A Novel Inhibitor of Human La Protein with Anti-HBV Activity Discovered by Structure-Based Virtual Screening and In Vitro Evaluation

Background: Over 350 million people worldwide are infected with hepatitis B virus (HBV), a major cause of liver failure and hepatocellular carcinoma. Current therapeutic agents are highly effective, but are also associated with development of viral resistance. Therefore, strategies for identifying other anti-HBV agents with specific, but distinctive mechanisms of action are needed. The human La (hLa) protein, which forms a stabilizing complex with HBV RNA ribonucleoprotein to promote HBV replication, is a promising target of molecular therapy. Aims: This study aimed to discover novel inhibitors of hLa that could inhibit HBV replication and expression. Methods: A multistage molecular docking approach was used to screen a Specs database and an in-house library against hLa binding sites. Sequential in vitro evaluations were performed to detect potential compounds with high scores in HepG2.2.15 cells. Results: Of the 26 potential compounds with high scores chosen for experimental verification, 12 had HBV DNA inhibition ratios of less than 50 % with P,0.05. Six had significant inhibition of HBV e antigen (HBeAg) levels, and 13 had significant inhibition of HBV surface antigen (HBsAg) levels by in vitro assays. Compounds HBSC-11, HBSC-15 and HBSC-34 (HBSC is system prefix for active compounds screened by the library) were selected for evaluation. HBSC-11 was found to have an obvious inhibitory effect on hLa transcription and expression

Short-term effects of repetitive transcranial magnetic stimulation on sleep bruxism:a pilot study

The purpose of this study was to investigate the effects of repetitive transcranial magnetic stimulation (rTMS) on patients with sleep bruxism (SB). Twelve patients with SB were included in an open, single-intervention pilot study. rTMS at 1 Hz and an intensity of 80% of the active motor threshold was applied to the ‘hot spot' of the masseter muscle representation at the primary motor cortex bilaterally for 20 min per side each day for 5 consecutive days. The jaw-closing muscle electromyographic (EMG) activity during sleep was recorded with a portable EMG recorder at baseline, during rTMS treatment and at follow-up for 5 days. In addition, patients scored their jaw-closing muscle soreness on a 0–10 numerical rating scale (NRS). Data were analysed with analysis of variance. The intensity of the EMG activity was suppressed during and after rTMS compared to the baseline (P = 0.04; P = 0.02, respectively). The NRS score of soreness decreased significantly during and after rTMS compared with baseline (P < 0.01). These findings indicated a significant inhibition of jaw-closing muscle activity during sleep along with a decrease of muscle soreness. This pilot study raises the possibility of therapeutic benefits from rTMS in patients with bruxism and calls for further and more controlled studies

Modulation of Bacterial Type III Secretion System by a Spermidine Transporter Dependent Signaling Pathway

10.1371/journal.pone.0001291PLoS ONE212

Impact of viral replication inhibition by entecavir on peripheral T lymphocyte subpopulations in chronic hepatitis B patients

<p>Abstract</p> <p>Background</p> <p>To investigate dynamic fluctuations of serum viral load and peripheral T-lymphocyte subpopulations of chronic hepatitis B patients and their correlation during entecavir therapy.</p> <p>Methods</p> <p>Fifty-five patients received entecavir 0.5 mg/d therapy. Serum HBV DNA load was measured by Real-Time-PCR, and the levels of peripheral T-lymphocyte subpopulations by flow cytometry biweekly, every four weeks and every eight weeks during weeks 1–12, 13–24 and 24–48, respectively. Multilevel modelling was used to analyse the relationship between these variables.</p> <p>Results</p> <p>Of the 55 patients, all HBeAg positive and with detectable HBV DNA, the majority (81.8%) had serum levels of HBV DNA over 10⁷copies per milliliter. HBV viral load dropped sharply during the first two weeks. In 28 and 43 patients, the level became undetectable from week 24 and 48, respectively. Using pre-therapy level as the reference, a significant decrease in CD8⁺T cells and increase in CD4⁺T cells were found from week 12. Both parameters and CD4⁺/CD8⁺ratio steadily improved throughout the 48 weeks. Multilevel analyses showed that the level of decrement of HBV DNA was associated with the increment of T-lymphocyte activities only in the later period (4–48 week). After 4 weeks of therapy, for each log₁₀scale decrement of HBV DNA, the percentage of CD4⁺lymphocyte was increased by 0.49 and that of CD8⁺decreased by 0.51.</p> <p>Conclusion</p> <p>T-lymphocyte subpopulations could be restored partially by entecavir treatment in patients with chronic hepatitis B concurrently with reduction of viremia.</p

