Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B

YMDD variants of hepatitis B virus (HBV) emerge in some patients with chronic hepatitis B who receive lamivudine. YMDD variants were examined in 794 patients in 4 controlled studies of 1 year's duration. The long-term effects of YMDD variants were examined in a subset of patients treated up to 4 years. YMDD variants were detected by polymerase chain reaction (PCR) and restriction fragment-length polymorphism assays. After 1 year, YMDD variants were detected in 81 (24%) of 335 patients. In these patients, the median serum HBV DNA concentration at 1 year was <20% of the baseline level, and serum alanine transaminase (ALT) levels and liver histologic findings had significantly improved. In patients with YMDD variants who were treated for up to 4 years, median HBV DNA and ALT levels showed improvements. Sex, baseline body mass index, and HBV DNA level were associated with emergence of YMDD variants. Patients with YMDD variants losing clinical response with a significant increase in the HBV DNA and ALT levels may require additional therapy.published_or_final_versio

Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor

PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes degradation of the LDLR [LDL (low-density lipoprotein) receptor] through an as-yet-undefined mechanism, leading to a reduction in cellular LDLc (LDL-cholesterol) and a concomitant increase in serum LDLc. Central to the function of PCSK9 is a direct protein–protein interaction formed with the LDLR. In the present study, we investigated a strategy to modulate LDL uptake by blocking this interaction using specific antibodies directed against PCSK9. Studies using surface plasmon resonance demonstrated that direct binding of PCSK9 to the LDLR could be abolished with three different anti-PCSK9 antibodies. Two of these antibodies were raised against peptide epitopes in a region of the catalytic domain of PCSK9 that is involved in the interaction with the LDLR. Such antibodies restored LDL uptake in HepG2 cells treated with exogenous PCSK9 and in HepG2 cells engineered to overexpress recombinant PCSK9. This latter observation indicates that antibodies blocking the PCSK9–LDLR interaction can inhibit the action of PCSK9 produced endogenously in a cell-based system. These antibodies also disrupted the higher-affinity interaction between the natural gain-of-function mutant of PCSK9, D374Y, and the LDLR in both the cell-free and cell-based assays. These data indicate that antibodies targeting PCSK9 can reverse the PCSK9-mediated modulation of cell-surface LDLRs

A T3 and T7 Recombinant Phage Acquires Efficient Adsorption and a Broader Host Range

It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress

Human Hepatitis B Virus Production in Avian Cells Is Characterized by Enhanced RNA Splicing and the Presence of Capsids Containing Shortened Genomes

Experimental studies on hepatitis B virus (HBV) replication are commonly done with human hepatoma cells to reflect the natural species and tissue tropism of the virus. However, HBV can also replicate, upon transfection of virus coding plasmids, in cells of other species. In such cross-species transfection experiments with chicken LMH hepatoma cells, we previously observed the formation of HBV genomes with aberrant electrophoretic mobility, in addition to the those DNA species commonly seen in human HepG2 hepatoma cells. Here, we report that these aberrant DNA forms are mainly due to excessive splicing of HBV pregenomic RNA and the abundant synthesis of spliced DNA products, equivalent to those also made in human cells, yet at much lower level. Mutation of the common splice acceptor site abolished splicing and in turn enhanced production of DNA from full-length pgRNA in transfected LMH cells. The absence of splicing made other DNA molecules visible, that were shortened due to the lack of sequences in the core protein coding region. Furthermore, there was nearly full-length DNA in the cytoplasm of LMH cells that was not protected in viral capsids. Remarkably, we have previously observed similar shortened genomes and non-protected viral DNA in human HepG2 cells, yet exclusively in the nucleus where uncoating and final release of viral genomes occurs. Hence, two effects reflecting capsid disassembly in the nucleus in human HepG2 cells are seen in the cytoplasm of chicken LMH cells

On the sensitivity of the HAWC observatory to gamma-ray bursts

We present the sensitivity of HAWC to Gamma Ray Bursts (GRBs). HAWC is a very high-energy gamma-ray observatory currently under construction in Mexico at an altitude of 4100 m. It will observe atmospheric air showers via the water Cherenkov method. HAWC will consist of 300 large water tanks instrumented with 4 photomultipliers each. HAWC has two data acquisition (DAQ) systems. The main DAQ system reads out coincident signals in the tanks and reconstructs the direction and energy of individual atmospheric showers. The scaler DAQ counts the hits in each photomultiplier tube (PMT) in the detector and searches for a statistical excess over the noise of all PMTs. We show that HAWC has a realistic opportunity to observe the high-energy power law components of GRBs that extend at least up to 30 GeV, as it has been observed by Fermi LAT. The two DAQ systems have an energy threshold that is low enough to observe events similar to GRB 090510 and GRB 090902b with the characteristics observed by Fermi LAT. HAWC will provide information about the high-energy spectra of GRBs which in turn could help to understanding about e-pair attenuation in GRB jets, extragalactic background light absorption, as well as establishing the highest energy to which GRBs accelerate particles

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein