GAIRDNER SYMPOSIUM June 6–9 2017

A Celebration of Excellence

PANTONE RED 485 MAINTAIN CLEAR SPACE AROUND THE LOGO EQUAL TO THE x-HEIGHT OF THE LETTER FORMS & ALWAYS USE A FUTURA FAMILY FONT FOR ASSOCIATED TEXTS. GAIRDNER SYMPOSIUM A Celebration of Excellence Welcome and congratulations on participating in the 2017 Canadian Student Health Research Forum, a unique venue offering students doing health research the opportunity to network, learn about cutting-edge research from internationally renowned experts and be recognized for their own outstanding scientific accomplishments.

BRIAN POSTL, MD The involvement of students in research creates National Research Poster Presentation and the DEAN the pipeline to the future of research in this Canadian National Medical Student Research country. We are grateful we can contribute to Symposium. We trust that the valuable Max Rady College their exposures through poster presentations, connections you make with your peers, judges, of Medicine, Rady a CIHR Career Development Workshop and AFMC and CIHR leadership will provide career- Faculty of Health numerous networking opportunities. In enhancing opportunities as well as a fun and Sciences partnership with the Gairdner Foundation, we enjoyable experience. Our thanks to all the are pleased to host an all-Gairdner Symposium student groups for their energetic support in and note the exceptional opportunities hosting our visitors. for mentorship from these internationally is an exciting and vibrant place to recognized expert speakers who will be sharing live, work and conduct state-of-the-art health their insights and experience. research. We hope you enjoy your visit with We extend warm greetings and a “friendly us at the University of Manitoba campus and Manitoba” welcome to the record number have the opportunity to explore all that our city of participants from across and has to offer. internationally who are taking part in the CIHR Gairdner Symposium Programme

SYMPOSIUM Thursday, 8:00 Registration & Refreshments (Concourse) June 8, 2017 9:00 Welcome and Opening Remarks Dr. Kevin Coombs, Assistant Dean (Research), Max Rady Frederic Gaspard College of Medicine, University of Manitoba PROGRAMME Lecture Theatre 9:10 Chair’s Overview Geoff Hicks 9:15 Dr. James Hogg Basic Medical – University of British Columbia Sciences Building “The “Achilles’ heel” of the Lung” (Introduced by Dr. Andrew Halayko) 2017 >>> 730 William Avenue 10:00 Coffee Break (Mezzanine) 10:30 Dr. Lea Harrington – Université de Montréal “Critically short chromosome ends impact stem cell fate" (Introduced by Dr. Hao Ding) 11:15 Dr. Antoine M. Hakim – University of “The Canadian Stroke Network: Bringing Knowledge to Society” (Introduced by Dr. Soheila Karimi) 12:00 Lunch and Poster Session (Brodie Atrium) 14:00 Dr. Janet Rossant – Hospital for Sick Children and University of “Establishing pluripotency and modelling disease” (Introduced by Dr. Geoff Hicks) 14:45 Coffee Break (Mezzanine) 15:15 Dr. William G. Kaelin, Jr. – Dana-Farber/Harvard Cancer Centre “New Approaches for Treating Anemia, Ischemia, and Cancer Emerging from Studies of the VHL Tumor Suppressor Protein” (Introduced by Dr. Mike Mowat) 16:00 Round Table Discussion 16:30 Awards Ceremony 17:15 Reception (Concourse) Apotex Fermentation Inc. Award – Molecular Biology Organizing James Gilchrist Lea Harrington Committee Canadian Institutes of Health Research – Poster Presentations Andrew Halayko Pingzhao Hu Meghan Azad Sabine Hombach-Klonisch Michael Jackson Caroline A. Cope Award – Oncology Patrick Choy Michael Jackson Bernard Jasmin Children’s Hospital Foundation Award – Child Health Kevin Coombs Tiina Kauppinen Soheila Karimi Dean of Graduate Studies – Poster Presentations Alysha Croker, CIHR Hassan Marzban Tiina Kauppinen Michael Czubryt Kirk McManus Forough Khadem Dean of Medicine – Poster Presentations Carla Dunton Don Miller Roger Mcleod 2017 Diagnostic Services Manitoba Awards 2017 Jessica Fraser Tooru Mizuno Tooru Mizuno – Translational Research Barbie Haddon Jim Nagy Nathan Nickel AWARDS – Imaging/Radiology AWARD Kent HayGlass Thomas Netticadan Karmin O COMMITTEES Geoff Hicks Mojgan Rastegar Trevor Pemberton E.L. Drewry Memorial Award – Health Research Ed Kroeger Miyoung Suh Garry Shen Graduate Students Association – Poster Presentations Yvonne Myal Bob Tate Bob Shiu Major Awards Hain, Emil and Lynette Scholarship – Excellence in Oncology Research Peter Nickerson Geoff Tranmer Louise Simard Committee Kimberly Ormiston Chris Siow Health Sciences Centre Foundation Inc. Award – Infection & Immunity/Neurobiology Chair – Yvonne Myal Nicole Szajcz-Keller, IMHA, CIHR/National Katinka Stecina Manitoba Medical Service Foundation Inc. Vern Dolinsky CIHR Poster Judging Panel Robert Tate – Poster Presentations Janice Dodd Terri Turner Co-Chairs – Jeff Wigle and – Awards for Excellence in Research Sachin Katyal Andrew Watson, AFMC Tara Beattie Teresa Zelinski Jeff Wigle Blake Ball Research Manitoba – Postdoctoral Fellow Poster Presentation Cynthia Ellison Kris Cowley Right Honourable Don Mazankowski Award - Excellence in Oncology Research Jeff Wigle Manitoba Poster Alysha Croker S.W. Prowse Memorial Award – Clinical Research Stephanie Booth Judging Panel Sanjiv Dhingra St. Boniface General Hospital Research Foundation Inc. Award – Cardiovascular Biology Chair- Michael Czubryt Janice Dodd 2017 Oncology Awards Blake Ball Jules Doré The Winnipeg Foundation – Travel Awards Committee Versha Banerji Christine Doucette Chair – Yvonne Myal Rajinder Bhullar Eftekhar Eftekharpour Sam Kung Kristine Cowley Paul Fernyhough Robert Tate Eftekhar Eftekharpour Jean-Eric Ghia Jeff Wigle Saeid Ghavami Sari Hannila GAIRDNER SYMPOSIUM Janet James William G. Rossant Hogg Lea Antoine Kaelin, Jr. Hospital for Sick University of British Harrington M. Hakim Dana-Farber/ Children and Columbia Université de University of Ottawa Harvard Cancer Montréal University of Toronto Gairdner Wightman Gairdner Wightman Center SPEAKERS Gairdner Wightman laureate Chair of the Gairdner laureate Gairdner laureate Medical Review Panel International 2017 >>> laureate William Kaelin obtained undergraduate and MD degrees from New Approaches Loss of the VHL tumor suppressor protein additional strategy for targeting transcription Duke University and completed his internal medicine training for Treating (pVHL) is the gatekeeper event in the most factors such as HIF2α (small molecule-mediated at the Johns Hopkins Hospital, where he served as chief common form of kidney cancer. pVHL is part target destabilization). Anemia, Ischemia, medical resident. He was a clinical fellow in medical oncology of an ubiquitin ligase that targets the alpha and Cancer subunits of the HIF transcription factor for Some cancers, such as a subset of acute at the Dana-Farber Cancer Institute and a postdoctoral fellow Emerging From proteasomal degradation. Binding of pVHL to leukemias and gliomas, are driven by IDH1 in the laboratory of David Livingston, during which time he HIFα requires that HIFα be prolyl hydroxylated or IDH2 mutations. These mutations cause Studies of the VHL was a McDonnell Scholar. He is currently a Professor in the by the EglN 2-oxoglutarate (2-OG)-dependent the accumulation of mM amounts of R-2 Tumor Suppressor dioxygenases, which act as oxygen sensors. hydroxyglutarate (R-2HG), which can interfere Department of Medicine at the Dana-Farber Cancer Institute HIF activates genes that promote adaptation with (usually inhibit) the activities of various Protein and the Brigham and Women's Hospital, Harvard Medical to hypoxia, including VEGF and EPO. EglN 2-OG-dependent enzymes, including the inhibitors are in clinical trials for anemia and JmjC histone demethylases, the TET DNA School. are promising in preclinical ischemia models. hydroxylases, and the EglN prolyl hydroxylases. Conversely, a HIF2 inhibitor is efficacious We showed that the effects of R-2HG in Dr. Kaelin is the Associate Director, Basic Science, for the Dana-Farber/Harvard Cancer Center. α in preclinical pVHL-defective kidney cancer leukemia are reversible and drugs that inhibit Doctor Kaelin is a member of the American Society of Clinical Investigation, the American College models and has entered phase 2 testing in man. R-2HG production are promising in early clinical of Physicians, the National Academy of Sciences and the National Academy of Medicine. He Our recent work with lenalidomide suggests an trials in leukemia patients. William G. recently served on the NCI Board of Scientific Advisors, the AACR Board of Trustees, and the IOM Kaelin, Jr. National Cancer Policy Board. He has received numerous awards including the MSKCC Paul Marks Dana-Farber/Harvard Prize, the AACR Richard and Hinda Rosenthal Prize, a Doris Duke Distinguished Clinical Scientist Cancer Center Award, the Canada International Gairdner International Award, the ASCI Stanley J. Korsmeyer Gairdner International Award, the Scientific Grand Prix of the Foundation Lefoulon-Delalande and the Institute of France, laureate and the Albert Lasker Prize. Janet Rossant, CC, PhD, FRS, FRSC, is President & Scientific Establishing Pluripotent stem cells provide a major resource Director of the Gairdner Foundation, SickKids Chief of Research Pluripotency and for studying human biology, modelling human Emeritus and a world-renowned expert in developmental disease and treating chronic illnesses. I Modelling biology. Widely known for her studies of the genes that will discuss how understanding the early Disease development of mouse and human embryos has control embryonic development in the mouse, Dr. Rossant helped identify the conditions that best maintain has pioneered techniques for following cell fate and altering the pluripotent state. I will also discuss the use genes in embryos. Her current research focuses on stem cell of pluripotent stem cells to develop assays that allow personalized therapies for diseases such development and cell differentiation in the developing embryo, as cystic fibrosis. important areas for the study of birth defects as well as regenerative medicine.

Dr. Rossant trained at the Universities of Oxford and Cambridge, UK and has been in Canada since 1977, at Brock University and then the Samuel Lunenfeld Research Institute in Toronto. She joined SickKids in 2005 and established it as a global forerunner in genetic research. Dr. Rossant Janet has been recognized for her contributions to science with many awards, including the ISDB Ross Rossant G. Harrison Medal, Killam Prize for Health Sciences, March of Dimes Prize in Developmental Hospital for Sick Biology, SDB Conklin Medal, and CIHR Michael Smith Prize in Health Research. She is a Fellow Children and of both the Royal Societies of London and Canada, and is a foreign Associate of the US National University of Toronto Academy of Science. Gairdner Wightman laureate Dr. Hogg earned an MD from the University of Manitoba in The 'Achilles’ The term Achilles' heel defines a focal point of bulk flow to diffusion by providing an exponential 1962, completed a rotating internship at the Winnipeg General Heel' of the weakness that creates an opportunity to destroy increase in total airway cross sectional area that Hospital in 1963 and served as a Medical Officer in the Royal a structure in spite of its overall strength. This slows the bulk flow and enhances the diffusion Lung Canadian Air force from 1963-66. He completed the PhD presentation will discuss the hypothesis that of gases in the periphery of the lungs. Moreover the transitional region between bulk flow and our working hypothesis is that the larger program in Experimental Medicine at McGill University in diffusion is the 'Achilles heel' of the lungs. particles suspended in the flowing gas develop 1969, received further training in anatomic pathology at the Respiration is driven by both the rhythmic sufficient momentum to deposit by impaction at Massachusetts General Hospital in Boston and Royal Victoria contraction and relaxation of the respiratory branch points in the central airway. Whereas the muscles that generate the force required to finer particles that remain suspended in the gas Hospital in and was appointed Assistant Professor of create pressure drop between the atmosphere preferentially deposit in the transitional region Pathology at McGill University in 1972, followed by promotion and alveoli that drives the bulk flow of gas in between bulk flow and diffusion because the and out of the lungs. In addition the uploading fine particles suspended in the air diffuse much to the Miranda Fraser professorship in Pathology at McGill in of O2 from the alveolar gas into the pulmonary more slowly than the gases they are suspended 1975. capillary blood and downloading CO2 from the in. Preliminary evidence in support of this circulating blood into the alveolar gas, create hypothesis that was obtained using a novel In September 1977 Dr. Hogg and Dr Peter Pare moved from McGill to the University of concentration gradients that move CO2 toward method that combines preoperative and post British Columbia where they founded the UBC Pulmonary Research Laboratory at St Pauls James the mouth and O2 toward the alveolar surface. operative specimen CT scans with micro CT and Hogg Hospital in . That was renamed the Center for Heart and Lung Innovation in Further the semi dichotomous branching system histology will be presented. 2010 and currently accommodates more than 20 principal investigators and supports University of British of the airways contributes to the transition from approximately 250 undergraduate, graduate and post doctoral students with their Columbia technical and administrative support staff. Dr Hogg has won several prestigious awards Gairdner Wightman including appointment as an officer of the Order of Canada 2005, Honorary Degrees from laureate UBC, McMaster and McGill Universities and the Gairdner Whiteman award for lifetime contributions to Canadian Medicine. Dr. Lea Harrington is a Professor in the Department of Medicine Critically Short Murine embryonic stem cells (mESCs) are well chemical inhibitors of the polycomb repressive at the University of Montreal, and a Visiting Professor at the Chromosome Ends suited to the study of stem cell fate as they can complex 2 (PRC2) further destabilized mTert-/- University of Edinburgh. Dr. Harrington studies the mechanisms be readily manipulated genetically and assessed ESC differentiation. Quantitative RNA profiling Impact Stem Cell by which chromosome ends, or telomeres, are elongated and for pluripotent properties. In mESCs lacking revealed that, despite the ability of mTert-/- Fate the telomerase reverse transcriptase mTert, ESCs to partially differentiate, the cells retained protected by the enzyme telomerase. Her laboratory employs telomere attrition normally serves to impede a propensity to re-acquire pluripotency, and several models, from yeast to mammals, to reveal unexpected cell division. Paradoxically, in differentiated formed teratomas in vivo with a remarkable consequences of limiting telomerase function in aging, mESCs that normally do not divide, short similarity to undifferentiated, wild-type ESCs. telomeres promote cell division. This aberrant Our data establish a direct link between two cancer, or stem cells. state is marked by global DNA hypomethylation, properties common to aging and cancer—short and is rescued by ectopic de novo DNA telomeres and methylome perturbations. If this Dr. Harrington obtained her M.Sc. at the University of Toronto (1990) and her Ph.D. at methyltransferase expression. Histone H3 relationship is conserved in humans, telomere SUNY, Stony Brook (1993). She led a research group at the Ontario Cancer Institute from methylation at lysine 27 (K27) is also reduced reparation may improve the ability of cancer 1995 until 2007, and in Edinburgh from 2007-2011. She has been honored with a Terry at pluripotency gene promoters in mTert-/- cells to respond to therapies that employ Fox Young Investigator award from the National Cancer Institute of Canada (2001), and ESCs, and inhibition of H3K27 methylation using differentiation induction. a Howard Hughes Medical Institute International Scholar Award (2007). Dr. Harrington Lea serves on the editorial boards of two scientific journals, and is a regular member of a Harrington National Institutes of Aging study section. She is editor of a textbook on the Basic Science of Oncology. Dr. Harrington has enjoyed the opportunity to work with over 50 students, Université de postdoctoral fellows, or associates, including several who have pursued careers in aca- Montréal demic, industry or governmental positions. Chair of the Gairdner Medical Review Panel Dr. Hakim was first an engineer and at the age of 29 began The Canadian The Canadian Stroke Network, working with his medical training. In 1979 he completed his residency in Stroke many Canadian partners, notably the Heart and Neurology at the Montreal Neurological Institute at which Stroke Foundation, was able to convert stroke Network: time his research career began. from a condition that handicapped the affected Bringing individual for life, into a preventable, treatable Since coming to Ottawa in 1992, Dr. Hakim has held the following positions: Knowledge to and repairable condition. I will describe how 1992 – Present Neurologist, The Ottawa Hospital and the University of Ottawa Canadian scientists, clinicians, paramedics, 1992 – 2001 Division Head, Division of Neurology, The Ottawa Hospital Society and municipal and provincial leaders in health 1992 – 2001 Director, Neuroscience Research Institute, University of Ottawa care all worked together to convert knowledge 1999 – 2014 CEO and Scientific Director, Canadian Stroke Network generated from laboratory and clinical research 2001 – 2008 Founding CEO & Scientific Director, Heart & Stroke Foundation to the public domain, and bring about a change Centre for Stroke Recovery in attitudes about this condition. The talk will 2001 – 2015 Director, Neuroscience Research Program, Ottawa Hospital Research Institute also describe the challenges still remaining in 2010 – 2014 Founding Director, University of Ottawa Brain & Mind Research Institute the field of stroke Dr. Hakim has received many honours during his career some of which are: 2007 – The Thomas Willis Lifetime Achievement Award, from the American Stroke Antoine Association & Appointed Officer of the Order of Canada M. Hakim 2009 – Heart and Stroke Foundation announced the "Dr Tony Hakim Innovative Stroke University of Ottawa Research Award” an annual grant of $100,000 Gairdner Wightman 2013 – Inducted into the Canadian Medical Hall of Fame laureate 2017 – Canada Gairdner Wightman Award Dr. Hakim has recently authored a book, titled 'Save Your Mind – Seven Rules to Avoid Dementia' which is published in both English and French. Alternatively Activated Macrophages Drive Browning of White Adipose Tissue Via IL-6 During Hypermetabolic States Abdikarim Abdullahi1, 2, Christopher Auger2, Mile Stanojcic1, Marc Jeschke1, 2, 3

1Faculty of Medicine, Introduction: The study of white adipose tissue (WAT) ‘browning’ has become a ‘hot topic’ in various POSTER University of Toronto; metabolic conditions (diabetes, obesity), because of the idea that WAT browning might be able to facilitate weight loss and improve metabolic health. This phenomenon called ‘browning’ entails a process 2 Sunnybrook Research by which white adipose tissue converts into a fat burning engine. However, this view cannot be translated Institute; 3Ross Tilley Burn into all areas of medicine. In hypermetabolic patients (e.g. burns, cancer) the browning of WAT has Centre, Sunnybrook presented substantial clinical challenges related to cachexia, atherosclerosis, and poor clinical outcomes. Although browning appears central in trauma, burn, or cancer induced hypermetabolic catabolism, the Hospital, Toronto, ON PRESENTATIONS mediators regulating this adverse switch in WAT is unknown. Using both clinical samples from burn patients and a mouse burn injury model of hypermetabolism, we sought to identify the efferent thermogenic circuitry that regulates burn injury induced browning. Methods: WAT and blood samples were collected from patients admitted to the Ross Tilley Burn Centre at Sunnybrook Hospital. Wild type, CCR2 KO, and IL-6 KO male mice were purchased from Jax laboratories 2017 >>> and subjected to a 30% total body surface area (TBSA) burn injury. WAT and serum collected was analyzed for browning markers and metabolic state via histology, gene expression, mitochondrial respiration, and resting energy expenditure (REE) assays. Results: Here, we show that burn-induced hypermetabolism is mediated by increased macrophage infiltration, interleukin 6, and M2 polarization in the fat of post burn rodents and humans. Genetic loss of the chemokine CCR2 (KO), responsible for macrophage migration to the adipose, and IL-6 (KO) signaling impairs burn-induced browning. Conversely, administration of recombinant IL-6 into mice genetically deficient in IL-6 production (IL-6 KO) recovers browning and induces hypermetabolism. Mechanistically, IL-6 alternatively activates macrophages recruited to burn-stressed subcutaneous white adipose tissue (scWAT), which induces tyrosine hydroxylase expression and catecholamine production, factors required for browning of scWAT. Lastly, pharmacological inhibition of the IL-6 receptor attenuates browning and improves postburn hypermetabolism. Countermeasures Against R-Loop-Driven Disease Mechanisms Identified Through Dual Interrogation of Yeast and Human Systems Karan J. Abraham1, Janet N.Y. Chan1, Jayesh S. Salvi1, Brandon Ho2, Amanda Hall1, Elva Vidya1, Samuel A. Killackey1, Jeffrey E. Lee1, 3, Grant W. Brown2, Karim Mekhail1,3

Conclusion: Together, our findings uncover IL-6 as a key signal for burn-induced browning through its 1Department of Background: Dietary calorie restriction (CR) promotes longevity and healthy aging from unicellular yeast effects on macrophage polarization, and designate IL-6 as a promising target for persistent Laboratory Medicine and to humans, globally rewiring metabolic and chromatin-modifying networks in ways that extend life. A hypermetabolism observed in burn patients. void exists, however, between these beneficial physiological shifts and the biochemical signals that Pathobiology, Faculty of ultimately coordinate such diverse processes. We recently reported that CR supports lifespan and Medicine, University of genome integrity of yeast cells by suppressing DNA-destabilizing associations between RNA and DNA Toronto; 2Department of called ‘Rloops’. Intriguingly, the toxic accumulation of R-loops is increasingly being linked to numerous debilitating diseases including neurodegenerative disorders and cancer, but countermeasures against Biochemistry and such processes have yet to be developed. Donnelly Centre, Methods/Results: Here, we combine yeast genetics with innovative approaches (CRISPR-Cas9, DNA-RNA University of Toronto; immunoprecipitation, confocal microscopy) in human cells to uncover a tunable and evolutionarily conserved mechanism that can be exploited to combat R-loops. Specifically, we show that CR induces 3Canada Research Chairs magnesium transporters (Alr1/2) and intracellular bivalent magnesium (Mg2+) ions, which promote Program, Toronto, ON genome integrity by suppressing R-loop pileups at fragile DNA loci. Importantly, exogenous magnesium supplementation is sufficient to mimic CR, and restores R-loop suppression, genome stability, and lifespan in yeast cells lacking Pbp1, a conserved RNA-binding protein linked to several fatal neurodegenerative disorders. Unexpectedly, Mg2+ exerts these beneficial effects independently of canonical chromatin and RNA control mechanisms, instead operating through Mg2+-sensitive enzymes directly targeted to R-loop-accumulating DNA loci. Similarly, human cells deficient in ATXN2, the human orthologue of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg2+ via analogous mechanisms, despite a billion years of evolutionary separation. Conclusion: We postulate that Mg2+ ions are biochemical effetors of beneficial CR and that Mg2+ or pharmacological CR mimetics may be used to counter R-loop-driven defects relevant to a wide-variety diseases. Effect of Stilbenoid Polyphenols on Aberrant Cerebral Artery Morphology and Mechanics in the Spontaneously Hypertensive Heart-Failure (SHHF) Rat C. Acosta1, 2, D.I. Lee2, 3, C.M. Anderson1, 4, H. Anderson2, 3

1Department of Introduction: Hypertension is an important risk factor for cognitive decline and dementia. Further Conclusion: SHHF middle cerebral arteries exhibited a combination of eutrophic and hypertrophic Pharmacology & adding risk of heart failure per se then cumulatively increases the possibility of cognitive decline and remodeling. Stilbenoids improved vascular morphology and geometry-independent stiffness promotes progression to bona fide dementia. This may be due, at least in part, to altered morphology and independently of blood pressure lowering, suggesting direct actions on the arterial wall. The ability of Therapeutics, University mechanical properties of cerebral arteries and arterioles. We hypothesized that hypertension combined pterostilbene to entirely normalize wall component stiffness may be related to its reported superior of Manitoba; 2Canadian with added risk of heart failure (as found in the SHHF rat) confer morphological and mechanical bioavailability. We conclude that further research is warranted on stilbenoid polyphenols as an adjunct Centre for Agri-Food aberrations in middle cerebral arteries. Resveratrol (trans-3, 5, 4’-trihydroxystilbene) is a stilbenoid to current cardiovascular treatment regimens that might reduce the risk of cognitive decline. polyphenol that is purportedly linked to improved longevity, cardiovascular health, and benefits in Research in Health and Alzheimer’s disease. Although resveratrol is well-tolerated in humans, it is readily metabolized and Medicine, St. Boniface exhibits low bioavailability. Thus, we also queried whether resveratrol analogues would exert greater Hospital Research Centre; vasculoprotective effects. Methods: Sprague-Dawley (SD) and SHHF rats (n=8) were treated for 8 weeks by gavage with vehicle 3College of Pharmacy, control (C) or low doses (2.5 mg/kg/d) of resveratrol (R), pterostilbene (P), and gnetol (G). Blood pressure University of Manitoba; (BP) was measured by tail-cuff plethysmography. Middle cerebral arteries were isolated and mounted on 4Neuroscience Research a pressure myograph. Lumen and media dimensions were measured and used to calculate various Program, Kleysen Institute parameters using previously described formulae to determine vascular geometry and mechanical properties of the arteries. for Advanced Medicine, Results: Systolic BP increased in the SHHF rat (196±3 mm Hg vs. SD 142±7 mm Hg, p<0.01), and was Winnipeg Health Sciences unaffected by stilbenoid treatment. SHHF arteries exhibited increased media-to-lumen ratios (15.9±2.4 Centre and College of vs. SD 9.0±0.5, p<0.05), and were associated with remodelling and growth indices of 58% and 44%, respectively. All three stilbenoids reduced media-to-lumen ratios (SHHF-R 12.3±1.7, SHHF-P 12.8±1.1, Medicine, University of SHHF-G 13.2±1.6, p<0.01). Wall component stiffness (i.e. slope of elastic modulus vs. stress) was Manitoba, Winnipeg, MB augmented in SHHF (18.2±2.9 vs. SD 6.1±0.9, p<0.01), and was attenuated in part by resveratrol and gnetol (SHHF-R 11.8±1.1, SHHF-G 11.5±1.0, p<0.05 vs. SD and p<0.01 vs. SHHF-C) and completely by pterostilbene (SHHF-P 9.4±1.1, not significant vs. SD and p<0.0001 vs. SHHF-C). Insulin Signalling Enhances AMPK Activity, Mitochondrial Function and Neurite The Mouse Cytomegalovirus M12 Glycoprotein Controls Innate Immune Recognition by Outgrowth in Adult Sensory Neurons Direct Engagement of the NKR-P1B and NKR-P1C (NK1.1) Receptors Mohamad Reza Aghanoori1, 2, Paul Fernyhough1 Oscar A. Aguilar1,2, Richard Berry3, Mir Munir A. Rahim4, Johanna J. Reichel5, Branka Popovic5, Miho Tanaka1,2, Zhihui Fu3, Gautham R. Balaji3, Timothy N.H. Lau1,2, Megan M. Tu4, Ahmad B. Mahmoud4, Christina L. Kirkham1,2, Aruz Mesci1,2, Astrid 1Division of Introduction: Depressed mitochondrial function has been proposed as a key mediator of peripheral Krmpotic5, David S.J. Allan1,2, Andrew P. Makrigiannis3, Stipan Jonjic5, Jamie Rossjohn3, James R. Carlyle1,2 Neurodegenerative neuropathy. There is down-regulation of AMPK (master regulator of cell bioenergetics), mitochondrial gene expression and function in dorsal root ganglia (DRG) in animal models of type 1 and type 2 diabetes. 1 Disorders, St Boniface We hypothesized that loss of direct insulin signaling in diabetes contributes to loss of AMPK activity and Dept. of Immunology, Introduction: Natural killer (NK) cells represent an innate lymphoid cell (ILC) subset responsible for the Hospital Albrechtsen mitochondrial function in DRG neurons leading to development of sensory neuropathy University of Toronto; early control of cells undergoing pathological changes, including cancer and infection. Such target cells are detected via alterations in self and non-self surface markers recognized by functionally paired Research Centre; Methods: Adult DRG neurons were cultured from age-matched control or streptozotocin (STZ)-induced 2Sunnybrook Research type 1 diabetic rats. Neurons treated with/without insulin underwent expression analysis of genes linked germline-encoded NK cell receptors. Murine cytomegalovirus (MCMV) is a herpesviral pathogen that has 2Department of Institute, Toronto, ON; to insulin signalling, assessment of mitochondrial respiration and quantification of neurite outgrowth. co-evolved to endure chronic and latent infections in mice, largely via the orchestrated production of Pharmacology & For in vivo work DRGs from age-matched control, STZ-induced and low dose insulin-implanted diabetic 3Dept. of Biochemistry immunoevasins that directly target NK cell receptor-ligand interactions. Methods: We employ a number of methods ranging from knockout mouse lines, protein biochemistry, Therapeutics, rat, where hyperglycemia was not affected, were analyzed for mitochondrial protein and mRNA and Molecular Biology, expression, cytochrome c oxidase activity and localization of mitochondrial Cox1 bioinformatics analysis, CRISPR-Cas9 systems, reporter cell assays, surface plasmon resonance (SPR), in University of Manitoba, Monash University, Results: Insulin treatment (10 or 100nM for 2-24h) of cultured neurons increased AMPK and P70S6K vitro and in vivo viral infection, and cytotoxicity assays to reveal the function of a newly discovered Winnipeg, MB phosphorylation. Insulin elevated mitochondrial gene expression, mitochondrial respiration and neurite Melbourne, Victoria, immunoevasin. outgrowth. Insulin-implanted diabetic animals exhibited correction of hind paw thermal insensitivity. In Australia; 4Dept. of Results: Here, we identify an MCMV-encoded m02 family member, m12, that restrains NK cell effector function by directly engaging the NKR-P1B inhibitory receptor. Remarkably, m12 also weakly interacts DRG of diabetic rats there was suppressed expression of mitochondrial genes, reduced cytochrome c Biochemistry, oxidase activity and abnormal mitochondrial distribution and all were restored by low dose insulin with the prototypical NK1.1 antigen and stimulatory orphan receptor, NKR-P1C. Notably, wild-type Microbiology and MW97 MW97 Smith therapy MCMV , an m12- deficient (Δm12 ) mutant, and an m12 revertant virus showed differential –/– Conclusion: Insulin elevates protein synthesis and mitochondrial function in sensory neurons leading to Immunology, University of viral titers in vivo upon infection of B6-strain wild-type (WT) but not NKR-P1B-deficient (Nkrp1b ) mice. In addition, polymorphisms in both the MCMV m12 gene product and host NKR-P1B/C alleles differentially neuronal growth and improved clinical neuropathy endpoints. Thus, insulin and its signaling should be Ottawa, Ottawa, ON; considered as a promising therapeutic target for prevention and even reversal of neurodegeneration in impact NK cell recognition, suggestive of functional co-evolution during host-pathogen interactions. 5Department for diabetic patients. Conclusion: The identification of m12 as a viral decoy that interacts with both the inhibitory NKR-P1B and Histology and stimulatory NKR-P1C receptors expands our understanding of innate immune evasion by MCMV, and Embryology, University of displays host-pathogen evolution at the molecular level. Rijeka, Rijeka, Croatia SMaRT Human Neural Stem Cells to Degrade CSPGs and Optimize Regeneration of the Food Insecurity May Lead to Incomplete HIV Viral Suppression and Less Immune Chronically Injured Cervical Spinal Cord Reconstitution Among HIV-HCV Co-Infected People Christopher S. Ahuja1, 2, 3, Mohamad Khazaei1, Priscilla Chan1, Jainetri Merchant1, Sara Baig1, Ahad Siddiqui1, Michael G. Wusiman Aibibula1, Joseph Cox1, 2, Anne-Marie Hamelin1, Ashley I Naimi3, Taylor Mclinden, Marina B Klein1, 4, Paul Brassard1, 4, 5 Fehlings1, 2, 3 1Department of Food insecurity (FI) is associated with unsuppressed HIV viral load and poor immune reconstitution among HIV infected people. It is unclear to what extent this association holds among HIVHCV co-infected 1Department of Genetics Introduction: Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) represent an Epidemiology, people. This study was conducted using data from the Food Security & HIV-HCV SubStudy of the Canadian and Development, exciting therapeutic approach to regenerate the traumatically injured spinal cord (SCI) by remyelinating Biostatistics and denuded axons, replacing neural circuits, and providing trophic support. Unfortunately, most individuals Co-Infection Cohort, which includes 17 treatment sites across Canada and follow up of co-infected Krembil Research are in the chronic phase of their injuries where dense perilesional chondroitin sulfate proteoglycan Occupational Health, people every six months. Fl was measured using the adult scale of Health Canada’s Household Food Institute, University (CSPG) scar significantly impedes both cell migration and neurite outgrowth. Scar-degrading enzymes McGill University; Security Survey Module (HFSSM) and was classified into three categories: food secure, moderate food insecurity and severe food insecurity. The association between Fl, HIV viral suppression, and CD4 count Health Network; have been found to enhance NSC-mediated recovery, however, nonspecific administration via intrathecal 2Public Health catheters can cause off-target effects. We aimed to generate a novel, genetically-engineered line of hiPS- was assessed using stabilized inverse probability weighting marginal structural model. A total of 725 2Department of Surgery, Department, CIUSSS du NSCs, termed Spinal Microenvironment Modifying and Regenerative Therapeutic (SMaRT) cells, capable HIV-HCV co-infected people with 1973 person-visits over 3 years of follow up contributed to this study. University of Toronto; of locally expressing a scar-degrading enzyme to improve functional recovery. Centre-Est-de-l ‘lie-de- At baseline, 23% of participants experienced moderate food insecurity and 34% experienced severe food insecurity. The proportion of people with undetectable HIV viral load was 75% and the median CD4 count 3Institute of Medical Methods: Using a non-viral transposon system, a scar degrading enzyme was genetically integrated into Montreal, Montreal, QC; hiPS-NSCs under a human promoter and a monoclonal line was generated by flow sorting. Enzyme was 480 (IQR 320-687). People experiencing severe food insecurity had 1.61 times (95% Cl : 1.27, 2.04) the Science, University of 3Department of expression and activity was extensively characterized by biochemical and cell culture assays. T-cell risk of having detectable HIV viral load and 0.897 fold (0.833, 0.965) increase in CD4 count compared to Toronto, Toronto, ON deficient RNU rats (N=60) with chronic C6-7 clip-contusion injuries were randomized to (1) hiPS-NSCs, (2) Epidemiology, University people who were food secure. Using novel statistical methods, these findings provide evidence of the negative impacts of food insecurity on HIV viral load and CD4 count among HIVHCV co-infected persons. enzyme-expressing hiPS-NSCs, (3) vehicle alone, (4) sham surgery (laminectomy alone). Behavioural of Pittsburgh, Pittsburgh, assessments are ongoing with an endpoint of 32 weeks. PA; 4Department of Results: Scar-degrading enzyme is expressed by transgenic hiPS-NSCs (SMaRT cells). The expressed enzyme appropriately degrades human CSPGs and allows neurons to extend into CSPG-rich regions in Medicine, McGill vitro. Conditioned SMaRT cell media also degrades rodent CSPGs in ex vivo injured cord cryosections. University; 5Division of While blinded behavioural assessments are ongoing, an interim histologic analysis of several animals Clinical Epidemiology, shows that grafted human cells are extending long (centimeter-scale) axons through rodent white matter. McGill University Health Conclusions: This work provides exciting proof-of-concept data that genetically-engineered SMaRT cells Centre, Montreal, QC can degrade CSPGs in vitro and that human NSC transplants can grow long axons in chronic cervical SCI. Effects of Resveratrol Analogues Gnetol and Pterostilbene on Cardiomyocyte Role of Scleraxis in Regulating Snail and Twist Gene Expression in Cardiac Fibroblasts Hypertrophy: Role of AMP-Activated Protein Kinase Danah S. Al-Hattab1 Hamza A. Safi1, Raghu S. Nagalingam2, Michael P. Czubryt1, 2 Bolanle Akinwumi1, 2, Neal Davies3 Hope Anderson1, 2 1Institute of Introduction: Cardiac fibrosis is one of the major consequences of heart disease. It is associated with 1College of Pharmacy, Introduction: Cardiac hypertrophy is the enlargement of the heart that occurs due to injury or stress and Cardiovascular Science; progressive cardiac stiffening due to abnormal deposition of extracellular matrix (ECM). This leads to inadequate filling capacity, pumping force, and improper electrophysiological rhythm of the heart, and Rady Faculty of Health has been identified as a potential target in the prevention of heart failure. Gnetol, pterostilbene and 2St. Boniface Hospital resveratrol belong to the group of polyphenols called stilbenes. Gnetol is found in the gymnospermous eventually heart failure. Upon injury, cardiac fibroblasts become activated to myofibroblasts, and deposit Sciences, University of plant genus Gnetum spp. while pterostilbene is found in Pterocarpus spp. and Vaccinum spp. (berries). Albrechtsen Research ECM elements, mainly fibrillar collagen type 1, as a part of the healing response process. We have Manitoba; 2Canadian Resveratrol is found in many plant species, most prominently, in Vitis spp. (grapes) and Vaccinum spp. Center; 3Department of examined the role of the transcription factor scleraxis in cardiac fibroblasts and found that scleraxis strongly up-regulates the expression of a variety of ECM proteins, including collagen1α2, the contractile Centre for Agri-Food (berries). Cardioprotective effects of resveratrol have been widely reported. However, reports about the Physiology and effects of resveratrol analogues such as gnetol and pterostilbene are lacking. AMP-activated protein protein αSMA (α-smooth muscle actin), the glycoprotein fibronectin, the basic helix-loop helix Research in Health and Pathophysiology, kinase is a key energy balance sensor that has been implicated in cardiac hypertrophy. Activation of transcription factor Twist1 and the zinc finger transcription factor Snail1. Both Snail1 and Twist1 are Medicine, St. Boniface AMPK via phosphorylation at THR-172 is protective and prevents the development of hypertrophy. Here University of Manitoba, highly up-regulated during fibrosis, and their activation induces N-cadherin up-regulation and down- regulates E-cadherin, which are the hallmarks of epithelial-to-mesenchymal transition (EMT). Our Hospital Research Centre, we queried first, the effects of gnetol and pterostilbene on cardiomyocyte hypertrophy and second, the Winnipeg, MB involvement of AMPK. published data has shown that overexpression of scleraxis induces the up-regulation of mesenchymal Winnipeg, MB; 3Faculty of Methods: Cardiomyocyte hypertrophy was induced in isolated neonatal ventricular cardiomyocytes markers (Twist1 and Snail1) in A549 epithelial cells through an unknown mechanism, as well as Pharmacy and using endothelin-1. The effects of gnetol and pterostilbene on two markers of hypertrophy were up-regulation of fibroblast markers including vimentin, fibronectin and collagen1α2, which are also Pharmaceutical Sciences, determined: cell size by computer-assisted planimetry and protein synthesis, measured using a Click-iT® induced in matrix remodeling in cardiac fibrosis. We therefore examined the role of scleraxis in inducing EMT progression through up-regulating Snail1 and Twist1 gene expression. University of Alberta, AHA (L-azidohomoalanine) Alexa Fluor® 488 Protein Synthesis HCS Assay kit. The role of AMPK was probed using compound C, which is an AMPK inhibitor, and lentiviral-based shRNA to achieve AMPK Methods: assaying Snail1 and Twist1 expression in relation to scleraxis overexpression in different types , AB knockdown. Levels of native AMPK and phosphorylated AMPK in cardiomyocytes were measured by of cell lines. Also, Identifying the E-Box(es) within the Snail1 and Twist1 gene promoters to which scleraxis conventional western blotting. binds using site-directed mutagenesis and luciferase reporter assay. Results: Similar to reported effects of resveratrol, gnetol and pterostilbene prevented cell size Results: Our data confirms that Scleraxis transactivates the Snail and Twist gene promoters through augmentation and protein synthesis induced by endothelin-1 in cultured neonatal cardiomyocytes. direct physical interaction with E-box sequences to govern gene expression. Gnetol and pterostilbene also activated AMPK by increasing phosphorylation at THR-172. Indeed, pre- Conclusion: This work shows how scleraxis may contribute to the induction of EMT and the progression treatment with compound C and shRNA knockdown of AMPK attenuated the ability of gnetol and of cardiac fibrosis. pterostilbene to inhibit cardiomyocyte hypertrophy. Conclusion: Gnetol and pterostilbene inhibit cardiomyocyte hypertrophy at least in part via activation of AMPK. Further study of the cardioprotective effects of these compounds is warranted. A Role of Semaphorin-3E in Regulating Natural Killer Functions in Breast Cancer? Elucidating the Role of Natural Killer Cell Derived IL-10 in Regulating T Cells D uring a Abdulaziz Alamri1, Abdel Gounni2, Sam K P Kung1 Sustained Viral Infection Alaa K. Ali, Seung-Hwan Lee 1University of Manitoba; Introduction: Breast cancer remains to be a leading cause of cancer death in Canadian women. 2Department of Semaphorin-3E (Sema-3E) signaling pathway has recently emerged to be an attractive therapeutic University of Ottawa, Introduction: Natural killer (NK) cells are innate immune cells directly involved in the killing of virus- approach in suppressing breast cancer cell. Recently, Sema-3E has been reported as a critical modulator Immunology, Department of infected cells and malignant cells. During a sustained viral infection, NK cells have been shown to of immune responses. Natural killer (NK) cells - an important innate cell that mediates direct anti-tumor undergo a functional switch. In the early phase, NK cells produce IFNγ, but as they get further activated Winnipeg, MB responses and shapes specific adaptive T cell responses. A fuller understanding of how Sema-3E regulates Biochemistry, and proliferate, they start producing IL-10 and become regulatory cells. Since IL-10 is a critical anti-tumor immune responses in breast cancers is therefore important in development of Sema-3E-based Microbiology and immunoregulatory cytokine to prevent immunopathology, I hypothesize that NK cell derived IL-10 is therapy. Immunology, Faculty of critical to protect the host from immune-mediated pathology elicited by an overwhelming T cell response Methods: Murine 4T1 breast cancer cells were injected subcutaneously into the breast region of the during an uncontrolled infection in perforin-deficient (PKO) mice. Medicine, Ottawa, ON Sema-3E +/+ and Sema-3E -/- mice. Phenotypes of NK cells were analyzed by FACS. NK-cell migrations Methods: Using a mouse model that has a perforin-deficiency in addition to an NK cell-specific IL-10- were examined in the Trans-well assays. deficiency (PKO-NKp46-Cre-il10fl/fl mice), I investigated the role of NK cell-derived IL-10 in the regulation Results: We observed that the 4T1 tumors developed significantly faster in the Sema-3E-/- mice when of T cell response during sustained murine cytomegalovirus (MCMV) infection. compared to Sema-3E+/+. The survival rate was lower in Sema-3E-/- mice when compared to their control Results: Our preliminary results demonstrated that during a persistent viral infection, NK cell derived mice. To examine whether NK cells percentage changed in the tumor microenvironment. Tumors were IL-10 is able to regulate the ability of T cells to produce IFNγ. Despite this increased expression of pro- collected from Sema-3E-/- mice and Sema-3E+/+ mice. We observed higher percentage of NK cells in inflammatory cytokine by T cells, the absence of NK cell derived IL-10 led to increased viral burden in the breast cancer microenvironment of Sema3E-/- compared to Sema-3E+/+ mice. In naïve mice (have no spleen. tumor), resting or IL-2 activated NK cells showed no apparent phenotypical changes in Sema-3E-/- mice Conclusion: This data suggest a model in which innate immunity regulates adaptive immunity to compared to Sema- 3E+/+. alleviate immunopathology caused by hyperactivated T cells. Our study will further elucidate the Conclusions and future direction: My data revealed a novel role of Sema-3E in suppressing tumor regulatory role of NK cells in infections in which immunopathology is prominent. growth in vivo. The tight regulation of Sema-3E signaling in NK and dendritic cells suggested that Sema-3E could a critical factor that regulates NK cells functions in anti-tumor immunity. Effect of Higher Vitamin D Dose on Osteopenia of Prematurity Neuregulin-1 Positively Modulates Glial Response and Improves Neurological Recovery Ebtihal Ali1, 2, John Baier1, Nada El Tobgy1, Mahmoud Torabi1 Following Traumatic Spinal Cord Injury Arsalan Alizadeh, Scott M. Dyck, Hardeep Kataria, Dung Nguyen, Kallivalappil Thomas Santhosh, Soheila Karimi-Abdolrezaee 1University of Manitoba; Background: Osteopenia of prematurity (OP) affects premature infants in NICU causing bone rarefaction 2Winnipeg Regional and spontaneous fractures. All neonates when compared with older individuals have higher relative Regenerative Medicine Introduction: Astrogliosis and neuroinflammation play pivotal roles in the secondary injury mechanisms requirements of calcium and phosphorus, which are critical components for bone structural integrity and Health Authority Program, Department of after spinal cord injury (SCI) with both pro- and anti-regenerative effects. We previously reported for the growth. Vitamin D is known to be protective in doses around 200-400 units per day. However, some first time that expression of Neuregulin-1 (Nrg-1) is dramatically and permanently downregulated in clinicians suggested that increasing the dose of vitamin D might results in more protection of infants, Physiology and acute SCI. Glial and immune cells express Nrg-1 receptors, ErbB2, 3, 4, suggesting the potential especially the premature ones. This study was conducted to investigate the effect of higher vitamin D Pathophysiology, ramifications of Nrg-1 dysregulation on glial activity and neuroinflammation. In this study, we sought to dosage on OP. University of Manitoba, unravel the role of Nrg-1 in regulating astrogliosis and immune response following SCI. We demonstrate Methods: A retrospective study include 109 infants less than 31 weeks gestational age. Vitamin D intake that Nrg-1 exerts anti-inflammatory and neuroprotective effects that can be culminated in improved Winnipeg, MB including the nutritional intake and the supplementations was collected. Chest X-rays were used to recovery of function following SCI. assess the OP. the data was collected at 8 weeks of post-natal age at which the x-rays are expected to Methods: In vitro, we used primary mixed culture of rat astrocytes and microglia activated by reveal the changes of OP and the infants are usually on full enteral feed and receiving full dose of vitamin lipopolysaccharide (LPS). In vivo, we employed a rat model of compressive SCI. We delivered recombinant D supplementation. Logistic regression model with ordinal outcome was used to detect the relation human Neuregulin-1 (rhNrg-1β1) intrathecally for up to 6 weeks. We conducted Western blotting, between the OP and vitamin D dose as categorical data. Vitamin D was used as categorical data <400 immunoprecipitation, gelatin zymography and stereology-based immunohistology at 1, 3, 7, 14 and 70 units, 400-800, 801-1000 units, 1000-1200 and >1200 units. days post-SCI. SCI rats were assessed for their locomotion and sensory recovery weekly. Results: Higher vitamin D >400 units, as a continuous data, is independently associated with increased Results: We show that Nrg-1 treatment attenuates several characteristics of activated glia such as OP (Odds ratio= 1.90, 95% CI =1.32-2.72, p =0.0005). While, vitamin D dose protective effect against the reduction in chondroitin sulfate proteoglycans (CSPGs), nitric oxide (NO), interleukin (IL)-1β and tumor OP is more when given as 400 unit than higher doses. For every one week increase in GA, the odds of OP necrosis factor (TNF)-α. In SCI, Nrg-1 promotes an anti-inflammatory immune response associated with improved toward milder category, being 2.22 times better for 400 unit and 1.86 times for 800 unit , 1.56 increased IL-10 and arginase-1 expression and a global decrease in pro-degenerative markers such as times for 1000 units and 1.3 times for 1200 and 0.09 times for infants getting higher than 1200 units per IL-1β, TNF-α and matrix metalloproteinases (MMP-2 and 9) after SCI. Nrg-1 also reduces astrocytic glial day. Lower gestational age is associated with higher OP while gender, parity, and the weight at 8 weeks scarring by decreasing GFAP and CSPGs expression in the SCI lesion. Mechanistically, we demonstrate of age was not a significant predictor of OP. that Nrg-1 effects on activated glia are mediated through ErbB2/3 heterodimer complex. Intracellualrly, Conclusion: Vitamin D is protective against OP in regular doses and higher doses are not Nrg-1 exerts its effects through downregulation of Myd88, a downstream adaptor of Toll-like receptors, and phosphorylation of Erk1/2 and STAT3. Importantly, Nrg-1 treatment significantly improves functional recovery following SCI. Conclusion: Our findings for the first time provide novel insight into the promising role of Nrg-1 in modulating activated glial and immune cells in repair and recovery following SCI. Autophagy Inhibition Directs the Epithelial to Mesenchymal Transition in Non-Small Motion Analysis for Video-Based Evaluation of Prosthetic Heart Valves Lung Cancer Cells Maryam Alizadeh, Alexandra Branzan Albu J. Alizadeh, A. Rezaei Moghadam, J. Thliveris, T. Klonisch, S. Ghavami Department of Electrical Heart disease is primarily caused by the malfunction in one or more of the four heart valves. One of the Dept. of Human Anatomy Introduction: Lung cancer is considered as one of the most important causes of cancer-related death & Computer Engineering, well-developed and effective therapeutic options is to replace the malfunctioned valve with a prosthetic heart valve (PHV). However, before implantation of these valves in human’s body, it is vital to control their and Cell Science, Max worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy University of Victoria, is a cellular process responsible for the recycling of damaged organelles and protein aggregates. quality to ensure their optimal characteristics. Quality control using visual data is a common approach for Rady College of Medicine, Transforming growth factor beta-1 (TGFβ1) is involved in Epithelial to Mesenchymal Transition (EMT) and Victoria, BC evaluation of the performance of the PHVs in comparison with the native valves -either in vivo or in vitro Rady Faculty of Health autophagy induction in different cancer models and plays an important role in pathogenesis of NSCLC. It environments. Such visual assessment provides the opportunity of studying some quality parameters that cannot be verified otherwise. However, manual visual assessment by an operator is prone to human Sciences, University of is not clear how autophagy can regulate EMT in NSCLC. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC cells. error and judgment. Therefore, it is of great interest to develop approaches for automatic visual analysis Manitoba, Winnipeg, MB Methods: Human NSCLC cell lines (A549 and H1975) were treated with TGFβ1 (5ng/ml) for 24, 48, 72 hrs. of heart valves. This study, therefore, aims to develop an automated, accurate and efficient framework for Western blots were done on cell lysates to investigate autophagy (LC3β lipidation, p62 degradation), EMT quantitative analysis of videos acquired from PHVs. The focus here is to develop/apply computer vision (E-cadherin and vimentin), and TGFβ1 canonical signaling (SMAD2 phosphorylation). Phenotype of the and image processing techniques to measure and evaluate critical quality parameters. This study cells were imaged upon TGFβ1 treatment by phase contrast microscopy. We confirmed autophagy and considers a variety of quality measures, such as detection of high frequency motion and motion EMT upon TGFβ1 treatment using immunocytochemistry (ICC) and Transmission Electron Microscopy symmetry. Our visual (video) data is acquired using a Pulse Duplicator Device built and performed by (TEM). We also used stable Atg7 knockdown and corresponding scramble NSCLC cells to investigate the ViVitro Labs (producer of cardiovascular device testing equipment). This widely-cited in-vitro cardiovascular role of autophagy inhibition in TGFβ1-induced EMT in NSCLC using immunoblotting and ICC. hydrodynamic testing system simulates the function of half of the human’s heart and produce visual data, Results: Our experiments showed that TGFβ1 can simultaneously induce both autophagy [as described while PHV is in function. Based on a modified deformable image processing model, we have developed by LC3β lipidation and p62 degradation using immunoblotting, autophagosome formation using TEM, an algorithm that is capable of detecting and tracking the free edges of valve leaflets by expanding the and punctuated LC3 using ICC] and EMT [as described by E-cadherin downregulation and vimentin detected opening area of the valve. Furthermore, we have studied fluttering (high frequency motion) and upregulation]. Furthermore, TGFβ1 treatment caused cells to acquire mesenchymal phenotype. Our symmetry of leaflets motion. The developed algorithm not only can be used for the purpose of heart results showed that EMT marker vimentin was significantly downregulated upon TGFβ1 treatment in Atg7 valve quality control, but also can be applied to various biological and non-biological motion analyses. knockdown cells [as described by downregulation of vimentin using immunoblotting and lower signal intensity in ICC] compared to corresponding scramble cells while E-cadherin was almost unchanged. Also, Atg7 knockdown cells treated with TGFβ1 had less migration compared to their scramble counterparts. Conclusion: Our results highlight the important role of autophagy as a positive regulator of TGFβ1- induced EMT in NSCLC cells. New autophagy blocking drugs may be useful in reducing NSCLC motility and metastasis in NSCLC. Effect of Maternal Antioxidant MitoQ Treatment on Vascular Function of Male and The Effects of Early Life Adversity on Epigenetic Regulation of the Oxytocinergic System: Female Offspring from a Rat Model of Intrauterine Growth Restriction (IUGR) From Childhood Maltreatment to Animal Models of Low Maternal Care Mais M. Aljunaidy1, 2, 3, Jude S. Morton2, 3, Raven Kirschenman2, 3, Patrick Case4, Christy-Lynn M. Cooke2, 3, Sandra T. Davidge1, 2, 3 Daniel Almeida1, 2, Laura Fiori1, Naguib Mechawaru3, Tie Yuan Zhang3, Michael Meaney3, Gustavo Turecki3

1Department of Introduction: A suboptimal environment in fetal life is linked to cardiovascular disease in adult life. 1McGill Group for Suicide Oxytocin is a mammalian neurohypophysial hormone, which acts primarily as a neuromodulator in the Physiology; 2Department Maternal hypoxia can lead to placental oxidative stress, which is associated with abnormal cardiovascular Studies, Douglas Hospital CNS. The early development of secure attachments, relationship quality, and the ability to regulate and function in the offspring. We hypothesize that maternal treatment with MitoQ, a mitochondrial manage emotions are all instances of psychological resources influenced by the oxytocinergic system. of Obstetrics and antioxidant, may prevent the development of cardiovascular disease in adult offspring. Research Center, Verdun, Previous studies have shown that early life adversity might act to disturb the oxytocinergic system during Gynecology; 3Women and Methods: Pregnant rats were injected with either MitoQ loaded nanoparticles (nMitoQ; 125 μM) or saline QC; 2Integrated Program critical developmental periods. A body of literature also supports alterations in the oxytocinergic system Children’s Health via tail vein injection on gestational day (GD) 15 (nanoparticles prevent MitoQ from crossing to the fetus). in Neuroscience, McGill in the aetiology of suicidal behaviour. Our research looks into the expression of the oxytocin receptor Rats then were subdivided into two groups exposed to either hypoxia (11% O ) or normoxia (21% O ) (OXTR) in the anterior cingulate cortex (ACC) of suicide completers who experienced childhood abuse, Research Institute, 2 2 University; 3Department from GD 15-21 (term; 22 days). Placentas were collected on GD 21 to assess placental oxidative stress level nonabused suicide completers, and non-psychiatric controls. As a second aim of our studies, we have University of Alberta, using 2’,7’-dichlorofluorescein (DCF) staining. In a different set of animals, rats were left to give birth and of Psychiatry, McGill investigated oxtr expression in the ACC of adult rats raised with low versus high maternal care. Similar to Edmonton, AB; aged to 7 months old. Vascular function was assessed in both male and female offspring using wire University, Montreal, QC our results in abused-suicide completers, rats raised with low maternal care are characterized by an myography. A 2-way ANOVA was used for data analyses. overexpression of oxtr in the ACC. Recently, several studies have identified the importance of epigenetic 4Musculoskeletal Results: nMitoQ treatment prevented placental oxidative stress at GD21 (normoxia+saline: 2.27 ± 1.55 mechanisms in the regulation of the oxytocinergic system, with an emphasis on DNA methylation. We are Research Unit, University arbitrary units (a.u.) vs. normoxia+nMitoQ: 3.15 ± 1.33 a.u. & hypoxia+saline: 13.34 ± 3.96 a.u. vs. therefore investigating methylation at the OXTR promoter via targeted bisulfite sequencing, expression of Bristol, Bristol, UK hypoxia+nMitoQ: 2.48 ± 1.06 a.u., , P=0.05). At 7 months of age, nMitoQ reduced mesenteric artery of miRNA’s involved in OXTR regulation, and protein expression of the receptor. sensitivity to the vasoconstrictor phenylephrine (PE) in male offspring (pEC50, PE: normoxia+saline: 5.76 ± 0.05 vs. normoxia+nMitoQ: 5.54 ± 0.02 & hypoxia+saline: 5.79 ± 0.02 vs. hypoxia+nMitoQ: 5.50 ± 0.05, P=0.0002); an effect which was not seen in female offspring. nMitoQ treatment did not affect mesenteric artery sensitivity to the vasorelaxant methacholine (MCh) in male offspring; however, nMitoQ reduced sensitivity to MCh in normoxic, but not hypoxic, female offspring (pEC50, MCh: normoxia+saline: 7.61 ± 0.05 vs. normoxia + nMitoQ: 7.26 ± 0.10, P= 0.01). Conclusion: nMitoQ treatment in mid gestation reduced sensitivity to adrenergic vasoconstriction in adult male offspring. In female offspring, maternal treatment with nMitoQ led to a reduction in sensitivity to MCh in the normoxic group only. This study suggests that maternal/placental treatment can alter vascular function of offspring later in life. Pharmacokinetics of Single Dose Creatine Monohydrate in Rats Brain Mechanisms of Associative Memory Deficits in Mild Cognitive Impairment Patients, Eman Alraddadi1, Ryan Lillico2, Jonathan L. Vennerstrom3, Ted M Lakowski2, Donald Miller1 With and Without Parkinson’s Disease T. Alrazi1, O. Monchi2 1Department of Background: Creatine is a natural ergogenic compound widely used by athletes to enhance performance Pharmacology and and increase muscle mass. In addition, supplementation with creatine monohydrate (CM) has been 1Departments of Clinical PD dementia (PDD) severely affects quality of life, interpersonal relationships, and independent activities suggested for musculoskeletal pathologies and neurological disorders. Although it is generally assumed Therapeutics, Neuroscience, Cumming of daily living; as well as is a huge economic burden to our society. Mild cognitive impairment (MCI) that the oral absorption of CM is nearly complete, little is known about its pharmacokinetic profile. defines patients with mild cognitive loss, but intact independent activities of daily living. It is usually a University of Manitoba; School of Medicine, Furthermore, as most therapeutic applications involve dietary supplementation with 20-­40 g/day of CM, transition phase from normal cognition to dementia, and is a major risk factor for the development of 2College of Pharmacy, consideration of potential dose-dependent effects on oral absorption is warranted. Objectives: To University of , AB, PDD2. MCI in PD (PD-MCI) is potentially important for early identification and intervention of PDD2. MCI determine the oral bioavailability of CM and the influence of dose on oral absorption. University of Manitoba; Hotchkiss Brain Institute patients show significant cognitive deficits in one or more cognitive domains, but are not demented. Yet, Methods: Pharmacokinetic studies were carried out in adult male Spraque Dawley rats. Rats were dosed the neural origins of memory, and particularly associative memory deficits in PD are not well known; nor Winnipeg, MB 13 Calgary, AB; 2Centre de orally (gavage) with either low dose (10 mg/kg) or high dose (70 mg/kg) of C-labeled CM. Blood is their impact on global cognition and decline; and their relationship with neuropsychiatric symptoms. 3Department of samples were removed at various time points (0-240 minutes), and muscle and brain tissue were collected Recherche de l’Institut Relationship between the associative memory deficits observed in PD-MCI and MCI-non-PD (MCI without at the conclusion of the study. Plasma and tissue levels of 13C-labeled creatine and 13C-labeled creatinine Pharmaceutical Sciences, Universitaire de Gériatrie PD), is not well understood. Tazrina will follow a total number of 200 participants (≥60 years), and were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The area under compare PD-MCI with PD-non-MCI/MCI-non-PD, and healthy controls, who will undergo a BOLD (blood University of Nebraska de Montréal, Montreal, the plasma concentration curve following oral dosing was compared to that obtained following i.v. oxygen level dependent) fMRI (functional magnetic resonance imaging) session while performing an 13 Medical Center, administration of C-labeled CM (10 mg/kg). QC; Departments of associative memory task (FAST=Face Associated Scene Task) that Tazrina has Omaha, NE Results: While the time to peak plasma concentrations were similar for both the high and low dose CM Clinical Neurosciences developed; a comprehensive neuropsychological assessment; as well as various clinical questionnaires of (60 minutes), peak concentrations were 13.6 ug/ml and 7.2 ug/ml, respectively. Absolute oral bioavailability and Radiology, University neuropsychiatric symptoms. Finally, correlations of BOLD fMRI activity patterns, and performance on the for low dose CM was approximately 47% while high dose CM was only approximately 15%. clinical and neuropsychological tests will be done. FAST can be a MCI diagnostic tool (20 minutes); and Conclusion: Our results suggest that the oral bioavailability of CM is less than complete and subject to of Calgary; Hotchkiss biomarkers found can be early predictors of PDD, considering MCI is the only effective therapy stage. dose. Brain Institute Calgary, AB; Department of Neurology and Neurosurgery, McGill University Montreal, QC Clonal Copy-Number Mosaicism In Insulin Reactive Cells In Type 1 Diabetes Somatostatin Receptor Subtype 2 Trafficking in Neuro-Endocrine Cells Maha Alriyami1, Quan Li 1, Luc Marchand1, Xiaoyu Du1, Martin Olivier2, Constantin Polychronakos1 Walaa Alshafie, Edward Pan and Thomas Stroh

1Endocrine Genetics Type 1 diabetes (TID) is due to the autoimmune destruction of the insulin-producing pancreatic beta McGill University, Introduction: Somatostatin Receptor Subtype 2 (SSTR2) is the major receptor subtype responding to Laboratory, McGill cells. Although Tl D depends on both inherited susceptibility and environmental factors, these cannot Montreal, QC Somatostatin (SST), an inhibitory neuropeptide widely expressed in the brain as well as in the periphery. explain that concordance in monozygotic twins is much less than 100% although by the age of onset, In central neurons, SSTR2 is well known to internalize to the Trans-Golgi Network (TGN) following agonist University Health Center, most twins share the same environment. Similarly, in the NOD mouse model, <50% of males have stimulation. For example in epileptic hippocampus, which is characterized by SSTergic hyperinnervation, Montreal Children’s diabetes, despite genetic identity and standardized environment. These observations point to stochastic SSTR2 is internalized for extended periods of time which desensitizes the neurons to the potentially Hospital Research determinants, one of which could consist of somatic mutations in the expanding autoreactive T -cell neuroprotective inhibitory effects of SST. In striking contrast, in pituitary endocrine cells, SST analogues lineages. In this study we test this hypothesis in both human patients and NOD mice by first concentrating treatment for various pituitary disorders i.e. acromegaly or pituitary tumors do not result in sst2 surface Institute, McGill University on copy-number aberration (CNAs) because they frequently occur in haematopoetic lineages, are easier down-regulation and there is no tolerance to SST analogues even after years of treatment. Recently, we 2Departments of to detect than point mutations and it is easier to deduce their functional consequences. We tested identified Somatostatin Receptor Subtype 5 (SSTR5) as a major regulator of SSTR2 trafficking and surface Medicine, Microbiology memory CD4+ cells from pancreatic lymph nodes of NOD mice and T-cells from blood of newly diagnosed expression in pituitary cells. In the Pituitary endocrine cell line, AtT20, that endogenously coexpresses TID patients, sorted for in vitro proliferation to proinsulin. CNAs were detected by high-resolution SSTR2 and SSTR5, we showed that the non-selective agonist [D-Trp8]-SST induces intracellular and Immunology, McGill comparative genomic hybridization using germline DNA as reference and confirmed by multiplex sequestration of SSTR2, whereas this receptor is maintained at the cell surface after treatment with the University, Montreal, QC ligation-dependent probe amplification (MLPA). In both the mouse and human samples we found CNAs SSTR2-selective agonist L-779,976. with highly non-random independent involvement of the same gene(s) across different samples. By the Experimental design and Results: In this study, we used immunolabeling and high resolution microscopy same methodology, CNAs were also found in lymphocytes expanded during normal host defense but to investigate the trafficking pathway of the endogenously expressed SSTR2 upon stimulation by either were significantly smaller. Moreover, Using TCR sequencing, the isolated lymphocytes are polyclonal, agonist in AtT20 cells. We found that both [D-Trp8]-SST and L-779,976 induce SSTR2 internalization via suggesting that the genomic alterations are happening in a marrow progenitor lineage prior to thymic transferrin-positive vesicles. However, SSTR2 internalized upon L-779,976 treatment undergoes fast selection. Our data strongly support a causal role for post-zygotic copy-number mutations in autoreactive recycling in the presence of the agonist, whereas receptors internalized by [D-Trp8]-SST recycle slowly T-cells in TID and, probably, other autoimmune diseases. Because they are not subjected to purifying only after washout of the agonist. By doing so, L- 779,976, also protects sst2A against the degradation selection through germline passage, we expect their biological effects to be much stronger than those observed under [D-Trp8]-SST stimulation as shown by western blotting. Importantly, unlike formerly of germ line risk variants and, being exclusive to the disease-causing cells, more amenable to reported, internalized SSTR2 does not reside entirely at the TGN. At least a large proportion is localized in individualized therapeutics. These findings challenge classical notions of autoimmunity and open roads a yet little defined syntaxin 6-positive non-TGN compartment as shown by dispersion of the TGN using to identify target genes for prevention and individualized therapeutics. Brefeldin A or Nocodazole. Conclusion: Our results indicate that L-779,976 protects against sst2A surface depletion by rapidly initiating sst2A recycling from a syntaxin 6-positive Juxta-TGN compartment. The identity of this compartment remained to be determined. Immune-Modulation by IL-17: Differential Alteration of IFN-γ and TNF-α Induced A Potential Lung Organoid Model to Study Abnormal Lung Development Resulting from Responses in Human Bronchial Epithelial Cells. Congenital Diaphragmatic Hernia Anthony Altieri1, 2, Hadeesha Piyadasa1, 2, Neeloffer Mookherjee1, 2, 3 Dustin Ameis1, 2, Naghmeh Khoshgoo1, 2, Phillip Snarr1, 2, Richard Keijzer1, 2, 3

1Manitoba Centre for Introduction: IL-17 is a critical pro-inflammatory cytokine that contributes to the severity and chronicity 1Department of Every day 150 babies are born with congenital diaphragmatic hernia (CDH) worldwide. CDH is a Proteomics and Systems of inflammatory diseases, including asthma. Previous studies have shown the synergistic effect of IL-17 Physiology and devastating disease resulting from a defect in the diaphragm causing the abdominal organs to herniate with other acute inflammatory cytokines. To better understand disease pathogenesis in chronic asthma, into the thoracic cavity, impairing lung development. This leads to two main pathologic features: Biology, Department of defining the effects of IL-17 on responses mediated by acute inflammatory cytokines such as IFN-γ and Pathophysiology, pulmonary hypoplasia and persistent pulmonary hypertension. Approximately one third of babies born Internal Medicine; TNF-α is essential. University of Manitoba; with CDH do not survive. Current understanding of the pathogenesis of CDH is poor, but it is known that 2Department of Methods: Human Bronchial Epithelial Cells (ATCC CRL-4051) were stimulated with recombinant human 2Children’s Hospital outcomes largely depend on the severity of their abnormally developed lungs. Improvements in TNF-α (20 ng/ml) and IFN-γ (30 ng/ml), in the presence and absence of IL- 17A/F (50 ng/ml). Quantitative outcomes and therapy should therefore come with a better grasp of the development of these lungs. The Immunology, University of Research Institute of real-time PCR (qRT-PCR) and specific ELISA assays were used to determine gene expression and protein animal models available today have yielded significant insights into the pathophysiology of CDH, and yet Manitoba, Winnipeg, MB; production respectively. Expression of transcription factors were monitored by Western Blots. Manitoba; 3Department they have largely failed to translate to clinical populations. These models induce diaphragmatic defects 3Canadian Respiratory Results: IL-17A/F synergistically enhanced TNF-α-induced pro-inflammatory cytokines e.g. IL- 6, of Surgery, Pediatrics and hernias either surgically or through nitrofen, a teratogen. Although these models phenotypically chemokines (Gro-α and IL-8), and antimicrobial peptide defensin hβd-2. In contrast, IFN-γ- induced IL-6 resemble human CDH, mechanistically there are critical differences. Abnormal lung development caused Research Network and Child Health, and chemokines were not altered by IL-17A/F. However, IFN-γ-induced hDB2 expression was enhanced in by CDH might be better recapitulated by a lung organoid model. Organoids are 3D tissue structures Ottawa, ON combination with IL-17. In contrast, IFN-γ-induced pro-inflammatory cytokine IL-33 was suppressed in Winnipeg, MB grown from stem cells, expressing multiple organ-specific cell types and self-organizing through spatially presence of IL-17. Modulation of IFN-γ-induced IL-33 production by IL-17 did not engage the canonical restricted lineage commitments. Additionally, they mimic some organ functions. Many organoids have IFN-γ signaling intermediates IRF1, IRF2, and IP10. been grown to resemble many organs, including the lungs. Stem cells derived from patients maintain Conclusion: IL-17A/F modulates TNF-α and IFN-γ induced pro-inflammatory responses disparately in disease-causing mutations and model disease when they differentiate and proliferate into lung human bronchial epithelial cells. organoids, and have applications including mechanistic studies of abnormal lung development and testing experimental drugs. Here, human and rat or mouse stem cells or progenitors will be cultured in a 3D media and driven in a stepwise manner by manipulating signaling pathways that mimic lung development. The procedure should produce airway and/or alveolar organoids, which will be validated by comparison to animal and human tissue. Investigating the Negative Impact of Embryonic Alcohol Exposure; Focus on Potential Unique Whole Blood MicroRNA Biosignature for Rheumatiod Arhtiris Biomarkers for Fetal Alcohol Spectrum Disorders (FASD) V. Anaparti1, 2, I. Smolik1, 2, X. Meng1, 2, N. Mookherjee1, 2, 3, H. El-Gabalawy1, 2, 3 Shayan Amiri1, 2, Shervin Pejhan1,2, Carl O. Olson1,2, Marc Del Bigio3, Wayne Xu2, Geoff Hicks1,2, James R. Davie2, Mojgan Rastegar1,2 1Manitoba Centre for Background: RA susceptibility risk is disproportionately high (~2-3fold) in Indigenous North American Proteomics and Systems (INA) tribes compared to other populations. Environmental, lifestyle & genetic factors account for <20% of observed disease variance suggesting contribution of additional risk determinants. Emerging evidence 1 2 Regenerative Medicine Introduction: Fatal alcohol spectrum disorders (FASD) are considered as common neurodevelopmental Biology; Rheumatic suggests small non-coding microRNAs (miRs) e.g. miR-155, miR-146a, miR-26b are key contributors to RA Program; 2Department of disorders in the Western countries affecting 2-5% of population in Canada. Focusing on the pathophysiology Diseases Unit, pathogenesis. In this project, we examined the role of miRs on RA incidence, and association with anti- of FASD, evidence indicates that prenatal exposure to ethanol can alter the DNA methylation status in Biochemistry and Medical Department of Internal citrullinated protein antibodies (ACPA), whose appearance precedes disease symptoms. We hypothesized neural stem cells (NSCs). Regarding that most people with FASD (about 80%) show no facial dysmorphic that differential expression of specific miRs associated with disease symptoms will facilitate RA transition Genetics; 3Department of Medicine; 3Department of features, finding a potential biomarker for the early diagnosis of FASD may open new avenues for in genetically susceptible first-degree relatives (FDRs). Pathology, Max Rady treatment strategies. Immunology, University of Methods: Whole blood and peripheral blood mononuclear cells (PBMCs) were obtained from age- Methods: The aim of this work is to establish and validate potential molecular biomarkers through College of Medicine, Manitoba, Winnipeg, MB matched ACPA+ RA patients (n=18), non-symptomatic ACPA+ FDRs (n=12) and ACPA- healthy controls genome-wide RNAseq studies by qRT-PCR, Western blot, and ChIP studies in vitro, and in vivo FASD model (n=12), who belonged exclusively to INA Cree-Ojibway communities of Northern Manitoba, Canada. Total Rady Faculty of Health systems, as well as human FASD samples (collaborations within a CIHR-funded team). Finally, using both RNA was isolated using miRVANA kit (Ambion). Expression of selected 32 miRs based on the published Sciences, University of in vivo and in vitro model systems, we will aim to identify and characterize methylation signatures in literature, and associated downstream mRNA targets, were monitored by quantitative real-time PCR, selected set of genes relevant to clinical aspects of FASD. Manitoba, Winnipeg, MB RNU48 and 18sRNA were used for input normalization for miRNA and mRNA expression respectively. Results: Our results showed that continuous (but not withdrawal or acute) exposure to ethanol is able to Results: Whole blood expression profiling identified 10 differentially expressed miRs in RA patients alter the expression of specific genes associated with cell fate commitment. Based on our genome wide compared to control subjects. Expression of miR-103a-3p was significantly up-regulated (~2.3-fold; RNA-seq studies, our data indicates that morphological changes are associated with altered expression p=0.0062), whereas that of imiR-16, miR-24, miR-29a, miR-125a-3p, miR-203, miR-222, miR-223, miR-150 of some specific genes in the cortical areas of brain during developing brain. We also have shown the and miR-346 were down-regulated in RA patients compared to controls. Increased miR-103a-3p potential role of sodium channels and some structural proteins in alcohol-related neurological disorders. expression was also confirmed in PBMCs from ACPA+ RA patients (~2 fold; p=0.0040) compared to Conclusion: Continuous exposure to ethanol during the development of brain is associated with the controls. Further, miR-103a-3p expression was increased in ACPA+ FDRs (>4 fold) compared to controls alteration of cellular morphology and the deregulation of genes associated with cell fate commitment. (p=0.0005), and ACPA+ RA patients (p=0.0149). miR-103a-3p expression was consistently elevated in Investigating the epigenetic marks on altered genes may result in finding biomarkers for early diagnosis ACPA+ FDRs, when we analyzed samples obtained at two independent time points (1year apart). and treatment of FASD. Consistent with this, expression of AGO1 and DAPK1 mRNA, downstream targets of miR-103a-3p, was decreased significantly (p<0.05) in ACPA+ FDRs compared to controls. Conclusion: This study defines a unique signature of dysregulated miRs amongst RA patients and their related FDRs within the INA cohort. Our results suggest a potential role of miR-103a-3p as a prognostic biomarker for pre-clinical RA. Does Moving More Improve Back Health?: Physical Activity and Chronic Back Disorders Maternal Well-Being and Equity in South India: Consulting the Community on What in Canadian Adults Really Matters for New Mothers in Underserved Regions Adriana Angarita-Fonseca1, 2, Catherine Trask2, Brenna Bath 2, 3 Dhiwya S. Attawar1, Prathibha Rai2, Arin Kar2, Mohan HL1, 2, Krishnamurthy Jayanna1, 2, James F. Blanchard1

