Kishore R. Sakharkar

Inactivated trivalent seasonal influenza vaccine induces limited cross-reactive neutralizing antibody responses against 2009 pandemic and 1934 PR8 H1N1 strains

BACKGROUND In June 2009, we conducted a prospective study in Singapore on 51 individuals to determine their serologic responses before and following receipt of the 2009 Southern Hemisphere seasonal influenza vaccine. MATERIALS AND METHODS Paired serum samples were obtained before and 3-4 weeks after vaccination. Virus microneutralization assays were performed to quantify antibodies against A/Brisbane/59/2007 vaccine, pandemic H1N1-2009 and A/Puerto Rico/08/34 H1N1 strains. RESULTS Post-vaccination, 43%, 12% and 24% of subjects displayed a 4-fold or greater rise in neutralizing antibody titers against the three strains, respectively. There was a positive correlation among individuals who showed increased titers to both pandemic H1N1-2009 and A/Puerto Rico/08/34 (p<0.001). However, this correlation was not observed for A/Brisbane/59/2007 with either strain. The relative conservation and accessibility of predicted B-cell epitopes may explain the limited cross-reactivity of the antibodies directed against common H1N1 epitopes. CONCLUSIONS These results suggest that seasonal influenza vaccination confers a certain degree of cross-protection to other H1N1 strains. The correlation in cross-reactive antibody titers to A/Puerto Rico/08/34 and pandemic H1N1-2009 implies that previous exposure to pre-1957 H1N1 strains may confer some protection against the 2009 pandemic strain.

Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species

Abstract Low density polythene (LDPE) is the most widely used packaging material primarily because of its excellent mechanical properties, barrier properties against water, light weight, low cost and high energy effectiveness. However, due to its ubiquitous nature, and resistance to biodegradability, the disposal strategies are crucial and need attention. Recently, microorganisms have become the focus of interest for environmental friendly disposal of plastic and polymer-based waste. This manuscript aims to investigate the extent of biodegradability of LDPE by four different strains of Pseudomonas bacteria—Pseudomonas aeruginosa PAO1 (ATCC 15729), Pseudomonas aeruginosa (ATCC 15692), Pseudomonas putida (KT2440 ATCC 47054) and Pseudomonas syringae (DC3000 ATCC 10862). Degradation of LDPE was determined by weight loss of the sample, morphological changes, mechanical and spectroscopic variations. The eluted compounds after degradation were analysed by gas chromatography coupled with mass spectroscopy. Our results show that Pseudomonas spp. can degrade LDPE films.

Functional and evolutionary analyses on expressed intronless genes in the mouse genome

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life – bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.

Targeting multiple targets in Pseudomonas aeruginosa PAO1 using flux balance analysis of a reconstructed genome-scale metabolic network

Constraint-based flux balance analysis (FBA) is a powerful tool for predicting target genes that can be engineered by analyzing the redistribution of metabolic fluxes on specific gene modifications. Specifically, the effects of metabolic gene deletions on flux distribution can be examined by forcing the fluxes of different reactions catalyzed by the corresponding gene product to zero. However, the target enzyme needs to be essential for survival of the organism to ensure that efficient chemical inhibition results in cell stasis or death. Here, we investigate the essentiality of enzymes in iMO1056 metabolic model of nosocomial pathogen Pseudomonas aeruginosa by performing in silico enzyme deletions using FBA. We identified 116/113 essential enzymes in rich medium in P. aeruginosa. These were then compared with human metabolic model to identify nonhomologous enzymes that could be possible drug targets. Here, we present a refined list of 41 novel potential targets for P. aeruginosa. These targets were then matched with the enzymes belonging to 97 correlated clusters through which we propose the concept of “one target per cluster.” Our approach relates to the “single drug multiple target (SDMT)” concept and has potential in efficient drug target discovery.

Biocomputational strategies for microbial drug target identification.

The complete genome sequences of about 300 bacteria (mostly pathogenic) have been determined, and many more such projects are currently in progress. The detection of bacterial genes that are non-homologous to human genes and are essential for the survival of the pathogen represent a promising means of identifying novel drug targets. We present a subtractive genomics approach for the identification of putative drug targets in microbial genomes and demonstrate its execution using Pseudomonas aeruginosa as an example. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This strategy enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. It should be recognized that there are limitations to this computational approach for drug target identification. Distant gene relationships may be missed since the alignment scores are likely to have low statistical significance. In conclusion, the results of such a strategy underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.

