Pyrosequencing method for sensitive detection of HBV drug resistance mutations.

Hepatitis B (HBV) drug resistance assay is important for guiding therapy after the development of virologic breakthrough for patients receiving nucleoside/-tide analog therapy. However, the existing genotyping tools are either costly or lack sensitivity to detect mixed genotypes, and an improved method of resistant mutation detection is needed. An assay protocol for clinical application using pyrosequencing method was developed, capable of detecting all known validated HBV polymerase gene mutations that impart resistance to lamivudine, adefovir, tenofovir, and entecavir. Sixty-eight serum samples with known HBV resistance genotypes, previously tested with either Sanger sequencing assay or commercial line probe assay, were used for validation. Where there were discrepancies between the two methods, clonal sequencing by Sanger's method was used for confirmation. The modified pyrosequencing method accurately identified all the cloned polymerase genotypes and was able to distinguish as little as 5% of the mutant populations. This assay can be performed on serum sample with HBV DNA as low as 13.5 IU/mL. The cost per test was less than existing commercial assay. HBV drug resistance pyrosequencing assay was accurate, more sensitive and cheaper compared with the existing methods. It can detect minor populations of drug-resistant clones earlier, before the drug resistant clones become dominant, allowing the opportunity for an earlier change of therapy.

Hepatitis B immunoprophylaxis failure and the presence of hepatitis B surface gene mutants in the affected children.

Hepatitis B virus (HBV) infection is usually vertically transmitted from the mother to child during birth in Asian countries. Despite immunization, immunoprophylaxis failure is well-documented. The aim of the study was to study immunoprophylaxis failure rate in the cohort of infants delivered by chronic HBV-infected mothers and to determine risk factors for failure. This was an observational study involving chronic hepatitis B infected mothers seen at a tertiary care center in Singapore between June 2009 and December 2013. Infants born to these mothers were recruited after they had completed the recommended vaccination schedule. Serological testing for the children was performed 3 months after completion of the last dose of vaccine. HBV surface gene sequencing was carried out if HBV DNA was detectable in the children. Among the 161 mothers enrolled, most were HBeAg negative. HBeAg positive mothers were younger and had a significantly higher viral load (6.5 log) as compared to HBeAg negative mothers (1.35 log) (P < 0.001). Four children (2.6%) were found to have immunoprophylaxis failure. Two occurred in children delivered by mothers with extremely high viral load of more than 5 × 10(7) IU/ml. HBV surface gene mutations were detected in most children (3 out of 4) with immunoprophylaxis failure. The overall effectiveness of the hepatitis B vaccination program was high. High maternal viral load and presence of surface gene mutants may be potential contributors.

SESN-1 is a positive regulator of lifespan in Caenorhabditis elegans.

Aging is a process of gradual functional decline leading to death. Reactive oxygen species (ROS) not only contribute to oxidative stress and cell damage that lead to aging but also serve as signaling molecules. Sestrins are evolutionarily conserved in all multicellular organisms and are required for regenerating hyperoxidized forms of peroxiredoxins and ROS clearance. However, whether sestrins regulate longevity in metazoans is still unclear. Here, we demonstrated that SESN-1, the only sestrin ortholog in Caenorhabditis elegans, is a positive regulator of lifespan. sesn-1 gene mutant worms had significantly shorter lifespans compared to wild-type animals, and overexpression of sesn-1 prolonged lifespan. Moreover, sesn-1 was found to play a key role in defense against several life stressors, including heat, hydrogen peroxide and the heavy metal copper; and sesn-1 mutants expressed higher levels of ROS and showed a decline in body muscle function. Surprisingly, loss of sesn-1 did not weaken the innate immune function of the worms. Together, these results suggest that SESN-1 is required for normal lifespan and its function in muscle cells prevents muscle degeneration over a lifetime.

Consensus sequence L/PKSSLL mimics crucial epitope on Loop III of Taiwan cobra cardiotoxin.

Phage display is effective in screening peptides that mimic venom's neutralizing epitopes. A phage display cyclized heptapeptide library (C7C library) was panned with purified divalent antivenin IgG, which neutralizes Naja naja atra venom (NAV) and Bungarus multicinctus venom (BMV). The selected heptapeptide sequences were aligned with known protein sequences of NAV and BMV in GenBank. One of the four consensus sequences, L/PKSSLL, mimicked the crucial epitope on Loop III of Taiwan cobra cardiotoxin that is associated with the venom's lethal potency. In dot blot analysis, several clones showed varying reactivities for NAV monovalent antivenin and lesser cross-reactions with BMV monovalent antivenin. The KSSLLRN-carrying phage occurred four times in selected clones and showed the strongest reactivity to NAV monovalent antivenin. Furthermore, the QDSLLPS-carrying phage also presented significant dot blot signal, indicating that the SLL sequence shared by these two clones may be a crucial antibody-binding site.