Novel Mouse Model Reveals Distinct Activity-Dependent and –Independent Contributions to Synapse Development

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ

Secretion of Genome-Free Hepatitis B Virus – Single Strand Blocking Model for Virion Morphogenesis of Para-retrovirus

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an “immature” nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a “maturation signal” that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, “Single Strand Blocking” model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis

Modeling the Basal Dynamics of P53 System

The tumor suppressor p53 has become one of most investigated genes. Once activated by stress, p53 leads to cellular responses such as cell cycle arrest and apoptosis.Most previous models have ignored the basal dynamics of p53 under nonstressed conditions. To explore the basal dynamics of p53, we constructed a stochastic delay model by incorporating two negative feedback loops. We found that protein distribution of p53 under nonstressed condition is highly skewed with a fraction of cells showing high p53 levels comparable to those observed under stressed conditions. Under nonstressed conditions, asynchronous and spontaneous p53 pulses are triggered by basal DNA double strand breaks produced during normal cell cycle progression. The first peaking times show a predominant G1 distribution while the second ones are more widely distributed. The spontaneous pulses are triggered by an excitable mechanism. Once initiated, the amplitude and duration of pulses remain unchanged. Furthermore, the spontaneous pulses are filtered by ataxia telangiectasia mutated protein mediated posttranslational modifications and do not result in substantial p21 transcription. If challenged by externally severe DNA damage, cells generate synchronous p53 pulses and induce significantly high levels of p21. The high expression of p21 can also be partially induced by lowering the deacetylation rate.Our results demonstrated that the dynamics of p53 under nonstressed conditions is initiated by an excitable mechanism and cells become fully responsive only when cells are confronted with severe damage. These findings advance our understanding of the mechanism of p53 pulses and unlock many opportunities to p53-based therapy

Hepatitis B Virus Alters the Antioxidant System in Transgenic Mice and Sensitizes Hepatocytes to Fas Signaling

Hepatitis B virus (HBV) is a major etiological factor of hepatocellular carcinoma (HCC). However, the precise pathogenetic mechanisms linking HBV infection and HCC remain uncertain. It has been reported that decreased antioxidant enzyme activities are associated with severe liver injury and hepatocarcinogenesis in mouse models. It is unclear if HBV can interfere with the activities of antioxidant enzymes. We established a HBV transgenic mouse line, which spontaneously developed HCC at 2 years of age. We studied the activities of the antioxidant enzymes in the liver of the HBV transgenic mice. Our results showed that the antioxidant enzymes glutathione peroxidase and superoxide dismutase 2 were down-regulated in HBV transgenic mice and correlated with JNK activation. HBV enhanced the Fas-mediated activation of caspase 6, caspase 8 and JNK without enhancing the activation of caspase 3 and hepatocellular apoptosis. As a proper redox balance is important for maintaining cellular homeostasis, these effects of HBV on the host antioxidant system and Fas-signaling may play an important role in HBV-induced hepatocarcinogenesis

Phylogenetic Relationships among the Colobine Monkeys Revisited: New Insights from Analyses of Complete mt Genomes and 44 Nuclear Non-Coding Markers

Background: Phylogenetic relationships among Asian and African colobine genera have been disputed and are not yet well established. In the present study, we revisit the contentious relationships within the Asian and African Colobinae by analyzing 44 nuclear non-coding genes (.23 kb) and mitochondrial (mt) genome sequences from 14 colobine and 4 noncolobine primates. Principal Findings: The combined nuclear gene and the mt genome as well as the combined nuclear and mt gene analyses yielded different phylogenetic relationships among colobine genera with the exception of a monophyletic ‘odd-nosed’ group consisting of Rhinopithecus, Pygathrix and Nasalis, and a monophyletic African group consisting of Colobus and Piliocolobus. The combined nuclear data analyses supported a sister-grouping between Semnopithecus and Trachypithecus, and between Presbytis and the odd-nosed monkey group, as well as a sister-taxon association of Pygathrix and Rhinopithecus within the odd-nosed monkey group. In contrast, mt genome data analyses revealed that Semnopithecus diverged earliest among the Asian colobines and that the odd-nosed monkey group is sister to a Presbytis and Trachypithecus clade, as well as a close association of Pygathrix with Nasalis. The relationships among these genera inferred from the analyses of combined nuclear and mt genes, however, varied with the tree-building methods used. Another remarkable finding of the present study is that all of our analyses rejected the recently proposed African colobine paraphyl