisomal fatty acid beta-oxidation. Taken together these data suggest that MFP has an important role in mRNA physiology in the cytoplasm, perhaps in regulating the localization or translation of mRNAs through an interaction with microtubules, in addition to its peroxisomal function.     

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

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