1Department of Background: Chronic back disorders (CBD) have been the leading cause of global years lived with a 1Centre for Global Public Introduction: Progress in maternal, newborn and child health (MNCH) is slow among poor populations Community Health and disability. Although increased physical activity (PA) is often recommended for the prevention and Health, University of worldwide. Many women and infants are missing out on life-saving care during the period after birth. management of back pain, the relationship to the amount and intensity of PA is unclear. The inconclusive Such inequities may be traced to social structures including social norms and values that position people Epidemiology, U of relationship between PA and CBD supports the need for further research to clarify this relationship. Manitoba, Winnipeg MB; unevenly. Community interventions using community health worker (CHW) platforms present viable Saskatchewan; 2Canadian Purpose: The objectives of this study are to: 1) To determine the prevalence of CBD in Canada over time 2Karnataka Health strategies to reach the most disadvantaged groups, but have variable impacts along the continuum of Centre for Health and by gender, age, and geographical location; 2) To assess the associations between self-reported PA and the Promotion Trust, maternal and newborn care if approached through a health systems lens. What happens during this prevalence of CBD over time; 3) To examine the association between directly-measured PA levels and continuum for women in underserved communities is not well understood. Safety in Agriculture Bengaluru, India CBD; and 4) To assess the effect of PA measurement method (direct vs self-reported) on the relationship Methods: Using a critical theory paradigm, this study explores the structural context of care for new (CCHSA) U of between PA and CBD. mothers and CHWs in Koppal – a district in Karnataka with some of the poorest MNCH outcomes in south Saskatchewan; Methods and procedures: To complete this project, we received approval from Statistics Canada’s India. A mixed methods approach will use regional survey data on MNCH services and ethnography to Network to access secure detailed data through the SKY Research Data Center at the University of examine how service use varies, and what are the structural drivers of care respectively within the district. 3School of Physical Saskatchewan. We will use all 2001-2014 cycles of the Canadian Community Health Survey (CCHS) for A community consultation process to inform study design was initiated in February, 2017, in collaboration Therapy, U of objective 1; all nine cycles of the National Population Health Survey (1994/1995 to 2010/2011) for with Karnataka Health Promotion Trust partners and program staff embedded in MNCH programs in Saskatchewan, objective 2; and the Canadian Health Measures Survey (2007 to 2015) for objectives 3 and 4. The northern Karnataka. Informal consultation meetings were held with local women and community population of interest will be the survey population aged 18 to 65 years. Multivariate modelling , SK decision-makers in 3 villages in Koppal to: define priority MNCH issues; and contextualize concepts of approaches using generalized estimating equation (GEE) (objective 2) and logistic regression (objectives well-being and equity in the community. 3 and 4) will be applied to achieve the research objectives. Results: Community members identified pressing social, economic and political issues as priorities in Relevance and implications: This research will address an important public health issue using cross- MNCH including: domestic violence; affordability of facility deliveries; and limited access to basic sectional and longitudinal data, as well as using direct measurement of PA. Another strength of this amenities such as water. Water access emerged as a cross-cutting issue at individual, family and research is applying GEE to model PA level and prevalence of CBD during 16 years, which has not done community levels. New mothers expressed multidimensional understandings of well-being in terms of before. Findings will provide researchers, policy makers, and clinicians with knowledge about the knowledge, family relationships and freedoms. A profound loss of freedoms marked the everyday lives of relationship between PA and CBD to guide clinical practice, health services, policies and programs aimed these young women. at reducing the magnitude of this public health issue. Conclusions: Participation of community stakeholders early in research planning informed conceptualization of maternal well-being, and identified key axes of equity and sites of power in the local community. This work contributes to ethical practices in reaching those most marginalized, and privileging their voices in research planning and action for social change in MNCH. Establishment of a Tissue Bank for Human Fetal Lungs of Congenital Diaphragmatic Identification of Novel Receptor Tyrosine Kinases (RTKs) Regulating the Hippo Signaling Hernia Patients Pathway in Tumorigenesis L. Ayoub1, 2, 3, 4, D. Liu1, C. Stevanovici1, 5, B. Iwasiow1, 2, P. Snarr1, 2, D. Mulhall1, M. Levesque1, R. Keijzer1, 2, 3 Taha Azad, Helena Janse van Rensburg, Yawei Hao, Yang X

1Children’s Hospital Background: Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm and is Department of Pathology Introduction: The Hippo signaling pathway is a novel intracellular network that is dysregulated in a wide Research Institute of associated with abnormal lung development. The molecular pathophysiological cause of the disease is and Molecular Medicine, variety of malignancies and plays critical roles in many aspects of cancer biology including tumorigenesis, unknown. Our laboratory investigates the role\ of microRNAs in abnormal lung development and CDH. cancer stem cell self-renewal, drug resistance and metastasis. As an emerging pathway, the connections 2 Manitoba; Department We have mainly used animal model studies and the aim of this project was to establish a tissue bank with Queen’s University, between the Hippo pathway core components (i.e. MST, LATS) and potential upstream kinase regulators of Surgery, University of human CDH lung tissues coupled to the clinical information to validate our findings from animal studies. Kingston, ON are not well understood. Like Hippo signaling, dysregulation of receptor tyrosine kinases (RTKs) plays a Manitoba; 3Department Methods: We reviewed the pathology records of all deceased patients with CDH from 1980 till 2016. We pivotal role in many cancers. However, there has been no comprehensive screen for RTKs regulating the stratified our patients into different groups, according to associated anomalies. A patient with an isolated Hippo pathway in part due to the challenge of measuring Hippo pathway activity in a high-throughput, of Physiology and CDH would be preferable to a patient with a syndrome, or chromosomal abnormality. We then evaluated quantitative manner. Pathophysiology, the paraffin lung tissue blocks of these patients and confirmed their tissue quality by Hematoxylin & Methods: In our study, we have established a biosensor accurately monitoring the activity of the LATS University of Manitoba, Eosin staining. We have started to analyze the expression of miR-200b through in situ hybridization kinase based on a split-luciferase complementation assay. We used this LATS biosensor to screen for new experiments. RTKs regulating the Hippo pathway by gain of function and kinase-inhibitors screening. Winnipeg, MB; Results: We have identified a total of 45 CDH patients, 24 of those are isolated CDH cases and the Results: Through our biosensor-based screening, we have identified several novel RTKs (e.g. FGFR, 4 Department of remaining are non-isolated. In the isolated group, the majority of the patients were full term (75%), males METRK, VEGFR, etc.) that negatively regulate the Hippo pathway. Moreover, we have investigated the Physiology, Rabigh, King (62%), with a mean age 2.43 days ±. 4.5 SD. Most of the patients (66.7%) have died within the first day of molecular mechanisms underlying the regulation of the Hippo pathway by these RTKs and their roles in Abdulaziz University, life due to pulmonary complications. tumorigenesis. Conclusion: CDH is a common and fatal disease. Understanding the molecular biology of the disease will Conclusion: In conclusion, we have developed and validated the first biosensor for studying the Hippo Saudi Arabia; lead to better treatment options. The establishment of this unique biobank will help us validate our signaling pathway. We have also demonstrated how this simple and powerful biosensor can be used for 5Department of findings from animal studies. quantitative and accurate monitoring of Hippo signaling activity in living cells. We have also identified Pathology, University of new interactions between RTKs and Hippo signaling. These findings will inform novel strategies for the treatment of cancer using combination therapies targeting both RTKs and Hippo. Manitoba, Winnipeg, MB Characterization of PA3284-3281 Operon That Affects Type Three Secretion System Elucidating the Role of DNMT3B Variant 6 in Tumor Formation and Early Brain Expression in Pseudomonas Aeruginosa Development Sara Badr1, Yanqi Li2, Kangmin Duan 1, 2 Andrea Bajic1, Claudia L. Kleinman1, 2, Jennifer A. Chan3, Damien Faur1,·4, Nada Jabado1, 4

1Department of Medical Introduction: Many extrinsic regulatory pathways influence type three secretion system (T3SS) gene 1Department of Human Background: Embryonal tumors with multilayered rosettes (ETMRs) are highly aggressive brain tumors Microbiology, University expression. Mechanisms by which they exert control over T3SS regulation and virulence is poorly Genetics, McGill that mainly affect infants and children below four years of age, with a survival of less than two years from understood. Further studies are needed to discover gene regulation by each of these pathways and diagnosis, despite intensive therapies. Until recently, ETMRs were considered to be a subtype of primitive 2 2 of Manitoba; Department identify the signals to which they respond to. Metal sensing networks such as fur (iron) and zur (zinc) have University; McGill neuroectodermal tumors of the central nervous system (CNS-PNETs), but are now recognized as a distinct of Oral Biology, shown to be crucial in the regulation of pathogenicity and virulence genes. Pseudomonas aeruginosa have University and Genome entity within embryonal neoplasms. Our group and collaborators have identified a striking overexpression Winnipeg, MB developed sophisticated strategies for metal acquisition during infections to aid in growth and toxicity. Quebec Innovation of DNMT3B, a gene that encodes a de novo DNA methyltransferase and is essential for mammalian Here, we identified operon PA3284-8, which can upregulate T3SS toxin and effector protein ExoS when development, in ETMRs. A brain-specific transcript variant of DNMT3B, called variant 6 (V6), is Centre, Montreal, QC; overexpressed in Pseudomonas aeruginosa (PAO1 strain). Additionally, a significant stimulation of overexpressed in ETMRs. This variant is normally only expressed during a short developmental window PA3284-81 is observed under different conditions and concentrations of zinc, iron, calcium, and peracetic acid. 3Department of in the fetal brain. The canonical isoform of DNMT3B, variant 1 (V1), is not overexpressed in ETMRs. Methods: To test regulatory pathways, virulence factors, and environmental cues (ions, peracetic acid), a Pathology & Laboratory Hypotheses: 1. Overexpression of DNMT3B V6 promotes ETMR tumor formation through reprogramming promoter -reporter construction of PA3284-81 and overexpression strains are constructed through of DNA methylation patterns in early neurogenesis. 2. DNMT3B V6 has a distinct role in early brain Medicine, University of molecular cloning. Promoter activity (Gene expression detection) of obtained stains pKD3284-81 development. reporter, and pAK 3284-81 overexpression strain was measured by inoculating PAO1 with one of the Calgary, Calgary, AB; Aim 1: Assess the oncogenic properties of DNMT3B V6 by overexpressing V1 and V6 in cell lines likely to reporter fusions. Overnight cultures are then diluted and inoculated in a 96-well to measure the 4Department of model the ETMR cell of origin and by silencing DNMT3B in an ETMR-derived cell line. To overexpress luminescence in a Microplate Reader. Pediatrics, McGill DNMT3B V1 and V6, as well as the control gene (LacZ), we are using a tetracycline (tet) repressor-based Results: PA3284-81 showed an increase in expression under zinc depletion, peracetic acid and iron expression system that allows inducible overexpression of the gene of interest. Expression constructs are limited condition. Moreover, a decrease in expression was observed in a calcium limited condition University and the McGill delivered to cells by lentiviral transduction. We have overexpressed DNMT3B V1 and V6, and LacZ, in a cell (condition required for activation of T3SS). Overexpression PA3284-81 resulted in a decreased expression University Health Centre line derived from a CNS-PNET tumor. Functional assays are in progress to assess the effects of of rsmY, rsmZ controlling the RsmAYZ regulatory pathway. In addition to a decrease of phzA2, rhlR, pilG Research Institute, overexpression on cells. The experiment will also be performed in human neural stem cells which are virulence factors. On the other hand, phzA1 displayed an increase under PA3284-81 overexpression, likely to model ETMRs, based on the timing of V6 expression in the fetal brain. DNMT3B will be knocked which was confirmed with pyocyanin production assay. Montreal, QC out in ETMR patient-derived cells that overexpress V6, using the CRISPR/Cas9 system. The effects of V1 Conclusion: Overexpression of operon PA3284 -PA3281 upregulates T3SS gene expression probably and V6 overexpression or DNMT3B knockout on DNA methylation patterns (lllumina lnfinium through RetS/GacAS/RsmAYZ pathway. Environmental signals-peracetic acid and ions (Zinc, iron and MethylationEPIC BeadChip array and whole genome bisulfite sequencing), oncogenic properties of cells calcium) can regulate PA3284-81 expression affecting T3SS regulation. (cell proliferation and migration assays), and tumor initiating properties (mouse xenografts) will be Understanding LAG-3-Associated Defects in iNKT Cell Function in HIV Infection Allison Balasko1, Andrew Stalker1, Colin Graydon1, Jennifer Juno1, Julie LaJoie1, 2, Yoav Keynan1, 2, 4, Keith Fowke1, 2, 3 assessed. We expect that V6 is driving the DNA methylation pattern of ETMRs, which is distinct from other 1Department of Medical Background: Human immunodeficiency virus (HIV) is currently a worldwide issue, with 36.7 million HIV- brain tumors and normal brain, and that only V6 will confer oncogenic and tumor initiating properties to Microbiology, University infected individuals and an annual new infection rate plateauing out at about 2 million/year over the past cells. decade. The hallmark of chronic HIV infection is immune exhaustion, characterized by a loss of immune 2 Aim 2: Examine the role of DNMT3B V6 expression in neural differentiation. DNMT3B V6 will be of Manitoba; Community system effectiveness. Immune exhaustion is observed even in the presence of effective anti-retroviral overexpressed in human embryonic stem cells and the effects of the overexpression on the ability of cells Health Sciences, therapies (ART). Lymphocyte-activation gene (LAG-3), an inhibitory exhaustion marker expressed on to differentiate into neural lineages will be assessed. ETMRs are tumors of primitive morphology, University of Manitoba, immune cells, is associated with immune dysfunction observed in HIV patients. Our lab has shown that therefore, we expect V6 overexpression to cause a differentiation block in cells. increased LAG-3 expression on invariant Natural Killer T (iNKT) cells correlates with decrease cell Winnipeg, MB; 3University Significance: Understanding the role of DNMT3B V6 overexpression in the mechanism of ETMR formation functionality in HIV infection. iNKT cells are critical contributors to a healthy immune response to viral could improve diagnosis and have direct implications for the treatment of these deadly tumors, as DNA of Nairobi, Nairobi, Kenya; infection, therefore, dysfunctional iNKT cells render the immune system of HIV-infected individuals less demethylating agents have shown anti-tumor effects in other cancer types. 4Department of Internal effective. Hypothesis: In HIV infection, we hypothesize that blocking LAG-3 via an antibody blockade will restore Medicine, University of iNKT cell function. Manitoba, Winnipeg, MB Approach: Ethics are approved from the University of Manitoba. Blood samples will be collected from 3 study groups (n=25 for each group of HIV uninfected, HIV infected (+) immunological responders and HIV+ immunological non-responders) from the Manitoba HIV program clinical cohort established at the Health Sciences Center. Our lab has experience designing a LAG-3 blockade system. We will conduct short-term and long-term iNKT cell stimulations of peripheral blood mononuclear cells from the 3 study groups either with or without the LAG-3 blockade system. Through our established flow cytometry protocols, we will assess the iNKT cell population and their expression of both LAG-3 and cytokines which are important signalling molecules that represent the functional ability of these iNKT cells. Summary: The long-term goal of this project is to implement a LAG-3 blockade system to restore iNKT immune cell function and the overall strength of the immune system, establishing if this leads to better control of infection in HIV-positive individuals. Transcriptome Based Analysis to Identify Estrogen Regulated Genes in Primary Breast Music Therapy and Percussion for Persons With Dementia: A Systematic Literature Review Tumor, Tumor Adjacent Breast Tissue and Healthy Breast Tissue Jaclyn Bell Pratima Basak Chatterjee1, 2, 3, 5, Sumanta Chatterjee 1, 2, 5, Janice Saffneck3, Ed Buschel4, Afshin Raouf 1, 2, 5 Concordia University, Introduction: As cognitive and verbal skills decline, persons with dementia may still be able to participate 1RIOH, CancerCare Introduction: Breast cancer is the 2nd leading cause of cancer-related death among the Canadian Montreal, QC, Canada in musical experiences. Many different professionals recognize the value of music for persons with dementia. This research is a systematic literature review of the use of percussion instruments for persons Manitoba; 2Regenerative women. Early detection has significantly improved overall patient survival, yet no molecular biomarkers are available to diagnose the most prevalent form estrogen-responsive breast cancer at a pre-cancerous with dementia in music therapy and non-music therapy contexts. Medicine Program, stage. Therefore, identification of early pre-malignant changes in estrogen signaling in otherwise healthy Objectives: The purpose of the review was to identify and describe the use of percussion instruments for University of Manitoba; women is urgently needed. The current understanding of estrogen signaling is limited to estrogen persons with dementia across professions, as well as the goals and outcomes of its use. Methods: Electronic databases, journals, and books were searched using relevant key words. Sources 3Department of receptor positive (ER+) breast cancer cell lines and due to gap in knowledge about estrogen signaling in healthy human breast cells and in primary human breast tumors has hampered the ability to identify were checked for relevance, quality, and suitability to the selection and exclusion criteria, and then Pathology, Faculty of cancer-associated changes to estrogen signaling. In this study normal human breast cells, primary human categorized using a template with 9 categories. Medicine, University of ER+ tumor cells (BCT) and cells from the matching tumor-adjacent breast tissue(TAT) have been Results: From 2005 to 2014, many resources were published on the use of percussion for persons with dementia in the fields of music therapy, occupational therapy, music, nursing, and drama therapy. All Manitoba; 4Department compared to obtain cancer-specific changes to estrogen signaling. Methods and Results: Primary cells placed in estrogen-reduced 3 dimensional cultures were subsequently stages of dementia were represented in the literature with a variety of targeted goals, intervention of Surgery, Faculty of treated with estrogen or vehicle control for 24hr to extract RNA used to generate RNA sequencing designs, percussion instruments used, group versus individual contexts, and outcomes. Medicine, University of libraries. Comparative analysis of these one-of-a-kind gene expression libraries identified significant Conclusions: The findings may serve as a resource for clinicians, students, and researchers, and has the Manitoba; 5Department differences in estrogen signaling networks in normal vs cancer cells which had not been appreciated potential to increase knowledgeable use of diverse percussion interventions for persons with dementia. of Immunology, before. Estrogens regulated genes above or below 1.5 folds expression change were analyzed. More emphasis was placed on genes showing similar expression changes in presence of estrogen in TAT and University of Manitoba, the matching BCT. The Cancer-specific targets of estrogen signaling were further examined for their Winnipeg, MB association with overall patient survival and various clinical outcomes using the cancer-wide gene expression database, SurvExpress. The genes that showed high association with poor overall survival were further analyzed with Ingenuity Pathway analysis and David Functional annotation software. Preliminary analysis done with genes commonly upregulated between TAT and BCT showed 14-15 high risk gene (p<0.05) that had poor patient outcome in terms of overall survival, recurrence free survival etc. Further gene signaling Network analysis revealed their association with cell cycle, endocrine system disorders etc. Conclusion: Identification of specific molecular signatures associated with high-risk breast tissue would help in predicting the risk of tumor occurrence and serving as a guide to initiate early preventive therapy. Anoxia and Mucin Act in Combinatorial Fashion to Activate PA1611 and Increase Biofilm The Effect of Antidepressants on the Thioredoxin System Formation in Pseudomonas Aeruginosa Veni Bharti1, 2, Jun-Feng Wang 1, 2 Anjali Bhagirath Y1, 3, Deepti Somayajula1, 3, Kangmin Duan1, 2, 3 1Kleysen Institute for Introduction: Antidepressants are the medications that are used to treat major depressive disorder 1Department of Oral Introduction: Pseudomonas aeruginosa is a key pathogen in Cystic fibrosis [CF] associated morbidity and Advanced Medicine; (MDD). Although short term treatment with antidepressants increases monoamines in brain, but in clinical condition it takes almost 4-6 weeks for a depressed patient to stabilize the mood. The exact Biology and Manitoba mortality. Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that 2University of Manitoba, P. aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. mechanism behind the lag phase between administration of antidepressants and clinical signs of Chemosensory Biology Classically, of the two infection states for Pseudomonas, the acute state is characterized by increased Winnipeg, MB improvement is not known yet. Studies have indicated that oxidative stress underlies the pathophysiology Research Group; motility and toxic effector secretion system via the type three secretion system [T3SS]; whereas, the of MDD. Oxidative stress is the imbalance between reactive oxygen and nitrogen species produced as a byproduct of our metabolism and endogenous antioxidants of the body to combat this stress. Oxidation 2Department of Medical chronic state is characterized by increased biofilm formation. Previously, we have identified PA1611, a hybrid histidine kinase, as a key regulator for biofilm formation. Thus far, no external signals for PA1611 of cysteine by reactive oxygen and nitrogen species lead to reversible cysteine sulfenylation and Microbiology, Faculty of have been identified. In this study, we identify mucin and oxygen limitation as triggers for PA1611 nitrosylation respectively. Thioredoxin as an oxidoreductase can reverse cysteine sulfenylation and Health Sciences, activation and biofilm formation. nitrosylation. Methods & Results: Using immunoblotting analysis, we have found that chronic treatment with University of Manitoba; Methods: First, luxCDABE: gene fusions were used to study the expression of type three secretion effector exoS and downstream small RNAs RsmA/Y/Z in the presence of mucin. Real time-quantitative PCR antidepressants namely venlafaxine, desipramine and fluoxetine for 5 days significantly increased protein 3Biology of Breathing analysis was used to study expression of PA1611 and other effectors under anaerobic conditions with and levels of thioredoxin in culture HT22 mouse hippocampal cells. Using biotin switch and dimedone Group, Children’s Hospital without the presence of mucin. Further, to examine the role of PA1611 in cell survival under anaerobic methods, we also found that chronic treatment with stress hormone corticosterone for 5 days increased Research Institute of conditions, a live-dead biofilm assay was performed in PAK, PA1611 deletion strain (ΔPA1611) and cysteine sulfenylation and nitrosylation in HT22 cells, while chronic antidepressant treatment significantly inhibited corticosterone-increased cysteine sulfenylation and nitrosylation. Manitoba, Winnipeg, MB, complementation strain. Results: T3SS effector exoS showed decreased promoter activity in presence of mucin in LB as compared Conclusion: Our findings suggest that antidepressants inhibit cysteine oxidation by upregulating Canada to control. Under anoxia however, exoS expression was higher but repressed when mucin and anoxia thioredoxin system. were combined. PA1611 demonstrated increased expression in both mucin as well as oxygen limited conditions as measured by qPCR. Biofilm formation was enhanced in PA1611 expression strain in anoxia as compared to aerobic controls. Biofilms visualized by live-dead stain demonstrated that ΔPA1611 had more dead cells than control. Significance: In conclusion, this study provides first evidence that PA1611 senses two key components of CF lung environment, mucin and anoxia in P. aeruginosa to form biofilms. Thus, we suggest that these factors represent targets for therapeutic intervention. PA1611 represents significant genetic target founded on the proposal that successful treatment of the disease may require the disruption of anaerobic respiration within the airway mucus. Non-Canconical WNT Signaling Through FZD7 Limits the Luminal Progenitor Pool Size Suppression of Avb6 Integrin Expression by Polymicrobial Oral Biofilms Vasudeva Bhat, Afshin Raouf Jiarui Bi, Leeni Koivisto, Gethin Owen, Ya Shen, Markus Haapasalo, Lari Häkkinen, Hannu Larjava

Regenerative Medicine Introduction: The proliferation and differentiation potentials of Breast Epithelial Stem and progenitors Department of Oral Objectives: Periodontal diseases manifest by the formation of deep pockets between the gingiva and Program, Department of Cells (BESPCs) could be hijacked to produce luminal type tumors which make up the majority of breast Biological & Medical teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Integrin αvβ6, cancer tumours. Evidence suggests that the evolutionarily conserved Notch and Wnt signaling pathways an activator of anti-inflammatory transforming growth factor-β1 (TGF-β1), is highly expressed in the Immunology, University of play an important role in regulating Self-renewal, proliferation and differentiation of BESPCs. Interestingly, Sciences, Faculty of junctional epithelium (JE) that connects the gingiva to the tooth enamel. However, αvβ6 is strongly Manitoba; CancerCare these signalling pathways also play a vital role in regulating Breast Cancer Stem Cells function, which are Dentistry, University of diminished in the pocket epithelium (PE) in periodontal disease, and Itgb6-/- mice spontaneously develop Manitoba, Winnipeg, MB thought to be responsible to tumour development and maintenance. Therefore, understanding the British Columbia, severe periodontal disease. In this study, we explored whether bacterial biofilms can suppress αvβ6 molecular mechanisms that regulate the proliferation and differentiation of the BESPCs could provide integrin expression, whether this change aggravates inflammation, and what cellular mechanisms are Vancouver, BC new insights into how their altered-functions could lead to the development of luminal type breast involved in ITGB6 downregulation. cancer. We previously reported that NOTCH3 receptor (NR3)-signaling was required for the commitment Methods: Integrin αvβ6 expression in human JE/PE was analyzed by immunostaining. Wildtype and of the human breast epithelial bipotent progenitors to the luminal cell fate. Recently, we showed that Itgb6-/- mouse jaw tissue sections were histologically analyzed for inflammation. Gingival epithelial cells NR3 signaling specifically and uniquely regulates the expression of a Wnt receptor, FRIZZLED7 (FZD7). We (GECs) were exposed to extracts of oral bacterial biofilms, and their gene expression was analyzed by now demonstrate that NR3-FZD7 signaling axis is important to luminal cell fate determination of the real-time quantitative PCR. Protein levels and activation of cell signaling were analyzed by Western bipotent progenitors blotting. Chemical inhibitors and siRNAs were used for exploring the mechanisms of β6 integrin Methods: Human breast epithelial cells were lentivirally transduced to lack expression of FZD7 and the regulation and its role in inflammation, respectively. Statistical analysis was performed (ANOVA, Tukeys, differentiation potential of progenitor cells were measured by in vitro Colony Forming Cell (CFC) assay in p<0.05). presence of different Wnt ligands. Results: GECs exposed to biofilm extracts showed significant downregulation of β6 integrin mRNA and Results: A non-canonical Wnt signaling activator, Wnt7A, enhanced the commitment of human breast protein levels and upregulation of pro-inflammatory interleukin-1β. Suppression of ITGB6 expression by epithelial bipotent progenitors to the luminal lineage while a canonical Wnt ligand, Wnt3A failed to siRNA knockdown or inhibition of TGF-β1 signaling also increased their interleukin-1β expression. produce the same effect. Interestingly however, we found that loss of FZD7 expression in the luminal Biofilm-induced activation of mitogen-activated protein kinases (ERK1/2, p38, and JNK) and epidermal progenitors increased luminal colony number. This finding is further supported by our observation that growth factor receptor (EGFR), and inhibitors of ERK, JNK, and EGFR blocked the biofilm-induced ITGB6 the Fzd7 null mouse mammary glands contained increased luminal progenitors and enhanced branch downregulation. Similar to biofilms, EGFR ligands suppressed ITGB6 expression, possibly by increasing morphogenesis. Our findings then indicate that Wnt7A commits the NR3+FZD7+ bipotent progenitors to the levels of TGF-β1 repressor TGIF. the luminal cell fate by blocking FZD7 signaling. Our work so far shows that signaling through NR3-FZD7 Conclusions: Oral biofilms can introduce acquired αvβ6 integrin deficiency in GECs by suppressing signaling axis regulates the size of the luminal progenitor pool. It is inviting to hypothesize that TGF-β1 signaling through EGFR and MAPKs and promote an epithelial cell-drive pro-inflammatory alterations to this signaling axis could lead to uncontrolled expansion of the luminal progenitor pool, a response in periodontal disease. possible antecedent event to the generation of luminal type tumours. Ferroptosis Can Be Induced in Glioblastoma and Lung Adenocarcinoma Cells by the West Central African Pygmy and Non-Pygmy Populations: Exploration of Anthropometric Combination of Siramesine and Lapatinib and Physiometric Data Anna R. Blankstein1, 2, Shumei Ma2, Spencer B. Gibson1, 2 A. Blant1, F. Ramirez-Rozzi3, A. Froment2, 4, P. Verdu2, 3, T. J. Pemberton1

1Department of Introduction: Ferroptosis, a morphologically and biochemically distinct cell death pathway, is 1University of Manitoba, Introduction: Adult body size is central to the biology of all living organisms, influencing physiological Biochemistry and Medical characterized by iron-dependent accumulation of reactive oxygen species (ROS). The combination of Winnipeg, MB; and fitness characteristics. It is a complex trait and reflects the cumulative effects between biological and siramesine, a lysosome disruptor, and lapatinib, a dual tyrosine kinase inhibitor, has been shown to environmental factors. Central African hunter-gatherer populations, traditionally referred to as “Pygmies,” 2 Genetics, University of synergistically induce death in breast cancer cells. This cell death had characteristics of ferroptosis: it was Muséum National are a fascinating example at the lower extreme of human body size, especially when contrasted to Manitoba; 2Research blocked by the ferroptosis inhibitor ferrostatin-1 (Fer-1) and the iron chelator deferoxamine (DFO). The D’Histoire Naturelle markedly taller neighboring agriculturalist populations living in the same tropical environment. The Institute in Oncology and objective of this study was to determine whether lysosome disruptors and tyrosine kinase inhibitors (MNHN); 3Centre National overarching aim of our work is to explore genetic and phenotypic differences between Central African induced synergic cell death via the ferroptotic pathway in additional types of cancer. Pygmies and non-Pygmies to identify the genetic and biological determinants of contemporary Pygmy Hematology, Winnipeg, de la Recherche Methods: U87 (glioblastoma) and A549 (lung adenocarcinoma) cells were treated with siramesine and body size. Our research is divided into two broad stages: the first involves a comprehensive phenotypic MB lapatinib, separately or in combination. Cell death was measured by trypan blue exclusion. To determine Scientifique (CNRS); description of Pygmy and non-Pygmy differences (the topic of this poster) while the second will pair this whether ferroptosis was the mechanism of cell death, cells were pretreated with either Fer-1 or DFO, or 4Institut de recherche data with high-resolution genetic data on the same individuals. with exogenous iron chloride (an inducer of ferroptosis) before treatment with the drug combination. Methods: This study is an international and interdisciplinary collaboration between the University of pour le dévelopemment Prussian Blue staining and Calcein-AM assays were used to quantify the intracellular labile iron pool Manitoba and the MNHN and CNRS in Paris, France. Research authorizations were obtained from the following drug treatment. DHE and BODIPY fluorescence were used to measure changes in ROS and lipid (IRD), Paris, France appropriate government agencies in Cameroon and informed consent was obtained from all participants. peroxidation, respectively. Western blotting of iron regulatory proteins was used to examine changes in In May 2016, our group collected DNA, family history data and body size data from 376 individuals from protein expression. Statistical significance was determined by unpaired tests, comparing siramesine/ four distinct ethnic groups from Central (Tikar, Bezan) and Southeastern (Nzime, Baka) Cameroon. Adult lapatinib to the other conditions. body size data involved 21 anthropometric and 6 physiometric measurements. Immigrants and relatives Results: Treatment of U87 and A549 cells with the drug combination produced synergic cell death. were removed prior to data analysis. Data analysis of measurements obtained from Central African Pretreatment with Fer-1 or DFO decreased drug-induced cell death by approximately 35%, while Pygmies (Bezan and Baka) and non-Pygmies (Tikar and Nzime) was completed using bivariate correlations, pretreatment with iron chloride increased cell death by approximately 45%. The drug combination also exploratory factor analysis, and logistic regression methods using the R statistical analysis software. caused increases in the labile iron pool (2-3-fold), in ROS (2-fold), and in lipid peroxidation (2-fold). Conclusion: This work improves our current understanding of the Central African pygmy phenotype by Expression levels of iron regulatory proteins, transferrin, ferritin, and ferroportin did not change, while providing the most detailed view to date of phenotypic differences between Pygmy and non-Pygmy expression levels of the transferrin receptor and DMT1 were increased by 20-30%. populations. Once paired with genetic data in stage 2, we expect this work to provide new insights into Conclusion: In U87 and A549 cells, the combination of siramesine and lapatinib caused synergic cell the evolutionary origins of the contemporary pygmy phenotype as well as its biological and genetic death via a ferroptotic mechanism. Inducing ferroptosis is a potential therapeutic strategy for these basis. Subsequent field missions visiting other Pygmy and non-Pygmy populations will also increase the cancers with limited available treatment options. sample size and diversity of our existing dataset. Examination of MicroRNA-128-3p Regulation of Gene Targets During Prion Induced Neurodegeneration Amrit S. Boese1, 2, Aileen Patterson, Kathy J. Manguiat, Stephanie A. Booth1, 2

1Medical Microbiology Introduction: Prion diseases are fatal neurodegenerative diseases that include Creutzfeld-Jakob Disease Conclusion: MiR-128-3p levels are altered in hippocampal neurons in RML Scrapie prion infected mice. In and Infectious Diseases, in humans, bovine spongiform encephalopathy in cows, scrapie in sheep, and chronic wasting disease in primary hippocampal neurons, miR-128-3p is involved in glutamate signaling pathways and directly cervids. The etiological agent is PrPSc, which is the misfolded form of the normal host cellular prion targets GRIN2D and GRM5 genes. Max Rady College of protein, PrPC. During prion infection, the loss of neuronal synapses commences before clinical symptoms Medicine, University of appear, this loss can be reversed to improve cognitive and behavioural symptoms in infected mice. Manitoba; 2Zoonotic MicroRNAs (miR) are modifiers of synaptic structure and could be used to rescue synaptic loss. Additionally, miR-128-3p is abundant at the synapse and is altered in abundance in Prion, Alzheimer’s, Diseases and Special and Huntington’s diseases, indicating that it may have an overarching role in neurodegeneration. The Pathogens, National goal of this study is to elucidate miR-128-3p neuronal function during early and late stages of prion Microbiology Laboratory, infection. Methods: Using laser capture microdissection for CA1 hippocampal neurons and gradient Public Health Agency of ultracentrifugation to isolate synaptoneurosomes of the forebrain and hippocampus, miR-128-3p levels Canada, Winnipeg, MB were determined temporally in RML Scrapie prion infected CD1 mice. Algorithm predicted targets of miR-128-3p were evaluated using Target Scan Human and a select few were validated using Luciferase reporter assays. Primary hippocampal neurons from mice were used to increase or knockdown miR- 128-3p levels to examine glutamate signaling pathways and genes. Lastly, the effects of increasing miR- 128-3p on the proteome of primary hippocampal neurons was examined using mass spectrometry. Results: The levels of miR-128-3p increased during early stages of infection in both CA1 neuronal cell bodies and hippocampal synapses but not forebrain synapses. The Target Scan Human algorithm indicated GRIN2D and GRM5 are gene targets of miR-128-3p, this was confirmed by Luciferase reporter assay using the 3` untranslated regions (UTR) of these genes. Increasing miR-128-3p in primary hippocampal neurons resulted in decreased GRIN2D and GRM5 mRNA while the proteome remained largely unchanged as detected by mass spectrometry. In primary hippocampal neurons, chronic neuroprotective calcium influx and excitotoxic glutamate levels decreased miR-128-3p levels. PCGF6 Controls Dendritic Cell Responses to Inflammatory Micro Environments Does Resveratrol Protect Against Gestational Diabetes and the Risk for Heart Disease in Giselle Boukhaled1, 2, Logan Smith3, Martin Richer3, Connie Krawczyk1, 2, 3 the Offspring? Gabriel M. Brawerman1, 2, Troy J. Pereira1, 2, Stephanie M. Kereliuk1, 2, Bo Xiang1, 2, Mario A. Fonseca1, 2, Vernon W. Dolinsky1, 2 1Department of Dendritic cells (DCs) are professional antigen presenting cells of the innate immune system. They are Physiology, 2Goodman finely tuned to sense invading pathogens and danger signals in their microenvironment and are 1Diabetes Research Introduction: Current rates of gestational diabetes mellitus (GDM) are about 5-10% of pregnant women. specialized to initiate antigen-specific T cell responses. Activation of DCs is accompanied by rapid Cancer Research Centre; Envisioned and GDM increases the risk of cardio-metabolic disease in mothers, and also predisposes offspring to obesity, transcriptional reprogramming mediated in large part by epigenetic modification downstream of signal insulin resistance and heart disease. Resveratrol (Resv), a polyphenolic molecule produced by plants, has 3Department of Accomplished in transduction pathways. Context determines the type and extent of DC responses. Certain pro anti-oxidant characteristics and positive metabolic health effects. We hypothesize that 150 mg/kg Microbiology and inflammatory cytokines can paradoxically induce both potent DC activation and immunosuppression. Manitoba (DREAM) administration of Resv during pregnancy will prevent maternal glucose intolerance caused by a GDM This phenomenon is evident during chronic infection and tumorigenesis. When exposed to a Immunology, McGill Research Theme of pregnancy and thus protect the offspring from GDM-induced obesity and heart disease. proinflammatory microenvironment in these contexts, a subset ofDCs (iregDCs) increase the expression Methods: 6 weeks prior to mating, female rats received a high fat and sucrose (HFS) diet (45% kcal fat) to University, Montreal, QC Children’s Hospital of immunosuppressive molecules PDLl and Ill 0. In the absence of proinflammatory stimuli intrinsic induce GDM, while lean control females received a low fat (LF) diet (10% kcal fat). At the beginning of the mechanisms exist to restrain DC activation. In particular, we have reported a role for polycomb group Research Institute of third trimester, the diets of a subgroup of pregnant HFS-fed rats was supplemented with Resv (150 mg/ factor 6 (PCGF6) as a regulator of DC quiescence by modifying chromatin accessibility. Downregulation Manitoba; 2Department kg), thus creating 3 experimental groups (GDM, Lean, GDM+Resv). After weaning, offspring were ofPCGF6 is required for optimal activation and function of DCs. However, whether PCGF6 plays a of Pharmacology and randomly assigned either a HFS or LF diet for 12 weeks. Glucose tolerance test was performed in third determining role during context-dependent inflammation warrants further study. We hypothesize that trimester of pregnancy. Functional and morphometric analyses of offspring hearts were assessed by PCGF6 expression determines whether DCs will generate a suppressive or activating response upon Therapeutics, University echocardiography using a Vevo 2100 ultrasound. stimulation. Through retroviral transduction of bone marrow derived DCs, we have found that over- of Manitoba, Results: Resv supplementation in the third trimester showed a trend towards improved maternal glucose expressing PCGF6 leads to enhanced PDLl and IL-l 0 expression. Antigen specific CD8+ T cells co-cultured Winnipeg, MB intolerance caused by a GDM pregnancy. In addition, GDM+Resv exposed neonates exhibited lower body with PCGF6-overexpressing DCs display reduced sensitivity to antigen, while PCGF6 deficient DCs elicit weights as well as lower heart weights than GDM exposed neonates (p<0.05). Resv significantly decreased enhanced T cell antigen sensitivity. iregDCs sorted from mice infected with the chronic LCMV strain the body weight of GDM+Resv 15 week-old offspring compared to the GDM offspring (p<0.05). + (clone 13) and co-cultured with CD4 antigen specific T cells also lead to reduced T cell antigen sensitivity. GDM+Resv exposed 15 week-old offspring exhibited a similar heart weight to that of lean exposed Preliminary data suggests that the generation of iregDCs is impaired in PCGF6 deficient mice infected offspring. 15 week-old offspring of GDM+Resv dams had a reduced left ventricular (LV) posterior wall with LCMV clone 13. These data suggest that PCGF6 expression promotes regulatory DC immune thickness when compared to GDM offspring (p<0.05). Functional parameters were unchanged among all functions that limit T cell activity. Understanding the mechanism by which PCGF6 regulates DC function 3 offspring groups at 15 weeks of age. can give insight into factors that lead to immune dysregulation in certain diseases and infections. Conclusion: Taken together, our results show that administration of Resv prevents two risk factors for heart disease, namely obesity and cardiac hypertrophy in offspring exposed to GDM. Therefore, Resv could potentially be administered to mothers diagnosed with GDM, in order to prevent negative health outcomes in their offspring. Design of A Low-Cost Kilovoltage Arc Radiation Therapy System Integrated Care Approaches for Hospital to Community Care Transitions: A Scoping Review D. Breitkreutz1, M. D. Weil2, M. Bazalova-Carter1 Cara Brown, Verena Menec

1Department of Physics Introduction: In 2012, 14.1 million people were diagnosed with cancer and 8.2 million people died from University of Manitoba, Background: Older adults are at risk of deterioration of overall health and function during hospitalization. and Astronomy, University cancer, worldwide. Of these deaths, 65% occurred in developing regions of the world. One of the primary Winnipeg, MB This puts them in a vulnerable position for post-hospitalization adverse events and readmission. forms of cancer treatment is external beam radiation therapy (RT), with 52% of new cancer patients being Integrated care approaches seem promising for improving care transitions for older adults, but this 2 of Victoria; Sirius recommended for RT. The gold standard of RT is MV x-rays produced by a medical linear accelerator concept is inconsistently defined and applied. Medicine LLC, Victoria, BC (linac) due to their excellent dosimetric characteristics. Unfortunately, treatment with a linac requires Purpose: To describe: 1) the size and nature of literature on integrated care approaches to hospital to expensive technology and infrastructure, resulting in a high cost-barrier to adequate cancer healthcare. community care transitions for older adults, and, 2) how this literature conceptualizes integrated care. For example, Great Britain spends $3000 per capita on healthcare but only provides for approximately Design and Methods: Scoping review methodology proposed by Levac, Colquhoun and O’Brien (2010) 50% of its demand for linacs. In a developing country, such as Kenya, where only $8 is spent per capita and The Joanna Briggs Institute (2015) guided this study. A systematic search strategy located both peer- on healthcare, the situation is significantly bleaker. Furthermore, cancer incidence trends show the reviewed and grey literature from the last 10 years. Two reviewers independently screened 899 majority of new cancer cases are arising in developing regions of the world. Thus, there is a need for the documents using eligibility criteria of population (65+ years of age), concept (explicit description of an development of a cheaper alternative to MV RT. integrated care approach), and context (transitions from an acute care environment to another care Methods: Monte Carlo simulations of our low-cost kilovoltage arc-therapy (KVAT) system have been environment, including home). Data were extracted on the characteristics of the documents, and the performed on phantom and breast patient cases for a variety of lesion sizes and depths. Dose metrics integrated care initiatives. Analysis included descriptive statistics and a content analysis approach to such as dose to isocentre, lesion-to-skin ratio and dose volume histograms were calculated to assess the summarize features of the integrated care initiatives. quality of treatments. KVAT treatments were compared to MV photon plans as a benchmark for clinical Results: The 48 included documents consisted of 23 peer-reviewed articles and 25 grey literature standards. Current efforts are focusing on simulation of more difficult patient cases through computer documents, describing 45 integrated care initiatives. They originated from 14 different countries with optimization. Future work will involve construction of a prototype and dosimetric verification of the more than 25% of the documents released in 2015. The most common outcome assessed in quantitative prototype. studies was health utilization (85.7%). Qualitative articles explored service provider’s experiences, in Conclusion: Our work has demonstrated the ability of the KVAT system to deliver conformal RT to a wide which challenges with collaboration described. Clinical program features were described for 100% of the range of lesion sizes and depths with outputs of up to 345 cGy/min (clinical radiation therapy is typically initiatives, and organizational features for 75.6%. The most common clinical features of integrated care delivered at 100 cGy/min). The prototype produced by the end of this project will go on to further design approaches were: specific strategies to support inter-agency care coordination, individual care as well as clinical testing, eventually leading to a commercial system. Development of this system will coordination, and multidisciplinary teams. improve healthcare worldwide by providing an order of magnitude cheaper, and therefore more Implications: This body of literature provides rich description of the clinical context and application of universally accessible, alternative to traditional RT. the integrated care initiatives. However, to promote international comparison of these initiatives, detailed descriptions of organizational context is also needed. Finally, full understanding of the impact of integrated care initiatives will not be achieved until there is appraisal and reporting on the extent to which the integration goals were achieved. Social Location and Mental Health Among Canadian Caregivers: Linking Intersectionality The Metalloporphyrin BMX-001 Augments Islet Function and Engraftment in a Murine, and Stress Process Frameworks Syngeneic, Marginal Transplant Model Sean D. Browning, Margaret J. Penning Antonio Bruni1, 2, 3, Andrew R. Pepper2, 3, Rena L. Pawlick2, 3, Boris Gala-Lopez1, 2, 3, A. Gamble1, 2, 3, A.M. James Shapiro1, 2, 3

University of Victoria, Introduction: Family and other informal caregivers provide the vast majority of long-term care to older 1Department of Surgery; Introduction: Transplantation of islets of Langerhans has become a well-accepted clinical therapy for Victoria, BC adults as well as others with chronic illnesses and disabilities. Although research suggests that caregiving 2Alberta Diabetes select patients with type-1 diabetes. Islet loss during isolation and the acute post-transplant period can is frequently stressful and has negative implications for health and well-being of caregivers, little is compromise long-term engraftment outcomes, attributed in part, to oxidative stress. Islets are particularly Institute; 3University of known regarding how social location factors intersect to influence such outcomes. Furthermore, it is susceptible to the deleterious events associated with oxidative stress due to their reduced antioxidant unknown how/whether these factors jointly effect the health and well-being of caregivers through Alberta, Edmonton, AB expression. Therapeutic strategies aimed to augment endogenous antioxidant levels, such as the primary and secondary stressors associated with caregiving recognized in stress process frameworks. The administration of metalloporphyrins, are an attractive approach to preserve beta-cell function and objective of this study was to draw on both intersectionality and stress process frameworks to examine improve transplant outcomes. Herein, we examine whether the administration of novel metalloporphyrin, the main and interactive effects of socioeconomic, gender and age inequalities in influencing caregiver BMX-001, during organ procurement, isolation and subsequent 24-hour culture could improve in vitro outcomes. It also assesses whether these effects reflect differential exposure to primary and secondary islet function and engraftment outcomes in a murine, syngeneic marginal transplant model. stressors associated with the provision of care. Methods: BALB/c murine pancreata were distended with collagenase supplemented with 0, 10 or 34μM Methods: Using data drawn from the 2012 Canadian General Social Survey, the study focuses on a BMX-001. Subsequent to isolation, islets were cultured for 24 hours in media supplemented with 0, 10 or national sample of people aged 15 and older who in the past year had provided care for someone with a 34μM BMX-001. Islets were harvested post-culture and either assessed for in vitro function by static long-term health condition or a physical or mental disability (n=7,082). glucose-stimulated insulin secretion (sGSIS) or transplanted under the renal capsule of diabetic BALB/c Results: Results from a series of multivariate regression models reveal significant main and interaction mice using a marginal dose of 150 islets. Post-transplant function was assessed through non-fasting effects (two-way, three-way), involving age, gender and education, in influencing caregivers’ levels of blood glucose measurements and intraperitoneal glucose tolerance testing (IPGTT) at 60 days perceived stress, life satisfaction, and mental health. Primary and secondary stressors also influence these post-transplant. outcomes but do not appear to account for the impact of social location factors on stress outcomes. Results: Islets cultured for 24 hours with 34μM BMX-001 exhibited a significant improvement in sGSIS Conclusion: The study points to the importance of addressing intersectionality issues when focusing on responses relative to control islets (p<0.05, one-way ANOVA). Furthermore, when transplanted at a caregiving outcomes as well as the need to link intersectionality and stress process frameworks in theory marginal dose under the renal capsule of diabetic recipients, 52.6% (10 of 19) and 57.8% (11 of 19) of 10 and research. The research also points to a need for intersectional and intersectoral social policy and 34μM BMX-001-treated islet recipients, respectively, became euglycemic as compared to 15.7% (3 of approaches to improving caregiver health and well-being. 19) control recipients (p<0.05 and p<0.01, log-rank). BMX-001-treated islet recipients exhibited improved daily blood glucose profiles relative to control recipients. When assessed for in vivo function by IPGTT, BMX- 001 treatment groups demonstrated improved physiological glucose clearance responses relative to control-islet recipients (p<0.05 and p<0.05, one-way ANOVA). Finding the Right Match for 89Zirconium: How to Keep the PET-Isotope on Target and Away from Bone Marrow Christian Buchwalder1, Cristina Rodríguez-Rodríguez1, Paul Schaffer2, Stoyan K. Karagiozov1, Katayoun Saatchi1, Urs O. Häfeli1

Conclusions: Administration of BMX-001 augments in vitro function and in vivo engraftment outcomes in 1Faculty of Introduction: Antibody-based positron emission tomography (immunoPET) is a rapidly advancing a murine, syngeneic marginal mass model. Further work evaluating the utility of BMX-001 in islet isolation Pharmaceutical Sciences, imaging method for the noninvasive diagnosis of many malignancies including cancer. Zirconium-89 and transplantation may lead to improved clinical outcomes. (89Zr) is an emerging radioisotope for immunoPET. Its physical half-life (3.3 days) matches the biological University of British halflife of antibodies (3-4 days) and its positron emission provides PET images of high spatial resolution. Columbia; 2Life Sciences Concerns over the stability of the clinically used 89Zr-chelator, desferrioxamine B (DFO), have led to 89 Division, TRIUMF, efforts to develop an alternative Zr-chelator with improved in vivo stability that can minimize radiation dose to the bone marrow. We synthesized a novel chelator to bind 89Zr, assessed its stability in vitro and Vancouver, BC in vivo, and compare it against DFO. Methods: We synthesized and characterized a new octadentate chelator, based on four bidentate binding units, and studied its complexation with ZrIV. The 89Zr-radiolabeling conditions were optimized and the radiochemical yields were determined in a dilution assay. The 89Zr-complex stability was challenged by incubation in blood serum, exposure to excess EDTA ligand, and in a direct transchelation challenge with DFO. Finally, the in vivo behaviour of the 89Zr-complex was assessed in mice in a biodistribution and PET/CT imaging study. Results: Our chelator quantitatively bound 89Zr within 10 min at room temperature and neutral pH using micromolar concentrations of the chelator. Mass spectrometry confirmed the formation of the 1:1 metal:ligand complex and computational studies suggested a favourable binding geometry. The radiochemical yields were comparable to those with DFO. The radiocomplex remained intact in human serum over a week. Moreover, it performed better than DFO in transchelation experiments when exposed to excess challenge ligands. When administered to mice, the radiocomplex was excreted rapidly via urine without signs of demetallation or significant organ uptake. Conclusion: A novel chelator was synthesized and was found to be a suitable ligand for the complexation of the PET-radioisotope 89Zr. Micromolar concentrations of the chelator provide fast and quantitative radiolabeling under mild conditions. This is ideal in conjunction with antibodies. Compared to the Adequacy of Recommended Cefazolin (CFZ) Prophylaxis in Cardiac Surgery: A Prospective Pharmacokinetic (PK) Study D. Calic1, R. E. Ariano1, 2, R. C. Arora1, 2, 3, H. P. Grocott1, 2, 3, T. M. Lakowsk1, R. G. Lillico1, S. A. Zelenitsky1, 2 current standard ligand DFO our chelator shows improved complex stability in challenge experiments. 1Rady Faculty of Health Although practice guidelines recommend the standard CFZ dosing for antimicrobial prophylaxis (AP) in Mice excreted the radiocomplex rapidly without signs of demetallation. Our chelator therefore appears Sciences, University of cardiac surgery, there is limited study as to whether adequate concentrations (concs) are achieved in this to be a promising alternative to the stability-troubled DFO. We are in the process of developing an population. The purpose of this PK study of CFZ AP in patients undergoing cardiac surgery was to 2 immunoconjugated version of the chelator that will further assess its long-term stability. Manitoba; St. Boniface determine the incidence of sub-therapeutic intra-op concs and to identify risk factors for failure to Hospital; 3Cardiac maintain target concs. Sciences Program, The study was conducted with consent in adults undergoing cardiac surgery with cardiopulmonary bypass and receiving CFZ AP as per institutional protocol (1-2 g IV pre-op based on body weight, Winnipeg Regional Health repeated every 4 h during surgery). Blood samples were collected 30 min after the pre-op dose, prior to Authority, Winnipeg, MB intra-op doses and within 15 min of wound closure. A quantitative liquid chromatography-tandem mass spectrometry assay was developed to measure total CFZ concs. Based on pharmacodynamic (PD) principles for AP, the therapeutic target was defined as maintaining total CFZ concs ≥ 40 mg/L during surgery (≥ 8 mg/L free concs, assuming 80% protein binding). Fifty-five subjects (69% male) with a mean age of 65 ± 10 yrs, weight of 90 ± 17 kg and Clcr of 80 ± 19 mL/min/72 kg completed the study. Twelve (22%) subjects received 1 g CFZ doses and 43 (78%) were given 2 g doses. Total CFZ concs at closure (Cclosure) were < 40 mg/L in 5 (9.6%) of the 52 evaluable cases, whereas levels below the target were observed at some point during surgery in 30.9% (17/55). A sub- therapeutic Cclosure was more frequently observed with 1 g doses for CFZ (P = 0.009), most commonly used in females (P=0.001) and those with increased age (P=0.037) and lower body weight (60-76 kg, P < 0.001). Under current dosing guidelines sub-therapeutic CFZ concs occur frequently during cardiac surgery. As antimicrobial concs at wound closure are a risk factor for surgical site infections, current dosing guidelines may not be adequate. Patient and Caregivers Experiences of Healthcare Errors Occur Outside of the Hospital Examination of Escherichia Coli Adapted to Various Cationic Antimicrobials to Identify Setting Altered Lipids and Membrane Proteins Diana Campbell Nicola H. Cartwright, George G. Zhanel, Denice C. Bay

University of Victoria, Introduction: The complex and turbulent nature of healthcare across the globe renders it vulnerable to University of Manitoba, Introduction: Cationic antimicrobial agents used as disinfectants and antiseptics are routinely used in Victoria, BC errors, resulting in harm, disability, injury and death. Initial research efforts focused on the acute care Department of Medical hospitals, food industries, and found in commercially available cleaning products. Despite the diverse setting. Research on errors in the home and community based healthcare settings is emerging, yet, there chemistry of these antimicrobial agents. Most cationic antimicrobial agents function by killing bacterial is a knowledge gap in experience of errors from patients/caregivers perspective. Microbiology and cells via membrane disruption. Overuse and accumulation of cationic antimicrobial agents within the Methods: This is a manuscript style dissertation consisting of three independent but linked studies. Two Infectious Diseases, environment is resulting in more antimicrobial-resistant bacteria, particularly within gram-negative of these studies are included here: (i) Interviews with Patient and Caregiver regarding their experiences Winnipeg, MB Enterobacteriaceae. Multidrug-resistant E. coli is one of the top five most commonly isolated infections in of errors outside of the hospital setting (ii) Patient experiences of healthcare errors occur outside of the Canadian hospital surveys since 2011. Cationic antimicrobial resistance mechanisms in E. coli have been hospital setting (survey) Data collection was through interviews and surveys. Invitations to be interviewed shown to require one or more of the following alterations at cell membrane level to reduce antimicrobial were sent by provincial and national organizations that (i) focused on quality and safety in healthcare and penetration: 1) increased efflux pump activity, 2) lipid modifications, and/or 3) porin down-regulation. (ii) had established processes to request participation. To date, ten phone interviews have been Cationic antimicrobial resistant bacteria often show reduced penetration of both antiseptics and conducted across Canada. The New Brunswick Health Council (NBHC) asks two questions specific to antibiotics, making a greater understanding of these resistance mechanisms an area that can no longer errors on their annual survey. The NBHC has provided responses to a question on their annual survey that be overlooked. focused on patients experience of harm or errors. There are approximately 800 responses to be analyzed. Rationale: Although correlations between E. coli adapted to cationic antimicrobial agents and antibiotic Preliminary data analysis has been started. resistance in E. coli has been reported, the identity of specific lipid and protein changes in cationic Preliminary Results: (i) Preliminary data analysis of patients/caregivers interviews were congruent with antimicrobial drug E. coli adapted membranes currently remains unknown. This study seeks to identify what is known from the literature (categorization of errors, anticipated response of the system). However, membrane lipid and protein alterations from the same E. coli strain adapted to three different cationic there are new themes identified (more than one episode of errors, impact on their relationship with the antimicrobial agents. The objective is to determine whether the same lipids and proteins are responsible healthcare system, potential solutions, concern over fiscal implications). (ii) Preliminary data analysis has for cationic antimicrobial agent adaptation and if different combinations of lipid and protein alterations been conducted on the data received by NBHC and initial findings indicate some similarities with current are required for different drugs. typologies, however, there errors identified by participants that would not fit within most of the existing Hypothesis: The same E. coli K12 strain repeatedly exposed to sub-inhibitory concentrations of different typologies. cationic antimicrobial agents will alter the same lipids, down-regulate porins and upregulate efflux Conclusion: Further analysis is needed for both the interview(s) and the surveys. There is a need to systems. conduct more interviews and pending HREB approval, recruitment will expand to include potential Approaches: Minimum inhibitory concentrations (MICs) for E. coli K12 strain BW25113 will be determined participants within organizations that focus on specific disease modalities and settings where caregivers for three cationic antimicrobials benzalkonium chloride, chlorhexidine and colistin. Sub-inhibitory MICs access (such as recreational centers). Family Attendance At Physical Activity Programs for Children and Youth With Neurodevelopmental Disabilities: Reinforcing Families Through Social Networks to Better Support Children Michelle Chakraborti, William McKellin, Anton Miller, JP Collet will be used to adapt E. coli to each of the antimicrobials. Lipid content alterations for each of three BC Children’s Hospital Introduction: The WHO’s International Classification on Functioning, Disability and Health acknowledges antimicrobial drug adapted strains will be identified using thin layer chromatography and fatty acid Research Institute, “family” as the most important contextual factor for the development of children and youth with methyl esterification mass spectrometry of phospholipids and lipopolysaccharides from isolated neurodevelopmental disabilities (CYND). Families are responsible for ensuring continuity of care membranes. Membrane protein alterations will be identified using 2-dimensional sodium dodecyl sulfate University of British throughout the lifespan. Family functioning is often destabilized by an ongoing need to adapt to CYND polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry. Columbia demands. Furthermore, common disruptive behavior among CYNDs contributes to the families’ social Outcomes: This project intends to improve our understanding of cationic antimicrobial adapted E. coli isolation, which further affects the child’s development. This highlights the need for programs that are resistance mechanisms in order to identify relevant drug targets for improved antimicrobial drug capable of supporting CYND development, and simultaneously, strengthening families through social development. A better understanding of membrane lipid and protein changes are critical to improve integration within the community. Community based physical activity programs (PAPs) such as Special drug delivery and combat antimicrobial resistance in Enterobacteriaceae. Olympics may be the solution. Besides the benefits the provide for CYNDs, PAPs have the potential to strengthen families by integrating social networks that provide family support, enhance social integration and help developing self-­‐efficacy feelings for childcare and building resilience. Objectives: This exploratory study investigates the PAPs’ potential for social network development; the impact of PAP-­‐ related networks on families’ integration; and the importance of PAP-‐networks­ in comparison to other support networks. Method: Mixed-­‐methods approach: prospective cohort study to collect information at baseline and after 3 months using standardized scales on social integration (SSI), quality of life (FQOL), resilience (FRAS), and empowerment (FES); structured interviews and ethnographic observation. Preliminary results: 50 families with CYND between 2 to 24 years were recruited from 24 PAPs across British Columbia. A content analysis of the baseline interviews conducted (n=25) illustrates that PAPs strengthen families by facilitating socialization through the formation of social networks. Emergent themes suggest these networks between families promote the sense of community, provide emotional support and exchange of information, reduces child-­‐related stress and promotes parental self-­‐efficacy. Furthermore, 75% families ranked PAPs as ‘most important support’ in comparison to other organizations Breastmilk Hormones and Infant Body Mass Index: Findings from the Canadian Healthy Infant Longitudinal Development (CHILD) Study Deborah Chan1, Susan Goruk2, Allan B. Becker1, Padmaja Subbarao3, Piushkumar J. Mandhane4, Stuart E. Turvey5, Malcolm R. Sears6, the CHILD Study Investigators7, Catherine J. Field 2 and Meghan B. Azad1 such as parent support groups, as they simultaneously benefit from socialization with families while their 1Department of Pediatrics and Introduction: Breastfeeding may protect against excessive weight gain. However, the breastmilk child benefits from the activity. Child Health, University of components responsible for this effect are unknown and some breastfed infants still become overweight. Conclusion: Preliminary evidence suggests PAPs represent a unique platform that can potentially Manitoba and Children’s Varying levels of hormones that influence weight gain are found in breastmilk. We hypothesize that strengthen both families and CYND simultaneously, enabling a continuous improvement in their health Hospital Research Institute of breastmilk hormones are associated with infant body mass index (BMI), independent of maternal BMI and outcomes. Ultimately, strengthened families provide better care to children. Manitoba, Winnipeg, MB; other obesity risk factors. 2Department of Agricultural Methods: This study involved a representative subset of 430 breastfed infants from the CHILD birth Food, and Nutritional Science, cohort born between 2009 and 2012. Breastmilk was collected at 3-4 months postpartum. Breastmilk University of Alberta, hormones were measured using the MesoScale Discovery System and classified in quintiles. Infant Edmonton, AB; 3Department anthropometrics were measured at 3 months and 1 year. BMI z-scores were calculated according to the of Pediatrics, Hospital for Sick World Health Organization reference standard. Maternal BMI and demographic characteristics were Children, University of Toronto, self-reported. Toronto, ON; 4Department of Results: Maternal BMI was positively correlated with breastmilk insulin (r=0.40, p<0.0001) and leptin Pediatrics, University of (r=0.71, p<0.0001), but not with adiponectin (r=-0.01, p=0.78). Breastmilk hormone concentrations were Alberta, Edmonton, AB; also associated with maternal age, ethnicity, and prenatal smoking. Independent of these factors and 5Department of Pediatrics, breastfeeding duration, higher breastmilk leptin was associated with lower infant BMI z-score at 3 Child and Family Research months (β -0.62, 95%CI -1.08,-0.16 for top vs. bottom quintile) and 1 year (β -0.59, 95%CI -1.02,-0.17). Institute and BC Children’s Insulin showed a U-shaped association, with intermediate concentrations predicting the lowest infant Hospital, University of British BMI z-score at 3 months (β -0.43, 95%CI -0.78,-0.09 for third vs. bottom quintile) and 1 year (β -0.33, 95%CI Columbia, Vancouver, BC; -0.65,-0.02). Breastmilk adiponectin was not associated with infant BMI. 6Department of Medicine, Conclusion: Our results confirm that breastmilk leptin and insulin concentrations are influenced by McMaster University, Hamilton, maternal BMI and identify novel associations with other maternal characteristics. High leptin and ON; 7Canadian Healthy Infant intermediate insulin concentrations in breastmilk were independently associated with lower infant BMI Longitudinal Development at 3 months and 1 year. This study provides new evidence regarding the specific breastmilk components Study responsible for the beneficial (yet variable) effect of breastfeeding on obesity prevention. Interphase H3S10ph in Mouse Embryonic Stem Cells The Validation of the SDQ for Measuring Child Mental Health and Impact Evaluation of Carol CL Chen, Preeti Goyal, Mohammad M. Karimi, Irina A. Maksakova, Hiroshi Kimura, Matthew C. Lorincz PAX GBG Program in Manitoba Xuan Chen1, 2, Depeng Jiang1, 2 Department of Medical Introduction: Phosphorylation of histone H3 at serine 10 (H3S10ph) act in dichotomous roles in Genetics, Life Sciences chromatin structure depending on cell cycle: it is necessary for the condensation chromosomes in 1Department of Introduction: PAX Good Behavior Game (short for PAX GBG) offers classroom teachers training and tools mitosis, and facilitates transcription activation at inducible genes in interphase cells. Biochemically, Institute, University of Community Health to help children develop social, emotional and self-regulation skills by incorporating social learning H3S10ph act as a strong deterrent for methylation of the adjacent lysine 9 (H3K9me2/3), a heterochromatin principles into normal classroom activities in a team-based, game-like context to reduce aggressive, British Columbia, mark deposited by G9a/Glp and Setdb1, respectively, in a conserved mechanism known as the “phospho- Sciences, University of disruptive, and off-task behavior. PAX GBG was launched by the government of Manitoba in 2012. The Vancouver, BC methyl switch”. Previous studies of interphase H3S10ph has been limited to a small number of genomic Manitoba; 2George Fay strength and difficulty questionnaire (short for SDQ), one of the most widely used and fundamental regions, and the role of this mark is not well defined. Yee Center for Healthcare instrument to assess mental health development throughout the world, was used in Manitoba for PAX Methods: We dissect cell cycle specific H3S10ph by deriving a mouse embryonic stem cell (ESC) line GBG program evaluation. However, the reliability and validity of SDQ have not studied in Canada. Innovation, University of harbouring an endogenous FUCCI fluorescent cell cycle reporter in combination with ChIP-seq. To detail Methods: In this study, we will examine the reliability and validity of SDQ for mental health assessment. the phospho-methyl crosstalk, H3S10ph ChIP-seq were also generated and intersected to H3K9me2 and Manitoba, Winnipeg, MB As students in the same classroom share the same environment and teachers, the dependency within the -/- RNA-seq from Glp ESCs. Finally, we then systematically knocked out each member of the MSK/RSK same class cannot be ignored. First, we will develop computer simulation model to illustrate the family in FUCCI ESCs, and assayed the contribution of each kinases by interphase H3S10ph ChIP and importance of considering the hierarchical data structure in the reliability study. Then, we will examine RNA-seq. the reliability of SDQ scales using PAX program data and validate the SDQ scales by linking PAX program Results: Interphase H3S10ph broadly demarcates gene-rich, early-replicating euchromatic regions in G1, data with health administrative data. Finally, we will examine the impact of PAX program using multilevel marking up to 30% of the ESC genome (manuscript in preparation). Reminiscent of JIL-1 kinase in structural equation model. Drosophila, interphase H3S10ph pervasively marks gene-dense regions to prevent the accumulation Results: The implications of these results and use of SDQ in mental health program evaluation in Canada H3K9me2 heterochromatin at actively transcribed genes in ESC. Surprisingly, the phospho-methyl will be discussed. antagonism acts reciprocally, with H3S10ph domain boundaries are restricted by G9a/Glp-mediated H3K9me2. Expansion of interphase H3S10ph in Glp-/- ESCs is accompanied by strand-biased aberrant transcription initiation of genic and repetitive transcripts at the phospho-methyl boundaries. CRISPR KO further reveals that several JIL-1 homologs serine kinases contributes to interphase H3S10ph in ESCs. Amongst of which, Msk2 ablation showed the greatest loss of interphase H3S10ph at active promoter/ enhancers, and selectively disrupted known H3K9me3 repressed targets - including imprinted genes, young endogenous retroviruses and germline genes. Conclusion: These experiments reveal that H3S10ph plays a previously unappreciated role in interphase chromatin architecture and facilitating appropriate genic transcription by countering repressive effects exerted by histone H3K9 methyltransferases. Exosomes As Carriers of Oncogenic DNA in the Tumour Microenvironment and Biofluids S. Chennakrishnaiah, TH. Lee, B. Meehan, L. Montermini, J. Rak

