An assessment of poly (ADP-ribose) polymerase-1 role in normal and cancer cells.

Poly (ADP-ribose) polymerase (PARP) is a superfamily of 18 proteins characterized by the PARP homology domain, the catalytic domain. This catalytic domain helps in the ADP-ribosylation of various acceptor proteins using nicotinamide adenine dinucleotide (NAD+) as a donor for ADP-ribose. PARP-1 and PARP-2 carry out 80% of poly-ADP-ribosylation of cellular protein. Hence, their combined knockout results in embryonic lethality of mice. PARP-1 consists of three major domains, namely, DNA binding domain, automodification domain, and a catalytic domain. These domains further consist of subdomains and motifs, which helps PARP-1 in a diverse function. PARP-1 is mainly involved in DNA damage detection and repair, but emerging evidence suggests its role in many other functions such as DNA synthesis, replication, apoptosis, necrosis, and cancer progression. Herein, we review the current state of the PARP-1 role in DNA damage repair and other biological processes including epithelial to mesenchymal transition (EMT). We have also observed the role of PARP-1 in modulating EMT regulators like E-cadherin, Vimentin, Claudin-1, Snail, Smad-4, Twist-1, and ß-catenin. Here, we have also attempted to relate the role of PARP-1 in EMT of cancer cells.

Emergence of Haitian variant genotype and altered drug susceptibility in Vibrio cholerae O1 El Tor-associated cholera outbreaks in Solapur, India.

It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance.

TGF-ß-mediated regulation of plasminogen activators is human telomerase reverse transcriptase dependent in cancer cells.

Telomerase is a specialized reverse transcriptase/terminal transferase enzyme that adds telomeric repeat sequences at the extreme end of a newly replicated chromosome. Apart from telomere lengthening, telomerase has many extracurricular activities. Telomerase is known to regulate the expression of many genes and helps in cancer progression and epithelial-to-mesenchymal transitions (EMTs). We have previously reported that human telomerase reverse transcriptase (hTERT) regulates the expression of plasminogen activator such as urokinase-type plasminogen activator (uPA) in cancer cells following a genome-wide transcriptomic study. Here, we present data substantiating these results in terms of real-time assays, western blots, and immunofluorescence. Another aim of this study is to find out the possible mechanism by which hTERT regulates the expression of plasminogen activators. We have used some molecular biology techniques such as quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence and some assays such as wound healing assay and colony formation assay to solve this question. In this study, we show a positive association between hTERT and uPA. We also demonstrate that hTERT enhances uPA expression concomitant with EMT. Knocking down of hTERT reduces uPA expression as well as reverses EMT in cancer cells. We have also found that uPA is a transforming growth factor beta (TGF-ß)-induced protein. Our observations establish that TGF-ß-induced uPA expression is hTERT dependent.

hTERT promotes tumor progression by enhancing TSPAN13 expression in osteosarcoma cells.

Telomerase complex maintains the length of the telome, cbre, and protects erosion of the physical ends of the eukaryotic chromosome in all actively dividing cells including cancer cells. Telomerase activation extends the lifespan of cells in culture by maintaining the length of the telomere. Compared to terminally differentiated somatic cells, telomerase activity remains high in over 90% of cancer cells. It has now become clear that the role of telomerase is much more complex than just telomere lengthening. The remaining 10% of cancers deploy ALT (alternative lengthening of telomeres) pathway to maintain telomere length. Telomerase inhibitors offer a good therapeutic option. Also, telomerase-associated molecules can be targeted provided their roles are clearly established. In any case, it is necessary to understand the major role of telomerase in cancer cells. Many studies have already been done to explore gene profiling of a telomerase positive cell by knocking down expression of hTERT (telomerase reverse transcriptase). To complement these studies, we performed global gene profiling of a telomerase negative cell by ectopically expressing hTERT and studied changes in the global gene expression patterns. Analysis of microarray data for telomerase negative cells ectopically expressing telomerase showed 76 differentially regulated genes, out of which 39 genes were upregulated, and 37 were downregulated. Three upregulated genes such as TSPAN13, HMGCS2, DLX5, and three downregulated genes like DHRS2, CRYAB, and PDLIM1 were validated by real-time PCR. Knocking down of TSAPN13 in hTERT overexpressing U2OS cells enhanced the apoptosis of the cells. TSPAN13 knockdown in these cells suppressed mesenchymal properties and enhanced epithelial character.

Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells.

Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.

Molecular epidemiology of Vibrio cholerae associated with flood in Brahamputra River valley, Assam, India.

Cholera is often caused when drinking water is contaminated through environmental sources. In recent years, the drastic cholera epidemics in Odisha (2007) and Haiti (2010) were associated with natural disasters (flood and Earthquake). Almost every year the state of Assam India witnesses flood in Brahamputra River valley during reversal of wind system (monsoon). This is often followed by outbreak of diarrheal diseases including cholera. Beside the incidence of cholera outbreaks, there is lack of experimental evidence for prevalence of the bacterium in aquatic environment and its association with cholera during/after flood in the state. A molecular surveillance during 2012-14 was carried out to study prevalence, strain differentiation, and clonality of Vibrio cholerae in inland aquatic reservoirs flooded by Brahamputra River in Assam. Water samples were collected, filtered, enriched in alkaline peptone water followed by selective culturing on thiosulfate bile salt sucrose agar. Environmental isolates were identified as V. cholerae, based on biochemical assays followed by sero-grouping and detailed molecular characterization. The incidence of the presence of the bacterium in potable water sources was higher after flood. Except one O1 isolate, all of the strains were broadly grouped under non-O1/non-O139 whereas some of them did have cholera toxin (CT). Surprisingly, we have noticed Haitian ctxB in two non-O1/non-O139 strains. MLST analyses based on pyrH, recA and rpoA genes revealed clonality in the environmental strains. The isolates showed varying degree of antimicrobial resistance including tetracycline and ciprofloxacin. The strains harbored the genetic elements SXT constins and integrons responsible for multidrug resistance. Genetic characterization is useful as phenotypic characters alone have proven to be unsatisfactory for strain discrimination. An assurance to safe drinking water, sanitation and monitoring of the aquatic reservoirs is of utmost importance for combating the impending epidemic threat in the flood affected areas. Further, the management of flood through multi-prong approaches and sustainable utilization of environmental resources would be effective in disease management.

Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug "Macugen" is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions.

Global expression profile of telomerase-associated genes in HeLa cells.

Telomerase is a specialized nucleoprotein enzyme complex that maintains the telomere length. The telomerase reverse transcriptase (TERT) is the catalytically active component of the telomerase complex. In humans, the protein component (hTERT) and RNA component (hTR) are found to differentially express in cancer cells. In contrast to differentiated cells, most of the cancer cells overexpress hTERT, which is needed to maintain the proliferative potential of cells. The overexpression of telomerase is not proportionate to telomere length in cancer cells, suggesting that the immortalizing phenotype can be mediated through other factors in addition to telomere length. To investigate the role of hTERT in immortalizing process, loss of gene function studies were carried out. Short interfering RNA (siRNA) and short hairpin RNA (shRNA) against hTERT showed the reduction of hTERT transcript, reduction of telomerase activity and alteration of gene expression in HeLa cells. The molecular basis of proliferative capacity of hTERT was investigated by gene expression microarray. Analysis of microarray data for HeLa cells following siRNA and shRNA mediated knockdown of hTERT showed that 80 genes were upregulated and 73 genes downregulated. Out of these, 37 genes are known to be involved in cancer. Further analyses of previously known genes involved in cancer like KLF4, FGF2, IRF-9 and PLAU by Real Time PCR showed their upregulation. We are documenting for the first time the effect of knocking down hTERT on expression of KLF4 and FGF2. Interestingly, it has been earlier reported that KLF4 and FGF2 up-regulate the expression of hTERT in cancer cells. This suggests that hTERT may be subject to its own auto-regulatory effects.

Expression of targeted ribozyme against telomerase RNA causes altered expression of several other genes in tumor cells.

Telomeres are tandem repeat sequences present at chromosome end that are synthesized by RNA-protein enzyme called telomerase. The RNA component (TR) serves as template for telomerase reverse transcriptase (TERT) for generating telomere repeats. TERT is overexpressed in actively dividing cells including cancerous cells, absent in differentiated somatic cells whereas human telomerase RNA (hTR) is present in normal as well as in cancer cells. Telomerase overexpression in cancer cells ensures telomere length maintenance that actually provides proliferative advantage to cells. Stable expression of ribozyme against hTR in HeLa cells results in reduction of hTR levels, telomerase activity, and telomere length which is accompanied by altered cell morphology and expression of several specific cellular genes. The altered genes deduced from differentially display PCR and 2D gel electrophoresis upon hTR knockdown have function in ribosome biogenesis, chromatin modulation, cell cycle control, and p63-dependant pathways. Our observations shows hTR participates in diverse cellular functions other than telomere maintenance, validates as a possible drug targets in p53- and pRB-negative status, and indicated possible cross-talks between telomerase and other cellular pathways.

