A novel strategy to inhibit the reproduction and translation of hepatitis C virus

Hepatitis C virus (HCV), a positive single-stranded RNA virus, is a major cause of liver disease in humans. Herein we report a novel strategy to inhibit the reproduction and translation of HCV using a short RNA, named an Additional RNA, to activate the endonuclease activity of Argonaute 2 (Ago2). In the presence of the Additional RNA, the HCV genome RNA has the requisite 12 nucleotides of base-pairing with microRNA-122. This activates the endonuclease activity of Ago2, resulting in cleavage and release of the HCV genome RNA from Ago2 and microRNA-122. The free HCV genome RNA would be susceptible to intracellular degradation, effectively inhibiting its reproduction and translation. This study presents a new method to inhibit HCV that may hold great potential for HCV treatment in the future.     

The Significance of Notch1 Compared with Notch3 in High Metastasis and Poor Overall Survival in Hepatocellular Carcinoma

Background

The prognosis for patients with hepatocellular carcinoma (HCC) is poor, and the mechanisms underlying the development of HCC remain unclear. Notch1 and Notch3 may be involved in malignant transformation, although their roles remain unknown.

Materials and Methods

HCC tissues were stained with anti-Notch1 or -Notch3 antibody. The migration and invasion capacities of the cells were measured with transwell cell culture chambers. RT-PCR was used to measure the expression of Notch1 and Notch3 mRNA. Additionally, western blot analysis was used to assess the protein expression of Notch1, Notch3, CD44v6, E-cadherin, matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA). RNA interference was used to down-regulate the expression of Notch1 and Notch3. Cell viability was assessed using MTT.

Results

Based on immunohistochemistry, high Notch1 expression was correlated with tumor size, tumor grade, metastasis, venous invasion and AJCC TNM stage. High Notch3 expression was only strongly correlated with metastasis, venous invasion and satellite lesions. Kaplan-Meier curves demonstrated that patients with high Notch1 or Notch3 expression were at a significantly increased risk for shortened survival time. In vitro, the down-regulation of Notch1 decreased the migration and invasion capacities of HCC cells by regulating CD44v6, E-cadherin, MMP-2, MMP-9, and uPA via the COX-2 and ERK1/2 pathways. Down-regulation of Notch3 only decreased the invasion capacity of HCC cells by regulating MMP-2 and MMP-9 via the ERK1/2 pathway.

Conclusions

Based on the migration and invasion of HCC, we hypothesize that targeting Notch1 may be more useful than Notch3 for designing novel preventive and therapeutic strategies for HCC in the near future.     

Tryptase Promotes Atherosclerotic Plaque Haemorrhage in ApoE-/- Mice

Tryptase, the most abundant mast cell (MC) granule protein, plays an important role in atherosclerosis plaque development. To test the hypothesis that tryptase participates directly in atherosclerosis plaque haemorrhage, the gene sequence and siRNA for tryptase were cloned into a lentivirus carrier and atherosclerosis plaque haemorrhage models in ApoE-/- mice were constructed. After a cuffing-cervical artery operation, the mice were randomly divided into 6 groups. Hematoxylin and eosin(HE) staining showed that the cervical artery plaque area was much larger in the tryptase overexpression group compared to the other groups, and there was greater artery stenosis. The artery stenosis from the cuff-side in all groups was more than 90%, except the siRNA group. Tryptase promotes plaque haemorrhage distinctively because 50% of the mice in the tryptase overexpression group had plaque haemorrhage, while only 10% in the siRNA group did. The immunohistochemistry of the cervical artery plaque showed that plasminogen activator inhibitor-1 (PAI-1) expression was the lowest while tissue plasminogen activator (tPA), CD31, CD34 and VEGF was the highest in the tryptase overexpression groups. This observation was completely contrary to what was observed in the siRNA group. Tryptase promoted bEnd.3 cell growth, migration and capillary-like tube formation, which suggests that tryptase can promote microvessel angiogenesis. PAI-1 expression was inhibited, while tPA expression was increased by tryptase in bEnd.3 cells. Our in vivo and in vitro studies suggest that trypase can promote atherosclerotic plaque haemorrhage by promoting angiogenesis and regulating the balance of PAI-1 and tPA. Thus, regulating tryptase expression in MCs may provide a potential target for atherosclerosis treatment.     

QC-Chain: Fast and Holistic Quality Control Method for Next-Generation Sequencing Data