Research Institute of the Cell-to-cell communication is essential for proper functional coordination among different cell types. disease even prior to cancer cell dissemination. One important aspect of oncogenic EVs is that they carry McGill University Health There are several types of intercellular communication including exchange of information through cell diagnostically important mutant cargo (e.g. oncogenic DNA). Indeed, we observed mutant HRAS, HER2 contact dependent mechanisms and soluble factors, as well as cell membrane dependent processes of and EGFR sequences in EVs isolated from blood of tumour bearing mice. This property of cancer EVs may Centre, Glen Site, McGill molecular exchange, such as tunneling nanotubes, cytoneme, and extracellular vesicles (EVs). EVs are add to the repertoire of liquid biopsy approaches that currently include circulating cell-free DNA University, Montreal, QC membrane-derived spherical structures that are emitted by virtually all cells and carry cell-specific (cf-DNA), and circulating tumour cells (CTCs) whereby the type of cancer and possible treatment could repertoires of bioactive lipids, proteins, microRNA, mRNA and DNA. Multiple cell types possess the ability be determined in a non-invasive manner. We propose that oncogenic pathways intersect with natural to specifically uptake EVs and respond to their cargo. Due to these properties EVs represent a unique processes of EV formation (vesiculation) in a number of ways, which contribute to cell autonomous and mechanism by which complex macromolecular packets can exit parental cells, traffic in the intercellular cell-non-autonomous aspects of malignant transformation and may provide new avenues of cancer space and biofluids, often over long distances, and impact multiple populations of recipient cells. Our therapy and diagnosis. laboratory has earlier uncovered that in cancer cells EV biogenesis is influenced by oncogenic pathways. Moreover, cancer derived small EVs (exosomes) may carry transforming oncoproteins, which can be transmitted to indolent and normal cellular populations, including endothelial cells altering their behavior. To extend these observations we asked whether similar horizontal transmission may apply to intercellular transfer of self-replicating forms of oncogenic macromolecules, such as DNA, and whether this process may permanently or transiently transform/alter normal cells exposed to cancer EVs. Indeed, we observed that in cancer cells oncogenic HRAS drives emission of EVs containing double stranded genomic DNA, which represents the entire cellular genome, and includes full length mutant HRAS sequences. The mechanism of this process is currently under study. The uptake of such oncogenic EVs (oncosomes) by indolent and normal cells causes morphological transformation, expression of angiogenic factors (VEGF), and formation of three dimensional colonies and foci. In transformation susceptible fibroblasts (RAT-I) such foci and exogenous (EV-derived) mutant DNA last approximately 3 weeks post EV exposure, but later disappear resulting in the reversal of the transformed phenotype. Higher exposure to oncosomes resulted in death of normal cells in cell culture. We study how oncogenic EVs that enter blood circulation come into contact with various types of blood cells and endothelial cells that form the lining of blood vessels, and how such cells could respond to this stimulus in ways that render cancer a systemic Recurrent Somatic Copy Number Alteration Analysis Identifies Risk Genes that Modulate Immunomodulatory Effects of Intravenous Immunoglobulin G on Neuroinflammation the Survival of Young Women with Breast Cancer After Cervical Spinal Cord Injury Chen Chi, Rasif Ajwad, Pingzhao Hu Jonathon Chio1, 3, Jian Wang1, Anna Badner1, 3, James Hong1, 3, Michael G. Fehlings1, 2, 3, 4

University of Manitoba, Breast cancer (BC) diagnosis in young women (<45 years old) has come forth as an independent factor 1Department of Genetics Introduction: Despite medical advances, spinal cord injury (SCI) continues to be a debilitating condition. Winnipeg, MB with higher recurrence risk and death than their older counterparts, and it has been suggested that it and Development, Following the initial trauma, neuroinflammation exacerbates the initial damage and hinders recovery. may exhibit its own unique biology. Copy number alterations (CNAs) have led many to consider them as Neutrophils and resident microglia constitute the early inflammatory response after SCI and propagate an alternate paradigm for the genetic basis of human diseases, as these large alterations may encompass Krembil Research neuroinflammation. By targeting this neuroinflammatory response, immunosuppressive drugs are key genes that contribute to carcinogenesis and disease progression. Although many complex diseases Institute, University currently in clinical use for SCI patients. Yet, as these agents increase risks of immune deficiency, have been linked to CNAs in the genomic DNA, prior studies have yet to document agerelated changes Health Network; modulation of the inflammatory response may be more attractive than immunosuppression. Human in somatic CNAs for young women with BC. We hypothesize recurrent somatic CNA regions uniquely intravenous Immunoglobulin G (hIgG) is FDA-approved for modulating inflammation. Although we have 2Department of Surgery, found in young women with BC harbor cancer susceptibility genes that modulate the survival of young shown that hIgG (0.4g/kg) improves functional recovery in rat SCI, the optimal dose of hIgG and its women with BC. We aim to find recurrent somatic CNA regions identified from BC microarray data and University of Toronto; modulatory effects on systemic and local immune response after SCI remain elusive. associate the CNA status of the genes harbored in the regions to the survival of young women with BC. 3Institute of Medical Methods: Female Wistar rats were subjected to clip-compression SCI at C7-T1 level. At 15 minutes We develop a new interval graph-based algorithm for identifying recurrent somatic CNAs in cancer using post-SCI, animals were randomized to receive a single intravenous bolus of hIgG (0.02, 0.2, 0.4, 1, 2g/kg), Science, University of a maximal clique detection technique. The algorithm guarantees that the identified CNA regions are the methylprednisolone or hIgG-free serum. To determine how treatments influence the local (spinal cord) 4 most frequent and identifies the delineated minimal regions. By using the algorithm on the Molecular Toronto; Faculty of and systemic immune response, blood, spinal cord and spleens were collected at 24 hours post-SCI for Taxonomy of Breast Cancer International Consortium CNA data consisting of 2000 breast tumour samples Medicine, University of various inflammatory readouts. Lesion volume of the injured spinal cord was determined using very high (equally divided into a Discovery set with 130 young women and a Validation set with 125 young Toronto, Toronto, ON resolution ultrasound. women), a total of 38 validated recurrent CNA regions with 39 protein encoding genes have been Results: A dose-dependent increase of hIgG was observed in the injured spinal cord after treatment. identified, along with 68 validated recurrent CAN regions that did not encompass any protein encoding Relative to hIgG-free serum, hIgG (2g/kg) significantly reduced various aspects of the local and systemic genes. CNA gain regions encompassing genes CAPN2, CDC73 and ASB13 are the top 3 with the highest acute neuroinflammatory responses. Locally, these include reduced neutrophil infiltration, resident occurring frequencies in both the young Discovery and Validation dataset, while gene SGCZ ranked top microglia activation, expression of inflammatory enzymes and greater expression of anti-inflammatory for the recurrent CNA loss regions. Of particular interest, the mutation status of two of these genes, ASB13 cytokines. Systemically, there was decreased systemic neutrophils and enhanced spleen mass. Altogether, and SGCZ, were also significant in the Kaplan Meier survival analysis. Patients with a mutated status in these changes were associated with reduced lesion volume. Importantly, relative to hIgG (0.4g/kg), hIgG both of these genes resulted in a worse survival outcome when compared to patients without the gene (2g/kg) achieved significant reductions on all readouts aside from microglia activation. mutations. Association and survival analyses demonstrated that the mutated CAN status in ASB13 seems Conclusion: This study demonstrates that hIgG have immunomodulatory effects on local and systemic to lead to correspondingly higher gene expression (binarized by mean), which is able to predict patient immune cell populations in a clinically-relevant rat model of SCI. Moreover, evidence suggests that hIgG survival outcome. Together, identification of the deletion and amplification events that may be prognostic in young women with breast cancer can be used in genomic-guided treatment. Cardiomyocyte VEGF Regulates Endothelial Cell GPIHBP1 to Relocate Lipoprotein Lipase to the Coronary Lumen During Diabetes Amy Pei-Ling Chiu, Brian Rodrigues

(2g/kg) is superior to hIgG (0.4g/kg) at modulating the acute neuroinflammatory response. Given the University of British Introduction: Lipoprotein lipase (LPL)–mediated triglyceride hydrolysis is the major source of fatty acid current use of hIgG in the clinic, our work to optimize hIgG dose and elucidate its immunomodulatory Columbia, Vancouver, BC for cardiac energy. LPL, synthesized in cardiomyocytes, is translocated across endothelial cells (EC) by its effects has potentially significant therapeutic value. transporter glycosylphosphatidylinositol-anchored high-density lipoprotein–binding protein 1 (GPIHBP1). Previously, we have reported an augmentation in coronary LPL, which was linked to an increased expression of GPIHBP1 following moderate diabetes mellitus. We examined the potential mechanism by which hyperglycemia amplifies GPIHBP1. Methods and Results: Exposure of rat aortic EC to high glucose induced GPIHBP1 expression and amplified LPL shuttling across these cells. This effect coincided with an elevated secretion of heparanase. Incubation of EC with high glucose or latent heparanase resulted in secretion of vascular endothelial growth factor (VEGF). Primary cardiomyocytes, being a rich source of VEGF, when cocultured with EC, restored EC GPIHBP1 that is lost because of cell passaging. Furthermore, recombinant VEGF induced EC GPIHBP1 mRNA and protein expression within 24 hours, an effect that could be prevented by a VEGF neutralizing antibody. This VEGF-induced increase in GPIHBP1 was through Notch signaling that encompassed Delta-like ligand 4 augmentation and nuclear translocation of the Notch intracellular domain. Finally, cardiomyocytes from severely diabetic animals exhibiting attenuation of VEGF were unable to increase EC GPIHBP1 expression and had lower LPL activity at the vascular lumen in perfused hearts. Conclusion: EC, as the first responders to hyperglycemia, can release heparanase to liberate myocyte VEGF. This growth factor, by activating EC Notch signaling, is responsible for facilitating GPIHBP1- mediated translocation of LPL across EC and regulating LPL-derived fatty acid delivery to the cardiomyocytes. Molecular Mechanisms and Effector Functions of the Human Cathelicidin Host Defence EsaSR : A Novel Two-Component System Involved in Growth and Antimicrobial Peptide LL-37: Modulation of Cytokine IL-32γ-Induced Responses and Inflammatory Resistance of Burkholderia Cenocepacia K56-2 Arthritis Matthew Choy1, April Gislason2, Silvina Stietz2, Silvia Cardona1, 2 Ka-Yee Grace Choi1, 2, Hemshekhar Mahadevappa1, Leola N.Y. Chow1, Neeloffer Mookherjee1, 2 1University of Manitoba, Introduction: Burkholderia cenocepacia is member of the Burkholderia cepacia complex, a group of Gram- negative opportunistic pathogens, especially to patients with cystic fibrosis or immunocompromised. 1Manitoba Centre The human host defence peptide (HDP) LL-37 selectively suppresses pathogen-induced inflammation, Department of Medical Bcc have intrinsic resistance to multiple clinical drugs used, and the ability to acquire resistances to all without compromising resistance to infections. The objective of this study was to investigate the effects Proteomics & Systems Microbiology and classes of antibiotics. With the use of a chemogenomic approach, previous work from our lab uncovered of LL-37 and its derivative peptide IG-19 in cytokine-mediated inflammation. I demonstrated that LL-37 Biology, Department of Infectious Diseases; a two-component system (TCS) that when under-expressed, resulted in hypersensitivity of B. cenocepacia and IG-19 significantly suppressed cytokine IL-32γ-induced pro-inflammatory responses, and induced K56-2 to different classes of antibiotics. TCSs are common signaling systems used by bacteria to sense Internal Medicine; the production of anti-inflammatory cytokine and chemokines in human PBMC and macrophage-like 2University of Manitoba, various environmental stimuli via the sensor kinase, and to elicit an appropriate response via the 2 THP-1 cells. However, there were significant quantitative differences between LL-37 and IG-19-mediated Deptartment of Department of response regulator. chemokine productions, suggesting that the mechanisms underlying the activity of these two peptides Immunology, University of Microbiology, Methods: To characterize the role of esaSR in Burkholderia cenocepacia the histidine kinase esaS was were different. I showed that both peptides suppressed IL-32γ-mediated phosphorylation of the Src deleted, and site-directed mutagenesis of the response regulator esaR was done. Growth of esaSR Manitoba, Winnipeg, MB kinase FYN(Y420), known to enhance inflammation. In contrast, phosphorylation of the dual phosphatase Winnipeg, MB mutants was studied with growth kinetics, and microscopic observation of mutants was carried out using MKP-1(S359), a negative regulator of inflammation, was enhanced in response to both peptides. a Live/Dead BacLight Bacterial Viability Kit. Antimicrobial assays were carried out to determine the MICs Consistent with this, both peptides increased the activity of p44/42 MAPK, which is known to for various compounds, and in addition to efflux activity assays using an alanine β-naphthylamide assay. phosphorylate and stabilize MKP-1. These results suggested that MKP-1 may be a critical mediator of the Results: Mutants of the esaSR TCS show reduced growth relative to the wild-type, in addition to showing immunomodulatory activity of these peptides. Bioinformatic interrogation revealed that direct interacting altered cell size and morphology. Genetic manipulation of esaSR suggests that esaR is essential for growth protein partners of MKP-1 were overrepresented in MAPK and NF-κB signalling pathways. I showed that of B. cenocepacia in rich media. Results from the Live/Dead kit suggest that esaR mutants have both peptides enhanced the phosphorylation of p38 MAPK. However in contrast to LL-37, IG-19 did not compromised cell envelopes. Antimicrobial screens show that esaR mutants are hypersensitive to mediate the phosphorylation of JNK MAPK and IKK-α, signaling intermediates involved in inflammation. novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, and meropenem, but not colistin, This was consistent with observations that chemokine production was significantly lower in response to hydrogen peroxide or dimethyl sulfoxide. IG-19 compared to LL-37. These results suggested that IG-19 may be a better immunomodulatory Conclusions: Characterization of this essential TCS, and the esaR response regulator in particular, could therapeutic candidate compared to LL-37. As cytokine-mediated inflammation plays a critical role in the aid in the discovery and a better understanding of antibiotic resistance and growth in B. cenocepacia, and disease pathogenesis of inflammatory arthritis, I examined the effects of exogenous administration of other members of the Bcc. esaSR represents an attractive target to study for antibacterial therapy or IG-19 in a murine model of collagen-induced arthritis (CIA). Administration of IG-19 decreased disease adjuvant therapies for the treatment of Bcc infections. severity, suppressed pro-inflammatory cytokines and anti-collagen antibodies, and mitigated cartilage destruction in the CIA mice. These results provide a rationale to further develop IG-19 as a therapeutic agent for chronic inflammatory arthritis. The advantage of HDP based therapy is the potential to control inflammation without compromising the patient’s ability to resolve infections. Src-Family Kinase Feline Gardner-Rasheed is Implicated in Chronic Lymphocytic Specific Inflammation-Associated Parameters and Clinical Comorbidities are Associated Leukemia Cellular Metabolic Regulation with Clonal Hematopoiesis of Aging Alexandra C. Ciapala1, James B. Johnston2, Spencer B. Gibson1, 2 Elina K. Cook1, Terumi Izukawa2, Dylan Johnson1, Eva Bain1, Jamie Hilland1, Brooke Snetsinger1, Bushra Momtaz2, Janika Francis3, Sherylan Young3, Gili Rosen2, Mina Jamali3, Jonah Buckstein1, Michael Rauh1, Rena Buckstein3 1University of Manitoba; Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease in terms of patient prognosis, cellular 2CancerCare Manitoba, microenvironment, and biochemistry. Broadly put, patients may experience either an aggressive or 1Department of Introduction: Bone marrow stem cells produce peripheral blood cells in a polyclonal fashion. With age, indolent form of the disease. Prognosis can be predicted based on whether the immunoglobulin heavy- Winnipeg, MB Pathology and Molecular this process of polyclonal hematopoiesis can become increasingly skewed toward a few clones containing chain (IgVH) of the B-cell antigen receptor has undergone somatic hypermutation. Mutated IgVH is a blood cancer-associated somatic gene mutations (e.g. in TET2, DNMT3A). This phenomenon (present in predictor for better prognosis than unmutated IgVH. CLL cells can exist in the peripheral blood, or they Medicine, Queen’s >10% among adults >65 years old) is called clonal hematopoiesis of indeterminate potential (CHIP), and can inhabit protective niches such as the lymph nodes and spleen. The metabolism of CLL cells varies University, Kingston, ON; is associated with increased risk of myeloid cancers, cardiovascular disease and diabetes. In vitro and in depending on their microenvironment: CLL cells in the peripheral blood prefer oxidative phosphorylation, 2Baycrest Health Sciences vivo models that we and others have used suggest that these mutated clones may contribute to or thrive while CLL cells in the lymph nodes, spleen, and bone-marrow favour anaerobic glycolysis despite the in an inflammatory environment. Indeed, aging, particularly poor aging, is associated with increased Centre; 3Odette Cancer presence of oxygen. The Src-family kinase Feline Gardner-Rasheed (Fgr) is not normally expressed in inflammation. Thus, we hypothesized that CHIP is associated with increased inflammation-associated peripheral B-cells, but is expressed in CLL. When expressed in myeloid-lineage cells, Fgr contributes to Centre, Sunnybrook factors and comorbid diseases. such processes as integrin signaling, degranulation, and cell-survival. In a mousemodel, Fgr expression Health Sciences Centre, Methods: We have analyzed blood genomic DNA, serum cytokines, blood labs and clinical comorbidity enhanced metabolic adaptation in response to stresses and stimuli. We hypothesized that Fgr plays a role data from 187 people >65 years old recruited at Baycrest/Sunnybrook (Toronto). Ion Proton next- Toronto, ON in the metabolic regulation of CLL cells. By western blot, we showed that Fgr expression is greater in CLL generation sequencing of 48 myeloid-cancer genes was used to assign CHIP status. Serum cytokine levels cells with an unmuated IgVH. By flow cytometry we demonstrated that Fgr+ BJAB cells are more likely to were quantified by multiplex assays and min-max-normalized. Mann- Whitney/Kruskal-Wallis tests were die in response to inhibition of nicotinamide adenine dinucleotide (NAD) synthesis than Fgr-, but less applied and data was fitted to logistic regressions to determine CHIP odds ratios (OR). likely to die after inhibition of glycolysis. Using an extracellular flux analyzer, we determined that Fgr Results: CHIP was evident in 26% of cases, with 17 mutant cases of TET2, 16 of DNMT3A, and 16 without expression enhances mitochondrial metabolic capacity. This metabolic alteration appears to be mediated TET2/DNMT3A mutations. The odds of CHIP was increased by male sex (OR=2.9, p=0.002), valvular heart by an increase in electron transport chain complex II activity, as determined by an enzyme activity assay. disease (OR=7.8, p<0.001), chronic symptomatic pulmonary disease (CPD) (OR=8.1, p<0.001) and This data suggests that Fgr plays a role in CLL cellular metabolic regulation. The ability of cells to cope diabetes with end-organ damage (OR=4.3, p=0.003). Interestingly, higher monocyte counts associated with stresses and stimuli is largely dependent on their ability to regulate their metabolism according to with the CHIP group (OR=4.5, p=0.039). Moreover, some monocyte-associated serum cytokines were energy demand. By interfering with their ability to do so, we could potentially augment existing increased in cases with TET2 mutants only (IP- 10/CXCL10, p=0.014), DNMT3A mutants only (eotaxin, therapies. p=0.028), or in cases where more than 2 mutations were found (IL1α, p=0.037). Conclusion: We confirm associations of CHIP with comorbidities such as cardio-metabolic disease, while uncovering links with CPD, male sex, and monocytosis. We suggest CHIP subtypes differentiate into Nek9 Exerts Transcriptional Control Over P53-Regulated Promoters through Novel Interaction with Adenovirus E1A Protein Leandro Crisostomo, Andrea Soriano, Jasmine Frost, Richard Jung, Sandi Radko, Peter Pelka biologically meaningful categories by genotype, with perhaps unique aspects of inflammation. University of Manitoba, Human adenovirus (HAdV) is a key model viral oncogenicity through host cell cycle control and Combinations of CHIP-associated factors may provide a novel screening score to identify CHIP. This may Winnipeg, MB transcriptional de-regulation. During infection, HAdV is capable of driving quiescent cells into S-phase in facilitate surveillance for cancer/co-morbid illness, while providing insight into clone-environment order for the virus to replicate efficiently via the Early region 1A (E1A) gene. On the other hand, p53 is a interactions that drive myeloid pathologies with aging. tumour suppressing protein responsible for activating and promoting DNA damage response proteins, arresting cell growth and inducing apoptosis in cells with severe DNA damage. P53 is a natural rival to E1A, as they contest each other by controlling the transcriptional activity of various key cellular pathways. My work focuses on an E1A associated cellular protein Nek9, a serine/threonine kinase whose primary function is to phosphorylate and aid in mitotic spindle formation. A previous study has cited that during HAdV infection, E1A is recruited by Nek9 unto the p53-regulated promoter of a DNA damage response protein GADD45A, resulting in its repression. It is then important to determine the role of Nek9 with regards to other p53 regulated genes to distinguish whether or not Nek9 is capable of imposing any regulatory control over them, similar to that of GADD45A. So far, we have observed in cells treated with Dactinomycin, an antibiotic capable of halting transcription and disrupting DNA replication, resulted in increased production Nek9 in infected cells. Levels of phosphorylated Nek9 in cells infected with mutant HAdV unable to bind Nek9 saw a decrease compared to cells infected with wild-type HAdV. During a transformation assay using mouse embryonic fibroblast (MEF) cells, a significant increase in the number of foci was observed in cells both infected with HAdV and induced for Nek9 expression, as compared to samples treated with only HAdV or Nek9 respectively. Although more work is required to fully understand Nek9’s role in HAdV infection, the results thus far have added more evidence pointing towards Nek9 as a potent oncogene in presence of E1A. Therapeutic Targeting of Skeletal Muscle Nix in Early-Onset Insulin Resistance Simone Cristina da Silva Rosa1, 2, 3, 5, Lucas Nguyen1 Donald Chapman1, Yan Hai1, Christ of Rampitsch5, Joseph W Gordon1, 2, 3, 4, 5

1Children’s Hospital Introduction: Fetal exposure to diabetes during pregnancy increases the risk for early-onset insulin Conclusion: Our data supports the hypothesis that Nix regulates mitochondrial function and insulin Research Institute of resistance in the offspring; however, the key molecular regulators responsible for fetal metabolic sensitivity in differentiated myotubes, and implicates PKA activating agents as possible therapeutic programming have not been characterized in muscle tissue. Previously, we demonstrated that the approach to restore mitochondrial dysfunction and insulin sensitivity in skeletal muscle of offspring 2 Manitoba; Department expression of a mitochondrial death gene Nix, was elevated in the skeletal muscle of rats exposed to exposed to gestational diabetes. of Human Anatomy and gestational diabetes. Here, we identify a novel phosphorylation residue, activated by clenbuterol Cell Science; 3Rady treatment that serves to prevent Nix-induced mitochondrial dysfunction in muscle cells. Methods: Using cell and biochemical approaches, the C2C12 skeletal muscle myotubes were exposed to Faculty of Health 200 μM palmitate, or vehicle control. To assess mitochondrial membrane potential, cells were stained Sciences; 4College of with TMRM, while macro-autophagy was determined by LC3-GFP aggregation into autophagosomes Nursing; 5University of (n=10). Plasmid-based PKA biosensor was used to identify clenbuterol activation. Cellular localization of Nix was determined by cell fractionation and protein expression by western blot. Phospho-peptide Manitoba mapping was performed by mass spectrometry and custom phopho-specific antibody was generated. One-way anova determined multiple comparisons between groups and student t-test compared mean differences. Results: Detailed phospho-peptide mapping of Nix, revealed a novel phosphorylation residue within the transmembrane domain of Nix. Mutational analysis of this novel phosphorylation site attenuated Nix- induced mitochondrial depolarization (p<0.05), without impacting Nix-induced autophagy, determined by LC3-GFP fluorescence. Exposure to palmitate and Nix expression during differentiation resulted in mitochondrial depolarization, compared to the control myotubes (p<0.05). Furthermore, mitochondrial depolarization was prevented by PKA-activating drug clenbuterol (p<0.05). Consistent with these findings, Nix-induced mitochondrial depolarization was inhibited by clenbuterol, or co-expression of PKA, and clenbuterol restored muscle glucose uptake levels. It has also been observed that phosphorylated version of Nix is located at the cytosol and not in mitochondria. Furthermore, we demonstrate that Nix translocation to cytosol is dependent on a physical interaction with the molecular chaperone 14- 3-3β, which is enhanced by PKA phosphorylation of Nix. The Effect of the Roots of Empathy Program on the Use of Psychotropic Medications Generating a Catalog of Variable Regions in the Transcriptomes of Human Tissues for the Among Youth in Manitoba: Research in Progress. Identification of Highly Specific Transcript Variants in Cancers Lindsey Dahl1, Randy Fransoo1, 2, Mariette Chartier1, 2, Bruce Tefft1 Urmi Das1, Yujia Wu1, 2, Hai Hoang Nguyen1, 2, Yangjun Chen2, Jiuyong Xie1

1University of Manitoba; Introduction: The use of psychotropic medications among youth has increased in recent decades. At the 1Department of Introduction: Pre-mRNA processing including splicing is often disrupted and cells use intronic sequences 2Manitoba Centre for same time, many youth requiring treatment for mental illnesses are not receiving appropriate treatment. Physiology & under abnormal conditions including cancer. The present study aims to identify cancer-specific variable Roots of Empathy (ROE) has been shown to effectively improve social skills and reduce bullying in regions (VRs) of the human transcriptome that are not present in most normal tissues under normal Health Policy, children. The objective of this study is to estimate the effect of the ROE program on being dispensed a Pathophysiology, Max conditions. Winnipeg, MB psychotropic medication. Rady College of Medicine, Methods: To assess the human transcriptome diversity among different normal tissues and to identify Methods: This quasi-experimental study used administrative health, education, and social service Rady College of Health cancer-specific aberrant variants, an accurate (>90% validation) in-house algorithm was used to predict records for youth in Manitoba to compare psychotropic medication dispensations between children who variants from RNA-Seq reads of 35 nominally normal human tissues. DEXSeq and MATS were used to Sciences, University of participated in the program and those who did not. Children were stratified into three grade strata (K - analyse VRs including alternative splicing, transcription start site and polyadenylation, and edgeR was Grade 3; Grade 4 - Grade 6; Grade 7 - Grade 8) and matched on key variables and propensity scores. Manitoba; 2Department used to identify differential expression of intronic and intergenic regions. Further, 32 human cancer Analyses were conducted separately for the three grade strata. Children were followed from the of Applied Computer transcriptomes will be compared to the normal transcriptome, to identify cancer-specific aberrant completion of the ROE program and observed for the first occurrence of psychotropic medication variants. Science, University of dispensation. Time to event analyses were performed for the dispensation of: (1) any psychotropic Results: From analysis of 6.2 billion of RNA-Seq raw reads of 35 normal human tissues, we have found that medication (2) antidepressants (3) stimulants (4) anxiolytics (5) antipsychotics (6) hypnotics & sedatives. Winnipeg, Winnipeg, MB >60% of the human transcriptome regions are variable. More than 90% of the genes identified in VR Cox regression models, controlling for annual physician visit rate, were performed using a robust variance analysis are protein-coding genes with ~18 VRs/gene. A group of the identified VRs by DEXSeq & MATS estimator to account for the clustering of the matched sets. were verified in IGV and validated by RT-PCR with >90% accuracy. Progress of the analysis of some cancer Results: Results are presented for the K - Grade 3 stratum only. Incomplete 10:1 matching without tissues will also be presented. replacement resulted the formation of 7430 matched pairs. Mean follow-up time was approximately 7 Conclusion: Our present results support a much more diverse human transcriptome, challenging the years. Although a greater proportion of ROE children received at least one psychotropic medication current concept of constitutive exons. The complete study will give full views of the diversity of human compared to the control children, the hazard ratio was not significant after controlling for annual tissue and cancer transcriptomes, and provide highly-specific diagnosis markers or potential therapeutic physician visit rate (HR = 0.964, 95% CI: 0.821-1.131). No significant hazard ratios were observed for the targets for personalized medicine. various classifications of psychotropic medications. Conclusion: The results indicate that there was no significant effect of the ROE program on K - Grade 3 children who received the program in Manitoba. However, the greater proportion of ROE children who received at least one medication may suggest that more children are accessing treatment. Results from the subsequent grade strata will contribute to the study by measuring the programs’ effect when delivered to older children. Lignans Target Cholesterol Trafficking to Modulate Endoplasmic Reticulum Stress and Sensitize Breast and Prostate Cancer Cells to Clinically Relevant Kinase Inhibitors Franklyn De Silva, Shelley Yang, Yunyun Di, Jane Alcorn

Drug Discovery & Introduction: The failures in breast/prostate cancer (BC/PC) targeted therapies focusing on the PI3K-Akt- findings warrant further investigations to support FlNs ability to enhance ER stress as the key mechanism Development Research mTOR pathway bespeak a need for a multi-target, yet safe approach that effectively thwarts cell survival involved in the disruption of cellular survival adaptations when combined with TKIs. Such combination adaptations. Tumor cells with endoplasmic reticulum (ER) stress following from oxidative stress, nutrient promises improved patient longevity and quality of life. Group, College of deprivation, DNA damage, altered glycosylation and energy modulation, evoke an adaptive mechanism, Pharmacy and Nutrition, the unfolded protein response, for survival. Overwhelming ER stress, though, can lead to slow tumor University of growth, release of cytochrome c and apoptosis through caspase-dependent cleavage of p53. Recent studies in our lab might suggest the flaxseed lignans (FlNs) enhance ER stress via alterations in intracelular Saskatchewan, cholesterol trafficking. Our lab also demonstrated safety and tolerability of FlNs-enriched products in Saskatoon, SK healthy and frail elderly with short and long-term daily administration of a pharmacological dose of FINs. We hypothesize that combination of FlNs with clinically relevant kinase inhibitors (KI) will increase ER stress and in turn enhance KI anti-tumor activity in-vitro. Methods: We have concluded some preliminary experiments and plan to identify/confirm key targets/ changes involved in energy metabolism, cholesterol synthesis, autophagy, reactive oxygen species, mitochondrial function, glucose uptake, cell survival, intracellular vesicular trafficking and apoptosis pathways using a battery of in-vitro assays in BC/PC cell lines. Results: The FINs modulate lipid metabolism in colon and BC/PC cell lines through modulation of FASN, SREBP-1, INSIG-1, PKM2, ACC-1, MAF-1 and PPARg at mRNA/protein level. ENL downregulated mTOR mRNA levels, reduced mitochondrial redox function (Alamar blue) and caused mitochondrial toxicity (galactose media-Cell Glo) in BC cell lines. Cytotoxicity evaluations with several KIs Ibrutinib (HER2), Dasatinib (Src), Temsirolimus (mTOR), Afuresertib (AKT), and VS-5584 (PI3K/mTOR)) show varying combined effects. We have also confirmed synergistic effects with taxol drugs (Docetaxel and Cabazitaxel). Further, the ENL and metformin with Docetaxel/Carboplatin/Doxorubicin caused a significant reduction in cell viability where individual treatments had little to no response. Conclusion: FINs with TKIs modulate cellular metabolic pathways and impair cancer cell survival. The Effect of Two Incubation Temperatures on the Proteome of Clinically Relevant Pathogen Effects of Acute Oxidative Stress on the Expression, Localization and Function of Cell Acinetobacter Baumannii ATCC17978 Death Regulatory Protein BNIP3 P. Malaka De Silva1, Patrick Chong3, Dinesh M. Fernando1, Garret Westmacott3, Ayush Kumar1, 2 Miriam Derksen1, 2, Spencer B. Gibson1, 2

1Department of Introduction: Acinetobacter baumannii is a notorious Gram-negative coccobacillus, which poses an 1University of Manitoba; Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) is a cell death regulator found at basal levels in Microbiology; 2Medical extremely potent threat in modern day health care setting due to multi-drug resistance mechanisms and 2Research Institute of skeletal muscle and in the nucleus of glial cells. BNIP3 can promote cell death or survival depending on expression of virulence factors. In this study, we aimed to explore the effect of incubation temperature expression level and localization. Nuclear localization prevents apoptosis by repressing transcription of Microbiology, University on the antibiotic resistance and virulence traits of A. baumannii ATCC17978. Oncology and pro-apoptotic proteins. In contrast, localization to the cytoplasm under stress conditions promotes cell of Manitoba; 3National Methods: A. baumannii ATCC17978 was subjected to a multitude of phenotypic assays for virulence traits Hematology, death in part by inducing autophagy, a form of self-digestion that can also be protective at lower levels Microbiology Laboratory, at both 28 ˚C and 37 ˚C along with determining minimum inhibitory concentrations for clinically relevant Winnipeg, MB or shorter periods of expression. Cell death by autophagy occurs due to excessive self-digestion of drugs at 28 ˚C and 37 ˚C. Late log phase A. baumannii cells grown at 28˚C and 37 ˚C were harvested by essential cellular components. BNIP3 is over-expressed in a variety of cancers, with up-regulation being Winnipeg, MB centrifugation. Proteins were extracted from each culture and trypsin-digested overnight using a filter- associated with poor prognosis of patients. Under hypoxic conditions, normally BNIP3 protein expression aided sample preparation protocol (FASP). The digested samples were then labelled using iTRAQ, and is increased and is localized to mitochondria, which promotes cell death by increasing reactive oxygen fractionated using reversed phase chromatography. Each fraction was analyzed using a nano-flow Easy species (ROS) and inducing autophagic cell death. Although hypoxic regions of varying severity are nLC II in-line to an LTQ Orbitrap Velos mass spectrometer. Mascot v2.5 was used for peptide identification common in solid tumors, BNIP3 expression in these cells has been found to promote cell survival and a searching against the sequence of the wild-type parent ATCC17978 as reference, and Scaffold Q+ v4 more aggressive disease, potentially due to a blockage of the cell death function of BNIP3. Hypoxic cells (Proteome Software, Portland, OR, USA) was used for protein filtering and quantitation of iTRAQ labels. may also experience oxidative stress, which is known to induce autophagy. However, the role of BNIP3 in Results: We observed that 632 proteins displayed differential expression levels in the two different regulating oxidative stress-induced cell death is unknown and is the focus of this study. We found that incubation temperatures where 367 proteins were up regulated while 265 proteins were down regulated treating the transformed embryonic kidney cell line HEK293 and the glioma cell line U87 with increasing when cultures were grown at 28 ˚C. Among these proteins, the csu operon, responsible for pili formation concentrations of hydrogen peroxide resulted in increased ROS and cell death as determined by flow and paa operon, responsible for phenyl acetic acid catabolism were respectively up regulated and down cytometer. Western blot analysis of BNIP3 protein levels after hydrogen peroxide treatment revealed a regulated. In addition, Resistant Nodulation Division efflux pump proteins were up regulated, which may three-fold increase in BNIP3 expression in HEK293 cells, with a modest increase in mRNA expression as cause the observed decreased susceptibility to aztreonam at 28 ˚C. These results suggest that there is a measured by qRT-PCR. Immunofluorescent staining for BNIP3 and mitochondria showed that BNIP3 correlation between the incubation temperature and antibiotic resistance and virulence of A. baumannii fluorescence was increased and localized to the cytoplasm and itochondria in both HEK293 and U87 cells ATCC17978. after hydrogen peroxide treatment. These results suggest that similar to hypoxia, BNIP3 expression is increased and localized to mitochondria under oxidative stress. However, knockdown of BNIP3 in HEK293 cells using siRNA provided minimal protection against cell death. The Anti-Inflammatory Action of MiR-146b Improves Ischemia-Induced Neovascularization in Atherosclerotic Conditions M. Desjarlais, S. Dussault, A. Rivard

Centre de Recherche Background: Atherosclerotic conditions are associated with impaired neovascularization in response to which leads to increased TRAF6 inflammatory signaling and inefficient neovascularisation in response to Hospitalier de l’Université ischemia. This is characterized by a pro-inflammatory state inhibiting endothelial cell angiogenic ischemia. The dual anti-inflammatory and pro-angiogenic actions of miR-146b could constitute a novel activities. Micro RNAs (miRs) are emerging as key regulators of several physiological processes, including therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions. de Montréal (CRCHUM), angiogenesis and inflammation. However, the precise mechanisms linking miRs, inflammatory responses Montreal, QC and neovascularisation are still poorly understood. This study sought to define the role of the anti- inflammatory miR146b in that pathophysiology. Methods and Results: Using next generation sequencing, we identified miR-146b as a microRNA which expression is significantly reduced in the ischemic muscles of ApoE knockout hypercholesterolemic mice (HC). miR-146b was previously documented to be an important inhibitor of inflammatory signaling, targeting TNFa-associated-factor-6 (TRAF6). qRT-PCR analyses confirmed that the expression of miR-146b is dramatically reduced in the ischemic muscles of HC mice, and also in HUVECs exposed to oxLDL. This is associated with increased expression of TRAF6 and downstream inflammatory factors including TNFa and VCAM1. To evaluate the function of miR-146b, HUVECs were transfected with an inhibitor of miR-146b (antimiR-146b) or a control antimiR. Similarly to oxLDL, we found that direct inhibition of miR-146b in HUVECs leads to increased TRAF6 expression and impairment of endothelial cell angiogenic functions (migration and tube formation). To confirm the important role of miR-146b in oxLDL-induced endothelial dysfunction, we rescued miR-146b expression using a miR mimic. We found that forced expression of miR-146b reduces TRAF6 expression and rescues the angiogenic functions of HUVECs exposed to oxLDL. In a mouse model of hindlimb ischemia, intramuscular injection of miR-146b mimic in HC mice leads to improved blood flow recuperation and increased vascular densities in ischemic muscles. Clinically, this is associated with reduced hindlimb ischemic damages at day 7 after surgery. Bone marrow-derived pro- angiogenic cells (PACs) have been shown to be important for postnatal neovascularisation. Interestingly, treatment of HC mice with miR-146b mimic increases the number and the angiogenic activities of PACs. Conclusions: Hypercholesterolemia and oxLDL are associated with reduced expression of miR-146b, The Use of “Scientific” and Anecdotal Evidence in the Promotion of a Popular Diet: Exploring the Definition and Meaning of Cancer-Related Fatigue from a Patient A Critical Qualitative Analysis of the Paleo Diet™ Perspective: A Small-Scale Qualitative Study Addressing Concepts of Energy and Lisa Vieira Dias Function in the Head and Neck Cancer Population Naomi Dolgoy1, Patricia O’Krafka2, Margaret McNeely3 Dalla Lana School of Introduction: Dietary advice is circulated through a variety of mediated sites of information. This includes Public Health, University nutritional guidelines produced by governmental organizations in the form of pamphlets, and books 1University of Alberta, Introduction: Lasting fatigue is the most prevalent ongoing health issue reported by head and neck written by proponents of popular or fad diets. Through these mediated sites, health consumers engage of Toronto Edmonton, AB; 2Alberta cancer survivors, negatively affecting physical, psychosocial, cognitive, and emotional functions. This not only with advice on what (and what not) to eat but also with ideas about health, lifestyle, and the ongoing fatigue limits their ability to participate in everyday activities and adversely impacts their overall Health Services, Calgary, body. This presentation shares the research findings from a critical qualitative study that examines the quality of life. Methods of diagnosing and treating cancer fatigue are still undergoing development, kinds of discourses that are mobilized in the promotion of a popular diet, The Paleo Diet™. AB; 3University of Alberta resulting in current challenges in detecting the condition. It is relevant to clinical practice in supportive Methods: Using a critical qualitative research approach informed by Foucauldian discourse analysis, this and Cross Cancer care in oncology to have qualitative and patient-directed research to better understand cancer related study analyzed the prevailing discourses of an “authoritative” website that promotes the Paleo diet. This fatigue from the patient perspective to cope with this growing condition. Institute, Edmonton, AB study entailed an initial mapping of the website and then a subsequent in-depth analysis of thirty-seven Purpose: Exploration of the lexicon used by patients in discussing cancer related fatigue symptoms, to blog posts and three podcasts with transcripts. inform clinical communications regarding cancer related fatigue. Results: My analysis examines the prevailing discourses mediating The Paleo Diet™ website and how Methods: A small-scale (n=25) qualitative research project, informed by interpretive description. these discourses have the effect of: i) constructing the Paleo diet not merely as an appropriate, but Results: Functional issues are the primary concerns of subjects experiencing fatigue symptoms “optimal” way of eating; ii) constructing the website as an authoritative text on the Paleo diet; and, iii) Conclusions: Two major suggestions emerge from this research: first, discussing cancer fatigue issues constructing the body as a site of personal transformation. My analysis also focuses on how both with patients may be an effective tool for determining symptoms and treatment; second, utilizing small- “scientific” and anecdotal evidence are used to make compelling and promote a popular diet that scale qualitative studies may be a successful means of highlighting the rationale for further research into challenges established governmental nutritional advice. development of protocols for assessment and treatment of cancer fatigue symptoms. It is beneficial to Conclusion: This study contributes to a sociological understanding of how knowledge practices work to capture the terminology that cancer survivors use in expressing their fatigue symptoms, in order to more construct authority. It also considers the social, political, and moral implications that the promotion of a effectively develop rehabilitative interventions for fatigue management. popular diet has for health consumers. Collateralization of Projections from the Paraventricular Nucleus of the Thalamus to the Predicting Survival After Pediatric Out-of-Hospital Cardiac Arrest Nucleus Accumbens, Bed Nucleus of the Stria Terminalis, and Central Nucleus of the Ian R. Drennan1, 2, Kevin Thorpe3, Sheldon Cheskes4, Muhammad Mamdani5, Damon Scales6, Anne Marie Guerguerian7, Amygdala Laurie Morrison1 Xinwen Dong1, Sa Li1, Gilbert J. Kirouac1, 2 1Rescu, Li Ka Shing Introduction: Pediatric out-of-hospital cardiac arrest (OHCA) is unique in terms of epidemiology, 1Department of Oral Introduction: The paraventricular nucleus of the thalamus (PVT) is a midline thalamic nucleus with dense Knowledge Institute, St. treatment, and outcomes. There is a paucity of literature examining predictors of survival to help guide resuscitation in this population. Biology, College of projections to the nucleus accumbens (NAc), dorsolateral region of the bed nucleus of the stria terminalis Michael’s Hospital; (BSTDL) and the lateral/capsular region of the central nucleus of the amygdala (CeL/CeC). Recent Objective: The primary objective was to examine predictors of survival to hospital discharge. The 2 2 Dentistry; Department of experimental evidence indicates that the PVT is involved in both appetitive and aversive behaviors. Institute of Medical secondary objective was to determine the probability of return of spontaneous circulation (ROSC) over Psychiatry College of However, it is unknown if subgroups of neurons in the PVT innervate different subcortical targets or if the Science, University of the duration of resuscitation. Methods: We performed a retrospective cohort study of non-traumatic OHCA (<18 years) treated by EMS Medicine, Rady Faculty of same neurons issue collaterals to multiple areas. Answers to this question will help us understand Toronto; 3Applied Health whether the PVT modulates different types of behaviors in a projection specific manner. from the Toronto Regional RescuNET Epistry-Cardiac Arrest database from 2006 to 2015. We used Health Sciences, Research Centre, St. Methods: We injected two different fluorescent retrograde tracers, cholera toxin subunit B conjugated to competing risk analysis to calculate the probability of ROSC over the duration of resuscitation. We then University of Manitoba, Alexa Fluor-488 or Alexa Fluor-594, into different pairs of the subcortical targets including different parts Michael’s Hospital; used multivariable logistic regression to examine the role of Utstein factors and duration of resuscitation in predicting survival to hospital discharge. We used area under the receiver operating characteristic Winnipeg, MB of the NAc (shell, core, dorsomedial shell and ventromedial shell), BSTDL and amygdala (basolateral 4Centre for Healthcare amygdala and CeL/CeC). The number of single and double labeled neurons in the PVT was counted on 50 (ROC) curve (AUC) to determine the predictive ability of our logistic regression model. Analytics Research and μm sections every 300 μm through the anterior - posterior extent of the PVT. Adjacent sections were also Results: A total of 658 patients met inclusion criteria. Survival to discharge was 10.2% with 70.1% of those stained for NeuN to estimate the proportion of neurons in the PVT that were labeled. Training, St. Michael’s children having a good neurologic outcome. The overall median time to ROSC was 23.9 min. (IQR 15.0, Results: There is a moderate to high level of collateralization of projections from neurons in the PVT to Hospital 5Department of 36.7). However, the median time to ROSC for survivors was significantly shorter than the time to ROSC for patients who died in hospital (15.9 (IQR 10.6 to 22.8) vs. 33.2 (IQR 22.0 to 48.6); P value <0.001). There was NAc, BSTDL and CeL/CeC (20% to 50%). We also observed a difference in the subcortical targets Critical Care, Sunnybrook innervated by the anterior PVT (aPVT) and posterior PVT (pPVT) showing that more neurons in the aPVT a decrease in the odds of survival of 14% per minute during the first 25 minutes of cardiac arrest. Older innervate the dorsal medial part of the NAc shell while more neurons in the pPVT innervate the ventral Health Sciences Centre; age (OR 0.9, 95% CI 0.86, 0.99), and longer duration of resuscitation (OR 0.9, 95% CI 0.88, 0.93) were medial NAc shell, BSTDL and CeL/CeC. 6Department of Critical associated with worse outcome while initial shockable rhythm (OR 5.8, 95% CI 2.0, 16.5), and witnessed arrests (OR 2.4, 95% CI 1.10, 5.30) were associated with improved patient outcome. The AUC for the Conclusion: The results suggest a potential importance of the PVT in simultaneously coordinating the Care, The Hospital for Sick activity of key regions of the brain involved in mediating emotional and motivational behaviors, and a Utstein factors was fair (0.77). Including duration of resuscitation improved the discrimination of the potential functional difference between the aPVT and pPVT. Children, Toronto, ON model to 0.85. Conclusion: Inclusion of duration of resuscitation improved the performance of our model compared to Utstein factors alone. However, our results suggest there are a number of other important factors for predicting patient outcome from pediatric OHCA. Differential Growth State-Dependent Activation of P38MAPK by Treament of Endothelial Cells with Docosahexaenoic Acid (DHA) Youjia Du1, 3, Carla G. Taylor1, 2, 3, Harold M. Aukema2, 3, Peter Zahradka1, 2, 3

1Department of Introduction: Endothelial cells line the luminal surface of blood vessels and play an essential role in as DHA in various disease states. Since activation of stress-stimulated protein p38MAPK, which is closely Physiology and regulating vascular function. Endothelial cells quiescence is a primary property of the healthy linked to pathological vascular remodeling, was observed in different growth conditions of cells, further endothelium, whereas cell proliferation is feature of vessel repair and progression of atherosclerosis. studies to delineate the physiological effects of DHA on p38MAPK signal transduction in endothelial cells Pathophysiology; Docosahexaenoic acid (DHA), an omega-3 fatty acid present in fish oil, is thought to exert an array of are warranted. 2Department of Human beneficial effects on vascular health by modulating endothelial function. However, a comparison of the Nutritional Science, effects of DHA on growing and confluent endothelial cells, which represent repairing and healthy states, respectively, has never been reported. University of Manitoba; Objective: To investigate the effects of DHA on viability and proliferation of subconfluent (growing) and 3Canadian Centre for confluent (quiescent) human endothelial cells (EA.hy926) in relation to p38 mitogen-activated protein Agri-Food Research in kinase (MAPK) activation. Methods: EA.hy926 endothelial cells were seeded on Matrigel-coated plates and grown up to 10 days. Health and Medicine, Cells were treated with DHA (1-150 μM) for 24 h when they were either subconfluent (4 d after seeding) St Boniface Albrechtsen or confluent (8 d after seeding). Viability and proliferation were assessed. Phosphorylation of p38MAPK Research Centre, and mitogen-activated protein kinase kinase (MKK3/MKK6) was assessed after treating subconfluent and confluent cells with 125 μM DHA over 8 h. Winnipeg, MB Results: Maximum cell number and DNA synthesis rate were reached on day 8, and the cells subsequently became quiescent. DHA >125 μM reduced viability of both growing and confluent cells after 24 h, while 40 μM slowed proliferation of both subconfluent and confluent cells. In subconfluent cells, 125 μM DHA activated p38MAPK and MKK3/MKK6 (a known upstream kinase), producing peaks at both 10 min and 4 h. In contrast, phospho-p38MAPK and phospho-MKK3/MKK6 peaks were only observed at 10 min in confluent cells. Conclusions: Our study is the first to show a dose-dependent effect of DHA on both proliferating and confluent endothelial cells with regards to viability and proliferation, as well as a growth state-dependent activation of p38MAPK. This is highly relevant to how blood vessels respond to a dietary constituent such Can Regular Physical Activity Reduce Anxiety in Adolescent Girls? – Protocol for a Influence of Human Papillomavirus E6 PDZ Binding Motif on Human Neonatal Systematic Review and Meta-Analysis Keratinocyte Immortalization and Transformation Anita Durksen1, 2, Jacqueline Hay1, 2, Nicole Askin3, Ahmed Abou-Setta1, Ryan Zarychanski1, Rasheda Rabbani1, 4, Sayma Malik1, Kerry Dust1, 3, Dana Cabilles2, Michael Carpenter 1, 2, Chris Grant2, Stuart McCorrister2, Alberto Severini 1, 2 Jonathan McGavock1, 2 1University of Manitoba, Objective: The presence of a PDZ binding motif on the C-terminus of the Human Papillomavirus (HPV) E6 1University of Manitoba; Background: Anxiety disorders are the most common psychiatric condition in adolescent girls, who at Department of Medical protein is a molecular feature unique to the cancer causing high risk HPV types. Many PDZ proteins are scaffolding proteins involved in cell to cell communication and maintenance of cell polarity and cell 2Children’s Hospital the same time are less likely than boys to meet physical activity guidelines. This systematic review will Microbiology, Winnipeg, summarize the evidence for the effectiveness of physical activity interventions for improving anxiety junctions. The ability to bind and degrade cellular PDZ proteins may allow the virus to immortalize and Research Institute of MB; 2Public Health outcomes in adolescent females. transform their host cells. Manitoba; 3 Seven Oaks Findings: To be included in the review interventions must be include a regular physical activity program Agency Canada; Methods: HEKn cells were transduced with lentiviral vectors carrying full length HPV16 E6 or a truncated HPV16 E6 that is missing the c-terminal PDZ binding motif (HPV16 E6ΔPBM); and HPV16 E7. HEKn cells General Hospital; of any intensity and a minimum of 4 weeks duration. The intervention must be administered to a 3Cadham Provincial population that is made of at minimum 80% females ages 11 – 20. A comprehensive, systematic, transduced with empty lentivectors were used as controls. To assess immortalization these cell lines were 4George and Fay Yee Laboratory electronic search strategy will be used to identify controlled studies from database inception to 23 monitored in culture for growth rate and longevity. Western Blots and Mass Spectrometry were used to Centre for Healthcare February 2017 in: Medline (via Ovid, including Epub ahead of print and other non-indexed materials), measure differences in protein expression among the cell lines. Results: Despite the absence of a PBM, the cell lines transduced with E6ΔPBM+E7 underwent more Innovation, Winnipeg, MB EMBASE (via Ovid), Cochrane CENTRAL (via Wiley), PsycINFO (via ProQuest), Scopus (web) and SPORTDiscus (via EBSCO). The search will be limited to randomized controlled trials using a modified population doublings and grew more quickly than the controls. All of the control cell lines were senescent Cochrane HSSS filter and no language restrictions. The search will use both subject heading and keyword by 50 population doublings (PD) while 72% of E6ΔPBM+E7 cell lines and 100% of E6+E7 cell lines searches to identify the population, intervention, and outcomes of interest – teenage girls, physical continued to grow and divide beyond 100 PD indicating that the PBM is not required for immortalization. activity, and improved anxiety/mental health. Where there are sufficient numbers of similar interventions, Supporting the longevity observations, telomerase (hTERT) was found to be expressed in both E6+E7 and we will calculate and report pooled risk ratios or standardised mean differences using meta-analysis. E6ΔPBM+E7 containing cell lines but not in the controls. The expression of hTert was higher in cell lines Discussion: This systematic review will provide recommendations on the use of physical activity for expressing full length E6 compared to the truncated E6. The degradation of the apoptotic protein p53 is prevention and/or treatment of anxiety among adolescent girls and will guide future work on intervention the hallmark of high risk HPV E6 proteins and as expected levels of p53 in the cell lines expressing full development and primary research in this field. length E6+E7 were low compared to the controls. While not as depressed as in the full length E6 cell lines, the levels of p53 in E6ΔPBM+E7 cell lines were also found to be lower than in the controls. This indicates that the PBM is not required for p53 degradation, but its absence hinders p53 degradation. Quantitative tandem mass spectrometry revealed that there were only a few proteins that were significantly different between E6ΔPBM+E7 and E6+E7 cell lines. Anchorage independent growth has not yet been observed. Inhibition of CSPGs Signaling Receptors Fosters a Pro-Regenerative Endogenous Response in Traumatic Spinal Cord Injury Scott Dyck1, Hardeep Kataria1, Santhosh Thomas1, Bradley Lang2, Jerry Silver2, Soheila Karimi-Abdolrezaee1

1Department of Introduction: Traumatic spinal cord injury (SCI) results in significant cell death in oligodendrocytes of microglia which was otherwise reduced upon their activation and by exposure to CSPGs in vitro. Physiology and the leading to demyelination and functional deficits. Resident neural precursor cells (NPCs) have a Conclusion: In conclusion, we demonstrate that CSPGs regulate the response of endogenous precursor tremendous potential to replace lost oligodendrocytes following SCI. However, the ability of NPCs to cells following SCI by LAR and PTPσ dependent mechanisms. Blocking these receptors by ILP and ISP Regenerative Medicine proliferate and differentiate into oligodendrocytes is challenged in the post-SCI microenvironment. We treatment improves oligodendrocyte differentiation in SCI likely by promoting a pro-regenerative Program, the Spinal Cord previously identified for the first time that injury-induced upregulation of chondroitin sulfate immune response following injury. CSPGs are a key component of the post-SCI milieu with a significant Research Center, proteoglycans (CSPGs) inhibits several properties of NPCs including their survival, proliferation and impact on the cell replacement process. Accordingly, our study has identified new clinically relevant differentiation which is meditated through signaling of LAR and PTPσ receptors and activation of therapeutic targets for enhancing cell-based therapies for the treatment of SCI. University of Manitoba, downstream Rho/ROCK pathway. Winnipeg, MB; Methods: In the present study, we evaluated the therapeutic potential of two functionally blocking 2Department of peptides against LAR (Intracellular LAR Peptide, ILP) and PTPσ (Intracellular Sigma Peptide, ISP) in promoting endogenous cell response after SCI. In a clinically-relevant model of compressive SCI in rats, Neurosciences, Case we delivered ISP and ILP treatment locally to the injured spinal cord in a sustainable manner for various Western Reserve time-points. Using BrdU incorporation, Western Blotting and immunohistochemistry, we identified University School of multiple beneficial effects of ISP and ILP therapy following SCI. Results: We show that ILP and ISP efficiently block CSPGs inhibitory effects on NPC survival, proliferation, Medicine, Cleveland, OH and oligodendrocyte differentiation. Our BrdU studies revealed that ILP and ISP therapy enhances endogenous cell response following SCI by promoting oligodendrocyte preservation and replacement. Conversely, there was a significant decrease in proliferating astrocytes associated with reduced astrocytic scarring. Moreover, ILP and ISP treatment fosters a neuroprotective and anti-inflammatory response in the post-SCI milieu characterized by induced interleukin-10 (IL-10) and arginase-1 expression. Using microglia conditioned media we then assessed the role in which microglia have on NPCs proliferation and differentiation. Pro-inflammatory M1 microglia were shown to promote astrocytic differentiation and limit NPC proliferation whereas anti-inflammatory M2 microglia promoted NPC proliferation and oligodendrocyte differentiation. Interestingly, ILP/ISP treatment also promoted the phagocytosis ability Falling Through the Cracks: The Characteristics and Ongoing Service Use of Patients Screened but Ineligible for Early Psychosis Intervention J. Edwards, R. Norman, A. MacDougall, L. Palaniyappan, P. Kurdyak, KK. Anderson

University of Western Introduction: Psychotic disorders have a major effect on patients’ quality of life and a significant services have high levels of ongoing mental health service use, potentially indicative of unmet needs. Ontario, London, ON economic impact. Over the past two decades there has been widespread implementation of early Linkages with more suitable service providers are warranted, and these people may benefit from the psychosis intervention programs, which have shown to assist with symptomatic and functional recovery. implementation of specialized follow-up and treatment plans. To date, there has been no research describing the characteristics and ongoing service use of people screened for early psychosis intervention (EPI) who do not meet the eligibility criteria of the program. These people may be at a high risk for unmet mental health care needs. The objectives of this study were: (i) to quantify the number of people screened for the EPI program who are not eligible and compare those persons characteristics with those admitted into EPI; and (ii) to describe health service use patterns between those screened for and admitted into EPI. Hypothesis: Our expectation was that screened persons would have dissimilar characteristics and ongoing health service use compared to individuals enrolled in EPI. Specifically, we predicted that screened clients would have higher rates of involuntary hospitalizations and emergency department use. Materials and Methods: Using health administrative data obtained from the Institute for Clinical Evaluative Sciences, we constructed a retrospective cohort of incident cases of psychotic disorder. We linked data from the Prevention and Early Intervention Program for Psychosis in London, Ontario from 1997 through 2014. Using 5 years of follow-up, we compared characteristics and ongoing service use between those who were screened and those admitted. Results: Compared to persons who were admitted into EPI (n=754), those who were screened (n=1059) were older, more likely to be female, and more likely to live in low-income neighbourhoods. A significant proportion (27.6%) were diagnosed with a personality disorder. Those screened were more likely to be involuntarily hospitalized (HR 1.37, 95% CI: 1.09, 1.72), use emergency department services (HR 2.33, 95% CI: 1.82, 2.94) and primary care services (HR 2.78, 95%CI: 2.13, 3.57) at two years, with trends persisting at five years. Discussion and Conclusions: Our findings suggest that people screened but found to be ineligible for EPI Pancreastatin (PTS): A Key Modulator of Ulcerative Colitis Nour Eissa1, 2, Laëtitia Kermarrec1, Charles N. Bernstein3, Jean-Eric Ghia1, 2, 3

1Department of Background: Chromogranin-A (CgA) is elevated in inflammatory bowel disease (IBD) and in murine however, a significant decrease of ARG-1, YM1, & TGFβ was demonstrated in both groups. Meanwhile, -/- Immunology; 2Children’s models of colitis. CgA produced by enteroendocrine cells can be cleaved in several peptides including PTSconditioned CgA & WT AMS expressed significantly less ARG-1, YM1, & TGFβ when compared to IL-4/ the immunoregulatory pancreastatin (PTS: CgA273-301). Macrophages (Mo) play a major role in IBD IL-13 control condition. In undifferentiated macrophages, PTS significantly increased macrophages Hospital Research through an impaired transition from a pro-inflammatory (classical activated Mo (CAMs)) to anti- migration. Institute of Manitoba; inflammatory (alternative activated Mo (AAMs)) phenotypes. Previously we demonstrated that in mouse, Conclusions: These findings suggest that PTS contributes to the pathogenesis of colitis and inflammatory 3Internal Medicine the lack of CgA (CgA-/-) induced a significant decrease of colitis that associated with a decrease and an process via the modulation of Mo phenotype and function. Targeting PTS may lead to novel therapeutic increase of CAM and AAM markers respectively. The aim of this study was to investigate the role of PTS in strategies in IBD. section of colitis using a pre-clinical and clinical approaches. Gastroenterology, Methods: Serum level of PTS and mRNA expression of CgA were quantified in active UC and healthy University of Manitoba, individuals & mice using ELISA & RT-qPCR. Colitis was induced in CgA-/- and wild type (WT) mice by administrating dextran sulfate sodium in drinking water (DSS 5%, 5 days), and PTS treatment (2.5mg/kg/ Winnipeg, MB day, 6 days) or vehicle started 1 day before induction. Disease activity index (DAI) was daily evaluated, and macroscopic scores at sacrifice day. The serum level of CRP was quantified using ELISA. Colonic MPO, IL-1β, IL- 6, TNF-α, MIP-1α, MIP-1β, ARG-1, YM1 & TGFβ were assessed using ELISA or RT-qPCR. Naïve peritoneal macrophages were isolated from CgA-/- and WT mice and treated with PTS (200ng/ml; 2, 24h) then exposed for 6 h to LPS (100 ng/ml) or to IL-4/IL-13 (20ng/ml) to promote CAMs or AAMs. CAMs markers (IL-6, IL-1β, TNF-α, MIP-1α, MIP-β) and AAMs markers (ARG-1, YM1, TGFβ) were quantified by using ELISA and/or RT-qPCR. In vitro chemotaxis activity of PTS (200 ng/ml) on naïve Mo was assessed by migration assay using MCP-1 (30ng/ml). Results: CgA & PTS levels significantly increased in human active UC and WT colitic mice. In colitic CgA-/- mice, we confirmed a significant increase of colitis through the regulation of all the pro and anti- inflammatory markers. PTS treatment significantly increased the onset and severity of colitis in CgA-/- and in WT mice. Macroscopic score, CRP, colonic MPO, IL-1β, IL-6, TNF-α, MIP-1α and MIP-1β increased significantly, while ARG-1, YM1, TGFβ decreased in colitic WT & CgA-/- mice treated with PTS. IL-6, IL-1β, TNF-α, MIP-1α & MIP-β were significantly increased in PTS-conditioned CgA-/- CAMs but not in WT, Tetrahydrobiopterin Prevents Coronary Artery Malformations Induced by Pregestational Diabetes Anish Engineer, Yong Lim, Sharon Lu, Qingping Feng

Department of Physiology Introduction: The heart is the first functional organ to form during embryogenesis. Angiogenesis in the through ROS probing and lipid peroxidation was significantly increased in diabetic hearts and reduced primitive heart begins at embryonic day (E)9.5 and involves coordinated epithelial to mesenchymal and Pharmacology, with BH4. transition (EMT) and cell differentiation, which form a complete coronary artery network by E15. Conclusions: The importance of BH4-mediated reduction in oxidative stress during cardiac angiogenesis Schulich School of Alterations in angiogenic gene expression, cell migration or proliferation can have implications on under diabetes provides insight into factors contributing to coronary artery defects. The therapeutic Medicine and Dentistry, vasculature and heart function, as seen during diabetic mbryopathy. Hyperglycemia-induced oxidative potential of BH4 during gestation can be extrapolated through these studies. University of Western stress can lead to inactivation of endothelial nitric oxide synthase (eNOS), vital for heart development. Tetrahydrobiopterin (BH4), an antioxidant, is the co-factor for eNOS, and when oxidized leads to dimer Ontario, London, ON uncoupling, which further increases oxidative environment. Hypothesis: BH4 treatment during the full gestational term of female mice with pregestational diabetes will reduce the incidence of coronary artery malformations via decreasing oxidative stress. Methods: Diabetes was induced by administering streptozotocin (STZ, 50 mg/kg, IP) to adult female C57BL/6 mice. BH4 (10 mg/kg/day) was orally administered to the pregnant female mice via peanut butter. Fetal hearts were collected at E18.5 for coronary artery analysis, through smooth muscle actin immunostaining and 3D reconstruction to view the vascular network. Embryonic markers of angiogenesis and EMT, as well as molecular regulators of artery development and oxidative stress were examined at E12.5 through immunostaining and qPCR. Results: Marked decreases in coronary artery diameter of both the left and right main branches for the coronary tree were found in hearts from diabetic mothers, which were prevented with BH4 administration. Coronary artery abundance and volume were also decreased in diabetic hearts, and recovered to normal levels with BH4 treatment. Additionally, a normal branching pattern and arborization of the vessels was abolished in hearts from diabetic mothers, and returned to normal with BH4. Molecular regulators of angiogenesis such as Snail1 and Slug, were altered with maternal diabetes and reestablished with BH4 supplementation. Additionally, more cell to cell connections, measured through E-cadherin expression were found in diabetic hearts, returned to control levels with BH4. Finally, oxidative stress, measured Towards a Shared Taxonomy for Health Technology Reassessment (HTR) and Knowledge Sleep, Sedentary Behaviour, Physical Activity, and Functional Ability After Inpatient Translation (KT) Stroke Rehabilitation Rosmin Esmail1, 2, Lesley J.J. Soril1, Fiona M. Clement1 Victor E. Ezeugwu, Patricia J. Manns

1Health Technology Introduction: The emerging field of HTR supports optimal use of technologies throughout their lifecycle. Department of Physical Introduction: The purpose of this study was to describe accelerometer-derived pattern of sleep, Assessment Unit, KT is required to translate HTR findings. However, no common taxonomy exists between the two leading Therapy, Faculty of sedentary behavior, physical activity and level of function and their association with demographic and to a gap in understanding. This work describes the common taxonomy of HTR and KT and identification clinical factors within the first month after inpatient stroke rehabilitation. Department of of related concepts between the two fields. Rehabilitation Medicine, Methods: This cross-sectional cohort study was conducted in the home environment and involved thirty Community Health Methods: A systematic review was undertaken. MEDLINE, EMBASE, PUBMED, and CINAHL databases were University of Alberta, people with stroke (mean ± SD; age: 63.8 ± 12.3 years; time since stroke: 3.6 ± 1.1 months. Participants Sciences and the O’Brien searched in March 2016 for taxonomy related review articles in HTR and KT. As a recent scoping review Edmonton, AB wore activPAL3 Micro accelerometer continuously for 7 days to measure 24-hour activity behavior. had been conducted for the HTR literature, the search strategy was limited from 2014 to current. For the Participants’ level of function was assessed using mobility and locomotion subdomain of Functional Institute for Public Health, KT literature the search was limited from 1993 to current. Text words and synonyms for HTR and Independence Measure. University of Calgary; de-adoption were combined with healthcare technologies. Search terms for KT were derived from Results: Sleep duration ranged from 6.6 to 11.6 hours per day. Fifteen participants (50%) engaged in long 2Alberta Health Services, identified terms for discriminating KT and non-KT articles. All titles, abstracts, full text articles were sleep > 9 hours per day. Participants were awake for a mean of 15.1 ± 1.3 hours and spent 74.8% of reviewed independently and compared in duplicate. Data was extracted in duplicate. Differences were waking hours in sedentary behavior, 17.9% standing, and 7.3% stepping. Of stepping time, only a median Calgary, AB resolved through consensus. A component based analysis was used to synthesize the evidence. of 1.1 (IQR: 0.3-5.8) minutes were spent walking at a cadence ≥ 100 steps/min. Sedentary time, stepping Results: Current analysis of the literature has identified two reviews: one that refers to de-adoption and time, and steps differed significantly by hemiparetic side (p<0.05), but not by sex or type of stroke. Sleep reassessment taxonomy with 43 words and the other which describes100 different terms for KT research. duration, sedentary time, gait speed, and cognitive scores were correlated with stepping at different Unique terms for reassessment include disinvestment, decrease use and discontinue. Unique terms that intensities (p<0.05). One third of participants could not perform a transfer to floor, 16.7% required describe KT include utilization, implementation, and adoption. Five terms were found to be common supervision with stairs, 26.7% assistance with bathroom transfers and 13.3% assistance with vehicle between HTR and KT: use, change, intervention, practice and service. These terms may illustrate how the transfers. two concepts are inter-related. HTR and the evidence from the re-evaluation of a technology may lead to Conclusion: People with stroke require follow-up support at home to reduce sedentary and increase a use or change in practice, intervention or service. KT could be used as a vehicle to compliment and activity behaviors, and possibly improve pattern of sleep after inpatient rehabilitation. facilitate the use of HTR evidence. Conclusion: This work provides a common taxonomy between HTR and KT leading to a better understanding of how these fields align. Both are complementary processes in putting technology- related evidence into practice. Future work will examine the development of a validated conceptual model that will provide a foundation for advancing and applying HTR knowledge in evidence-informed decision making through KT. Deficiency of PEPCK in Leishmania Major Leads to Attenuated Pathology and Impaired The Pancreatic Specific Isoform of SPCA2 Affects Store-Operated Ca 2+ Entry Immunity Against Secondary Virulent Challenge Melissa Fenech1, 2, 5, Christopher Pin1-4 Aida Feiz Barazandeh1, Zhirong Mou1, Wen Wei Zhang2, Greg Maltashewski2, Jude E. Uzonna1 1Department of Introduction: Digestive enzymes are released from pancreatic acinar cells through regulated exocytosis, 1Department of Recovery from primary natural or experimental infection with Leishmania major induces long-term Pediatrics; 2Department which involves a signalling network that regulates the spatial and temporal accumulation of cytosolic Ca2+ within the cell. Cytosolic Ca2+ levels are regulated, in part, by a series of Ca2+ATPases that move Ca2+ Immunology, College of protection to reinfection collectively known as infection-induced resistance. However, it is not well of Physiology and known what antigens induce and maintain this resistance and whether these antigens preferentially back into specific organelles or out of the cell. Our laboratory has previously identified a novel isoform of 2+ Medicine, University of favor the development of memory T cells. Recently, we identified phosphoenolpyruvate carboxykinase Pharmacology; secretory pathway Ca -ATPase 2 (SPCA2) that appears to be pancreas specific. However, this isoform, Manitoba, Winnipeg, MB; (PEPCK) as an immunodominant antigen of Leishmania and showed that PEPCK-specific CD4+ T cells 3Department of which we have designated SPCA2C (C for cytoplasmic) contains only the last 136 amino acids of SPCA2, 2+ 2 produce polyfunctional cytokines (IL-2, IFN-γ and TNF) and undergo expansion, effector activities, 4 making it unlikely that SPCA2C functions as a Ca -ATPase. Other studies report a separate function of Department of Oncology; Department 2+ contraction and stable maintenance following lesion resolution. In addition, vaccination with PEPCK SPCA2 demonstrating its ability to increase cytosolic Ca concentrations via interaction with Orai1, a Microbiology and of Obstetrics and 2+ peptide, DNA expressing full length PEPCK or rPEPCK induced strong durable cross-species protection in Ca channel on the plasma membrane. The goals of this study were to determine if SPCA2C could affect 2+ 2+ Immunology, McGill both resistant and susceptible mice. To further study the role of PEPCK in infection-induced resistance, Gynaecology, University cytosolic Ca levels and Ca movement in response to GPCR-mediated signalling. 2+ we generated PEPCK deficient (PEPCK KO) Leishmania major using CRISPR/cas9 genome editing system. Hypothesis: Secretory Pathway Ca -ATPase 2C (SPCA2C) functions in pancreatic acinar cells to maintain University, Montreal, QC of Western Ontario; 2+ 2+ We confirmed PEPCK gene deletion in vitro byWestern blot and in vivo by complete absence of PEPCK- cytosolic Ca homeostasis by regulating entry of Ca into the cytosol. 5Children’s Health specific CD4+ T cell response in PEPCK KO L. major-infected mice. The growth of PEPCK KO promastigotes Materials and Methods: The pancreas-specific isoform of SPCA2 tagged with FLAG (SPCA2C-FLAG) was in axenic culture was compromised during the late log phase as well as infectivity and proliferation in Research Institute, transiently transfected into HEK293A cells with and without the stable expression of Orai1 in combination infected macrophages in vitro. Both BALB/c and C57BL/6 mice infected with PEPCK KO L. major showed London, ON with GFP. Co-immunofluorescence analysis (IF) for FLAG and cell organelle markers was performed to smaller lesion sizes and reduced parasite burden although there were no significant differences in localize SPCA2C within the cell. Additionally, transfected cells were prepared for Fura2 ratiometric 2+ cytokine responses. Interestingly, C57BL/6 mice that healed primary PEPCK KO L. major infection showed imaging, identified by GFP expression, and then assessed for cytosolic Ca levels before and after weak delayedtype hypersensitivity (DTH) response and harboured higher parasite burden than those that stimulation with carbachol. healed wild-type infection following virulent L. major challenge. Collectively, these findings show that Results: IF analysis revealed SPCA2C localization to both the endoplasmic reticulum and golgi apparatus PEPCK is a virulent factor of Leishmania and that PEPCK-induced immune responses contribute to in HEK 293A cells. Transient expression of SPCA2C in HEK293A cells, both with and without the stable 2+ protective memory immunity. expression of Orai1, resulted in elevations in resting cytosolic Ca levels and altered responses to carbachol stimulation, indicating a functional role for SPCA2C in Ca2+ homeostasis. Conclusions: Our findings suggest SPCA2C plays a role in maintaining Ca2+ homeostasis and may interact with Orai1. Understanding how SPCA2C functions during acinar cell differentiation will provide insight into factors that affect acinar cell function. Inhibitory Effect and Molecular Mechanism of Curcumin and 5-Fluorouracil on Gastric Why Are Adolescent Athletes Hiding Concussion Symptoms? A Multimodal Approach Cancer MKN45 Cells Towards Understanding Reporting Behaviours Ting Feng, Shaoqiu Huang, Ling Zhao Kaleigh Ferdinand Pennock, Lynda M. Mainwaring

Department of Introduction: Gastric carcinoma is one of the highest morbidity of malignant tumor, and the incidence is University of Toronto, Introduction: Sport-related concussions (SRC) are a health concern for Canadian athletes of all ages and Pharmaceutical Sciences, fourth worldwide. Asian countries are with a high incidence of gastric cancer and low five-year survival. Toronto, ON levels of play. Adolescents may have an increased risk of sustaining a concussion and protracted recovery. Therefore, it is necessary to find a novel combination chemotherapeutic agents for gastric cancer Despite these risks, research indicates adolescent athletes often fail to disclose concussion symptoms. College of Pharmacy, treatment. As is known to all, 5-fluorouracil was wildly applied into gastric cancer treatment, but its Uncovering their motivations to hide SRC can be achieved by examining the literature on injury reporting Southwest Medical efficacy was poor. Curcumin is isolated from turmeric rhizome with multiple anticancer properties and behaviours, and qualitative inquiry. Therefore, the purpose of this research is to synthesize the current University, Jiangyang Qu, high safety. It was reported that combination of 5-fluorouracil and curcumin improved effectiveness on concussion reporting literature and to develop a substantive theory that explains concussion breast and colon cancer. In this study, inhibitory effect and mechanism of curcumin and 5-fluorouracil an underreporting in female adolescent athletes. Luzhou Shi, Sichuan gastric cancer MKN45 cells were investigated to provide an experimental basis for gastric cancer in clinic. Method: This research will be conducted in two complimentary phases: the first will critically examine the Sheng, China Methods: MTT assay was used to examine inhibitory effect on MKN45 cells by treating curcumin and existing under-reporting literature in adolescent athletes through a systematic review. The second phase 5-fluorouracil separately or simultaneously after culturing 48 h and 72 h. Western blot analysis was used will develop a substantive theory to explain under-reporting in female adolescent athletes using to detect COX-2 and NF-κB protein expression for exploring molecular mechanism. constructivist grounded theory methodology. The social complexity of the sport environment will be Results: Curcumin and 5-fluorouracil (1:2, mol/mol) showed 2.3 times and 2.2 times enhanced cytotoxic addressed, and the research will be informed by theories from psychology of athletic injury, adolescent effect of curcumin at 48 h and 72 h, respectively in comparison with curcumin alone. Meanwhile, the development, decision making and normative sport ethic. In line with Charmaz’s approach, important combination enhanced 3.9 times at 48 h and 3.5 times of cytotoxic effect at 72 h for 5-fluorouracil. sensitizing concepts from these areas will serve as points of departure for study development. According to the exponential curve, curcumin and 5-fluorouracil generated synergistic effect. Western Research Significance: As concussion diagnosis primarily relies on symptoms, athletes failing to report is blot analysis showed that COX-2 expression decreased by 47.17%, 44.79% and 60.21%, respectively and a serious concern. Considerable research has focused on the development and implementation of NF-κB decreased by 48.21%, 37.67% and 62.44%, respectively by treating with curcumin, 5-fluorouracil educational initiatives to improve concussion knowledge. However, findings on the effectiveness of and the combination, respectively. educational programs have been mixed, and increased concussion knowledge has not consistently Conclusion: These data suggested that curcumin and 5-fluorouracil significantly inhibited the growth of shown to improve SRC reporting behaviours in athletes. If, as the evidence suggests, athletes are aware gastric cancer effect (P < 0.05). It might be that curcumin and 5-fluorouracil could inhibit NF-κB channel of concussion symptoms and associated risks, yet remain reluctant to report, then other influences activation together leading to down-regulating COX-2 protein expression in gastric cancer MKN45 cells. warrant investigation. Preliminary research examining sport norms and SRC reporting appears promising, but there is a need for a comprehensive understanding of adolescent reporting behaviours, particularly in female athletes. Conclusion: There are multiple factors beyond education to consider when investigating why athletes do not report concussions. Elucidating the barriers and facilitators of concussion reporting is critical for ensuring the health and well-being of adolescent athletes. Scleraxis Expression in Early Phases of Wound Healing Post-Myocardial Infarction The Impact of Prenatal Alcohol Exposure on the Dynamic Range of Synaptic Plasticity K.L. Filomeno1, M.R. Zeglinski1, S.G. Rattan1, S.L. Bage2, D.Y. Cheung1, D.S. Jassal1, M.P. Czubryt1, J.T. Wigle1, I.M.C. Dixon1 Christine J. Fontaine, Cristina Pinar, Brian R. Christie

1Department of Cardiac fibrosis is causal to heart failure and it affects chronic wound healing. Wound healing and fibrosis Division of Medical Introduction: Prenatal alcohol exposure (PAE) can have detrimental effects for the developing fetus, and Physiology and is mediated by myofibroblasts, the interstitium of the myocardium. Ski has been identified as a repressor Sciences, University of depending on the timing of exposure and dosage, can produce some form of Fetal Alcohol Spectrum of the fibroblast activation. Scleraxis has been implicated in enhancing the profibrotic effects of Disorders (FASDs). The prevalence of FASD is difficult to track given current diagnostic criteria, however Pathophysiology, Institute myofibroblasts. We now investigate how Scleraxis contributes to wound healing in vivo. Our first step in Victoria recent estimates place the incidence as high as 5% of first grade schoolchildren. FASDs are characterized of Cardiovascular this series of studies is to measure mRNA and protein expression of Scleraxis in post-myocardial infarction by cognitive impairments that include learning and memory, executive function and decision-making Sciences, Faculty of (MI) hearts. The left anterior descending (LAD) coronary artery of male Sprague-Dawley rats was ligated (Patten et al., 2014; Popova et al., 2012). Animal models of FASD are used to investigate the cellular to induce a myocardial infarction. Control (sham-operated) animals underwent surgery without ligation mechanisms underlying these behavioural impairments particularly in relation to learning and memory Health Sciences, College of the LAD artery. Animals were sacrificed at 48 hours, 4 days, 1, 2, 4 and 8 week(s) post-MI, and the tissue (Gil-Mohapel et al., 2010). These animal studies have focused on synaptic plasticity in hippocampus, a of Medicine, University of was collected for analysis. Scleraxis mRNA expression within the infarct scar area was significantly brian region critical for some behavioural learning and memory processes. Synaptic plasticity is the Manitoba; 2The Burrell elevated vs. sham controls at 8 weeks post-MI. Scleraxis protein expression increased within the infarct ability for neurons to modify synaptic transmission in response to previous activity and experience, such scar at 4 days, 1 week and 2 weeks vs. controls post-MI. In future studies, we will compare Scleraxis as long term potentiation (LTP) which is the resulting response following high-frequency stimulation and Laboratory, St. Boniface expression to other regulatory genes in pathological wound healing to identify novel targets for the long term depression (LTD) which is the product of prolonged low-frequency stimulation. It is likely that Hospital Albrechtsen treatment of fibrotic disease. both LTP and LTD are essential for normal learning and memory to occur, however the majority of work Research Centre, on PAE to date has focused on LTP (Fontaine et al., 2016). Previous work from our laboratory has shown Winnipeg, MB that PAE reduces the magnitude of LTP in adult offspring (Helfer et al., 2012; Patten et al., 2013), however few studies have examined LTP and LTD in PAE juveniles. Together these data will better elucidate how PAE impacts the dynamic range for synaptic plasticity in the developing brain. Methods: In the present study we examine both hippocampal LTP and LTD using in vitro field electrophysiology in the juvenile brain following administration of an alcohol-containing liquid diet (Weinberg, 1985) throughout gestation. Results: Our data from juvenile PAE offspring indicate that the magnitude of LTP and LTD is restricted when compared to control offspring. Conclusions: PAE may restrict the dynamic range of synaptic plasticity in the hippocampus and negatively impact cognitive flexibility during this critical period. The Interaction of Adenovirus E1A with the Mammalian Protein Ku70/XRCC6 Clinical, Genetic and Functional Study of ABCA4 Mutations in Stargardt Patients Jasmine Rae Frost1, Oladunni Olanubi1, Stephen Ka-Hon Cheng2, Andrea Soriano1, Leandro Crisostomo1, Alennie Lopez1, Fabian Garces1, Kailun Jiang2, Laurie L Molday1, 2, Heidi Stohr3, Bernhard Weber3, David Maberley1, 2 , Robert S. Molday1, 2 Peter Pelka1 1Department of Introduction: ABCA4 is an ABC importer that is predominantly expressed in the rod and cone 1Department of Early region 1A proteins (E1A) are the first proteins expressed upon viral infection. E1A is therefore Biochemistry and photoreceptor cells of the retina where it plays a crucial role in vision. ABCA4 helps to regulate the turnover of all-trans-retinal to the light-sensitive pigment 11-cis-retinal, while at the same time prevents Microbiology, University responsible for the remodeling of the intracellular environment to promote viral replication. It is also the Molecular Biology; major transactivator of viral early gene expression. E1A carries out its functions predominantly by the formation and accumulation of harmful bisretionids in the outer-disc membranes of photoreceptors. 2 of Manitoba; binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A Department of To date, more than 1000 point-mutations in ABCA4 have been directly linked to the development of 2General Byng School, enables it to bind to a variety of proteins and form new molecular complexes with novel functions. Ku70 Ophthalmology and Stargardt macular degeneration (STGD) and related retinopathies. How mutations in the ABCA4 gene affect the structure and function of ABCA4 and lead to the development of STGD is not well understood. Winnipeg, MB has recently been identified as a novel binding partner of E1A. Ku70, encoded for the by the XRCC6 gene Visual Sciences, University is 70kDa in size and often found in a heterodimer state with its homolog Ku80. This relatively conserved Methods: DNA from Stargardt patients was screened for mutations in the ABCA4 using MiSeq – Illumina. of British Columbia, protein is a main component of the Non-Homologous End Joining and DNA damage response pathways. Mutations in the gene responsible for STGD were identified and were cloned by site-directed mutagenesis. Ku70 has also been shown to play a role in telomere maintenance in mammalian cells, as well as having Vancouver, B.C; 3 Institute Mutant isoforms of ABCA4 were immunopurified using a 1D4-column against a 1D4-tag placed at the C-terminus. Purified ABCA4 was used for structure function studies looking at: 1. Cellular localization of a role in V(D)J recombination for the formation of genetic diversity in the immune response of vertebrates. of Human Genetics, We have identified Ku70 as a novel E1A binding protein that interacts with the CR4 region of E1A. ABCA4 mutants in transiently transfected COS7 cells. 2. Protein expression levels from lysate of transiently University of Regensburg, Depletion of Ku70 lead to a reduction in E1As ability to induce S-phase in quiescent human fibroblasts, transected HEK293T cells. 3. Functional activity of ABCA4 mutants using substrate binding assays and indicating Ku70 is important for virally-induced S-phase entry. We have also found that Ku70 is localized Regensburg, Germany ATPase assays. to viral replication centers, and it was found to associate with the viral genome. The association with the Results: Illumina-sequencing-results revealed that 11 of the 13 patients diagnosed with STGD had viral genome is a potential viral mechanism to prevent the detection of foreign genomes within the missense mutations in the ABCA4 gene. Localization of ABCA4 mutants in COS7 cells revealed some infected cell. Depletion of Ku70 inhibited the virus’ ability to replicate, but had minimal effects on viral ABCA4 mutants localized to the ER where as some mutants localized to vesicles similar to WT. 2. genome replication and viral gene transcription. Interestingly we have shown that Ku70 may play a role Expression levels for the majority of ABCA4 mutants is similar to WT but some mutants express at 50% or in the expression of cell cycle genes, as upregulation of CDKN1A, PCNA and CDK6 were observed upon below WT levels. Substrate-binding activity and ATPase activity are severely affected in some ABCA4 Ku70 depletion. This is supported by data showing Ku70 is recruited together with E1A to cell cycle mutants and only moderately affected in others. promoters after adenovirus infection. Overall, we show that Ku70, a novel E1A C-terminus binding Conclusion: This is the first study to directly compare clinical and genetic data from STGD patients with protein is important in the viral life cycle. biochemical structural and functional data from purified ABCA4 protein. Our results reveal that protein misfolding is strongly correlated with attenuated protein expression levels, and a severely decreased functional activity of purified ABCA4. At the clinical level, misfolded mutants also showed stronger phenotypes in STGD patients than properly folded mutant isoforms. Targeting Post-Surgical Breast Cancer Metastasis Through Combining Natural Killer Transcriptome-Wide Adaptive Changes of Gene Expression upon Repeated Stimulation T Cell Immunotherapy and Low Dose Chemotherapy by Dopamine Simon Gebremeskel1, 4, Kaitlyn Tanner1, Lynnea Lobert1, 4, Brent Johnston1, 2, 3, 4 Maryam Ghandizadeh Dezfouli, Urmi Das, Jiuyong Xie

1Department of Breast cancer is the most common cancer in Canadian women and the second leading cause of cancer Department of Physiology Introduction: Gene expression in the brain’s reward circuitry undergoes remarkable adaptive changes Microbiology & deaths. Given that most mammary tumors are surgically resectable and over 90% of breast cancer- and Pathophysiology, Max upon repeated exposure to external signals. A key step in the expression of most genes is alternative associated deaths are due to metastasis, new therapeutic strategies targeting metastasis are required. pre-mRNA splicing, which controls the splice variants that are critical for adaptive responses. Dopamine Immunology; Natural killer T (NKT) cells are a rare population of immune cells that have been shown to limit primary Rady College of Medicine, is increased by drugs of abuse and most types of rewards. In this study we sought to obtain a 2Department of tumor growth and target distant metastatic disease in various animal models. We have shown that NKT Rady College of Health transcriptomic view of repeated dopamine-induced changes in gene expression compared to the first Pediatrics; 3Department cell activation improves survival in a model of post-surgical metastatic breast cancer. We are now Sciences, University of exposure in striatal cells. expanding this work to determine whether NKT cell activation can be combined with chemotherapies to Methods: Mouse striatum-derived cells were divided into three groups: those treated just once with of Pathology, Dalhousie Manitoba, Winnipeg, MB improve outcomes. In our model, 4T1 mammary carcinoma cells were injected into the mammary fatpad dopamine (10nM) for 3 hours, those treated up to 7 times with 21 hours of interval between each University; 4Beatrice of syngeneic BALB/c mice. Tumors were resected at day 12, and mice were treated with cyclophosphamide treatment, and a group of non-treated controls. Total RNA were extracted, and whole-transcriptome Hunter Cancer Research or gemcitabine. On day 17, NKT cells were activated by transfer of dendritic cells loaded with the RNA-Seq sequencing was carried out. Differential levels of total transcripts were determined using edgeR, glycolipid antigen α-GalCer. We also examined whether gemcitabine or mafosphamide (active component and alternative splicing, alternative transcription start site and polyadenylation were analyzed using Institute, Halifax, NS of cyclophosphamide) would induce immunogenic cell death of 4T1 cells in culture. Chemotherapeutics DEXSeq and/or MATS. did not affect NKT cell activation as measured by serum IFNγ levels. Treatment with cyclophosphamide, Results: We identified 591 pre-mRNA splicing and 254 transcript level changes in response to repeated gemcitabine, or α-GalCer-loaded dendritic cells alone reduced metastasis and prolonged survival. versus single stimulation by dopamine. Interestingly, we found that the two schemes of dopamine Combined treatments significantly enhanced survival. NKT cell activation decreased the frequency and stimulation caused changes in different clusters of genes at the total transcript level. In particular, ‘mRNA immunosuppressive function of myeloid derived suppressor cells (MDSCs). Treatments resulted in splicing’ genes are most significantly enriched in the ones with splicing changes after repeated dopamine enhanced tumor specific immunity as surviving mice exhibited slower tumor growth following secondary exposure, while genes in ‘transcription’ were enriched most significantly after single treatment. tumor challenge. Gemcitabine and mafosphamide also increase the immunogenicity of cancer cells in Conclusions: This study provides a novel molecular dimension of adaptive changes across cell vitro by increasing the exposure/release of MHCI, MHCII, CD1d, Calreticulin, HMGB1 and ATP. NKT cell generations, and suggests that alternative splicing and/or expression of specific genes are regulated by activation therapy can successfully be combined with low doses of gemcitabine or cyclophosphamide to repeated and single dopamine exposure differently. We speculate that splicing changes of splicing enhance protection against tumor metastasis and recurrence. This work provides a clear rationale for factors affect their function, and consequently control alternative splicing of their target genes, among combining chemotherapy with NKT cell immunotherapy to target metastatic disease in the clinical other changes, leading to adaptive splicing. Understanding the control of adaptive splicing by dopamine setting. will not only help us understand basic cell physiology but also provide us better approaches to design novel strategies to treat addiction and other dopamine-related disorders such as Parkinson disease and schizophrenia. Sumoylation in Lysine 126 Regulate c-Myc Tumorigenic Activity Mohammad Ghorbani

McGill University, Sumoylation is a reversible post-translational modification that targets a variety of proteins to modify machinery is critical for the tumorigenic activity of c-Myc. Notably, inhibition of sumoylation in cancer Montreal, QC protein-protein interaction, subcellular localization and cellular functions. This modification involves cells containing high expression of c-Myc promotes apoptosis. However, the underlying mechanism of covalent conjugation of the SUMO (small ubiquitin-related modifier) peptide to the lysine residue of the the sensitivity of c-Myc-expressing tumor cells to SUMO inhibition is unknown. c-Myc increases target protein through an isopeptide bond. Similar to ubiquitination, surnoylation requires series of transcription of SUMO-activating enzyme 1 (SAEl). Based on these studies, we hypothesized that direct enzymatic activities. The El activating enzyme activates SUMO through an ATP dependent process. El sumoylation of cMyc regulates its tumorigenic functions. Several research groups tried to identify the consists of two subunits, SUMO-activating enzyme 1 (SAE1) and SUMO-activating enzyme 2 (SAE2). The c-Myc sumoylation site but unfortunately there was not any clear sumoylation site reported. The sole SUMO-conjugating enzyme, Ubc9, mediates the ligation of activated SUMO to the ε-amino group of conclusion was there is more than one sumoylation site presented in c-Myc. Given all the information the lysine residue within the Sumoylation consensus motif of substrate proteins. The sumoylation together we ended up recognising the c-Myc major sumoylation site which is Kl26. Mutation in this consensus motif consists of ΨKx(D/E), where Ψ is a large hydrophobic amino acid, x is any amino acid and Lysine showed major reduction in this modification. Using the mutagenesis version of gene showed DIE is an acidic residue. The role of SUMO E3-ligases is not very clear; however, there are some examples, interesting effect on c-Myc tumorigenic ability. Further experiments showed highly effect on c-Myc in which SUMO E3-ligases accelerate sumoylation or contributes to target specificity. Accordingly, transcription factor activity as-well. In conclusion, Mutation in lysine 126 of c-Myc reduce the sumoylation E3-ligases promote the interaction between Ubc9 and its substrate proteins. Several proteins, such as level and induce the c-Myc transcription and tumorigenic activity. SAP and Mizl domain proteins, have been identified to display SUMO E3-ligase activity. Despite the significant advance in our understanding of the role of sumoylation in variety of cellular processes, little is known about its role in cancer progression. Recent studies have demonstrated that inhibition of sumoylation of microphthalmia-associated transcription factors (MITF) promotes the formation of melanoma and renal cell carcinoma. Point mutation of the lysine residue within the SUMO consensus motif enhances MITF binding to downstream targets and stimulates transcription of MRT and HIFlA, two MITF regulated genes involved in transformation of melanocytes and kidney cells. In my research, I have recently obtained preliminary data showing that c-Myc itself is sumoylated and thus provided another line of evidence for the involvement of sumoylation in cancer. The proto-oncogene product c-Myc is a master regulator of cell growth and differentiation. Overexpression and stabilization of this protein has been related to several human cancers for instance high expression of this protein has been reported in about 25% of the breast cancers. Given the dose-dependent regulatory roles of Myc in cancer precise regulation of c-Myc expression is critical for mammalian cells. These results suggest that blocking of c-Myc oncogenic function can inhibit tumor progression. Recent study showed that the sumoylation A Comparative Study of Host Cell Proteomic Change After Zika Virus Infection Neonatal Antibiotic Exposure Skews the Developing Microbiota and Mucosal T Cell Kathleen K.M. Glover1, 2, Kevin M. Coombs1, 2 Populations Resulting in Exacerbated Colitis in Nod2-/- Mice Ashleigh Goethel1, Williams Turpin2, Sandrine Rouquier3, Galliano Zanello3, Dana Philpott1, Kenneth Croitoru1, 2, 3, 4 1Department of Medical Introduction: Zika virus, a mosquito-borne Flavivirus was first isolated in rhesus monkeys in Uganda in Microbiology, Faculty of 1947.This virus has emerged as a new type of public health threat due to increase in the spread of the 1Department of Background: Early life represents a ‘critical window’ of development for the gut immune system and the virus, microcephaly cases in babies born to Zika infected mothers as well as Guillain–Barré syndrome. Health Sciences, Immunology, University of microbiota. From birth, we are colonized with trillions of bacteria and other microorganisms, which Appropriate interventions and management strategies are being sought to control Zika associated influence and co-develop with the mucosal immune system. Antibiotic exposure during early life is University of Manitoba; Toronto; 2Zane Cohen diseases. One strategy is to understand the mechanism of replication of Zika virus in an infected cell in associated with development of auto-inflammatory diseases, including Crohn’s disease, but the 2Manitoba Centre for order to identify targets sites for future interventions such as antivirals and vaccine production as well as Centre for Digestive mechanism remains unclear. We hypothesized that early life antibiotic exposure would have a prolonged rapid kit for early diagnosis. Thus, this study seeks to employ proteomic techniques to identify proteins Proteomics & Systems Diseases, Lunenfeld- impact on both microbiota composition and T cell function within the gut, and that this would influence significantly induced and regulated during Zika infection. colitis severity, particularly in a genetically susceptible host (Nod2-/- mice). Biology, Winnipeg, MB Tanenbaum Research Methods: Somalogic and TMT-based mass spectrometric analysis of Vero and Hek 293 cells infected at an Methods: Heterozygous Nod2+/- breeders were used to generate littermates. Dams and littermates MOI of 3 at selected time points will help us define proteins that are actively induced during Zika virus Institute, Sinai Health received water or amoxicillin [200mg/L] in the drinking water until weaning. Amoxicillin passes through infection. The effect of significantly regulated proteins on Zika virus replicative cycle will be accessed by System; 3Department of the milk from dam to pups. Fecal samples collected at weaning and at 5 weeks old were analyzed by 16S q RT-PCR and immunofluorescence. sequencing. At 8 weeks old, mucosal lymphocyte populations of the small and large intestine were Medicine, University of Results and Conclusion: Study is currently ongoing and successful completion will add up to existing analyzed via flow cytometry. Colitis was induced by five days of 3.5% DSS in the drinking water. 4 knowledge about the mechanism of replication, transcription and translation as well as potential target Toronto; Division of Results: Neonatal amoxicillin treatment resulted in a significant shift of the gut microbial community sites for future interventions towards control and management of diseases caused by Zika virus. Gastroenterology, structure at weaning, with a significant reduction of Bifidobacterium and Erysipelotrichaceae and a Mount Sinai Hospital, bloom of Enterobacteriaceae, which was irrespective of Nod2 genotype or sex. At 5 weeks old, antibiotic- treated Nod2-/- mice had reduced microbial diversity and maintained a loss of Bifidobacterium and Toronto, ON Erysipelotrichaceae compared to antibiotic-treated WT mice and water-treated controls. Antibiotic- treated Nod2-/- mice had an increase of Foxp3+, RORγt+, and Foxp3+RORγt+ T cells in the gut lamina propria. These mice also showed exacerbated DSS colitis compared to WT mice and water treated controls, with increased weight loss, increased levels of fecal lipocalin-2 and greater colonic shortening. Analysis of colonic lymphocytes after DSS identified a significant increase in Gata3+ Tregs and increased IL4, IL6 and IL33 expression. Conclusion: These results suggest that neonatal antibiotic exposure has lasting effects on microbiota composition and alters microbe-driven immune cell development via Nod2 signalling. Moreover, these cells may be involved in the regulation of colitis and could be potential targets for therapeutics. LAG-3 Inhibition of T Cell Activation Exercise in Pregnancy and Children’s Cardiometabolic Risk Factors: A Systematic Review Colin G Graydon1, Andrew T Stalker1, Jen A Juno1, Genevieve Boily-Larouche1, Julie Lajoie1, Keith R Fowke1, 2, 3 and Meta-Analysis Laetitia Guillemette1, 2, 4, D. Scott Kehler1, 3, 4, Naomi C. Hamm1, 3, 4, Jonathan M. McGavock1, 2, Todd A. Duhamel1, 3 , 4 1Department of Medical Background: Persistent antigen stimulation of lymphocytes can lead to impaired cytokine production Microbiology and and cellular proliferation; this is known as ‘exhaustion’. Exhausted cells express proteins that contribute to 1University of Manitoba; Introduction: Prenatal exercise might reduce offspring cardiometabolic risk in childhood. We conducted this dysfunctionality; LAG-3 is one such protein. When antigen is presented to a T cell, LAG-3 binds to Infectious Diseases, 2Children’s Hospital a systematic review and meta-analysis to clarify this relationship. MHC class II on the antigen presenting cell and impairs T cell activation; however, the mechanism of Methods: We searched Pubmed, MEDLINE, EMBASE, and CENTRAL up to May 2016. Two reviewers University of Manitoba; LAG-3-mediated impairment is unknown. Additionally, LAG-3 is upregulated on T cells latently infected Research Institute of independently identified observational studies and randomized controlled trials (RCTs) evaluating 2Department of with HIV. LAG-3 blockade may enhance the ability of latently infected cells to become activated and Manitoba; 3Health, Leisure prenatal exercise and offspring cardiometabolic outcomes and extracted trial-level data with piloted simultaneously, restore functionality of exhausted cells. In this way, LAG-3 blockade could encompass Community Health & Human Performance forms. The primary outcome was birthweight; secondary outcomes included large-for-gestational age both arms of a “shock and kill” strategy, but a greater understanding of how LAG-3 inhibits cell function status, fat and lean mass, dyslipidemia, and blood pressure. We included 47 studies and companion Sciences, University of Research Institute; is needed. articles of the 10 574 citations initially identified and evaluated their internal validity. Data was pooled Manitoba, Winnipeg, MB; Objective: To clarify the effects of LAG-3 blockade on human T cells. 4Institute of using random effects models. Approach and Results: We stimulated human PBMC with the superantigen SEB with or without antibody 3Department of Medical Cardiovascular Sciences, Results: We included 12 original observational studies (n=165 230 children) and 24 original RCTs (n=4930 blockade of LAG-3. We found that LAG-3 blockade enhances T cell proliferation, in support of similar children). Observational studies were highly heterogeneous and had discrepant conclusions. Meta- Microbiology, University Winnipeg, MB findings by other groups. analyzed RCTs indicated that prenatal exercise did not significantly impact birthweight (mean difference of Nairobi, Nairobi, Kenya We also evaluated cross-linking methods for assessing the LAG-3 mechanism of inhibition. [MD]: −45.8g, 95% confidence interval [CI] −156.1 to 64.5g) or large-for-gestational age status (risk ratio: Future Directions: We will use cross-linking and blockade methods to assess the intracellular effects of 1.45, 95%CI 0.97 to 2.17) compared to no exercise. Sensitivity analyses of RCTs showed that prenatal LAG-3. Specifically, we will assess the impact of LAG-3 on the T cell receptor signaling pathway. This will exercise reduced birthweight only in women with a body mass index <25 kg/m2 (MD: −238.5g, 95% CI: inform how LAG-3 inhibits T cell activation and its potential to reverse HIV latency. −475.1, −1.8g). Other outcomes were too scarcely reported to be meta-analyzed. Conclusion: Prenatal exercise does not significantly impact birthweight or large-for-gestational-age status and is not sufficient to reduce birthweight in offspring of women with overweight. Longer follow up of offspring exposed to prenatal exercise is needed along with measures of relevant metabolic variables (eg. fat mass). Comparing the Health-Related Quality of Life of Indigenous and Non-Indigenous Peoples Eliminating the Luminal Grey Zone: Using a Novel Estrogen Receptor DNA-Binding Living in Canada Signature to Describe Luminal A Breast Cancers Christina L. Hackett1, David Feeny2, Gillian Mulvale3 Lacey Haddon, Hosna Jabbari, Xiuying Hu, Kirstin Arnold, Judith Hugh

1Department of Clinical Background: Although there is substantial variation in health outcomes among certain demographically, University of Alberta, Introduction: Estrogen receptor positive (ER+) breast cancer has been divided into two subtypes, luminal Epidemiology & culturally and ethnically delineated populations in Canada, Indigenous peoples living in Canada face Edmonton, AB A and luminal B, which differ in their ER expression and response to hormone therapy. Luminal A patients unique disparities in health and mental as well as in the social determinants of health (SDH) they often have higher ER levels, excellent response to hormone therapy and occur predominately in post- Biostatistics, McMaster experience. We investigate population health differences with respect to health-related quality of life menopausal women, when serum estrogen (E2) levels are undetectable. Currently, molecular testing is University; 2Department (HRQL). only 75% accurate at differentiating between the luminal subtypes, causing many luminal A patients to of Economics, McMaster Methods: Using a nationally representative Canadian survey, we employ multivariate regression analyses receive unnecessary chemotherapy. Since ER is a major regulator of gene transcription in breast cancer to estimate the variation in the Health Utility Index Mark 3 (HUI3) scores for Indigenous and non- cells, we hypothesized that increased ER expression enables binding of distal enhancer regions and University; 3DeGroote Indigenous peoples living in Canada. We then decompose this variation between Indigenous and non- promotes the chromatin reconfiguration required for gene activation in the absence of estrogen. School of Business, Indigenous peoples within HUI3 disability levels, and across various points of the HUI3 distribution using Methods: MCF-7 cells were transduced with a doxycycline inducible, mEmerald fluorescent, lentiviral McMaster University, unconditional quantile regression methods. We test the degree to which control variables account for the plasmid with (MCF7-ER) or without (MCF7-EM) the estrogen receptor. Cells were adapted for 3 days in difference in HRQL between groups, to understand how the experience of being Indigenous may extend E2-free media, induced with 0.5μg/ml doxycycline for 24 hours and then treated with 10nM E2 or ethanol Hamilton, ON beyond the differences in access to structural resources and SDH documented for these two groups. control. Next Generation Sequencing (NGS) was done for DNA-binding by chromatin immunoprecipitation Results: After controlling for several sets of SDH and factors related to HRQL, Indigenous peoples living (ChIP-seq) experiments using an anti-ER antibody after 1 hour E2 exposure and for gene expression off-reserve in Canada, report lower HRQL than non-Indigenous Canadians. This difference remains (RNA-seq) using RNA extracted after 24 hours E2 exposure. NGS libraries were run on the Illumina statistically significant and clinically important. At the 10th and 30th percentile of the HUI3 distribution, NextSeq 500 and analyzed using standard bioinformatics techniques. ChIA-PET data for long-range ER this difference is mostly driven by the discrepant levels exogenous, socio-demographic, health chromatin interactions from MCF-7 cells treated with E2 was obtained from the NCBI Gene Expression behavioural and structural covariates Omnibus (GEO) database. Conclusion: As at least 80% of Indigenous peoples in high-income countries rely on non-Indigenous, or Results: Our ChIP-seq results show increased ER expression leads to novel ER peaks at several well- dominant culture health systems as their primary source of healthcare, thus health policy and service defined ER target genes, in the absence of E2. Furthermore, differential gene expression analysis for these practice must be directed towards culturally informed care to address health disparities. Covariates, genes show an increase in basal activation in the MCF7-ER cell line. Comparison of our ChIP peaks against controlling for various SDH, account for the majority of the variation between Indigenous and non- the published MCF-7 ChIA-PET dataset show these novel ER DNA binding sites correspond to long-range Indigenous peoples’ HRQL; investing in reducing inequities in these factors could have a significant chromatin interactions and may mark distal enhancers. impact on reducing inequities in HRQL for Indigenous Canadians. Conclusion: Our results suggest that increased ER expression promotes transcriptional activation in the absence of ligand through the reconfiguration of chromatin. These findings highlight a novel biological mechanism that could serve as a diagnostic test to better define the luminal A subtype. Epithelial Polarity Remodeling and Luminal Collapse in Early Mammary Tumourigenesis Ruba Halaoui1, 2, Joseph Szymborski1, 2, Carlis Rejon1, Sarkis Meterissian4, 5, 6, William J. Muller1, 3, Atilla Omeroglu6, 7, Luke McCafTrey1, 2, 4

1Rosalind and Morris Introduction: Breast cancer is a leading cancer type in women and although breast cancer mortality has cells exhibited an apical patch at cell-cell contacts. Furthermore, non-polarized cells exhibited a Goodman Cancer reduced, incidence is increasing. A greater understanding of the events relating to initiation and the proliferative advantage over polarized cells. Re-establishment of tension through finally, de-induction of transition to malignant disease is therefore necessary to reduce the incidence of breast cancer. Breast the tumour in mice leads to not only a regression of the tumour, but also to reversion of polarity in the Research Centre, McGill cancer progresses through benign and malignant stages, associated with altered epithelial organization, resulting ducts. University; 2Division of with loss of ductal lumen and cell polarity being major phenotypes. How apical-basal polarity is Conclusions: Therefore, there exist different modes of polarity loss in early breast cancer progression. The Experimental Medicine, disrupted, however, is incompletely understood. Here, our aim is to better characterize early stages of most prevalent mode is a progressive loss through different pre-malignant to malignant stages, through progression from a polarity point of view, focusing on describing the mechanisms by which luminal filling the cooperation of multiple mechanisms such as mis-oriented divisions, increased proliferation, and McGill University; with non-polarized cells occurs. loosened apical tension, leading to luminal collapse, rather than filling. Notably, this series of 3Department of Methods: We examined cell polarity loss in biopsies from human breast that contained pre-invasive morphological steps leading to the formation of solid ducts is in fact reversible, and ducts are able to Biochemistry, McGill breast lesions and tumors from mice expressing the PyMT oncogene, by immuno-staining with different re-establish proper organization and polarity upon cessation of oncogenic activity. polarity and proliferation markers. We then moved on to an ex-vivo culture model, where we extracted University; 4Department primary cells from the PyMT mouse model and when stated, knocked-down genes by lenti-viral infection. of Oncology, McGill We proceeded to grow them on an extra-cellular matrix allowing them to form cysts, inducing oncogene University; 5Department expression after cyst formation with Doxycycline. We then validated our culture results by performing of Surgery, McGill orthotopic transplants using these PyMT primary cells and injecting them into a cleared mammary fat pad of syngeneic mice. 6 University; McGill Results: In our human samples, there were different polarity phenotypes present at DCIS, each having University Health Centre; different modes of progression towards polarity loss. In fact, apical polarity was lost progressively in the 7Department of absence of intra-luminal filling on these early stages of breast cancer progression. Collective invasion of polarized cells initiates luminal filling to form secondary lumen. Further investigations using the PyMT Pathology, McGill mouse model showed that instead of luminal filling, asymmetric cell divisions produced multilayered University, Montreal, QC ducts with a population of basally positioned cells lacking cell polarity. The remaining polarized cells were subsequently eliminated following collapse of luminal integrity, with defects in apical tension marked by reduced activity of myosin II and RhoA-GTP. At later stages, the luminal space disappeared and Early Childhood Antibiotics Exposure and the Risk of Autism Spectrum Disorders Efficacy of Treatments for Pain Associated With Trapeziometacarpal (Thumb Base) Amani Hamad1, I fan Kuo1, Silvia Alessi-Severini1, Salah Mahmud1, 2, Marni Brownell2 Osteoarthritis: A Systematic Review (Part 1 Qualitative Overview) Tokiko Hamasaki1, 2, 3, Sylvain Laprise4, Patrick Harris3, 5, Nathalie J. Bureau1, 6, 7, Nathaly Gaudreault8, 9, Lyne Lalonde1, 10, 1College of Pharmacy, Background: Autism spectrum disorders (ASD) are a leading cause of disabilities in children and adults. Daniela Ziegler11, Manon Choinière1, 12 University of Manitoba; Changes in microbiota composition induced by antibiotics use in early life can impair the gut-brain axis and thus, have been proposed as a possible etiology of ASD. 1Centre de recherche du Centre Introduction: Trapeziometacarpal osteoarthritis (TMO) causes persistent pain, handicapping a large 2Department of Objectives: We examined the association between antibiotic use during the first year of life and the risk hospitalier de l’Université de number of individuals. Despite the fact that the thumb is solicited in 50% of hand function and essential Community Health of ASD. Montréal (CRCHUM); 2School of for daily activities, this type of chronic pain is understudied. This systematic review (SR) thus aims to Methods: A population-based cohort study was conducted using the Manitoba Population Research Rehabilitation, Faculty of Sciences, University of Medicine, L’Université de document the efficacy of all the existing pharmacological, nonpharmacological, and surgical interventions Data Repository. Children born in Manitoba between 1998 and 2015 were followed until migration, 3 for TMO in terms of pain, function, psychological well-being, quality of life, and/or treatment satisfaction. Manitoba, Winnipeg, MB Montréal; Hand Center, Centre death, age 18, or end of study period. Antibiotics use to age 1 was identified using the Drug Program hospitalier de l’Universite de Methods: The protocol was developed based on the recommendations of the Preferred Reporting Items for Information Network. Standard diagnostic algorithm was used to identify ASD diagnoses from hospital Montreal (CHUM), Montreal, QC; Systematic Reviews and Meta-Analyses, registered with PROSPERO, and published in BMJ Open. In Part 1, 4 abstracts, medical claims and educational special needs database. Logistic regression was used to Department of Physiotherapy, the methodological quality of existing SRs was appraised by using Assessment of Multiple Systematic Hôpital Hôtel-Dieu d’Amos, examine the association between antibiotic use and ASD diagnosis while adjusting for potential Amos, QC; 5Department of Reviews (AMSTAR). In Part 2, the Cochrane Effective Practice and Organization of Practice Risk of Bias Tool and confounding by infections, maternal and child characteristics in addition to factors previously reported Surgery, Faculty of Medicine, the Effective Public Health Practice Project Tool are being used to assess the methodological quality of to have an association with ASD or with microbiota colonization. L’Universite de Montreal; randomized controlled trials (RCT) and observational studies (OS) not included in earlier SRs or included Results: Compared to children who did not use antibiotics during infancy, those who received one 6Department of Radiology, in those having poor quality ratings. antibiotic course or more did not have higher rates of ASD: Odd ratios (OR) 0.99 (95% CI 0.90-1.09). This Radio-Oncology and Nuclear Results: 4916 references were identified through bibliographic electronic databases and an additional 70 Medicine, Faculty of Medicine, estimate did not change appreciably after adjusting for confounding (0.89; 95% CI 0.80-1.00). Types of L’Universite de Montreal; through the eligible ones. Of these, 21 SRs, 52 RCTs, and 53 OSs met the selection criteria, documenting antibiotic and number of antibiotic courses also showed no statistically significant association with ASD 7Department of Radiology, the efficacy of the following 18 interventions: naproxen, topical medications, steroid injections, diagnosis. Several covariates including sex, receipt of income assistance, region (rural versus urban), CHUM, Montreal, QC; 8School of hyaluronate injections, saline injections, dextrose prolotherapy, acupuncture, leech therapy, nettle sting, gestational age, mothers age at delivery, and maternal psychiatric disorders were found to be significant Rehabilitation, Faculty of orthoses, exercises, joint protection, laser therapy, manual therapy, surgery, combination of orthosis- Medicine and Health Sciences, predictors of ASD. exercises, of orthosis-exercises-joint protection, or of exercises-manual therapy. The critical appraisal of Université de Sherbrooke; Conclusion: Preliminary results suggest that antibiotics use during the first year of life is not associated 9Centre de recherche du Centre Part 1 revealed that only 2 SRs were of high quality while 7 SRs of low quality. The mean AMSTAR score of with increased risk of ASD. Sensitivity analysis will be conducted to test the robustness of study estimates. hospitalier universitaire de all the SRs was 4.7 ± 2.5/11. The results of these SRs support with varying levels of evidence the use of Additional analysis using a sibling control group will be conducted to limit residual confounding. Sherbrooke, Sherbrooke, QC; the following interventions for TMO: steroid/hyaluronate injections, prolotherapy, orthoses, manual 10 Faculty of Pharmacy, Universite therapy, combination of exercise-manual therapy or of exercise-orthoses, and surgery. The superiority of de Montreal; 11CHUM Library; 12Department of Anesthesiology, a particular type of orthosis or of surgical intervention has not been demonstrated. Faculty of Medicine, Universite Conclusion: This qualitative overview of SRs allowed the identification of the TMO interventions whose de Montreal, Montreal, QC efficacy was relatively well documented in the literature. An Examination of Youth’s Chronic Disease Risk Behaviours and their Associations with Bone Morphogenetic Protein 4 Regulates Endogenous Cell Response and Astrocyte Obesity: Cross-Sectional Analyses Reactivity Following Spinal Cord Injury Nour Hammami, Scott Leatherdale, Philip Bigelow, Ashok Chaurasia Christopher G. Hart, Scott M. Dyck, Hardeep Kataria, Kavillapilli T. Santhosh, Soheila Karimi-Abdolrezaee

School of Public Health Introduction: The prevalence of overweight and obesity among Canadian children and adolescents has University of Manitoba, Introduction: Reactive astrocytes modulate the spinal cord injury (SCI) microenvironment by producing and Heath Systems, reached 9% and 29% respectively in 2007, up from 3% and 14% in 1979. A report released in 2016 from Winnipeg, MB a plethora of inhibitory factors. In this study, we demonstrate that activated astrocytes influence the the Senate of Canada prioritized obesity on a federal and provincial level. Chronic disease risk factors (e.g. properties of neural stem/progenitor cells (NPCs) by upregulating bone morphogenetic protein 4 (BMP4). University of Waterloo, physical activity, nutrition intake, sedentary status and substance use) and excess weight are seen to BMP4 is a well-known morphogen in the developing CNS with an established role in modulating Waterloo, ON track from adolescence into adulthood, making adolescence an optimal phase for interventions. This astrocyte differentiation. In a clinically-relevant model of compressive SCI in rats, we found that BMP4 is study examined latent classes of health behaviours among adolescents and their associations with robustly upregulated in the acute phase of SCI between 1 and 3 days post injury. To date, the role of BMP4 weight status (normal, overweight, or obese). in acute SCI has remained largely unknown. BMP4 is a key morphogen in neural development and Methods: This study used year 2013 data of the COMPASS (2013-2021) Study. Data from 33,836 therefore understanding its role in injury mechanisms will aid in developing repair strategies for CNS adolescents in grades 9-12 was used to identify information on student’s health behaviour practices and injuries. weight status. Latent Class Analysis was used to determine subgroups based on chronic disease risk Methods: To investigate the impact of BMP4, we delivered an endogenous inhibitor of BMP ligands, factors and observe the nature of the latent classes among adolescents. Mixed effects regression model noggin, into our clinically-relevant SCI rat model via mini-osmotic pumps at the time of injury. We was used to assess the association between adolescent latent class and weight status. performed tissue analysis at 1, 3 and 7-day time-points following SCI and noggin treatment. We used in Results: Three latent classes were identified: obesogenic (OB) youth, high-risk substance users and sedentary vitro primary cultures including rodent NPCs and astrocyte-microglia to assess the direct effects of BMP4. (HRSS) youth and moderately active screenagers (MAS). The regression of weight status on latent classes Assessments were conducted by immunocytochemistry, Greiss assay, slot blot, and Western blot. adjusting for grade ethnicity and gender suggests that HRRS youth are at higher odds of obesity than Results: BMP4 protein expression was significantly and transiently upregulated following compressive OBS youth (odds ratio=1.15; CI: 1.07, 1.24) relative to MAS. SCI in rats at 1 and 3 days post injury (dpi). Reactive astrocytes showed to be a significant source of BMP4 Conclusions: Three latent classes of adolescents were identified to have similar chronic disease risk in vitro. NPCs and astrocyte cultures responded to BMP4 by Smad1/5/8 phosphorylation, the canonical factors. These classes were significantly associated with weight status and suggest that obesogenic youth, pathway for BMP signaling. NPCs treated with BMP4 (30-100ng/ml) decreased their proliferation, and high-risk youth are at higher odds of obesity compared to their more active peers. Public health increased their viability while predominantly differentiating into astrocytes. These effects were inhibited interventions targeting obesity may benefit from addressing chronic disease risk factors that youth when BMP4 was co-treated with noggin, in a concentration-dependent manner. In cultures of reactive engage in simultaneously with the acknowledgment that certain subgroups are more prone to an astrocytes, BMP4 had no significant effect on GFAP expression, but increased CSPG production. obesity state than others. Conclusion: Our findings indicate that BMP4 is upregulated following SCI and promotes astrogliosis in mature astrocytes. BMP4 also promotes the differentiation of activated NPCs into astrocytes that contribute to scar formation. Our data has identified a new role for BMP4 in pathophysiology of SCI that requires further elucidation. Presence of Diabetes Autoantigens in Extracellular Vesicles Derived from Human Islets Craig Hasilo1, 2, 5, Sarita Negi1, 2, 5, Islabelle Allaeys3, Nathalie Cloutier3, Alissa Rutman1, 2, 5, Marco Gasparrini1, 2, Eric Bonnei1, 4, Pierre Thibault4, 5, Eric Boilard3, 5, Steven Paraskevas1, 2, 5

1Department of Background: The role of extracellular vesicles (EV) in the etiology of type I diabetes is poorly understood. proteins capable of eliciting an immune response. Genuine EV were identified with DAA by FC, with Experimental Surgery, We set out to determine if human islets, isolated using clinical protocols, produce EV in culture and ifthey significantly greater GAD65, ZnT8 and Glut2 in non-treated ICM versus ICM treated with 100,000 g McGill University; 2Human contain diabetes autoantigens (DAA). centrifugation, EDTA, and Triton-XIOO. DAA presence on EV was confirmed with immunogold-EM. A wide Islet Transplantation Methods: Islet conditioned media (ICM) was collected from human islets isolated from nondiabetic variation in antigen levels between all ICM (n= IO donors) and in the total percentage of AnnV+CT+EV Laboratory, Department donors (n=IO) under research consent using clinical protocols. Fractions of~80% purity were cultured up with detected antigens on the surface was observed. of Surgery, McGill to 72hrs and the ICM centrifuged at I, 200 g for I5 min. for analysis of EV production. Nanoparticle tracking Conclusions: Morphologically diverse EV with a consistent size profile, but great variability between University Health Centre, analysis (NTA) and flow cytometry (FC) were used to determine particle concentration and profile of size non-diabetic donors, were detected in human ICM. DAA GAD65, ZnT8, and Glut2 were identified in ICM distribution. ICM (n= lO) were labeled as follows: cytosolic esterase marker CeliTracker Deep Red; + Montreal, QC; 3Centre de EV with similar variability to that of total Ann V EV alone. Proteomic evaluation reflected an islet-specific membrane phosphatidylserine marker AnnexinV (AnnV)-V450, or AnnV-FITC; DAA anti-GAD65-PE, anti- protein signature capable of promoting an islet-specific immune response. EV biomarkers were found in Recherche en IA-2-APC, and antiZnT8- FITC; βcell markers anti-Giut2-PE and anti-HSC70-APC; membrane vesicle common with islet cells, indicating they may be strong targets for early diagnostic markers of β-cell injury Rhumatologie et markers antiCD9- BV421 and anti-CDI4-PE, on a FACS Canto II with small particle analyzer. Controls for and interventional strategies in diabetes and islet transplantation. lmmunologie, Centre de antibody specificity and genuine EV detection included ICM (n=4 donors) centrifuged at IOO,OOO x g for Recherche du Centre 1 hour, 50 mM EDTA, or 0.3% Triton X-IOO, and compared to untreated ICM. Fractions of ICM were Hospitalier Universitaire generated by serial centrifugation at 50 000 g and 200 000 g (ICM-50K and ICM-200K, respectively). de Quebec, Faculte de Double-labeled immunogold TEM was performed on ICM-50K and ICM-200K using anti-AnnV plus anti- Medecine de I’Universite GAD65, anti-ZnT8 or anti-Giut2 primary antibodies and I2nm or I8nm colloidal gold conjugated secondary Laval, Quebec City, QC; IgG. Proteomic analyses were performed by mass spectrometry (LC-MS/MS) on ICM-50K and ICM-200K 4Institut de Recherche en (n=5 donors), and islet cell lysate (ICL), then separated by SDS-PAGE. Tryptic digests were analyzed by Immunologie et en LC-MS/MS on a LTQ-Orbitrap Elite. Values obtained in Jot-matched control, unconditioned media were Cancerologie, Universite subtracted from all experimental values. de Montreal, Montreal, Results: We detected varying levels of particles and AnnV+CT+EV between donor ICM, but a consistent QC; 5Canadian National profile of size distribution, with a peak density in the I00-1,000 nm range, by NTA and FC, respectively. Transplant Research Proteomics of ICM-50K and ICM-200K revealed over 56% homology with islet cell lysates, but not with an Program endothelial cell line, indicating an islet-specific protein signature. Enrichment analyses identified proteins of exosome lineage associated with the metabolism of lipids and lipoproteins, diabetes pathways, and TC-PTP Regulates the IL-7 Transcriptional Response During Murine Early T Cell Neuraminidase-1 Modulates the Host Pro-Inflammatory and Angiogenic Cytokine Profile Development in Pancreatic Tumorigenesis T. Hatzihristidis1, K.A. Pike1, S. Bussieres-Marmen1, 3, F. Robert1, N. Desai1, 3, D. MirandaSaavedra4, 5, J. Pelletier1, 2, M.L. Tremblay1, 2, 3 Fiona Haxho, Myron R. Szewczuk

1Rosalind and Morris Cytokines play a critical role in directing the discrete and gradual transcriptional changes that define T Queen’s University, Introduction: Neuraminidase-1 (Neu1) has been shown to positively regulate the activation of several Goodman Cancer Centre, cell differentiation. The interleukin-7 receptor (IL-7R), via its activation of the JAKSTA T pathway, promotes Department of receptors, including the epidermal growth factor receptor, insulin receptor, and a number of Toll-like gene programs that change dynamically as cells progress through T cell differentiation. The molecular receptors. Notably, these receptors each play profound roles in cancer progression via promotion of cell McGill University; mechanism(s) directing differential gene expression downstream of the IL-7R are not fully elucidated. Biomedical and Molecular survival and proliferation, growth and metabolism, and immune-mediated tumorigenesis, respectively. 2Division of Experimental Here, we have identified T cell protein tyrosine phosphatase (TC-PTP) as a negative regulator of IL-7R-STA Sciences, Kingston, ON Indeed, treatment with Neu1 inhibitor oseltamivir phosphate (OP) has been reported to reduce tumor Medicine, Department of T signaling in T cell progenitors, contributing to both the quantitative and qualitative nature of STAT volume, neovascularization and metastasis in mouse models of breast, ovarian and pancreatic cancer. -gene targeting. Novel genetic strategies used to modulate TC-PTP expression demonstrate that However, the mechanisms associated with these broad therapeutic effects are not fully understood. Here, Medicine, McGill depletion of TCPTP expression heightens the phosphorylation of STAT family members, causing aberrant we investigate the role of Neu1 in the modulation of host pro-inflammatory and angiogenic cytokines University; 3Department expression of an interferon-response gene profile. Such molecular re-programming results in deregulation during pancreatic tumor development. of Biochemistry, McGill of early development checkpoints culminating in inefficient differentiation of CD4+CD8+ double positive Methods: MiaPaca-2 human pancreatic cancer cells were grown to 80% confluence and resuspended in cells. TC-PTP is therefore shown to be required to safeguard the dynamic transcriptome necessary for sterile saline. 106 cells were implanted subcutaneously into immunodeficient RAG2xCγ double mutant University, Montreal QC; efficient T cell differentiation. BALB/c mice. Tumor volume measurements were calculated twice weekly and OP treatment (100 mg/kg/ 4Centro de Biologia daily intraperitoneal) started on day 35 postimplantation. Whole blood was collected throughout the Molecular Severo Ochoa, experiment and serum was isolated and frozen. Serum analyte levels were quantified in pg/mL using CSIC/Universidad Luminex Mouse Cytokine/ Chemokine/Growth Factors multi-plex analysis kits. Luminex results were analyzed using BioPlex Manager 6.0. Statistical analyses were conducted using GraphPad Prism 6. Results Autonoma de Madrid, were compared using a one-way ANOVA and multiple comparisons at 95% confidence. Madrid, Spain; Results: OP treatment regimen resulted in a significant reduction in tumor volume in RAG2xCγ mice 5Department of bearing tumor xenografts when compared to untreated mice. OP treatment also resulted in a significant reduction in the circulating concentration of serum proinflammatory cytokines (IL-1β, IL-10, GM-CSF, Computer Science, IFN-Υ and TNF-α). OP treatment also modulated the concentration of serum angiogenic signaling University of Oxford, molecules (IL-15, IL-18, FGF, LIF, M-CSF, MIG, MIP-2, PDGF-BB and VEGF). Our findings indicate that Oxford, UK inhibition of Neu1 using OP can therapeutically modulate the host pro-oncogenic inflammatory and angiogenic cytokine profile of pancreatic tumorigenesis in vivo. Conclusion: Neu1 modulates host serum pro-inflammatory and angiogenic signaling molecules and presents a novel approach in the systemic targeting of pancreatic cancer. The Prevalence of Legionella Species in HIV-Associated Pneumonia Accessibility to Primary Care: A Cross-Provincial Comparative Health Policy Analysis Breanne M. Head1, Adriana Trajtman1, Kathy Bernard1, 2, Tamara Burdz2, Heidi Wood2, Zulma V. Rueda3, 4, Lazaro Velez4, Nicole Herpai Yoav Keynan1 University of Manitoba, Introduction: At the turn of the millennium, satisfaction with the healthcare system and accessibility to 1University of Manitoba; Introduction: Despite successful antiretroviral therapy, HIV-infected individuals are 10 times more likely Winnipeg, MB health services in Canada fared poorly compared with other OECD countries, attributable to years of funding retrenchment and unprecedented changes to health care delivery systems. In response to 2National Microbiology to contract bacterial pneumonia compared to the general population. Although HIV-associated lung infections are most often due to Pneumocystis jiroveci, Mycobacterium tuberculosis and other typical inferior access to services and a decline in citizen satisfaction, two high profile reports were commissioned, Laboratory, Winnipeg, MB; microorganisms, atypical bacteria, such as Legionella species, must also be considered. However, due to The Health of Canadians – The Federal Role and The Royal Commission on the Future of Health Care in Canada, 3Universidad Pontificia poor performance of diagnostics, decreased sensitivity of serology in the setting of advanced which emphasized that enhancing access to primary care services was essential to improving the health care system. The First Ministers struck an accord, created a strategic plan and pledged significant funding Bolivariana 4Universidad immunosuppression, the lack of clinical suspicion and the increased chance of co-infection in these immunocompromised individuals, Legionella pneumonia is currently believed to be under reported. to improving access to primary care services among other objectives. Considering the commitments de Antioquia, Antioquia, Thus, this study aims to determine the prevalence of Legionella in HIV-associated pneumonia. made to improving access to primary care services, considerable gains would be expected. Regrettably, Columbia Methods: Bronchoalveolar lavage (BAL) from immunocompromised patients who were hospitalized with recent data reflects: a continual lag of primary care access relative to other OECD countries; access for pneumonia at Hospital San Vicente Fundación in Medellin, Colombia were assayed for the presence of same-day and next day primary care services; and, inequity in access. PAN Legionella and L. pneumophila. Microbial DNA was extracted from BAL and subsequently amplified via Objective: This project is conceptualized through a political economy lens. Its purpose is to identify the qPCR. qPCR results were confirmed by agarose gel electrophoresis. key interests, ideas and institutions that have been influential in making policy reform around accessibility Results: Of the 70 immunocompromised patients enrolled in the study, 57 had HIV, 12 were transplant to primary care successful or not. recipients and 1 had immune mediated disease. Initial HIV pneumonia diagnoses were M. tuberculosis Methods: The objective will be assessed by first examining data from two quantitative cross-sectional (47.4%), P. jiroveci (33.3%) and others. Majority of Legionella infections were found in BAL of HIV and M. data sources collected across the provinces from 2013 to 2014: the Quality and Costs of Primary Care and tuberculosis or P. jiroveci-infected patients. Preliminary results indicated that 12 of 18 M. tuberculosis- the Canadian Community Health Survey. The results of this analysis will serve to structure the second infected BAL were positive for PAN Legionella with 4 positive for L. pneumophila. Of the 10 P. jiroveci- analysis: a systematic documentary analysis of health policy literature and publicly available primary infected BAL, 7 were positive for PAN Legionella with none positive for L. pneumophila. Further speciation healthcare policies. for all positive PAN Legionella results are currently pending. Contribution: This study will bring a novel perspective to the analysis of the issue of accessibility to Conclusion: Our study indicates that Legionella species are prevalent in the BAL of HIV and M. tuberculosis primary care in Canada and add to the growing literature that applies a political economy lens to the or P. jiroveci co-infected patients. Clinicians should be aware of the possibility of the presence of Legionella study of health policy. The results of this project will contribute to understanding the factors that have in HIV-associated pneumonia. The role Legionella plays in clinical presentation, disease severity or influenced the policy arena around access to primary care services, which may prove useful for policy inflammation remains to be determined. Improved molecular diagnostics can lead to more effective makers concerned with primary care reform and draw lessons for improving the effectiveness of future therapeutic interventions resulting in an improved quality of life for the HIV-infected population. health policy making. Health and Social Predictors of Moving Out of Public Housing Using Linked Population- Enhanced Sensitivity to the Novel Antimicrobial Benzothiadiazole C109 Is Caused by Based Administrative Data Underexpressing the Essential Cell Division Protein, FtsZ Aynslie M. Hinds, Brian Bechtel, Jino Distasio, Leslie L. Roos, Lisa M. Lix Andrew M. Hogan1, April S. Gislason1, Silvia T. Cardona1, 2

University of Manitoba, Introduction: People move into and out of public housing for a variety of reasons with length of tenancy 1University of Manitoba, Introduction: Antibiotic targets can be determined by quantifying the depletion of mutants with Winnipeg, MB varying. Our objective was to test the association between moving out of public housing with Department of enhanced antibiotic sensitivity. We have developed an Illumina-based chemogenomics assay using a demographic, geographic, economic, health status, and health service use characteristics while library of transposon mutants in Burkholderia cenocepacia each underexpressing one or more essential Microbiology; 2University accounting for length of tenancy. Additionally, we determined whether there were differences in genes. One mutant in the division and cell wall (dcw) gene cluster (CGdcw) was hypersensitive to C109, a associations for voluntary and forced moves. of Manitoba, Department novel antimicrobial benzothiadiazole. Subsequent in vitro analysis suggests the essential cell division Methods: We used linked population-based administrative data from Manitoba. The cohort consisted of of Medical Microbiology protein FtsZ, encoded at the end of the dcw cluster, is the target of C109. However, the in vivo target is individuals who moved into public housing in 2007 and 2008. Demographic and geographic characteristics unknown; therefore, the goal of this research is to determine if the enhanced sensitivity of CGdcw to & Infectious Disease, were obtained from the population registry. Economic measures included area-level income and receipt C109 is due to underexpression of FtsZ. of income assistance. Measures of health status and health service use were obtained from hospital, Winnipeg, MB Methods: Homologous recombination was used to place the rhamnose-inducible promoter upstream of physician, emergency department, and prescription databases. These variables were defined in the year the ftsZ gene in B. cenocepacia. This (CGftsZ) and other mutants were treated with various concentrations before the move-in date. Lengths of tenancy and move-out status were determined from move-in and of C109 in sensitizing conditions and growth was measured by OD600nm. These data were supported by move-out dates. Censoring events were loss of health coverage, death, or study end date (March 31, DIC microscopy. Transcription start sites in the dcw cluster were mapped with the program TSSAR. 2013). Multivariable Cox proportional hazards regression produced adjusted hazard ratios and 95% Results: In sensitizing conditions, the CGftsZ mutant forms long filamentous cells reminiscent of the confidence intervals. phenotypes caused by depletion of other cell division components. Both the CGdcw and CGftsZ mutants Results: The cohort consisted of 3131 public housing residents; 62.8% moved out during the 7-year show equal enhanced sensitivity to C109 versus the wild-type. Supporting these findings, both mutants observation period. Voluntary movers (48.6%) and the evicted (14.2%) resided an average of 720 days and show more extensive filamentous morphology with lower concentrations of C109. Additionally, seven 674 days, respectively. Both mover groups differed from non-movers on age, region of residence, receipt transcription start sites were identified in the dcw cluster, elucidating the transcriptional landscape of the of income assistance, and prior residential mobility. Voluntarily moving out was associated with a mutants. younger age, a lower likelihood of residing in Winnipeg and receiving income assistance, and a greater Conclusions: The equal sensitivity of the CGdcw and CGftsZ mutants to C109 demonstrates that the entire likelihood of prior residential mobility. Eviction was associated with a greater likelihood of comorbid sensitivity phenotype caused by underexpressing the dcw cluster can be reproduced by underexpressing conditions (i.e., injury, chronic physical illness, substance abuse), higher health service use (i.e., ftsZ alone. Combining this with the disruption of all start sites for ftsZ in the CGftsZ mutant suggests FtsZ hospitalization), and a lower likelihood of receiving care from the same family physician. is the in vivo target of C109. Conclusion: Socioeconomic factors may be helpful in identifying who will move out of public housing voluntarily, and by extension, who will have short versus long tenancies. Physicians may have a role to play in identifying at-risk individuals. Tenants should be supported to become self-sufficient, and policies and programs should address factors that contribute to housing instability. Endothelial NMDA Receptors are Involved in Astrocyte-Mediated Cortical Vasodilation Adam D. Hogan-Cann1, 2, Lingling Lu1, 2, Andrea K. Globa3, Shernaz X. Bamji3, James I Nagy4, Christopher M. Anderson1, 2

1Neuroscience Research Functional hyperemia (FH) is a regulatory mechanism that ensures active brain regions receive driver line. This strategy resulted in greater than 50% loss of endothelial GluN1 (eGluN1) expression in proportional delivery of blood flow. FH requires both local neurotransmitter-mediated effects and Program, Kleysen Institute cultures and commensurate reduction in NO production. In acute cortical slices, conditional eGluN1 loss coordinated upstream conduction of vasodilatory signals in an endothelium-dependent manner. Brain of function significantly attenuated endothelial NO generation and vasodilatory responses in situ, for Advanced Medicine, glutamatergic neurotransmission results in local vasodilation and enhanced blood flow to active areas. produced by stimuli including TP astrocytic Ca2+ uncaging, direct vascular application of NMDA receptor Health Sciences Center; There is strong literature consensus that this hyperemic response is mediated by neuronal N-methyl-D- agonists and bath-applied metabotropic glutamate receptor agonist (t-ACPD). In agreement, endothelial- 2Department of aspartate (NMDA) receptors and neuronal NO synthase (nNOS) and release of vasodilatory gliotransmitters selective GluN1 silencing was associated with altered metabolism of arachidonic acid, favouring from perisynaptic astrocytes. However, neither of these mechanisms is endothelium-dependent, leaving enhanced tissue levels of the smooth muscle constrictor, 20-HETE, relative to the vasodilatory metabolite, Pharmacology & neuro-endothelial coupling as a key conceptual deficit in understanding FH. We have observed that PGE2. Our results identify a novel mechanism of coupling between astrocytes and brain endothelial cells, Therapeutics, University isolated middle cerebral artery segments free of neurons dilate in response to NMDA receptor agonists raising the possibility that endothelial NMDA receptors mediate activity-dependent, glutamatergic of Manitoba, Winnipeg, in a manner that requires functional endothelium and endothelial NOS (eNOS). We also found that direct neurovascular signaling. stimulation of astrocytes by two-photon (TP) flash photolysis of caged Ca2+ in mouse cortical slices led MB; 3Department of to NMDA receptor and eNOS-dependent vasodilation. The current study was designed to test the Cellular and Physiological possibility that NMDA receptors are expressed by brain endothelial cells and participate in astrocyte- Sciences, University of mediated vasodilation in cortical slices. Primary mouse brain microvascular endothelial cultures expressed the pan-NMDA receptor subunit, GluN1, and NO production in response to NMDA receptor British Columbia, co-agonists, glutamate and D-serine, in a manner sensitive to NMDA receptor antagonists, channel Vancouver, BC; activity, chelation of intracellular Ca2+ and eNOS inhibition. Using immuno-electron microscopy, GluN1 4Department of immunoreactivity was also detected in brain endothelium in situ, with preferential localization to brain- Physiology and facing (abluminal) endothelial membranes. Direct exposure of cortical slice arterioles to glutamate and D-serine by local pressure ejection and two-photon (TP) flash photolysis of NP-EGTA (o-Nitrophenyl EGTA; Pathophysiology, Ca2+ uncaging) in astrocytes adjacent to cortical slice arterioles both produced increases in endothelial University of Manitoba, NO production and lumen diameter. These effects were significantly mitigated by competitive antagonists Winnipeg, MB of NMDA receptor glutamate (AP5) or co-agonist (DCKA) binding sites, and by eNOS loss of function. To distinguish between neuronal and endothelial NMDA receptors, we created conditional endothelial NMDA receptor loss of function mice by crossing “floxed” Grin1 animals with a Tie-2 Cre recombinase Dual RNA-Seq of Cervical and Thoracic Spinal Cord Injury Reveals Level-Specific Absence of Evidence Supporting the Contribution of Rare Genetic Variants in STK32B, Heterogeneity of Neurovascular Response. PPARGC1A, CTNNA3 to Essential Tremor in a Cohort of Canadians of European Decent. James Hong1, 2, Mahmood Chamankhah2, Anna Badner1, 2, Jian Wang2, Yang Liu2, Priscilla Chan1, Michael Fehlings1, 2 Gabrielle Houle1, 2, Amirthagowri Ambalavanan1, 2, Jean-Francois Schmouth1, 3, Claire S.Leblond1, 2, Dan Spiegelman1, Sandra B. Laurent1, Cynthia V. Bourassa1, Michel Panisset4,Sylvain Chouinard4, Nicolas Dupre5, 6, Carles Vilarino-Guell7, Alex Rajput8, Simon 1Institute of Medical Introduction: Despite the fact that over 65% of traumatic spinal cord injury (SCI) occur in the cervical L.Girard9, Patrick A. Dion1, 2, 3, Guy A. Rouleau1, 2, 3 Science, Faculty of region (cSCI), and startling differences between the response of cSCI and thoracic SCI (tSCI) to trial drugs such as cethrin, spatiotemporal differences between the injury milieu of cSCI and tSCI remains elusive. As 1 Introduction: Essential tremor (ET) is a common neurological disorder characterized by postural, kinetic Medicine, University of Montreal Neurological Institute secondary injury after initial trauma is driven by the neurovascular and neuroimmune axis, we posit that and Hospital; 2Department of and intention tremors of a body part. ET is known to cluster in families and it is believed to be influenced Toronto; 2Division of the increased vascularity and grey-white matter ratio of the cervical cord will result in an injury response Human Genetics,McGill by several genetic and environmental factors. Previous studies have suggested genetic variants and loci 3 Genetics and with an earlier onset, severity and chronicity. University; Department of associated with the condition which provides valuable insights into the complexity of the genetic Methods: A rat model of moderate clip compression injury was used in the C6-7 and T6-7 region after Neurology and Neurosurgery, architecture of the disorder. Despite these recent advances, causative variants yet explain only a small Development, Krembil McGill University; 4Centre laminectomy, respectively. This was followed by time-course (3, 7, 14, and 56 days with time-matched proportion ET cases. Recently, a two-stage genome-wide association study (GWAS) that included 2,807 Research Institute, Hospitalier Universitaire de laminectomy controls) RNA-sequencing, 3D ultrasound Doppler imaging, and classical techniques Montreal (CHUM)-Notre-Dame, cases and 6,441 controls of European descent recently identified associations between essential tremor Toronto Western Hospital. including RT-qPCR, Western blot, ELISA and immunohistochemistry. Andre Barbeau Movement (ET) and specific intronic variants within three genes: a serine/threonine kinase (STK32B), a transcriptional Results: RNA-sequencing revealed striking differences in the onset and temporal profile of fibrotic and coactivator (PPARGC1A) and a cell-adhesion molecule (CTNNA3) . The present study aims to establish if Toronto, ON Disorders Unit, Montreal, QC; gliotic processes with canonic stat3-dependent gliosis markers--lcn2, gfap and serpina3n—being 5Department of Medicine, coding variants from these genes might be associated with ET. differentially expressed across time. Further, 3D ultrasound revealed that these processes correspond to Faculty of Medicine, Laval Methods and Results: To further assess the role of STK32B, PPARGC1A, CTNNA3 as ET predisposing factors, 6 rapid tissue loss and hemorrhage starting as early as 3 days post-cSCI which was validated with increased University; Departement des we first screened the coding regions of these genes for rare variants. Looking at exome and whole Sciences Neurologiques, CHU de gfap and cspg4 staining in the cord. Moreover, western blotting showed a comparatively greater loss of genome sequencing data, no rare deleterious variant were found to co-segregate with the disease in 14 Quebec (Enfant-Jesus), Quebec and —two crucial markers of the blood-spinal cord-barrier –after cSCI from 3 days lasting up to tjp1 ocln City, QC; 7Department of Medical autosomal dominant multiplex ET families. Next we used a targeted massive parallel sequencing 56 days. Genetics, University of British approach to examine the protein-coding region of these genes in 269 ET cases and 287 control Conclusion: Taken together, this data demonstrates—for the first time—the level-specific heterogeneity Columbia, Vancouver, BC; individuals. Thirty-four variants were identified but none had a significantly different allelic distribution of SCI with cSCI having a quicker onset and chronicity in its neurovascular disruption compared to tSCI. 8Division of Neurology, between cases and controls. Finally, we performed a gene-based, variance-component test (SKAT-0) to As the majority of clinical trials on the cSCI population is largely ineffective, this study reconciles the Saskatchewan Movement assess the cumulative impact of rare variants. No difference in variant distribution for any of the genes potential reasons behind why tSCI-derived preclinical paradigms may not be suited--in both strategy and Disorders Program, University of was observed between the ET cases and the controls. timing—to cSCI, and aims to engage clinicians and scientists to employ level-specific study designs. Saskatchewan, Saskatoon Health Conclusion: No difference emerged regarding the distributions of individual variants (or gene} between Region, Saskatoon, SK; 9Departement des Sciences cases and controls. Thus no rare mRNA altering variant further validated one of these genes as a risk Fonda mentales, Universite du factor for ET. The recent GWAS offers promising avenues but the genetic heterogeneity of ET is Quebec a Chicoutimi, nonetheless challenging for the validation of risk factors, ultimately larger cohorts of cases should help Saguenay, QC to overcome this task. The Prognositic Value of 3.0T MR in Evaluating Extrahepatic Cholangiocarcinoma Exploiting Broadly Protective Immune Responses Against Influenza Strains for Xin-Qiao Huang, Jian Shu, Tie-Jun Zhou, Peng Li, Ming-Li Jin Development of a Universal Influenza Vaccine Marnie Hustins1, 2, Mable Hagan1, 2, Anders Leung2, Darwyn Kobasa1, 2 Southwest Medical Introduction: To assess the tumor (T) stage, node (N) stage and pathological differentiation grade in the University, Luzhou patients with extrahepatic cholangiocarcinoma (EHCC) using diffusion-weighted imaging (DWI) at 3.0-T 1Department of Medical Influenza Type A is a significant health threat to individuals of all ages. The annual influenza vaccine MR. 646000, Sichuan Microbiology, University provides protection against Influenza Type A subtypes H1N1, H3N2, and Type B viruses that are endemic Methods: Eighty-five patients who were clinically and histologically diagnosed with EHCC underwent in humans. For each of these viruses, the vaccine has a separate component that matches the dominant 2 Province, P. R. China abdominal DWI within 2 weeks before surgery. The lesion apparent diffusion coefficient (ADC) value in of Manitoba; Special strain in circulation. Strain selection for the vaccine is decided one year in advance, which can lead to different T stage, N stage and pathological differentiation grade were retrospectively analyzed. Pathogens, Public Health vaccine failure if a different dominant strain emerges during that time. Our pilot study showed vaccination Results: In these patients, 10 cases were T1 (11.76%), T2: 30 cases (35.29%), T3: 43 cases (50.60%), and T4: Agency of Canada, with a 2009 pandemic H1N1 (pH1N1) vaccine, but not a seasonal 2007 H1N1 (sH1N1) vaccine, provided 2 cases (2.35%). There showed no statistical difference between the lesion ADC value and T stage (P = complete protection against lethal challenge with highly antigenically divergent avian and human Winnipeg, MB 0.781). Fifty-seven patients showed no evidence of lymph node metastasis (N0, 67.06%) and 28 patients viruses in mice. We propose to further develop this model of heterosubtypic immunity provided by the showed lymph node metastasis (N1, 32.94%). The median ADC value of N0 group and N1 group were pH1N1 virus, initially focusing on cross-protection provided by pH1N1 against the other human seasonal -3 2 -3 2 1.316×10 mm /s and 1.220×10 mm /s, separately. As the ADC value descended, the possibility of Type A virus subtype, the H3N2 viruses. Our infection model will use a human H3N2 virus, A/Hong 2 lymph node metastasis increased (x =11.268, P = 0.001). These EHCCs displayed hyper-intensity on DWI Kong/1/1968 (HK68), which has been adapted to cause lethal infection in mice (MA-HK68). The MA-HK68 in 97.65% (83/85) of patients. Hyper-intensity lesions were found in 96.55% (28/29) of patients with good- strain will be obtained by introducing previously published mutations into HK68 plasmids encoding each differentiation, in 97.56% (40/41) with moderate-differentiation and in 100% (15/15) with poor- influenza gene, then rescued using reverse genetics to generate the live virus. Influenza viruses contain differentiation. The median ADC value of 2 the well-differentiated, moderately-differentiated and highly conserved domains in the M2 ion channel, hemagglutinin, and neuraminidase proteins, which are -3 2 -3 2 -3 2 poorly-differentiated EHCCs were 1.528×10 mm /s, 1.256×10 mm /s and 1.148×10 mm /s, located on the viral surface, and may contribute to the observed cross-protection, as these regions may respectively. As the pathological differentiation grade decreased, the lesion ADC value of EHCC gradually be bound by neutralizing antibodies. In this study we will confirm that pH1N1 vaccination protects 2 declined (x =67.546, P = 0.000). against lethal H3N2 challenge in mice. We will then generate recombinant viruses with pH1N1 genes and Conclusion: The N stage and histopathological differentiation grade of EHCC can be detected non- the sH1N1 virus that does not provide cross-protection. These recombinant viruses will be used as invasively using DWI at 3.0-T MR. vaccines to identify the pH1N1 protein(s) that provide the cross-protective responses and then we will map crossprotective antibody determinants using a B-cell ELISPOT assay. By identifying which constituents of influenza contribute to heterosubtypic cross-protection, we propose to enhance the efficacy of the current influenza vaccine against antigenic variant viruses and to contribute to the development of a Universal Flu Vaccine that provides protection against all Type A influenza subtypes. Regulatory Evolution Drives Immune Evasion by Salmonella Typhimurium Characterization of Haemophilus Influenzae Serotype a Population Structure Using Bushra Ilyas1, 2, David T. Mulder1, 2, Brian K. Coombes1, 2 Whole Genome Sequencing Mariam Iskander1, 2 1Department of Bacterial pathogens encounter a wide variety of host niches which require niche-specific gene expression. Biochemistry and The bacterial pathogen Salmonella Typhimurium tightly regulates gene expression to subvert the 1University of Manitoba; Haemophilus influenzae is a gram negative coccobacillus that can cause disease such as pneumonia and immune system and to activate its intracellular virulence program. SsrA-SsrB is a horizontally-acquired Biomedical Sciences, 2Public Health Agency of meningitis. Some H. influenzae strains have a polysaccharide capsule and are classified into six serotypes, two component system which detects a variety of environmental stimuli and has been identified as a a through f. The polysaccharide capsule genes are organized in the cps operon, which is flanked by McMaster University, Canada, Winnipeg, MB master regulator of this intracellular lifestyle. Research in our lab has shown that mutations in cis- insertion sequence IS1016. Historically, H. influenzae serotype b (Hib) had been the major cause of Hamilton, ON; 2Michael regulatory genetic elements have led to these genes being regulated by SsrB, and that these genes are invasive disease worldwide. However, the development of the Hib conjugate vaccine in the late 1980s important for Salmonella virulence. To identify novel pathways under the control of SsrB, we conducted DeGroote Institute for drastically decreased Hib disease incidence. Duplication in the cps operon along with a partial deletion comparative RNA-sequencing of S. Typhimurium and the closely related, SsrBnaive species Salmonella in one of the copies has been implicated in the increased virulence of Hib. In recent years serotype a (Hia) Infectious Disease bongori, in the presence of SsrB under infection relevant conditions. These data identified numerous has emerged as a significant cause of invasive disease in the indigenous populations of North America Research, McMaster functional groups of genes that are differentially regulated in the SsrBadapted S. Typhimurium, as and Australia. Some virulent Hia strains harbour a cps operon duplication-deletion event similar to that compared to S. bongori, thus identifying novel infection relevant genetic regulation. In particular, we have University, Hamilton, ON found in Hib. Little else is known about Hia, and no whole genome-based studies have been published identified a strong repression of genes involved in flagellar-based swimming motility. Here we have to date. In this project, we investigate the presence and distribution of the cps throughout the Hia confirmed that swimming motility is differentially regulated in S. Typhimurium expressing constitutively population structure. In addition, we develop a whole genome MLST schema for H. influenzae and active SsrB compared to a knockout strain, and that this difference does not exist when SsrB is introduced compare it to the traditional 7-gene MLST classification scheme. into S. bongori. We have also found that immune activator flagellin (FliC) is differentially regulated by SsrB using transcriptional reporter data and in vitro secretion assays. Future work will focus on demonstrating that this regulation is important for virulence by exploring the ability of these strains to activate the flagellin-sensing inflammasome complex in macrophages, which has been shown to be important in limiting Salmonella infections. These results will further our understanding of regulatory evolution and pathogenesis of S. Typhimurium. Furthermore, these results can be used as a model understanding how other organisms evolve in response to horizontally-acquired transcriptional regulators. A High Protein Diet Alters Rat Kidney and Liver Oxylipins The Role of Bitter Taste Receptors (T2Rs) in Cystic Fibrosis Ariful Islam1, Harold M Aukema2 Appalaraju Jaggupillia1, Nisha Singha2, Renaud Sylvain Robert3, John W. Hanrahan3, Kangmin Duan1, 2, Prashen Chelikania,2

