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Haryana, India

Laddha S.V.,CSIR - Central Electrochemical Research Institute | Nayak S.,All India Institute of Medical Sciences | Paul D.,CSIR - Central Electrochemical Research Institute | Reddy R.,Premas Biotech Pvt. Ltd. | And 7 more authors.
Biology Direct | Year: 2013

Background: MicroRNAs (miRNAs) are non-uniformly distributed in genomes and ~30% of the miRNAs in the human genome are clustered. In this study we have focused on the imprinted miRNA cluster miR-379/miR-656 on 14q32.31 (hereafter C14) to test their coordinated function. We have analyzed expression profile of >1000 human miRNAs in >1400 samples representing seven different human tissue types obtained from cancer patients along with matched and unmatched controls.Results: We found 68% of the miRNAs in this cluster to be significantly downregulated in glioblastoma multiforme (GBM), 61% downregulated in kidney renal clear cell carcinoma (KIRC), 46% in breast invasive carcinoma (BRCA) and 14% in ovarian serous cystadenocarcinoma (OV). On a genome-wide scale C14 miRNAs accounted for 12-30% of the total downregulated miRNAs in different cancers. Pathway enrichment for the predicted targets of C14 miRNA was significant for cancer pathways, especially Glioma (p< 3.77x10-6, FDR<0.005). The observed downregulation was confirmed in GBM patients by real-time PCR, where 79% of C14 miRNAs (34/43) showed downregulation. In GBM samples, hypermethylation at C14 locus (p<0.003) and downregulation of MEF2, a crucial transcription factor for the cluster was observed which likely contribute to the observed downregulation of the entire miRNA cluster.Conclusion: We provide compelling evidence that the entire C14 miRNA cluster is a tumor suppressor locus involved in multiple cancers, especially in GBM, and points toward a general mechanism of coordinated function for clustered miRNAs.Reviewers: Reviewed by: Prof. Gregory J Goodall and Dr. Alexander Max Burroughs. © 2013 Laddha et al.; licensee BioMed Central Ltd.

Cytochrome P450 (CYP450) isozymes play an important role in the study of drug metabolism and drug discovery. A number of reports are available that describe recombinant expression of CYP450 isozymes. In this paper, human CYP2C9 and human cytochrome P450 reductase cDNAs were cloned and expressed in Premas proprietary yeast episomal and integrative vectors respectively under the influence of GAL1 promoter. Yeast cells were grown and induced at optimal parameters to make microsomal membranes. Isolated microsomal membranes were analyzed for CYP2C9 and cytochrome P450 reductase activity, CYP2C9 content and inhibition properties. We report heterologous expression of human CYP2C9 along with human cytochrome P450 reductase in protease deficient S. cerevisiae at a 5 litre scale resulting in high yields (8-10 nmols/litre) of enzyme with higher specific activity (2-3 fold higher). This yields a superior enzyme and makes it amenable to miniaturization of screening assays with concomitant lowering of costs.

Vishal M.,CSIR - Central Electrochemical Research Institute | Sharma A.,CSIR - Central Electrochemical Research Institute | Sharma A.,Academy of Scientific and Innovative Research AcSIR | Kaurani L.,CSIR - Central Electrochemical Research Institute | And 17 more authors.
BMC Medical Genomics | Year: 2016

Background: Glaucoma is the largest cause of irreversible blindness affecting more than 60 million people globally. The disease is defined as a gradual loss of peripheral vision due to death of Retinal Ganglion Cells (RGC). The RGC death is largely influenced by the rate of aqueous humor production by ciliary processes and its passage through the trabecular meshwork (TM) in the anterior part of the eye. Primary open angle glaucoma (POAG), the most common subtype, is a genetically complex disease. Multiple genes and many loci have been reported to be involved in POAG but taken together they explain less than 10 % of the patients from a genetic perspective warranting more studies in different world populations. The purpose of this study was to perform genome-wide search for common variants associated with POAG in an east-Indian population. Methods: The study recruited 746 POAG cases and 697 controls distributed into discovery and validation cohorts. In the discovery phase, genome-wide genotype data was generated on Illumina Infinium 660 W-Quad platform and the significant SNPs were genotyped using Illumina GGGT assay in the second phase. Logistic regression was used to test association in the discovery phase to adjust for population sub-structure and chi-square test was used for association analysis in validation phase. Publicly available expression dataset for trabecular meshwork was used to check for expression of the candidate gene under cyclic mechanical stress. Western blot and immunofluorescence experiments were performed in human TM cells and murine eye, respectively to check for expression of the candidate gene. Results: Meta-analysis of discovery and validation phase data revealed the association of rs7916852 in MPP7 gene (p = 5.7x10-7) with POAG. We have shown abundant expression of MPP7 in the HTM cells. Expression analysis shows that upon cyclic mechanical stress MPP7 was significantly down-regulated in HTM (Fold change: 2.6; p = 0.018). MPP7 protein expression was also found to be enriched in the ciliary processes of the murine eye. Conclusion: Using a genome-wide approach we have identified MPP7 as a novel candidate gene for POAG with evidence of its expression in relevant ocular tissues and dysregulation under mechanical stress possibly mimicking the disease scenario. © 2016 Vishal et al.

Kokatam S.,Premas Biotech Pvt. Ltd. | Pandey D.,Premas Biotech Pvt. Ltd. | Gupta R.,Premas Biotech Pvt. Ltd. | Krishna R.,Premas Biotech Pvt. Ltd. | Soni R.,Premas Biotech Pvt. Ltd.
Combinatorial Chemistry and High Throughput Screening | Year: 2011

Angiogenesis is a promising area of research that targets key therapeutic areas like cancer; wound healing, inflammatory diseases, etc. There is an increasing demand for screening of potential angiogenic and anti-angiogenic agents using sensitive, robust cell-based assays. We have developed a reporter vector containing cis-acting elements that respond to growth factors/angiogenic ligands for use in a cell-based luciferase reporter assay. We performed transient transfection of our reporter gene vector in MCF-7 cells to establish its application for screening of potential pro/antiangiogenic agents. Reporter gene transactivation studies with different concentrations of fetal bovine serum clearly indicated that the vector is functionally responsive to the angiogenic signals mediated by serum growth factors. We also used endostatin to inhibit transactivation and prove responsiveness to the anti-angiogenic agent. This vector is a promising tool for studying angiogenesis using cell-based reporter gene assays. © 2011 Bentham Science Publishers Ltd.

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