A Novel Strategy To Develop a Robust Infectious Hepatitis C Virus Cell Culture System Directly from a Clinical Isolate

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 10⁵ focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.     

Effects of Land Use,Topography and Socio-Economic Factors on River Water Quality in a Mountainous Watershed with Intensive Agricultural Production in East China

Understanding the primary effects of anthropogenic activities and natural factors on river water quality is important in the study and efficient management of water resources. In this study, analysis of Variance (ANOVA), Principal component analysis (PCA), Pearson correlations, Multiple regression analysis (MRA) and Redundancy analysis (RDA) were applied as an integrated approach in a GIS environment to explore the temporal and spatial variations in river water quality and to estimate the influence of watershed land use, topography and socio-economic factors on river water quality based on 3 years of water quality monitoring data for the Cao-E River system. The statistical analysis revealed that TN, pH and temperature were generally higher in the rainy season, whereas BOD₅, DO and turbidity were higher in the dry season. Spatial variations in river water quality were related to numerous anthropogenic and natural factors. Urban land use was found to be the most important explanatory variable for BOD₅, COD_Mn, TN, DN, NH₄⁺-N, NO₃⁻-N, DO, pH and TP. The animal husbandry output per capita was an important predictor of TP and turbidity, and the gross domestic product per capita largely determined spatial variations in EC. The remaining unexplained variance was related to other factors, such as topography. Our results suggested that pollution control of animal waste discharge in rural settlements, agricultural runoff in cropland, industrial production pollution and domestic pollution in urban and industrial areas were important within the Cao-E River basin. Moreover, the percentage of the total overall river water quality variance explained by an individual variable and/or all environmental variables (according to RDA) can assist in quantitatively identifying the primary factors that control pollution at the watershed scale.     

Interaction of Notch signaling modulator Numb with α-Adaptin regulates endocytosis of Notch pathway components and cell fate determination of neural stem cells

The ability to balance self-renewal and differentiation is a hallmark of stem cells. In Drosophila neural stem cells (NSCs), Numb/Notch (N) signaling plays a key role in this process. However, the molecular and cellular mechanisms underlying Numb function in a stem cell setting remain poorly defined. Here we show that α-Adaptin (α-Ada), a subunit of the endocytic AP-2 complex, interacts with Numb through a new mode of interaction to regulate NSC homeostasis. In α-ada mutants, N pathway component Sanpodo and the N receptor itself exhibited altered trafficking, and N signaling was up-regulated in the intermediate progenitors of type II NSC lineages, leading to their transformation into ectopic NSCs. Surprisingly, although the Ear domain of α-Ada interacts with the C terminus of Numb and is important for α-Ada function in the sensory organ precursor lineage, it was dispensable in the NSCs. Instead, α-Ada could regulate Sanpodo, N trafficking, and NSC homeostasis by interacting with Numb through new domains in both proteins previously not known to mediate their interaction. This interaction could be bypassed when α-Ada was directly fused to the phospho-tyrosine binding domain of Numb. Our results identify a critical role for the AP-2-mediated endocytosis in regulating NSC behavior and reveal a new mechanism by which Numb regulates NSC behavior through N. These findings are likely to have important implications for cancer biology.     

DNMT3A rs36012910 A>G polymorphism and gastric cancer susceptibility in a Chinese population

DNA-methyltransferase (DNMT)-3A plays a crucial role in embryonic development and aberrant DNA methylation in carcinogenesis. Polymorphisms of the DNMT3A gene may influence its enzymatic activity and its contribution to susceptibility to cancer. This study evaluated the association of DNMT3A rs36012910 A>G with susceptibility to gastric cancer (GC) in a Chinese population. Genomic DNA was extracted from samples taken from 340 patients with GC and 251 healthy control subjects. The genotype frequency of DNMT3A rs36012910 A>G in all subjects was detected by polymerase chain reaction–restriction fragment length polymorphism and confirmed by sequencing. Stratification analyses were used to study subgroups by age and gender and to evaluate the association of rs36012910 A>G polymorphism with genetic susceptibility to GC. All patients and control individuals were successfully genotyped for the DNMT3A rs36012910 A>G polymorphism. The frequency of DNMT3A rs36012910 allele G is 3.39?% in healthy individuals and 7.78?% in GC patients, respectively. The rs36012910 AG genotype was significantly more common in the GC group than in the controls, although the rs36012910 GG genotype was only one case in GC patients. Further stratification indicated that AG+GG genotypes were associated with susceptibility to GC in males older than 60, but this polymorphism has no significant association with GC susceptibility in females. Male individuals who carried AG+GG genotypes had a 2.362-fold increased risk of GC compared to those who carried the AA genotype. The rs36012910 allele G was associated with an increased risk of GC compared to the rs36012910 allele A. This is the first report to investigate the distribution and evaluate the association of a rare SNP in DNMT3A with genetic susceptibility to GC. DNMT3A rs36012910 A>G might become a potential biomarker for use in GC prediction, although further studies in larger groups and different populations are needed for confirmation.     