Coffee component hydroxyl hydroquinone (HHQ) as a putative ligand for PPAR gamma and implications in breast cancer

BackgroundCoffee contains several compounds that have the potential to influence breast cancer risk and survival. However, epidemiologic data on the relation between coffee compounds and breast cancer survival are sparse and inconsistent.ResultsWe show that coffee component HHQ has significant apoptotic effect on MDA-MB-231 and MCF-7 cells in vitro, and that ROS generation, change in mitochondrial membrane permeability, upregulation of Bax and Caspase-8 as well as down regulation of PGK1 and PKM2 expression may be important apoptosis-inducing mechanisms. The results suggest that PPARγ ligands may serve as potential therapeutic agents for breast cancer therapy. HHQ was also validated as a ligand for PPARγ by docking procedure.ConclusionThis is the first report on the anti-breast cancer (in vitro) activity of HHQ.

PPARγ disease gene network and identification of therapeutic targets for prostate cancer.

Peroxisome proliferator-activated receptor (PPAR) belongs to the nuclear hormone receptor superfamily. Recently published reports demonstrate the importance of a direct repeat 2 (DR2) as a PPARγ-responsive element in addition to the canonical direct repeat 1 (DR1) Peroxisome proliferator response elements (PPREs). However, a comprehensive and systematic approach to constructing de novo disease-specific gene networks for PPARγ is lacking, especially one that includes PPARγ target genes containing either DR1 or DR2 site within their promoter region. Here, we computationally identified 1154 PPARγ direct target genes and constructed the PPARγ disease gene network, which revealed 138 PPARγ target genes that are associated with 65 unique diseases. The network shows that PPARγ target genes are highly associated with cancer and neurological diseases. Thirty-eight PPARγ direct target genes were found to be involved in prostate cancer and two key (hub) PPARγ direct target genes, PRKCZ and PGK1, were experimentally validated to be repressed upon PPARγ activation by its natural ligand, 15d-PGJ2 in three prostrate cancer cell lines. We proposed that PRKCZ and PGK1 could be novel therapeutic targets for prostate cancer. These investigations would not only aid in understanding the molecular mechanisms by which PPARγ regulates disease targets but would also lead to the identification of novel PPARγ gene targets.

Therapeutic implications of targeting energy metabolism in breast cancer.

PPARs are ligand activated transcription factors. PPARγ agonists have been reported as a new and potentially efficacious treatment of inflammation, diabetes, obesity, cancer, AD, and schizophrenia. Since cancer cells show dysregulation of glycolysis they are potentially manageable through changes in metabolic environment. Interestingly, several of the genes involved in maintaining the metabolic environment and the central energy generation pathway are regulated or predicted to be regulated by PPARγ. The use of synthetic PPARγ ligands as drugs and their recent withdrawal/restricted usage highlight the lack of understanding of the molecular basis of these drugs, their off-target effects, and their network. These data further underscores the complexity of nuclear receptor signalling mechanisms. This paper will discuss the function and role of PPARγ in energy metabolism and cancer biology in general and its emergence as a promising therapeutic target in breast cancer.

Gene fusion in Helicobacter pylori: making the ends meet

Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era.

Novel phytochemical–antibiotic conjugates as multitarget inhibitors of Pseudomononas aeruginosa GyrB/ParE and DHFR

Background There is a dearth of treatment options for community-acquired and nosocomial Pseudomonas infections due to several rapidly emerging multidrug resistant phenotypes, which show resistance even to combination therapy. As an alternative, developing selective promiscuous hybrid compounds for simultaneous modulation of multiple targets is highly appreciated because it is difficult for the pathogen to develop resistance when an inhibitor has activity against multiple targets. Methods In line with our previous work on phytochemical–antibiotic combination assays and knowledge-based methods, using a fragment combination approach we here report a novel drug design strategy of conjugating synergistic phytochemical–antibiotic combinations into a single hybrid entity for multi-inhibition of P. aeruginosa DNA gyrase subunit B (GyrB)/topoisomerase IV subunit B (ParE) and dihydrofolate reductase (DHFR) enzymes. The designed conjugates were evaluated for their multitarget specificity using various computational methods including docking and dynamic simulations, drug-likeness using molecular properties calculations, and pharmacophoric features by stereoelectronic property predictions. Results Evaluation of the designed hybrid compounds based on their physicochemical properties has indicated that they are promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereoelectronic properties such as HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital), and MEP (molecular electrostatic potential) maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for multitarget inhibition. Furthermore, docking and dynamics simulations revealed that the designed compounds have favorable binding affinity and stability in both the ATP-binding sites of GyrB/ParE and the folate-binding site of DHFR, by forming strong hydrogen bonds and hydrophobic interactions with key active site residues. Conclusion This new design concept of hybrid “phyto-drug” scaffolds, and their simultaneous perturbation of well-established antibacterial targets from two unrelated pathways, appears to be very promising and could serve as a prospective lead in multitarget drug discovery.