Determination of the Human Antibody Response to the Neutralization Epitopes Encompassing Amino Acids 313–327 and 432–443 of Hepatitis C Virus E1E2 Glycoproteins

It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regionsencompassing amino acids 313–327 and 432–443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of whichwere from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes.The remaining 126 samples were isolated from patients who had spontaneously clearedHCV infection.In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313–327 and 432–443was 24.29%(51 of 210) and4.76%(10 of 210),respectively. In thespontaneousclearance group (SC group),the prevalence was 0.79%(1 of 126) and 12.70%(16 of 126), respectively.The positive serum samples that contained antibodies reactive to epitope 313–327 neutralizedHCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313–327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432–443 had cross-genotype neutralizing activities. Theneutralizing activityof SC38, SC86, SC92 and CHC75waspartiallyinhibited by peptide 432–443. However,the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVppwere notinhibited by the peptide.This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope432–443as sources for future antibody therapies.     

N-Substituted Benzyl Matrinic Acid Derivatives Inhibit Hepatitis C Virus (HCV) Replication through Down-Regulating Host Heat-Stress Cognate 70 (Hsc70) Expression

Heat-stress cognate 70 (Hsc70) is a host factor that helps hepatitis C virus (HCV) to complete its life cycle in infected hepatocytes. Using Hsc70 as a target for HCV inhibition, a series of novel N-substituted benzyl matrinic/sophoridinic acid derivatives was synthesized and evaluated for their anti-HCV activity in vitro. Among these analogues, compound 7c possessing N-p-methylbenzyl afforded an appealing ability to inhibit HCV replication with SI value over 53. Furthermore, it showed a good oral pharmacokinetic profile with area-under-curve (AUC) of 13.4 µM·h, and a considerably good safety in oral administration in mice (LD₅₀>1000 mg/kg). As 7c suppresses HCV replication via an action mode distinctly different from that of the marketed anti-HCV drugs, it has been selected as a new mechanism anti-HCV candidate for further investigation, with an advantage of no or decreased chance to induce drug-resistant mutations.     

TGF-b Superfamily Cytokine MIC-1/GDF15 Is a Physiological Appetite and Body Weight Regulator

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1^−/−) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1^−/− mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1^−/− mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.     

Bmi-1 Promotes Glioma Angiogenesis by Activating NF-κB Signaling

Angiogenesis in glioma is associated with the poor prognosis of the disease and closely correlates with the highly invasive phenotype of glioma cells, which represents the most challenging impediment against the currently glioma treatments. Bmi-1, an onco-protein, has been implicated in the progression of various human cancers, including gliomas, whereas its role in glioma angiogenesis remains unclear. Our current study examined the effects of Bmi-1 on glioma angiogenesis in vitro as well as in vivo. We found that overexpression of Bmi-1 enhanced, whereas knockdown of Bmi-1 diminished, the capability of glioma cells to induce tubule formation and migration of endothelial cells and neovascularization in chicken chorioallantoic membrane. In vivo, Bmi-1 overexpression and knockdown, respectively, promoted and inhibited angiogenesis in orthotopically transplanted human gliomas. Furthermore, NF-κB activity and VEGF-C expression was induced by Bmi-1 overexpression, whereas Bmi-1 knockdown attenuated NF-κB signaling and decreased VEGF-C expression. Additionally suppression of NF-κB activity using a specific chemical inhibitor abrogated the NF-κB activation and the pro-angiogenic activities of glioma cells. Together, our data suggest that Bmi-1 plays an important role in glioma angiogenesis and therefore could represent a potential target for anti-angiogenic therapy against the disease.     

Large-Scale Screens of miRNA-mRNA Interactions Unveiled That the 3′UTR of a Gene Is Targeted by Multiple miRNAs

Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3′ untranslated regions [3′UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3′UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3′UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3′ end targeted regions, as well as the 5′ end (seed region) binding sites.     

The Futile Cycling of Hexose Phosphates Could Account for the Fact That Hexokinase Exerts a High Control on Glucose Phosphorylation but Not on Glycolytic Rate in Transgenic Potato (Solanum tuberosum) Roots

The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O₂ uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using ¹⁴C-glucose as precursor showed the formation of ¹⁴C-fructose and ¹⁴C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.     

Community Based Promotion on VCT Acceptance among Rural Migrants in Shanghai,China

Objective

Voluntary counseling and testing (VCT) plays an important integral role in response to the HIV/AIDS epidemic. However, VCT service has not been effectively utilized among rural migrants, a high risk group in China. In this study, we developed a community based intervention to examine if community mobilization with comprehensive VCT is more effective than current HIV preventions with routine VCT service in promoting VCT acceptability among rural migrants in Shanghai, China.

