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Background & Aims Assembly of infectious hepatitis C virus (HCV) particles is known to involve host lipoproteins, giving rise to unique lipo-viro-particles (LVPs), but proteome studies now suggest that additional cellular proteins are associated with HCV virions or other particles containing the viral envelope glycoprotein E2. Many of these host cell proteins are common markers of exosomes, most notably the intracellular adaptor protein syntenin, which is required for exosome biogenesis. We aimed to elucidate the role of syntenin/E2 in HCV infection. Methods Using cell culture-derived HCV, we studied the biogenesis and function of E2-coated exosomes in both hepatoma cells and primary human hepatocytes (PHHs). Results Knockout of syntenin had a negligible impact on HCV replication and virus production, whereas ectopic expression of syntenin at physiological levels reduced intracellular E2 abundance, while concomitantly increasing the secretion of E2-coated exosomes. Importantly, cells expressing syntenin and HCV structural proteins efficiently released exosomes containing E2 but lacking the core protein. Furthermore, infectivity of HCV released from syntenin-expressing hepatoma cells and PHHs was more resistant to neutralization by E2-specific antibodies and chronic-phase patient serum. We also found that high E2/syntenin levels in sera correlate with lower serum neutralization capability. Conclusions E2- and syntenin-containing exosomes are a major type of particle released from cells expressing high levels of syntenin. Efficient production of E2-coated exosomes renders HCV infectivity less susceptible to antibody neutralization in hepatoma cells and PHHs. Lay summary This study identifies a key role for syntenin in the regulation of E2 secretion via exosomes. Efficient production of E2-coated exosomes was shown to make hepatitis C virus less sensitive to antibody neutralization. These results may have implications for the development of a hepatitis C virus vaccine.

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies and inflicts high mortality worldwide. The effect of tumor microenvironment components on HCC oncogenesis remains unknown. In particular, the nonleukocyte portion of the stromal fraction (SF) is poorly understood. Methods We comprehensively evaluated the proportional cell counts and gene expression data from The Cancer Genome Atlas (TCGA) to examine the contributions of cell components to the tumor microenvironment. Single-cell sequencing data from the Gene Expression Omnibus (GEO) were also analyzed to verify the association between the nonleukocyte SF and genes. We classified HCC using a hierarchical clustering method based on diversity of nonleukocyte SF–related gene expression among different components, and we used an appropriate GEO dataset to verify the clusters with a support vector machine (SVM) model. The prognosis of subtypes and their relationship with tumor microenvironmental cell proportions, clinicopathogenesis factors, and other indicators were evaluated. Results Based on linear regression, 711 genes related to nonleukocyte SF were selected from the TCGA dataset. We classified HCC into three subtypes using genes related to the nonleukocyte SF. Additionally, the GEO single-cell sequencing data confirmed the relationship between genes and the nonleukocyte SF. The tumor microenvironment of Type 2 contained the most significant mutually reinforcing interaction between the nonleukocyte SF and tumor cells. Meanwhile, Type 2 patients had the poorest prognosis and the most severe tumor-node-metastasis (TNM) stages, histological grades, etc. The analysis based on the GEO dataset verified the classification results with an SVM model. Type 2 was associated with worse clinicopathological characteristics, including tumor grading and staging, than the other types. In addition, the pathway analysis revealed that signals related to the SF and cell proliferation were significantly enhanced in Type 2 compared to the other group, which consisted of Types 1 and 3. Conclusion The nonleukocyte SF in the tumor microenvironment contributed greatly to HCC oncogenesis. We can use these HCC classification criteria to stratify patients into subtypes for personalized treatment.

