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Breast cancer mortality in women is largely attributed to the metastasis of primary breast tumors. We have analysed the function of EphB6, a kinase-deficient receptor, in the invasive phenotype of breast cancer cell lines. We have demonstrated the loss of EphB6 protein in invasive breast carcinoma cell lines and absence of EphB6 transcript in a metastatic breast tumor specimen. The function of EphB6 in invasiveness was confirmed by the ability of EphB6 protein to decrease the in vitro invasiveness of MDA-MB-231, MDA-MB-435 and BT549 cells transfected with an EphB6 expression construct. In MDA-MB-231 cells, the decreased invasiveness appeared to be mediated by decreased transcript levels of matrix metalloproteinase (MMP)7 and MMP19, and increased transcript levels of tissue inhibitors of metalloproteinase 2. In addition to affecting invasiveness phenotype, EphB6 overexpression was also responsible for altering the growth rate and colony-forming efficiency of MCF-7 and MDA-MB-231 cells in a cell-line-specific manner. We suggest that the significant decrease in the invasiveness of MDA-MB-231 and other cell lines transfected with EphB6 is likely occurring by the ability of EphB6 to transduce signals to the nucleus and altering relevant gene expression.

Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.

We have characterized comprehensive transcript and proteomic profiles of cell lines corresponding to normal breast (MCF10A), noninvasive breast cancer (MCF7) and invasive breast cancer (MDA-MB-231). The transcript profiles were first analysed by a modified protocol for representational difference analysis (RDA) of cDNAs between MCF7 and MDA-MB-231 cells. The majority of genes identified by RDA showed nearly complete concordance with microarray results, and also led to the identification of some differentially expressed genes such as lysyl oxidase, copper transporter ATP7A, EphB6, RUNX2 and a variant of RUNX2. The altered transcripts identified by microarray analysis were involved in cell–cell or cell–matrix interaction, Rho signaling, calcium homeostasis and copper-binding/sensitive activities. A set of nine genes that included GPCR11, cadherin 11, annexin A1, vimentin, lactate dehydrogenase B (upregulated in MDA-MB-231) and GREB1, S100A8, amyloid β precursor protein, claudin 3 and cadherin 1 (downregulated in MDA-MB-231) were sufficient to distinguish MDA-MB-231 from MCF7 cells. The downregulation of a set of transcripts for proteins involved in cell–cell interaction indicated these transcripts as potential markers for invasiveness that can be detected by methylation-specific PCR. The proteomic profiles indicated altered abundance of fewer proteins as compared to transcript profiles. Antisense knockdown of selected transcripts led to inhibition of cell proliferation that was accompanied by altered proteomic profiles. The proteomic profiles of antisense transfectants suggest the involvement of peptidyl-prolyl isomerase, Raf kinase inhibitor and 80 kDa protein kinase C substrate in mediating the inhibition of cell proliferation.

Diabetic retinopathy (DR) is a serious complication of diabetes, which is fast reaching epidemic proportions worldwide. While tight glycemic control remains the standard of care for preventing the progression of DR, better insights into DR etiology require understanding its genetic basis, which in turn may assist in the design of novel treatments. During the last decade, genomic medicine is increasingly being applied to common multifactorial diseases such as diabetes and age-related macular degeneration. The contribution of genetics to the initiation and progression of DR has been recognized for some time, but the involvement of specific genes and genetic variants remains elusive. Several investigations are currently underway for identifying DR susceptibility loci through linkage studies, candidate gene approaches, and genome-wide association studies. Advent of next generation sequencing and high throughput genomic technologies, development of novel bioinformatics tools and collaborations among research teams should facilitate such investigations. Here, we review the current state of genetic studies in DR and discuss reported findings in the context of biochemical, cell biological and therapeutic advances. We propose the development of a consortium in India for genetic studies with large cohorts of patients and controls from limited geographical areas to stratify the impact of the environment. Uniform guidelines should be established for clinical phenotyping and data collection. These studies would permit identification of genetic loci for DR susceptibility in the Indian population and should be valuable for better diagnosis and prognosis, and for clinical management of this blinding disease.

Eph receptors constitute the largest family of receptor tyrosine kinases, which are activated by ephrin ligands that either are anchored to the membrane or contain a transmembrane domain. These molecules play important roles in the development of multicellular organisms, and the physiological functions of these receptor-ligand pairs have been extensively documented in axon guidance, neuronal development, vascular patterning, and inflammation during tissue injury. The recognition that aberrant regulation and expression of these molecules lead to alterations in proliferative, migratory, and invasive potential of a variety of human cancers has made them potential targets for cancer therapeutics. We present here the involvement of Eph receptors and ephrin ligands in lung carcinoma, breast carcinoma, prostate carcinoma, colorectal carcinoma, glioblastoma, and medulloblastoma. The aberrations in their abundances are described in the context of multiple signaling pathways, and differential expression is suggested as the mechanism underlying tumorigenesis.

We describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric,and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo 1-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotide. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)n microsatellites with this approach contained clones with inserts containing CA repeats. We have also used this protocol for enrichment of (CAG)n and (AGAT)n sequence repeats and for Not I jumping clones. We have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.

The ionotropic glutamate receptor 6 (GluR6 or GRIK2) gene is transcribed by two cell-type-specific promoters in neuronal and non-neuronal cells, which results in five different transcript variants. The purpose of this study was to explore cell-type-specific silencing of these promoters by epigenetic mechanisms. The neuronal and non-neuronal promoter sequences were cloned upstream of the luciferase gene in the pGL3 luciferase reporter vector. Promoter susceptibility to methylation was confirmed by 5-azacytidine and trichostatin treatment, and the status of CpG dinucleotides was determined by bisulfite sequencing of the promoter was determined by bisulfite sequences. GluR6A transcript variant was expressed in the brain, and GluR6B was most abundant in tumor cell lines. The neuronal promoter was methylated in non-neuronal cell lines. The treatment with 5-azacytidine and trichostatin upregulated transcription of the GluR6 gene, and methylation of the GluR6 promoter sequence in the luciferase reporter system led to downregulation of the luciferase gene transcription. Bisulfite sequencing revealed methylation of 3 and 41 CpG sites in non-neuronal and neuronal promoters, respectively. The differential activation/silencing of GluR6 promoters suggests that the transcript variants of GluR6 are involved in tissue-specific biological processes and their aberrant regulation in tumor cells may contribute to distinct properties of tumor cells.

National physical laboratory India (NPLI) has five commercial cesium atomic clocks. Till recently one of these clocks had been used to maintain coordinated universal time (UTC) of NPLI. To utilize all these clocks in an ensemble manner to generate a smoother time scale, it has been essential to inter-compare them very precisely. This has been achieved with an automatic measurement system with well-conceived software. Though few laboratories have developed such automatic measurement system by themselves based on the respective requirements; but they are not reported. So keeping in mind of the specific requirement of the time scale generation, a new system has been developed by NPLI. The design has taken into account of the associated infrastructure that exists and would be used. The performance of the new system has also been studied. It has been found to be quite satisfactory to serve the purpose. The system is being utilized for the generation of time scale of NPLI.

A strong demand of a separate time zone by northeast populace has been a matter of great debate for a very long period. However, no implementable solution to this genuine problem has yet been proposed. The CSIR-National Physical Laboratory, CSIR-NPL (the National Measurement Institute, NMI, of India and custodian of Indian Standard Time, IST) proposes an implementable solution that puts the country in two time zones: (i) IST-I (UTC + 5 : 30 h, represented by longitude passing through 82°33′E) covering the regions falling between longitude 68°7′E and 89°52′E and (ii) IST-II (UTC + 6 : 30 h, represented by longitude passing through 97°30′E) encompassing the regions between 89°52′E and 97°25′E. The proposed demarcation line between IST-I and IST-II, falling at longitude 89°52′E, is derived from analyses of synchronizing the circadian clocks to normal office hours (9 : 00 a.m. to 5 : 30 p.m.). This demarcation line passes through the border of West Bengal and Assam and has a narrow spatial extension, which makes it easier to implement from the railways point of view. Once approved, the implementation would require establishment of a laboratory for ‘Primary Time Ensemble – II’ generating IST-II in any of the north-eastern states, which would be equivalent to the existing ‘Primary Time Ensemble-I’ at CSIR-NPL, New Delhi.

We have previously shown by chromosome transfer technique that chromosome 6 alters the phenotype of a variety of tumour cells and SV40 immortalized cells. We present here the phenotypic effects of the ectopic expression of RNaseT2, a highly conserved ribonuclease encoded by chromosome 6q27, in SV40 immortalized cell lines. We contrast our findings with those reported for ovarian carcinoma cell lines and an SV40 immortalized cell line transfected with RNaseT2. Although RNaseT2 expression is elevated in normal diploid fibroblasts approaching senescence (passage 64), forced expression of the gene in immortalized cells does not cause them to senesce. A significant reduction was observed in colony forming efficiency, anchorage independence and growth rate of cells transfected with RNaseT2. The levels of transcripts involved in Akt signalling pathway, cell cycle control and pathways related to cell proliferation decreased 2–10‐folds in SV40 immortalized cells in response to RNaseT2 expression. Interestingly, some immortalized cells expressed alternatively spliced transcript variants instead of the full‐length RNaseT2 transcript. Our results are consistent with the conclusion that RNaseT2 is a cell growth regulator and it does not induce senescence in SV40 immortalized cell lines.