1Human Nutritional Introduction: High protein (HP) diets are increasing in popularity, but their effects on tissues including 1Manitoba Chemosensory Introduction: Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. In Sciences, University of the kidney and liver remain unclear. Oxylipins are a group of bioactive lipids formed via oxidative Biology (MCSB) Research humans, 25 T2Rs perform a chemosensory function, however very little is known regarding their metabolism of polyunsaturated fatty acids via cyclooxygenase and lipoxygenase enzymes, and the expression and function in extraoral tissues. Recent studies showed that T2Rs recognize quorum-sensing 2 2 Manitoba; Canadian epoxygenase and hydroxylase activities of cytochrome P450 enzymes. They are involved in both the Group; Department of molecules (QSMs) secreted by bacteria. Cystic fibrosis (CF) is a genetic disease caused by mutations in the Centre for Agri-Food maintenance of normal renal and hepatic functions, as well as being involved in pathological processes. Oral Biology, University of gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The opportunistic Research in Health and For example, the vasodilatory effect of arachidonic acid derived prostaglandins (cyclooxygenase Manitoba, Winnipeg, MB; pathogen Pseudomonas aeruginosa colonizes CF airways and secretes QSMs, which increase intracellular oxylipins) are involved in maintaining normal renal function, while linoleic acid derived cytochrome P450 calcium and modulate innate immune responses through unidentified cell surface receptors. We Medicine, St. Boniface 3Department of epoxygenase oxylipins such as epoxyoctadecadienoic acid (EpOME) and their dihydroxy metabolites hypothesized that T2Rs are expressed in epithelial cells of human airways, where they mediate QSM Hospital Research Centre, (DiHOME) have been reported to be cytotoxic in renal cells and pro-inflammatory in a number of tissues. Physiology and CF responses and modulate immune responses in CF. Winnipeg, MB HP diets have been shown to alter renal prostaglandins, but effects on other oxylipins in the kidney or Translational Research Methods and Results: We monitored the expression of T2Rs in primary CF and non-CF bronchial epithelial the liver are not known. cells. Cells were studied at both the mRNA and protein levels using qPCR and FACS analysis, respectively. centre, McGill University Methods: Normal adult male Sprague Dawley rats were provided isocaloric diets with LP (low protein, 8% We found similar expression of T2Rs in non-CF and CF cells. Next we treated these cells with a number of protein by weight), NP (normal protein, 14% protein) or HP (50% protein) diets for two weeks. Kidney Health Centre Research QSMs and measured the intracellular calcium mobilized. We found that the calcium mobilized was (cortex and medulla) and liver were analyzed and quantified by UPLCMS/ MS. Institute, Montreal, QC sensitive to the G-protein βγ subunit inhibitor gallein and PLC inhibitor U-73122 suggesting that the Results: Kidney (p<0.0001) and liver weights (p< 0.0021) were higher in HP compared to NP and LP rats. signaling in these cells occurs predominantly through the QSM-T2R-Gαβγ-PLC pathway. To further Medullary 9,10 DiHOME and 12,13 DiHOME in HP rats were 2-3 times higher than in the NP and LP rats, characterize the candidate T2Rs that may recognize specific QSMs and modulate signaling in CF, a 12,13-EpOME was similarly higher in HP rats but only significantly different than the LP group, while heterologous expression system was used to stably express selected T2Rs and study the effects of QSMs. 9,10-EpOME followed the same trend, but was not significant. Several renal oxylipins from the We found that a single QSM (for example, 3-oxo-C12-AHL) can activate more than one T2R, and that cyclooxygenase and lipoxygenase pathways that may have protective effects were lower in the HP multiple QSMs can activate the same T2R. compared to the NP and/or LP diets. In liver, several oxylipins were higher in rats given HP compared to Conclusions: T2Rs are expressed in bronchial epithelial cells and are functional. Although expression was LP diets, including 9, 10 EpOME and 12, 13 EpOME (2.5 times higher). not altered in CF cells, these results suggest there is a synergistic effect of QSMs on T2R signaling in CF Conclusion: HP diets result in oxylipin alterations in kidney and liver, including increased linoleic acid airways and provides a rationale for further research on the pathophysiological function of T2Rs in derived cytochrome P450 epoxygenase products. These findings warrant further investigation of their extraoral tissues. These mechanistic studies should improve our understanding of host response potential effects in these tissues. mechanisms and may lead to new therapeutic approaches for CF that target T2Rs. Arginine Methylation in Determining Chromatin Fate Prenatal Alcohol Affected Epigenetic Modifications in Human and Non-Human Primate Sanzida Jahan, Tasnim Hussain, Wayne Xu, Aleksandar Ilic and James R. Davie Hippocampus Jessica S. Jarmasz1, 2, Duaa Basalah2, 3, Albert E. Chudley2, 3, James R. Davie2, 4, Mojgan Rastegar2, 4, Marc R. Del Bigio2, 3 University of Manitoba, Chicken polychromatic erythrocytes are nucleated and transcriptionally active. These cells are terminally Winnipeg, MB differentiated and lack replication. Therefore, it is a suitable system to study the mechanism of 1University of Manitoba, Introduction: One of the major hypotheses addressing the adverse effect of alcohol on the developing transcription and transcription related events in absence of replication. In higher eukaryotes, genomes Department of Human brain relates to the ability of alcohol to cause epigenetic changes. Epigenetics is the study of heritable are organized into chromosomal domains. The combination of histone modifications and histone changes in gene expression that do not result in DNA sequence changes. Instead, DNA and its surrounding modifying enzymes along with DNase sensitive regions ensure the conformation of the active Anatomy and Cell Science; components such as histones and chromatin are reversibly modified in order to influence gene chromosomal locus. It was demonstrated for the well-characterized chicken erythroid β-globin domain 2Children’s Health expression. Our objective is to demonstrate that prenatal alcohol exposure (PNAE) causes abnormal that the histone modifying enzyme protein arginine methyltransferase 1 (PRMT1), which is a major type Research Institute of epigenetic changes in human brain cells which in turn, persist throughout that individual’s life. I PRMT, plays a critical role in establishing and maintaining active histone marks. On the other hand, type Methods: Autopsy records at Health Sciences Centre (Winnipeg, Manitoba) were retrospectively Manitoba (CHRIM); II PRMT, PRMT5 demonstrate the crosstalk with lysine methyltransferase WDR5 to establish active reviewed. PNAE and/or an FASD diagnosis was identified in 173 human autopsy reports. Of these cases, chromatin signature to the site. However, PRMT5 mediated arginine methylation can lead to repressed 3University of Manitoba, 17 human hippocampal/medial temporal brain samples will be examined at an epigenetic level along chromatin state as well. Therefore, we hypothesize that recruitment of H4R3me2a and H3R2me2s by Department of Pathology; with age, sex and post-mortem delay matched controls (n=17). Samples are split up into 2 age groups: PRMT1 and 5 to the active chromosomal domains is a critical event in maintaining active domain fetuses (8) and infants (9). As a relative comparison, a PNAE Macaque Primate brain model (~4-6 months 4University of Manitoba, structure. It is of interest as these enzymes can confer both active and inactive arginine modifications in of age, N=12) developed by Clarren, Bowden and Astley will also be included in this study in order to a context-dependent manner and can change the fate of chromatin structure. We recently reported the Department of identify epigenetic modifications truly affected by PNAE. genome-wide profiles of chromatin signatures (H3K4me3, H3K27ac, salt-soluble domains) in relation to Biochemistry & Medical Results: Using immunohistochemistry, both human and macaque primate hippocampus samples are expression levels in chicken polychromatic erythrocytes (PMID: 27226810). We identified and mapped all stained with antibodies targeting 16 epigenetic modifications (4 DNA, 12 histone). Preliminary data in the Genetics, Winnipeg, MB transcriptionally active chromosomal domain in chicken polychromatic erythrocytes. In the current PNAE Macaque brain tissues showed decreases in 7 (5mC, 5hmC, 5caC, H3K4me3, H3K27me3, H3K36me3, study, we explored the distribution of arginine modifications and the potential mechanism of recruitment H3K9ac), increases in 2 (H4K5ac, Total H4) and no effect in 2 (H3K27ac, H4K12ac) of the 13 epigenetic of PRMT1 and 5 in the active chromosomal domain. The findings will provide novel insight into the modifications assessed so far: Preliminary data in the PNAE Human autopsy brain tissue showed a mechanisms of how PRMT1 and 5 can regulate the complex network of gene expression. This will decrease in 1 (H3K27me3), an increase in 1 (5hmC) and no effect in 2 (H3K4me3, H4K12ac) of the 4 contribute to the knowledge of using PRMT1 and 5 inhibitors which are under consideration to use in epigenetic modifications assessed so far. cancer therapy. Conclusion: This will be the first PNAE study to utilize human and non-human primate brain tissue for the analysis of epigenetic changes. Our findings will not only validate previous epigenetic findings in rodent models, it will also have the potential to identify diagnostic tools (biomarkers) for early identification of FASD if the modifications can also be detected in accessible tissue such as mucosal (buccal) epithelium from the mouth, which will allow for the provision of better care to affected children. Interaction of TAPPS With PI (3,4)-P2 Regulates B Cell Metabolism and Autoantibody The Gut, the Brain, and HIV-1 Infection: Inflammation Processes in Humanized BLT Mice Production Virginie S. E. Jean-Baptiste, Marc S. Horwitz Nipun Jayachandran, Ivan Landego, Sen Hou, Edgard Mejia, Kimia Sheikholeslami, Grant M Hatch, Aaron J. Marshall University of British Background: Highly active antiretroviral therapy (HAART) has lowered the incidence of HIV-dementia, Departments of PI3K signaling is essential for regulating B cells metabolism and antibody production. Membrane Columbia, Vancouver, BC the most severe form of HIV-associated neurocognitive disorders (HAND). However, ~50% of HAART- treated individuals still experience some form of HAND impacting many aspects of life, including work Immunology and phosphoinositides such as PI (3,4) P2 and PI (3,4,5)P3, are second messengers generated downstream of PI3K activation that promote recruitment of various proteins to the membrane. Although the role of PI efficiency and social agility. HAND are gradually becoming a global issue as the number of people Biochemistry, University (3,4,5)P3 interacting proteins and their significance are extensively studied, PI (3,4)P2 dependent accessing HAART increases. Recent research has indicated bidirectional interactions between gut and of Manitoba, mechanisms are not fully understood. TAPPs (Tandem PH domain containing proteins) are PI (3,4) P2 brain, coined “gut-brain axis”. The gut-brain axis holds significance for HAND, as the largest loss of cells in early HIV infection occurs in the gut. Winnipeg, MB binding proteins that get recruited to the membrane on PI3K activation. To investigate the role of TAPP- PI (3,4) P2 interaction in B cell signaling, we performed studies on mice bearing knock in (KI) mutation Aims: We focus on understanding how HIV establishes inflammation in the gut/brain to better determine within the C terminal PH domain of TAPP 1 and TAPP 2 that disrupts their membrane targeting function. how we can control inflammation to prevent HAND. Using the bone marrow liver thymus (BLT) mouse TAPP KI mice exhibited lupus like characteristics such as hypergammaglobulinemia, splenomegaly and model, we aim to 1) determine how infection alters the interferon (IFN) response in the gut and brain, and immune complex deposition within the kidneys. These mice progressively developed anti-dsDNA 2) evaluate the neurological involvement of infection by assessing human immune cell infiltration in antibody with age that corresponds to the generation of chronic germinal centres (GCs). Blocking the brains. development of GCs by disrupting the GC B cell – T follicular helper cell interactions reversed Methodology: BLT mice were generated by surgically implanting human fetal liver and thymus tissues + autoimmunity in these aging TAPPKI mice. Our mixed bone marrow chimera approach has demonstrated under the kidney capsule of NSG mice, with the injection of autologous CD34 stem cells. When + the B cell intrinsic role of TAPP mutation in the generation of chronic GC and autoimmunity. TAPP KI B peripheral blood was reconstituted with ≥ 20% human (h) CD45 cells, BLT mice were infected cells under in vitro stimulation activating PI3K signaling exhibited B cell hyperresponsiveness. TAPP KI B intraperitoneally (i.p.) with live HIV-1. Levels of human immune cells in brains were determined by flow cells showed metabolic abnormalities displaying elevated levels of mitochondrial respiration and cytometry. Inflammatory profiles of brains and intestines were assessed using a human type 1 IFN qPCR glycolysis. These cells exhibited increased expression of Glut-1 and enhanced rate of glucose uptake array. + which was reversed by inhibiting PI3K. We targeted glycolysis in aging TAPP KI mice by treating them with Preliminary Results: Brains of infected mice trended towards higher levels of NK cells, CD4 and CD8+ T + 2-DG that blocked the development of chronic GC and autoimmunity in these animals. Together these cells, despite an overall decrease in infiltrating hCD45 cells. While serum p24 levels varied, infected mice findings suggest that TAPP -PI(3,4)P2 interaction is important for regulation of metabolic defect driven by had similar profiles of type 1 IFN response in both brains and intestines following i.p. infection, suggesting enhanced PI3K signaling and thus mediating protection from B cell hyperactivation and autoimmunity. a direct link between gut and brain. Of 84 genes arrayed, 30+ genes had fold-changes indicative of biologically meaningful effects, with the non-classical HLA-E gene showing up to 2902-fold-change, dependent on infection status. The high up-regulation of HLA-E and its previously reported involvement in activation and expansion of NK cells suggests a potential role for NK cell infiltration in establishment of inflammation in BLT brains. Characterizing Chromosome Instability in USP22-Depleted Colorectal Cancer Cell Lines The Study of Sonic Hedgehog Signaling Pathway in Development of Cerebellar Granule Lucile M. P. Jeusset, Kirk J McManus Cells Xiaodan Jiao, Niloufar Ashtari, Maryam Rahimi Balaei, Saeid Ghavami, Hassan Marzban Department of Introduction: Chromosome instability (CIN) is an increase in the rate at which chromosomes or large Biochemistry and chromosome fragments are gained or lost. CIN is an aberrant phenotype observed in virtually all cancer Department of Human Introduction: The cerebellar cortex consist of three layers forming with different cell types, such as types, including colorectal cancer. It is suspected to drive oncogenesis by increasing the rate at which Medical Genetics, Anatomy and Cell Science, granule cells (gcs), Purkinje cells (Pcs) and Bergmann glial cells. During the development, the granule cell oncogenes are gained and tumor suppressors are lost. CIN is associated with aggressive tumors, multi- precursors arise from the cerebellar rhombic lip and form the external germinal zone (egz). Thereafter, University of Manitoba; drug resistance and consequently poor patient prognosis. Despite these associations, the genetic defects Faculty of Health Sciences, the granule cell precursors (gcps) proliferate and then move to granule layer (gl) and differentiate into Research Institute in and aberrant molecular mechanisms underlying CIN remain poorly understood. Preliminary data from University of Manitoba, mature gcs. In this process, sonic hedgehog (Shh) secreted by Pcs is an important promoter of granule our laboratory suggests that decreased USP22 (Ubiquitin-Specific Peptidase 22) expression induces CIN. Oncology and Winnipeg, MB cell precursors proliferation. A lysosomal acid phosphatase 2 (Acp2) mutant mouse (nax) shows a Importantly, hypomorphic USP22 expression occurs in numerous cancer types, including colorectal significant gcs reduction in the cerebellum. We hypothesize that in nax mouse the elevated proteasome Hematology, cancer. activity decrease N-myc expression and cause the interruption of Shh pathway and thereafter the Winnipeg, MB Methods: We aim to determine whether a karyotypically stable colorectal cancer cell line (HCT116) can cerebellar gc neurodevelopmental disorders. be converted into an unstable line following transient USP22-silencing with siRNAs, or stable knockout Methods: We used the nax and the wild-type sibling mice as control in this study. In vivo and in vitro (KO) using a CRISPR-Cas9 approach. The effects decreased USP22 expression has on the development of immunohistochemistry and Western-blotting are used to detect the molecular expression. CIN are assessed by single cell, imaged-based analyses that monitor surrogate markers for CIN, including Results: In nax mouse the number of gcs is around 80% reduced both in cerebellar vermis and two changes in nuclear area, micronucleus formation and chromosome numbers. hemispheres at P13 compared with WT sibling mice. The Shh expression is down regulated from P10 in Results: To date, we have efficiently silenced USP22 and preliminary results revealed significant changes WT mouse, while in nax is delayed and higher than WT after P12. The immunostaining shows strikingly in nuclear area and micronuclei formation, as well as increases in the frequency of chromosomes gains or reduced expression of N-Myc in nax MEF cells. Gcps express N-Myc in WT egl while reduced in nax losses, which are indicative of underlying CIN. In addition, we have generated two stable USP22-KO cerebellum at P13. The N-myc expression is increased since P10 in WT, while in nax is downregulated from HCT116 clones and preliminary data also showed changes in nuclear area, micronuclei formation and P1 to P15. Proteasome both expresses in WT and nax gcs at P12 but significantly increases in nax MEF chromosome numbers relative to controls. cells. Conclusion: Further analysis of USP22-silenced and -KO cells is ongoing to validate USP22 as a CIN gene Conclusion: The deregulated Shh pathway in nax mouse ceases gcs proliferation during cerebellar and investigate the mechanism through which USP22-depletion induces CIN. Characterizing the effect development. The significant reduction in the proliferation and probably differentiation of gcs in nax diminished USP22 expression has on CIN is the first step towards the development of therapeutic mouse reveals that Acp2 mutation affects the activity of proteasome and then disorganizes the important strategies specifically targeting USP22-deficient cancer cells. factor N-myc expression in Shh pathway. Social Network Analysis as a Tool to Describe Egocentric Networks of Integrated Identifying Mir-200b Targets in Normal and Hypoplastic Lungs Due to Congenital Treatment Programs in Five Ontario Communities Diaphragmatic Hernia Chantele Joordens, Karen Urbanoski Shana Kahnamoui Zadeh, Naghmeh Khoshgoo, Barbara Iwasiow, Phillip Snarr, Richard Keijzer

The Centre for Addictions Background: The majority of women who seek treatment for substance use and addiction issues are of Children’s Hospital Introduction: Every 10 minute a baby is born with Congenital Diaphragmatic Hernia (CDH). Failure of the Research of BC, University child-bearing age, therefore the likelihood that they will also be dealing with pregnancy/parenting issues Research Institute of diaphragm to fuse properly during embryonic development results in CDH, which allows the abdominal is quite high. While there has been a shift to integrate substance use and mental health services, the organs to migrate up into the chest cavity. This results in complications after birth such as pulmonary of Victoria, Victoria, BC Manitoba, University of integration of substance use and prenatal services has been slower to gain traction. Women would hypertension and pulmonary hypoplasia. Improvement in management techniques has not significantly benefit from integration or coordination of services beyond prenatal and substance use, such as Manitoba (Department of influenced the mortality rate of CDH babies. Our research group has shown that CDH likely results from collaborative mental health, trauma, primary care, child-minding, and housing supports. Physiology and abnormal expression of epigenetic gene regulators especially, microRNAs that are essential for normal Objective(s): (1) Investigate the structure and composition of networks across different communities that organogenesis during embryonic development. We found higher miR-200b expression in hypoplastic Pathophysiology, offer integrated substance use treatment and prenatal/parenting support. (2) Test whether network lungs from preterm infants with CDH. We hypothesize that hypoplastic CDH lungs have disrupted structure and composition varies according to ECD program characteristics. Department of Surgery), miR-200b signaling which results in disrupted target gene expression in lung epithelial and fibroblast Methodology: Using UCINet, we investigated the structure of five purposefully selected egocentric Winnipeg, MB cells. healthcare networks in Ontario. The agencies were selected based on variation in size, geography, and Methods: Primary epithelial and fibroblast cells were isolated from E19 control and nitrofen-induced breadth of partners. Network-level measures of density and centrality and node-level measures of hypoplastic rat lungs. Cells were transfected with biotinylated miR-200b mimics and cel-67. After 20 constraint, brokerage roles and structural holes were employed to indicate the overall functioning and hours, the cytoplasmic lysate was extracted and combined with prepared Streptavidin beads, which has cohesiveness of the network. NetDraw was used to present the networks visually. high affinity to biotin. Next, bound mRNA targets were pulled down from total RNA. After RNA Results: Ego basic measures were run for each of the five networks and results describe the structure and precipitation and RNA clean up, samples will be sent for Next Generation Sequencing to analyze data for composition of each network in terms of network-level and node-level measures. comparison between control and nitrofen-induced hypoplastic lung epithelial and fibroblast cells. Conclusions: The results from the social network analysis demonstrate the diversity in partnered Results: First, E19 rat lungs were chosen because of the high concentration of mRNA targets of miR-200b. providers in each ECD network. These exploratory findings will hopefully lead to increased health and Second, I optimized the protocol of isolating the epithelial and fibroblast cells from these lungs. Third, I well-being for women and their children through better communication and delivery of substance use optimized the number of cells to seed in 6 well plates to get 70% confluency on the day of transfection. and prenatal treatment. Fourth, transfection efficiency was tested on cell function under different conditions. Finally, the entire experiment was performed for three control and three nitrofen rats and after cleaning up RNA samples, we will send the samples for Next Generation Sequencing in the next step. Significance: Our studies will provide the capacity and information to build a foundation for future studies in which we will determine the contribution of miR-200b and its target genes to lung development. DNA Damage-Induced Poly [ADPribose] Polymerase -1 (PARP1) Activity Is Enhanced by Potential Therapeutic Effect of Chromofungin, on Experimental Colitis High Mobility Group A2 (HMGA2) Protein Kunal Kapoor Forouh Kalantari1, Manoj Reddy Medapati1, Idunnu Adeojo1, Farhana Begum1, Marek Los4, Thomas Klonisch1, 2, 3, Sabine Hombach-Klonisch1 University of Manitoba, Introduction: Inflammatory Bowel Disease (IBD) involves primarily two disorders: Crohn’s Disease (CD) Winnipeg, MB and Ulcerative Colitis (UC). Uninhibited inflammation of intestinal mucosa, which can affect any part of the gastrointestinal tract, is the hallmark of IBD. A lot of therapies have been developed but most of them 1 Human Anatomy and Introduction: The non-histone chromatin binding protein HMGA2 is expressed in embryonic stem cells are not long lasting or effective. Hence new treatment needs to be developed. Cell Science; 2Medical and cancer cells, including triple-negative breast cancer cells (TNBC). HMGA2 enhances resistance to Hypothesis: Chromofugin (CHR) regulates DC functions during the development of colonic inflammation, chemotherapeutics by enhancing base excision repair (BER) capability and decreases apoptosis by Microbiology & Infectious via a modulation of T-cell priming. modulating the ATM and ATR DNA damage signaling pathways. Here we investigated the role of HMGA2 Preliminary Data: Since September 2016, my research project targets the role of dendritic cells (DC) Diseases; 3Surgery, in DNA damage-induced PARP1 activity and determined its influence on cell survival and apoptosis. during the development of colitis and how CHR could modulate the DCs functions. I extracted naïve DCs University of Manitoba, Methods: Expression of HMGA2 and PARP-1 was determined by Western blot. Co-immunoprecipitation from spleen and Bone marrow (BM) and optimised the LPS dose with IL-6 as readout. In in-vitro study, I from nuclear protein extracts was used to study the interaction between HMGA2 and PARP-1. DNA Winnipeg, MB; 4Dept. of have even checked the role of CHR on BMDC’s surface markers to check their activation. CD40 and CD80 damage was induced using the alkylating agent methyl methanesulfonate (MMS), was quantified by were reduced in groups where CHR was given prior to LPS. But a clear trend was not visible in MHC-II and Clinical and Experimental detection of γH2AX, and PARylation activity of PARP1 was detected by Western blot. HMGA2 AT hook CD86 surface markers. Medicine, Division of mutant constructs were transiently transfected to assess PARP activity. We used WST assays to quantify AIM1: To determine whether preventive in vivo Chr treatment regulates DC maturation and cytokine cell survival and caspase3/7 assays to determine apoptosis. Otorhinolaryngology, release in the context of acute experimental colitis. Results: HMGA2 reduced MMS-induced DNA damage, increased cell survival and reduced apoptosis in Linköping University, Experimental Design: CD11c+ DCs will be extracted from colitis induced C57BL/6 mice. The organs triple-negative cancer cells. We identified PARP1, an ADP-ribosylating enzyme recruited to DNA damage considered for extraction are spleen, Bone Marrow, colon and mesenteric lymph nodes. The surface Sweden sites, as a novel HMGA2-interaction partner and showed that PARP1 activity was increased in the markers will be checked for their activation (MHCII+, CD40+, CD11b+, CD80+ and CD86+) with the help presence of HMGA2. This required functional AT hook domains of HMGA2. HMGA2 reduced the sensitivity of flow cytometry. The supernatants will be collected and will be checked for a range of cytokines (CCL21, of triple-negative breast cancer cells towards PARP1 inhibitors. Silencing of HMGA2 resulted in decreased TNF-α, IL-1β, IL-6, IL-23, IL-12p40 and IL-12p70) with ELISA. The same experiment will also be done in-vitro cell survival and increased apoptosis following treatment with MMS and Olaparib. and the stimulation to DCs will be given with LPS (100ng/ml, 12h) Conclusion: we identified HMGA2 as a novel regulator of PARP1 activity in cancer cells causing a reduced AIM2: To examine the effect of CHR protein treatment on DCs subsequent T-cell priming sensitivity towards PARP1 inhibitors. Experimental Design: CD11c+ DCs extracted from spleen, Bone Marrow, colon and mesenteric lymph nodes will be pulsed with or without LPS and with or without CHR. These cells will then be co-cultured with splenic CD4+ T-cells. The supernatants will be collected to analyse the type of T-cell response i.e. Th1, Th2 or Th17. This will be judged with cytokine profile analysis (IFN-γ, IL-4 and IL-17) with ELISA. TILRR is a Potential Enhancer of Inflammation and HIV-1 Vaginal Infection through Interacting with NFκβ Signaling Pathway M. A. Kashem1, 2, P. Lacap2, H. Li1, 2, W. Omange1, 2, N. P. Toledo1, 2, F. A. Plummer1, 3, M. Luo1, 2

Overall anticipated results and future studies: We postulate that there will be a significant decrease in 1Department of Medical Background: TILRR, a transcript variant of FREM1, is a novel regulatory component, which stimulates host DC activation after CHR treatment and subsequently decrease in T cell priming. Identification of the Microbiology, University defense against infection through binding to IL-1R1 and TLR complex and enhancing the recruitment of cellular mechanisms underlying the protective role of CHR may lead to novel therapeutic targets in IBD. MYD88 in the Ras-dependent NFκβ signal transduction pathway. Our previous study identified FREM1 as 2 Our future direction is to target the effect of CHR on mucosal healing process in IBD experimental models of Manitoba; National a novel candidate gene in correlation with HIV-1 resistance/susceptibility in the Pumwani Sex worker and its effect on the plasticity of DCs. Additionally, targeting gut microbiota composition under effect of HIV and Retrovirology cohort. In this study, we investigated the effect of TILRR on the global gene expression profile of the NFκB CHR. Laboratory, JC Wilt signaling pathway by over-expressing it into human cell lines. Methodology: TILRR was overexpressed in HeLa and VK2 cells using eGFP tagged plasmid construct. Infectious disease Transfection efficiency was confirmed by Confocal microscopy and Flow cytometry. TILRR RNA Research Laboratory, overexpression was confirmed by qRT-PCR and Western blot analysis. The effect of TILRR on the expression National Microbiology of 84 genes linked to NFκβ pathway was subsequently investigated by qRT-PCR. Conditioned media were also tested for the protein level expression of important pro- and anti-inflammatory cytokines/ Laboratory; 3National chemokines using Bioplex multiplex cytokine/chemokine bead assay. Microbiology Laboratory, Results: Overexpression of TILRR significantly up-regulated 32- and 52-genes, as well as down-regulated Winnipeg, MB 15- and 4-genes in IL-1β treated and non-treated cells, respectively. We observed that most of the pro- inflammatory cytokine/chemokine encoded genes, like IL-1β, IL-6, TNFα, and IL-8 (chemokine) were significantly increased at mRNA level expression. We further noticed that cytokines/chemokines, those were assessed for mRNA level expression by qRT-PCR, were also increased at the protein level expression in conditioned media using multiplex bead assay. Data described here are the representative of three independent experiments using female genital tract cell lines, which suggest a potential link of TILRR in HIV-1 vaginal infection through enhancing the NFκβ and inflammatory responses. Conclusion: Although how TILRR influence the expression of these genes needs to be further studied, our study is the first to show that TILRR may influence the expression of genes directly involved in HIV-1 infection in addition to its role in enhancing NFκβ and inflammatory responses. NFκβ and inflammatory response pathways are extremely important in HIV-1 transmission; therefore, further study of the role of TILRR may identify novel intervention targets and strategies against HIV-1 vaginal infection. Neuregulin-1 Promotes Oligodendrocytes Replacement and Accelerates Remyelination in Focal Demyelinating Lesions of the Rat Spinal Cord Hardeep Kataria, Arsalan Alizadeh, Ghazaleh M. Shahriary, Shekoofeh S. Rizi, Ryan Henrie Kallivalappil T. Santhosh, Soheila Karimi-Abdolrezaee

Regenerative Medicine Introduction: Oligodendroglial cell death and demyelination are hallmarks of central nervous system significant reduction in the expression of scar associated chondroitin sulfate proteoglycans (CSPGs) that Program, Department of (CNS) injuries and multiple sclerosis resulting in axonal damage and functional impairments. Restoration are known to inhibit remyelination. Our complementary in vitro studies also provided direct evidence of myelin sheath (remyelination) remains a major obstacle as the ability of endogenous neural precursor that Nrg-1 significantly increases the number of myelin basic protein expressing cells and overall Physiology and cells (NPCs) for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating myelination in DRG-NPC co-cultures. Mechanistically, we demonstrate that Nrg-1 specifically mediates it Pathophysiology, conditions. Our recent evidence shows that Neuregulin-1 (Nrg-1), a critical growth factor for effects through ErbB2 and ErbB4 receptor activation. University of Manitoba, oligodendrocytes, is dysregulated in demyelinating lesions that may underlie the poor endogenous Conclusion: Our work provides new evidence suggesting an impact for Nrg-1 in promoting replacement of oligodendrocytes and remyelination. Currently the role of Nrg-1 in demyelinating oligodendrocyte replacement, recruitment of myelinating cells and axonal remyelination in demyelinating Winnipeg, MB conditions is largely undefined. Here, we have used in vivo and in vitro models to unravel the impact of lesions of the CNS. Nrg-1 downregulation in focal demyelinating lesions of the spinal cord. Methods: We employed an in vivo rat model of lysolecithin (LPC)-induced focal demyelination. Recombinant human Nrg-1β1 (rhNrg-1β1) was delivered intraspinally at the time of LPC injections using biodegradable poly lactic-co-glycolic acid (PLGA) microcarriers for various times. Spinal cord tissues were analyzed with ELISA, Western blotting, slot blotting, immunohistology and electron microscopy at different time-points after LPC injections. For in vitro studies, we used brain derived primary rat NPCs to assess the role of Nrg-1 in oligodendrocyte differentiation and maturation. For myelination studies, rat dorsal root ganglion neurons (DRGs) were co-cultured with NPCs under control or Nrg-1 treated conditions. Differentiation, maturation and myelination were analysed using immunocytochemistry. Co-immunoprecipitation and Western blotting was performed to identify Nrg-1 mediated activation of signaling pathways involved in oligodendrocyte regulation. Results: Our in vivo findings show that rhNrg-1β1 treatment promotes oligodendrocyte generation and maturation in LPC induced demyelinating lesions. Additionally, Nrg-1 treatment enhanced infiltration of myelin forming Schwann cells into demyelinated lesions, and increased the number of remyelinated axons. We also found an overall significant improvement in myelin thickness. Nrg-1 therapy resulted in a What’s Right, What’s Wrong, and What’s New The McGill Augmentation Mammoplasty Operation (MAMO) Simulator: Validation Study Navaldeep Kaur Roy Kazan, Alex Viezel-Mathieu, Shantale Cyr, Thomas M Hemmerling, Mirko S Gilardino

McGill University, Introduction: Funding agencies encourage investigators to have pilot data prior to submitting a proposal Division of Plastic and Introduction: Given the private aspect and the focus on aesthetic outcome, residents’ exposure to Montreal, QC for an intervention study. The Canadian Institutes of Health Research (CIHR) has had around 100 Reconstructive Surgery, augmentation mammoplasty procedures remains limited. The development of the MAMO simulator competitions in the last 10 years where pilot projects were sought. Pilot studies are meritorious for aimed to provide an alternative training method as well as an assessment tool, much needed in a determining the feasibility of a definitive clinical trial in terms of conduct and potential for efficacy, but Department of competency-based education. The purpose of this study is to complete face, content and construct their possible applications for planning a future trial are not always realized. The recent publication of Experimental Surgery, validations of the MAMO simulator for subpectoral breast augmentation procedures. CONSORT reporting guideline for randomized pilot and feasibility trials has revolutionized the rigour McGill University, Method: The simulator represents the external features of a female chest and incorporates the essential with which pilot studies should be reported. We applied this novel guideline to pilot trials published over anatomical structures relevant to such procedure. Using an electromagnetic tracking system, an Montreal, QC 30 years in a rehabilitation journal with the aim to estimate the extent to which pilot/feasibility studies: assessment tool was developed and incorporated into the simulator to objectively assess subpectoral (i) addressed the needed objectives; (ii) led to definitive trials; and (iii) whether the strength of the pocket dissection. Plastic surgeons and residents were recruited to perform a mammoplasty procedure evidence of outcome improvement influenced the subsequent undertaking of a definitive trial. on the simulator. Following an instructional video, participants completed the essential steps of the Methods: Trials published in Clinical Rehabilitation, since 1987, were eligible if the word ‘pilot’ or ‘feasibility’ procedure, then experts evaluated its different parameters and their overall experience. Video recordings was specified somewhere in the article. A total of 191 studies were reviewed, results were summarized of participants’ performance were blindly reviewed and assessed by an expert using three scales: descriptively, and between-group effect sizes were computed. modified OSA TS score, surgery-specific score and a checklist. Data will be presented as Mean (SO). Results: As required, the specific purposes of piloting were stated in only 58% (n = 110) of the studies. Results: Preliminary results including six participants (3 residents and 3 experts) has shown an average The most frequent purpose was to estimate the potential for efficacy (85%), followed by testing the OSATS score of 22.3 (5) vs 38 (1.7), an average surgery-specific score of 30.3 (2.1) vs 41.7 (2.5) and an feasibility of the intervention (60%). Only 12% of the studies were followed by a definitive trial; <4% of average checklist score of 11 (1.7) vs 12.7 (1.2) between residents and experts respectively. The average studies had a main study underway or a published study protocol. No association was observed between time to complete the procedure was 20.5 (3.2) min for residents and 17 (2.7) min for experts. Face and effect size and follow-up of pilot studies, although the confidence intervals were very wide owing to content validations results showed excellent results among parameters evaluated, with an overall mean small number of trials that followed on. score of 4.8 (0.3) on 5 (96%). Discussion: At least in one journal, labelling and reporting of pilot studies needs to be improved to be Conclusions: The MAMO simulator has shown upon preliminary results to be an excellent assessment concordant with the advent of CONSORT guidelines. These findings are likely to generalize. CIHR should tool of novices’ performance and has the potential to become a standard method to determine residents’ reinforce following these guidelines when requesting and reviewing proposals for feasibility studies and competence in a competency-based educational system. results should be reported rigorously prior to committing considerable human and monetary resources to a full scale trial. Using Yeast to Study the Pathobiology of SIFD Exposure to Gestational Diabetes in Utero Impairs Mitochondrial Bioenergetics and Erin Kennedy1, Martin Holcik2, 3, Pranesh Chakraborty3, 4, Adam Rudner1 Cardiac Function in the Rat Offspring Stephanie M. Kereliuk1, 2, Kyle G. Cheung1, 2, Bo Xiang1, 2, Laura K. Cole1, 2, Mario A. Fonseca1, 2, Grant M. Hatch1, 2, Jonathan 1Ottawa Institute of tRNA nucleotidyl transferase (TRNT1 in mammals, Cca1 in yeast) is the RNA polymerase that adds the 3’ McGavock1, 3, Vernon W. Dolinsky1, 2 Systems Biology, CCA nucleotides to all tRNA and is required for translation, tRNA quality control, and protection of the translation machinery. Mutations of TRNT1 cause SIFD, Sideroblastic Anemia with B-Cell Immunodeficiency, 1 Department of Periodic Fevers, And Developmental Delay, a rare disease that has hallmarks of mitochondrial diseases. Diabetes Research Introduction: Gestational diabetes mellitus (GDM) is the most common complication of pregnancy. Biochemistry, Faculty of Defects in TRNT1 could impact mitochondrial function by altering the translation and abundance of a Envisioned and Children of mothers with GDM are at increased risk for the development of cardiometabolic diseases later in life. Though the mechanisms responsible are unknown, mitochondrial dysfunction is associated with Medicine, University of subset of proteins or by impairing a non-canonical function of TRNT1. We have shown that yeast surviving Accomplished in on wild type or mutant human TRNT1, or analogous CCA1 mutants, share phenotypes with patient- cardiovascular disease. The heart depends on mitochondrial energy production and the energy-requiring Ottawa; 2Molecular Manitoba (DREAM) derived cells. My preliminary data has shown that activation of the osmosensing HOG-MAP kinase process of calcium transport to maintain adequate contractility. We hypothesize that GDM induces fetal Biomedicine Program, pathway or inhibition of TOR signaling counteracts the deleterious effects of reduced TRNT1 or Cca1 Research Theme of the cardiomyocyte mitochondrial dysfunction and impairs calcium handling, conditioning the offspring for the development of heart disease later in life. CHEO Research Institute; function, suggesting a model in which an imbalance of mitochondrial and cytosolic translation may be Children’s Hospital the primary defect in SIFD, and that inhibition of cytoplasmic translation may be an effective therapeutic Methods: To induce GDM, female rats were fed a high fat (45% kcal) and sucrose diet prior to mating, 3Department of Research Institute of target. throughout pregnancy and lactation. Lean control females received a low fat (10% kcal) diet. Fetal rat Pediatrics, Faculty of Manitoba; 2Department ventricular cardiomyocytes (FRVCs) were isolated from the hearts of e20 offspring for the analysis of mitochondrial respiration, reactive oxygen species production and calcium transport. To assess cardiac Medicine, University of of Pharmacology and function over the entire life course of the offspring, serial echocardiography was performed at e18 and at 4 Ottawa; Newborn Therapeutics, University 3, 6, 9 and 12-months of age using a Vevo 2100 ultrasound. Screening Ontario, of Manitoba; 3Department Results: Basal and maximal mitochondrial oxygen consumption was reduced for glucose (35% & 68%) Section of Metabolism of Pediatrics and Child and fatty acid (49% & 52%) substrates in FRVCs isolated from GDM offspring (p<0.05). Mitochondrial reactive oxygen species production was increased 1.4-fold (p<0.05) in FRVCs from GDM dams. Integrated and Newborn Screening, Health, University of calcium flux was 1.5-fold greater and calcium re-uptake was 1.6-fold longer in FRVCs isolated from GDM Department of Pediatrics, Manitoba. Winnipeg, MB offspring (p<0.05). Fetal and 3-month old offspring exposed to GDM exhibit increased left ventricle CHEO, Ottawa, ON posterior wall thickness (p<0.05), a marker of cardiac hypertrophy. From 6 to 12-months of age offspring exposed to GDM exhibit increased isovolumetric relaxation time (p<0.05), indicating impaired diastolic heart function. Conclusions: GDM induced fetal cardiomyocyte mitochondrial dysfunction as indicated by reduced substrate oxidation and ATP production, elevated reactive oxygen species production and altered Semaphorin3E (Sema3E) and Intestinal Inflammation: Clinical and Experimental Approaches Laëtitia Kermarrec1, Nour Eissa1, 2, Charles N. Bernstein3, 4, Jean-Eric Ghia1, 2, 3, 4 calcium transport. These GDM-induced effects on fetal cardiomyocyte mitochondrial bioenergetics and 1Department of Introduction: Inflammatory bowel diseases (IBD) involve an increase of dendritic cells (DC) infiltration calcium transport were associated with early-life cardiac hypertrophy and later in life diastolic Immunology, University of and cytokines production. Recently, semaphorins have been implicated in inflammation and cell dysfunction. These mechanisms put offspring of GDM mothers at greater risk of developing heart disease. migration and has emerged as an essential axis in DC immune responses. This study aims to determine 2 Manitoba; The Children’s the role of Sema3E on DC regulation during colitis using, clinical rectal biopsies from active ulcerative Hospital Research colitis (UC), a murine model of colitis and, DC cell culture. Institute of Manitoba; Methodology: mRNA expression level of Sema3E was determined in human rectal biopsies collected from healthy (n=7) and active UC patients (n=7) using RT-qPCR, and an absolute correlation analysis was 3Internal Medicine conducted against pro- and anti-inflammatory markers. Bone marrow-derived DCs (BMDC) were isolated section of from naïve C57BL/6-deficient (Sema3E-/-) and wild type (WT) mice, and differentiation, IL-12p40 and Gastroenterology; 4IBD interferon (IFN)-γ production were quantified by flow cytometry and/or ELISA. Colitis was induced by dextran sulfate sodium (DSS 5%) for 5 day in Sema3E-/- and WT mice treated or not with recombinant Clinical and Research Sema3E-Fc. Disease activity index (DAI), macro- and microscopic scores were determined. Colonic Centre, University of myeloperoxidase (MPO) activity, Sema3E, TNF-α, IL-1β, IL-6, and IL-12p40 were quantified using ELISA. Manitoba, Winnipeg, MB Splenocytes and splenic CD11C+ cells were isolated from colitic groups then treated with Sema3E (10-6M) or vehicle, and IL-12p40 and IFN-γ levels were assessed. Results: In active UC biopsies, Sema3E mRNA was significantly decreased and levels of Sema3E were negatively and positively correlated with pro- and anti-inflammatory cytokines. In naïve mice, Sema3E deficiency resulted in an increase of DC differentiation, migration and antigen uptake, and CD11C+ BMDC showed an increase of the intracellular IL-12p40. In colitic WT mice, Sema3E level was significantly decreased. In colitic Sema3E-/- mice, DAI, macro- & microscopic scores, colonic MPO activity, TNF-α, IL-1β, IL-6, IL-12p40, IFN-γ were significantly increased compared to wild type and treatment with rSema3E-Fc abolished that deleterious effect. When compared to WT, in colitic conditions, SemaE-/- splenocytes and splenic CD11C+ cells showed an increased production of IL-12p40 and IFN-γ, and rSema3E-Fc treatment of splenic CD11C+ decreased them. Inhibitory Influence of Denosumab, a Monoclonal Antibody to Receptor Activator of Nuclear Factor-Kappa B Ligand (RANKL), in the Context of Experimental Colitis. A Khafipour1, H Derakhshani2, M F Rabbi1, N Eissa1, E Khafipour3, JE Ghia1, 4

Conclusion: Sema3E signaling is critical in the pathogenesis of inflammation in both the clinical and 1Department of Background: The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) experimental setting through the modulation of DC migration and activation. These novel findings may Immunology, University of plays a major role in the development of rheumatoid arthritis; however, its role in inflammatory bowel pave the road toward novel therapeutic strategies in IBD targeting Sema3E. disease is unknown. Genome-wide associations scan meta-analysis for Crohn’s disease (CD) identified a 2 Manitoba; Department variant, near the gene TNFSF11 that encodes RANKL, moreover CD risk allele increased expression of of Animal Science, RANKL in specific cell lines. This study aims to elucidate how the RANKL inhibitor (Denosumab) by University of Manitoba; modifying gut microbiota dysregulation can be harnessed to diminish experimental olitis and ultimately serve as a safe therapeutic target in IBD. 3Department of Medical Methods: CD-like colitis was induced through intrarectal administration of dinitrobenzenesulfonic acid Microbiology, University (DNBS, 4mg/kg) dissolved in ethanol (30%) to C57Bl/6 mice. One day before colitis induction, daily of Manitoba 4Internal injection of denosumab (10mg/kg/day, i.p.) was initiated and continued over four days. Control mice received whether ethanol (30%) or PBS (1%). On the sacrifice day, inflammation was evaluated clinically. Medicine section of Colonic tissue and fecal samples were amplified and subjected to Illumina sequencing for microbiome Gastroenterology, analysis (V4 regions of bacterial 16S-rRNA). Alpha- and beta-diversities were calculated in QIIME and University of Manitoba, subjected to SAS and PERMANOVA, respectively. Differences in clustering at the genus level were Winnipeg, MB determined using Bray-Curtis dissimilarity clustering analysis. Differences (P < 0.05) between treatments were tested using two-way ANOVA followed by the Tukey’s multiple comparisons in Prism. Results: Disease severity, macroscopic score and proinflammatory cytokines (Il-6, IL-1β and TNF-α) were increased in DNBS/ethanoltreated vs. control mice and denosumab-treatment significantly decreased only the proinflammatory cytokines. Treatment also modified the alpha and beta-diversity of colonic and fecal microbiota toward controls. DNBS/ethanol-treated animals showed a significant different clustering pattern in comparison with control group while denosumab moved such clustering toward controls. The predicted functional capacity of bacterial community was significantly altered after denosumab injection in DNBS/ethanol-treated group. Conclusions: The development of colitis in DNBS-ethanol model was accompanied by a disruption of the Parents and Clinicians’ Opinions About Probiotic Therapy in Prevention of Pediatric Antibiotic-Associated Diarrhea Samaneh Khanpour Ardestani1, Joan Robinson1, Hien Q. Huynh1, Hsing Jou1, Levinus A. Dieleman 2, Sunita Vohra1 gut microbiota and preventative treatment with Denosumab modulates intestinal inflammation and gut 1Department of Introduction: Probiotic therapy may be effective in preventing pediatric antibiotic-associated diarrhea microbiota dysbiosis in a murine model of colitis. This provides a rationale for considering denosumab as Pediatrics, University of (AAD). Some limitations in previous studies include lack of patient input in the determination of minimal a therapy in CD. important difference (MID) and selection of relevant outcomes. Our objectives were to establish the MID 2 Alberta Department of that would prompt parents and clinicians to use probiotics to prevent AAD and to obtain parents and Medicine, Faculty of clinicians’ opinion about the most important outcomes in clinical trials of AAD. Medicine & Dentistry, Methods: In this survey, parents of children presenting to the emergency department of a Canadian tertiary care children’s hospital and pediatricians working in that hospital were approached. A range of University of Alberta, potential MIDs were presented and participants selected the MID that they would require to use Edmonton, Alberta, probiotics for AAD prevention. Additionally, participants were asked to rate a list of outcomes they would Canada consider to be important in clinical trials of AAD. Results: In total, 127 parents and 45 pediatricians were recruited. Sixty four parents (56%) and 21 clinicians (51%) reported they would use probiotics if it would reduce the risk of AAD by 40% (i.e. reduce the risk of AAD from 19% to 12%; yielding a Number Needed to Treat of 13 and a relative risk of 0.61). In comparison to parents, pediatricians more frequently “agreed” or “strongly agreed” that probiotics were effective (34 (77%) clinicians vs. 60 (48%) parents, p=0.002) and safe (43 (98%) clinicians vs. 76 (62%) parents, p=0.001) for prevention of AAD. Three (2.5%) parents and none of the clinicians “disagreed” or “strongly disagreed” that probiotics were safe for prevention of AAD. The most important outcomes to parents in descending order were need for hospitalization, prevention of dehydration, disruption of normal daily activities, diarrhea duration and physician revisit. Pediatricians considered need for hospitalization along with physician revisit as the most important outcomes. They rated prevention of dehydration, diarrhea duration and stool frequency as important outcomes as well. Conclusion: There is good agreement between parents and clinicians regarding how effective probiotics would need to be in preventing AAD in order to warrant use. This information, along with outcomes they perceived important, will help designing future trials. MicroRNA-200b Regulates Distal Airway Development in Congenital Diaphragmatic Responses of KIR3DS1+ Versus KIR3DS1- NK Cells to HLA-Null Cell Stimulation Hernia Zahra Kiani1, 2 Franck P. Dupuy2, Elise Jackson2, Irene Lisovsky1, 2, Julie runeau\Nicole F. Bernard 1, 2 Naghmeh Khoshgoo1, 2, 3, Robin Visser1, 2, Ramin Kholdebarin1, 2, Thomas Mahood1, 3, Patricia Terra1, 6, Arzu Öztürk4, 5, Barbara Iwasiow1, 2, Molly Pind4, 5, Geoff Hicks4, 5, Andrew Halayko1, 3, Richard Keijzer1, 2, 3 1Division of Experimental Background: KIR3DS 1 (3DS 1) is an activating Natural Killer (NK) cell receptor (NKR) implicated in several Medicine, McGill viral infection, autoimmunity and cancer outcomes. In the context ofHIV, co-carriage of3DS1 and HLA- Bw4*80I alleles is associated with slower time to AIDS while 3DS1 homozygotes (hmz) have a reduced risk 1Children’s Hospital 2 Introduction: MicroRNAs are important epigenetic factors in development and disease. They are non- University; Research of HIV infection. The ligand for 3DS1 is HLA-F, a non-classical MHC-1 b antigen expressed on the HLA-null Research Institute of coding RNAs that regulate gene expression by directing their target mRNAs for degradation or Institute of the McGill 721.221 (721) cell line and on HIV-infected CD4+ T cells (iCD4). Manitoba, University of translational repression. We recently reported that higher microRNA miR- 200b expression is associated University Health Center; Methods: 9 3DS 1hmz donors were studied. PBMC from these were stimulated with 721 cells. The Manitoba; 2Department of with better response to prenatal lung growth-promoting therapy in preterm infants with congenital frequency of 3DS 1 +/-CD3-CD56dim NK cells exhibiting 1) any combination of IFN -y, CD 1 07 a, or CCL4 diaphragmatic hernia (CDH). We hypothesized that miR-200b regulates airway branching and epithelial 3Centre de Recherche du Surgery, University of functionality (total), function characterized by 2) total IFN-y, 3) total CCL4 secretion and 4) total CD107a cell homeostasis. Here, we aimed to delineate the role of miR-200b in normal and abnormal lung Centre Hospitalier de Manitoba; 3Department of expression was assessed using gating strategies that included and excluded NK cell subsets coexpressing development associated with CDH. other NKRs that may contribute to 3DS 1 + NK cell function. For results using inclusive gating to examine Physiology and -/- l’Universite de Montreal, Methods: We generated miR-200b mice and used lung function studies, micro-CT, surfactometry and the frequency of total, IFN-y+ and CD107a+ cells, PBMC from 7 3DS1hmz were tested twice (16 Montreal, QC pathophysiology, University immunostaining to evaluate the lungs. We complemented these studies and assessed miR-200b observations). N=8 subjects were examined using inclusive gating for total CCL4+ cells and for all 4 of Manitoba; Manitoba expression using RT-qPCR and in situ hybridization in a rat model of lung hypoplasia and CDH. We functional subsets examined using exclusive gating. 3DS 1-Fc chimeric protein was used to block the Institute of Cell Biology performed loss- and gain-of-function studies to determine the effects of miR-200b on lung explants. interaction between 3DS1 and HLA-F. Finally, we used prenatal miR-200b therapy to improve abnormal lung development in vivo. Results: We confim1ed that 721 cells expressed HLA-F. Acid pulsing 721 cells, which favors formation of University of Manitoba; -/- Results: MiR-200b mice displayed reduced distal airway branching, thickened alveolar walls and less + 5Department of HLA-F open conformers that bind 3DS1 , did not improve their ability to stimulate 3DS 1 NK cells. A septation. They had higher VIMENTIN and TWIST abundance and lower PPC resulting in significantly significantly higher frequency of inclusively gated 3DS1+ than 3DS1 NK cells responded to 721 stimulation Biochemistry & Medical higher tissue damping and elastance and lower hysteresivity in the lung. Normalizing miR-200b for total, IFN-y and CCL4 functions (p$0.05, Wilcoxon matched-pairs test), A higher frequency of Genetics, University of abundance reduced CDH incidence and significantly improved lung hypoplasia ex vivo and in vivo. exclusively gated 3DS1+ than 3DS1- NK cells responded to 721 stimulation for all functional subsets Manitoba, Winnipeg, MB; Conclusion: MiR-200b is required for normal distal lung branching and alveolarization. The knockout analyzed though differences did not achieve statistical significance. Blocking the interaction of3DS1 and 6Life and Health Sciences lung recapitulates lung hypoplasia in CDH. Prenatal therapy with miR-200b has the potential to rescue HLA-F reduced the frequency of functional 3DS 1 + NK cells. abnormal lung development in CDH. Research Institute (ICVS), Conclusion: The interaction of3DS1 on ex vivo NK cells with HLA-F on 721 cells is responsible for activating + - School of Health Sciences, a higher frequency of 3DS 1 than 3DS 1 NK cells. 3DS1hmz may have a reduced risk of HIV infection because HLA-F on iCD4 activates 3DS 1 + NK cells for anti-viral functions. University of Minho, Braga, Portugal The Role of Microglia in Cognitive and Metabolic Impairments in Offspring of Dams with Gestational Diabetes Jin Hee Kim1, 2, Abbas Rezaeian Mehrabadi1, 2, Lori Tessler1, 2, Tooru Mizuno3, 4, Vernon Dolinsky1, 4, Tiina M. Kauppinen1, 2, 4

1Department of Introduction: Gestational diabetes mellitus (GDM) is a common complication in pregnancy, affecting microglial PARP-1 was depleted in offspring. This suggests that inflammation/microglia mediates health Pharmacology and ~10% of expectant mothers. Epidemiological data shows that offspring of mothers with GDM are at (metabolic and cognitive) impacts, thus serving as a potential target in prevention of health complications higher risk for obesity and diabetes. GDM has also been suggested to impair cognitive performance in in GDM offspring. Therapeutics, Rady the offspring. Hyperglycemia promotes systemic inflammation triggering activation of brain immune Faculty of Health Sciences, cells, microglia. Chronic microglial pro-inflammatory responses can impair fetal nervous system University of Manitoba; development. We have recently demonstrated that GDM induces persistent microglial activation in hippocampus and hypothalamus, brain areas associated with regulation of cognitive functions and 2Neuroscience Research appetite. Our previous studies have indicated that poly(ADP)ribose-polymerase-1 (PARP-1) has a key role Program, Kleysen Institute in the regulation of microglial functions. Thus we hypothesize that microglial PARP-1 depletion attenuates for Advanced Medicine, GDM-induced cognitive and metabolic impairments in offspring. Methods: GDM was induced by providing mouse dams a diet high in fat (45%) and sucrose (HFS) for 6 Health Sciences Centre; weeks before mating and through the pregnancy resulting mild hyperglycemia at mid pregnancy. “Lean” 3Department of control dams received a balanced diet low in fat (LF; 10% fat). We utilized conditional PARP-1 KO mice Physiology, University of model, in which microglial PARP-1 depletion was induced in half of the offspring by mating PARP-1-lox dams with CD11b-cre sire. The offspring went through longitudinal body weight measurements, analysis Manitoba; 4Children of lean and fat tissue composition (by DXA), energy expenditure/calorimetric analysis (metabolic cage), Hospital Research glucose tolerance test, and behavioral tests (open field and novel object recognition) until the 15 weeks Institute of Manitoba, of age. Winnipeg, MB Results: Male GDM offspring showed increased weight gain (8.4%, n=10 compare to Lean male offspring), while female offspring weight was not affected. GDM induced reduced energy expenditure, metabolic activity and glucose tolerance in offspring of both sex. Microglial PARP-1 depletion reduced all these metabolic changes. GDM induced changes in explorative activity and impaired recognition memory in offspring were also prevented by microglial PARP-1 depletion. Conclusion: Fetal GDM exposure induced metabolic and cognitive impairment that were reduced when Prebiotics Prevent Antibiotic-Induced Obesity in Pregnant/Lactating Dams and their Improved Forensic Detection Methods for Abrin Toxin Offspring Matthew W. Klassen 1, 2, Cindi R. Corbett 1, 2 T. Klancic1, A. C. Choo1, N. A. Cho1, R. A. Reimer1, 2 1University of Manitoba; Introduction: Abrin is a toxin derived from the seeds of the plant Abrus precatorius. It is nearly identical 1Faculty of Kinesiology, Background: Antibiotics are the most widely prescribed therapeutic agents. Even though they are life- 2National Microbiology to the well known ricin toxin, both in structure and function. This Type II Ribosome-Inhibiting Protein (RIP) exerts its toxicity through inhibition of polypeptide translation within host cells, resulting in an University of Calgary; saving drugs, recent research suggests that early life exposure to antibiotics (during pregnancy and/or in Laboratory, Public Health the first year of life) may increase infant’s risk of obesity. On the other hand, non-digestible prebiotics extremely low lethal dose. While not as widely available as ricin, abrin poses a significant biological 2 Department of improve metabolic health resulting in lower body weight and fat mass. Our aim was to examine whether Agency of Canada, threat. There is great potential to improve abrin diagnostic assays beyond what currently exists. Biochemistry & Molecular prebiotic supplementation during pregnancy/ lactation reduces antibiotic-associated obesity risk in Winnipeg, MB Methods: This project encompasses development of new monoclonal antibodies, multiplexed bead- based detection strategies, as well as validation of mass spectrometry (MS) applications. Due to freely Biology, Cumming School Sprague-Dawley rats and their offspring. Methods: 10 week old female Sprague-Dawley rats (n=60) were mated and randomized to: 1)control [C] available crude toxin isolation literature, a wide variety of preparation types will be analyzed for critical of Medicine, University of (water + AIN-93G diet), 2)antibiotic [A] (low dose penicillin (LDP) + AIN- 93G diet), 3)prebiotic [P] (water forensic signatures. Calgary, Calgary, AB + 10% oligofructose (OFS) diet) or 4)antibiotic+prebiotic [A+P] (LDP+10%OFS diet). Mothers received Conclusion: Abrin toxin is one of the deadliest toxins available. The ability to disperse active toxin with LDP via drinking water from the third week of pregnancy through lactation. At 10 weeks of age, offspring relative ease requires a comprehensive diagnostic toolkit in order to conduct proper detection and underwent an 8-week high fat, high sugar metabolic challenge to unmask obesity risk. Dams and forensic analysis. Improved immunological detection and MS applications will be developed to meet this offspring underwent oral glucose and insulin tolerance tests and DXA. need. Results: Dams given antibiotics had higher body weight during lactation than C, P and A+P groups (p<0.05); with higher body fat % compared to P (p=0.09) and A+P (p=0.01). Male offspring (A) were heavier after metabolic challenge than C (p=0.04). This phenotype was preventable with prebiotics; A+P offspring were lighter throughout the challenge with lower fat mass. Female offspring (A) were heavier throughout the metabolic challenge than P. Adding prebiotics (A+P) prevented the weight gain, but only in the first 4 weeks of the challenge. Before the metabolic challenge, males (A) were insulin resistant; insulin sensitivity improved in A+P. Prebiotics improved insulin resistance in female offspring, but only after the metabolic challenge. Conclusion: Antibiotic consumption during pregnancy/lactation impairs “normal” postpartum weight loss in dams. This is the first demonstration that co-administering prebiotics with antibiotics mitigates the obesity risk of dams and their offspring, but has sex-specific effects. CD40L Primes Platelet Via CD40 Activation of NF-κB Fibroblast Growth Factor-2 Protection from Cancer Drug Cardiotoxicity Is Mediated by K. Kojok1, S. El Akoum1, M. Mohsen1, W. Mourad1, 2, Y. Merhi1, 2 the MTOR/Nrf2/Hemoxygenase-1Pathway Navid Koleini1, 2, Barbara E. Nickel1, Jie Wang2, Robert R. Fandrich1, 3, Peter A. Cattini2, Elissavet Kardami1, 2, 3 1Montreal Heart Institute; Introduction: CD40 ligand (CD40L), a member of the Tumor Necrosis Factor superfamily, is released 2Department of principally by activated platelets in the circulation and considered as a thrombo-inflammatory molecule 1Institute of Introduction: Cardiotoxic side effects pose limits to the use of Doxorubicin (Dox), a potent that predicts cardiovascular events, related to enhanced platelet reactivity and thrombosis. We have Medicine, Université de Cardiovascular Sciences, chemotherapeutic drug. There is need of cardioprotective strategies to prevent Dox toxicity. Fibroblast shown that CD40L is a strong activator of NF-κB in platelets that primes and enhances platelet activation Growth Factor‐2 (FGF‐2), especially the low molecular weight (Lo) isoform is well known to protect Montréal, QC and aggregation in response to thrombotic stimulus. In addition to its main receptor CD40, platelets Albrechtsen Research cardiomyocytes from a variety of stresses. Less is known about the properties of the high molecular express two other CD40L receptors, αIIbβ3 and α5β1. The present study was designed to identify the Centre; 2Department of weight (Hi) FGF-2 isoform. We have now compared the ability of FGF-2 isoforms to prevent Dox‐induced receptors of CD40L involved in platelet NF-κB activation and its implication in platelet aggregation. Physiology and cardiomyocyte cell death in vitro, and investigated signal transduction mediating FGF-2protection. Methods: Using washed human platelets; the presence of the different receptors of CD40L was Results: Primary cultures of neonatal rat cardiomyocytes provided an in vitro model to study acute Dox Pathophysiology, determined by flow cytometry and WB. The release of CD40L was analyzed by ELISA. Activation of NF-κB toxicity. Pre-incubation with either FGF-2 isoform significantly decreased Dox- induced cell damage and was assessed by determining the phosphorylation of IκBα and P65 in human and mouse platelets. University of Manitoba; death assessed by LDH release and the Live/Dead assay and prevented the Dox‐induced downregulation Platelet aggregation was measured optically. 3Department of Human of active mTOR.Dox caused a significant decrease in the mRNA and protein level of Nrf2, a master Results: We showed that platelets release CD40L in response to thrombin and CD40L stimulation and transcription factor for antioxidant genes, as well as relative levels of Nrf2 targets such as Hemoxygenase‐1 Anatomy and Cell express three CD40L receptors, CD40, αIIbβ3 and α5β1. CD40L, dose- and time- dependently, induced (HO‐1) and p62/SQSTM1. These effects were prevented by both FGF‐2 isoforms. Rapamycin, a selective platelet NF-κB activation as revealed by IκBα and P65 phosphorylation. Activation of NF-κB by CD40L was Sciences, University of inhibitor of mTOR, abrogated protection from either Hi or Lo FGF‐2, and also prevented the FGF‐2 -/- absent in CD40 mouse platelets and inhibited by CD40 blockade in human platelets, but unaffected by Manitoba, Winnipeg, MB induced upregulation of Nrf‐2; HO‐1; and to some extent, p62/SQSTM1. αIIbβ3 or α5β1 blockade. CD40L alone had no effect on platelet aggregation but potentiated the Conclusion: We have shown that not only Lo‐FGF‐2, but also Hi‐FGF‐2 pre‐treatment protects aggregation response in the presence of priming doses of thrombin; an effect that was abolished by cardiomyocytes against acute Dox damage. FGF‐2 protection requires mTOR activation, and the CD40 blockade. downstream upregulation of antioxidant defenses by Nrf2 and HO‐1. Conclusion: This study demonstrates that CD40L triggers activation of NF-κB and primes platelets exclusively through the CD40 receptor. This suggests that CD40L is a platelet primer via a signaling pathway involving CD40/NF-κB activation, which predisposes platelets to enhanced activation and aggregation in response to thrombotic stimuli. Using a Multivariate, Non-Parametric Approach to Reveal Spatiotemporal Brain- Comparison of Breast Tumor Size by Photoacoustic Tomography, Magnetic Resonance Behaviour Differences Across Learning Phases in Schizophrenia Imaging and Pathology Michele Korostil 1, 2, 3, Gary Remington 1, 3, Anthony Randal McIntosh2, 3 Ivan Kosik1, 2, Muriel Brackstone3, 4, Anat Kornecki1, 5, Astrid Chamson-Reig1, Philip Wong1, 2, J.J.L. Carson1, 2, 4