Multiple lines of evidence implicate that aberrant activation of Hedgehog (Hh) signaling is involved in a variety of human cancers. However, the molecular mechanisms underlying how cancer cells respond to Hh inhibition remain to be elucidated. In this study, we found that blockade of Hh signaling suppresses cell proliferation in human cancer cells. Microarray analysis revealed that differentially expressed genes (DEGs) in human cancer cells are enriched in autophagy pathway in response to the inhibition of Hh signaling. Interestingly, inhibition of Hh signaling induced autophagy, whereas activation of Hh signaling by ligand treatments prevented the induction of autophagy. In addition, inhibition of autophagy by 3-methyladenine (3-MA) partially suppressed cytotoxicity induced by inhibition of Hh signaling. Finally, in autophagy deficient cells, cytotoxic effect triggered by inhibition of Hh signaling was partially reversed, indicating the modulation of autophagy by Hh signaling is autophagy-specific. These results suggest that inhibition of Hh signaling impedes cancer cell proliferation in part through induction of autophagy.

Viral quasispecies analysis is important for basic and clinical research. This study was designed to detect hepatitis B virus (HBV) genome-wide mutation profiling with detailed variant composition in individual patients, especially quasispecies evolution correlating with liver disease progression. We characterized viral populations by massively parallel pyrosequencing at whole HBV genome level in 17 patients with advanced liver disease (ALD) and 30 chronic carriers (CC). An average sequencing coverage of 2047× and 687× in ALD and CC groups, respectively, were achieved. Deep sequencing data resolved the landscapes of HBV substitutions and a more complicated quasispecies composition than previously observed. The values of substitution frequencies in quasispecies were clustered as either more than 80% or less than 20%, forming a unique U-shaped distribution pattern in both clinical groups. Furthermore, quantitative comparison of mutation frequencies of each site between two groups resulted in a spectrum of substitutions associated with liver disease progression, and among which, C2288A/T, C2304A, and A/G2525C/T were novel candidates. Moreover, distinct deletion patterns in preS, X, and C regions were shown between the two groups. In conclusion, pyrosequencing of the whole HBV genome revealed a panorama of viral quasispecies composition, characteristics of substitution distribution, and mutations correlating to severe liver disease.

For transcriptome analysis, it is critical to precisely define all the transcripts across the whole genome. More and more digital gene expression (DGE) scannings have indicated the presence of huge amount of novel transcripts in addition to the known gene models. However, almost all these studies still depend crucially on existing annotation. Here, we present Gene2DGE, a Perl software package for gene model renewal with DGE data. We applied Gene2DGE to the mouse blastomere transcriptome, and defined 98,532 read-enriched regions (RERs) by read clustering supported by more than four reads for each base pair. Taking advantage of this ab initio method, we refined 2,104 exonic regions (4% of a total of 48,501 annotated transcribed regions) with remarkable extension into un-annotated regions (>50bp). For 5% of uniquely mapped reads falling within intron regions, we identified 13,291 additional possible exons. As a result, we renewed 4,788 gene models, which account for 39% of a total of 12,277 transcribed genes. Furthermore, we identified 12,613 intergenic RERs, suggesting the possible presence of novel genes outside the existing gene models. In this study, therefore, we have developed a suitable tool for renewal of known gene models by ab initio prediction in transcriptome dissection. The Gene2DGE package is freely available at http://bighapmap.big.ac.cn/.

The chromosome 17q21.31 inversion is a 900-kb common structural polymorphism found primarily in European population. Although the genetic flux within inversion region was assumed to be considerable suppressed, it is still unclear about the details of genetic exchange between the H1 (non-inverted sequence) and H2 (inverted sequence) haplotypes of this inversion. Here we describe a refined map of genetic exchanges between pairs of gene arrangements within the 17q21.31 region. Using HapMap phase II data of 1,546 single nucleotide polymorphisms, we successfully deduced 96 H1 and 24 H2 haplotypes in European samples by neighbor-joining tree reconstruction. Furthermore, we identified 15 and 26 candidate tracts with reciprocal and non-reciprocal genetic exchanges, respectively. In all 15 regions harboring reciprocal exchange, haplotypes reconstructed by clone sequencing did not support these exchange events, suggesting that such signals of exchange between two sister chromosomes in certain heterozygous individual were caused by phasing error regions. On the other hand, the finished clone sequencing across 4 of 26 tracts with non-reciprocal genetic flux confirmed that this kind of genetic exchange was caused by gene conversion. In summary, as crossover between pairs of gene arrangements had been considerably suppressed, gene conversion might be the most important mechanism for genetic exchange at 17q21.31.

