01 College Street

Toronto, Canada

01 College Street

Toronto, Canada
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Higgins G.A.,01 College Street | Breysse N.,01 College Street | Undzys E.,01 College Street | Derksen D.R.,01 College Street | And 7 more authors.
Psychopharmacology | Year: 2010

Rationale: Antiepileptic drugs (AEDs) have been available for many years; yet, new members of this class continue to be identified and developed due to the limitations of existing drugs, which include a propensity for cognitive impairment. However, there is little preclinical information about the cognitive effects they produce, which clinically include deficits in attention and slowing of reaction time. Objectives: The purpose of this study was to profile two first-generation AEDs, phenytoin and valproate, and three second-generation AEDs, levetiracetam, pregabalin and lacosamide. Initially, each drug was examined across a range of well characterised preclinical seizure tests, and then each drug was evaluated in the five-choice serial reaction time test (5-CSRTT) based on efficacious doses from the seizure tests. Materials and methods: Each AED was tested for anti-seizure efficacy in either (1) the maximal electroshock seizure test, (2) s.c. PTZ seizure test, (3) amygdala-kindled seizures and (4) the genetic absence epilepsy rat of Strasbourg model of absence seizures. On completion of these studies, each drug was tested in rats trained to asymptotic performance in the 5-CSRTT (0.5 s SD, 5 s ITI, 100 trials). Male rats were used in all studies. Results: Each AED was active in at least one of the seizure tests, although only valproate was active in each test. In the 5-CSRT test, all drugs with the exception of levetiracetam, significantly slowed reaction time and increased omissions. Variable effects were seen on accuracy. The effect on omissions was reversed by increasing stimulus duration from 0.5 to 5 s, supporting a drug-induced attention deficit. Levetiracetam had no negative effect on performance; indeed, reaction time was slightly increased (i.e. faster). Conclusions: These results highlight somewhat similar effects of phenytoin, valproate, pregabalin and lacosamide on attention and reaction time, and comparison to efficacious doses from the seizure tests support the view that there may be a better separation with the newer AEDs. Levetiracetam had no detrimental effect in the 5-CSRTT, which may be consistent with clinical experience where the drug is considered to be well tolerated amongst the AED class. © 2009 Springer-Verlag.

Nanau R.M.,University of Toronto | Neuman M.G.,01 College Street
Digestive Diseases and Sciences | Year: 2012

In vitro and animals models have long been used to study human diseases and identify novel therapeutic approaches that can be applied to combat these conditions. Ulcerative colitis and Crohn's disease are the two main entities of inflammatory bowel disease (IBD). There is an intricate relationship between IBD features in human patients, in vitro and animal colitis models, mechanisms and possible therapeutic approaches in these models, and strategies that can be extrapolated and applied in humans. Malnutrition, particularly protein-energy malnutrition and vitamin and micronutrient deficiencies, as well as dysregulation of the intestinal microbiota, are common features of IBD. Based on these observations, dietary supplementation with essential nutrients known to be in short supply in the diet in IBD patients and with other molecules believed to provide beneficial anti-inflammatory effects, as well as with probiotic organisms that stimulate immune functions and resistance to infection has been tested in colitis models. Here we review current knowledge on nutritional and probiotic supplementation in in vitro and animal colitis models. While some of these strategies require further fine-tuning before they can be applied in human IBD patients, their intended purpose is to prevent, delay or treat disease symptoms in a non-pharmaceutical manner. © Springer Science+Business Media, LLC 2012.

Senisterra G.,01 College Street | Wu H.,01 College Street | Allali-Hassani A.,01 College Street | Wasney G.A.,01 College Street | And 20 more authors.
Biochemical Journal | Year: 2013

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3-9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys 4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers. © 2013 The Author(s).

Tarhonskaya H.,University of Oxford | Szollossi A.,University of Oxford | Leung I.K.H.,University of Oxford | Bush J.T.,University of Oxford | And 8 more authors.
Biochemistry | Year: 2014

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual "ping-pong" mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases. © 2014 American Chemical Society.

