Identification of polymorphic microsatellite loci in the queenless, ponerine ant Diacamma ceylonense

Diacamma ceylonenseis a queenless, ponerine ant whose colonies are headed by a single, mated, egg-laying worker referred to as the gamergate. Thus, new colonies are a result of dispersal by wingless gamergates. This is expected to influence patterns of colony dispersal and spatial distribution of genetic variablity. In order to facilitate the study of population genetic structure we have identified six unique, polymorphic, microsatellite loci. We have used fluorescence tagged primers to detect polymorphism at these loci

A STUDY OF METHOD DEVELOPMENT, VALIDATION AND FORCED DEGRADATION FOR SIMULTANEOUS QUANTIFICATION OF CABOZANTINIB AND NIVOLUMAB IN BULK AND PHARMACEUTICAL DOSAGE FORM BY RP-HPLC

Objective: The present paper describes a simple, accurate, and precise reversed-phase high-performance liquid chromatography (HPLC) method for rapid and simultaneous quantification of cabozantinib (CZT) and nivolumab (NVM) in bulk and pharmaceutical dosage form. Methods: The chromatographic separation was achieved on Luna C18 (150 mm×4.6 mm, 3.5 μm). Mobile phase contained a mixture of 0.1% orthophosphoric acid and acetonitrile in the ratio of 50:50 v/v, flow rate 1.0 ml/min, and ultraviolet detection at 222 nm. Results: The proposed method shows a good linearity in the concentration range of 20–300 μg/ml for CZT and 5–75 μg/ml for NVM under optimized conditions. Precision and recovery study results are in between 98 and 102%. In the entire robustness conditions, percentage relative standard deviation is <2.0%. Degradation has minimum effect in stress condition and solutions are stable up to 24 h. Conclusion: This method is validated for different parameters such as precision, linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), ruggedness, robustness, and forced degradation study were determined according to the International Conference of Harmonization (ICH) Q2B guidelines. All the parameters of validation were found to be within the acceptance range of ICH guidelines. Since there is no HPLC method reported in the literature for the estimation of CZT and NVM in pharmaceutical dosage forms, there is a need to develop quantitative methods under different conditions to achieve improvement in sensitivity, selectivity, etc. The author declares the interest to develop a validation and forced degradation for simultaneous quantification of CZT and NVM

Relationship between morphological and amplified fragment length polymorphism (AFLP) marker based genetic distance with heterosis in hot pepper (Capsicum annuum L.)

Identification of potential parents that produce the hybrids with superior yield is the most important step in developing hybrids to save the substantial resources. The present study was carried out to assess the morphological and amplified fragment length polymorphism (AFLP) marker based genetic diversity, to estimate mid parent heterosis and to correlate the estimated parental genetic diversity with heterosis chilli. Five CMS B - lines and 30 testers were used for morphological and AFLP marker genetic divergence analysis. 150 hybrids were synthesized through Line × Tester (5 × 30) mating design and were used to estimate the mid-parent heterosis for nine characters at two locations. 35 parents were examined for nine morphological traits and were grouped in to six clusters. These parents were also examined for eight AFLP primers combinations and were grouped into seven clusters. More than 50% of hybrids showed significant mid-parent heterosis for both green and red fruit yield plant-1. Hence, there is a much potential for development of good yielding hybrids. The positive significant correlation was found between morphological and AFLP marker distance of the parents with heterosis for plant height (r = 0.17 and 0.38), green fruit yield plant-1 (r = 0.19 and 0.25) and red fruit yield plant-1 (r = 0.20 and 0.34); however, the correlation coefficients were not strong in these traits. Genetic distance between parents was not strong enough to predict the performance of the hybrids and proved to be of no predictive value.Keywords: Correlation, molecular markers, genetic diversity, chill

Assessing unrealized yield potential of maize producing districts in India

The projected demand of maize production in India in 2050 is 4–5 times of current production. With the scope for area expansion being limited, there is need for enhancement of yield. This calls for identifying areas where huge unrealized yield potential exists. With a view to address the issue, the present study delineates homogeneous agro-climatic zones for maize production system in India taking district as a unit and using the factors production, viz. climate, soil, season and irrigated area under the crop. There are 146 districts in India that grow maize as a major crop. They were divided into 26 zones using multivariate cluster analysis. Study of variation in yield between districts within a zone vis-à-vis crop management practices adopted in those districts was found useful in targeting the yield gaps. These findings can have direct relevance to the maize farmers and district level administrators

Potential and Challenges of Rainfed Farming in India

India ranks first in rainfed agriculture globally in both area (86 Mha) and the value of produce. Rainfed regions in India contribute substantially toward food grain production including 44% of rice, 87% of coarse cereals (sorghum (Sorghum bicolor), pearl millet (Pennisetum glaucum), maize (Zea mays)), and 85% of food legumes, 72% of oilseeds, 65% of cotton, and 90% of minor millets. Overall, the rainfed areas produce 40% of the food grains, support two-thirds of the livestock population, and are critical to food security, equity, and sustainability..

Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point

Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Despite extensive research, the five-year survival rate of oral squamous cell carcinoma (OSCC) patients has not improved. Effective treatment of OSCC requires the identification of molecular targets and signaling pathways to design appropriate therapeutic strategies. Several genes from the mTOR signaling pathway are known to be dysregulated in a wide spectrum of cancers. However, not much is known about the involvement of this pathway in tumorigenesis of OSCC. We therefore investigated the role of the tumor suppressor genes, <it>TSC1 </it>and <it>TSC2</it>, and other members of this pathway in tumorigenesis of OSCC.</p> <p>Methods</p> <p>Expression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively. Loss of heterozygosity was studied using matched blood and tumor DNA samples and microsatellite markers from the <it>TSC1</it>, <it>TSC2 </it>and <it>PTEN </it>candidate regions. The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine. Methylation status of the <it>TSC2 </it>promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).</p> <p>Results</p> <p>The semi-quantitative RT-PCR analysis showed downregulation of <it>TSC1</it>, <it>TSC2</it>, <it>EIF4EBP1 </it>and <it>PTEN</it>, and upregulation of <it>PIK3C2A</it>, <it>AKT1</it>, <it>PDPK1</it>, <it>RHEB</it>, <it>FRAP1</it>, <it>RPS6KB1</it>, <it>EIF4E </it>and <it>RPS6 </it>in tumors. A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level. Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively. No mutation was found in TSC genes. A low LOH rate of 13% was observed at the PTEN locus. Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in the expression of TSC genes, suggesting methylation of their promoters. However, the 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in the expression of the <it>TSC2 </it>gene only. In order to confirm the results in patient tumor samples, the methylation status of the <it>TSC2 </it>gene promoter was examined by COBRA. The results suggested promoter hypermethylation as an important mechanism for its downregulation. No correlation was found between the presence or absence of LOH at the TSC1 and TSC2 loci in 50 primary tumors to their clinicopathological variables such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.</p> <p>Conclusion</p> <p>Our study suggests the involvement of TSC genes and other members of the mTOR signaling pathway in the pathogenesis of OSCC. LOH and promoter methylation are two important mechanisms for downregulation of TSC genes. We suggest that known inhibitors of this pathway could be evaluated for the treatment of OSCC.</p

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.Cathepsin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27(Kip1) and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by alphaVbeta3/PI3K/AKT/FOXO pathway as observed by the decreased alphaVbeta3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27(Kip1) and FOXO3a when treated with Ly294002 (10 microM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27(Kip1) was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27(Kip1) accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells