Description: We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection. IMPORTANCE Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.

Description: Apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3) proteins are interferon (IFN)-inducible antiviral factors that counteract various viruses such as hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) by inducing cytidine (C)-to-uracil (U) mutations in viral DNA and inhibiting reverse transcription. However, whether APOBEC3 proteins (A3s) can hypermutate human papillomavirus (HPV) viral DNA and exhibit antiviral activity in human keratinocyte remains unknown. Here we examined the involvement of A3s in the HPV life cycle using cervical keratinocyte W12 cells, which are derived from low-grade lesions and retain episomal HPV16 genomes in their nuclei. We focused on the viral E2 gene as a potential target for A3-mediated hypermutation because this gene is frequently found as a boundary sequence in integrated viral DNA. Treatment of W12 cells with beta interferon (IFN-β) increased expression levels of A3s such as A3A, A3F, and A3G and induced C-to-U conversions in the E2 gene in a manner depending on inhibition of uracil DNA glycosylase. Exogenous expression of A3A and A3G also induced E2 hypermutation in W12 cells. IFN-β-induced hypermutation was blocked by transfection of small interfering RNAs against A3G (and modestly by those against A3A). However, the HPV16 episome level was not affected by overexpression of A3A and A3G in W12 cells. This study demonstrates that endogenous A3s upregulated by IFN-β induce E2 hypermutation of HPV16 in cervical keratinocytes, and a pathogenic consequence of E2 hypermutation is discussed.

Description: The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS ( p hosphatidic a cid biosensor with s uperior s ensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.

Description: A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to ( S )-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli , and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg –1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg –1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP + , suggesting the nature of being an aldehyde reductase.

Description: The BZLF1 gene controls the switch between latent and lytic infection by Epstein-Barr virus (EBV). We previously reported that both the ZV and ZIIR elements within the BZLF1 promoter, Zp, are potent transcription silencers within the context of an intact EBV genome. We report here identification of another sequence element, ZV', which synergized with ZV in repressing Zp via binding ZEB1 or ZEB2. We then determined the phenotype of a variant of EBV strain B95.8 in which the ZV, ZV', and ZIIR elements were concurrently mutated. HEK293 cell lines infected with this triple mutant (tmt) virus spontaneously synthesized 6- to 10-fold more viral BZLF1 , BRLF1 , BMRF1 , and BLLF1 RNAs, 3- to 6-fold more viral Zta, Rta, and EAD proteins, 3- to 5-fold more viral DNA, and 7- to 9-fold more infectious virus than did 293 cell lines latently infected with either the ZV ZV' double mutant (dmt) or ZIIR mutant (mt) virus. While ZV ZV' ZIIR tmt EBV efficiently infected human primary blood B cells in vitro , it was highly defective in immortalizing them. Instead of the nearly complete silencing of BZLF1 gene expression that occurs within 4 days after primary infection with wild-type EBV, the ZV ZV' ZIIR tmt-infected cells continued to synthesize BZLF1 RNA, with 90% of them dying within 9 days postinfection. BL41 cells infected with this "superlytic" virus also exhibited increased synthesis of BZLF1 and BMRF1 RNAs. Thus, we conclude that the ZV, ZV', and ZIIR silencing elements act synergistically to repress transcription from Zp, thereby tightly controlling BZLF1 gene expression, which is crucial for establishing and maintaining EBV latency.

Description: Objective There have been suggestions that currently recommended waist circumference (WC) cut-off points for Australians of European origin may not be applicable to Aboriginal people who have different body habitus profiles. We aimed to generate equivalent WC values that correspond to body mass index (BMI) points for identifying absolute cardiovascular disease (CVD) risks. Design Prospective cohort study. Setting An Aboriginal community in Australia's Northern Territory. Participants From 1992 to 1998, 920 adults without CVD, with age, WC and BMI measurements were followed-up for up to 20 years. Outcome measures Incident CVD, coronary artery disease (CAD) and heart failure (HF) events during the follow-up period ascertained from hospitalisation data. We generated WC values with 10-year absolute risks equivalent for the development of CVD as BMI values (20–34 kg/m 2 ) using the Weibull accelerated time-failure model. Results There were 211 incident cases of CVD over 13 669 person-years of follow-up. At the average age of 35 years, WC values with absolute CVD, CAD and HF risks equivalent to BMI of 25 kg/m 2 were 91.5, 91.8 and 91.7 cm, respectively, for males, and corresponding WC values were 92.5, 92.7 and 93 cm for females. WC values with equal absolute CVD, CAD and HF risks to BMI of 30 kg/m 2 were 101.7, 103.1 and 102.6 cm, respectively, for males, and corresponding values were 99.2, 101.6 and 101.5 cm for females. Association between WC and CVD did not depend on gender (p=0.54). Conclusions WC ranging from 91 to 93 cm was equivalent to BMI 25 kg/m 2 for overweight, and 99 to 103 cm was equivalent to BMI of 30 kg/m 2 for obesity in terms of predicting 10-year absolute CVD risk. Replicating the absolute risk method in other Aboriginal communities will further validate the WC values generated for future development of WC cut-off points for Aboriginal people.

Description: Objective To use Monte Carlo simulation to assess the uncertainty and variability of tobacco consumption through wastewater analysis in a city. Methods A total of 11 wastewater treatment plants (WWTPs) (serving 2.2 million people; approximately 83% of urban population in Dalian) were selected and sampled. By detection and quantification of principal metabolites of nicotine, cotinine (COT) and trans-3'-hydroxycotinine (OH-COT), in raw wastewater, back calculation of tobacco use in the population of WWTPs can be realised. Results COT and OH-COT were detected in the entire set of samples with an average concentration of 2.33±0.30 and 2.76±0.91 µg/L, respectively. The mass load of absorbed NIC during the sampling period ranged from 0.25 to 4.22 mg/day/capita with an average of 1.92 mg/day/capita. Using these data, we estimated that smokers in the sampling area consumed an average of 14.6 cigarettes per day for active smoker. Uncertainty and variability analysis by Monte Carlo simulation were used to refine this estimate: the procedure concluded that smokers in Dalian smoked between 10 and 27 cigarettes per day. This estimate showed good agreement with estimates from epidemiological research. Conclusions Sewage-based epidemiology may be a useful additional tool for the large-scale monitoring of patterns of tobacco use. Probabilistic methods can be used to strengthen the reliability of estimated use generated from wastewater analysis.

Description: Objectives We compared the differences in median age at spermarche among 11 ethnic minorities in 2010, estimated the trends regarding age at spermarche in different ethnic minorities from 1995 to 2010, and explored the association of spermarche with body mass index (BMI). Methods We used four cross-sectional Chinese National Surveys on Students’ Constitution and Health (CNSSCH, 1995, 2000, 2005 and 2010), and the total sample size was 40 113 children aged 11–18 years. The median age at spermarche of each ethnic minority was determined by using probit analysis. Logistic regression was used to assess the association of spermarche with BMI. Results In 2010, the ethnic minorities with earliest age at spermarche were Qiang (12.03 years), Zhuang (12.91 years) and Kirghiz (13.17 years); the three ethnic minorities with latest age at spermarche were Dong (14.73 years), Yao (14.60 years), and Naxi (14.36 years). From 1995 to 2010, age at spermarche showed a decline in almost each minority group except Yao and Dong. A higher BMI was associated with an increased likelihood of having reached spermarche after adjusting for age, regions or ethnic minorities. Conclusions A large variation in age at spermarche was observed among different ethnic minorities. The age at spermarche showed a downward shift in almost each of the 11 ethnic minorities with different patterns over time, and the children with higher BMI are more likely to enter puberty early.