1Centre for Addiction and Background: Learning impairments are core symptoms of schizophrenia and impact directly on 1Imaging Program, Introduction: Breast cancer is one of the most common cancers to affect women, presenting a lifetime Mental Health; 2Rotman functional outcome in the disorder. Elucidating the underlying large-scale brain substrates underlying Lawson Health Research risk of 1 in 8. Diagnosing and staging the cancer is critically dependent on medical imaging technology, which facilitates tumor detection and disease extent assessment. Furthermore, by monitoring the learning impairments using fMRI has been limited by common approaches to analysis of fMRI data. We Institute; 2Department of Research Institute of addressed these gaps in the literature with a multisession fMRI experiment in which participants with efficacy of neoadjuvant therapy, medical imaging often continues to play a vital role in the treatment of Baycrest Health Sciences; schizophrenia learned a novel lexicon and used multivariate non-parametric and direct brain-behaviour Medical Biophysics, stage 3 or higher breast cancers. Currently, X-ray mammography, Ultrasonography and Dynamic Contrast 3Institute of Medical analytic techniques to better understand the spatiotemporal unfolding of functional brain networks Schulich School of Enhanced Magnetic Resonance Imaging (DCE-MRI) are the most common modalities used in this field. underlying learning. Medicine and Dentistry, Unfortunately, the complexity of implementing multiple imaging methods, combined with their Science, University of Methods: Participants with schizophrenia were compared with healthy control participants as they Western University; sensitivity and specificity deficits, particularly in dense breasts, indicate an unmet need in this area. Toronto, Toronto, ON learned a novel 30-word lexicon during two fMRI scans over a several day period. Participants were Photoacoustic tomography (PAT) is a relatively new imaging modality which uses safe near-infrared laser 3Department of Oncology, trained to equal task proficiency prior to scanning in an MRI-simulator. Behavioral-Partial Least Squares, illumination to generate 3-D images of soft tissues to a depth of up to several cm. Hypothesis: breast a multivariate analytic approach, was used to analyze the imaging data. Permutation testing was used to Schulich School of tumor size determined by PAT is positively correlated to, and is not significantly different from, size determine statistical significance and bootstrap resampling to determine the reliability of the findings Medicine & Dentistry, determined by pre-operative DCE-MRI and post-operative pathology. Results: Healthy participants showed a typical two-stage brain-behaviour pattern. Persons who Western University; Methods: A raster scanning PAT system was constructed on a portable transport cart and deployed in a transitioned between an early learning network to a late learning network that included dorsostriatal university-affiliated hospital surgical suite. One-hundred freshly excised lumpectomy specimens were 4Department of Surgery, regions were more successful learners. The schizophrenia participants did not make the same late- scanned prior to formalin fixation. Patients who had received a pre-operative DCE-MRI scan and who learning transition despite successfully learning the lexicon. Better learners with schizophrenia were Schulich School of were found by post-operative pathologic examination to have at least an invasive component were differentiated primarily on the basis of greater engagement of perceptual and perceptual-integration Medicine & Dentistry, included in this study. Tumor size was measured volumetrically for PAT and DCE-MRI, and separately using brain regions. For both groups, the dominant multivariate brain-accuracy patterns showed positive Western University; maximum diameters for pathology, DCE-MRI and PAT. Comparisons were performed using Pearson’s correlations with cognitive measures out of the scanner. 5Department of Medical correlation coefficients, and the non-parametric Wilcoxon signed-rank test, with significance set to Conclusions: Our experiment demonstrates that utilizing a multisession, multivariate approach to p=0.05. Imaging, Schulich School studying learning reveals a different spatiotemporal unfolding of brain-behavior relationships in Results: Twelve patients met the criteria and were included in the study. The image acquisition procedure schizophrenia. Given the same amount of practice and comparable learning curves, participants with of Medicine and Dentistry, took less than 20 minutes to complete and did not interfere with the standard of care. PAT derived schizophrenia did not move from networks suggestive of effortful learning toward subcortically-driven Western University, volumetric tumor size was strongly positively correlated to DCE-MRI (r=0.78, p=0.003) and not procedural networks similar to the healthy controls. Accuracy in schizophrenia was instead driven by the London, ON significantly different (p=0.97). On the other hand, the maximum diameters were only moderately relative degree of engagement in perceptual regions, suggesting that perception itself is impaired in the disorder and this may impact downstream, “higher level” cognition. Investigation into the Effects of Repeated Solvent Use on the Immune System Monika Kowatsch1, Margaret Ormond2, Genevieve Boily-Larouche3, Julie Lajoie1, 4, John Wylie5, 6, Javier Mignone5, Keith R. Fowke1, 4, 5 positively correlated (r=0.54, p=0.07) and borderline not significantly different (p=0.064). In comparison 1University of Manitoba Background: Solvent use is defined as the use of a broad group of readily available lipid soluble to pathology, PAT maximum tumor diameters were very strongly positively correlated (r=0.87, p=0.0002) Department of Medical substances resulting in psychoactive effects. Examples include lacquer thinner, hairspray or rubbing but significantly different (p=0.0015). Comparison of DCE-MRI and pathology revealed only a borderline alcohol. Solvents are generally sniffed, huffed, bagged or sprayed into the mouth to cause psychoactive 2 significant moderate positive correlation (r=0.56, p=0.06) but no significant difference in size (p=0.55). Microbiology; Sunshine effects. In 2010 the Canadian community epidemiology network on drug use reported that 5% of adults Conclusion: The results indicate that PAT volumetric-measurement performance of invasive breast House; 3University of and 9% of Winnipeg youth use solvents, although numbers are higher in the homeless population. In tumors is similar to that of DCE-MRI. On the other hand, maximum diameter measurements are less Manitoba, Winnipeg, MB; 4 collaboration with Sunshine House, a community-based organization working with street-involved reliable. A similar trend is seen in comparing both PAT and DCE-MRI maximum diameters to pathology. In Winnipeggers, we engaged with solvent using community members who told us they were interested in University of Nairobi, contrast, examination of the very high correlation between PAT and pathology maximum diameters, determining effects of solvent use on their health. Additionally, there have been clinical reports of combined with a significant difference in size, indicate the presence of an apparent systematic over- Nairobi, Kenya; 5University inflammation in the trachea of solvent users, as our lab’s expertise is in cellular immunology, we were estimate of maximum diameters by PAT. We hypothesize that pathologically undocumented DCIS- of Manitoba Department interested in assessing the effect of long-term solvent use on the immune system. Excess inflammation components, combined with compression of the ex-vivo lumpectomy samples by the specialized is of concern as we know that an activated immune system enhances susceptibility to chronic and of Community of Health lumpectomy holder, are responsible for the maximum diameter discrepancy. infectious diseases such as HIV. Sciences; 6Cadam Hypothesis: Solvent users will have greater immune activation profiles than matched non-solvent users. Provincial Laboratory, Objectives: Assess the impact of solvent use on microbial translocation, immune activation, Immune cell Winnipeg, MB phenotype and function. Methods: 27 Individuals were recruited and enrolled in the pilot study, 14 solvent users and 13 matched controls (solvent non-users). Cytokines, chemokines and immune modulatory hormones were assessed by bead assay. Immune cell activation was assessed using flow cytometry. Results: Our study found that solvent users had increased innate immune activation characterized by activated natural killer cells (lower mean fluorescent intensity of CD38 on their NK cells along with fewer CD38 positive NK cells). Solvent users also exhibited increased pro-inflammatory chemokines (cell recruitment molecules) MIP-1β and IL-8 compared to controls. We also found that solvent users had lower levels of the thyroid hormones, T3 and T4, required for efficient metabolism necessary for efficient immune response The Transcriptional Regulator TBX3 Promotes Progression from Non-Invasive to Invasive Breast Cancer Milica Krstic1, 2, Carl O. Postenka2, 3, Joseph Andrews2, 3, Hon S. Leong1, 4, Muriel Brackstone4, 5, Ann F. Chambers1, 2, 3, Alan B. Tuck1, 2, 3

Conclusion: Solvent users exhibit higher levels of innate immune activation than solvent non-users. 1Department of Pathology Introduction: In cell lines derived from the same breast cancer patient at different phases of progression, Understanding the effects of solvent use on the immune system is an important first step in improving and Laboratory Medicine, we have shown that TBX3 is abundant in the invasive 21MT-1 cells, and minimally expressed in the non- the health of this underserved community. Schulich School of Medicine invasive, DCIS-like 21NT cells. There are two isoforms of TBX3 (TBX3iso1 and TBX3iso2), with a 20 amino and Dentistry, The University acid addition in the DNA binding domain of TBX3iso2; functional differences between these two isoforms, of Western Ontario; and their role in cancer progression has not been reported in existing literature. 2The Pamela Greenaway- Methods: TBX3 isoforms were overexpressed in 21NT cells. Functional changes and gene expression Kohlmeier Translational changes were assessed. ChIP-array and RNA-Seq studies were employed to examine the mechanism of these associated changes. Cells were injected into the chick embryo vasculature to examine extravasation Breast Cancer Research Unit, and invadopodia formation. Cells also were implanted into nude mice. In vitro angiogenesis (i.e. tubule London Regional Cancer formation) was assessed. Patient samples of early breast cancer cases (186) from the London Breast Program, London Health Cancer Database were examined for TBX3 expression by immunohistochemistry. Sciences Centre; Results: Overexpression of TBX3iso1 or TBX3iso2 in non-invasive 21NT cells resulted in increased survival, 3 Department of Oncology, growth and invasiveness in vitro, with increased extravasation and invadopodia formation in the chick Schulich School of Medicine embryo. Through genome-wide ChIP-array studies coupled to RNA-Seq we have mined the direct and Dentistry, The University transcriptional targets of both isoforms. Our results indicate that both TBX3 isoforms promote of Western Ontario; invasiveness through altered expression of epithelial-mesenchymal transition (EMT)-related genes, 4Department of Surgery, including the direct up-regulation of EMT-inducing transcription factor SLUG. SLUG expression is required Schulich School of Medicine for TBX3-induced migration and invasion of breast cancer cells. Assessing TBX3 levels in early stage breast and Dentistry, The University cancer by immunohistochemistry revealed that expression was high in low-grade lesions, suggesting of Western Ontario; 5Division TBX3 involvement in progression through the low-grade DCIS molecular pathway. We will continue to of Surgical Oncology, validate markers from our genomic studies by immunohistochemistry to assess whether they are London Regional Cancer correlated with increased risk for developing invasive cancer. Interestingly, only TBX3iso1 overexpressing Program, London, ON cells exhibited significant tumorigenic potential in nude mice. We have performed data mining and A c-Di-GMP Metabolizing Domain Gene, BCAL1069, Regulates the Motility of Burkholderia Cenocepacia in Cystic Fibrosis Sputum Nutritional Conditions Brijesh Kumar, John L. Sorensen, Silvia T. Cardona supporting functional studies that suggest this is likely due to the promotion of angiogenesis and Department of Introduction: Cystic fibrosis (CF) patients are susceptible to chronic lung infection by opportunistic secretion of cancer-associated cytokines upon TBX3iso1 overexpression. Microbiology and bacterial pathogens, such as Burkholderia cenocepacia, one of the most prevalent and multidrug resistant Discussion: This work may have clinical potential in identifying patients with high-risk lesions, and/or as species of the B. cepacia complex (Bcc). Previously, we showed that CF sputum nutritional conditions, rich potential direct/indirect therapeutic targets to prevent disease progression. Chemistry, University of in amino acids, induce motility of the clinical isolate B. cenocepacia K56-2. Cyclic dimeric guanosine Manitoba, Winnipeg, MB monophosphate (c-di-GMP) is an intracellular second messenger that regulates bacterial virulence, including motility in bacterial pathogens. The aim of this study is to investigate role of putative c-di-GMP metabolizing domain genes in B. cenocepacia motility during growth in CF sputum nutritional conditions. Methods: To demonstrate that c-di-GMP can modulate B. cenocepacia motility, the c-di-GMP metabolizing domains genes wspR and rocR from Pseudomonas aeruginosa were cloned and introduced in the K56-2 (WT) strain. C-di-GMP levels and their effect in motility were evaluated by HPLC and swimming assays. To detect flagellin expression and flagellar biosynthesis, Western blot and electron microscopy were performed, respectively. Further, eleven putative c-di-GMP metabolizing domains genes were inactivated by insertional mutagenesis. The mutants were examined for motility on 0.3% agar semi-solid plates containing a CF sputum-mimicking medium or a defined medium with individual amino acids. Results: Using HPLC, we confirmed increased c-di-GMP levels in the WT/pWspR strain. These high levels of c-di-GMP caused a significant reduction in motility, whereas low c-di-GMP levels displayed similar motility to WT strain. The increase of c-di-GMP did not affect flagellin expression or flagellar biosynthesis, indicating that c-di-GMP regulates motility at a post-translational level. Screening of the c-di-GMP- related insertional mutants for defective motility led us to identify a gene, BCAL1069, encoding a protein with putative signalling and c-di-GMP degrading domain. Further, the reduced motility was evident in CF conditions and in the presence of arginine and glutamate. Conclusion: These results suggest that BCAL1069 affects the motility of B. cenocepacia K56-2 in response to arginine and glutamate by changing c-di-GMP levels. Importance of Cellular and Plasma Fibronectin in Maintaining Vessel Wall Integrity and Effects of Glycogen Synthase Kinase 3 Beta (GSK3β) Inhibition on P-Glycoprotein Function Expression and Activity in Cell Culture Models of the Blood-Brain Barrier Heena Kumra1, Laetitia Sabatier1, Amani Hassan1, Karina Zeyer1, Elaine C. Davis1, Deane Mosher2, Pierre Chambon3, Marlyn D. Laksitorini1, 2, Donald W. Miller 1, 2 Takao Sakai4, Dieter P. Reinhardt1, 5 1Department of Wnt/β catenin signaling is important for maintaining blood-brain barrier (BBB) function. One of the target 1Faculty of Medicine, Introduction: Fibronectin (FN) is an alternatively spliced glycoprotein which exists in two major forms- Pharmacology and genes regulated by the canonical Wnt/β-catenin pathway is the drug efflux transporter, P-glycoprotein cellular FN (cFN) and plasma FN (pFN). cFN is secreted by various cells, including smooth muscle cells (Pgp). As GSK3β-mediated phosphorylation of β-catenin controls the level of gene expression, we Department of Anatomy Therapeutics, University and Cell Biology, McGill (SMC), and is assembled into fibers. pFN is synthesized exclusively by hepatocytes and secreted into the hypothesized that GSK3β inhibitors could alter Pgp activity through a β-catenin dependent process. of Manitoba; 2Kleysen Using Madine Darby Canine Kidney cells transfected with MDR1 gene (MDCK-MDR1) and immortalize University, Montreal, QC; blood. FN plays a crucial role in development as indicated by early embryonic lethality (E9.5) of the FN human cerebral microvascular endothelial cell (hCMEC3) line as in vitro models of the BBB, we examined 2Departments of knockout (KO) mouse. Although the role of FN in embryonic development of blood vessels is well Institute of Advanced established, its function in postnatal development is poorly understood. alterations in PgP expression and activity resulting from modulation of Wnt signaling pathways. Biomolecular Chemistry and Medicine, Health Sciences Methods: To determine the postnatal role of FNs in blood vessels, we have employed three conditional Treatment of MDCK-MDR1 and hCMEC3 with BML 284 (Wnt activator) increased PgP expression and Medicine, University of KO mouse models: 1) a newly generated smooth muscle-specific and tamoxifen-inducible cFN KO mouse Center, Winnipeg, MB enhanced activity as assessed by the cellular accumulation of the Pgp fluorescent probe, rhodamine 123 Wisconsin, Madison, WI; that allows timed postnatal cFN deletion; 2) an established mouse that lacks pFN postnatally; and 3) a (R123). In contrast, the Wnt inhibitor (WntC59) resulted in decreased Pgp expression in both cell lines. 3Institut de Génétique et de novel double KO (dKO) mouse lacking both cFN and pFN. Next we assessed the effects of various GSK3β inhibitors on Pgp expression and activity. Of the Biologie Moléculaire et Results: We observed that postnatal deletion of cFN in SMCs leads to SMC disorientation in the media of pharmacological agents examined, both quetiapine (QTP) and lithium chloride (LiCl) were found to Cellulaire, CNRS UMR7104, vascular walls and upregulation of collagen deposition in the adventitia, but does not affect the life span. increase PgP expression at both the messenger and protein level (approximately 30 and 50%, Inserm U964, Strasbourg, Unexpectedly, huge amounts of pFN was observed to translocate from the plasma to the aortic wall in respectively). In addition, both QTP and LiCl enhanced Pgp activity as demonstrated by the reduced France; 4MRC Centre for the cFN KO (also observed in control mice) as observed by immunohistochemistry, and with fluorescently accumulation of R123 in cells. Both QTP and LiCl treatment increased total β-catenin and reduced phosphorylation of β-catenin in cells consistent with inhibition of GSK3β. These results suggest that Drug Safety Science, labelled, systemically injected pFN. Contrary, systemically injected dextran of the same molecular mass as of a pFN monomer (250kDa) did not translocate to the aortic vessel wall, demonstrating a specific GSK3β inhibitors can modulate Pgp expression and activity in the BBB and as such may have both Department of Molecular mechanism for the pFN uptake. Unlike cFN, postnatal deletion of pFN did not show a histological aortic therapeutic and adverse consequences. and Clinical Pharmacology, phenotype. However, complete deletion of FNs in dKO mice resulted in postnatal lethality starting as Institute of Translational early as P10, indicating a safeguard role of pFN in vascular stability. The safeguard function of pFN also Medicine, University of became evident in cell culture where on deletion of cFN, pFN could still be assembled and also support Liverpool, Liverpool, UK; the assembly of other elastic fiber related matrix proteins although in a less complete manner. 5Faculty of Dentistry, McGill Conclusions: Overall, our data show that pFN play an important role in safeguarding vascular organization University, Montreal, QC and health. Studies on the Roles of Viral Envelope Surface Proteins in Cell Attachment and Entry Anti-Fibrotic Ski Regulates ZEB2 Via a Hippo-Dependent Mechanism Lindsey L. Lamboo1, 2, Daniel R. Beniac1, Christine G. Golding1, Shannon L. Hiebert1, Tim F. Booth1, 2 Natalie M. Landry1, 2, Mark Hnatowich1, Sunil Rattan1, 2, Jeffrey T. Wigle1, 3, Ian M.C. Dixon1, 2

1National Microbiology Introduction: Type 1 viral fusion proteins are a group of trimeric surface glycoproteins that are 1Institute of Background: Cardiac fibrosis is the pathological deposition of scar tissue in the myocardial interstitium Laboratory, Public Health responsible for binding to the host cell receptor and the membrane fusion that follows. Hypothetical Cardiovascular Sciences, which results in the abnormal thickening of the heart muscle and decreased cardiac output. Quiescent membrane fusion mechanisms have been suggested, but several details regarding entry and fusion cardiac fibroblasts are required for tissue homeostasis of the myocardium, which is delimited by the rate Agency of Canada; remain unknown. The Ebola virus is an enveloped virus characterized by an external layer of glycoprotein St-Boniface Hospital of breakdown and synthesis of extracellular matrix proteins. Upon activation, cardiac fibroblasts exhibit 2Department of Medial (GP) spikes that bind to the cellular receptor Niemann-Pick C1 (NPC1) during infection. Albrechtsen Research the hyper-secretory, hyper-proliferative myofibroblast phenotype. We have shown that overexpression of Microbiology, University Methods: This project studies the interaction of GP with NPC1, as well as the interaction of the virus and Centre; 2Department of the endogenous TGF-β1 repressor, Ski, reverts activated myofibroblasts into a more fibroblast-like host cell membranes, using a virus-like particle (VLP)/liposome model system. Liposomes are comprised phenotype. In addition, Ski downregulates the expression of Hippo pathway effectors and the expression of Manitoba, Physiology & of 18:1 PC, 18:1 DGS-NTA (Ni), and 18:1 Lissamine/Rhodamine PE; while the VLPs are comprised of the of pro-fibrotic Zinc finger E-box Binding homeobox 2 (ZEB2). Previous studies have shown that the Hippo Winnipeg, MB Ebola viral protein VP40, and GP. The NPC1 receptor is being produced via a stable 293TN cell line. Once Pathophysiology, pathway regulates ZEB2 expression; thus, we hypothesize that Ski’s downregulation of ZEB2 expression interaction has been established between GP and NPC1, intermediate fusion structures will be captured University of Manitoba; is mediated by the Hippo signalling pathway. using cryo-electron microscopy. Methods: We studied primary rat cardiac fibroblasts cultured on compressible silicone substrata with an 3Department of Results: To date, Ebola VLPs have been constructed by co-transfecting 293T cells and NPC1 has been elastic modulus biomimetic to healthy myocardium (E = 5 kPa). Cells were then transduced with purified from stable cell line culture media by FPLC. Liposomes and NPC1 have been mixed and binding Biochemistry & Medical adenoviral overexpression vectors to induce the expression of Hippo signaling components, as well as has been confirmed by Western blot. I am currently working towards capturing cryo-EM images of the Genetics Max Rady anti-fibrotic Ski. Conversely, pro-fibrotic gene expression was downregulated using siRNA-mediated NPC1-decorated liposomes and will soon be adding VLP’s will be added to the NPC1-decorated liposomes. College of Medicine, Rady gene knockdown. We used immunoblotting, fluorescence immunocytochemistry and qPCR to examine Once interaction between the GP and NPC1 is observed, cryo-EM of the structures will take place. resulting pro- and anti-fibrotic gene expression. Conclusion: Upon completion, this project will enable us to clearly understand the roles that envelope Faculty of Health Sciences, Results: Culturing primary cardiac fibroblasts on compressible substrata maintained a fibroblast-like surface proteins play in cell attachment and entry during a viral infection. We will also be able to elucidate University of Manitoba, phenotype prior to the first passage (P0). Overexpression of Hippo effectors in cardiac fibroblasts induced the possible triggers involved in how virus-to-cell fusion occurs. By determining how the GP spike protein Winnipeg, MB the expression of myofibroblast markers and decreased the expression of TCF21 on 5 kPa substrata. is involved in the fusion process, new avenues for drugs and antibodies to combat this deadly infection Knockdown of ZEB2 prevented the activation of myofibroblasts with exogenous Hippo activation. may be revealed. Conclusions: Our data suggest that cardiac fibrogenesis is contingent on the Ski-Hippo-ZEB2 relationship and that this axis of cellular phenotype regulation may be a relatively selective antifibrotic therapeutic target. A Qualitative Exploration of Tuberculosis Treatment-Seeking and Treatment-Taking Experiences in South Africa: Patient and Provider Perspectives Stephanie Law

Epidemiology, McGill Background: The growing incidence of drug resistant forms of tuberculosis (TB), including multidrug- Discussion: Many patients and providers have reported significant improvements in care provided at University, Montreal, QC resistant (MDR) and extensively drug-resistant (XDR) TB, is jeopardizing progress made to halt the global public facilities, including shortened wait times and improved follow-up systems for outpatient care. TB epidemic. This qualitative study seeks to identify ways to improve healthcare-related services for TB However, they also identified many areas where improvements can be made. Recommendations for patients in order to reduce delays in starting effective TB treatment, and to increase treatment completion changes to healthcare-related service include, but are not limited to: improving training and retention of rates. community care workers; increasing follow-up of patients who transition between hospitals and private Methods: Purposive sampling of participants - including TB patients, private and public healthcare clinics to the public TB clinics; reducing nurse rotations in TB clinics and aligning nurse training with these workers, community care workers, and traditional healers - took place between December 2015 and rotations; establishing facilities for vulnerable patients to stay during treatment; and providing better February 2017 in Cape Town, South Africa. Participants were recruited from four public TB clinics and their assistance to patients applying for disability grants. catchment areas, and one TB hospital, Open-ended, semi-structured interviews were conducted with consenting participants. Qualitative content analysis identified themes emerging from the interviews. Findings: Interviews were conducted with 33 adult TB patients - 11 (33%) with drug sensitive TB, 13 (40%) with MDR-TB, 5 (15%) with pre-XDR-TB, and 4 (12%) with XDRTB. Of these patients, 12 (36%) had HIV co-infection, and their ages ranged from 20 to 69 years. Patient delays in seeking care ranged from 1 to 16 weeks, with the majority of patients (76%) reporting visiting a public facility first (i.e. TB clinic or public hospital). Other patients reported visiting private clinics or hospitals, or chemists prior to visiting public facilities, and no patients reported seeking care from traditional healers. A total of 32 healthcare workers were interviewed, including community care workers, nurses, public and private doctors, as well as traditional healers. Six main themes affecting treatment-seeking behaviours and retention-in-care emerged from the interviews: 1) socioeconomic issues, which include TB-related stigma, poverty and vulnerable employment, and lack of social support; 2) provider and patient attitudes and behaviours, which include the lack of rapport-building and trust between providers and patients; 3) health system issues, such as staff burnout and problems with staff rotation and training; 4) issues affecting access to disability grants, including the lack of involvement of clinic staff in aiding sick patients to apply for grants; 5) TB knowledge and understanding among patients and non-public healthcare workers; and 6) problems with TB medications. Exploring Skin Tear Prevalence, Incidence and Associated Risk Factors Among the Elderly Effect of Stilbenoid Polyphenols on Resistance Artery Structure and Mechanical Long-Term Care Population Properties in the Spontaneously Hypertensive Heart Failure (SHHF) Rat Kimberly LeBlanc, Kevin Woo Danielle I. Lee1, 2 Crystal Acosta2, 3, Hope D. Anderson1, 2, 3

School of Nursing, Faculty Background: Skin tears (STs) are among the most prevalent wounds found in long-term care (LTC) 1College of Pharmacy, Purpose: Small resistance arteries affect total peripheral resistance (TPR). Increased blood pressure (BP) of Health Sciences settings. Given our aging population, the burden related to STs will further increase. Skin tears are University of Manitoba; in hypertension is proportional to changes in cardiac output (CO) and TPR. Therefore, it is imperative to frequently under-recognized and under-treated as they are often misunderstood as expected outcomes examine compounds that affect microvascular parameters. Resveratrol (trans-3,5,4’-trihydroxystilbene), a Queen’s University, 2Canadian Centre for of normal skin changes associated with aging. When coupled with age related co-morbidities, STs may stilbenoid polyphenol, is linked to improved longevity and cardiovascular health. Although resveratrol is Kingston, ON exhibit prolonged healing times leading to complications such as infection. Emerging evidence suggests Agri-food Research in well-tolerated in humans, it is readily metabolized and exhibits low bioavailability. We queried whether that pain is a common symptom associated with STs affecting people’s ability to function and quality of Health and Medicine, St. resveratrol analogues would produce greater vasculoprotective effects in an experimental model of life. While many factors have been purported to be associated with ST development, there is little human heart disease. Boniface Research Centre; evidence to corroborate their roles in ST risks. The primary purpose of this study was to examine risk Method: Sprague-Dawley (SD) and SHHF rats (n=5-8) were treated for 8 weeks by gavage with vehicle factors associated with ST development in the Ontario LTC population. 3Department of control (C) or low doses (2.5 mg/kg/d) of resveratrol (R), pterostilbene (P), and gnetol (G). Blood pressure Methods: A prospective study design was used to determine the risk factors associated with ST Pharmacology and (BP) was measured biweekly by tail-cuff plethysmography. Animals were anesthetized and 3rd order development. Prior to the study, a systematic literature review was conducted to identify previously mesenteric resistance arteries were isolated. Vascular structure and mechanical properties were evaluated Therapeutics, University identified risk factors and inform the study. A total of 380 individuals over the age of 65 years from 4 LTC by pressure myography. Signaling effectors were investigated via western blot, and oxidative stress was facilities in Ontario were followed over 4 weeks. The participants were examined for STs at the beginning of Manitoba; Winnipeg, assessed through malondialdehyde (MDA) assay. of the study and at week 4 to determine if STs had occurred and to record the ST type, location, and MB Results: Systolic BP increased in SHHF rats (196±3 mm Hg, vs. SD 142±7 mm Hg, p<0.01), and was associated factors. unaffected by stilbenoid treatment. SHHF media:lumen (M:L) increased (17.4±1.2 vs. SD 8.9±0.5, p<0.01), Results: The study demonstrated a ST prevalence of 20.8% and an incidence of 18.9%. History of a while media cross-sectional area was unchanged. All stilbenoids reduced M:L toward normal (SHHF-R previous ST (p=.012) and presence of non-modifiable skin changes (p<.001) were identified as key risk 13.8±0.8, SHHF-P 13.7±0.5, SHHF-G 13.0±0.3, p<0.01). Intrinsic stiffness of the arterial wall was factors associated with ST development and are supported by the literature review. The study also comparable between groups, demonstrating unchanged geometry-independent stiffness (SHHF identified two key modifiable risk factors; requiring assistance with activities of daily living (p<.001) and 4.97±0.27 vs SD 4.36±0.18). AMPKα activation was elevated in SHHF small arteries and attenuated by resisting care (p =.001). gnetol. No differences in ERK1/2 nor p38 signaling were detected in mesenteric resistance arteries. Conclusion: Our study results provide much needed Canadian data for benchmarking the burden of STs Conclusion: Mesenteric resistance arteries of the SHHF rat exhibit eutrophic remodeling, as found in in the LTC population. By identifying modifiable and non-modifiable risk factors, healthcare professionals human essential hypertension. Stilbenoids failed to lower BP; this suggests that vascular improvement can establish prevention programs targeted at reduction of risks for ST development. This study is an was not secondary to BP lowering, but rather a result of direct actions on the arterial wall. Further important first step towards developing a risk predictor scale for ST development in the LTC population. research on stilbenoid polyphenols as an adjunct to current anti-hypertensive therapy is warranted. Improving Clostridium Difficile Infection Surveillance Through Mathematical Modelling Measuring Performance in Vascular Disease Management in Primary Care Settings Based Seungwon Lee1, Michael Li2, Elizabeth Henderson1, 3 on the Canadian Cardiovascular Harmonization of National Guidelines Endeavour (C-­CHANGE) 1Department of Introduction: Clostridium difficile is a major hospital associated infection responsible for most cases of Theresa M. Lee1, 2, Sheldon Tobe3, 4, 5, Noah Ivers1, 2, 3, 6, Liisa Jaakkimainen1, 2, 3, 4, Debra Butt3, 7, Kimberly Walker5, Peter Liu3, 8, Community Health hospital associated diarrhea. The overall CDI (Clostridium difficile infection) rate in Canada peaked in 2008 Karen Tu1, 2, 3, 9 but remained stable since then. CDI can be broken down into two types: hospital-acquired CDI (HA-CDI) Sciences, University of and community-acquired CDI (CA-CDI). Traditionally, HACDI was the dominant form but CA-CDI is on the 1 Calgary, Calgary, AB; rise and now makes up a significant proportion of identified CDI in the acute inpatient settings. CDI Institute of Health Policy, Introduction: This project aims to use electronic medical record (EMR) data to assess the current Management and performance by primary care physicians in the screening for, diagnosis, and management of vascular 2Department of became a nationally notifiable disease in 2009. Our primary objective was to use mathematical modelling to link the relationship between these two forms. Evaluation, Dalla Lana School disease and vascular disease risk factors against national harmonized guidelines set forth by the Canadian Mathematical and Methods: We reviewed the microbiology of CDI and generated Susceptible - Asymptomatic - Infectious of Public Health, University Cardiovascular Harmonization of National Guidelines Endeavour (C-­‐CHANGE). Statistical Sciences, -Isolated - Recovery - Susceptible mathematical model of differential equations (DEs) to reflect currently of Toronto; 2Institute for Methods: We conducted an exploratory retrospective cross-­‐sectional study using Electronic Medical Record Administrative data Linked Database (EMRALD®) housed at the Institute for Clinical Evaluative University of Alberta, ongoing healthcare practices. We created a specialized compartment specific for asymptomatic infections Clinical Evaluative Sciences; Sciences in Ontario. We developed a set of measurable quality indicators (QI) based on the 74 C-­CHANGE 3 as we theorized these cases could contribute to the changing epidemiology of CA-CDI. The surveillance 3Faculty of Medicine, Edmonton, AB; Alberta published guidelines to measure performance in the screening for and management of vascular disease population consisted of all hospitalized patients in AHS/COV (Covenant Health) healthcare facilities who University of Toronto; Health Services risk factors; and to identify the gaps in performance based on QIs. We measured the rate of adherence to were> 1 year of age. We used AHS Case definitions for HA-CDI and CA-CDI. 4Sunnybrook Health Results and Conclusion: Initial review of currently existing surveillance data confirmed changing trend each QI. Multilevel multivariable logistic regression models were constructed for each QI to assess Sciences Centre, Toronto, ON; in CA-CDI distribution in the community. The initial model revealed that there is an inseparable association between adherence and patient-‐level­ characteristics (sex, age, income quintile, comorbidity, 5Canadian Cardiovascular relationship between HA-CDI and CA-CDI which supports the potential role in transmission played by body mass index [BMI]) and physician-‐level­ characteristics (physician sex, time in practice, clinic size, asymptomatic carriage. There is an implication that current CA-CDI estimation obtained through current Harmonization of National rurality). practice underestimates the true prevalence of CA-CDI. Next steps require integrating AHS/COV Guidelines Endeavour; Results: Our study population consisted of 325 Ontario family physicians and 233,418 patients. We surveillance data to estimate parameters of CDI and simulate appropriate intervention strategies from 6Women’s College Hospital; assessed 22 Qis over different time periods (12 months, 18 months, and all time). There is variance in rates the model. 7Scarborough Hospital, of adherence to Qis related to screening rates for cardiovascular disease. Lowest adherence to C-­CHANGE Toronto, ON; 8Ottawa guidelines were seen in administration of 2hPG oral glucose tolerance tests for pre-­‐diabetic patients (9.5%), measuring waist circumference (2.2%) and recording children’s BMI measurements annually Hospital Research Institute, (27.5%). Highest adherence to the guidelines was seen in reaching treatment targets for antihypertensive Ottawa, ON; 9Toronto medication, use of Angiotensin-‐converting­ enzyme (ACE) inhibitors and/or Angiotensin II Receptor Western Hospital, Blockers (ARBs), statin therapy for patients with diabetes, and using lipid profile tests for risk factor University Health Network, screening. Patient age, presence of chronic conditions, resource utilization and higher BMI category were Toronto, ON most frequently associated with adherence to C-­CHANGE. A Comparison of Eccentric and Concentric Upper Limb Strength in Elderly Women in the Sixth, Seventh and Eighth Decade: A Preliminary Investigation H. S. Legg1, 3, C. Arnold1, 2, J. P. Farthing1, J. Lanovaz1

Conclusion: EMR data can be used to measure adherence to C-­‐CHANGE recommendations. There are 1College of Kinesiology, Introduction: Strength capacity has been strongly linked to functional ability in an ageing population. varying levels of adherence among the measurable QIs. Some differences can be accounted for by University of Universally, upper limb strength is assessed via hand-grip dynamometry (HG), providing an isometric patient-­, and physician‐level variability. There is room for improvement in quality of primary care assessment that has been associated as a predictor of frailty and disability in the elderly. An understanding 2 management of vascular disease in Ontario. Saskatchewan; School of of dynamic strength changes in elderly women is limited, particularly for the upper limb. Physical Therapy, Methods: 35 older women (74 ± 8 yrs., 1.6 ± 0.1 m, 68.7 ± 11.4 kg) were stratified into age groups: 60-69 University of (n = 12), 70-79 (n = 13) and 80-89 (n = 10). Multi-joint dynamic functional strength was assessed utilising a novel dynamometer-controlled single-arm press with separate concentric (CON) and eccentric (ECC) Saskatchewan, Saskatoon, movements. In addition, isometric strength was assessed via HG. Following a series of increasing intensity SK; 3 St Mary’s University, familiarization repetitions, participants performed three maximal efforts of each strength measure with London, UK an average of the three peak values for the dominant (DA) and nondominant (NA) arms being utilized for analysis. Results: A 3-way (contraction type) repeated measures ANOVA was utilized to assess each arm independently. The results demonstrated 80-89 year olds had significantly lower strength in the NA compared to 60-69 (p = 0.042) and 70-79 year olds (p = 0.005). These differences were not apparent in the DA (p = 0.061). There were no strength measure x age interactions for either the NA or DA. Conclusion: Some preservation of ECC strength has previously been shown in elderly populations. The current findings suggest preservation of upper limb ECC, CON and HG strength in the DA across age groups, while for the NA this preservation is seen in the sixth and seventh decades but not in the eighth decade. The difference in strength preservation between arms is a new result and requires further investigation. The novel arm-press strength measure requires increased co-ordination, possibly more so for the NA, which may be a contributing factor to the results. The functional multi-joint nature of the isokinetic single-arm press strength measurement may be indicative of functional capacity and ability in an ageing population. Future research is needed to establish the relationship of strength preservation and functional capacity. The Planar Cell Polarity Protein Vangl2 Is Essential for Retinal Axon Guidance and Optic A Fast and Easy to Use Framework for Automatic Biological Knowledge Base Nerve Development Construction Vicki Leung1, 2, Alexandra Iliescu1, 3, Christine Jolicoeur4, Michel Gravel3, Sergio Apuzzo3, Elena Torban5, Michel Cayouette4, 6, Jake Lever, Steven Jones Philippe Gros1, 2 University of British Introduction: Modern biomedical research relies heavily on bioinformatics knowledge bases in order to 1McGill Research Centre Introduction: Vangl2 plays a critical role in the establishment of planar cell polarity (PCP) and is well Columbia and Canada’s easily understand the relationship between different genes, drugs and diseases. Without knowledge bases, researchers would have to read enormous numbers of paper to gain the same level of information. on Complex Traits; characterized for its role in vertebrate neural tube development. In humans, variants in VANGL2 have Michael Smith Genome been associated with familial and sporadic cases of NTD. Vangl2~dependent PCP activities are required The key problem is that knowledge bases require substantial investment of time, money and expertise to 2Department of Human Sciences Centre for patterning of not only neural, but also a number of additional structures. This is apparent as Vangl2 keep them well maintained. Automated methods for generating knowledge bases are becoming possible Genetics, 3Department of mouse mutants display developmental defects in many other tissues such as the heart, kidneys, lungs, through advances in text mining technologies. Methods: We present a framework for easily building a knowledge base for a variety of biomedical Biochemistry, McGill and reproductive system. Previously, expression ofVangl2 was detected in the developing retina during late embryogenesis, which led to the investigation of the potential role ofVangl2 in retinal development. problems. Our framework allows simple annotation of sentences to be used as example data. These University; 4Cellular Methods: A Vangi2βGeo knock-in reporter mouse, and a polyclonal isoform-specific antibody were examples are then used by our award-winning text mining tool VERSE. This approach allows us to process Neurobiology Research generated to evaluate Vangl2 mRNA and protein expression, respectively. To further investigate the role the entirety of PubMed and the Open Access Subset of Pubmed Central to generate a highly robust and valuable biomedical resource. Unit, Institute de of Vangl2 in retinal development, retinas of embryos homozygous for mutant Vangl2 alleles (Lp) were examined. Results: As an example of the power of this framework, we have created a useful resource that identifies Recherches Cliniques de Results: Vangl2 mRNA and protein are dynamically expressed in the developing retina with expression a gene as a driver, oncogene or tumor suppressor in a specific type of cancer. Furthermore this approach 5 Montreal; Department of becoming progressively restricted to the ganglion cell layer and optic nerve as the retina matures. The will be used to assist curation of the CIViC knowledge base for personalised cancer medicine. Medicine, McGill expression ofVangl2 is most prominent at the plasma membrane and the axons of retinal ganglion cells Conclusions: The use of text mining is essential in creating and maintaining knowledge bases for biology Lp/Lp research. Modern machine learning methods coupled with expert knowledge of the domain give a University Health Centre; (RGCs). Additionally, Vang12 is essential for retinal and optic nerve development as Vangl2 mutant embryos display reduced ocular size, thickening of the retina, and optic nerve hypoplasia. Notably, in flexible approach to generate a robust knowledge base that can be easily updated as new research is 6 Faculty of Medicine, Vangl2Lp/Lp mutants, axon bundles were observed to traverse throughout the entire retina without published. Universite de Montreal specific orientation. These ectopic axons ultimately become trapped within the sub-retinal space resulting in optic nerve hypoplasia. Conclusion: Taken together, these results identify a severe intraretinal pathfinding defect of RGC axons in Vangl2Lp/Lp embryos leading to severe optic nerve hypoplasia, highlighting a novel and essential role for Vangl2 in retinal axon guidance. UGT2B17 Promotes Castration-Resistant Prostate Cancer Progression Through BRPFl Regulates Hematopoietic Stem Cell Development Enhancing Ligand Independent AR Signaling Lin Li, Linya You, Xiang-Jiao Yang Haolong Li1, Ning Xie1, Ruiqi Chen1, Mélanie Verreault2, Ladan Fazli1, Martin Gleave1, Olivier Barbier2, Xuesen Dong1 Division of Experimental Introduction: Epigenetic regulators such as histone acetyltransferases, methyltransferases, chromatin- 1Vancouver Prostate Background: Castration resistant prostate cancer (CRPC) is characterized by a shift in androgen receptor Medicine, McGill remodeling enzymes change the way specific genes function without altering their DNA sequence. One of these epigenetic regulations is governed by histone acetylation, which is tightly controlled by various Centre, Department of (AR) signaling from ligand dependent to ligand-independent. Defining mechanisms that control AR University, Montreal, QC signaling transformation is important to develop therapies for disease control. UDP-glucuronosyltransferase histone “modifiers”, “readers” and “erasers”, and functions in transcriptional regulation. Bromodomain- Urologic Sciences, 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into and PHD finger-containing protein 1 (BRPFl) possesses four histone-binding domains and activates three University of British inactive forms. Although enhanced UGT2B17 expression by antiandrogens was reported in androgen- lysine acetyltransferases (MOZ, MORF and HBOl). BRPFl and their two binding partners, MOZ and MORF, are found frequently mutated in cancers especially in leukemia. However, the importance and mechanism Columbia; 2Laboratory of dependent prostate cancers, its role in regulating AR signaling transformation and CRPC progression remain unknown. of BRPFl in hematopoietic development and disease are not clear. Thus, we explored the role of BRPFl in Molecular Pharmacology, Method and Results: We first evaluated the UGT2B17 protein expression levels by immunohistochemistry hematopoiesis using hematopoietic-specific knockout mice. CHU de Québec Research (IHC) on Vancouver Prostate Centre tissue microarrays. We show that higher UGT2B17 protein expression Methods: Brpfl gene is deleted in the hematopoietic system in loxP flanked C57BL/6.SJL mice by Vav-iCre which confers hematopoietic-specific iCre expression. Cellular populations and characteristics were Centre, Laval University in prostate tumors is associated with higher Gleason score, metastasis and CRPC progression. The expression and activity of UGT2B17 are also higher in androgen-independent compared to androgen- assessed with various markers using flow cytometric approach using cells harvested from bone marrows dependent cell lines. Interestingly, enzalutamide-resistant MR49F cells expressed low UGT2B17 mRNA or fetal livers at different stages from hematopoietic-specific knockouts and their wild-type littermates. but strong protein expression, suggesting that posttranslational mechanisms may enhance UGT2B17 Specific histone acetylation marks were detected with immunofluorescence and Western blotting. Gene protein stability in these cells. We then constructed PCa cell lines with gain-of-function UGT2B17 by expression levels in different populations were checked with whole transcriptome sequencing and/or lentivirus. UGT2B17 stimulates cancer cell proliferation, invasion, and xenograft progression to CRPC after validated by RT-PCR. prolonged androgen deprivation. To further decipher the molecular mechanisms by which UGT2B17 Results: RT-PCR indicated that Brpfl is highly expressed in hematopoietic system especially in enhances PCa cell growth independent of androgen, we performed gene microarray using LNCaP(mock) hematopoietic stem cells, and knockout efficiency in various populations is high in fetal livers as early as and LNCaP(UGT2B17) cells cultured under the regular serum condition or prolonged androgen E12.S. Flow cytometry on fetal livers at E12.5 and bone marrows at P7 showed that hematopoietic stem deprivation condition. The analysis of the results showed that UGT2B17 suppresses androgen-dependent cells (HSCs) are dramatically reduced in Brpfl deficient mice, accompanied with increased apoptosis, AR transcriptional activity, while enhancing androgen (ligand)-independent AR transcriptional activity. senescence and reactive oxygen species level. Immunofluorescence and Western blotting confirmed that The latter targets genes associated with cell mitosis. These UGT2B17 actions are mainly mediated by histone acetylation marks such as H3K9Ac, H3K14Ac, H3K18Ac, H3K23Ac are decreased significantly. activating the c-Src kinase, and in CRPC tumors, UGT2B17 expression is associated positively with c-Src RNA-seq on hematopoietic stem cells and LSK cells showed that multiple HSC signature genes are activation. dysregulated. Conclusion: These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand- Conclusion: The study shows for the first time that BRPFl, as an epigenetic reader, is important in independent AR signaling that activates predominantly cell mitosis in cancer cells. Histone Deacetylase Inhibitor RGFP109 Overcomes Temozolomide Resistance by Blocking NF-κB-Dependent Transcription in Glioblastoma Cell Lines Qingzhong Li, Weiping Li Introduction: Glioblastoma multiforme (GBM) demonstrates an unsatisfactory clinical prognosis due to hematopoietic stem cell development. Loss of which will lead to loss of multiple histone acetylation Department of Clinical the intrinsic or acquired resistance to temozolomide (TMZ). As a major onco-pathway in glioma, the transcription of NF-κB subunits is highly regulated by several post-translational nuclear modifications, marks, altered cellular properties and changes in multiple HSC signatu re genes. This also provides Medicine, Shantou insights into roles of epigenetic readers in hematopoietic diseases such as leukemia. especially acetylation, a bidirectional process mediated via histone acetyltransferase and histone University Medical deacetylase (HDAC), implying that HDAC regulation might be a target for overcoming resistance in GBM College, Shantou, cells by mediating the NF-κB transactivation. Thus, we aim to determine whether HDAC inhibitor RGFP109 would be a candidate reagent for combination therapy with TMZ for patients with temozolomide- Guangdong, China. resistant (TR) GBM. Materials & Methods: Firstly, we established TR/U251 GBM cell lines and screened the inhibitory effect of RGFP109 in combination with temozolomide on cell viability via CCK-8 assay. Then, cell apoptosis were detected by annexin V staining and TUNEL in TR/U251 cells. Expression of NF-κB and NF-κB-Regulated Genes were measured by immunoblotting assay, immunofluorescence microscopy and RT-PCR. Finally, interactions between protein-protein and DNA–protein were detected by co-immunoprecipitation assays and Electrophoretic Mobility Shift Assay. The cells were harvested after transfection to western blotting for target proteins. Results: RGFP109 leaded to apoptosis and down-regulated the expression of NF-κB-regulated pro- survival genes in TR/U251 cells. However, RGFP109 did not alter the phosphorylation levels and the nuclear translocation of NF-κB/p65, but induced hyperacetylation of NF-κB/p65 and blocked interactions between NF-κB/p65 and its coactivators, p300 and p300/CBP-associated factor (PCAF).RGFP109 also reduced NF-κB/p65 binding to κB-DNA and the transcriptional level of κB-mediated genes, suggesting that RGFP109-induced hyperacetylation leaded to attenuated transcription of the κB gene. In addition, RGFP109 elevated the expression of inhibitor of growth 4 (ING4) and enhanced ING4 recognition and binding to NF-κB/p65, which may be correlated with reduced interactions between NF-κB/p65 and p300 and PCAF, thereby effecting transcription of the κB gene. Finally, we showed that knockdown of ING4 abolished the effect of RGFP109. Small Organic Compound Mimicking the L1 Cell Adhesion Molecule Promotes Functional Recovery After Spinal Cord Injury in Zebrafish Rong Li, Melitta Schachner

Conclusion: The study concludes that RGFP109 overcomes TMZ resistance by inhibiting NF-κB dependent Department of Introduction: Tacrine is a small organic compound that was discovered to mimic the functions of the transcription and regulating ING4 and NF-κB pathway interaction. We show that RGFP109 in combination Neurobiology, Shantou neural cell adhesion molecule L1 by promoting the cognate functions of L1 in vitro, such as neuronal with temozolomide may be a therapeutic candidate for patients with temozolomide-resistant GBM. survival, neuronal migration, neurite outgrowth and myelination. Based on studies indicating that L1 University Medical enhances the functional recovery in disease paradigms of different central and peripheral nervous College, Shantou, system of rodents, the current research is focused to examine the beneficial role of tacrine in the zebrafish Guangdong, China by investigating the functional recovery after spinal cord injury. Materials & Methods: Two-photon laser injury and L1.1 translation blocking morpholino microinjection were performed on larvae. Locomotor activities were conducted in adult and larval zebrafish. L1.1and L1.2 mRNA expression levels were detected by qRT-PCR. L1.1 protein level and its intracellular signal transduction pathways were determined by Western blot analysis. Immunohistology and anterograde tracing were performed to evaluate axonal regeneration after SCI in adult zebrafish. Results: Tacrine treatment enhanced recovery of locomotor activities following SCI in larval and adult zebrafish. L1.1 mRNA but not L1.2 mRNA expression levels in the spinal cord of larvae after SCI were increased following treatment with tacrine. In addition, MAPK, PI3 kinase/Akt and CK2 signaling pathways were activated by tacrine treatment. Also, tacrine treatment reduced the astrogliosis in the lesioned spinal cord after SCI. L1.1 and MBP expression were enhanced following tacrine treatment after SCI. Re-growth of severed axons were noticed especially at days 20 and 28 after SCI in the tacrine-treated group. Conclusion: Tacrine, as a mimetic for L1 stimulates L1 functions that promote locomotor recovery and axonal regrowth after spinal cord transection in larval and adult zebrafish. Together with possibly other functions of tacrine, application of this molecule at concentrations that are orders of magnitude lower than those previously used in humans in Alzheimer’s disease may ameliorate by topical application the functional deficits in spinal cord injured patients. Identification and Characterization of New Target of Pseudomonas Aeruginosa Type CD271 (p75NTR) As a Novel Diagnostic Marker and Therapeutic Target for SHH Three Secretion System Master Transcriptional Activator ExsA-Fem System Medulloblastoma Yanqi Li1, Sara Badr2, Kangmin Duan 1, 2 Lisa L. Liang1, Ludivine Coudiere-Morrison1, Nazanin Tatari1, Vijay Ramaswamy2, Timothy Ryken4, Marc Del Bigio5, Michael Taylor2, Cynthia Hawkins2, Jennifer Chan6, Tamra Werbowetski-Ogilvie1 1Department of Oral Introduction: Pseudomonas aeruginosa is one of the predominant causes of nosocomial infections in 1 Biology, University of immunocompromised individuals and is responsible for chronic respiratory disease of patients with Regenerative Medicine Introduction: Medulloblastoma (MB) is the most common malignant primary brain tumor and is currently cystic fibrosis (CF). P. aeruginosa manipulates host cells by using Type three secretion system (T3SS)- Program, Department of classified into 5 distinct molecular subtypes based on genomic alterations, gene expression profile, Manitoba; 2 Department hallmark of acute infection- that can translocate toxic proteins directly into host cell and cause damage, Biochemistry and Medical response to treatment and cell of origin. This extensive heterogeneity has revealed a critical need for of Medical Microbiology, as the exclusively reported function so far. Our previous data showed that T3SS mediates inter- and Genetics, University of subtype-specific, functionally validated biomarkers and therapeutic strategies. Using an unbiased high Winnipeg, MB intra-species interaction but the mechanism remains unclear. The Fem system is predicted to be regulated Manitoba, Winnipeg, MB; throughput flow cytometry screen and gain/loss of function studies, we previously identified CD271/ 2 by T3SS master transcriptional activator exsA, and is one of cell-surface signal system (CSS) regulating the Arthur and Sonia Labatt p75NTR as a candidate stem/progenitor cell marker, specifically in SHH MB. Here, we show that CD271+ uptake of iron-chelating product named mycobactin and carboxymycobactin produced by Mycobacterium Brain Tumour Research and CD271- subpopulations do not exhibit a hierarchal organization; but are rather two co-existing species. The Fem system consists of an outer membrane-localized TonB-dependent receptor-FemA, a Centre and Program in cellular subsets. cytoplasmic membrane-localized anti-sigma factor-FemR (PA1911) and an extracytoplasmic function Developmental and Stem Methods: FACS sorting followed by re-culturing of CD271- and CD271+ cells from low passage primary Cell Biology, The Hospital for (ECF) sigma factor-FemI (PA1912). cultures demonstrated that both cell populations can recapitulate the parental phenotype. Global gene Sick Children, Toronto, ON; Methods: 1Construction of CTX-FemA reporter strain and overexpression of sigma factor of Fem System expression profiling by RNA sequencing was performed to further evaluate the molecular heterogeneity 3Cancer Care Manitoba pAK-1912-1911; 2 Construction of knockout mutant ΔFemA and measurement of pyocyanin production; between these two subpopulations. Immunohistochemical (IHC) staining was also performed to evaluate (CCMB), Winnipeg, MB; 3 measurement of promoter activity of virulence factors (exoS, rsmY, rsmZ, phzA1, phzA2, rhlR, rhlA, CD271 expression levels across MB subgroups. 4Department of MexAB, pilG and algD) in addition to mycobactin and upon overexpression of pAK1912-1911. Results: RNA sequencing revealed that genes/pathways associated with cell survival, proliferation and Neurosurgery, University of Results: 1 Fem System was identified as new target of exsA by sequence alignment of exsA-dependent Kansas, Kansas City, Kansas; motility are downregulated in CD271- vs. CD271+ subpopulations. Cell cycle analysis supported these promoters; 2 The pyocyanin production of ΔexsA and ΔFemA were significantly increased when 5Department of Pathology, findings and demonstrated that CD271- cells exhibited a decrease in proliferation compared to CD271+ compared with wild type PAO1; 3 the promoter activity of virulence factors (exoS, rsmY, rsmZ, phzA1, University of Manitoba and cells. Moreover, PI3K/AKT, JAK/STAT, and NFkB, are downregulated in CD271- vs. CD271+ cells, while phzA2, rhlR, rhlA, MexAB, pilG and algD) were affected in addition to mycobactin or under overexpression Manitoba Institute of Child pathways such as RAS/MYC are upregulated in CD271- vs. CD271+ cells. IHC analysis of CD271 levels of pAK1912-1911. Health, Winnipeg, MB; across the MB subtypes demonstrated that nearly all SHH tumors express CD271, while Group 3 tumors Conclusion: 1 new target of exsA-Fem system was identified and exsA activates Fem System to regulate 6Department of Pathology exhibit moderate staining. In contrast, Group 4 MB and WNT do not express CD271. Importantly SHH MB the expression of other virulence factors; 2 novel function of T3SS was observed mediating interaction and Laboratory Medicine, exhibit a nodular CD271 staining pattern, further underscoring the heterogeneity within these tumors with Mycobacterium species and probably other microbiota through Fem system. University of Calgary, and supporting our findings that CD271- and CD271+ cells are distinct, yet clinically relevant, Calgary, AB subpopulations. Differential Methylation Profile of Rat Fetal Brains Chronically Exposed to Moderate Levels of Alcohol During Pregnancy Xavier Lieben Louis1, Songyan Liu2, Yoonsik Park1, Fatemeh Ramezani Kapourchali1, Wayne Xu2, 3, Michael N. Eskin1, Geoff Hicks2, James Davie2, 3, Miyoung Suh1, 3

Conclusions: Our results suggest that CD271, in combination with other markers, could be effectively 1Department of Human Introduction: In Canada, prevalence of fetal alcohol spectrum disorder (FASD) is closer to 1% and it is the utilized as a diagnostic tool for SHH MB and that therapeutic strategies concomitantly targeting both Nutritional Sciences; leading preventable developmental disability. This untreatable condition is a huge social and economic CD271+ and CD271- subpopulations would be the most effective in SHH MB treatment. burden to the country. Alcohol is a teratogenic compound and DNA methylation could be a potential University of Manitoba; mechanism through which genes involved in neurogenesis are suppressed during fetal stages, leading to 2Department of cognitive and intellectual deficits. This study is aimed to identify differentially methylated regions in rat Biochemistry & Medical fetal brains exposed to chronic moderate levels of alcohol. Methods: Sprague Dawley female rats (9-10 week old) received either ethanol 3g/kg body weight or Genetics, University of isocaloric dextrose twice a day by gavage. On gestational day 20, fetal brains were harvested and Manitoba; 3Children’s genomic DNA was extracted. DNA library was prepared by fragmentation, bead based enrichment of Hospital Research methylated fragments, adaptor ligation and amplification, emulsion PCR and sequenced on the SOLiD5500 platform. Institute of Manitoba, Results: DNA methylation was significantly higher in alcohol groups, when compared to control group. Winnipeg, MB A number of differentially methylated regions were identified for validation and gene expression analyses. A number of these regions were in close proximity to genes associated to neuronal and brain development. Conclusion: The current finding confirms that prenatal alcohol exposure does increase DNA methylation in brains. Methylation patterns could determine the severity of FASD phenotype in children with prenatal alcohol exposure. Blocking or minimizing methylation changes in brain using nutritional supplements such as DHA could be a potential treatment strategy against FASD. Melanin-Concentrating Hormone Neurons are Activated During Diet-Induced Obesity Interactions of the Syphilis Spirochete, Treponema Pallidum, with the Vascular Victoria Linehan, Lisa Fang, Michiru Hirasawa Endothelium Karen V. Lithgow1, Wei-Chien Kao2, Rebecca Hof1, Helena Petrošová2, Yi-Pin Lin3, Martin J. Boulanger1, John Leong3, John E. Hirasawa Lab., Division of Introduction: Over half of Canadians are either overweight or obese, increasing the risk of many obesity- Burke1, Tara J. Moriarty2, Caroline E. Cameron1 BioMedical Sciences, related diseases and reducing quality of life. During obesity, chronic inflammation within the hypothalamus, the brain region responsible for body weight regulation, is thought to promote weight 1 Introduction: Treponema pallidum (Tp) is the causative agent of syphilis, a human-specific sexually Memorial University, gain. However, it is unknown whether inflammatory signaling directly activates weight-promoting Department of transmitted infection characterized by multi-stage disease and diverse clinical manifestations. Despite St. John’s, NL neurons. Melanin concentrating hormone (MCH)-expressing neurons are thought to be a key factor in Biochemistry and continued sensitivity to penicillin, Tp remains a major global health burden for which no vaccine is obesity as they promote weight gain by increasing appetite and reducing energy expenditure. Therefore, Microbiology, University available. Of utmost concern are the predominance of congenital syphilis and increased risk of HIV we tested the hypothesis that inflammation induced by high-fat diet directly activates MCH neurons to of Victoria, Victoria, BC; transmission during co-infection. Tp undergoes rapid vascular dissemination, accessing distant organ promote weight gain. sites where organisms cross tissue, placental, and blood-brain barriers. Tp0751 is a cell surface Tp adhesin Methods: Male Sprague Dawley rats were fed a palatable high-fat Western Diet (WD) or a control low fat 2Matrix Dynamics Group, that interacts with host factors in the vasculature; Tp0751 immunization provides protection against Tp chow diet. Acute hypothalamic brain slices from these rats were used for whole-cell patch clamp University of Toronto, dissemination in an animal model. This study explores molecular interactions and functional outcomes recordings of MCH neurons. Also, the role of MCH in the physiological response to WD was tested in vivo. Toronto, ON; 3Department of Tp0751 adhesion to the vascular endothelium to characterize the role of this promising vaccine Results: While acute (1 week) WD feeding had little effect on MCH neurons, prolonged feeding (4 - 11 candidate in Tp dissemination. weeks) activated MCH neurons. Specifically, this activation comprised increased excitatory synaptic of Molecular Biology and Methods: Plate-based binding assays quantified recombinant Tp0751 (rTp0751) attachment to human transmission onto MCH neurons, depolarization of resting membrane potential and increased firing Microbiology, Tufts primary endothelial cells (ECs). Tp0751, heterologously expressed in non-infectious Borrelia burgdorferi frequency. The depolarization a strong indicator of the level of MCH neuron activation and therefore we University School of (Bb-Tp0751), was evaluated for gain-of-function attachment to ECs and used in competitive inhibition tested if this was due to inflammation. We found that the inflammatory mediator prostaglandin E2 (PGE2) studies with synthetic peptides from Tp0751 host-binding regions. Live Tp harvested from in vivo growth is induced by WD, which depolarizes MCH neurons by inhibiting the Na+/K+ ATPase . This in vitro finding Medicine, Boston, MA in an animal model was used in EC attachment assays with blocking antiserum. Using immobilized is physiologically significant as the intraperitoneal administration of an MCH antagonist, GW-803430, rTp0751, we applied affinity chromatography and mass spectrometry to isolate and identify interacting decreases food intake and weight gain in WD-fed rats only at the time point that displays MCH neuron proteins from EC membrane extractions. Immunofluorescence allowed for evaluation of EC junctional depolarization. Interestingly, activation of MCH neurons can be reversed when rats are switched from WD architecture following rTp0751 exposure. to a low-fat diet. However, cycling between WD and the low-fat diet shortens the latency for MCH neuron Results: Here we demonstrate that rTp0751 attaches to microvascular and macrovascular ECs. Further, activation on WD, suggesting that the sensitivity to WD increases with repeated exposures, which our heterologous expression model, Bb-Tp0751, adheres to ECs and interaction specificity is confirmed implicates MCH neurons not only in obesity but in the effects of yoyo dieting. through competitive inhibition with Tp0751 peptides whereby one discrete region inhibits EC attachment Conclusion: Our study suggests that diet-induced inflammation in the hypothalamus, specifically PGE2, of Bb-Tp0751. Tp attachment to ECs can be disrupted with Tp0751-specific antiserum. LamR (67 kDa causes activation of MCH neurons and contributes to the onset and maintenance of obesity. Additionally, laminin receptor), a brain EC for other neutrotropic bacterial pathogens, was identified as a candidate EC MCH neurons become more sensitive to high-fat diet with repeated exposures, which may have implications for dieting strategies to lose weight. Adaptive Changes in Alternative Splicing Upon Repeated Membrane Depolarization Ling Liu, Urmi Das, Jiuyong Xie receptor for Tp0751. Disruption of intercellular junctional protein VE-cadherin was observed following EC Department of Physiology Introduction: Epigenetic modifications, genetic alterations as well as post-translational modifications are exposure to Tp0751. Current investigations aim to validate the Tp0751-LamR interaction and characterize & Pathophysiology, Max critical for the establishment of cellular memory. However, little is known about the role of alternative Tp0751-induced alterations in endothelial barrier integrity. splicing – a major contributor for protein diversity. Here we examine transcriptome-wide alternative Conclusion: Tp0751 mediates EC attachment, a fundamental step in Tp dissemination. Understanding Rady College of Medicine, splicing to identify adaptive changes upon repeated stimulation by membrane depolarization. this process will provide opportunities to prevent host attachment and facilitate syphilis vaccine Rady College of Health Methods: For repeated KCl stimulation, we treated cells (GH3, Lan-5, N1E and PC12 cells, respectively) development. Sciences, University of using 50mM KCl for 6h, then washed off KCl and added back fresh medium till the next KCl treatment 18h later. We treated the cells 6 times in total, and extracted RNA 6h after the 1st or 6th KCl treatment. RNA-seq Manitoba, Winnipeg, MB was carried out in the Génome Québec Centre (McGill University). Others: cell culture, RT-PCR, agarose gel electrophoresis, RNA-seq data analysis by MATS, DEXseq or edgeR. Results: In total, 1880 alternative exons showed significant changes between 1st and 6th KCl treatment of GH3 cells. Twenty-four of these exons belong to splicing factor genes. The changes of the exons after the 1st KCl treatment were mostly reversed back to different extents by the 6th KCl treatment. Present RT-PCR results of 3 out of 4 exons (STREX, Upf3b, Rps24) are consistent with RNAseq predictions. Particularly for the STREX exon in GH3 cells, its index level was significantly reduced from 100 in the non-treated group to 62 upon the 1st treatment, and further to 35 upon the 6th treatment. Such an enhanced reduction by repeated stimulation was also seen in PC12 but not LA-N-5 or N1E cells. In the latter two, the STREX reduction upon 1st treatment was instead reversed after repeated stimulation. Conclusion: There are trans-generational, transcriptome-wide, adaptive changes of alternative splicing upon repeated stimulation by depolarization. The STREX adaptive changes are cell-specific. Further detailed examination of these changes would allow us to elucidate the molecular mechanisms for adaptive splicing and its role in cell physiology or pathophysiology. Elucidating the Function of Neomorphic IDH Mutations in Acute Myeloid Leukemia A. Lorzadeh1, C. Hommond2,4, J. Parker3, D. Knapp2,4, M. Bilenky3, M. Moksa1, G. Paliouras3, D. Hogge4,5, M. A. Marra2,3, C. Eaves2,4,5, A. Karsan3,5, M. Hirst1,3

1Department of Introduction: Neomorphic isocitrate dehydrogenase (IDH) mutations are recurrent genetic lesions Conclusions: Our results support a model where IDH neomorphic mutations lead to localized Microbiology and observed in Acute Myeloid Leukemia (AML) that drive the accumulation of the metabolite D-2- reprogramming of the epigenetic landscape of primary AML through the inhibition 2-OGDD histone hydroxyglutarate (D2-HG). D2-HG is an inhibitor of 2- oxoglutarate-dependent dioxygenases (2-OGDD), demethylases. Immunology, University of a family of enzymes that include a class of epigenetic modifiers that are responsible for histone and DNA British Columbia; demethylation. In mouse models neomorphic IDH mutations have been shown to reversibly transform 2Department of Medical progenitor hematopoietic populations harboring FMS-like tyrosine kinase 3 internal tandem duplications (FLT3ITD). To investigate the role D2-HG might play in remodelling the epigenome of primary FLT3ITD AML Genetics, University of blast cells we performed native ChIP-seq on cord blood derived CD34+ cells and primary AML cells British Columbia; harboring FLT3ITD in the presence and absence of neomorphic IDH mutations. 3Canada’s Michael Smith Methods: We applied an optimized native ChIP-seq protocol to profile five histone modifications (histone3 lysine 4 tri-methylation (H3K4me3), H3K4me1, 3K27me3, H3K36me3, and H3K9me3). ChIP-seq Genome Science Centre, was conducted on aliquots of 10,000 CD34+CD38- bone marrow, CD34+ cord blood, cord blood purified 4 BCCA; Terry Fox CMP, GMP, and MEP cells, and 10,000 AML cells harboring the FLT3ITD allele and IDH2R140, IDH2R172, Laboratory, BC Cancer IDH1R132, or IDH2Wt alleles. The resulting libraries prepared from these cells were sequenced on an Association; 5Department Illumina HiSeq platform in paired-end mode and integrated with existing matched RNA-seq datasets. Results: Comparisons of H3K27me3 (a repressive histone modification) landscape across normal blood of Pathology and cells and AML-IDH2Wt-FLT3ITD and AML-IDHMu-FLT3Wt revealed a unique H3K27me3 profile in AML- Laboratory Medicine, IDH2Mu - FLT3WT cells compared to that of healthy blood and AML-IDH2WT-FLT3ITD cells. Matched University of British RNA-seq datasets confirmed transcriptional repression of genes with H3K27me3 gains in their promoter. Our results are consistent with a model where inhibition of 2-OGDD histone demethylases, such as the Columbia, Vancouver, BC UTX (H3K27me3 demethylase) by 2D- HG lead localized gains of H3K27me3 and gene repression. In addition, comparison of regulatory landscape identified by H3K4me1 across IDHMu, IDHWt, and normal blood cells revealed an IDHMu unique regulatory regions enriched in PU.1 binding site that promotes leukemia genesis. A 4-Season Longitudinal Study Examining the Association Between Seasonality and Revisiting Colistin Combination Therapy in the Era of Mobile Colistin Resistance Sedentary Behaviour in 9-15 Year Old Canadian Children Craig R. MacNair, Jonathan M. Stokes, Lindsey A. Carfrae, Eric D. Brown Larisa Lotoski1, Nazeem Muhajarine1, 2, Daniel Fuller3, Tarun Katapally4, Kevin Stanley1, Daniel Rainham5 Michael G. DeGroote Introduction: The emergence of plasmid-mediate colistin resistance (mcr-1) has magnified the antibiotic 1University of Introduction: The average Canadian youth spends 8.6 waking hours of their day in a sedentary state, and Institute for Infectious resistance threat by further limiting available treatment options. Colistin’s documented ability to synergize with antibiotics provides an attractive treatment opportunity as potentiation is conserved Saskatchewan; consistently do not meet recommended leisure time sedentary limits of 2hrs per day. Greater than 2 Disease Research, hours of sedentary behaviour (SED) per day is associated with an increased risk of overweight, obesity, within chromosomally mediated resistance, yet uncharacterized for mobile resistance mechanisms. 2 Saskatchewan and cardiovascular disease. This study seeks to establish how seasonal changes affect weekday school McMaster University, Methods and Results: Colistin resistant strains of E. coli, K. pneumoniae, S. Typhimurium, E. aerogenes and E. Population Health and hour/leisure time, weekend and total SED in children and how the relationship between season and SED Hamilton, ON cloacae generated through the expression of a pGDP2:mcr-1 plasmid were screened for antibiotic potentiation in the presence of clinically relevant levels of colistin (2μg/mL). Gram-positive antibiotics Evaluation Research Unit; effects are moderated by children’s and parent’s view of SED and urban design. Method: Families with children aged 9-15 were recruited from the city of Saskatoon, Saskatchewan within the rifamycin and macrolide classes account for the overwhelming majority of synergistic partners, 3Memorial University of (n=800). Location-specific SED was measured in children over three time frames (Sept-Dec; Jan-April; with patterns of potentiation conserved within all pGDP2:mcr-1 expressing Enterobacteriaceae spp. as Newfoundland, St. Johhn’s, May-July) using GPS equipped accelerometers. Questionnaires developed specifically for children and well as nine mcr-1 positive environmental E.coli isolates. Importantly, susceptibility to several gram- positive antibiotics in the presence of colistin demonstrates growth inhibition below the corresponding NF; 4University of Regina, parents collected data on perceptions of seasonal changes, SED and demographic information. Using the random intercept model a multilevel modeling approach will be taken to understand seasonal changes clinical breakpoint of these gram-positive drugs. In vivo evaluation Clarithromycin (200mg/kg) and Regina, SK; 5Dalhousie in mean daily, weekday and weekend SED of children, the location of activities. Colistin (7.5mg/kg) significantly reduced (p<0.001) bacterial load of mcr-1 expressing K. pneumoniae University, Halifax, NS Results: After the final (third) round of data collection 631 participants remained (343 females (54.3%), when used in combination. 288 males (45.6%)). Almost one third of children were overweight or obese (31% of girls, 30% boys), and Conclusion: Our data suggests the use of colistin in combination with gram-positive antibiotics may 12% of boys and 13% of and girls were considered obese. Children reported the greatest amount of mean represent a viable treatment option for mcr-1 expressing gram-negative infections. daily sedentary time in winter months (January to April, 483 min) in comparison to fall and spring seasons (September to December, 463 min; April to June, 435 min). The highest amount of inactive trips to and from school were reported in colder months, from September to December (31%) and January to April (32%) (vs 13% from April to June). Children living in grid pattern neighbourhoods accumulated the least amount of sedentary time, but living within the same neighbourhood as one’s school did not improve sedentary outcomes. Results from multilevel multivariable models will be presented. Conclusions: Forming a clearer understanding of SED in children is necessary for future successful intervention implementation. Disruptive positive interventions to physical activity in both boys and girls may provide the greatest benefit when done so in our youngest populations. Differential Regulation of Host Defence Peptide LL-37-Mediated Immune Response by Changes in Lung Tissue Proteome in an Allergen Challenge Mouse Model of Asthma CDC42/RAC1 GTPASES Thomas H Mahood1, 2, 3, Chris D Pascoe1, 2, 3, Aruni Jha1, 2, 3, Peyman Ezzati4, Sujata Basu1, 2, Victor Spicer4, Hemshekhar Mahadevappa1, Grace Choi Ka-Yee1, 2, Mookherjee Neeloffer1, 2 Neeloffer Mookherjee2, 3, 4, 5, Andrew J Halayko1, 2, 3

