Using the Integrated Genome Viewer to reveal amplicon-derived polymorphism enriched at the phenylthiocarbamide locus in the teaching lab.

Due to its distinct phenotype and relatively simple inheritance pattern, the phenylthiocarbamide (PTC) loci is frequently utilized in teaching laboratories to demonstrate genetic concepts such as Mendelian inheritance and population genetics. We have developed a next-generation sequencing and bioinformatics approach to analyze the PTC gene locus to reveal single nucleotide polymorphism (SNP) variation at nucleotide position 785 that predicts tasting ability in humans. Here students purify DNA from their own cheek cells, perform polymerase chain reaction (PCR) amplification of the PTC gene followed by cleaved amplified polymorphic sequence (CAPS) testing. Students perform a second PCR on the PTC loci using high-fidelity Taq to create bar-coded amplicons for next-generation sequencing on the Ion Torrent Personal Genome Machine. Bioinformatic verification reveals polymorphic variation by aligning the entire class PTC PCR fragment sequence to the human gene using Bowtie2 and visualizing the results in the Integrated Genome Viewer. This exercise presents a learning opportunity for students to use next-generation sequencing to predict their own PTC taste sensitivity phenotype coupled with the standard CAPS method. This approach brings the PTC teaching method into the genomics era.

Umbilical cord blood-selected CD133(+) cells exhibit vasculogenic functionality in vitro and in vivo.

BACKGROUND AIMS: Current clinical trials utilize non-selected bone marrow (BM) mononuclear cells (MNC) to augment vasculo genesis within ischemic vascular beds. Recent reports have identified a diminished number and function of hemat-opoietic stem cells (HSC) from aged and diseased patients. Umbilical cord blood (UCB) provides a potential robust allo-geneic source of HSC for therapeutic vasculogenesis. METHODS: MNC and magnetically isolated CD133(+) cells were assessed for viability (trypan blue) and surface phenotype (flow cytometry). To test in vivo functionality of the cells, NOD/SCID mice underwent ligation of the right femoral artery followed immediately by cell injection. Blood flow recovery, necrosis, BM engraftment of human cells and histologic capillary density were determined. Cells were tested for potential mechanisms mediating the in vivo effects, including migration, cytokine secretion and angiogenic augmentation (Matrigel assays). RESULTS: Surface expression analysis showed CD31 (PECAM) expression was greatly increased in UCB CD133(+) cells compared with BM MNC. At 28 days, perfusion ratios were highest in animals receiving UCB CD133(+) cells, while animals receiving BM CD133(+) cells and BM MNC demonstrated perfusion ratios statistically higher than in animals treated with cytokine media alone. Animals receiving CD133(+) cells showed a statistically higher capillary density, reduced severe digit necrosis and increased engraftment in the BM than animals treated with unselected BM MNC. In vitro studies showed equivalent migration to stromal-derived factor-1 (SDF-1), increased production of tumor necrosis factor alpha (TNF-alpha) and increased branch points with the co-incubation of CD133(+) cells with human umbilical vein endothelial cells (HUVEC) in the Matrigel angiogenesis assay. CONCLUSIONS: Taken together, UCB CD133(+) cells exhibit robust vasculogenic functionality compared with BM MNC in response to ischemia.

Synteny and candidate gene prediction using an anchored linkage map of Astyanax mexicanus.

The blind Mexican cave tetra, Astyanax mexicanus, is a unique model system for the study of parallelism and the evolution of cave-adapted traits. Understanding the genetic basis for these traits has recently become feasible thanks to production of a genome-wide linkage map and quantitative trait association analyses. The selection of suitable candidate genes controlling quantitative traits remains challenging, however, in the absence of a physical genome. Here, we describe the integration of multiple linkage maps generated in four separate crosses between surface, cave, and hybrid forms of A. mexicanus. We performed exhaustive BLAST analyses of genomic markers populating this integrated map against sequenced genomes of numerous taxa, ranging from yeast to amniotes. We found the largest number of identified sequences (228), with the most expect (E) values <10(-5) (95), in the zebrafish Danio rerio. The most significant hits were assembled into an "anchored" linkage map with Danio, revealing numerous regions of conserved synteny, many of which are shared across critical regions of identified quantitative trait loci (QTL). Using this anchored map, we predicted the positions of 21 test genes on the integrated linkage map and verified that 18 of these are found in locations homologous to their chromosomal positions in D. rerio. The anchored map allowed the identification of four candidate genes for QTL relating to rib number and eye size. The map we have generated will greatly accelerate the production of viable lists of additional candidate genes involved in the development and evolution of cave-specific traits in A. mexicanus.

The safety of autologous intracoronary stem cell injections in a porcine model of chronic myocardial ischemia.

BACKGROUND: Intracoronary mononuclear cell therapy may produce angiogenesis in chronic myocardial ischemia. Potential complications include periprocedural infarction secondary to: reduced coronary blood flow; hyperviscosity from the cell preparation; or microvascular dysfunction. To date, no studies to evaluate these potential complications have been reported. The objective of this report was to study the safety and feasibility of intracoronary injections of autologous bone marrow mononuclear cells in a porcine chronic myocardial ischemia model. METHODS: Domestic pigs (n = 5) underwent ameroid cuff placement of the left circumflex artery. Bone marrow-derived mononuclear cells [15 x 10(6) cells] labeled with CM dioctadecyl tetramethylindocarbocyanine were given by intracoronary injection. Animals were sacrificed, and hearts and vital organs were inspected grossly and by histopathology, and bone marrow underwent immunofluorescence microscopy. RESULTS: Troponin I levels, gross inspection and histopathology did not reveal evidence of myocardial infarction. Labeled cells were observed in perivascular structures in myocardium at the injection site in all animals and in the spleen from one animal. Bone marrow aspirates indicated labeled cells. CONCLUSIONS: Intracoronary injection of autologous mononuclear cells in a porcine chronic myocardial ischemia model appears safe. Intracoronary injection resulted in cell localization in the perivascular areas of myocardium supplied by the injected vessel. Cell localization was observed only in the spleen in just one animal. Labeled cells were identified in bone marrow aspirates from three animals following injection, suggesting a role for bone marrow engraftment and repopulation as a possible mechanism for progenitor cell localization in myocardium.