Donnelly Center for Cellular and Biomolecular Research

Toronto, Canada

Donnelly Center for Cellular and Biomolecular Research

Toronto, Canada
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Van Bakel H.,Donnelly Center for Cellular and Biomolecular Research | Tsui K.,University of Toronto | Li J.,Donnelly Center for Cellular and Biomolecular Research | Morris Q.D.,Donnelly Center for Cellular and Biomolecular Research | And 3 more authors.
Genome Research | Year: 2014

Identifying genes in the genomic context is central to a cell's ability to interpret the genome. Yet, in general, the signals used to define eukaryotic genes are poorly described. Here, we derived simple classifiers that identify where transcription will initiate and terminate using nucleic acid sequence features detectable by the yeast cell, which we integrate into a Unified Model (UM) that models transcription as a whole. The cis-elements that denote where transcription initiates function primarily through nucleosome depletion, and, using a synthetic promoter system, we show that most of these elements are sufficient to initiate transcription in vivo. Hrp1 binding sites are the major characteristic of terminators; these binding sites are often clustered in terminator regions and can terminate transcription bidirectionally. The UM predicts global transcript structure by modeling transcription of the genome using a hidden Markov model whose emissions are the outputs of the initiation and termination classifiers. We validated the novel predictions of the UM with available RNA-seq data and tested it further by directly comparing the transcript structure predicted by the model to the transcription generated by the cell for synthetic DNA segments of random design. We show that the UM identifies transcription start sites more accurately than the initiation classifier alone, indicating that the relative arrangement of promoter and terminator elements influences their function. Our model presents a concrete description of how the cell defines transcript units, explains the existence of nongenic transcripts, and provides insight into genome evolution. © 2014 de Boer et al.

van de Bunt M.,University of Oxford | Gaulton K.J.,University of Oxford | Parts L.,Wellcome Trust Sanger Institute | Parts L.,Donnelly Center for Cellular and Biomolecular Research | And 9 more authors.
PLoS ONE | Year: 2013

Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis. © 2013 van de Bunt et al.

News Article | November 19, 2016

TORONTO, ONTARIO--(Marketwired - Nov. 19, 2016) - The Canadian-Croatian Chamber of Commerce is pleased to be welcoming the President of the Republic of Croatia, Kolinda Grabar-Kitarovic, during her first visit to Canada in her capacity as President. The Chamber and its members will be hosting a number of events featuring the President in Toronto and surrounding areas during her visit from November 20-22, 2016. The Croatian President will, among other activities, be meeting with Croatian-Canadian community and business leaders and professionals; addressing Croatian-Canadian students at a youth mentorship event in Norval; meeting with Mayor Berry Vrbanovic in Kitchener; touring the innovation ecosystem (including the University of Waterloo, the Lazaridis Quantum Nano computer centre, Communitech, and Velocity) that is part of the Toronto-Waterloo Region Corridor; meeting with representatives and students of Croatian language studies at the University of Waterloo; meeting with Ontario Premier Kathleen Wynne and Ontario Lieutenant-Governor Elizabeth Dowdeswell in Toronto; visiting the Donnelly Center for Cellular and Biomolecular Research at the University of Toronto; engaging in a roundtable discussion with leading female executives in Canada; and meeting with business leaders on Bay Street in Toronto to discuss potential investment opportunities in Croatia. To conclude her visit to Canada, the Croatian President will deliver a keynote address to nearly 1,000 members and friends of the Croatian community in Canada at a sold-out gala dinner and fundraiser being organized by the Chamber at the Burlington Convention Center. The event will benefit the restoration of the Vukovar water tower, one of the most famous symbols of Vukovar and the suffering of that heroic city as well as the Croatian War of Independence during the early 1990s. "We are honoured to host the Croatian President during her visit to Canada," said Ivan Grbesic, a member of the board of directors of the Chamber and one of the coordinators of the visit. "Significant time and effort was invested in ensuring that this working visit would contribute to expanding existing ties and exploring new opportunities between Canada and Croatia, especially given the recent signing of the CETA trade deal. The visit will also be a historic one for members of our Croatian-Canadian community in general and one that will be remembered for years to come given that it is the first time that a sitting Croatian President will visit our community since Croatia declared its independence 25 years ago", he added. "We also expect that this visit will lay the groundwork for an official visit to Ottawa by the Croatian President or recently elected Croatian Prime Minister in the near future". President Grabar-Kitarovic became the first female and youngest president of the Republic of Croatia in 2015 and is one of seven female heads of state in the world today. Her election as President capped a two-decade career in politics and diplomacy, including key roles as: the country's first female Minister of Foreign Affairs and European Integration, Croatia's Ambassador to the U.S., and the Assistant Secretary General for Public Diplomacy at NATO (the first woman Assistant Secretary-General ever in the history of NATO and the highest-ever ranking female official to have served within NATO's governing structure). Prior to visiting Norval, Kitchener, Toronto, and Burlington, the President is attending the Halifax International Security Forum on November 19-20, 2016. Founded in 1995, the Canadian-Croatian Chamber of Commerce is a not-for-profit network of Croatian-Canadian businesses, professionals and organizations that has emerged as the voice of Croatian-Canadian business in Canada. Canada has one of the largest and most successful Croatian communities outside of Croatia and the Chamber brings together businesses, professionals and organizations with strategic relationships (economic, commercial, political, and cultural) in both Canada and Croatia.

