Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

Replies to my critics

This paper contains replies to the reviews of my book by Steven Downes, Massimo Pigliucci and Deborah Shelton & Rick Michod.     

First Report of Rhizoctonia solani AG‐7 on Cotton in Egypt

Eighty‐two isolates of Rhizoctonia solani were recorded from roots of naturally‐infected seedlings of the Egyptian cotton (Gossypium barbadense L.). Anastomosis groups (AGs) of the isolates were determined by using 13 different AGs testers. Three (3.7%) of the isolates were identified as R. solani AG7, while the remaining isolates were belonging to the AG 2‐1, AG4 and AG5. The identification of the three isolates was based on the frequency of the C2 reaction with the AG7 tester isolate. No fusion was observed between AG7 and isolates representing the other 13 AGs. Colonies of AG7 isolates grown on potato dextrose agar (PDA), malt yeast agar (MYA) and melt peptone agar (MPA) were brown to dark brown with aerial mycelium and sclerotia. The isolates had pitted sclerotial clusters and brownish exudates after 21 days of culturing on PDA, but without clear zonation. Pathogenicity test under greenhouse conditions revealed that AG7 caused the common symptoms of damping–off, which included seed rot, lesions on the hypocotyls and root rot.     

Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. HCV genome replication occurs in the replication complex (RC) around the endoplasmic reticulum membrane. However, the mechanisms regulating the HCV RC remain widely unknown. Here, we used a chemical biology approach to show that estrogen receptor (ESR) is functionally associated with HCV replication. We found that tamoxifen suppressed HCV genome replication. Part of ESRalpha resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESRalpha reduced HCV replication. Mechanistic analysis suggested that ESRalpha promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESRalpha regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESRalpha to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics.     

Involvement of chromosomally-encoded genes in malathion utilization by Pseudomonas aeruginosa AA112

Malathion is an organophosphate insecticide that has been widely used for both domestic and commercial agricultural purposes. However, malathion has the potential to produce toxic effects in mammalian systems. In this study, Pseudomonas aeruginosa AA 112 which was isolated from soil using enrichment technique could utilize the malathion as a sole carbon source and a source of energy. Pseudomonas aeruginosa AA112 was able to grow in MSMPY medium containing 42.75 mg/ml malathion. However, the optimum concentration of malathion which supported the maximum bacterial growth was found to be 22. 8 mg/ml. Malathion was used as an initial source of energy and carbon when it was found without additional carbon sources (in MSM medium) while it was utilized as second source of energy and carbon in a nutrient-supplemented medium (in MSMPY medium). Moreover, lead acetate test indicated that malathion was first attacked at a sulphur site 1-2 hours after the start of incubation. TLC and IR analysis indicated that malathion was completely degraded into diethyl succinate, hydrogen sulphide and phosphates. Therefore a malathion degradation pathway was proporsed. The degradation of malathion is attributed to the genes located on the chromosome and at least three proteins of high molecular size might be involved in malathion utilization. Bacteria able to use malathion as a food source or metabolize its residues in the environment to inactive, less toxic, and harmless compounds, could be used in bioremediation of an environmental pollution caused by the pesticide.     

In vitro infection of immortalized primary hepatocytes by HCV genotype 4a and inhibition of virus replication by cyclosporin

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide. We previously reported that cyclosporin A (CsA) inhibits HCV-1b replication. However, its inhibition of JFH-1 (HCV-2a) was much less. Since HCV genotype clearly affects the in vitro and in vivo response to anti-viral therapy, we wished to examine the effect of CsA and its non-immunosuppressive derivative NIM811 on HCV genotype 4a replication. We first established an in vitro system supporting HCV-4a infection and replication using immortalized human hepatocytes, HuS-E7/DN24 (HuS) cells, and these cells were infected with sera obtained from Egyptian patients with chronic HCV-4a infection. HuS cells supported more robust HCV-4a replication than both HuH-7.5 and PH5CH8 cells, and HCV-4a infection and replication were completely inhibited by 3 mug/ml CsA and 0.5 mug/ml NIM811. Thus, HuS cells are a good model system supporting the infection and high-level replication of HCV-4a, and both CsA and NIM811 effectively inhibit HCV-4a replication in this system.     

FTIR Assessment of the Effect of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> Leave Extract (EGb 761) on Mammalian Retina

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this study, we investigated the effect of prolonged administration of EGb 761, up to 10 weeks, on mammalian retina using Fourier transform infrared spectroscopy (FTIR). Two main groups were involved in this study: the normal group (n = 10); and EGb-administrated group (n = 50) that received—orally—a dose of 40 mg/kg/day EGb 761. The results demonstrated that EGb administration was associated with different beneficial effects on the retinal constituents especially the underlying amide I protein secondary structure components as well as the NH-OH region. It concluded that the optimum daily administration period of EGb (40 mg/kg) for ophthalmic applications that targeting the retina ranges from 5 to 8 weeks.     

Synthesis of selenium-containing amino acid analogues and their biological study

Synthesis of selenium-containing amino acid analogues is described. These compounds were prepared in a concise and short synthetic route in good yields by nucleophilic substitution reaction of pyridineselenol and quinolineselenol derivatives with N-phthaloylglycyl chloride followed by hydrazinolysis. The newly synthesized compounds were screened against different strains of bacteria and fungi.     

Emergence of epidemic Neisseria meningitidis serogroup X meningitis in Togo and Burkina Faso

Serogroup X meningococci (NmX) historically have caused sporadic and clustered meningitis cases in sub-Saharan Africa. To study recent NmX epidemiology, we analyzed data from population-based, sentinel and passive surveillance, and outbreak investigations of bacterial meningitis in Togo and Burkina Faso during 2006-2010. Cerebrospinal fluid specimens were analyzed by PCR. In Togo during 2006-2009, NmX accounted for 16% of the 702 confirmed bacterial meningitis cases. Kozah district experienced an NmX outbreak in March 2007 with an NmX seasonal cumulative incidence of 33/100,000. In Burkina Faso during 2007-2010, NmX accounted for 7% of the 778 confirmed bacterial meningitis cases, with an increase from 2009 to 2010 (4% to 35% of all confirmed cases, respectively). In 2010, NmX epidemics occurred in northern and central regions of Burkina Faso; the highest district cumulative incidence of NmX was estimated as 130/100,000 during March-April. Although limited to a few districts, we have documented NmX meningitis epidemics occurring with a seasonal incidence previously only reported in the meningitis belt for NmW135 and NmA, which argues for development of an NmX vaccine.