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Hinton A.,University of California at San Diego | Afrikanova I.,University of California at San Diego | Wilson M.,Asuragen, Inc. | King C.C.,University of California at San Diego | And 4 more authors.
Stem Cells and Development

Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs. © Copyright 2010, Mary Ann Liebert, Inc. Source

Kolb P.,University of California at San Francisco | Kolb P.,University of Marburg | Phan K.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | Gao Z.-G.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | And 4 more authors.

G protein-coupled receptors (GPCRs) are attractive targets for pharmaceutical research. With the recent determination of several GPCR X-ray structures, the applicability of structure-based computational methods for ligand identification, such as docking, has increased. Yet, as only about 1% of GPCRs have a known structure, receptor homology modeling remains necessary. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A1 adenosine receptor (A1AR) and docked ~2.2 M lead-like compounds. High-ranking molecules were tested on the A1AR as well as the close homologs A2AAR and A3AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. These findings have implications for future screens. Source

Cannon B.,University of Texas at Austin | Pan C.,University of Texas at Austin | Chen L.,Asuragen, Inc. | Hadd A.G.,Asuragen, Inc. | Russell R.,University of Texas at Austin
Molecular Biotechnology

Fragile X syndrome is the leading cause of inherited mental impairment and is associated with expansions of CGG repeats within the FMR1 gene. To detect expanded CGG repeats, we developed a dual-mode single-molecule fluorescence assay that allows acquisition of two parallel, independent measures of repeat number based on (1) the number of Cy3-labeled probes bound to the repeat region and (2) the physical length of the electric field-linearized repeat region, obtained from the relative position of a single Cy5 dye near the end of the repeat region. Using target strands derived from cell-line DNA with defined numbers of CGG repeats, we show that this assay can rapidly and simultaneously measure the repeats of a collection of individual sample strands within a single field of view. With a low occurrence of false positives, the assay differentiated normal CGG repeat lengths (CGGN, N = 23) and expanded CGG repeat lengths (CGGN, N = 118), representing a premutation disease state. Further, mixtures of these DNAs gave results that correlated with their relative populations. This strategy may be useful for identifying heterozygosity or for screening collections of individuals, and it is readily adaptable for screening other repeat disorders. © 2012 Springer Science+Business Media, LLC. Source

Sah S.,Asuragen, Inc. | Eveleigh D.,Asuragen, Inc. | Wilson M.,Asuragen, Inc.
BMC Research Notes

Background. microRNAs (miRNA) are short, endogenous transcripts that negatively regulate the expression of specific mRNA targets. The relative abundance of miRNAs is linked to function in vivo and miRNA expression patterns are potentially useful signatures for the development of diagnostic, prognostic and therapeutic biomarkers. Finding. We compared the performance characteristics of four commercial miRNA array technologies and found that all platforms performed well in separate measures of performance. Conclusions. The Ambion and Agilent platforms were more accurate, whereas the Illumina and Exiqon platforms were more specific. Furthermore, the data analysis approach had a large impact on the performance, predominantly by improving precision. © 2010 Irizarry et al; licensee BioMed Central Ltd. Source

Smith D.L.,Asuragen, Inc. | Lamy A.,University of Rouen | Beaudenon-Huibregtse S.,Asuragen, Inc. | Sesboue R.,University of Rouen | And 3 more authors.
Archives of Pathology and Laboratory Medicine

Context. - Current clinicopathologic assessment of malignant neoplastic diseases entails the analysis of specific genetic alterations that provide diagnostic, prognostic, or therapy-determining information. Objective. - To develop and validate a robust molecular method to detect clinically relevant mutations in various tissue types and anatomic pathology specimens. Design. - Genes of interest were amplified by multiplex polymerase chain reaction and sequence variants identified by liquid bead array cytometry. The BRAF assay was fully characterized by using plasmids and genomic DNA extracted from cell lines, metastatic colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissues, and thyroid nodule fine-needle aspirates. Results. - Qualitative multiplex assays for 22 different mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were established. The high signal-to-noise ratio of the technology enabled reproducible detection of BRAF c.1799T>A (p.V600E) at 0.5% mutant allele in 20 ng of genomic DNA. Precision studies with multiple operators and instruments showed very high repeatability and reproducibility with 100% (98.7%-100%) qualitative agreement among 292 individual measures in 38 runs. Evaluation of 1549 representative pathologic specimens in 2 laboratories relative to independent reference methods resulted in 99.0% (97.6%-99.6%) agreement for colorectal FFPE tissues (n = 416) and 98.9% (98.2%-99.4%) for thyroid fine-needle aspiration specimens (n = 1133) with an overall diagnostic odds ratio of 10 856 (2451-48 078). Conclusions. - The multiplex assay system is a sensitive and reliable method to detect BRAF c.1799T>A mutation in colorectal and thyroid lesions. This optimized technology platform is suitable for the rapid analysis of clinically actionable genetic alterations in cytologic and histologic specimens. Source

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