Methods

A comprehensive intervention with community mobilization and comprehensive VCT services including community-based VCT and mobile VCT was implemented during 2007–2009. Three communities in Minhang District of Shanghai were randomly selected and were designed to receive community mobilization and comprehensive VCT, traditional VCT and none intervention, respectively. After 24 months intervention, effects were evaluated by comparing outcome indicators between the baseline (2,690 participants) and follow-up surveys (1,850 participants).

Findings

A substantial increase in VCT acceptance was observed among community mobilization group (94.9% vs. 88.5%, P<0.001), whereas the reverse effect was seen in the traditional VCT group (86.1% vs. 94.6%, P<0.001) and control group (69.0% vs. 91.7%, P<0.001). Rural migrants from community mobilization group were more likely to accept VCT (OR = 2.91, 95% CI 1.69–4.97). Rural migrants from community mobilization group also showed significant increase in HIV/AIDS knowledge, positive attitude towards HIV positive individuals and condom use.

Conclusion

Community mobilization with comprehensive VCT has significant impact on promotion of VCT acceptance and utilization among rural migrants in Shanghai. These findings provide evidence to support community mobilization as a suitable strategy for VCT promotion among rural migrants in Shanghai, China.     

Gene Expression Profiling of Ampullary Carcinomas Classifies Ampullary Carcinomas into Biliary-Like and Intestinal-Like Subtypes That Are Prognostic of Outcome

Background

Adenocarcinomas of the ampulla of Vater are classified as biliary cancers, though the exact epithelium of origin for these cancers is not known. We sought to molecularly classify ampullary adenocarcinomas in comparison to known adenocarcinomas of the pancreas, bile duct, and duodenum by gene expression analysis.

Methods

We analyzed 32 fresh-frozen resected, untreated periampullary adenocarcinomas (8 pancreatic, 2 extrahepatic biliary, 8 duodenal, and 14 ampullary) using the Affymetrix U133 Plus 2.0 genome array. Unsupervised and supervised hierarchical clustering identified two subtypes of ampullary carcinomas that were molecularly and histologically characterized.

Results

Hierarchical clustering of periampullary carcinomas segregated ampullary carcinomas into two subgroups, which were distinctly different from pancreatic carcinomas. Non-pancreatic periampullary adenocarcinomas were segregated into two subgroups with differing prognoses: 5 year RFS (77% vs. 0%, p = 0.007) and 5 year OS (100% vs. 35%, p = 0.005). Unsupervised clustering analysis of the 14 ampullary samples also identified two subgroups: a good prognosis intestinal-like subgroup and a poor prognosis biliary-like subgroup with 5 year OS of 70% vs. 28%, P = 0.09. Expression of CK7+/CK20- but not CDX-2 correlated with these two subgroups. Activation of the AKT and MAPK pathways were both increased in the poor prognostic biliary-like subgroup. In an independent 80 patient ampullary validation dataset only histological subtype (intestinal vs. pancreaticobiliary) was significantly associated with OS in both univariate (p = 0.006) and multivariate analysis (P = 0.04).

Conclusions

Gene expression analysis discriminated pancreatic adenocarcinomas from other periampullary carcinomas and identified two prognostically relevant subgroups of ampullary adenocarcinomas. Histological subtype was an independent prognostic factor in ampullary adenocarcinomas.     

Occurrence of Regulated and Emerging Iodinated DBPs in the Shanghai Drinking Water

Drinking water chlorination plays a pivotal role in preventing pathogen contamination against water-borne disease. However, chemical disinfection leads to the formation of halogenated disinfection by products (DBPs). Many DBPs are highly toxic and are of health concern. In this study, we conducted a comprehensive measurements of DBPs, including iodoacetic acid (IAA), iodoform (IF), nine haloacetic acids and four trihalomethanes in drinking waters from 13 water plants in Shanghai, China. The results suggested that IAA and IF were found in all the water treatment plants, with maximum levels of 1.66 µg/L and 1.25 µg/L for IAA and IF, respectively. Owing to deterioration of water quality, the Huangpu River has higher IAA and IF than the Yangtze River. Our results also demonstrated that low pH, high natural organic matter, ammonia nitrogen, and iodide in source waters increased IAA and IF formation. Compared to chlorine, chloramines resulted in higher concentration of iodinated DBP, but reduced the levels of trihalomethanes. This is the first study to reveal the widespread occurrence of IAA and IF in drinking water in China. The data provide a better understanding on the formation of iodinated disinfection byproducts and the findings should be useful for treatment process improvement and disinfection byproducts controls.