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Lam H.Y.K.,Yale University | Mu X.J.,Yale University | Stutz A.M.,Genome Biology Unit | Tanzer A.,University of Vienna | And 8 more authors.
Nature Biotechnology | Year: 2010

Structural variants (SVs) are a major source of human genomic variation; however, characterizing them at nucleotide resolution remains challenging. Here we assemble a library of breakpoints at nucleotide resolution from collating and standardizing ∼2,000 published SVs. For each breakpoint, we infer its ancestral state (through comparison to primate genomes) and its mechanism of formation (e.g., nonallelic homologous recombination, NAHR). We characterize breakpoint sequences with respect to genomic landmarks, chromosomal location, sequence motifs and physical properties, finding that the occurrence of insertions and deletions is more balanced than previously reported and that NAHR-formed breakpoints are associated with relatively rigid, stable DNA helices. Finally, we demonstrate an approach, BreakSeq, for scanning the reads from short-read sequenced genomes against our breakpoint library to accurately identify previously overlooked SVs, which we then validate by PCR. As new data become available, we expect our BreakSeq approach will become more sensitive and facilitate rapid SV genotyping of personal genomes. © 2010 Nature America, Inc. All rights reserved. Source

Odedra D.,University of Toronto | Chiu L.L.Y.,University of Toronto | Shoichet M.,University of Toronto | Shoichet M.,Terrence Donnelly Center for Cellular and Biomolecular Research | Radisic M.,University of Toronto
Acta Biomaterialia | Year: 2011

A key challenge in tissue engineering is overcoming cell death in the scaffold interior due to the limited diffusion of oxygen and nutrients therein. We here hypothesize that immobilizing a gradient of a growth/survival factor from the periphery to the center of a porous scaffold would guide endothelial cells into the interior of the scaffold, thus overcoming a necrotic core. Proteins were immobilized by one of three methods on porous collagen scaffolds for cardiovascular tissue engineering. The proteins were first activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/sulfo N-hydroxysuccinimide and then applied to the scaffold by one of three methods to establish the gradient: perfusion (the flow method), use of a source and a sink (the source-sink method) or by injecting 5 μl of the solution at the center of the scaffold (point source method). Due to the high reproducibility and ease of application of the point source method it was further used for VEGF-165 gradient formation, where an ∼2 ng ml-1 mm-1 gradient was formed in a radial direction across a scaffold, 12 mm in diameter and 2.5 mm thick. More endothelial cells were guided by the VEGF-165 gradient deep into the center of the scaffold compared with both uniformly immobilized VEGF-165 (with the same total VEGF concentration) and VEGF-free controls. All scaffolds (including the controls) yielded the same number of cells, but notably the VEGF-165 gradient scaffolds demonstrated a higher cell density in the centre. Thus we concluded that the VEGF-165 gradient promoted the migration, but not proliferation, of cells into the scaffold. These gradient scaffolds provide the foundation for future in vivo tissue engineering studies. © 2011 Acta Materialia Inc. Source

Ito C.Y.,University of Toronto | Kirouac D.C.,University of Toronto | Madlambayan G.J.,University of Florida | Yu M.,University of Toronto | And 3 more authors.
Blood | Year: 2010

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture, better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs), we demonstrated that the rhodamine-low phenotype was lost, whereas AC133 expression was retained throughout culture. Furthermore, the AC133+CD38- subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number, and limiting dilution analysis in NOD/SCID mice, showed a 43-fold expansion of the AC133+CD38- subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation, respectively. Thus, AC133+CD38 - is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures. © 2010 by The American Society of Hematology. Source

Zuberi K.,Terrence Donnelly Center for Cellular and Biomolecular Research | Morris Q.,Terrence Donnelly Center for Cellular and Biomolecular Research | Morris Q.,University of Toronto | Hughes T.R.,Terrence Donnelly Center for Cellular and Biomolecular Research | Hughes T.R.,University of Toronto
Nucleic Acids Research | Year: 2011

The RNA-Binding Protein DataBase (RBPDB) is a collection of experimental observations of RNAbinding sites, both in vitro and in vivo, manually curated from primary literature. To build RBPDB, we performed a literature search for experimentalbinding data for all RNA-binding proteins (RBPs) with known RNA-binding domains in four metazoan species (human, mouse, fly and worm). In total, RPBDB contains binding data on 272 RBPs, including 71 that have motifs in position weight matrix format, and 36 sets of sequences of in vivo-bound transcripts from immunoprecipitation experiments. The database is accessible by a web interface which allows browsing by domain or by organism, searching and export of records, and bulk data downloads. Users can also use RBPDB to scan sequences for RBP-binding sites. RBPDB is freely available, without registration at http://rbpdb.ccbr.utoronto.ca/. © The Author(s) 2010. Source

Garrido-Urbani S.,Aix - Marseille University | Garrido-Urbani S.,University Hospitals Geneva Medical Center | Garg P.,Terrence Donnelly Center for Cellular and Biomolecular Research | Garg P.,University of Toronto | And 11 more authors.
FEBS Letters | Year: 2016

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance. © 2015 Federation of European Biochemical Societies. Source

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