Senisterra G.A.,01 College Street | Ghanei H.,Ontario Cancer Institute | Ghanei H.,University of Toronto | Khutoreskaya G.,01 College Street | And 5 more authors.
Journal of Biomolecular Screening | Year: 2010

Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg++ channel), and CpxA (a histidine kinase) as model proteins and show that DSLS is not sensitive to the presence of detergents or protein hydrophobicity and can be used to monitor thermodenaturation of membrane proteins, assess their stability, and detect ligand binding in a 384-well format. © 2010 Society for Biomolecular Sciences.

Neuman M.G.,University of Toronto | Neuman M.G.,01 College Street | Neuman M.G.,Institute of Drug Research | Schneider M.,Alcohol and Drug Abuse Research Unit | And 4 more authors.
AIDS Research and Treatment | Year: 2012

The present paper describes the possible connection between alcohol consumption and adherence to medicine used to treat human deficiency viral (HIV) infection. Highly active antiretroviral therapy (HAART) has a positive influence on longevity in patients with HIV, substantially reducing morbidity and mortality, including resource-poor settings such as South Africa. However, in a systematic comparison of HAART outcomes between low-income and high-income countries in the treatment of HIV-patients, mortality was higher in resource-poor settings. Specifically, in South Africa, patients often suffer from concomitant tuberculosis and other infections that may contribute to these results. Alcohol influences the use of medicine for opportunistic infections (e.g., pneumonia, tuberculosis), or coinfections HIV-hepatitis viruses-B (HBV) and C (HCV), cytomegalovirus, or herpes simplex virus. Furthermore, alcohol use may negatively impact on medication adherence contributing to HIV progression. The materials used provide a data-supported approach. They are based on analysis of published (2006-2011) world literature and the experience of the authors in the specified topic. Intended for use by health care professionals, these recommendations suggest approaches to the therapeutic and preventive aspects of care. Our intention was to fully characterize the quality of evidence supporting recommendations, which are reflecting benefit versus risk, and assessing strength or certainty. © 2012 Manuela G. Neuman et al.

Hojjat S.-P.,Sunnybrook Research Institute | Foltz W.,01 College Street | Wise-Milestone L.,Sunnybrook Research Institute | Whyne C.M.,Sunnybrook Research Institute
Medical Physics | Year: 2012

Purpose: Multimodal microimaging in preclinical models is used to examine the effect of spinal metastases on bony structure; however, the evaluation of tumor burden and its effect on microstructure has thus far been mainly qualitative or semiquantitative. Quantitative analysis of multimodality imaging is a time consuming task, motivating automated methods. As such, this study aimed to develop a low complexity semiautomated multimodal μCT/μMR based approach to segment rat vertebral structure affected by mixed osteolytic/osteoblastic destruction. Methods: Mixed vertebral metastases were developed via intracardiac injection of Ace-1 canine prostate cancer cells in three 4-week-old rnu/rnu rats. μCT imaging (for high resolution bone visualization), T1-weighted μMR imaging (for bone registration), and T2-weighted μMR imaging (for osteolytic tumor visualization) were conducted on one L1, three L2, and one L3 vertebrae (excised). One sample (L1-L3) was processed for undecalcified histology and stained with Goldner's trichome. The μCT and μMR images were registered using a 3D rigid registration algorithm with a mutual information metric. The vertebral microarchitecture was segmented from the μCT images using atlas-based demons deformable registration, levelset curvature evolution, and intensity-based thresholding techniques. The μCT based segmentation contours of the whole vertebrae were used to mask the T2-weighted μMR images, from which the osteolytic tumor tissue was segmented (intensity-based thresholding). Results: Accurate registration of μCT and μMRI modalities yielded precise segmentation of whole vertebrae, trabecular centrums, individual trabeculae, and osteolytic tumor tissue. While the algorithm identified the osteoblastic tumor attached to the vertebral pereosteal surfaces, it was limited in segmenting osteoblastic tissue located within the trabecular centrums. Conclusions: This semiautomated segmentation method yielded accurate registration of μCT and μMRI modalities with application to the development of mathematical models analyzing the mechanical stability of metastatically involved vertebrae and in preclinical applications evaluating new and existing treatment effects on tumor burden and skeletal microstructure. © 2012 American Association of Physicists in Medicine.