1Manitoba Centre Introduction: Rho GTPases are GTP-binding proteins that promote fundamental immune functions e.g. 1Department of Introduction: A leading animal model for asthma uses repeated intranasal (i.n.) challenge with house Proteomics & Systems chemokine activity for recruitment of leukocytes. However, recent studies have also shown that Rho Physiology & dust mite (HDM) in adult mice, which promotes airway remodelling and hyperresponsiveness. GTPases Rac1 / Cdc42 regulates TNF-α trafficking. The human host defence peptide LL-37 plays a role in Comprehensive analysis of the changes in the lung proteome with HDM challenge is sparse. The objective Biology, Department of Pathophysiology, both immune activation (mediates induction of chemokine production, and promotes differentiation of this study was to use a proteomics approach to characterize and quantify global protein changes in Internal Medicine, and polarization of T-cells), and anti-inflammatory responses such as suppressing the production of University of Manitoba; the lung following HDM challenge. University of Manitoba; endotoxin-induced inflammatory cytokines in myeloid cells. Therefore, the aim of this study was to 2Biology of Breathing Methods: Female, BALB/c mice (6-8 weeks, n=4) were subjected to HDM i.n. challenge (25μg/mouse) 5 interrogate the involvement of Rho GTPases in LL-37-mediated immunomodulation in mononuclear cells. times per week for two weeks. Lung function and airway responsiveness to methacholine was measured 2Department of Group, Children’s Hospital Methods: Human macrophage like THP-1 cells and human mononuclear cells were used in the study. The using a small animal ventilator. Immune cell counts and differentials in lung lavage were performed. Lung Immunology, University of LL-37-induced activation of Cdc42/Rac1 was measured by GLISA and production of chemokines/ Research Institute of tissue from age-matched allergen-naïve and HDM challenged mice were homogenized prior to Manitoba, Winnipeg, MB cytokines were determined by ELISA. Phosporylation of JNK was determined by immunoblot and gene Manitoba, Winnipeg, MB; processing by filter centrifugation and trypsinization, before analysis by HPLC-MS/MS. Proteins were expression of chemokines/cytokines was measured by qRT-PCR. Human neutrophil and THP-1 monocytic identified and quantified using X!Tandem. Statistical (MEV) and pathway analysis (IPA) was conducted. 3Canadian Respiratory cell migration was measured through Transwell cell migration assay. Results: HDM challenge induced an increase in airway resistance (p<0.001), tissue dampening (p<0.0001) Results: We demonstrated that LL-37-mediated the activation of Cdc42 and Rac1 Rho GTPase, and Research Network; and tissue elastance (p<0.0001) in response to 50mg/mL methacholine (two-way ANOVA, n=4). Compared phosphorylation of downstream kinase p21-activating kinase (PAK), in macrophages. Consistent with 4Manitoba Centre for to allergen-naïve mice, we observed a 148% increase in BALF total inflammatory cell number (p<0.05), this, we showed that LL-37-induced production of chemokines Gro-α and IL-8 was inhibited in the Proteomics and Systems consisting of ~36% eosinophils and ~16% neutrophils. X!Tandem analysis identified 1541 non-redundant presence of a pharmacological inhibitor of Cdc42/Rac1 activation (ML141). Furthermore, LL-37-induced proteins per sample. We identified 158 proteins that were significantly regulated by allergen challenge, phosphorylation of JNK MAPK was also inhibited in the presence of ML141. However, LL-37-induced Biology, Department of with 128 up- and 30 down-regulated compared to lungs from allergen-naïve mice (MEV; welch’s t-test, production of anti-inflammatory cytokine IL-1RA was not inhibited in the presence of ML141. Internal Medicine, p<0.05). Consistent with an inflammatory signature, top regulated proteins included eosinophil Conclusion: This study suggests that LL-37-mediated pro-inflammatory responses such as induction of University of Manitoba; peroxidase, eosinophil cationic protein 2, and cathepins B, D and S. Top up-regulated pathways included chemokines is promoted by Rho GTPase Rac1/Cdc42 via JNK phosphorylation. Whereas, anti-inflammatory protein translation (p=9.69x10-11) and antigen presentation (p=5.84x10-8). Among down regulated 5Department of activity of LL-37 e.g. production of anti-inflammatory cytokine IL-1RA is independent of Rac1/Cdc42 pathways we detected reduced apolipoprotein A1 (APOA1) abundance indicating that oxidized lipid signaling. Immunology, University of turnover may be compromised. Significance: As LL-37 is enhanced in chronic inflammatory diseases such as rheumatoid arthritis, results Manitoba, Winnipeg, MB Conclusion: Using system’s biology we have defined changes in the lung tissue proteome upon HDM from this study suggest that Cdc42/Rac1 may be a useful target to selectively control LL-37-associated challenge that alters lung function and produces an eosinophil and neutrophil rich inflammation that recruitment to the site of inflammation, without compromising its anti-inflammatory activity. Human Amniotic Stromal Cells Encapsulated in a Chitosan/Hyaluronic Acid Based Platform Proliferate and Increase Cardiac Function in a Rat Myocardial Infarction Model G. Makhoul1, B. Yu2, J. Ghulam3, P. K. Jaiswal1, M. Cerrut3, A. Schwertani2, R. Cecere1 mimics severe asthma. We describe an inflammatory profile replete with eosinophil and neutrophil 1Department of Introduction: Cardiac myocytes are understood to possess a limited regenerative capacity. Thus, a associated proteins, increased antigen presentation, and altered lipid metabolism. Our platform provides Experimental Surgery; myocardial insult can lead to an irreversible injury and an eventual cardiac failure, one of the leading an approach to decipher pathobiological mechanisms and response to new therapies in chronic lung causes of mortality worldwide. Stem cells stand as one of the leading experimental therapies. Nonetheless, 2 disease. Department of an ideal stem cell that could replenish the injured site is lacking. Furthermore, stem cell based cardiac Experimental Medicine; repair suffers from low engraftment rates and modest improvements in ventricular function. Here, we 3Chemical Engineering, investigated the cardia-protective potential of a novel composite inserting human amniotic stromal cells (hASCs) in a chitosan/hyaluronic acid (C/HA) based platform to increase cellular retention rates and McGill University, combat heart degeneration. Montreal, QC Methods: A re-designed C/HA scaffold crosslinked with β-glycerophosphate and genipin and mixed with hASCs culture medium was synthetized. Thermo-gelling properties reflecting its structural characteristics were determined. To examine its cellular preservation, Alamar blue assay was performed on hASCs encapsulated in the C/HA scaffold. The cardiac impact of hASCs + CIHA composite was then assessed in vivo. Following an induced myocardial infarction, 40 female Lewis rats divided into 4 groups were injected with either 3x105 hASCs, C/HA, 3xl05 hASCs + CIHA, or control (infarction only). Animals were followed with transthoracic echocardiography for a period of 5 weeks then euthanized on week 6 with further histological analysis conducted. Results: Mechanical characterization of the C/HA platform indicated in a temperature sweep assay a swift elastic conversion at 40°C. Moreover, at 37°C, the sol-gel transition time of C/HA occurred fairly rapidly. Alamar blue metabolic activity presented a healthy and multiplying hASCs 15 days after their mixture with C/HA. In vivo, on week 5, the groups injected with C/HA and hASCs + CIHA had significantly higher ejection fraction, fractional shortening, and left ventricular end systolic diameter compared to the hASCs alone and control groups. Six weeks following their intra-myocardial implantation, the hASCs in the hASCs + CIHA group were abundantly detected. Furthermore, immunofluorescence labeling of these encapsulated cells showed the co-expression of cardiac proteins such as connexin 43 and myosin heavy Protecting the Hypoxic Neonate: Misoprostol-Induced Repression of Bnip3 Through NFκ-B Matthew Martens, Jared Field, Yan Hai, Wajihah Mughal, Simone da Silva Rosa, Tammy Ivanco, William Diehl-Jones, Joseph Gordon chain. Interestingly, labelling additionally revealed the co-expression of the proliferative marker Ki67 in Children’s Hospital Introduction: Systemic hypoxia affects more than 50% of preterm infants and has been implicated in the injected hASCs. Cross-sectional Masson Trichome’s staining of the myocardium showed a thinner and Research Institute of conditions associated with both intestinal and brain injury. While the mechanism for hypoxic injury more fibrotic left ventricular wall in the control rats compared to the hASCs + C/HA injected hearts. remains unclear, it appears that the genetically conserved, pro-death Bnip3 pathway may play a central Manitoba, John Buhler Moreover, the hASCs + C/HA composite triggered a significant and massive neovascularization of role. We hypothesize that Misoprostol, an FDA approved prostaglandin receptor agonist, may activate CD31+cells at the infarction site. Research Centre, Health NFk-B, which represses Bnip3, in turn protecting infants from hypoxic injury. Conclusion: The limited and ineffective cell-based cardiac regeneration therapies incite the pursuing of Sciences Centre, Methods: Both environmental hypoxia (10% oxygen) and drug treatments were applied to a neonatal rat novel and advanced cellular delivery platforms. Despite low injected quantities at baseline, our findings model (n=5) to assess the effect of Misoprostol and hypoxia on hippocampal, and intestinal Bnip3 Winnipeg, MB indicate that the hASCs encapsulated in C/HA were abundantly retained at the infarction site and expression. Gene and protein expression were determined by RT-PCR and protein immunoblot, and increased cardiac functional parameters. Moreover, this platform provided an active milieu for the hASCs compared to control treatments (normoxia and/or drug vehicle). The secondary outcome of this study to proliferate and induce new vessel formation via secretion of paracrine factors. Thus, this novel used a human cell line (HCT-116) (n>45 cells) to focus on the underlying mechanism, assessed through composite of hASCs encapsulated in a C/HA biological platform is a conceivable candidate that could fluorescent imaging with a plasmid-based PKA biosensor, as well as expression of wildtype and non- restore cardiac function and reduce remodeling. phosphorylatable mutants of NFκ-B. Results: In the animal study, hypoxia induced a several-fold increase (p<0.05) in Bnip3 protein expression in both intestine and hippocampus, which was mirrored in mRNA expression. When Misoprostol was added in hypoxic conditions, Bnip3 protein was repressed by 87.5% (p<0.05). In parallel in vitro studies, overexpression of wild-type NFκ-B causes considerable reduction in Bnip3 protein expression, however when NFκ-B phosphorylation was inhibited with a neutral alanine mutation, Bnip3 expression was unchanged. Finally, we show that Misoprostol induces a 3-fold increase (p<0.01) in intracellular PKA activation and a 1.8-fold increase (p<0.01) in nuclear localization of NFκ-B. Conclusion: Taken together, both animal and cell data suggests that Misoprostol activates PKA/cAMP, resulting in nuclear accumulation of NFκ-B, repressing Bnip3 protein expression, which may serve to protect the developing neonate. Development of a Reporter System Allowing Real-Time Monitoring of The Cortisol Response to Stress: Gender Differences in Depressed Adolescents Neurodegeneration in Prion Disease Raegan Mazurka1, Katherine Wynne-Edwards2, Kate L. Harkness1 Matthew J. Martin 1, Stephanie A. Booth 1, 2 1Queen’s University, Introduction: Depression is twice as common among women compared to men, and this gender 1University of Manitoba; Introduction: Prion diseases are a relatively unknown neurodegenerative disease. Current methods of Kingston, ON; difference first emerges in mid- to late-adolescence. In addition to gender, another important risk factor for depression is exposure to life stress. One possible mechanism to explain the high prevalence of 2National Microbiology studying these diseases require sacrificing cells at specific time-points to allow for further analysis. In vivo 2University of Calgary, imaging is a promising method which allows the real-time monitoring of gene expression of cells in vitro depression among women is a gender difference in reactivity to stress. Of particular interest is Laboratory, Public Health or in vivo. Previous research from Stephanie Booth’s lab has identified genes that are significantly Calgary, AB neurobiological stress reactivity, as stress response systems such as the hypothalamic-pituitary-adrenal Agency of Canada, upregulated in neurons during the early stages of prion disease. Reporter constructs will be produced axis have been shown to be dysregulated among depressed adolescents. The objective of this study was to assess gender differences in neuroendocrinological stress reactivity by measuring the cortisol Winnipeg, MB using the regulatory regions of these genes which control a reporter gene. Reporter constructs will be delivered into primary neurons in culture, and to mice in vivo, to allow real-time monitoring of prion response to a laboratory stress paradigm. disease progression. Additionally, previous research by White et al. has shown that delivery of a short Methods: We compared cortisol trajectories in response to the Trier Social Stress Test as a function of hairpin RNA against the prion protein, shPrP, is able to silence prion protein expression. This silencing has gender and diagnostic status in a community sample of depressed and non-depressed adolescents (age been shown to protect mice from infection, and promote the recovery of prion-infected mice when shPrP 12 to 18). is delivered in the early stages of the disease. Using this reporter system, the mechanisms behind the Results: In contrast to the typical cortisol response pattern to stress shown by non-depressed participants protection of shPrP will be studied. and depressed males (i.e., a rise to peak cortisol followed by a decrease back to baseline), depressed Methods: HEK293T cells will be triple transfected to produce Rh10 serotype adeno-associated viruses females showed blunted cortisol reactivity. These results held controlling for relevant clinical and containing the reporter constructs. These virus vectors will then be used deliver the constructs to primary demographic variables and current stress exposure. neuron cultures, or live mice. Subjects will be exposed to brain homogenate from a prion infected mouse, Conclusion: This study provides the first evidence of a gender difference in stress reactivity to a or healthy uninfected mouse as a control. Progression of neurodegeneration will be observed using the psychosocial stressor in a clinically-relevant and developmentally important sample of depressed In vivo Imaging System from Perkin Elmer, and confirmed with Western blots and immunohistochemistry. adolescents. Possible mechanisms to explain this gender difference will be discussed, as well as etiological Intended Outcome: A reporter system allowing real-time monitoring of the neurodegeneration of and treatment implications. neurons in prion disease will be produced. This system will allow further analysis of affected neurons, as well as a rapid assay of potential therapeutics against prion disease. Further study of the shPrP protective mechanisms may allow discovery of pathways that can be targeted in future treatments. Impact of ICU Structure and Processes of Care on Outcomes After Traumatic Brain Injury: Reproductive Justice As Defined by Urban Indigenous Women: A Collaborative, Action A Multicenter Cohort Study Based Project Victoria A. McCredie1, 2, 3, Aziz S. Alali1, Damon C. Scales1, 3, 4, Gordon D. Rubenfeld1, 4, Brian H. Cuthbertson1, 4, 5, Avery B. Holly A. McKenzie Nathens3, 6 University of British Introduction: Refusing colonial narratives that continue to frame Indigenous women as inherently 1Interdepartmental Division Introduction: It is uncertain whether dedicated neurocritical care units are associated with improved Columbia, Vancouver, BC irresponsible and vulnerable, this research centers Indigenous women’s understandings of reproductive of Critical Care Medicine, outcomes for critically ill neurologically injured patients in the era of collaborative protocol-driven care. justice and health and wellness. Women of colour and Indigenous women have long been articulating and doing reproductive justice work. Reproductive justice is often defined as the rights a) to determine Department of Medicine, We examined the association between dedicated neurocritical care units and mortality, and the effects of standardized management protocols for severe traumatic brain injury (TBI). whether or not to have children, b) to determine birthing conditions, and c) to raise children women do University of Toronto; Methods: We surveyed trauma medical directors from centers participating in the American College of have in safe and healthy environments, free from violence. There is a need for further conversations 2Division of Critical Care Surgeons Trauma Quality Improvement Program (TQIP) to obtain information about ICU structure and around Indigenous women’s reproductive justice. This research seeks to contribute to these conversations. Medicine, Department of processes of care. Survey data were then linked to the TQIP registry, and random-intercept hierarchical Methods: This three-phase, collaborative, action-based project drew on post-critical ethnographic and Medicine, University Health multivariable modeling was used to evaluate the association between dedicated neurocritical care units, Indigenous storytelling approaches. This poster is grounded in the qualitative findings from the semi- Network; 3Institute of Health the presence of standardized management protocols and mortality. structured interviews, research circles, and meetings with a diverse group of 32 collaborators (Indigenous Policy, Management and Results: Data was analyzed from 9,773 adult patients with isolated severe traumatic brain injury admitted women and allies) across the Métis homelands and Treaty 1 (Winnipeg), Treaty 4 (Regina) and Treaty 6 (Saskatoon) territories. Evaluation, University of to 134 TQIP centers between 2011 to 2013. Only 50 ICUs (37%) were dedicated neurocritical care units, whereas 84 (63%) were general ICUs. Rates of using standardized TBI management protocols and order Findings: Within this project, Indigenous women and allies emphasized the relationships among cultural Toronto; 4Department of sets were similar comparing dedicated neurocritical care units and general ICUs. Among severe TBI practices and teachings, community relationships, rights and access to Indigenous lands, and Indigenous Critical Care Medicine, patients admitted to trauma centres, care in a dedicated neurocritical care unit did not improve risk- women’s bodily autonomy over a) sexual experiences and gender and sexual expression, b) reproduction, Sunnybrook Health Sciences adjusted in-hospital survival (odds ratio [OR] 0.97; (95% confidence interval [CI] 0.80-1.19; P= 0.79). including determining whether to become, continue, or end a pregnancy, c) birthing conditions, and d) Centre; 5Department of However, the presence of a standardized TBI management protocol was associated with lower risk- living and housing conditions. Collaborators discussed how supportive individuals, families, organizations, Anesthesia, University of adjusted in-hospital mortality (OR 0.77; 95% CI 0.63-0.93; P=0.009). and communities, as well as Indigenous teachings and practices foster Indigenous women’s reproductive Toronto; 6Department of Conclusion: In the largest study to date of specialized ICU care for TBI, standardized TBI management justice and self-determined health and wellness. Conclusion: Collaborators understandings of reproductive justice can inform policies, practices, and Surgery, Sunnybrook Health protocols for severe traumatic brain injured patients are low-cost process-targeted intervention strategies that may improve clinical outcomes, compared to dedicated neurocritical care models. Until further conversations across diverse contexts. Further, we recommend that healthcare practitioners, policy Sciences Centre and the rigorous observational studies or randomized trials are conducted, we recommend utilization of makers, and researchers engage with local Indigenous women in long-term, meaningful relationships in University of Toronto, standardized TBI management protocols in centers aiming to improve their outcomes. order to center local understandings of reproductive justice within programming and research. Toronto, ON Antenatal Midwifery Care and Reduced Prevalence of Small-for-Gestational-Age Birth Using a ‘Two-Eyed Seeing’ Framework to Support a New Dialogue of Water Wellness in and Preterm Birth for Women with Low Socioeconomic Position Indigenous Communities Daphne McRae1, Nazeem Muhajarine1, Saraswathi Vedam2, Maureen Mayhew3, Debbie Mpofu1, 4, Ulrich Teucher5, Andrea Mellor1, Denise Cloutier1, 2 Patricia Janssen3 1University of Victoria; Introduction: The current Federal government has a tremendous opportunity to support Indigenous 1Department of Background: There are few recent studies comparing perinatal outcomes of antenatal midwifery-led care 2Institute on Aging and self-determination and reconciliation by responsibly engaging with communities who will benefit from the $673 million dollars earmarked for water and wastewater improvements in their communities. Community Health and to physician-led care for women with low socioeconomic position (SEP). Available studies are limited by Lifelong Health, methodological weaknesses, and report inconsistent results. However, approaches to these ‘improvements’ remain fixed within Western worldviews that separate Epidemiology, University Purpose: To determine among women with low SEP, if antenatal midwifery vs. obstetrician or general Victoria BC environment and wellness virtually ignoring the views of Indigenous Elders who note ‘there is no greater of Saskatchewan, practitioner care significantly reduced the odds of small-for-gestational-age birth and/or preterm birth. medicine than water’, The knowledge that guides Indigenous water governance and water resource management is situated within different worldviews and these differences can be viewed as a gap. We Saskatoon, SK; 2UBC Methods: Eligibility for this retrospective cohort study was based on low to moderate risk pregnancy, to B.C. residents, who had singleton births (2005- 2012), no more than two provider-types involved in care, propose viewing this gap as a ‘liminal space’, one that can be a place of transformation from dichotomy Midwifery, University of and received medical insurance premium assistance (n=57,872). Maternity data were supplied by (the historical relationship) to unity (harmony and wholeness). In this way, this research explores how this British Columbia; 3School Perinatal Services B.C. and merged with billing, neighbourhood, and community data from the B.C. liminal space can support a holistic framework within which health and wellness can be seen as equally central to the sustainability of water as a resource. of Public and Population Ministry of Health, Population Data B.C., and B.C. Statistics. Logistic regression models were used to control for confounding. Methods: To enter this liminal space, transitioning from ‘what was’ to ‘what can be’, a Two-Eyed Seeing Health, University of Results: Odds of small-for-gestational-age birth were reduced by 40% for midwives’ vs. obstetricians’ framework will be used. Two-Eyed Seeing is a partnered approach that aims to provide balanced British Columbia, patients (adjusted OR 0.60, 95% CI: 0.51-0.70), and by 27% compared to GPs’ patients (AOR 0.73, 95% CI: contributions from Indigenous and non-Indigenous worldviews. In this context, Two-Eyed Seeing will provide a safe space to ‘decolonize’ water by exploring embedded assumptions in the non-Indigenous Vancouver, BC; 4 0.63-0.84). Likelihood of preterm birth was reduced by 47% for midwives’ vs. obstetricians’ patients (AOR relationship with the environment and support bringing this knowledge in to a more holistic framework. Saskatoon Health Region, 0.53, 95% CI: 0.45-0.62), and by 26% compared to GPs’ patients (AOR 0.74, 95% CI: 0.64-0.86). In sub-group analyses, substance using midwifery patients were less likely to have preterm births (AOR 0.25, 95% CI: Further, use of Two-Eyed Seeing will reposition the research in order to explore how solutions to water 5 Saskatchewan; 0.12-0.52), vs. obstetrician patients, as were substance using midwifery patients with mental health quality and access can be community based, benefit and reflect the needs of each community, and honor Department of conditions (AOR 0.21, 95% CI: 0.07-0.65). tribal knowledge. Conclusion: By removing the domination of one way of seeing over another, questions of importance to Psychology, University of Conclusions: For women with low SEP, who have low to moderate medical and/or obstetric risk, antenatal midwifery care is associated with a reduction in the odds of small-for-gestational-age birth and preterm Indigenous and non-Indigenous communities about water and wellness can be respectfully integrated Saskatchewan, birth compared to obstetrician or GP care. This association is stronger among women with low SEP and and co-constructed. Ideally, this re-creation of knowledge will support a new dialogue where place, Saskatoon, SK other socially complex conditions. culture, and wellness are integral to Canada’s water narrative. Inferring Axon Diameter Sizes Using Monte Carlo Simulations of Magnetic Resonance Programmed Death Ligand 1 (PD-L1) Immune Checkpoint-Mediated Stimulation of Oscillating Gradient Spin Echo Sequences Malignant Phenotypes in Tumour Cells Morgan Mercredi1, Sheryl Herrera1, Christopher Bidinosti1, 2, Melanie Martin1, 2 Lori Minassian1, Shannyn MacDonald-Goodfellow1, Peter Truesdell2, Abdi Ghaffari1, 2, Andrew Craig1, 2, Madhuri Koti1, 2, 3, Robert Siemens2, 4, Charles H. Graham1, 4 1Physics & Astronomy, Introduction: Diffusion-weighted magnetic resonance imaging (MRI) can be used to determine axon University of Manitoba; diameter distributions in brain tissue. Current MRI axon diameter measurements rely on the pulsed 1Department of Introduction: The interaction between the Programmed Death Ligand 1 (PD-L1) immune checkpoint on gradient spin echo sequence which cannot provide short enough diffusion times to measure small axon 2Physics, University of Biomedical and Molecular the tumour cell surface with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes (CTLs) diameters. Oscillating gradient spin echo sequences (OGSE) probe the smallest diffusion times to infer leads to CTL inactivation, thereby promoting tumour cell escape from adaptive immunity. We previously Winnipeg, Winnipeg, MB micron-sized axon diameters. Here we analyze data generated from Monte Carlo simulations of OGSE Sciences, Queen’s demonstrated that signalling by PD-L1/PD-1 is bidirectional and leads to activation of oncogenic sequences using AxCaliber and ActiveAx, two parametric models for determining mean axon diameters University; 2Cancer pathways as well as drug resistance in tumour cells. Based on these findings, we hypothesize that PD-1/ and axon diameter distributions. Research Institute, PD-L1 signalling in tumour cells is a survival mechanism leading to the stimulation of malignant Methods: Monte Carlo computer simulations were conducted using a gamma distribution of non- phenotypes, such as invasiveness and metastasis, in tumour cells. overlapping parallel cylinders surrounded by extra-axonal water with lattice periodicity. This geometry Queen’s University; Methods: Human breast (MDA-MB-231) and mouse mammary (4T1) cancer cells were engineered to aims to model the axon environment in healthy white matter regions. A cosine gradient spin echo 3Department of stably express PD-L1. To test for activation of oncogenic pathways, tumour cell PD-L1 signalling was sequence was used to generate 100 signals with different OGSE frequencies (50 to 1000 Hz) and gradient Obstetrics and induced by exposing MDA-MB-231 cells to recombinant PD-1 (rPD-1) for 10, 30 or 60 minutes. strengths (0 to 909 mT/m). Data were fitted to AxCaliber/ActiveAx models using least-squares Phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian Gynaecology, Queen’s minimization. The simulations were programmed in CUDA C/C++ and run on a HP Z240 workstation target of rapamycin (mTOR) was determined by western blotting. The effect of PD-1/PD-L1 signalling on containing a Tesla C2075 (Fermi 2.0) graphics card for dedicated CUDA computation and a Quadro 600 University; 4Department tumour cell invasion was determined using a Matrigel® transwell invasion assay. To test whether PD-1/ (Fermi 2.1) graphics card handling the display. of Urology, Kingston PD-L1 interaction increases metastasis, alymphoid Rag2-/-γc-/- mice bearing 4T1 tumours were treated Results: The fitted diameters and distributions agree fairly well with the simulated values indicating our with rPD-1-Fc or control IgG-Fc. The effect of PD-1/PD-L1 signalling on tumour cell gene expression was General Hospital, method can be used to infer axon diameters from 1 μm to 5 μm using gradient strengths available on the assessed by NanoString® analysis using RNA extracted from MDA-MB-231 cells exposed to rPD-1 for 3, 6, Bruker BG6 gradient set. Either a careful selection of frequency ranges needs to be selected for the Kingston, ON 9, or 12 hours. methods to model extra-axonal water correctly or the current models need to be improved. Results: Exposure of human breast cancer cells to rPD-1 increased ERK and AKT phosphorylation in a Conclusion: This work is the first step toward combining oscillating gradient measurements with axon time-dependent manner. In addition, rPD-1 increased in vitro MDA-MB- 231 cell invasiveness. The mouse diameter distribution models to infer distributions of small axon diameters in tissues. Further studies will and NanoString studies are still underway. Based on our results showing an increase in oncogenic be directed towards determining of the range of feasible diameter sizes necessary for the model to pathway activation and invasiveness, we expect that mice exposed to rPD-1 will develop more accurately work. Distributions of non-parallel axons and more diffusion gradient directions will be metastastes than those exposed to IgG. In addition, we expect to see differences in oncogenic pathway needed to make a more complete model. gene expression in cells treated with rPD-1 compared to controls. The Controlling Role of Autophagy in Regulating Temozolomide-Induced Apoptosis in Rhabdomyosarcoma Cells Adel Rezaei Moghadam, Javad Alizadeh, Simone Cristina da Silva Rosa, Jared Field, Saeid Ghavami, Joseph W. Gordon

Conclusions: Our results show that PD-1/PD-L1 bidirectional signalling can alter the phenotype of Department of Human Introduction: Rhabdomyosarcoma (RMS) is a muscle-derived tumor and is the most common pediatric tumour cells to promote malignant progression via activation of oncogenic pathways as well as by Anatomy and Cell Science, soft tissue sarcoma representing 5% of all childhood cancers. RMS is a major clinical problem in pediatric increasing tumour cell invasiveness. oncology. Treatment of RMS with the oral alkylating agent temozolomide (TMZ), alone or in combination Rady Faculty of Health with other drugs, has recently received considerable interest. However, the mechanism of action of TMZ Sciences, Max Rady remains unclear. The aim of this investigation was to determine if autophagy modulates TMZ-induced cell College of Medicine, death in RMS cell lines. Methods: MTT assay and Nicoletti flow cytometry analysis were used to measure cell death and apoptosis University of Manitoba, in SJCRH30 (RH30) human RMS cells and the non-transformed mouse myoblast cell line C2C12, following Winnipeg, MB TMZ treatment (100 μM). Results: We monitored autophagy using transmission electron microcopy (autophagosome and autophagolysosome formation) and immunoblotting (LC3 lipidation, Atg15-12 conjugation, p62 degradation). The effect of bafilomycin A1 on TMZ-induced cytotoxicity was also evaluated. We showed that TMZ decreased the viability of RMS cells in a dose/time-dependent manner and induced accumulation of sub-G1 cell population, representing apoptotic cells. Interestingly, TMZ induced apoptosis by 16-fold in the RH30 cells, but only increased apoptosis by 63% in C2C12 cells. In RH30 cells, TMZ decreased the expression of antiapoptotic proteins Bcl-XL and Mcl-1, and increased the expression of the death gene Nix. Moreover, we showed that TMZ altered biochemical markers of autophagy, such as LC3 lipidation, Atg5-12 conjugation, and p62 degradation, and induced morphological evidence autophagy, including accumulation of autophagosomes and autophagolysosome, determined by transmission electron. Finally, treatment of RMS cells with the autophagy flux inhibitor (Bafilomycin A1, 4 and 6 nM) had no significant effect TMZ-induced cell death in either cell line. Conclusion: Our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both RH30 and C2C12 cells; however, cell death induction by TMZ does not appear to be dependent on Bafilomycin A1 inhibited processes, such as autophagosome-lysosome fusion or autolysosome acidification. Neuregulin-1 Positively Regulates Neuroinflammation in Demyelinating Conditions Cyclooxygenase (COX) Derived Oxylipins Are Elevated in Diverse Models of Cystic Kidney Ghazaleh M. Shahriary1, 2, Hardeep Kataria1, 2, Arsalan Alizadeh1, 2, Kallivalappil Thomas Santhosh1, 2, Diseases, Indicating Potential for COX Inhibition to Treat Disease Soheila Karimi-Abdolrezaee1, 2 Md Monirujjaman1, 2, Jessay G. Devassy1, 2, Tamio Yamaguchi1, 2, 3, Nikhil Sidhu1, 4, Masanori Kugita5, Melissa Gabbs1, 2, Shizuko Nagao5, Jing Zhou6, Amir Ravandi7, Harold M. Aukema1, 2, 4 1Regenerative Medicine Introduction: Activation of resident microglia in concert with infiltrating leukocytes initiate a pro- 1 Program, University of inflammatory process that plays pivotal role in oligodendroglial death and myelin damage in demyelinating Department of Human Introduction: Cystic kidney diseases are characterized by multiple renal cysts and are the leading cause conditions such as spinal cord injury and multiple sclerosis. We have shown that the neuronally-derived Nutritional Sciences, University of inherited renal disease. Oxylipins are bioactive lipids derived from fatty acids formed via cyclooxygenase, Manitoba; 2Department growth factor, neuregulin-1 (Nrg-1), is acutely downregulated in demyelinating lesions. Our goal in this of Manitoba; 2Canadian Centre lipoxygenase and cytochrome P450 activity, and are important regulators of renal health and disease. The of Physiology and study was to unravel the ramification of Nrg-1 decline on immune response using in vitro and in vivo for Agri-Food Research in oxylipin profile is altered in the Han:SPRD-Cy rat and the pcy mouse models of nephronophthisis, a rare Pathophysiology, models. Currently, the immunomodulatory role of Nrg-1 in demyelinating conditions is poorly defined. Health and Medicine, St type of renal cystic disease. In these models, oxylipins formed via the COX pathway are elevated, while Methods: Primary microglia were harvested from rat cerebral cortex and cultured in DMEMHam’s F12 Boniface Hospital Research those produced via the LOX and CYP pathways are reduced. Further, inhibition of COX oxylipin formation University of Manitoba, containing 10% FBS. Lipopolysaccharide (LPS) was used to activate cells. The cells were treated with Centre, Winnipeg, MB; reduces disease progression in Han:SPRD-Cy rat, suggesting its potential for the treatment of these rare 3 Winnipeg, MB recombinant human Nrg-1β1 (rhNrg-1β1) for 24 and 72 hours. For in vivo studies, lysolecithin (LPC) was Department of Clinical disorders. injected into the rat spinal cord to induce focal demyelination. We delivered rhNrg-1β1 intraspinally at Nutrition, Suzuka University of Methods: To further investigate and to determine whether other cystic renal diseases share these the time of LPC injections using poly lactic-co-glycolic acid (PLGA) microparticles for 7 and 14 days. We Medical Science, Suzuka, Mie, abnormalities, a targeted lipidomic analysis of renal oxylipins was performed in orthologous models of 4 used immunohistochemistry, Western blotting and ELISA to analyze inflammatory response at different Japan; Children’s Hospital autosomal dominant polycystic kidney disease 1 (Mx1Cre+ Pkd1flox/flox mouse) and 2 (Pkd2ws25/- time-points after LPC-demyelination. Research Institute of Manitoba, mouse), autosomal recessive polycystic kidney disease (PCK rat) and nephronophthisis (jck/jck mouse). Winnipeg, MB; 5Education and Results: Our in vitro assays demonstrated a significant reduction in the secretion of proinflammatory Over 100 oxylipins were analyzed by HPLC-MS/MS and quantified using the stable isotope dilution Research Center of Animal cytokine, Interleukin-1β, and nitric oxide from activated microglia treated with Nrg-1. Our in vivo studies technique. Models for Human Diseases, in LPC-demyelinating lesion also showed that Nrg-1 therapy promoted an anti-inflammatory phenotype Results: Kidney cyclooxygenase oxylipins were consistently higher in all diseased kidneys, even in very Fujita Health University, in microglia and T cells characterized by increased expression of IL-10, FoxP3 and CD4. early stage disease. On the other hand, cytochrome P450 epoxygenase derived oxylipins were lower only Toyoake, Aichi, Japan; 6Renal Conclusion: We demonstrate that Nrg-1 is a positive regulator of neuroinflammation seemingly through in the autosomal recessive polycystic kidney disease and nephronophthisis models, while lipoxygenase Division, Department of IL-10 dependent mechanisms. IL-10 is a key pro-regenerative factor that supports oligodendrocyte and cytochrome P450 hydroxylase derived oxylipins were lower only in nephronophthisis. Sex effects on Medicine, Brigham and differentiation and myelin repair in CNS injuries. Hence, Nrg-1 may constitute a beneficial therapy with Women’s Hospital, Harvard renal oxylipin alterations were observed but they did not always coincide with sex effects on disease. For immunomodulatory and regenerative roles in demyelinating conditions. Medical School, Boston, MA; oxylipins with sex effects, arachidonic acid derived oxylipins formed via cyclooxygenases and 7Institute of Cardiovascular lipoxygenases were higher in females, while oxylipins from other fatty acids and via cytochrome P450 Sciences, St. Boniface Hospital enzymes were higher in males. Research Centre, Winnipeg, MB Conclusion: The consistent and unique patterns of oxylipin alterations in the different models indicates Maternal and Early Life Factors Influencing the Human Milk Microbiota in the CHILD Cohort S. Moossavi1, 2, E. Khafipour1, 3, S. Sepehri3, B. Robertson4, L. Bode4, A. B. Becker3, 5, P. J. Mandhane6, P. Subbarao7, S. E. Turvey8, D. L. Lefebvre9, M. R. Sears9, the CHILD Study Investigators10, M. B. Azad3, 5

1 the importance of these bioactive lipids in cystic renal diseases, suggesting that pharmacological agents Department of Medical Background: Previous studies have suggested that human milk contains a complex microbial community (e.g. cyclooxygenase inhibitors) may be useful in treating these disorders, for which effective treatment Microbiology, University of that likely influences infant health and development. However, the maternal and early life factors Manitoba, Winnipeg, MB; determining the composition of milk microbiota is not well understood. remains elusive. 2 Digestive Disease Research Methods: We studied the milk microbiota of 395 lactating mothers in the Canadian Healthy Infant Institute, Tehran University of Longitudinal Development (CHILD) study by sequencing the V4 region of the 16S rRNA gene on Illumina Medical Sciences, Tehran, MiSeq. Human milk oligosaccharides (HMOs) were analysed by rapid high-throughput HPLC. Maternal Iran; 3Children Hospital Research Institute of factors were self-reported or documented from hospital records; 2010 Healthy Eating Index (HEI) was Manitoba, Winnipeg, MB; derived from a modified food frequency questionnaire. Effects of single factors on the β-diversity (Bray- 4Pediatrics, University of Curtis dissimilarity matrix) was estimated with PERMANOVA. Multivariate associations of maternal and California San Diego, San early life factors with the milk microbial community was also assessed by redundancy analysis. The overall Diego, CA; 5Pediatrics and and by term effects were determined by permutation testing. Samples were clustered into groups based Child Health, University of on hierarchical clustering with ward sum of square algorithm and multivariable logistic regression was Manitoba, Winnipeg, MB; used to identify factors associated with cluster membership. 6Pediatrics, University of Results: While maternal intrapartum antibiotics, secretor status, ethnicity and method of delivery were Alberta, Edmonton, AB; not associated with milk microbiota β-diversity; parity (p=0.019) and infant sex (p=0.037) significantly 7 Pediatrics & Physiology, affected the community structure. In multivariate analyses, HMO diversity, maternal HEI, birth weight, University of Toronto, lactation stage, maternal age, maternal BMI, infant sex, and duration of exclusive breastfeeding Toronto, ON; 8Pediatrics, collectively accounted for 7% of the variability in microbiota composition. Although this overall model University of British Columbia, Vancouver, BC; was not significant (p>0.05), analysis by term revealed that duration of exclusive breastfeeding (p=0.019), 9Medicine, McMaster maternal BMI (p=0.041), and infant sex (p=0.038) were independently associated with the overall University, Hamilton, ON; structure. We identified three clusters with distinct microbiota profiles; of all factors evaluated, only the 10Canadian Healthy Infant duration of exclusive breastfeeding was significantly associated with cluster membership. Longitudinal Development Conclusion: Our findings identify several novel maternal, infant, and early life factors that may influence Study milk microbiota composition. Inhibitory Control and Impulsivity in Tourette Syndrome: A Meta-Analysis Genome-Wide Association Study of Heel Bone Mineral Density Identifies 153 Novel Loci Simon Morand-Beaulieu1, 2, 3, Stéphanie Grot2, 4, Jacob Lavoie2, 3, Julie B. Leclerc2, 5 David Luck2, 3, 4, Marc E. Lavoie1, 2, 3, 4 and Implicates Functional Involvement of GPC6 in Osteoporosis John A Morris1, 2, JP Kemp3, 4, C Medina-Gomez5, V Forgetta1, NM Warrington3, 6, SE Youlten7, J Zheng4, CL Gregson4, 1Cognitive and social Introduction: Tourette syndrome (TS) is a neuropsychiatric disorder characterized by motor and phonic E Grundberg2, K Trajanoska5, JG Logan8, AS Pollard8, PC Sparks8, EJ. Ghirardello8, R Allen8, D. Leitch8, NC Butterfield8, psychophysiology lab; tics. TS often comes with important comorbid disorders, which can be more debilitating than the disorder DSK Komla-Ebri8, A-T Adoum8, KF. Currl, JK White9, F Kussy9, K Greenlaw1, C Xu10, NC Harvey11, C Cooper1, DJ Adams9, itself. Some of these comorbidities involve impulsive behavior: attention deficit hyperactivity disorder, 2 1, 2, 15, 16 12 13 5 7 14 Centre de recherche de oppositional defiant disorder, explosive outbursts, etc. Even though an important proportion of TS CMT. Greenwood , MT Maurano , S Kaptoge , F Rivadeneira , JH. Tobias, PI. Croucher , CL. Ackert-Bicknell , l’Institut universitaire en patients are affected by those impulsive comorbidities, there is still no consensus regarding the inhibitory JHD Bassett8, GR illiams8, JB. Richards1, 2, 16, 17, DM. Evans3, 4 santé mentale de control of TS patients, and important discrepancies exist throughout the literature. Therefore, the goal of the current meta-analysis is to evaluate if impulsive and disinhibited behavior is present in 1Lady Davis Institute, McGill Montréal; 3Département Introduction: Bone mineral density (BMD) is a highly heritable trait and is used as the primary diagnostic neuropsychological tasks, and to study the factors modulating those behaviors. University; 2Department of Human and prognostic marker for osteoporosis and fracture risk, and is conventionally measured at the hip or de neurosciences, Methods: To this end, we performed a systematic search through MEDLINE and PsycINFO databases to Genetics, McGill University; Montreal, QC; 3University of Queensland, AUS; spine by dual-energy X-ray absorptiometry. Quantitative ultrasound (QUS) is a non-invasive technique Université de Montréal; retrieve studies that used a neuropsychological task involving the inhibition of a motor or verbal 4University of Bristol, Bristol, UK; that measures the speed and attenuation of ultrasound through bone, and estimates bone mineral response in TS patients. 5 4Département de Erasmus Medical Center, Rotterdam, density (eBMD). Previous genome-wide association studies (GWAS) of BMD/QUS measures (N=35,000) Results: Sixty-one studies and 1717 TS patients were included in our meta-analysis. Our analyses revealed NLD; 6University of Western Australia, identified up to 68 loci but explained less than 5% of trait heritability, therefore missing heritability could psychiatrie, Université de Crawley, AUS; 7Garvan Institute of that TS patients had significant impairment in inhibitory control (d = .33). Also, inhibitory deficits were be explained by loci remaining to be discovered at greater sample sizes. 5 more important in TS+ADHD patients (d = .51) than in pure TS patients (d = .26). Medical Research, Darlinghurst, AUS; Montréal; Département 8Imperial College London, London, Methods: To identify genetic loci associated with BMD variation, we undertook a GW AS of heel eBMD on de psychologie, Université Conclusion: It appears that impulsivity and inhibitory deficits are core symptoms of TS. The presence of UK; 9Wellcome Trust Sanger Institute, 142,487 individuals of European descent (76,067 females and 66,420 males) from the UK Biobank Study. comorbid ADHD seems to potentiate this problematic. Therefore, treating comorbid ADHD symptoms Hinxton, UK; 10University of Toronto, To investigate underlying mechanisms of genetic loci, we undertook: 1) bioinformatic, functional du Québec à Montréal, 11 might be the best way to reduce impulsivity and improve inhibitory control in TS patients. Toronto, ON; University of genomic annotation, and human osteoblast expression studies; 2) gene function prediction; 3) skeletal Montreal, QC Southampton, Southampton, ENG; 12NYU Langone Medical Center, New phenotyping of 120 knockout mice with deletions of genes proximal to the identified loci; and 4) analysis York, NY; 13University of Cambridge, of gene expression in mouse osteoblasts, osteocytes and osteoclasts. Cambridge, UK; 14University of Results: We identified 307 conditionally independent SNPs attaining genome-wide significance Rochester, Rochester, NY; (P<6.6xi0-9) at 203 loci, explaining 11.8% of the phenotypic variance. These included 153 novel loci, and 15Department of Oncology, McGill University; 16Department of several rare variants with large effect sizes. Linkage disequilibrium score regression revealed genetic Epidemiology, Biostatistics & correlations between eBMD, BMD measures at other skeletal sites, and fracture. Our study of underlying Occupational Health, McGill mechanisms strongly implicated GPC6 as a novel determinant of BMD and also identified abnormal University, Montreal, QC; 17King’s skeletal phenotypes in knockout mice for a further 100 prioritized genes. College London, London, UK Modular Foams Derived from Decellularized Adipose Tissue Enhance the Survival and Angiogenic Response of Fibroblasts Isolated from Human Chronic Wounds in an in vitro Chronic Wound Model Pascal Morissette Martin, Cody Brown, Douglas W. Hamilton, Lauren E. Flynn

Conclusion: Overall, we have almost tripled the number of BMD-associated loci, doubled the variance in University of Western Introduction: With a high prevalence in the elderly and diabetic populations, chronic wounds cause BMD explained by genetic factors, and identified new potential drug targets for the prevention and Ontario, London, ON significant morbidity in industrialized countries. Frequent failures of existing treatments demonstrate the treatment of osteoporosis that can be tested directly in disease models. need for new strategies to stimulate cutaneous tissue repair. To address this challenge, our team is developing modular biomaterials termed “bead foams”, which are comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive 3-D network. In the current study, human wound edge dermal fibroblasts (weDF) were cultured on bead foams fabricated from human decellularized adipose tissue (DAT) or purified collagen (COL) under chronic wound-simulating conditions to explore the effects of ECM composition on the cellular response. Hypothesis: Human weDF will demonstrate enhanced survival, pro-angiogenic, and pro-fibrotic properties when cultured on DAT-derived scaffolds in an in vitro chronic wound model. Methods: Following fabrication using an established method (Yu et al., 2013), the structure and composition of the DAT and COL bead foams were assessed. Primary human weDF were cultured on scaffolds or tissue culture plastic controls under serum-free conditions, 2% oxygen tension, and ±1 ng/ mL of TNF-α. At 1 and 7 days, samples were harvested for total DNA quantification, cell cytotoxicity assays, and RT-qPCR. Cell distribution and apoptosis were investigated by immunolabelling for vimentin and cleaved caspase-3. Results: Imaging and porosity measurements showed that DAT and COL bead foams displayed similar structural features, while immunohistochemistry for ECM components and glycosaminoglycan quantification demonstrated that each had a unique biochemical composition. weDF abundance and survival were significantly increased on the DAT bead foams as compared to the COL bead foams (p-value<0.01 and<0.001, respectively). When treated with TNF-α, mRNA levels of HGF and VEGFA were elevated in the weDF cultured on the DAT scaffolds (p-value<0.05 and<0.001, respectively), while mRNA levels of COL1A2, CTGF and TGFB1 were unchanged between scaffold groups. Enhancing Chemotherapeutic Responses in CNS Malignancy Through Suppression of Hyperactive DNA Damage Repair Pathways Marina Mostafizar, Sachin Katyal

Discussion and Conclusions: Taken together, these results suggest that the unique composition of the Dept. of Pharmacology & Introduction: Current methods to treat childhood medulloblastoma are highly intrusive leading to poor DAT bead foams may promote the survival and pro-angiogenic phenotype of weDF within our in vitro Therapeutics, University quality of life while the three-year survival rate of patients afflicted with malignant glioma remains chronic wound model. Future studies will focus on more fully assessing the impact of these cell instructive abysmal. Recurrence of these malignant tumors is pervasive as they can adopt several mechanisms to properties on tissue repair processes. of Manitoba, Research resist anti-cancer therapeutics including activation and up-regulation of DNA repair pathways that act to Institute in Oncology & resolve DNA damage elicited by chemo-radiotherapy. DNA repair inhibitors, such as Poly (ADP-Ribose) Hematology, CancerCare Polymerase (PARPi), DNA-dependent protein kinase (DNA-PKi) and Ataxia Telengiectasia Mutated (ATMi), have shown promise to sensitize tumors to DNA damaging chemo-radiotherapeutics. Differing tumors Manitoba, Winnipeg, MB have variable expression of these enzymes and/or their corresponding repair pathway(s), therefore; identification, characterization and new strategies to modulate their expression can enhance current treatment efficacy in combination with anti-tumor agents. Method: Using novel high-throughput DNA repair assays to interrogate DNA repair inhibitors and genotoxic agents, I have identified synergistic combinations that sensitize CNS tumors to chemoradiotherapeutics. Using gene expression methodology with the use of “DNA damage repair response” PCR arrays, I will identify specific differentially-regulated DNA repair enzymes to understand mechanisms underlying therapeutic resistance to genotoxic agents. From these findings, I will inhibit DNA repair enzymes via shRNA or enzyme-specific inhibitors, in an effort to use a targeted approach to chemosensitize MB and MG cells. Extent of DNA damage will be quantified using additional high- throughput DNA damage measurement assays combined with unique cellular DNA damage reporters. Result: In comparing subsets of MB and MG cell lines with existing DNA repair inhibitors, I have found that MB cells rely on DNA-PK/SSBR-dependent pathways and MG, ATM/SSBR pathways to resolve induced genotoxicity. High-throughput DNA damage repair assays reveal enhanced tumor cell death in MB lines (DAOY&D283) in presence of PARPi+CPT, PARPi+DNAPKi and PARPi+ATMi. Gene expression analysis will be carried to compare the expression levels of individual DNA repair enzymes in MB lines. Conclusion: Using high-throughput genotoxcitity assays, I have identified unique combinations of DNA Altered Fatty Acid and Mitochondrial Metabolism in the Liver of Pregnant Adiponectin- Deficient Mice Contributes to Insulin Resistance and Gestational Diabetes Mellitus Brittany L. Moyce, Laura K. Cole, Bo Xiang, Mario A. Fonseca, Christine A. Doucette, Grant M. Hatch, Vernon W. Dolinsky repair pathway inhibitors that sensitize CNS tumors to resistant chemoradiotherapy. An expanded University of Manitoba, Introduction: Gestational diabetes mellitus (GDM) is a common pregnancy-related health condition. analysis is currently underway to further delineate these pathways to better understand tumor DNA Winnipeg, MB While genetics, lifestyle and diet contribute to development of GDM, evidence suggests that low levels repair mechanisms and therapeutic responses. of adiponectin increases the risk for GDM. Adiponectin is a fat derived hormone that improves the sensitivity of tissues to insulin. We hypothesize that adiponectin deficiency causes fatty liver during pregnancy, ultimately contributing to the development of GDM. Methods: We compared the glucose and insulin tolerance of pregnant (3rd trimester) adiponectin-/- (strain B6;129-Adipoqtm1Chan/J) and wild-type mice, and assessed parameters of hepatic metabolism, including mitochondrial function and fatty acid metabolism. We assessed the impact of adiponectin supplementation by administering adenovirus mediated full length adiponectin at the end of the second trimester of pregnancy, and comparing to control containing GFP. Results: In the third trimester, pregnant adiponectin-/- mice exhibited fasting hyperglycemia regardless of diet (9.2mmol/L vs. 7.7mmol/L in controls, p<0.05). These mice display impaired glucose and insulin tolerance relative to wild-type controls. Pregnant adiponectin-/- mice develop hepatic steatosis, including a 3-fold elevation in hepatic triglycerides (p<0.05). This was associated with altered hepatic lipid metabolism, including a 2.5 fold increase in fatty acid synthase expression (p<0.05), elevated circulating free fatty acids, triglycerides and cholesterol. Nearly 2-fold reduction (p<0.05) in maximal mitochondrial respiration was observed via oxidative flux analyzer in hepatocytes of adiponectin -/- mice. Hepatocytes from pregnant adiponectin-/- mice dramatically reduced respiratory capacity when using fatty acids alone, and display elevated synthesis and secretion of triglycerides and cholesterol. Gestational weight gain and food consumption were similar in knockout and wild-type mice. Adiponectin supplementation to pregnant adiponectin-/- mice significantly improved glucose tolerance, prevented fasting hyperglycemia, and attenuated fatty liver development. Conclusion: Results show that adiponectin deficiency is associated with altered hepatic lipid metabolism and hepatic steatosis during pregnancy. Consequently, adiponectin deficiency contributes to The Art of Preventing Broken Hearts in Colorectal and Renal Cell Cancer Viktoriya Mozolevska1, 2 David Cheung1, James Thliveris3, Pawan Singal1, 2, Piotr Czaykowski4, Davinder S. Jassal1, 4, 5 med-gestation insulin resistance and hyperglycemia characteristic of GDM. Moreover, adiponectin 1St. Boniface Hospital Background: Despite the beneficial effects of chemotherapy agents in increasing overall survival of supplementation rescues the effects of adiponectin deficiency on insulin sensitivity and hepatic lipid Albrechtsen Research cancer patients, cardiotoxicity remains a serious complication of many anti-cancer therapies. Two types metabolism. of targeted therapy currently in use for colorectal (CRC) and renal cell cancer (RCC) are the monoclonal 2 Centre; Department of antibody Bevacizumab (BVZ) and the tyrosine kinase inhibitor Sunitinib (SNT). An unexpected side effect Physiology and of these two anti-cancer drugs is the increased risk of cardiotoxicity in up to 25% of patients. The Pathophysiology; objective of this study is to investigate whether prophylactic administration of renin-angiotensin system (RAS) antagonists will attenuate the cardiotoxic side effects of BVZ or SNT in a chronic in vivo murine 3Department of model. Pathology; 4Department Methods: A total of 166 wild-type C57Bl/6 male mice received: i) 0.9% saline (i.p. weekly; n=33); ii) BVZ of Internal Medicine; (10mg/kg i.v. weekly; n=67) or iii) SNT (40mg/kg/d orally, n=66) for 4 weeks. Within each arm, mice were administered prophylactic treatment either with water, Aliskiren (50mg/kg/d), Perindopril (4mg/kg/d), or 5Department of Valsartan (2mg/kg/d) via oral gavage for the entire study period. Following serial echocardiographic and Radiology, Rady Faculty of hemodynamic assessments for 4 weeks, mice were euthanized, and the hearts were analyzed for Health Sciences, histological injury. University of Manitoba, Results: At baseline, the left ventricular ejection fraction (LVEF) was similar between all groups. In mice treated with BVZ, LVEF decreased from 72±3% at baseline to 41±2% at day 28 (p<0.05). As compared to Winnipeg, MB BVZ alone, prophylactic administration of Aliskerin, Perindopril, or Valsartan was partially cardioprotective with LVEF values of 57±2%, 50±2%, and 51±3% at day 28, respectively (p<0.05). Similarly, in mice treated with SNT, LVEF decreased from 73±4% at baseline to 34±3% at day 28 (p<0.05). As compared to SNT alone, prophylactic treatment with Aliskerin, Perindopril, or Valsartan was also partially cardioprotective with LVEF values of 53±3%, 45±2%, and 44±3% at day 28, respectively (p<0.05). Finally, BVZ and SNT treated animals demonstrated increased mean arterial pressure, a loss of cellular integrity, and myofibril disarray, which were diminished by the prophylactic administration of RAS antagonists. Conclusion: The cardiotoxic side effects of BVZ or SNT were partially attenuated by the prophylactic administration of RAS antagonists in a chronic in vivo murine model. Myocardin Regulates Mitochondrial Permeability Transition and Cell Death During Cardiac Development and Heart Failure Wajihah Mughal1, 2, Jared Field1, Matthew Martens1, 2, Donald Chapman1, 2, Yan Hai1, 2, Richard Keijzer1, 3, Sunil Rattan4, Ian Dixon4, 1, Jianhe Huang5, Michael S Parmacek5, Joseph W Gordon1, 2

1Children’s Hospital Introduction: Myocardin is a transcriptional co-activator required for cardiovascular development such observed reduced Myocardin expression within the infarcted border zone following coronary ligation, Research Institute of that genetic inhibition of Myocardin results in congenital heart defects associated with increased reduced miR-133a levels and elevated Nix expression, in comparison to sham animals. Manitoba, John Buhler programmed cell death (PCD). Studies show the mitochondrial permeability transition has been Conclusion: Our results describe a genetic pathway linking Myocardin activation of miR-133a to prevent implicated in cardiomyocyte development. We hypothesize that Myocardin regulates mitochondrial Nix-induced cell death, restoring mitochondrial function and cardiomyocyte survival. Research Centre, Health permeability to oppose PCD during cardiac development and heart failure. Sciences Centre; Methods: Using primary rat cardiomyocytes and immortalized rat cardiac myocytes (H9C2), genetic 2Department of Human manipulation and/or exposure to drug treatments (18 hours) were used to assess cell viability by Anatomy and Cell Science; fluorescent microscopy (n>200 cells counted/condition). Fluorescent dyes assessed mitochondrial 3Department of Surgery; function (n=10): permeability transition measured by calcein-AM/cobalt chloride; membrane potential 4Institute of Cardiac by TMRM, and respiration measured by metabolic flux analyzer. Genetically encoded fluorescent plasmids assessed organelle-targeted calcium flux. Gene and protein expression was determined by qPCR and Sciences, St. Boniface protein immunoblots, respectively. Lentiviral particles were used to transduce primary cardiomyocytes Hospital Albrechtsen while empty vector or scrambled vector was used for transfection efficiency in H9C2s. An in vivo model Research Centre, of myocardial infarction by left coronary artery ligation was used for protein and RNA analysis (n=4). Winnipeg, MB; 5Penn Results: At embryonic day 9.5, Myocardin knock out mice display an increase in cardiac Nix expression Cardiovascular Institute, and cardiac necrosis in comparison to wild type, as determined by immunofluorescence. Cell culture Hospital of the University studies illustrate that loss of Myocardin function reduces cell viability by 25% (p<0.001), mitochondrial function and expression of miR- 133a in comparison to control. Conversely, loss of Myocardin increases of Pennsylvania, expression of a mitochondrial death protein Nix, which is reversed by miR-133a inhibition. Gain of Department of Medicine, function studies demonstrates that Myocardin or miR-133a expression independently restore cell University of viability (p<0.01), mitochondrial function (p<0.01) and reduce Nix protein. Our data demonstrates the Pennsylvania, protective role of Myocardin and miR-133a in opposing mitochondrial calcium uptake and sarcoplasmic Philadelphia, PA reticulum (SR) calcium release induced by protein kinase A activating agents, Forskolin and IBMX. We The Effect of Interferon- and Mycobacterium Tuberculosis-Specific Antigen Stimulation Impact of Sex Work on the Mucosal Immune Milieu of Young Women in Mombasa, Kenya on Transcript Gene Expression in Mycobacterium Tuberculosis Infection R. Mwatelah1, A. Sivro1, 2, C. Kambaran2, H. Gebremedhin1, N. Klatt3, P. McLaren1, E. Cheuk1, S. Mishra4, M. Becker1, L. McKinnon1, 2 F. Mutua1, R-C. Su1, 2, S. Kiazyk1, 2, T. B. Ball1, 2 1Univeristy of Manitoba, The mechanisms of HIV acquisition in the female genital tract are not yet completely understood, 1Department of Medical Background: Interferon (IFN) signaling pathways play an important role in the immune response and Winnipeg, MB; 2Centre for however, these are believed to be key for the design of biomedical HIV prevention interventions that can reduce systemic spread of the virus. The mucosal immune milieu of the female genital tract is thought to Microbiology, University outcome of infection with Mycobacterium tuberculosis (Mtb). Type II IFN has a protective role in infection; the AIDS Programme of the role of type I IFNs is not well defined. In TB, type I IFN signatures have been shown to differentiate be an important determinant of HIV acquisition, and is likely influenced by sexual activity. Here we 2 of Manitoba; National active TB (ATB) from latent TB infection (LTBI), and predict TB reactivation. This signature resolves on Research, South Africa; characterized FGT inflammation and dysbiosis, both of which have been associated with higher risk of Laboratory for HIV treatment accompanied by IFN-γ up-regulation. 3University of HIV acquisition. We used a biological and behavioural cross-sectional survey of young women aged 14-24 who were screened into three groups according to their sexual behaviours; female sex workers (FSW), Immunology, National HIV Rationale: The drivers of the skewed type I IFN stimulated gene (ISGs) profile, its correlation to IFNα/β Washington, Seattle, WA; levels, whether this response is stimulus-specific, and the effect of skewed ISGs on immune functions are and participants who practiced transactional and casual sex. We randomly selected 162 samples from a and Retrovirology 4University of Toronto, TM largely unknown factors. There is little overlap between transcriptional profiles from different studies. larger cohort of 768 participants who consented to collection of vaginal secretions using a SoftCup . Laboratory, JC Wilt Mtb-specific antigen stimulation may identify transcriptional profiles specific to TB. Toronto, ON We analysed cytokine levels in a panel of 39 cytokines and characterized the vaginal microbiome via 16 rRNA sequencing. Increased levels of IL7 (p=0.004) and IFNa2 (p=0.018) were observed in FSWs compared Infectious Diseases Hypothesis: Type I IFN activity correlates with an altered pattern of ISG expression and cytokine/ chemokine profile in ATB and LTBI that is skewed towards an ATB-like profile in HIV to the other groups. The levels of MIG (p=0.095) and IL17 (p=0.095) trended to be higher among the Research Centre, Objectives: Our goal is to evaluate the response of ISGs in peripheral blood mononuclear cells (PBMCs) transactional group and casual group respectively. Within all groups, a higher number of sexual partners Winnipeg, MB from individuals with ATB and LTBI stimulated with Mtb or with interferons and correlate this to plasma in the last month correlated positively with IL7 (R=-0.28, p=0.003) and IFNa2 (R=-0.28, p=0.002). Recent type I interferon activity and soluble immune response patterns. sex was associated with two diverse vaginal microbiome groups (p=.003). From this pilot study we report Methods: Using RT-qPCR we evaluated the expression of 15 selected IFN-stimulated genes on PBMCs positive correlation between sexual practices and both cytokine and microbiome levels in the female from healthy donors stimulated with IFN-α, IFN-β and IFN-γ to optimize stimulation concentration and reproductive tract. These data might have important implications for understanding heterogeneity in duration. IFNα/β activity was assessed using a reporter cell assay in unstimulated PBMCs from ATB, LTBI early HIV acquisition across high-risk adolescent women. and TB-negative individuals. Results: We have defined optimum IFN concentrations and time course for our future experiments in ATB and LTBI. Preliminary data shows no statistically significant difference in IFN activity between the groups. Conclusion and Future directions: Currently type I IFN activity seems not to be the driver of type I IFN signature in TB. We will run the reporter cell assay and cytokine/chemokine assays in IFN- and Mtb- stimulated plasma from HIV-negative ATB and LTBI, and TB-negative HIV-positive individuals. We will extend the gene expression experiments to IFN- and Mtb-stimulated PBMCs from the same individuals and investigate for correlations to IFNα/β activity and immune functions. Pre-Injury Variables and Risk of Sport Concussion Involvement of Thioredoxin Interacting Protein in Induction of Neuronal Cell Death Sandhya Mylabathula1, Lynda Mainwaring1, Michael Hutchison1, Doug Richards1, 2, Paul Comper1, 3 Under Oxidative Stress Conditions Pandian Nagakannan, Mohamed Ariff Iqbal, Eftekhar Eftekharpour 1Faculty of Kinesiology Introduction: Concussions are a public health concern in Canada. This injury can involve physiological and Physical Education, and neuropsychological symptoms and signs. However, research on risk factors is not extensive and Department of Physiology Rationale: Redox imbalance has been linked to wide range of neurological disorders such as stroke, many questions remain unanswered. University of Toronto; and Pathophysiology, traumatic brain and spinal cord injury and diabetic neuropathy. Thioredoxin (Trx) is an endogenous Objective: This study examined whether cognitive functioning, history of concussion (HOC), and sex antioxidant defence system having roles in cell survival and homeostasis. Failure of Trx system is well 2 MacIntosh Sports predicted risk of sport concussion. Spinal Cord research documented in number of neurological disorders, where Trx is either oxidised or inhibited by its natural Medicine Clinic; 3Toronto Design: Retrospective study design. Predictive models were used to determine the predictive ability of Centre and Regenerative inhibitor Thioredoxin Interacting Protein (TXNIP). This protein is upregulated in dementia, diabetic each variable. Rehabilitation Institute, Medicine Program, Max neuropathy and other neurological conditions. Glutamate excitotoxicity is a major contributor of cell Setting: Canadian University. death in traumatic nervous system injuries that mediates its toxicity through oxidative stress. In this Toronto, ON Rady Faculty of Health Participants: 708 data observations from 701 varsity athletes (41.2% female), representing 14 sports. study, we aimed to investigate the potential protective role of TXNIP in neurons when exposed to Assessment of Risk Factors: Two measures of cognitive functioning (mean reaction time and throughput Sciences, University of glutamate. [speed and accuracy]) were assessed using the Automated Neuropsychological Assessment Metrics Manitoba, Winnipeg, MB Hypothesis: Upregulation of Txnip interferes with the protective role of Trx under oxidative stress, and testing battery. Sex, and categorical and continuous measures of HOC were examined. therefore promotes cell death. Outcome Measures: Occurrence of concussion after baseline testing. Experimental Model: A cellular model of oxidative stress was used in this study: mouse immortalized Main Results: HOC was a significant predictor for both sexes. For every previous concussion, the odds of hippocampal cell line (HT-22) have been used to study the effect of glutamate on cell viability. These cells sustaining another concussion increased by 1.5 (95% Confidence Interval [CI]: 1.1, 2.1 [females]; 1.2, 1.9 lack the ionotropic calcium channels that commonly mediate cell death in neurons. Additionally, [males]). Females with a HOC had twice the odds of sustaining another concussion than those without a exposure to glutamate leads to depletion of glutathione and therefore makes this model an optimal HOC (CI: 1.1, 4.0). For males, the odds were three times (CI: 1.7, 5.6). Cognitive functioning and sex were model for investigation of Trx system. not meaningful predictors. Results: Glutamate exposure in HT-22 cells caused a time dependent cell death and a corresponding Conclusions: This study supports previous findings that HOC is a risk factor and suggests that pre-injury increase in TXNIP protein levels and depletion of Trx antioxidant system, indicating enhancement of cognitive functioning is not a risk factor for sport concussion. These results can inform current oxidative stress. This was associated with reduced levels of the anti-apoptotic protein Bcl-2, induced management protocols and policies in varsity sport. Specifically, assessing HOC in all athletes prior to cytochrome C release into the cytosol and depletion of ATP, indicating a failure of mitochondrial energy their competitive season would provide information regarding their risk of future sport concussion. Thus, production and apoptosis. Genetic deletion of TXNIP, using CRISP Cas9 system, prevented these changes it is important for clinicians to record HOC, and to encourage athletes to report concussions to ensure and enhanced cell viability. Our results showed that TXNIP knockout cells showed higher resistance to accurate recording. Pre-injury cognitive screening of athletes is not recommended for assessing risk of these effects of glutamate. future concussion. Significance and Impact: This research expands our knowledge and provides new tools for treatment of The Role of Scleraxis in Pressure Overload-Induced Cardiac Fibrosis Raghu S. Nagalingam, David Y. Cheung, Nina Aroutiounova, Davinder S. Jassal, Michael P. Czubryt many diseases. Considering the involvement of glutamate system in CNS diseases and conditions Institute of Cardiovascular Cardiac fibrosis decreases the myocardial compliance via increased ventricular wall stiffness, resulting in including brain and spinal cord injury and diabetic neuropathy, our findings may be applicable to any Sciences, St. Boniface heart failure and sudden death in patients with several cardiac pathologies including pressure overload diseases associated with neuronal cell death. typically due to hypertension. However, treatment options to reduce the progression of myocardial Hospital Albrechtsen fibrosis are severely limited. Fibrosis occurs when cardiac fibroblasts convert to myofibroblasts and Research Centre and greatly increase their output of extracellular matrix proteins such as collagen. Restricting the activation Department of Physiology of myofibroblasts could be of clinical significance in the prevention and treatment of fibrosis. We have shown that the transcription factor scleraxis, present in cardiac fibroblasts and myofibroblasts, is a key and Pathophysiology, regulator of the production of major matrix proteins such as collagen 1α2, α-smooth muscle actin and University of Manitoba, fibronectin, and controls fibroblast to myofibroblast phenoconversion. Hence, in the present study, we Winnipeg, MB hypothesize that gene deletion of scleraxis will attenuate pressure overload-induced cardiac interstitial fibrosis. To identify the involvement of cells other than resident fibroblasts in cardiac fibrosis, we performed ubiquitous (pan-Cre) or cardiac fibroblast-specific (Tcf21-iCre) deletion of scleraxis gene via administration of tamoxifen to the adult mice (to activate Cre) and corn oil used as control. At 8 weeks of age, pressure overload was surgically induced in wild type (WT) and scleraxis conditional null mice by transverse aortic banding (n=10 per group). Sham-operated animals served as the control (n=10 per group). Animals were euthanized at two time points: 4 weeks post-surgery (compensated heart failure) or 8 weeks post-surgery (decompensation). Using qPCR we observed that loss of scleraxis significantly reduced the mRNA expression of major fibrillar collagens including collagen 1α1, collagen 1α2 and collagen 3α1 compared to WT TAC mice. In conclusion, our results emphasize the crucial role of scleraxis in governing multiple processes that contribute to fibrosis, including ECM expression and remodeling. Predictive Factors for Survival in Surgical Series of Symptomatic Metastatic Epidural Derivation of a Novel Mural Trophectoderm Cell Line by Overexpression of Trophoblast- Spinal Cord Compression: A Prospective North American Multicenter Study in 142 Enriched MicroRNAs in Murine Embryonic Stem Cells Patients Ursula Nosi, Brian Cox A. Nater1, L. A. Tetreault1, B. Kopjar2, P. M. Arnold3, M. B. Dekutoski4, J. A. Finkelstein5, C. G. Fisher6, J. C. France7, Z. L. Gokaslan8, L. D. Rhines9, P. S. Rose10, A. Saghal5, J. M. Schuster11, A. R. Vaccaro12, M. G. Fehlings1 University of Toronto, Introduction: microRNA involvement in the execution of the first cell fate decision resulting in the Department of specification of the extraembryonic trophectoderm (TE) and embryonic inner cell mass (ICM) lineages of the early pre-implantation embryo remains controversial. We postulated that perturbations of microRNA 1University of Toronto, Introduction: Symptomatic Metastatic Epidural Spinal Cord Compression (MESCC) afflicts up to 10% of Physiology, Toronto, ON expression affect early cellular fate, and that identification of lineage-enriched microRNAs, in conjunction Toronto Western Hospital, all cancer patients and is associated with shortened survival and worsened quality of life. This study aims with target analyses would provide a window into the molecular networks that regulate these cell fates. University Health Network, to identify the key survival predictive factors in MESCC patients who were surgically treated for a single 2 Methods: By employing high-throughput transcriptomic analysis of stem cells representative of the TE Toronto, ON; University of symptomatic lesion. Washington, Seattle, WA; and ICM (trophoblast stem cells (TSCs) and embryonic stem cells (ESCs) respectively), we identified three Methods: 142 surgical MESCC patients enrolled in a prospective, multicenter, North American, cohort 3University of Kansas, Kansas TSC-enriched candidate microRNAs predicted to target 56 mRNAs differentially enriched to ESCs with study, were followed postoperatively for at least 12 months or until death. Kaplan-Meier, log-rank tests City, KS; 4The CORE Institute, pluripotency-associated function. Transgenic ESC lines with inducible expression of each candidate and Cox proportional hazards regression were used. Of the 17 potential preoperative predictors of Sun City West, AZ; microRNA were derived. Induction was then performed by exposure to doxycycline in the presence of an 5 survival considered in univariable analysis, non-collinear factors with <10% missing data, ≥10 events per Sunnybrook Health HDAC2 inhibitor, and culture conditions conducive to TSC maintenance. Sciences Center, Toronto, ON; stratum, and p<0.05 were tested through a backward stepwise selection process; proportional hazard Results: Six-day microRNA-induced ESCs not only adopt a trophoblast morphology, but also more closely 6University of British assumption was assessed. resemble the global transcriptional profile of wild-type TSCs than ESCs. Multiple pathways required for Columbia and Vancouver Results: The median survival (MS) was 7 months. A total of 88 patients died and 54 were censored. maintaining ESC and TSC fate, including signaling and epigenetics were significantly affected, indicating Coastal Health, Vancouver, Univariate analyses yielded seven significant predictors for survival: growth of primary tumor (Tomita 7 that induction of these microRNAs has widespread affects resulting in the acquisition of a new cell fate. BC; West Virginia University, Grade I vs. II/III), sex, presence of lymph node/other organ metastasis, BMI, and SF-36v2 physical 8 We have demonstrated the in vivo competence of these cells by performing transplantation experiments Morgantown, WV; Johns component, EQ-5D and Oswestry disability index (ODI) scores. Since the ODI*time term was significant, into pre-implantation embryos. Induced cells contribute exclusively to the TE of pre-implantation Hopkins University School of it was included in the multivariable model. Tomita tumor Grade I (HR: 0.361, 95% CI: 0.199-0.794, Medicine, Baltimore, MD; blastocysts, and the Reichert’s membrane of the postimplantation embryo which is comprised of primary p=0.0009), absence of lymph node/other organ metastasis (HR: 0.489, 95% CI: 0.301-0.794, p=0.0038), 9MD Anderson Cancer trophoblast giant cells (pTGCs) deriving from the mural TE, and parietal endoderm cells. Moreover, size- and SF-36v2 physical component score (HR: 0.945, 95% CI: 0.920-0.970, p<0.0001) had an independent Center, University of Texas, dependent microsphere uptake assays reveal that they retain phagocytic properties of the mural TE. 10 effect on overall survival. Houston, TX; Mayo Clinic, Within the in vivo environment these cells retain developmental potential and differentiate into TGCs. 11 Conclusions: Slow growing tumor (Tomita tumor Grade I), absence of lymph node/other organ Rochester, MN; University Conclusion: We have shown for the first time, that TSC-enriched microRNAs are sufficient to induce of Pennsylvania; 12Thomas metastasis, and lower degree of preoperative physical disability, as reflected by a higher score on he embryonic to extraembryonic cross-lineage conversion by shutting down transcriptional networks Jefferson University, SF-36v2 physical component questionnaire, are preoperative clinical factors associated with longer required for the maintenance of pluripotency, and indirectly relieving suppression of trophoblast Philadelphia, PA survival in surgical MESCC patients. Rat Fetal Brain Gene Expression Changes Induced by Prenatal Alcohol Exposure and DHA Supplementation Elaheh Nosratmirshekarlou1, Alemu Regassa1, Xavier Lieben Louis1, Fatemeh Ramezani1, Woo-Kyun Kim2, Michael N Eskin1, Miyoung Suh1 transcriptional networks to promote mural trophectoderm fate in a time-dependent manner. This 1Department of Human Introduction: Prenatal alcohol exposure is known to affect genes involved in signal transduction and observation is significant as mural trophoblast cells have never been cultured. These cell lines could be Nutritional Sciences, neurological system processes, thus affecting normal fetal brain development. Omega-3 docosahexaenoic developed into an in vitro model of embryo implantation to address pathologies of infertility. acid (DHA) is a membrane structural component of brain. Alcohol decreases DHA levels in brain, justifying University of Manitoba, its provision if there is exposure to alcohol during pregnancy. However, how DHA impacts on global gene Winnipeg, MB; expressions in fetal brain is not known, especially with prenatal ethanol exposure. This study investigated 2Deptartment of Poultry whether DHA supplementation during pregnancy affects global gene expressions in fetal brain with prenatal ethanol exposure. Science, University of Methods: Pregnant Sprague Dawley rats were raised on one of the 3 experimental diets; Control (gavaged Georgia, Athens, GA with dextrose iso-caloric to ethanol), Ethanol (3g/kg BDWT, twice a day via gavage), and DHA with ethanol throughout the gestational period. The diet was semi-purified, nutritionally rich and energy dense (4.3g/kg diet). Global gene expression in the fetal brains (n=5 each group, 1-2 from each dam) at gestational day 20 were analysed by microarray. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for validating selected genes from microarray experiment. Further, protein expressions were also analysed by western blot. Results: No significant differences were identified in the fetal brains and body weights between the groups. Microarray analysis revealed that none of the transcripts were significantly altered in fetus brain (Log2(fold change) ≤ ± 2, and P > 0.05). qRT-PCR, agreed with microarray data in fold changes (FC<2 or FC>0.5). EtOH significantly (p<0.05) increased the expression of protocadherin beta 6 (PCDHB6) whereas, DHA decreased it to the levels in control group. EtOH also decreased WD repeat domain 92 (WDR92) regardless of a DHA supplementation in comparison to the control. Western blot revealed that protein expressions were also not significantly different among the groups. Conclusion: In this study, moderate ethanol exposure or DHA supplementation during pregnancy have minor impact on rat fetal brain gene expressions. The nutrient dense diet provided in this study may have mitigated the effects of ethanol. Perspectives on Health and Support Services for People With Young Onset Dementia: Investigating the Role of Antibiotics and Adherent-Invasive E. Coli in the Pathogenesis of Some Preliminary Results Crohn’s Disease Sheila Novek, Verena Menec Alexander M. Oberc1, Aline Fiebig-Comyn1, Brian K. Coombes1