van Bakel H.,Mount Sinai School of Medicine | Tsui K.,Donnelly Center for Cellular and Biomolecular Research | Tsui K.,University of Toronto | Gebbia M.,Donnelly Center for Cellular and Biomolecular Research | And 6 more authors.
PLoS Genetics | Year: 2013

Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies. © 2013 van Bakel et al.

Maria S.,University of Toronto | Lilien R.H.,University of Toronto | Lilien R.H.,Donnelly Center for Cellular and Biomolecular Research
Journal of Computational Chemistry | Year: 2010

Dead-end elimination (DEE) has emerged as a powerful structure-based, conformational search technique enabling computational protein redesign. Given a protein with n mutable residues, the DEE criteria guide the search toward identifying the sequence of amino acids with the global minimum, energy conformation (GMEC). This approach does not restrict the number of permitted mutations and allows the identified GMEC to differ from, the original sequence in up to η residues. In practice, redesigns containing a large number of mutations are often problematic when taken into the wet-lab for creation via site-directed mutagenesis. The large number of point mutations required for the redesigns makes the process difficult, and increases the risk of major unpredicted and undesirable conformational changes. Preselecting a limited subset of mutable residues is not a satisfactory solution because it is unclear how to select this set before the search has been performed. Therefore, the ideal approach is what we define as the κ -restricted redesign problem in which any k of the n residues are allowed to mutate. We introduce restricted dead-end elimination (rDEE) as a solution of choice to efficiently identify the GMEC of the restricted redesign (the kGMEC). Whereas existing approaches require n-choose-K individual runs to identify the kGMEC, the rDEE criteria can perform the redesign in a single search. We derive a number of extensions to rDEE and, present a restricted form of the A* conformation search. We also demonstrate a 10-fold speed-up of rDEE over traditional DEE approaches on three different experimental systems. © 2009 Wiley Periodicals, Inc.

Weirauch M.T.,Donnelly Center for Cellular and Biomolecular Research | Hughes T.R.,Donnelly Center for Cellular and Biomolecular Research | Hughes T.R.,University of Toronto
Trends in Genetics | Year: 2010

Regulatory regions with similar transcriptional output often have little overt sequence similarity, both within and between genomes. Although cis- and trans-regulatory changes can contribute to sequence divergence without dramatically altering gene expression outputs, heterologous DNA often functions similarly in organisms that share little regulatory sequence similarities (e.g. human DNA in fish), indicating that trans-regulatory mechanisms tend to diverge more slowly and can accommodate a variety of cis-regulatory configurations. This capacity to 'tinker' with regulatory DNA probably relates to the complexity, robustness and evolvability of regulatory systems, but cause-and-effect relationships among evolutionary processes and properties of regulatory systems remain a topic of debate. The challenge of understanding the concrete mechanisms underlying cis-regulatory evolution - including the conservation of function without the conservation of sequence - relates to the challenge of understanding the function of regulatory systems in general. Currently, we are largely unable to recognize functionally similar regulatory DNA. © 2009 Elsevier Ltd. All rights reserved.