Ceccarelli D.F.,Samuel Lunenfeld Research Institute | Tang X.,Samuel Lunenfeld Research Institute | Pelletier B.,University of Montréal | Orlicky S.,Samuel Lunenfeld Research Institute | And 26 more authors.
Cell | Year: 2011

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF Skp2 substrate p27Kip1. CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS. © 2011 Elsevier Inc.

PubMed | University of Toronto, 01 College Street and Sunnybrook Research Institute
Type: | Journal: BMC research notes | Year: 2016

Earlier studies by our group have demonstrated that a transgenic animal engineered to express Tie2 under the control of the Tie2 promoter produced animals with a scaly skin phenotype that recapitulated many of the hallmarks of atopic dermatitis (AT-Derm). To test the hypothesis that this model of AT-Derm is driven by dysregulated Tie2-signalling, we have bred AT-Derm transgenic (TG) animals with TG-animals engineered to overexpress Angiopoietin-1 or -2, the cognate Tie2 ligands. These two ligands act to antagonize one another in a context-dependent manner. To further evaluate the role of Ang1-driven-Tie2 signalling, we examined the ability of Vasculotide, an Ang1-mimetic, to modulate the AT-Derm phenotype.AT-Derm+Ang2 animals exhibited an accentuated phenotype, whereas AT-Derm+Ang1 presented with a markedly reduced skin disease, similarly VT-treated AT-Derm animals present with a clear decrease in the skin phenotype. Moreover, a decrease in several important inflammatory cytokines and a decrease in the number of eosinophils was noted in VT-treated animals. Bone marrow differentiation in the presence of VT produced fewer CFU-G colonies, further supporting a role for Tie2-signalling in eosinophil development. Importantly, we demonstrate activation of Tie2, the VT-target, in lung tissue from nave animals treated with increasing amounts of VT.The AT-Derm phenotype in these animals is driven through dysregulation of Tie2 receptor signalling and is augmented by supplemental Ang2-dependent stimulation. Overexpression of Ang1 or treatment with VT produced a similar amelioration of the phenotype supporting the contention that VT and Ang1 have a similar mechanism of action on the Tie2 receptor and can both counteract the signalling driven by Ang2. Our results also support a possible role for Tie2-signalling in the development of eosinophilic diseases and that activation of Tie2 may directly or indirectly modulate the differentiation of eosinophils, which express Tie2. In summary, these data support the hypothesis that this AT-Derm mouse model is driven by dysregulation of the Tie2 signalling pathway and increased Ang2 levels can aggravate it, whereas it can be reversed by either Ang1-overexpression or VT treatment. Moreover, our data supports the contention that VT acts as an Angiopoietin-1 mimetic and may provide a novel entry point for Tie2-agonist-based therapies for atopic diseases.

Winer D.,Toronto General Research Institute | Winer D.,King's College | Winer D.,01 College Street | Luck H.,Toronto General Research Institute | And 8 more authors.
Cell Metabolism | Year: 2016

Obesity and insulin resistance are associated with chronic inflammation in metabolic tissues such as adipose tissue and the liver. Recently, growing evidence has implicated the intestinal immune system as an important contributor to metabolic disease. Obesity predisposes to altered intestinal immunity and is associated with changes to the gut microbiota, intestinal barrier function, gut-residing innate and adaptive immune cells, and oral tolerance to luminal antigens. Accordingly, the gut immune system may represent a novel therapeutic target for systemic inflammation in insulin resistance. This review discusses the emerging field of intestinal immunity in obesity-related insulin resistance and how it affects metabolic disease. © 2016 Elsevier Inc.

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