p-Toluenesulfonyl Chloride Catalysed Facile Synthesis of O-benzyl-l-amino Acids and Their In Vitro Evaluation

International Journal of Peptide Research and Therapeutics - Protection and subsequent deprotection of amino acid functional groups play a key role in regioselective peptide synthesis. For...     

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.     

Distribution of HCV genotypes and HCV RNA viral load in hepatitis infected patients of Kolar region,Karnataka, India

Decisions and disease progression prediction, understanding the distribution of the hepatitis C virus (HCV) genotype and its association with viral load is significant for treatment. Therefore, it is of interest to document the distribution pattern of HCV genotypes and their association with viral load among HCV infected patients in Kolar, Karnataka. Seventy-four HCV-positive patients and not on antiviral therapy were enrolled from R.L. Jalappa hospital in Kolar, Karnataka. Blood samples were taken and demographics were recorded. HCV RNA was isolated after plasma was separated. qPCR was performed to measure the viral load, and RT-PCR was performed to determine the genotype. Genotype 3 was the prevalent (n=11, 40.7%) followed by genotype 4 (n=8, 29.6%), 2 (n=6, 22.2%), 1 (n= 13.7%), and mix (n=1, 13.7%). The median viral load of genotype 3 was a 2,87,835 IU/mL (IQR 10, 780-3, 71, 66) , genotype 2 was 81,030 IU/mL (IQR 66,495-95,565), genotype 4 was 43, 410 IU/mL (IQR 38, 355-48, 465) belongs to viral load less than 8,00,000 IU/mL. The median viral load genotype 3 was a 1, 05, 19, 500 IU/mL (IQR 49, 37, 250-2, 36, 71, 500), genotype 2 was 2,55,99,000 IU/mL (IQR 2,00,10,000-32,725,500), genotype 4 was 1,67,40,000 IU/mL (IQR 1,45,50,000-17,493,000) belonging to viral load more than 8,00,000 IU/mL category. A correlation between genotype and viral load was observed (p =1.5x10-12), of which genotype 3 showed a high viral load. Thus, HCV genotypes 1 2, 3, 4, and mixed genotype was observed in the patients studied. HCV genotype was associated with viral load in patient plasma. This data finds use in the treatment and prevention of hepatitis C in Kolar, Karnataka.     

IFN-alpha beta secreted during infection is necessary but not sufficient for negative feedback regulation of IFN-alpha beta signaling by Mycobacterium tuberculosis

IFN-alphabeta functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-alphabeta secretion and down-regulation of IFN-alphabeta signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-alphabeta prevents inhibition of IFN-alpha signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-alphabeta. First, greater inhibition of IFN-alpha-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-alphabeta pretreatment. Second, LPS also induces IFN-alphabeta secretion and causes IFN-alphabeta-dependent down-regulation of IFN-alpha signaling, yet the inhibition differs from that caused by infection. Third, IFN-alpha signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-alphabeta is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-alphabeta receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-alpha stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-alpha signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.     

Primate-specific evolution of an LDLR enhancer

Background  

Sequence changes in regulatory regions have often been invoked to explain phenotypic divergence among species, but molecular examples of this have been difficult to obtain.     

Simultaneous analysis of multiple data types in pharmacogenomic studies using weighted sparse canonical correlation analysis

Variation in drug response results from a combination of factors that include differences in gender, ethnicity, and environment, as well as genetic variation that may result in differences in mRNA and protein expression. This article presents two integrative analytic approaches that make use of both genome-wide SNP and mRNA expression data available on the same set of subjects: a step-wise integrative approach and a comprehensive analysis using sparse canonical correlation analysis (SCCA). In addition to applying standard SCCA, we present a novel modification of SCCA which allows different weighting for the various pair-wise relationships in the SCCA. These integrative approaches are illustrated with both simulated data and data from a pharmacogenomic study of the drug gemcitabine. Results from these analyses found little overlap in terms of genes detected, possibly detecting different biological mechanisms. In addition, we found the proposed weighted SCCA to outperform its unweighted counterpart in detecting associations between the genomic features and phenotype. Further research is needed to develop and assess new integrative methods for pharmacogenomic studies, as these types of analyses may uncover novel insights into the relationship between genomic variation and drug response.     

Automatic segmentation and supervised learning-based selection of nuclei in cancer tissue images

Analysis of preferential localization of certain genes within the cell nuclei is emerging as a new technique for the diagnosis of breast cancer. Quantitation requires accurate segmentation of 100-200 cell nuclei in each tissue section to draw a statistically significant result. Thus, for large-scale analysis, manual processing is too time consuming and subjective. Fortuitously, acquired images generally contain many more nuclei than are needed for analysis. Therefore, we developed an integrated workflow that selects, following automatic segmentation, a subpopulation of accurately delineated nuclei for positioning of fluorescence in situ hybridization-labeled genes of interest. Segmentation was performed by a multistage watershed-based algorithm and screening by an artificial neural network-based pattern recognition engine. The performance of the workflow was quantified in terms of the fraction of automatically selected nuclei that were visually confirmed as well segmented and by the boundary accuracy of the well-segmented nuclei relative to a 2D dynamic programming-based reference segmentation method. Application of the method was demonstrated for discriminating normal and cancerous breast tissue sections based on the differential positioning of the HES5 gene. Automatic results agreed with manual analysis in 11 out of 14 cancers, all four normal cases, and all five noncancerous breast disease cases, thus showing the accuracy and robustness of the proposed approach. ? Published 2012 Wiley Periodicals, Inc.     

Induction of experimental autoimmune Graves' disease in BALB/c mice.

We immunized BALB/c mice with M12 cells (H-2d) expressing either mouse (mM12 cells) or human thyrotropin receptor (TSHR) (hM12 cells). Immunized mice developed autoantibodies to native TSHR by day 90 and, by day 180, showed considerable stimulatory Ab activity as measured by their ability to enhance cAMP production (ranging from 6. 52 to 20.83 pmol/ml in different treatment groups relative to 1.83 pmol/ml for controls) by TSHR-expressing Chinese hamster ovary cells. These mice developed severe hyperthyroidism with significant elevations in both tetraiodothyronine and triiodothyronine hormones. Tetraiodothyronine levels in different experimental groups ranged from a mean of 8.66-12.4 microg/dl, relative to 4.8 microg/dl in controls. Similarly, mean triiodothyronine values ranged from 156.18 to 195.13 ng/dl, relative to 34.99 ng/dl for controls. Next, we immunized BALB/c mice with a soluble extracellular domain of human TSHR (TBP), or TBP expressed on human embryonic kidney cells (293 cells) (293-TBP cells). These mice showed severe hyperthyroidism in a manner very similar to that described above for mice immunized with the mouse TSHR or human TSHR, and exhibited significant weight loss, with average weight for treatment groups ranging from 20.6 to 21.67 g, while controls weighed 24.2 g. Early after onset of the disease, histopathological examination of thyroids showed enlargement of colloids and thinning of epithelial cells without inflammation. However, later during disease, focal necrosis and lymphocytic infiltration were apparent. Our results showed that conformationally intact ectodomain of TSHR is sufficient for disease induction. Availability of a reproducible model in which 100% of the animals develop disease should facilitate studies aimed at understanding the molecular pathogenesis of Graves' disease.