Restoration of ultrastructural and biochemical changes in alloxan-induced diabetic rat sciatic nerve on treatment with Na3VO4 and Trigonella--a promising antidiabetic agent.

Vanadium has been reported to have broad pharmacological activity both in vitro and in vivo. Vanadium compound, sodium orthovanadate, Na3VO4, is well known for its hypoglycaemic effects. However, Na3VO4 exerts these effects at relatively high doses (0.6 mg/ml) and exhibit several toxic effects. In the present study lower doses of Na3VO4 (0.2 mg/ml) are combined with Trigonella foenum graecum seed powder (TSP), another hypoglycaemic agent, to reduce its toxicity without compromising its antidiabetic potential. The efficacy of the lower doses of Na3VO4 has been investigated in restoring the altered glucose metabolism and histological structure in the sciatic nerves in 21 and 60 days alloxan diabetic rats. A portion of the glucose was found to be channelled from the normal glycolytic route to polyol pathway, evident by the reduced hexokinase activity and increased polyol pathway enzymes aldose reductase and sorbitol dehydrogenase activity causing accumulation of sorbitol and fructose in diabetic conditions. Ultrastructural observation of the sciatic nerve showed extensive demylination and axonal loss after eight weeks of diabetes induction. Blood glucose levels increased in diabetic rats were normalized with the lower dose of vanadium and Trigonella treatment. The treatment of the diabetic rats with vanadium and Trigonella prevented the activation of the polyol pathway and sugar accumulations. The sciatic nerves were also protected against the structural abnormalities found in diabetes with Trigonella foenum graecum as well as Na3VO4. Results suggest that lower doses of Na3VO4 may be used in combination with TSP as an efficient antidiabetic agent to effectively control the long-term complications of diabetes in tissues like peripheral nerve.

Efficacy of lower doses of vanadium in restoring altered glucose metabolism and antioxidant status in diabetic rat lenses.

Vanadium compounds are potent in controlling elevated blood glucose levels in experimentally induced diabetes. However the toxicity associated with vanadium limits its role as therapeutic agent for diabetic treatment. A vanadium compound sodium orthovanadate (SOV) was given to alloxan-induced diabetic Wistar rats in lower doses in combination with Trigonella foenum graecum, a well-known hypoglycemic agent used in traditional Indian medicines. The effect of this combination was studied on lens morphology and glucose metabolism in diabetic rats. Lens, an insulin-independent tissue, was found severely affected in diabetes showing visual signs of cataract. Alterations in the activities of glucose metabolizing enzymes (hexokinase, aldose reductase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase) and antioxidant enzymes (glutathione peroxidase, glutathione reductase) besides the levels of related metabolites, [sorbitol, fructose, glucose, thiobarbituric acid reactive species (TBARS) and reduced glutathione (GSH)] were observed in the lenses from diabetic rats and diabetic rats treated with insulin (2 IU/day), SOV (0.6 mg/ml), T. f. graecum seed powder (TSP, 5%) and TSP (5%) in combination with lowered dose of vanadium SOV (0.2 mg/ml), for a period of 3 weeks. The activity of the enzymes, hexokinase, aldose reductase and sorbitol dehydrogenase was significantly increased whereas the activity of glucose-6-phosphate dehydrogenase, glutathione peroxidase and glutathione reductase decreased significantly in lenses from 3 week diabetic rats. Significant increase in accumulation of metabolites, sorbitol, fructose, glucose was found in diabetic lenses. TBARS measure of peroxidation increased whereas the levels of antioxidant GSH decreased significantly in diabetic condition. Insulin restored the levels of altered enzyme activities and metabolites almost to control levels. Sodium orthovanadate (0.6 mg/ml) and Trigonella administered separately to diabetic animals could partially reverse the diabetic changes, metabolic and morphological, while vanadate in lowered dose in combination with Trigonella was found to be the most effective in restoring the altered lens metabolism and morphological appearance in diabetes. It may be concluded that vanadate at lowered doses administered in combination with Trigonella was the most effective in controlling the altered glucose metabolism and antioxidant status in diabetic lenses, these being significant factors involved in the development of diabetic complications, that reflects in the reduced lens opacity.