Neuropathic pain is the most difficult type of pain to cure. The P2X(2/3) receptors play a crucial role in facilitating the transmission of pain at neuropathic pain states. Emodin is a natural anthraquinone in rhubarb. The present research investigated the effects of emodin on the pain transmission in neuropathic pain states that was mediated by P2X(2/3) receptor in primary sensory neurons. Chronic constriction injury (CCI) model was used as neuropathic pain model. Emodin was dissolved in 0.5% sodium carboxymethyl cellulose (CMC) as vehicle. Sprague-Dawley male rats had been randomly divided into Sham+vehicle group, CCI+emodin group, and CCI+vehicle group. Mechanical withdrawal threshold and thermal withdrawal latency were measured. P2X(2/3) expression in L4/L5 dorsal root ganglion (DRG) was detected by immunohistochemistry, in situ hybridization (ISH) and RT-PCR. The mechanical withdrawal threshold and thermal withdrawal latency in CCI+vehicle group were lower than those in Sham+vehicle group and CCI+emodin group (p

Population genomic approaches, which take advantages of high-throughput genotyping, are powerful yet costly methods to scan for selective sweeps. DNA-pooling strategies have been widely used for association studies because it is a cost-effective alternative to large-scale individual genotyping. Here, we performed an SNP-MaP (single nucleotide polymorphism microarrays and pooling) analysis using samples from Eurasia to evaluate the efficiency of pooling strategy in genome-wide scans for selection. By conducting simulations of allelotype data, we first demonstrated that the boxplot with average heterozygosity (HET) is a promising method to detect strong selective sweeps with a moderate level of pooling error. Based on this, we used a sliding window analysis of HET to detect the large contiguous regions (LCRs) putatively under selective sweeps from Eurasia datasets. This survey identified 63 LCRs in a European population. These signals were further supported by the integrated haplotype score (iHS) test using HapMap II data. We also confirmed the European-specific signatures of positive selection from several previously identified genes (KEL, TRPV5, TRPV6, EPHB6). In summary, our results not only revealed the high credibility of SNP-MaP strategy in scanning for selective sweeps, but also provided an insight into the population differentiation.

Sporadic amyotrophic lateral sclerosis (sALS) is a severe neurodegenerative disease that causes progressive motor neuron death. Although the etiology of sALS remains unknown, genetic variants are thought to predispose individuals to the disease. Several recent genome-wide association studies have identified a number of loci that increase sALS susceptibility, but these only explain a small proportion of the disease. To extend the current genetic evidence and to identify novel candidates of sALS, we performed a pooling genome-wide association study by 859,311 autosomal single-nucleotide polymorphisms of IlluminaHumanOmniZhongHua-8 combining pathway analysis in 250 typical sALS cases precluding age, clinical course, and phenotype interference and 250 control subjects from Chinese Han populations (CHP). The results revealed that 8 novel loci of 1p34.3, 3p21.1, 3p22.2, 10p15.2, 22q12.1, 3q13.11, 11q25, 12q24.33, and 5 previously reported loci of CNTN4 (kgp11325216), ATXN1 (kgp8327591), C9orf72 (kgp6016770), ITPR2 (kgp3041552), and SOD1 (kgp10760302) were associated with sALS from CHP. Furthermore, the pathway analysis based on the Gene Set Analysis Toolkit V2 showed that 10 top pathways were strongly associated with sALS from CHP, and among them, the 7 most potentially candidate pathways were phosphatidylinositol signaling system, Wnt signaling pathway, axon guidance, MAPK signaling pathway, neurotrophin signaling pathway, arachidonic acid metabolism, and T-cell receptor signaling pathway, a total of 39 significantly associate genes in 7 candidate pathways was suggested to involve in the pathogenesis of sALS from CHP. In conclusion, our results revealed several new loci and pathways related to sALS from CHP and extend the association evidence for partial loci, genes, and pathways, which were previously identified in other populations. Thus, our data provided new clues for exploring the pathogenesis of sALS.