Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.     

贵州省野生火棘果营养成分与气候因子相关性分析

野生火棘在贵州省的资源分布广泛，由于省内各地区气候因子的不同，导致野生火棘果实的营养性成分存在差异。为探究贵州省野生火棘果的营养性成分差异，明确影响营养成分积累的气候因子，筛选出适宜生长区域，采用硫酸-苯酚法、全自动凯氏定氮仪、索氏提取法和福林-酚比色法对火棘果粗多糖、总蛋白、总脂肪和总多酚含量进行测定，并进行差异性分析、主成分分析和聚类分析；同时结合气候因子，进行灰色关联度的相关性分析。结果显示，不同产地之间4种营养成分含量存在明显差异，可将10个不同产地火棘分成3大类；灰色关联度分析表明，相对湿度是影响贵州野生火棘果营养成分含量的主要影响因子；降低相对湿度利于火棘果中营养性成分的积累。     

Study of dried blood spots technique for the determination of dextromethorphan and its metabolite dextrorphan in human whole blood by LC–MS/MS

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.     

The mating system of the Sichuan snub‐nosed monkey (Rhinopithecus roxellana)

This article reports the first genetic study of the mating system of the Sichuan snub‐nosed monkey (Rhinopithecus roxellana), an endemic and endangered species in China. The investigation was carried out in a population (WRT) in the Qinling Mountains using data from both field observation and paternity analysis through microsatellite DNA profiling. During a mating season, a male on an average copulated with 5.7 females. Approximately 18% of the females were observed to copulate with more than one male over the study period. The majority of copulations (94.5%) were initiated by females. Twenty‐eight of 430 observed matings were extra‐unit copulations. Eight polymorphic microsatellite loci were used for paternity analysis. The number of alleles at each locus ranged from 3 to 7 (mean=4.3). Observed heterozygosity ranged between 0.32 and 0.79. None of the loci showed significant deviation from Hardy–Weinberg equilibrium. Results from paternity exclusion showed that 12 of 21 (57.1%) immature individuals were sired by extra‐unit males. Although the basic social unit of snub‐nosed monkeys is consistent with a polygynous mating system, both field observation and genetic data suggests that their mating system is polygamous. Infanticide and inbreeding avoidance are the most likely explanations for the promiscuity of female snub‐nosed monkeys. Am. J. Primatol. 72:25–32, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.     

Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic DeltapO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the DeltapO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the DeltapO157 mutant. The DeltapO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the DeltapO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the DeltapO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed approximately 50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.