Next-generation sequencing (NGS) technologies have been widely used in life sciences. However, several kinds of sequencing artifacts, including low-quality reads and contaminating reads, were found to be quite common in raw sequencing data, which compromise downstream analysis. Therefore, quality control (QC) is essential for raw NGS data. However, although a few NGS data quality control tools are publicly available, there are two limitations: First, the processing speed could not cope with the rapid increase of large data volume. Second, with respect to removing the contaminating reads, none of them could identify contaminating sources de novo, and they rely heavily on prior information of the contaminating species, which is usually not available in advance. Here we report QC-Chain, a fast, accurate and holistic NGS data quality-control method. The tool synergeticly comprised of user-friendly tools for (1) quality assessment and trimming of raw reads using Parallel-QC, a fast read processing tool; (2) identification, quantification and filtration of unknown contamination to get high-quality clean reads. It was optimized based on parallel computation, so the processing speed is significantly higher than other QC methods. Experiments on simulated and real NGS data have shown that reads with low sequencing quality could be identified and filtered. Possible contaminating sources could be identified and quantified de novo, accurately and quickly. Comparison between raw reads and processed reads also showed that subsequent analyses (genome assembly, gene prediction, gene annotation, etc.) results based on processed reads improved significantly in completeness and accuracy. As regard to processing speed, QC-Chain achieves 7–8 time speed-up based on parallel computation as compared to traditional methods. Therefore, QC-Chain is a fast and useful quality control tool for read quality process and de novo contamination filtration of NGS reads, which could significantly facilitate downstream analysis. QC-Chain is publicly available at: http://www.computationalbioenergy.org/qc-chain.html.     

Association of Cerebral Networks in Resting State with Sexual Preference of Homosexual Men: A Study of Regional Homogeneity and Functional Connectivity

Recent imaging studies have shown that brain morphology and neural activity during sexual arousal differ between homosexual and heterosexual men. However, functional differences in neural networks at the resting state is unknown. The study is to characterize the association of homosexual preference with measures of regional homogeneity and functional connectivity in the resting state. Participants were 26 healthy homosexual men and 26 age-matched healthy heterosexual men in whom we collected echo planar magnetic resonance imaging data in the resting state. The sexual orientation was evaluated using the Kinsey Scale. We first assessed group differences in regional homogeneity and then, taking the identified differences as seed regions, we compared groups in measures of functional connectivity from those seeds. The behavioral significances of the differences in regional homogeneity and functional connectivity were assessed by examining their associations with Kinsey Scores. Homosexual participants showed significantly reduced regional homogeneity in the left inferior occipital gyrus, right middle occipital gyrus, right superior occipital gyrus, left cuneus, right precuneus, and increased regional homogeneity in rectal gyrus, bilateral midbrain, and left temporal lobe. Regional homogeneity correlated positively with Kinsey scores in the left inferior occipital gyrus. The homosexual group also showed reduced functional connectivity between left middle temporal gyrus, left supra-marginal gyrus, right cuneus and the seed region, i.e. left inferior occipital gyrus. Additionly, the connection between the left inferior occipital gyrus and right thalamus correlated positively with Kinsey scores. These differences in regional homogeneity and functional connectivity may contribute to a better understanding of the neural basis of male sexual orientation.     

Drug Use and Sex Work: Competing Risk Factors for Newly Acquired HIV in Yunnan,China

Objective

To investigate the HIV incidence and its related factors among female sex workers (FSWs) in a high prevalence area where injection drug use is also widely documented.

Method

A cross-sectional study of 1642 female sex workers (FSWs) was conducted in Honghe Prefecture of Yunnan Province. Interviewed-questionnaires were administrated to collect information on sexual partnerships, condom use and illicit drug using behaviors etc. Blood samples were collected to test for HIV antibodies, and all HIV seropositive specimens were tested with the BED IgG capture-based enzyme immunosorbent assay (BED-CEIA) to distinguish between new and established HIV infection (<153 days).

Results

15.9% (261/1642) of participants reported ever having used drugs, and 7.4% had injected in recent 3 months. The overall HIV prevalence was 10.2% (168/1642), among which 16.7% (28/168) were identified as recent infections using BED-CEIA. The crude HIV incidence estimated from BED-CEIA results was 4.4 (95%CI 2.8–6.0) /100 person years (PY). Multivariate logistic analysis showed that an illicit drug using history (by either self-reporting or urine opiates testing) was both significant risk factors both for HIV established and recent infection (each p<0.05). Drug using FSWs (DU-FSW) reported more male clients in the previous week, and had significantly higher prevalence of HIV, chlamydia trachomatis and HSV-2 as compared to non DU-FSW (each p<0.05).

Conclusion

Our results show that a history of drug use poses significant risks for both new and established HIV infection among FSWs, and that HIV-incidence among Honghe FSWs is relatively high compared to similar populations. Comprehensive interventions targeted at DU-FSWs'' injection drug using and high risk sexual behaviors are urgently needed to reduce the rapid spread of HIV epidemic.     

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Background

Low efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed.

Methodology/Principle Findings

EGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep.

Conclusions/Significance

Injection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification.     

植物叶片化石气孔参数与古海拔定量重建: 方法与进展

古海拔的定量重建在推演地球动力学模型、大气循环模式、古气候变化以及地球化学循环过程方面都具有十分重要的作用,但是迄今为止在古植物学和古环境研究领域中古海拔的定量重建仍然存在许多难点。本文在介绍目前古海拔定量重建领域几种方法的基础上,重点对植物叶片化石气孔参数及其应用与进展进行了讨论,着重介绍:(1)如何利用植物叶片化石气孔参数法恢复古海拔;(2)方法实践过程中的误差来源与分析;(3)应用实例及展望。