Terrence Donnelly Center for Cellular and Biomolecular Research

Donnelly, Canada

Terrence Donnelly Center for Cellular and Biomolecular Research

Donnelly, Canada
SEARCH FILTERS
Time filter
Source Type

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.


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.


Kittanakom S.,Terrence Donnelly Center for Cellular and Biomolecular Research | Arnoldo A.,Terrence Donnelly Center for Cellular and Biomolecular Research | Brown K.R.,Terrence Donnelly Center for Cellular and Biomolecular Research | Wallace I.,Terrence Donnelly Center for Cellular and Biomolecular Research | And 13 more authors.
G3: Genes, Genomes, Genetics | Year: 2013

The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl2positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness bymore general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug2target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug2target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings. © 2013 Kittanakom et al.


Pu S.,Hospital for Sick Children | Turinsky A.L.,Hospital for Sick Children | Vlasblom J.,Hospital for Sick Children | Vlasblom J.,University of Toronto | And 13 more authors.
PLoS ONE | Year: 2010

Chromatin modification (CM) plays a key role in regulating transcription, DNA replication, repair and recombination. However, our knowledge of these processes in humans remains very limited. Here we use computational approaches to study proteins and functional domains involved in CM in humans. We analyze the abundance and the pair-wise domaindomain co-occurrences of 25 well-documented CM domains in 5 model organisms: yeast, worm, fly, mouse and human. Results show that domains involved in histone methylation, DNA methylation, and histone variants are remarkably expanded in metazoan, reflecting the increased demand for cell type-specific gene regulation. We find that CM domains tend to co-occur with a limited number of partner domains and are hence not promiscuous. This property is exploited to identify 47 potentially novel CM domains, including 24 DNA-binding domains, whose role in CM has received little attention so far. Lastly, we use a consensus Machine Learning approach to predict 379 novel CM genes (coding for 329 proteins) in humans based on domain compositions. Several of these predictions are supported by very recent experimental studies and others are slated for experimental verification. Identification of novel CM genes and domains in humans will aid our understanding of fundamental epigenetic processes that are important for stem cell differentiation and cancer biology. Information on all the candidate CM domains and genes reported here is publicly available. © 2010 Pu et al.


Ng A.H.C.,University of Toronto | Ng A.H.C.,Terrence Donnelly Center for Cellular and Biomolecular Research | Li B.B.,University of Toronto | Li B.B.,Terrence Donnelly Center for Cellular and Biomolecular Research | And 4 more authors.
Annual Review of Biomedical Engineering | Year: 2015

Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-Art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. © 2015 by Annual Reviews. All rights reserved.


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 12 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.


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.


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.


Choi K.,University of Toronto | Choi K.,Terrence Donnelly Center for Cellular and Biomolecular Research | Ng A.H.C.,University of Toronto | Ng A.H.C.,Terrence Donnelly Center for Cellular and Biomolecular Research | And 4 more authors.
Annual Review of Analytical Chemistry | Year: 2012

Digital microfluidics (DMF) is an emerging liquid-handling technology that enables individual control over droplets on an open array of electrodes. These picoliter- to microliter-sized droplets, each serving as an isolated vessel for chemical processes, can be made to move, merge, split, and dispense from reservoirs. Because of its unique advantages, including simple instrumentation, flexible device geometry, and easy coupling with other technologies, DMF is being applied to a wide range of fields. In this review, we summarize the state of the art of DMF technology from the perspective of analytical chemistry in sections describing the theory of droplet actuation, device fabrication and integration, and applications. Copyright © 2012 by Annual Reviews. All rights reserved.


PubMed | Aix - Marseille University, Terrence Donnelly Center for Cellular and Biomolecular Research and Uppsala University
Type: Journal Article | Journal: 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.

Loading Terrence Donnelly Center for Cellular and Biomolecular Research collaborators
Loading Terrence Donnelly Center for Cellular and Biomolecular Research collaborators