Candida pruni sp. nov. is a new yeast species with antagonistic potential against brown rot of peaches

Brown rot caused by Monilinia spp. is among the most important postharvest diseases of commercially grown stone fruits, and application of antagonistic yeasts to control brown rot is one promising strategy alternative to chemical fungicides. In this research, new yeast strains were isolated and tested for their activity against peach brown rot caused by Monilinia fructicola. Three yeast strains were originally isolated from the surface of plums (cv Chinese Angelino) collected in the north of China. In artificially wounded inoculation tests, the yeast reduced the brown rot incidence to 20 %. The population of the yeast within inoculated wounds on peaches significantly increased at 25 °C from an initial level of 5.0 × 10⁶ to 4.45 × 10⁷ CFU per wound after 1 day. The antagonistic strains were belonging to a new species of the genus Candida by sequence comparisons of 26 S rDNA D1/D2 domain and internal transcribed spacer region. The strains are most closely related to C. asparagi, C. musae and C. fructus on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA. However, the strains are notably different from C. asparagi, C. musae and C. fructus, in morphological and physiological characteristics. Therefore, the name Candida pruni is proposed for the novel species, with sp-Quan (=CBS12814^T = KCTC 27526^T = GCMC 6582^T) as the type strain. Our study showed that Candida pruni is a novel yeast species with potential biocontrol against brown rot caused by M. fructicola on peaches.     

Identification of species in the angiosperm family Apiaceae using DNA barcodes

Apiaceae (Umbelliferae) is a large angiosperm family that includes many medicinally important species. The ability to identify these species and their adulterants is important, yet difficult to do so because of their subtle fruit morphological differences and often lack of diagnostic features in preserved specimens. Moreover, dried roots are often the official medical organs, making visual identification to species almost impossible. DNA barcoding has been proposed as a powerful taxonomic tool for species identification. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL+matK as the core plant barcode. Recently, the China Plant BOL Group proposed that the nuclear ribosomal DNA internal transcribed spacer (ITS), as well as a subset of this marker (ITS2), be incorporated alongside rbcL+matK into the core barcode for seed plants, particularly angiosperms. In this study, we assess the effectiveness of these four markers plus psbA‐trnH as Apiaceae barcodes. A total of 6032 sequences representing 1957 species in 385 diverse genera were sampled, of which 211 sequences from 50 individuals (representing seven species) were newly obtained. Of these five markers, ITS and ITS2 showed superior results in intra‐ and interspecific divergence and DNA barcoding gap assessments. For the matched data set (173 samples representing 45 species in five genera), the ITS locus had the highest identification efficiency (73.3%), yet ITS2 also performed relatively well with 66.7% identification efficiency. The identification efficiency increased to 82.2% when using an ITS+psbA‐trnH marker combination (ITS2+psbA‐trnH was 80%), which was significantly higher than that of rbcL+matK (40%). For the full sample data set (3052 ITS sequences, 3732 ITS2 sequences, 1011 psbA‐trnH sequences, 567 matK sequences and 566 rbcL sequences), ITS, ITS2, psbA‐trnH, matK and rbcL had 70.0%, 64.3%, 49.5%, 38.6% and 32.1% discrimination abilities, respectively. These results confirm that ITS or its subset ITS2 be incorporated into the core barcode for Apiaceae and that the combination of ITS/ITS2+psbA‐trnH has much potential value as a powerful, standard DNA barcode for Apiaceae identification.     

Fibroblast growth factor (FGF)-21 signals through both FGF receptor-1 and 2

Fibroblast growth factor (FGF)-21 is a member of the FGF superfamily based on sequence homology. However, unlike most members of this family it does not show any mitogenic activity in all cell types tested. The objective of this study is to identify and characterize receptors for this molecule. Sequencing of the cDNA clones from 3T3-L1 adipocytes indicates that the only isoforms for FGFR-1 and 2 expressed in 3T3-L1 cells are 1IIIc and 2IIIc, respectively, suggesting that FGF-21 regulates glucose metabolism in 3T3-L1 adipocytes through FGFR-1IIIc and FGFR-2IIIc.