CoPS: Comprehensive Peptide Signature database

We present the development of a Comprehensive database of 12 076 invariant Peptide Signatures (CoPS) derived from 52 bacterial genomes with a minimum occurrence in at least seven organisms. These peptides were observed in functionally similar proteins and are distributed over nearly 1250 different functional proteins. The database provides function, structure and occurrence in biochemical pathways of the proteins containing these signature peptides. It houses additional information on the signature peptides, such as identical match in other motif/pattern (e.g. PROSITE, BLOCKS, PRINTS and Pfam) databases and the database of interacting proteins, human proteome and mutation effect on these signature peptides. There is a wide applicability of this database in the identification of critical functional residues in proteins. The database also facilitates the identification of folding nucleus/structural determinants in proteins and functional assignment to yet unknown proteins. We demonstrate functional assignment to 2605 hypothetical proteins in bacterial genomes and 112 unknown proteins in human using this database. AVAILABILITY: The database can be freely accessed through the following URL: http://203.195.151.46/copsv2/index.html or http://203.90.127.70/copsv2/index.html     

MassWiz: a novel scoring algorithm with target-decoy based analysis pipeline for tandem mass spectrometry

Mass spectrometry has made rapid advances in the recent past and has become the preferred method for proteomics. Although many open source algorithms for peptide identification exist, such as X!Tandem and OMSSA, it has majorly been a domain of proprietary software. There is a need for better, freely available, and configurable algorithms that can help in identifying the correct peptides while keeping the false positives to a minimum. We have developed MassWiz, a novel empirical scoring function that gives appropriate weights to major ions, continuity of b-y ions, intensities, and the supporting neutral losses based on the instrument type. We tested MassWiz accuracy on 486,882 spectra from a standard mixture of 18 proteins generated on 6 different instruments downloaded from the Seattle Proteome Center public repository. We compared the MassWiz algorithm with Mascot, Sequest, OMSSA, and X!Tandem at 1% FDR. MassWiz outperformed all in the largest data set (AGILENT XCT) and was second only to Mascot in the other data sets. MassWiz showed good performance in the analysis of high confidence peptides, i.e., those identified by at least three algorithms. We also analyzed a yeast data set containing 106,133 spectra downloaded from the NCBI Peptidome repository and got similar results. The results demonstrate that MassWiz is an effective algorithm for high-confidence peptide identification without compromising on the number of assignments. MassWiz is open-source, versatile, and easily configurable.     

An in situ collagen-HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC-derived vascular smooth muscle cells

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.     

Electrozymographic evaluation of the attenuation of arsenic induced degradation of hepatic SOD,catalase in an in vitro assay system by pectic polysaccharides of Momordica charantia in combination with curcumin

Momordica charantia (MC) fruit known as bitter gourd, is of potential nutritional and medicinal value. The objectives of the present in vitro study were to evaluate the efficacy of bioactive pectic polysaccharides (CCPS) of MC along with another well-known bioactive compound curcumin in the abrogation of hepatocellular oxidative stress persuaded by sodium arsenite. Electrozymographic method was developed for the assessment of superoxide dismutase (SOD) and catalase activities of liver tissues maintained under an in vitro system. A significant association of CCPS of MC in combination with curcumin was found in the alleviation of oxidative stress induced by sodium arsenite in liver slice. Generated data pointed out that CCPS of MC and curcumin separately or in combination can offer significant protection against alterations in malondialdehyde (MDA), conjugated diene (CD) and antioxidative defense (SOD, CAT) markers. Furthermore, results of hepatic cell DNA degradation strongly supported that both these co-administrations have efficacy in preventing cellular damage. This is the first information of extracted polysaccharides from MC preventing arsenic induced damage in a liver slice of rat.     

Formulation and Optimization of Doxorubicin and Biochanin A Combinational Liposomes for Reversal of Chemoresistance

Circumvention of drug resistance still remains a challenge in the development of anticancer therapeutics. Combinational nano-formulations provide many avenues for effective cancer therapy and reversal of drug resistance. In the current study, combination of biochanin A (BioA) and doxorubicin (DOX) in liposomes were prepared and studied for its potential to reverse DOX resistance in COLO205 cells. After development and validation of DOX resistant cells of COLO205 (ColoR), dosing ratio of DOX and BioA for reversal of DOX resistance was determined by co-treatment in ColoR cells. As limited solubility and analytical data available for BioA, therefore solubility was studied for BioA and analytical method was developed for the combination. Combinational liposomes were prepared and optimized for both lipid content and surface charge by evaluating size, polydispersity index, zeta potential, and encapsulation efficiency. The optimized formulation had a size about 125 nm; zeta potential of ?19.5 mV and 70% encapsulation efficiency (EE) for BioA. Thus, prepared combinational liposomes of DOX and BioA were evaluated for its cellular uptake and efficacy to reverse DOX resistance. From the study, increased DOX uptake and promising effect for reversal of DOX resistance was observed.     

Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates

Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.     

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.