Publications (87) View all
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Article: Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity.
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ABSTRACT: Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.Journal of Viral Hepatitis 02/2012; 19(2):e81-8. · 4.09 Impact Factor -
Article: Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response.
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ABSTRACT: Aberrant activation of the epidermal growth factor receptor (EGFR) has been found in human cancers of various origins, and has been implicated in cancer pathogenesis. The therapeutic anti-EGFR antibodies cetuximab and matuzumab inhibit both ligand-induced receptor activation and growth of EGFR-expressing tumor cells. The efficacy of such EGFR-targeted therapies may be further enhanced by induction of functionally equivalent endogenous antibody responses. Here we describe novel peptide sequences selected from random peptide libraries for binding to single-chain antibody fragments of cetuximab or matuzumab. Two of these peptides characterized by KTL and YPLG motifs are recognized equally well by cetuximab and matuzumab, although nonoverlapping epitopes were previously reported for these antibodies. Immunization of experimental animals with synthetic KTL- and YPLG-containing peptides led to induction of antibodies that cross-react with human EGFR, and prevent binding of natural EGFR ligands, ligand-induced receptor activation and tumor cell growth in a manner similar to cetuximab and matuzumab. Our findings show that these peptide mimotopes can induce anti-EGFR antibodies with antitumoral activity, which may have implications for EGFR-specific cancer immunotherapy.Oncogene 08/2010; 29(32):4517-27. · 6.37 Impact Factor -
Article: Antibody isolation from immunized animals: comparison of phage display and antibody discovery via V gene repertoire mining.
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ABSTRACT: Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of V(H) and V(L) genes obtained from lymphocytes of immunized animals. Recently, a different approach to antibody isolation that circumvents library screening and instead relies on the mining of the V(H) and V(L) gene repertoires obtained by high throughput sequencing of cDNAs from bone marrow antibody-secreting cells was reported. Here we compared the antibodies obtained via phage library screening or via repertoire mining of V gene cDNAs obtained from total splenocytes of mice immunized with the hapten trinitrophenyl (TNP) conjugated to carrier proteins. We show that, despite the large heterogeneity of B lymphocytes in the spleen, the most abundant V genes encoded antigen-specific antibodies, indicating that total splenocytes can be used in place of bone marrow plasma cells for antibody discovery at least in high titer animals. While both phage display and repertoire mining yielded antigen-specific antibodies showing comparable affinities by enzyme-linked immunosorbent assay analysis, clones obtained by the latter approach displayed higher selectivity towards TNP relative to control haptens. Interestingly, the antibody genes isolated by phage display were of low abundance or absent from the V gene repertoire obtained by 454 sequencing. Similarly, the highly abundant V genes identified by repertoire mining, that as soluble antibodies were antigen-specific, were found to be poorly displayed on phage and were not enriched by phage panning. Thus, our results reveal that phage display and repertoire mining of immune repertoires are complementary technologies that can yield different antigen-specific antibody clones.Protein Engineering Design and Selection 09/2012; 25(10):539-49. · 2.94 Impact Factor -
Article: Insights into the bovine rumen plasmidome.
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ABSTRACT: Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses.Proceedings of the National Academy of Sciences 03/2012; 109(14):5452-7. · 9.68 Impact Factor -
Article: Removal of hepatitis C virus-infected cells by a zymogenized bacterial toxin.
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ABSTRACT: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named "zymoxins". These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the "first generation zymoxins" by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases.PLoS ONE 01/2012; 7(2):e32320. · 4.09 Impact Factor