Ng A.H.C.,University of Toronto | Ng A.H.C.,Donnelly Center for Cellular and Biomolecular Research | Lee M.,University of Toronto | Lee M.,Donnelly Center for Cellular and Biomolecular Research | And 6 more authors.
Clinical Chemistry | Year: 2015

BACKGROUND: Whereas disease surveillance for infectious diseases such as rubella is important, it is critical to identify pregnant women at risk of passing rubella to their offspring, which can be fatal and can result in congenital rubella syndrome (CRS). The traditional centralized model for diagnosing rubella is cost-prohibitive in resource-limited settings, representing a major obstacle to the prevention of CRS. As a step toward decentralized diagnostic systems, we developed a proof-of-concept digital microfluidic (DMF) diagnostic platform that possesses the flexibility and performance of automated immunoassay platforms used in central facilities, but with a form factor the size of a shoebox. METHODS: DMF immunoassays were developed with integrated sample preparation for the detection of rubella virus (RV) IgG and IgM. The performance (sensitivity and specificity) of the assays was evaluated with serum and plasma samples from a commercial antirubella mixed-titer performance panel. RESULTS: The new platform performed the essential processing steps, including sample aliquoting for 4 parallel assays, sample dilution, and IgG blocking. Testing of performance panel samples yielded diagnostic sensitivity and specificity of 100% and 100% for both RV IgG and RV IgM. With 1.8 μL sample per assay, 4 parallel assays were performed in approximately 30 min with <10% mean CV. CONCLUSIONS: This proof of concept establishes DMF powered immunoassays as being potentially useful for the diagnosis of infectious disease. © 2014 American Association for Clinical Chemistry.

Luk V.N.,University of Toronto | Luk V.N.,Donnelly Center for Cellular and Biomolecular Research | Fiddes L.K.,University of Toronto | Luk V.M.,Donnelly Center for Cellular and Biomolecular Research | And 3 more authors.
Proteomics | Year: 2012

Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 μg trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a single sample was simultaneously digested on multiple gel discs bearing different enzymes. We propose that these methods represent a useful new tool for the growing trend toward miniaturization and automation in proteomic sample processing. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Eberhard Y.,Ontario Cancer Institute | Gronda M.,Ontario Cancer Institute | Hurren R.,Ontario Cancer Institute | Datti A.,Mount Sinai Hospital | And 5 more authors.
Oncotarget | Year: 2011

Evasion of death receptor ligand-induced apoptosis contributs to cancer development and progression. To better understand mechanisms conferring resistance to death ligands, we screened an siRNA library to identify sequences that sensitize resistant cells to fas activating antibody (CH-11). From this screen, we identified the Sterol- Regulatory Element-Binding Protein 1 (SREBP1), a transcription factor, which regulates genes involved in cholesterol and fatty acid synthesis including fatty acid synthase. Inhibition of SREBP1 sensitized PPC-1 and HeLa to the death receptor ligands CH-11 and TRAIL. In contrast, DU145 prostate cancer cells that are resistant to death ligands despite expressing the receptors on their cell surface remained resistant to CH-11 and TRAIL after knockdown of SREBP1. Consistent with the effects on cell viability, the addition of CH-11 activated caspases 3 and 8 in HeLa but not DU145 cells with silenced SREBP1. We demonstrated that knockdown of SREBP1 produced a marked decrease in fatty acid synthase expression. Furthermore, genetic or chemical inhibition of fatty acid synthase with shRNA or orlistat, respectively, recapitulated the effects of SREBP1 inhibition and sensitized HeLa but not DU145 cells to CH-11 and TRAIL. Sensitization to death receptor ligands by inhibition of fatty acid synthase was associated with activation of caspase 8 prior to caspase 9. Neither silencing of SREBP1 or fatty acid synthase changed basal expression of the core death receptor components Fas, caspase 8, FADD, caspase 3 or FLIP. Thus, inhibition of SREBP1 or its downstream target fatty acid synthase sensitizes resistant cells to death ligands. © Eberhard et al.

Nissar A.A.,Institute of Biomaterials and Biomedical Engineering | Martowirogo A.,Institute of Biomaterials and Biomedical Engineering | Gilbert P.M.,Donnelly Center for Cellular and Biomolecular Research
Current Opinion in Solid State and Materials Science | Year: 2016

Regenerative medicine aims to restore form and function to aged, injured, and diseased tissues. One strategy that is gaining traction is in vivo regenerative medicine. Many adult tissues possess stem and progenitor cells that are enlisted to repair minor tissue damage after insult. However, in the setting of disease, aging, or criticalsized injuries, the microenvironment may lack structural elements, physical, and/or chemical cues required to drive repair to completion. Natural and synthetic materials offer an opportunity to facilitate the repair process by restoring the natural reparative capacity of adult tissues. Using design criteria selected based upon an understanding of the inductive niche cues that naturally instruct stem and progenitor cell behavior and fate, it is possible to elicit tissue repair by implementing a cell-free approach. This review highlights recent studies that assess biomaterials for in vivo regenerative medicine applications and demonstrate a capacity for controlling stem and progenitor cell behavior to restore tissue form and function. © 2016.

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