Hepatitis C virus (HCV) is the causative agent of end-stage liver disease. Recent advances in the last decade in anti HCV treatment strategies have dramatically increased the viral clearance rate. However, several limitations are still associated, which warrant a great need of novel, safe and selective drugs against HCV infection. Towards this objective, we explored highly potent and selective small molecule inhibitors, the ellagitannins, from the crude extract of Pomegranate (Punica granatum) fruit peel. The pure compounds, punicalagin, punicalin, and ellagic acid isolated from the extract specifically blocked the HCV NS3/4A protease activity in vitro. Structural analysis using computational approach also showed that ligand molecules interact with the catalytic and substrate binding residues of NS3/4A protease, leading to inhibition of the enzyme activity. Further, punicalagin and punicalin significantly reduced the HCV replication in cell culture system. More importantly, these compounds are well tolerated ex vivo and‘no observed adverse effect level' (NOAEL) was established upto an acute dose of 5000 mg/kg in BALB/c mice. Additionally, pharmacokinetics study showed that the compounds are bioavailable. Taken together, our study provides a proof-of-concept approach for the potential use of antiviral and non-toxic principle ellagitannins from pomegranate in prevention and control of HCV induced complications.

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Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of b-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.Huh7 and HCV replicon cells were seeded at 80% confluency in 35-mm dishes and allowed to grow for 24 h. Huh7 cells were treated with 6 mM Nondimensional Mechanical Nuclear Model

A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse 'off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated region (UTR) of HCV RNA using a liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand (DG). Physico-chemical characterization revealed finer details of complexation of DG with siRNA, whereas molecular dynamic simulations demonstrated sugar moieties projecting "out" in the complex. Preferential delivery of siRNA to the liver was achieved through a highly specific ligand-receptor interaction between dendritic galactose and the asialoglycoprotein receptor. The siRNA-DG complex exhibited perinuclear localization in liver cells and co-localization with viral proteins. The histopathological studies showed the systemic tolerance and biocompatibility of DG. Further, whole body imaging and immunohistochemistry studies confirmed the preferential delivery of the nucleic acid to mice liver. Significant decrease in HCV RNA levels (up to 75%) was achieved in HCV subgenomic replicon and full length HCV-JFH1 infectious cell culture systems. The multidisciplinary approach provides the 'proof of concept' for restricted delivery of therapeutic siRNAs using a target oriented dendritic nano-vector.

DNA and RNA based antiviral strategies using nonviral vectors have shown better potential over the viral pathway due to the fewer chances of gene recombination and immunogenicity. In this work a mesoporous silica nanoparticle (MSN) based carrier system has been used for targeted delivery of shDNA molecule against the conserved 5′-untranslated region (UTR) in the RNA of a hepatitis C virus to inhibit its replication. The MSNs coated with amine and galactose could specifically target liver cells. Significant reduction (about 94%) of viral RNA level was achieved in HCV-JFH1 infectious cell culture compared to the control RNA levels directed the successful delivery and action of the shDNA. This study showed that Gal-AMSN can be used as a synthetic delivery vector to deliver the shDNA effectively for the treatment of HCV infection.

Abstract-The rbcL gene and protein sequences have been used in addressing systematic questions among the few selected members of the family Cucurbitaceae has been investigated. In order to elucidate the systematic positions, a set of chloroplast-rbcL nucleotide sequences (from 42 taxa of 7 genera) and aminoacid sequences (from 52 taxa of 10 genera) were withdrawn from GenBank and GenPept databases, respectively. The evolutionary distance was inferred from these sequences by employing Bootstrap method of UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and MP (Maximum Parsimony) using MEGA (Molecular Evolutionary Genetic Analysis) software. From the separate analysis produced almost similar although not identical results, no strongly supported incongruent results. The members of the genus Austrobryonia showed strictly monophyletic, Trichosanthes, Luffa, Momordica and Coccinia are found to be paraphyletic. But the members of the genus Cucumis are distributed throughout these hiraeoid clades, confirming the polyphyly of this large genus observed in both the family trees. From the results, it is also clear that, the chloroplast-rbcL gene and aminoacid sequences resolved the relationships, as well as provided a good indication of major supra-generic groupings among the selected members of the family Cucurbitaceae. These results provide the necessary frame work and explicit phylogenetic hypotheses from which further reversionary and other systematic studies can proceed.

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The present study was undertaken to assess the role of plastid-rbcL (ribulose-1, 5-bisphosphate carboxylase large-subunit) gene sequences in addressing the evolutionary relationships within the angiosperms at inter and intra-familial levels using computational experiment. In order to elucidate the relationships, a set of 92 chloroplast rbcL sequences representing from 90 taxa of 12 genera and 10 angiospermic plant families (dicot and monocots) were withdrawn from the GenBank database. The multiple sequence alignment was performed using Genebee-ClustalW service to findout the regions of conserved or indels among the sequences. The phylogenetic tree was inferred from these sequences by employing Bootstrap method of UPGMA (Unweighted Pair Group Method with Arithmetic Mean) using MEGA (Molecular Evolutionary Genetics Analysis) software. The consistency of these generic-wise groupings was further confirmed by the MP (Maximum-Parsimony), ME (Minimum-Evolution) and NJ (Neighbor-Joining) methods. The analysis of these studies strongly indicates that, out of the 12 selected genera Trichosanthes (Cucurbitaceae), Phyllanthus (Phyllanthaceae), Austrobryonia (Cucurbitaceae), Solanum (Solanaceae), Piper (Piperaceae) and Saxifraga (Saxifragaceae) are grouped into separate clusters and exhibiting monophyletic conditions. Where as, Drypetes (Putranjivaceae), Asparagus (Asparagaceae), Cassia (Caesalpinioideae, sub-family), Canna (Cannaceae), Mentha (Lamiaceae), are paraphyletic and the members of the Salvia (Lamiaceae) are distributed throughout these hiraeoid clades, confirming the polyphyly of this large genus. Similar observations were noticed in all four methods. Thus, chloroplast rbcL gene sequences can unambiguously resolve the relationships, as well as provided a good indication of major supra-generic groupings among the selected angiospermic plant families and also gave many clues for future studies.