Department of Background: In contrast to the growing global spotlight on dementia and aging populations, young 1McMaster University, Introduction: Crohn’s disease (CD) is an immune-mediated intestinal illness that is a significant health Community Health onset dementia (YOD) (onset before 65) has received relatively little attention. The predominant view of Hamilton, ON concern in many developed countries. CD is believed to have a complex etiology consisting of both host dementia as a disease of old age, coupled with concerns about population aging, has overshadowed the susceptibility factors and environmental insults. Multiple epidemiological studies have linked antibiotics Sciences, University of presence of dementia in younger age groups and the needs and experiences of this population. with subsequent CD diagnosis. CD is also associated with increased abundance of an unusual phenotypic Manitoba, Winnipeg, MB Specialized services for people with YOD are extremely limited across Canada and around the world. As group of Escherichia coli known as adherent-invasive E. coli (AIEC) in many patients. AIEC are characterized a result, people with YOD must navigate through health and support systems designed for older adults, by their ability to adhere and invade various cell types, to stimulate the production of inflammatory and it remains unclear whether these services meet their needs. cytokines, and also tend to be resistant to multiple classes of antibiotics. Our lab has found that chronic Purpose: This poster will present preliminary results from an ongoing study examining the existing colonisation of conventional mice with AIEC leads to intestinal inflammation and fibrosis. Our objective health and support services for people with YOD from the perspectives of people living with YOD, family was to use this mouse model to investigate the impact of antibiotics on AIEC colonisation, pathology, and members and providers. The study aims to identify barriers and facilitators to care, areas of unmet need, immune responses. and promising practices. These finding will be used to develop recommendations to improve and expand Methods: Mice were treated with various antibiotics in drinking water or by oral gavage. These mice were services for people with YOD and their families. infected with various doses of AIEC either before or after antibiotic treatment. Methods: In-depth, semi-structured interviews with people living with YOD and their family members Results: We found that certain antibiotics administered prior to infection greatly reduced the infectious will explore participants’ experiences of health and support services, areas of unmet needs, and dose of bacteria required and led to greater bacterial burden. Administering antibiotics post AIEC preferences for service delivery. Interviews with health practitioners, service providers and administrators infection similarly showed an expansion of AIEC in the feces and tissues and penetration of AIEC into will identify barriers and facilitators to diagnosis and care, gaps in services, and areas of good practice. underlying tissues. We are continuing to investigate how antibiotics alter AIEC colonisation by studying Significance: This study aims to address a critical gap in our understanding of the care experiences, the metabolic and immune consequences of antibiotic treatment. barriers to care and service needs of people with YOD and their families. Conclusion: These results show that antibiotics may create a favourable environment for AIEC colonisation in CD patients. Future work will continue investing how antibiotics impact the gut environment in the context of CD. Natural Killer Cells are Critical for Optimal Immunity to Trypanosoma Congolense Using C. Elegans for Amyotrophic Lateral Sclerosis (ALS) Drug Discovery Infection Sulin Oré Rodriguez1, 2, Claudia Maios2, Alex Parker1, 2 Chukwunonso Onyilagha1, Shiby Kuriakose1, Sam Kung1, Jude Uzonna1, 2 1Université de Montréal; Introduction: ALS is an aggressive neurodegenerative disease characterized by the loss of motor neurons 1Department of Introduction: Worldwide, several millions of people die each year from parasitic diseases, with the bulk 2Centre de recherche du in the brain and the spinal cord, leading to a progressive degeneration of the motor system, culminating in paralysis and the patient’s death within 1 to 5 years following diagnosis. Although the onset of the Immunology, University of of those deaths coming from parasitic protozoa. African trypanosomiasis (AT), also called sleeping CHUM, Montréal, QC sickness in man, is a deadly parasitic disease of humans and livestock caused by blood parasites disease still remains to be further elucidated, multiple genes have been linked to ALS, including Fused in 2 Manitoba; Department belonging to the genus Trypanosoma. Trypanosoma congolense is considered the most important sarcoma (FUS). No cure is known for ALS, and riluzole is administered only as a palliative treatment, of Medical Microbiology, pathogen for livestock and animal trypanosomiasis poses huge agricultural and economic problems in extending the patient’s lifespan by only 3 months. Hence, it is urgent to find a treatment to considerably increase patient’s survival. College of Medicine, the affected regions. Understanding the mechanisms that regulate resistance and/or susceptibility to AT could reveal novel targets for interventions that might lead to effective disease control and prevention. Aim: Our objective is to use a FUS C. elegans model of ALS to rapidly identify new neuroprotective drugs Faculty of Health Sciences, Natural killer (NK) cells are key innate immune cells that play critical roles in immunity to infectious and to gain insight into the mechanisms underlying neurodegeneration in ALS. University of Manitoba, diseases by regulating the adaptive immune response. Although NK cells are known to regulate immunity Methods: A drug screen of ~4000 compounds allowed us to identify hits that supress the ALS motordeficient motility problem phenotype of FUS-(S57Δ) mutant worms. To better assess these drugs, Winnipeg, MB to other protozoan parasites, their role in immunity to African trypanosomiasis has not been investigated. NK cells are vital source of IFN-γ and TNF-α, key cytokines that have been shown play important role in we will further investigate whether they restore neuronal morphology and/or neuronal connectivity and early control of African trypanosomes. if they extend the lifespan of the mutant worms. Afterwards, various double mutants of FUS-(S57Δ) and Methods and Results: Here, we show for the first time that mice infected with T. congolense show genes participating in proteostasis, e.g., insulin / IGF-1 signaling pathway (daf-2, daf-16), heatshock increased levels of NK cells in multiple tissue compartments. Depletion of NK cells with anti-NK1.1 response (hsf-1), protein degradation (uba-1), or the unfolded protein response mediated by the ER (ire- 1) monoclonal antibody led to increased parasitemia that was accompanied by significant reduction in will be generated to asses any phenotypic changes in the mutant FUS worm. IFN-γ production by immune cells in the spleen and liver of infected mice, suggesting that NK cells may Results: The drug screen identified 5 promising drugs that increase the motility and supresses the be contributing to optimal resistance following infection. Indeed, infected NFIL-3 deficient mice (which progressive paralysis in FUS-(S57Δ) worms. Further characterization will allow for a better assessment of genetically lack functional NK cells), on the resistant background were highly susceptible to T. congolense these drugs for potential ALS treatment. infection. These mice developed fulminating parasitemia and died significantly earlier (13 +/- 1 days) Conclusion: Because of its short life cycle, C. elegans is a great tool to rapidly discover molecules for ALS than their wild type (WT) controls (106 +/- 26 days). The enhanced susceptibility of NFIL-3 deficient mice drug discovery and development. Additionally, the elucidation of the mode of action of the compounds to infection was accompanied by significantly impaired cytokine (IFN-γ and TNF-α) response by CD4+ T may allow for a better understanding of the neuronal dysfunction of the disease. cells in the spleens and liver. Adoptive transfer of WT NK cells into NFIL-3 deficient mice before infection rescued these mice from acute death in a perforin-dependent mechanism. Conclusion: Collectively, our studies show that NK cells are critical for optimal resistance to T. congolense and its deficiency leads to enhanced susceptibility in infected mice. A Systematic Review on Hyperlexia and Its Relation to Autistic Neurocognition The Safety and Efficacy of Ketamine for Treatment-Resistant Depression: A Systematic Alexia Ostrolenk1, 2, Patricia Jelenic1, Fabienne Samson1, Baudouin Forgeot d’Arc1, 2, Laurent Mottron1, 2 Review and Meta-Analysis. (Protocol) Michael Paillé1, Abdellah Bezzahou2, Orvie Dingwall3, Rasheda Rabbani1, 4, Ahmed Abou-Setta1, 4, Ryan Zarychanski1,4 1Hôpital Rivière-des- Introduction: Four features describe hyperlexia: the presence of an accompanying neurodevelopmental Prairies, Centre disorder, advanced reading skills relative to comprehension skills, the early acquisition of reading skills 1University of Manitoba, Introduction: In Canada, 21.7% patients with depression fail to respond to at least two courses of without explicit teaching, and a strong orientation toward written material. Hyperlexia is considered one d’excellence en Troubles Winnipeg, MB; 2University antidepressants. Failing to respond to multiple antidepressants increases odds of suicide by 52% of the special abilities often observed in autism. However, little information is available on its compared to patients with responsive depression. Recent studies have shown that when administered at envahissants du of Ottawa, Ottawa, ON; neurocognitive basis. The aim of this literature review was to further our understanding of the causes and sub-anaesthetic doses, ketamine may reduce symptoms of depression and suicide ideation in humans. développement, Montreal, mechanisms involved in hyperlexia. We asked: Is the hyperlexic profile associated with autism? Are 3Neil John Maclean Ketamine’s most valuable quality is that the onset of its effects appear within 48 hours, in stark contrast perceptual strengths often observed in autism involved in the emergence of hyperlexia? Does hyperlexia QC; 2Département de Health Sciences Library, to the several weeks necessary for traditional antidepressants to take effect. However, research into this impact future development? therapy is still early, with many studies being of very small scale. Psychiatrie, Université de University of Manitoba; Methods: We conducted a systematic review on hyperlexia, including detailed information on 81 Methods: We will search CENTRAL (the Cochrane Library – Wiley), EMBASE (Ovid), and PubMed/Medline Montréal, Montréal, QC individual cases, and 22 group studies including 912 subjects, of which 315 hyperlexic. To estimate the 4George & Fay Yee Centre (National Library of Medicine), and PsychINFO (ProQuest) from inception to present using individualized proportion of autistic cases among hyperlexics, we classified the cases into four categories based on their for Healthcare Innovation, search strategies prepared for each database. In addition to electronic searching, we will search abstracts autistic traits. We described and compared reading mechanisms and acquisition in typical and autistic and conference proceedings for the following societies (2011 – 2017): the American College of Winnipeg, MB individuals. Based on the perceptual strengths observed in autism, we decided to further investigate the Neuropsychopharmacology, and the Society of Biological Psychiatry. The reference lists of relevant cerebral structures involved in reading in autism, focusing our analysis on the visual word form area narrative and systematic reviews as well as all included trials will be hand-searched for possible relevant (VWFA). We compared the regions showing superior activity in autistic relative to control groups for citations. Initially, two reviewers will independently screen the titles and abstracts of search results to word-related tasks with the location of the VWFA, established through an automated meta-analysis of 35 determine if each study meets inclusion criteria. Next, the two reviewers will independently assess the studies. full text of each report, employing a standardized data extraction tool that outlines the predetermined Results: The reported prevalence of hyperlexia in autism varies between 6-21% depending on the inclusion and exclusion criteria. The internal validity of RCTs will be assessed by using the Cochrane stringency of the criteria. Furthermore, a majority (84%) of hyperlexic cases were on the autistic spectrum. Collaboration Risk of Bias tool. Finally, a formal meta-analysis will be conducted if the data are sufficiently Autistic children often start reading before they speak. Follow-up shows examples of development of statistically and clinically homogeneous. language but the impact of hyperlexia on further communication remains unknown. The region of Discussion: This review will provide ketamine researchers with high quality data regarding its efficacy superior activity during word processing in autistics compared to controls overlaps with the VWFA. and safety, which will either encourage future, larger-scale trials, or potentially dissuade further study on Conclusion: There is a strong co-occurrence of autism and hyperlexia. The perceptual expertise system the therapy. seems strongly involved in reading in autistics. Hyperlexia often precedes the development of language in autism and there is no evidence that it prevents language acquisition or comprehension. We conclude by discussing how hyperlexic skills might be assessed in intervention strategies. Adults With Cerebral Palsy Use of Physician and Hospital Services Oxylipins Derived from the Plant Omega-3 Lipid α-linolenic Acid Can Directly Modulate Krysten A. Patrick1, Nancy L Young1, Anna McCormick2, Bruce E Oddson1, Elizabeth F. Wenghofer1 Macrophage Function Samantha D. Pauls1, 3, Peter Zahradka1, 2, 3, Carla G. Taylor1, 2, 3, Harold M. Aukema1, 3 1Laurentian University, Introduction: Cerebral Palsy (CP) is the most common cause of complex and chronic physical disability Sudbury, ON; 2Children’s of childhood. Life expectancy extended, with the majority living into adulthood. A natural gap occurred 1University of Manitoba, Uncontrolled inflammation is a causative or perpetuating factor in many human disorders including between the current knowledge and the required supports to care for the adult population with CP. Thus, Hospital of Eastern Department of Human obesity and associated metabolic diseases. There is evidence that dietary polyunsaturated fatty acids care for adults with CP often falls outside the scope of existing health care paradigms. There is an (PUFAs) can modulate inflammatory pathways and therefore provide an attractive therapeutic strategy, Ontario, Ottawa, ON imperative need to better understand how adults with CP are presently using health services. Nutritional Sciences; but a comprehensive understanding of their overall effects and specific modes of action is required. In Methods: The objective of this project is to examine patterns of health care utilization of adults with CP 2University of Manitoba, blood and tissues, PUFAs can be converted to a variety of oxygenated metabolites known as oxylipins. and compare to the rates of the age-matched general population. A secondary analysis of health Department of Physiology There has been much interest and investigation into the specific bioactivities of oxylipins derived from insurance data was completed using outpatient physician visits and hospital admissions for a 4-year marine (fish oil) omega-3 PUFAs. Conversely, until recently, the effects of plant omega-3 PUFA α-linolenic and Pathophysiology; period (2011—2014 inclusive). Participants were originally recruited from six children’s treatment centers acid (ALA) were thought to be mediated through its conversion to longer chain fatty acids rather than to in Ontario, Canada. The sample included 1007 adults with CP identified from health records. Age groups 3Canadian Centre for oxylipins. Evidence pointing to very low long chain conversion rates as well as to the relatively high were 25 to 29 and 35 to 44 at the end of the data range. Agri-Food Research in abundance of ALA oxylipins in human plasma alludes to potential direct effects. Here, we investigate Results: Annual rates of outpatient physician visits and hospitalizations per 1000 persons were computed, these effects in an important innate immune cell type: the macrophage. We have established cell culture Health and Medicine, and compared between the age groups, across sex, and with the general population by using the models of resting (M0-like), IFNγ/LPS-treated (M1-like) and IL-4/IL-13-treated (M2-like) macrophages. We negative binomial distribution. The most common reasons for hospital admission, relative frequencies of Winnipeg, MB investigate the differential effects and relative potencies of the commercially available ALA oxylipins, admissions for each reason, and mean lengths of stay were reported. Analysis is ongoing at the time of comparing them to other omega-3 and omega-6 oxylipins derived via the same pathways. A brief abstract submission. It will be updated with the complete results at the end of April 2017. exposure (30 min) to low concentrations (10nM) of the ALA oxylipin 12,13-DiHODE was sufficient to Conclusions: The project estimated the use of physician and hospital services by adults with CP. This promote the phagocytic function of M2-like macrophages. Other ALA oxylipins had no measurable effect information is not available elsewhere and is needed to guide the development of adult services and to on phagocytosis and 12,13-DiHODE itself had no effect on other macrophage subtypes, suggesting a educate clinicians regarding what to expect when a person with CP presents to their practice. specific and regulatable role. Investigations into other functional effects of ALA oxylipins and the signaling mechanisms involved are ongoing. Through this study we hope to identify and describe novel biological mechanisms by which food influences immune function, inflammation and health. The HIV-1 Accessory Proteins Nef and Vpu Hijack Membrane Trafficking to Downregulate the Immune Cell Surface Receptor CD28 E. N. Pawlak, J. D. Dikeakos

Department of Introduction: Despite tremendous effort put forth to combat Human Immunodeficiency Virus Type 1 interactions with specific host cell membrane trafficking proteins were also tested for their effect on CD28 downregulation. Similar to Nef-mediated downregulation, mutation of motifs critical for the ability Microbiology and (HIV-1), it remains fundamentally unknown how this deadly pathogen thrives in the face of host antiviral responses. The HIV-1 accessory proteins Nef and Vpu lack any known enzymatic and structural functions, of Vpu to interact with membrane trafficking adaptor proteins resulted in decreased CD28 downregulation. Immunology, Schulich but improve the ability of the virus to replicate and persist within the host by interacting with host Conclusion: These novel finding suggests that the HIV-1 virus evolved multiple means to eliminate the School of Medicine & cellular proteins, including members of the membrane trafficking machinery. One way in which Nef and immune cell activating receptor CD28 within infected cells. Moreover, our data suggests that Nef and Vpu Dentistry, Western Vpu increase the success of the virus is by downregulating cell surface receptors, such as the key immune hijack membrane trafficking, specifically adaptor proteins, to reduce CD28 in HIV-1 infected cells. This cell activator CD28. However, the mechanisms utilized by these viral proteins to downregulate CD28 project will shed light on basic cellular trafficking mechanisms, as well as increase our understanding of University, London, ON remain unknown. Nef and Vpu, attractive therapeutic targets. Hypothesis: HIV-1 Nef and Vpu are known to co-opt host cellular membrane trafficking proteins to alter the subcellular localization or levels of various host cellular receptors. We therefore hypothesize that Nef and Vpu subvert membrane trafficking pathways to downregulate CD28. Methods: In order to establish the trafficking pathways used by Nef and Vpu to downregulate CD28, CD4+ T cells were infected with pseudotyped HIV-1 viruses expressing Nef, Vpu, or mutated forms of these viral proteins. A combination of flow cytometry and immunoblotting was utilized to determine the fate of endogenous CD28 in the presence of these HIV-1 accessory proteins. Results: Upon investigating the mechanism of HIV-1 mediated CD28 downregulation, we observed a decrease in both cell surface and total CD28 protein levels, which were attributed to both Nef and Vpu. Furthermore, impaired CD28 downregulation occurred upon introduction of mutations that inhibit the ability of Nef to bind to the vacuolar ATPase, an enzyme essential for lysosomal acidification. This suggests that Nef may facilitate the transport of CD28 to a degradative compartment and is supported by an increase in total CD28 observed upon treatment with ammonium chloride, which blocks lysosomal acidification. Additionally, mutation of a motif in Nef known to facilitate interactions with the membrane trafficking protein adaptor protein 2 (AP-2), resulted in decreased downregulation of cell surface CD28, implicating AP-2 in the transport of CD28 away from the cell surface. Mutations in Vpu known to inhibit FDA-Approved Drugs Targeting EEF1A2 to Upregulate Utrophin A Translation for the B Cells Contribute to Intracellular Bacterial Infection-Mediated Inhibition of Allergic Treatment of Duchenne Muscular Dystrophy Responses Christine Péladeau, Nadine Adam, Hasanen Al-Rewashdy, Bernard J. Jasmin Ying Peng, Sai Qiao, Chunyan Zhang, Shuhe Wang, Xi Yang

Department of Cellular Introduction: Duchenne Muscular Dystrophy (DMD) is the most common genetically-inherited form of University of Manitoba, To experimentally examine the hygiene hypothesis, here we studied the effect of chlamydial infection on muscular dystrophy. One strategy to counteract the detrimental effects of DMD is upregulating utrophin and Molecular Medicine, Winnipeg, MB the development of allergic responses induced by OVA and the involvement of B cells in this process in dystrophic muscle. Multiple studies have focused on underlining transcriptional regulatory mechanisms using a mouse model of airway inflammation. The aim of this study was to examine the role of B cells in and Centre for that act upon utrophin expression in muscle. However, recent evidence lead towards the importance of Chlamydia muridarum (Cm) infection-mediated inhibition of established allergy to OVA. The results Neuromuscular Disease, studying translational modulation of utrophin. In fact, our laboratory has shown that utrophin contains showed that adoptive transfer of B cells from Cm-infected mice (B cell [Cm]), in contrast to B cells from Faculty of Medicine, an internal ribosomal entry site (IRES) in its 5’UTR that can drive cap-independent translation. Specifically, naïve mice (B cell [naïve]), significantly inhibited established allergic airway eosinophilia and mucus over the IRES trans-activating factor, eEF1A2, can regulate the translation of utrophin through this site. Thus, production. The inhibitory effect was correlated with alterations of allergen-driven cytokine and University of Ottawa, we hypothesize that eEF1A2 could be a potential target for increasing utrophin translation in dystrophic chemokine production in the lung. The inhibition was also correlated with alteration of allergen driven Ottawa, ON muscle. The objective of this study is to determine the therapeutic potential of activating translation of cytokine-producing patterns of T cells. We demonstrated that B cells were activated following chlamydial utrophin through eEF1A2 in dystrophic muscle and identifying FDA-approved drugs that could activate infection, showing both cell expansion and cytokine secretion. Moreover, B cell(Cm) produced these pathways. significantly higher levels of IFN-r and TNF than did B cell(naïve). Furthermore, blockade of IFN-r or TNF Methods: Accordingly, we designed a high-throughput screen to determine the ability of 262 FDA- significantly reversed the inhibitory effect of B cell (Cm) on established allergic airway inflammation and approved drugs to activate eEF1A2 and utrophin expression in myoblasts. The top eEF1A2 and utrophin Th2 cytokine responses. These findings suggest that B cells play a crucial role in infection-mediated activating drugs were used for a 7-day treatment of transgenic mice harbouring a bicistronic utrophin inhibition of established allergic responses, and IFN-r and TNF production by these B cells may be a major 5’UTR-IRES reporter construct. mechanism for the inhibition. Results: The screen revealed 11 drugs that could increase eEF1A2 and utrophin protein levels in myoblast cells. Based on these results, we treated our transgenic mice with the drugs that upregulated both eEF1A2/utrophin in our initial screen. Our results show that the top drugs stimulate utrophin IRES reporter activity independent of changes in utrophin mRNA levels, suggesting regulation of utrophin expression through translational events. In addition, western blotting revealed the ability of these drugs to increase endogenous eEF1A2 and utrophin protein levels in the tibialis anterior muscle of the transgenic mice. Conclusion: Altogether, this study provides better understanding of the mechanisms regulating utrophin translation in skeletal muscle. Ongoing work consists of treating dystrophin-deficient mice with the top drugs to highlight their potential for the treatment of DMD. Proteomic Methods to Simultaneously Monitor Host Immunity and Its Modulation by Microbiome Composition and Function Michelle Perner1, 2, Laura Noël-Romas1, 2, Kenzie Birse1, 2, Max Abou1, 2, John Schellenberg1, 2, Jennifer Butler1, Stuart McCorrister3, 1, Lyle McKinnon2, Garrett Westmacott3, Adam Burgener1, 2, 4

1National HIV and Background: The composition of the microbiome has a large impact on mucosal barriers and crispatus CST’s, including increased leukocyte movement, decreased cell proliferation, and changes to Retrovirology Labs, JC Wilt consequences for opportunistic co-infections and mucosal health. However advanced tools to bacterial metabolic pathways. These effects may have important implications in reproductive health simultaneously evaluate host-bacterial interactions, which could be relevant for therapeutic efficacy, are outcomes, including HIV infection and therapeutics targeting mucosal surfaces. Infectious Disease Centre, not readily available. We developed a mass spectrometry (MS)- based workflow to simultaneously Public Health Agency of evaluate host immune parameters in relation to the microbiome in a large-scale study. Canada; 2University of Method: 688 cervicovaginal lavage samples from the CAPRISA-004 trial were analyzed by label-free tandem-MS in 18 separate runs. Women were assigned community state types (CST’s) by proportion of Manitoba, Winnipeg, MB; bacterial proteins. Unpaired t-tests, hierarchical clustering, and pathways analysis were used to identify 3Mass Spectrometry Core host differences between CST’s using the L. crispatus CST as the reference. Facility, National Results: Batch reproducibility of the 18 MS runs was high (p<0.00001, rs 2=0.915), with 5.4% covariance between runs. Of 2,102 host factors detected, 576 (27.4%) were identified in all MS runs for downstream Microbiology Laboratory, analysis, including pathways important for innate immunity, leukocyte migration, and wound healing. In Public Health Agency of total, 3,334 bacterial proteins representing 188 species/strains were quantified, resulting in 7 major CST’s, Canada 4Karolinska dominated by L. crispatus (9.3%), L. iners (41.4%), Gardnerella (23.7%), Prevotella (8.1%), Gardnerella & Institutet, Stockholm, SE Lactobacillus (6.7%), Mobiluncus (3.2%), and Pseudomonas (2.6%). Overall, 184 host pathways were differentially expressed in non-L. crispatus CST’s. Neutrophil movement pathways were most significantly increased, in CST’s with Gardnerella (p=8.3E-5), then Prevotella (p=1.2E- 6), Mobiluncus (p=0.0049), and Pseudomonas (p=2.4E-5). Cell proliferation pathways decreased most with Pseudomonas (p=1.8E-6), then Gardnerella & Lactobacillus (p=5.2E- 5), Mobiluncus (p=1.5E-6), and Prevotella (p=1.1E-10) CST’s. Of 402 KEGG pathways identified, significant alterations in bacterial metabolism were evident in non- Lactobacillus women, including a 36.5 fold increase in detection of pentose phosphate pathway proteins (p=2.0E-80). Conclusion: Utilizing an integrated proteomics approach, we identified novel pathways altered in non-L. Vitamin A Deficiency Induces FAS-Like Craniofacial and Neurodevelopmental Malformations: A New Molecular Etiology of FASD in vivo B. Petrelli1, A. Ozturk1, M. Pind1, H. Ayele1, A. Fainsod2, GG. Hicks1

1Department of Introduction: Prenatal alcohol exposure (PAE) resulting in Fetal Alcohol Spectrum Disorder (FASD) is the Conclusion: Taken together, our data provide in vivo evidence that strongly supports retinoic acid Biochemistry & Medical most common cause of neurodevelopmental impairments in the western world, with a prevalence of deficiency as a major molecular etiology of craniofacial malformations associated with FASD. The finding 1-2% in Canada. It is well established in Xenopus FASD models that a single exposure to alcohol during suggests Vitamin A supplementation may significantly reduce or prevent FASD outcomes in children with Genetics; Regenerative gastrulation is sufficient to induce the developmental defects associated with FAS. PAE. Medicine Program, Hypothesis: Acute ethanol exposure overwhelms the aldehyde metabolic enzymes that would normally Faculty of Medicine, convert retinol (Vitamin A) to retinoic acid (RA). We hypothesize that PAE reduces RA levels during critical developmental stages in early gastrulation and this aberration drives the later craniofacial malformations University of Manitoba, associated with FAS. Winnipeg, MB; Methods: To biochemically mimic the alcohol-induced RA deficiency in vivo, we genetically engineered a 2Department of mouse expressing Cyp26A1-eGFP from the endogenous Goosecoid (Gsc) promoter. The Gsc promoter dictates spatial-temporal expression to the Spemann Mangold Organizer at the start of gastrulation. Developmental Biology Cyp26A1 degrades endogenous RA in these cells, mimicking the reduced RA levels induced by acute and Cancer Research, alcohol exposure and dysregulating the induction of neural crest cells. Institute for Medical Results: Gsc:Cyp26A1 mice were derived by germline transmission; F1 mice are born with a Mendelian Research Israel–Canada, ratio of 0.75:1 (het:wt , n=208). This loss of mutant embryo viability may reflect the prevalent miscarriages observed in human pregnancies with PAE. Gsc:Cyp26A1xRARE-LacZ E8.5 embryos show reduction in RA Faculty of Medicine, activity in the frontonasal prominence region (LacZ expression in early face and forebrain). Mutant Hebrew University of embryos also demonstrate body-axis developmental variation, indicating early developmental Jerusalem, Jerusalem, perturbation of retinoic acid pathways in FASD relevant tissues (n=48). E18.5 embryos were next examined using scanning electron microscopy to demonstrate mutant embryos have sentinel FASD Israel craniofacial malformations: larger philtrum-to-philtrum-lip length ratio, smaller bigonial line width, and smaller whisker pad area compared to WT littermates (n=66). Gsc:Cyp26A1 mice also develop craniofacial malocclusions at significantly higher rates than WT littermates (12.5% vs 0.04%; n=208 and 3711, respectively). Adeno-Associated Virus Type 6 (AAV6) Vector Transduction of Human Dipeptidyl Oxysterol-Binding Protein-Related Protein 4 (ORP4) Is Required for Maintenance of TGN Dipeptidase 4 (DPP4) into Mice for Development of a Small Animal Model for Middle East Morphology and is Regulated by Phosphorylation Respiratory Syndrome Coronavirus (MERS-CoV) Antonietta Pietrangelo1, Neale Ridgway1, 2 Yvonne Pho1, 2, Sarah Wootton3, Anders Leung2, Darwyn Kobasa1, 2 1Department of Introduction: Oxysterol binding protein (OSBP) and related proteins (ORPs) constitute a 12-member family of mammalian lipid-binding and transport proteins. ORP4 is expressed as a fulllength protein 1Department of Medical Introduction: Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic virus that has Biochemistry and (ORP4L) as well as two N-terminally truncated variants (ORP4M and ORP4S) that differ from ORP4L in the emerged in the last 5 years causing respiratory disease in humans ranging from mild cold-like symptoms Microbiology, University Molecular Biology, absence of an intact pleckstrin homology domain (PHD). Cholesterol, oxysterols and phosphatidylinositol- to severe pneumonia. Since it was first identified in 2012, 1,905 laboratory confirmed cases with 677 of Manitoba; 2Special Dalhousie University; 4-phosphate (PI4P) compete for binding to the ORP4 C-terminal lipid-binding domain (OHD). ORP4 is deaths have been reported, many of which resulted from nosocomial spread between infected patients required for cell proliferation and is a target for steroidal antineoplastic drugs called ORPphilins. The Pathogens, Public Health and health care staff. At a lethality rate of over 30%, development of vaccines and treatments against 2Department of primary function of ORP4 is unknown. Agency of Canada, MERS-CoV is vital. Assessment of these potential intervention strategies requires an adequate animal Pediatrics, Dalhousie Methods: Confocal immunofluorescence microscopy was performed on HeLa cells transiently expressing model. Previous studies observed clinical symptoms of infection in rhesus macaques and common Winnipeg, MB; University, Halifax, NS ORP4 cDNAs or a lentiviral ORP4 shRNA. Quantitative fluorescence image analysis was performed using marmosets, which develop mild to severe disease, respectively. However, using primate models for 3 ImageJ. Recombinant wild-type and mutant ORP4L were expressed and purified from insect sf21 cells. Department of vaccine and therapeutic evaluation presents significant ethical and economic challenges and may not be Lipid extraction assays were performed with PC:PE:PS:lactosyl- PE:[3H]cholesterol (59:20:10:10:1) or Pathobiology, University available to all institutions. In contrast, smaller animal models such as wild type mice and hamsters are PC:PE:PS:lactosyl-PE:[32P]PI4P (59.5:20:10:10:0.5) liposomes made by extrusion. not permissive to MERS-CoV infection, which may be due to low levels or lack of functional interaction of , Guelph, ON Results: A pool of ORP4L localizes to the trans-Golgi network (TGN) in a PI4P-dependent manner between DPP4 cellular receptor and MERS-CoV spike, the viral receptor protein. We are interested in following oxysterol treatment. Sterol-binding deficient ORP4L (Δ501-505) displays increased sterol- transducing the airway in mice with human DPP4 in order to raise receptor levels and provide a DPP4 that induced TGN localization, suggesting that ORP4L may play a role in PI4P egress from the TGN. ORP4 is permissive to Spike binding to allow efficient infection. silencing induced mild TGN vesiculation but no defect in cholera toxin trafficking. The ORP4 OHD is Methods: In this study, we inserted a human DPP4 gene into an Adeno-associated virus serotype 6 phosphorylated at 4 serine residues in a MAPK consensus motif. A phosphomimetic mutant (ORP4L-S (AAV6) vector (AAV-hDPP4) and were able to purify and obtain high titers of the virus following 762- D) displayed increased PI4P and cholesterol extraction from liposomes and enhanced vimentin transfection in 293T cells. 768 interaction, resembling the truncated ORP4 variant ORP4S. The corresponding alanine mutant displayed Expected results: We intend to intranasally inoculate Balb/C mice with AAV-hDPP4 virus and then wild-type ORP4L lipid binding and localization, suggesting that the N-terminal PHD has an autoinhibitory challenge with MERS-CoV four weeks post transduction. If transduction proves to be successful, we role that is relieved by phosphorylation at the OHD. would observe MERS-CoV infection in the mice. Conclusion: ORP4 could be a sterol-regulated lipid-transport protein or PI4P coincidence detector at the Conclusion: This result would offer a new strategy for evaluating newly developed vaccines and Golgi. Phosphorylation of the MAPK consensus motif regulates lipid binding and localization, possibly by treatments. relieving autoinhibition. Identifying the ORP4 kinase will elucidate the function of ORP4 and its role in growth regulation. The Rare Congenital Anomaly Omphalocele: A Review of 25 Years of Management An Innate Defence Regulator Peptide Alleviates Allergic Airway Inflammation by Chantalle Pitre Regulating the Cytokine IL-33 Hadeesha Piyadasa1,2, Mahadevappa Hemshekhar1, Anthony Altieri1,2, Sujata Basu3,4, Anne M. van der Does5, University of Manitoba, Introduction: Omphalocele is a rare congenital anomaly that occurs approximately once in every 5,000 Andrew J. Halayko3,4,6, Pieter S. Hiemstra5, Neeloffer Mookherjee1,2,4,6 Winnipeg, MB births and is defined by herniation of abdominal contents into the base of the umbilical cord. Omphalocele is often categorized as ‘small’ versus ‘giant’, where giant omphalocele is defined as a defect 1Manitoba Centre for Asthma is one of the most common chronic inflammatory respiratory diseases. Increased risk of ≥ 5cm and containing liver. The main treatment options include: (i) Primary fascial closure, (ii) Staged Proteomics and Systems pulmonary infections in chronic asthma is associated with altered expression of antimicrobial host closure (silo, skin flap, or mesh), and (iii) Delayed closure. Biology, Department of defence peptides (HDPs) in the lungs. Small synthetic derivatives of HDPs, known as innate defence Objectives: 1. Evaluate the various techniques chosen for management of small versus giant omphalocele Internal Medicine, University regulator (IDR) peptides, exhibit immunomodulatory functions and control infections. In this study, we & the complications that arose for each 2. Compare the above outcomes with current published literature of Manitoba, Winnipeg, MB, examined the effects of exogenous administration of an IDR peptide, IDR-1002, in a house dust mite Study Design: Cases of omphalocele that underwent evaluation between January 1990 and December Canada. 2Department of (HDM)-challenged murine model of allergic asthma, and in human primary bronchial epithelial cells 2015 at Health Sciences Centre (HSC) Children’s Hospital. Inclusion criteria was restricted to live births Immunology, University of (PBEC). Administration of IDR-1002 significantly blunted HDM challenge-induced airway with a plan for postnatal repair and patients who had their initial treatment for the defect at HSC. hyperresponsiveness (AHR) to methacholine in mice. IDR-1002 significantly suppressed lung leukocyte Results: Among the 28 cases, 14 patients were categorized as having a small omphalocele. Of these 14 Manitoba, Winnipeg, MB, 3 infiltration by ~40 %, in particular eosinophils (>45%) and neutrophils (> 35%), and reduced airway patients, 78.6% underwent primary closure, 7.1% staged repair, and 14.3% delayed repair. No complications Canada. Department of Physiology and goblet cell hyperplasia. Concomitant with these effects, IDR-1002 significantly suppressed the HDM occurred among the small omphalocele patients. 14 patients were categorized as having a giant challenge-induced IL-33 protein production in the lungs. Inhibition of IL-33 production in HDM- Pathophysiology, University omphalocele. Of these 14 patients, 14.3% underwent primary closure where 50% of these patients challenged mice was essential for the anti-inflammatory activity of IDR-1002, as exogenous administration of Manitoba, Winnipeg, developed complication. 57.1% had a staged repair where 37.5% of these patients developed of recombinant IL-33 mitigated this effect. In contrast, exogenous administration of IL-33 was not Manitoba, Canada. 4Biology complication. 28.6% were treated with a delayed repair technique, 0% of these patients developed sufficient to mitigate the ability of IDR-1002 to protect against HDM-induced AHR, suggesting that the of Breathing, The Children’s complication. peptide controls AHR by mechanisms independent of IL-33 production. Further interrogation in human Conclusion: Primary fascial closure is the preferred technique for small omphalocele reduction at HSC Hospital Research Institute PBEC cultures showed that IFNγ/TNFα-induced IL-33 protein production was abrogated in the presence and in current published literature. Primary fascial closure is an excellent technique for small omphalocele of Manitoba, Winnipeg, MB, of IDR-1002. In contrast, a related peptide IDR-1 did not suppress the production of IL-33, thus 5 as it requires only one procedure and few complications arise. Staged closure is the preferred technique Canada. Department of demonstrating the specificity of IDR-1002 in regulating IL-33. Moreover, further mechanistic studies for giant omphalocele reduction at HSC and in current published literature. This technique is often Pulmonology, Leiden showed that IFNγ-induced IP-10 or IRF-1 production was not suppressed by IDR-1002, suggesting that chosen as it produces less complications than that seen with primary fascial closure in giant omphalocele. University Medical Center, mechanisms underlying the ability of IDR-1002 to suppress IL-33 production maybe independent of the Delayed repair was less frequently employed for giant omphalocele despite having the lowest Leiden, The Netherlands. canonical IFN-γ pathway. This is the first study to demonstrate the ability of an IDR peptide in the control complication rates of all three techniques. 6The Canadian Respiratory of allergic asthma in-vivo. As IL-33 is essential in steroid-refractory severe asthma, the results from this Research Network, Ottawa, study suggest that the peptide IDR-1002 has the potential to control steroid-refractory asthma ON, Canada pathobiology. The Effects of Oxytocin on the Immune Cytokine Profile of Vaginal, Ectocervical, and B Cells from Chlamydia Infected Mice Inhibits OVA Induced Acute Allergic Airway Endocervical Epithelial Cells Inflammatory Responses Via IFN-r–Secreting Mechanisms Andrew M. Plesniarski1, 2, T. Blake Ball1, 2, Ruey-Chyi Su1, 2 Sai Qiao, Ying Peng, Chunyan Zhang, Shuhe Wang, Xi Yang

1Department of Medical Introduction: Oxytocin, a neural hormone, is released during sexual activity to help with the contraction University of Manitoba, Our previous study has shown that Chlamydia lung infection can inhibit local eosinophilic inflammation Microbiology & Infectious of smooth muscle, and facilitate intimacy. This study examined how oxytocin may impact female Winnipeg, MB induced by OVA sensitization and challenge. In this study, we explored the role of B cells in chlamydial reproductive health by modulating immunologic regulation at the female genital tract (FGT). As infection-mediated modulation of OVA-induced acute allergic responses. The results showed that B cells Diseases, University of inflammation can predispose susceptibility to infection, this study tested the hypothesis that oxytocin isolated from chlamydia infected mice (IBCs), comparing to those from naive mice (NBCs), could Manitoba; 2National HIV promotes a less inflammatory environment at the FGT, and, hence, reduces cellular susceptibility to effectively inhibit airway eosinophilia and mucus overproduction in acute allergic responses to OVA. the and Retrovirology sexually transmitted infections such as HIV. remission caused by IBC was also accordant with the change of Th1/Th2 balance. We also demonstrated Methods: Cell lines (Vk2 (Vaginal), Ect1 (Ectocervical), and End1 (Endocervical)) of FGT origin were the changes of B cells subpopulation and proliferation after chlamydial lung infection which showed a Laboratories, JC Wilt treated with oxytocin in conjunction with an immune stimulus to analyse inflammation. Cells were grown Be1 dominant phenotype following chlamydia challenge. Moreover, as IBCs were examined to produce Infectious Diseases as monolayers in Keratinocyte Serum Free Medium (Gibco). Oxytocin (a titration of 1-10 000 pg/mL) was higher level of IFNγ and TNF than NBCs, which played a positive role in protect host from allergic Research Centre, Public used in conjunction with either LPS (400 ng/mL), Poly(I:C)/LyoVec (1 μg/mL), or alone. Culture response, it suggest that B cells from chlamydia lung infected mice could inhibit OVA-induced acute supernatants were collected at 1, 3, 8, and 18 hours post-treatment, and cells were lysed for RNA allergic response via developing to Be1 phenotype. Health Agency of Canada, extraction. Supernatants were tested with bead-based cytokine assays (Milliplex), and RNA was tested by Winnipeg, MB qPCR after reverse transcription. Results and Discussion: Oxytocin alone had no effect on the expression of immunologic genes. Following 3 hours of stimulation cytokines such as IL-6 and IL-1β (among the 30 genes examined) were up regulated by high levels of oxytocin. Contrarily, low levels of oxytocin, more similar to that found in vivo, seemed to downregulate the expression of the pro-inflammatory cytokines IL-6 and IL-1β but only in the context of an immune stimulus. Significance: These results provide the first evidence that oxytocin may have modulatory effects on the immunologic environment at the FGT and a potential impact on female reproductive health and cellular susceptibility to sexually transmitted infections. Catestatin: A Novel Anti-Inflammatory Peptide Against Gut Inflammation MF Rabbi1, N Eissa1, PM Munyaka2, L Kermarrec1, A Khafipour1, E Khafipour2, JE Ghia1, 3

1Department of Introduction: While catestatin (CTS), a peptide derived from pro-hormone Chromogranin A (Chga), is cytokine (IL-6, IL-1β, TNF-α) expression through the STAT3 pathways were abrogated in the presence of Immunology and Internal widely known as an anti-microbial peptide in vitro, the role of this peptide on the immune system is CTS. Both DSS and CTS therapy resulted in distinct microbial composition (β diversity) when compared unknown. In the intestine, enterochromaffin cells (EC) are the major source of Chga and its derived to vehicle treated mice, and the effect was more prominent in fecal samples. However, CTS was not able Medicine section of peptides. EC cells number and ChgA levels are alerted during ulcerative colitis (UC) and Crohn’s disease to alter the gut microbial composition in colitic mice. Gastroenterology; (CD). Using two models mimicking UC and CD, the aim of the study was to determine 1) whether CTS Conclusion: These findings suggest that CTS possess anti-inflammatory effect through the downregulation 2Department of Animal influences acute or chronic reactivation of colitis; 2) whether the CTS can regulate the macrophages and/ of M1 macrophage population and pro-inflammatory cytokine secretion associated with a moderate or the gut microbiota. effect on physiological gut microbiota composition. CTS might open a novel therapeutic avenue in the Sciences; 3Inflammatory Methods: Acute gut inflammation was induced in C57BL/6 mice (7-8 weeks) by administrating 5% near future for UC and CD patients. Bowel Disease Clinical & dextran sulfate sodium (DSS) in their drinking water for 5 days or by intrarectal administration of 4 mg of Research Centre, 2, 4 dinitrobenzene sulfonic acid (DNBS) in 30% Ethanol for 3 days. Chronic reactivation of colitis was induced using 4 cycles (DSS) (4%, 2%, 2%, 4%, 5 days each followed by 11 days of rest). Preventive or University of Manitoba, prophylactic CTS (1.5 mg/kg/day) treatments and/or its distal and proximal portion of the peptide along Winnipeg, MB with the scrambled peptide was injected intrarectally one or two days before colitis induction or reactivation and continued till sacrifice. Disease activity index was determined daily and upon sacrifice macro-, microscopic score, pro-inflammatory cytokine (IL-6, IL-1β, TNFα and/or anti-inflammatory cytokine (IL-10, TGF-β) level and STAT-3 intracellular pathway were determined. M1 (iNOS and MCP-1) and M2 (ARG-1 and YM-1) macrophage marker expression were determined. In vitro, naïve peritoneal macrophages isolated from naïve mice and treated with CTS (10-5M) were exposed to LPS (100ng/ml) or IL-4/13 (20ng/ml) for 12h to polarize M1 or M2 macrophages respectively. Pro and anti-inflammatory cytokine secretion, the intracellular pathway along with M1 and M2 macrophages marker expression were determined. Feces and mucosa-associated microbiota (MAM) samples were collected and the V4 region of 16s rRNA was subjected to Miseq Illumina sequencing. Results: CTS treatment significantly improved gut inflammation in both acute colitis models which was further validated using the reactivation model. CTS treatment significantly reduced M1 macrophage marker (iNOS, MCP-1) expression. In vitro, M1 macrophage markers along with pro-inflammatory Exploring Potential Consequences of Obesity Prevention on Psychological Health Global Interactome Analysis of Influenza A Virus NS1 Reveal Essential Host Factors in Amanda Raffoul, Sharon I. Kirkpatrick Viral Replication Niaz Rahim, Kevin M. Coombs University of Waterloo, Introduction: Rates of overweight and obesity among Canadian adults have nearly doubled since 1981. Waterloo, ON A dominant weight-centric framework has led to significant emphasis on reducing excess weight and Department of Medical Introduction: Influenza A virus (IAV) is an intracellular parasite. It is assumed that viral proteins interact mitigating its physical consequences, but less attention has been paid to the psychosocial implications Microbiology, Manitoba with different host factors to favour viral replication process and evade host cell defense systems. The of being affected by overweight or obesity. Research suggests that individuals with obesity experience Influenza A viral non-structural protein 1 (NS1) is a multifunctional protein expressed to high levels in greater body dissatisfaction, increased psychological stress and anxiety, poorer quality of relationships, Centre for Proteomics and infected cells. Previous studies identified some NS1 interacting host proteins by expressing recombinant and increased risk of engaging in harmful behaviours. Nevertheless, obesity prevention initiatives in Systems Biology, NS1 protein. We performed global screening for additional host factors that interact with native NS1 and Canada primarily focus on outcomes related to weight and associated physical conditions. The potential University of Manitoba, determined how some of these proteins affected IAV growth. of these interventions to elicit stronger societal weight bias, through increased blame on overweight and Methodologies: Three different broadly cross reactive anti-NS1 monoclonal antibodies generated in our obese individuals, is unknown, but concerning to researchers in public health. We aim to investigate Winnipeg, MB lab were used to co-immunoprecipitate (co-IP) NS1 from IAV-infected cell lysates. NS1 interacting experiences of weight bias and stigma and psychological health and well-being among young Canadian proteins were detected by mass spectrometry (MS) analysis. The roles of these interacting factors in viral adults in relation to population-level obesity prevention strategies. Using longitudinal data from a cohort replication were determined by RNAi experiments. RT-PCR, super-resolution structured illumination study, we will investigate the prevalences of internalized weight bias and experienced weight stigma and microscopy and electron microscopy were performed to determine the mechanism of NS1- interacting associations with stress, depression, and anxiety; changes over a three-year period in experiences of host factor involved in viral replication. weight bias and stigma in relation to weight-related policies; and changes over time in weight-related Results: 183 NS1-interacting host factors were consistently identified by co-IP and MS in at least two perceptions and attitudes. biological replicates. NS1 interacting host factors were analyzed with the Database for Annotation, Methods: This research will be conducted using data from an ongoing cohort study of young adults aged Visualisation and Integrated Discovery (DAVID). Most proteins clustered into different cellular pathways, 16 to 30 years residing in Vancouver, Edmonton, Toronto, Montreal, and Halifax. Participant data, collected molecular functions and biological processes such as mRNA splicing, processing of capped intron- online, include measures of weight bias and experienced stigma, psychological health, and weight- containing pre-mRNA and gene expression. Among these, we detected 124 novel NS1-interacting related behaviours and attitudes. These individual-level data are contextualized by on-going scans of proteins not previously identified. We targeted 107 NS1 interacting host factors based on their novelty, weight-related municipal-, provincial- and federal-level policies. We will employ multilevel longitudinal functions and high scores in MS to check their effects in viral replication by RNA interference (RNAi). RNAi regression analyses to assess changes over time in experiences of weight bias/stigma and related screening then identified 11 NS1-interacting host factors as vital for IAV replication, knocking down of behaviours among subgroups characterized by gender and race/ethnicity in relation to policies. which inhibited viral replication >70%. Among these, Nuclear mitotic apparatus protein 1 (NUMA1) was Implications: No known study has investigated the potential impact of weight-related policies on overall involved in IAV maturation steps. psychological health, experienced weight stigma, and weight-related behaviours among young adults, Conclusion: Novel and essential NS1-interacting host factors were identified in this study where NUMA1 both overweight/obese and ‘normal’ weight. Through the use of a systems lens to investigate potential interacts with IAV-NS1 to facilitate viral maturation. This study may identify alternative non-viral targets consequences of a weight-centred landscape on these constructs, this study will inform future health to develop new antiviral therapies. policy. The Role of a Novel Subset of Mesencephalic Neural Crest Derived Cells in Cerebellar Delineating the Role(s) of the JLP Scaffolding Protein in Regulating NK-Cell Functions Nuclei Development in Mice Rahmat Rahman1, Manli Zhang1, Aparajita Pathak1, Jolly Hipolito2, Francis Lin2, Sam K P Kung1 Maryam Rahimi Balaei, Karen Bailey, Niloufar Ashtari, Xiaodan Jiao, Hassan Marzban 1Departments of Introduction: Natural Killer (NK) cells are effector cells of the innate immune system. These lymphocytes Department of Human Introduction: During cerebellar development, cerebellar nuclei (CN) neurons and Purkinje cells are the Immunology, University of mediate spontaneous cytotoxicity, and produce cytokines and chemokines upon recognition of cancerous or viral infected cells. Mitogen-activated protein kinase (MAPK) is an important signaling Anatomy and Cell Science, earliest born among the different neuronal subtypes. Purkinje cells are the sole output of the cerebellar Manitoba; 2Physics and cortex and they project to the CN. The CN represents the main output of the cerebellum, which is pathway that regulates cellular growth and differentiation. The MAPK kinase 4-ERK-p38-JNK signaling The Children’s Hospital generated from the rhombic lip. In this study, we investigated new origin for part of the CN neurons Astronomy, University of pathways contribute to the induction of NK-cell cytotoxicity and cytokine (IFN-γ, TNF)/chemokine Research Institute of during early cerebellar development. Manitoba, Winnipeg, MB releases. Scaffold proteins act as interaction platform or signal processing hubs to control the flow of cellular signaling networks in specific cellular responses. The JNK-associated leucine zipper protein (JLP) Manitoba (CHRIM), Max Methods: We used whole mount/section immunohistochemistry, cerebellar culture, Western blot, and embryonic cultures to examine the origin of a new subset of CN neurons from the mesencephalon during is a scaffold protein in MAPK signalling. JLP tether upstream signaling molecules JNK and p38 and also Rady College of Medicine, early cerebellar development. interacts with transcriptional factors Max and c-Myc. Delineation of any scaffolding protein in the Rady Faculty of Health Results: Our results show that a subset of CN neurons, which are immunopositive for α- synuclein (SNCA) regulation of these cellular processes has not been elucidated in NK cells. Rationale and Hypothesis: Recent publications from the Kung laboratory reported for the first time the science, University of and orthodenticle homeobox 2 (Otx2, mesencephalic marker), originate from the mesencephalon and cross the isthmus toward the rostral end of the nuclear transitor zone. Interestingly, double immunostaining importance of JLP in regulating B and dendritic cell functions, thus supporting its boarder roles in Manitoba, Winnipeg, MB of the SNCA with Otx2 or p75 neurotrophin receptor (p75ntr) suggests that these cells are probably regulating immune cell functions. We hypothesized that the JLP scaffolding protein is a critical modulator derived from neural crest cells. We also showed that this population of neurons with nerve fibers of NK-cell functions. terminates at the subpial surface of putative lobules VI/VII. The SNCA+/Otx2+/p75+ cells, which divide Methods: We used established flow cytometry, NK cytotoxicity, cytokine and migration assays in the the cerebellar primordium into rosterodorsal and caudoventral compartments, show increased cleaved analyses of NK cell development, and NK-cell effector functions in the JLP+/+ and JLP-/- mice. caspase-3 (CC3+) activation. Results: Our preliminary analyses suggested that NK-cell development was intact in the JLP-deficient Conclusion: These results suggest that early CN neurons originate from the mesencephalic neural crest mice. Both JLP+/+ and JLP-/- NK cells could be activated by IL-2 and IL-15 in vitro, and be further activated population; contrary to popular opinion that Otx2 has been shown to be involved in prosencephalon and to induce cell-mediated cytotoxicity and IFN-g responses upon interaction with a prototypic NK-sensitive mesencephalon establishment, but not the rhombencephlon. The p75+immunopositive cells which tumor target. However, we observed that JLP-/- NK cells were impaired in IFN-g responses upon PMA/ show activation of caspase-3 during embryonic stage suggest their role in proliferation, differentiation, ionomycin stimulation, and also in migratory responses toward soluble factors produced by mature survival and axonal guidance. The presence of migratory mesencephalic derived neural crest cells in the dendritic cells. nuclear transitory zone suggests that these neurons/fibers have a regulatory role as a signaling center Conclusions and future direction: I will examine JLP-/- NK-cell development, cytotoxicity and cytokine that may play as an intrinsic organizer during early cerebellar development. responses in details to delineate the role of JLP in regulating NK-cell functions. Understanding the Future Direction: In future, TUNEL assay plus the cooperative role of sortilin and p75ntr will be signaling mechanisms underlying fine regulation of differential NK effector function may reveal novel investigated to show the fate of this subset of neurons and also neuronal migration in mice embryo will therapeutic targets for NK-based immunotherapy. be followed by Qtracker injection in mesencephalon with track tracing. The Effect of Docosahexaenoic Acid Supplementation During Development on Male The Role of Mast Cells in Bone Healing Reproductive Function in Rat Exposed to Prenatal Alcohol Jose Ramirez-GarciaLuna1, 2, Daniel Chan1, 4, Robert Samberg1, Mira Abou-Rjeili 1, 3, Timothy Wong1, 4, Ailian Li1, Thorsten B. Fatemeh Ramezani Kapourchali, Xavier Lieben Louis, Elaheh Nosratmishekarlou, Michael Eskin, Miyoung Suh Feyerabend5, Hans-Reimer Rodewald5, Janet E Henderson1, 2, 3, Paul A. Martineau1, 2

Department of Human Although male infertility is present in adult life, strong body of evidence supports its possible fetal 1Bone Engineering Labs, Introduction: There is a pressing need for innovative approaches to promote bone repair in cases where Nutritional Science, determinants. Both alcohol and dietary docosahexaenoic acid (DHA) are known to modulate the Research Institute-McGill healing is predicted to be compromised, as an estimated 25% of fractures in otherwise healthy individuals testicular lipid composition, influencing normal sperm and testosterone production, thereby affecting fail to heal. Mast cells are a subset of immune cells with widespread functions in wound healing that University of Manitoba, University Health Centre; male fertility. This study investigated the effect of prenatal alcohol and dietary DHA on testicular include modulation of inflammation and angiogenesis, and might therefore represent a therapeutic Winnipeg, MB maturation and function in a rat model. Forty pregnant Sprague-Dawley rats randomly assigned to 2Experimental Surgery, target to promote bone repair. The objective of this study was to investigate bone healing in Cpa3Cre/+ receive either ethanol (3g/kg, twice a day by gavage) or dextrose, isocaloric to ethanol, throughout McGill University; mast cell deficient mice, which lacks mast cells in the absence of any other cellular deficiency. Our pregnancy. Half of each group was fed either control or DHA supplemented diet (1.4%, w/w fatty acids). hypothesis is that mast cells play a pivotal role in bone repair by coordinating the recruitment and 3Experimental Medicine, Respective diets were continued for the pups. Samples were collected from fetus, neonates, weaned, activity of vascular and skeletal cells to the site of healing. pre-puberty, and young adult, at gestational day 20 (G20), postnatal day 4 (P4), P21, P49 and P90, Faculty of Medicine, McGill Methods: Bone repair in Cpa3Cre/+ and wild type (WT) mice was quantified in uni-cortical defects drilled respectively. Dietary DHA significantly augmented serum levels of testosterone at G20, accompanied by University, Montreal, QC; in both femurs. Cohorts were euthanized at 5, 14, 28 and 56 days post-operative (PO). Quantity and an improved normal sperm morphology at P90. Although DHA diet showed a positive effect on testicular quality of the mineralized tissue and re-vascularization of the defect were analyzed using micro-CT. The 4Biotechnology Program, histological markers until puberty (P49), the height of seminiferous epithelium diminished significantly bones were then embedded in PMMA plastic for structural analyses or decalcified and embedded in at P90. Compared to the control groups, the changes in the pattern of testosterone levels during University of British paraffin for cell/molecular analyses. Acidified toluidine blue (aTB) was used to identify mature mast cells development from P4 to P90 in alcohol group indicated a delay in the surge of testosterone in puberty. Columbia; Vancouver, BC; 5 with purple granules and mast cell (MC) tryptase immunohistochemistry to identify cells committed to Prenatal alcohol induced a significant increase in expression of genes involved in testicular polyunsaturated Division of Cellular the mast cell lineage. Thin sections from undecalcified femurs were stained with Von Kossa/Toluidine fatty acids and seminolipid synthesis at P90. There were no significant changes in sperm function in both blue (VK/TB) to distinguish mineralized from un-mineralized tissue. The quantity and spatial distribution treatments at P90. Immunology, University of of osteoblasts, osteoclasts, vascular endothelium, and macrophages was assessed using alkaline In conclusion, it is indicated that early dietary DHA during development may have a positive effect on Heidelberg, Heidelberg, DE phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), anti-CD34 and anti-F4/80 antisera male fertility with impacting on sperm morphology in adulthood, probably by influencing the respectively. Comparisons were made by Wilcoxon-sum rank test at 95% confidence level. testosterone levels in fetal period. Prenatal alcohol exposure affects minimally on sperm parameters in Results: The data indicate 1) in WT mice, aTB positive mature mast cells were seen on the periosteal adult life. surface proximal to the defect at 5d PO and in bone marrow adjacent to vascular channels of regenerating bone from 14d onwards. aTB positive cells were never seen in bones harvested from Cpa3Cre/+ mice; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organized in Cpa3Cre/+ mice compared with WT mice; 3) the defects in Cpa3Cre/+ mice were associated with little HIV Envelope Enhances Progeny Virus Infectivity Through Downregulating HDAC10 Expression Xiozhuo Ran1, Zhujun Ao1, Abdulaziz Almuqruin1, Adriana Trajtman2, Yoav Keynan2, Xiojian Yao1 change in bone anabolic activity, and 4) biphasic alterations in osteoclast and macrophage activity were 1Laboratory of Molecular Introduction: HIV infection is initiated by the viral glycoprotein Envelope binding to CD4 receptor and coreceptors (CCR5 and CXCR4). The binding is not only essential for virus entry, but also important for identified. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice Human Retrovirology; and fibrous mal-union in most Cpa3Cre/+ mice. inducing downstream cascade of cell signals to benefit viral infection/replication. Histone deacetylase 10 2 Conclusion: The results indicate that mast cells promote bone healing, possibly by recruiting vascular Department of Medical (HDAC10) is a member of HDAC families and has a great homology to HDAC6. Recent studies have endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during Microbiology, Faculty of demonstrated that HDAC6 inhibits HIV mediated target cell fusion and entry through inhibiting the tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair. Medicine, University of acetylation status of cortical tubulin and it also inhibits Vif-mediated A3G degradation to reduce virus The capacity to isolate mast cells from bone marrow and grow them ex-vivo identify them as an attractive infectivity. However, the impact of HDAC10 during HIV infection and replication is still not known. This Manitoba, Winnipeg, MB target for therapeutic intervention aimed at modifying bone repair in individuals at risk of failed healing, study investigated the role of cellular protein HDAC10 in HIV infection/replication and initially explore such as the elderly or diabetic patients. the underlying mechanism. Methods: Through whole transcriptome sequencing, we identified the genes that were significantly regulated upon the HIV Envelope treatment. Among these genes, we further detected the change of HDAC10 in both J-Lat 6.3 cells and PBMCs by RT-PCR and western blots. To understand the role of HDAC10 in HIV infection, HDAC10 knock down cells were produced by transducing with specific shRNA and then HIV infection in HDAC10 KD cells was detected through measuring viral protein expression in supernatant. Results: The results of RNA-Seq analysis showed that there were 1,349 differentially expressed genes whose expression levels had significant changes (p<0.05) in the Env treated group. In the heat map, the hierarchial clustering of RNA-Seq, we validated the downregulation of HDAC10 in J-Lat 6.3 T cells and PBMCs upon Env treatment. The known down assay showed that the downregulation of HDAC10 benefits of HIV infection/replication with or without the presence of AZT. Futher, we found that one of the mechanisms to explain the phenomenon is the enhanced progeny virus’s infectivity in HDAC10 known down cells. Role of Fibronectin-Interacting Cellular Proteins in Influenza A Virus Infection in Human Studying Mechanisms and Novel Therapies Using Omics-Based Approaches in Ischemia Lung Cells Reperfusion Injury Associated with Kidney Transplant Mahamud-ur Rashid, Kevin Coombs Shelby Reid1, Peter Urabenellis1, Ramanuja Simha2, Igor Jurisica2, Lisa Robinson1, 3, Markus Selzner1, 4, James W. Scholey1, 5, Ana Konvalinka1, 5 University of Manitoba, Introduction: Influenza A virus (IAV) is one of the most serious human pathogens causing about 3 to 5 Winnipeg, MB million cases of severe illness, and approximately 500000 deaths worldwide every year. Designing 1Institute of Medical Introduction: Kidney transplantation remains the optimal treatment for end stage kidney disease, as it effective vaccine has proven difficult and treatment has become more challenging, as the Influenza A Sciences, University of prolongs life, improves the quality of life and results in cost savings, compared to staying on dialysis. virus mutates quickly against antiviral drugs and vaccines. As viruses are obligate parasites they use the However, the continuous organ shortage remains a serious problem leading to the use of marginal grafts. 2 host cell machinery to facilitate replication and evade the immune response during infection. These Toronto; Department of These grafts tolerate cold storage poorly, resulting in more severe ischemiareperfusion injury (IRI) leading changes are reflected by dysregulation of host cellular proteins, involved in different cellular pathways. Medical Biophysics and to a higher rate of delayed graft function (DGF), defined as the need for dialysis in the first week after So, virus replication could be potentially prevented inside the host cell by blocking cellular signaling Computational Biology; transplant. The increased usage of marginal grafts has led to the development of alternative preservation pathways important for viral replication but not needed for the cells. Previous proteomic studies have techniques to reduce the IRI. Normothermic exvivo kidney perfusion (NEVKP) is a storage method that 3Department of found that 1000s of host proteins are dysregulated in IAV infected cells and many of these interact with results in improved graft outcomes in porcine model of IRI and kidney transplantation. We sought to Fibronectin, is an extracellular matrix glycoprotein, plays important role in cell adhesion, growth, Pediatrics, Division of determine the molecular mechanisms behind the beneficial effects of NEVKP. migration, and differentiation. In a recent study we found that knockdown of several fibronectin Nephrology, the Hospital Methods: Kidneys from pigs were explanted, subjected to 30-minute warm ischemia time, and then interacting proteins in A549 cells significantly dysregulated virus replication. More specifically knockdown placed in static-cold-storage (SCS) or NEVKP for 8 hours, followed by reimplantation into the pig. Kidney for Sick Children; of a few proteins, BST1, CLIC1, FUBP1, PSMA2, EIF4A3 and HSPA5 reduced virus replication >3-fold. So far, biopsies were taken at time of explant, 30 minutes following anastomosis and on day 3 post-transplant 4 no study has investigated how these proteins are involved in Influenza virus replication cycle. Department of Surgery, from 5 pigs in NEVKP group and 5 in SCS group. Biopsy tissue was subjected to proteomic analysis on Methods: In this study we will investigate the knockdown effect (by siRNA) of these genes in IAV life cycle Division of General state-of-the-art mass spectrometer. by comparing different viral proteins, viral RNA, mRNA and virus yield in IAV infected and mock infected Surgery, University Health Results: 6179 proteins in total were quantified. Sixty-nine and 160 proteins were differentially expressed A549 cells. (p<0.05) between NEVKP and SCS groups at 30 minutes and on day 3 post-transplant respectively, with 5 Conclusion: A clear understanding of the role of these proteins in the IAV life cycle during human Network; Department of many proteins relating to oxidative stress and metabolism. infection may direct us to develop new antiviral treatment strategy in future. Medicine, Division of Conclusion: We will next validate top protein candidates and determine the key pathways and protein- Nephrology, University protein interaction networks. The identification of key proteins and mechanisms involved in IRI may be useful when selecting donor organs. Improved preservation of grafts may lead to utilization of grafts that Health Network, would be wasted. Toronto, ON Pan-Microbial Profiling of Female Genital Tract Microbiome Using Cutting Edge Illumina The Controlling Role of Autophagy in Regulating Temozolomide-Induced Apoptosis in MiSeq Sequencing Platform Rhabdomyosarcoma Cells Neil Reyes1, Omari Henry2, Emma Lee2, Binhua Liang3, Eric Enns3, Gary Van Domselaar1, 3, T. Blake Ball1, 2, Hezhao Ji 1, 2 Adel Rezaei Moghadam, Javad Alizadeh, Simone Cristina da Silva Rosa, Jared Field, Saeid Ghavami, Joseph W. Gordon

1Department of Medical Introduction: The female genital tract (FGT) microbiome is a complex organization of an array of Department of Human Introduction: Rhabdomyosarcoma (RMS) is a muscle-derived tumor and is the most common pediatric Microbiology, University microbial species including bacteria, viruses, fungi, and parasites that play a role in the host susceptibility Anatomy and Cell Science, soft tissue sarcoma representing 5% of all childhood cancers. RMS is a major clinical problem in pediatric to exogenous pathogens such as HIV. Commensal microbes establish the first line of defense against oncology. Treatment of RMS with the oral alkylating agent temozolomide (TMZ), alone or in combination of Manitoba; 2National Rady Faculty of Health infection. A pan-microbial profiling approach helps understand the microbial composition in a sample. with other drugs, has recently received considerable interest. However, the mechanism of action of TMZ Microbiology Laboratory Next-generation sequencing (NGS) and metagenomics characterize millions of microbial species Sciences, Max Rady remains unclear. The aim of this investigation was to determine if autophagy modulates TMZ-induced cell at JC Wilt Infectious sequences without prior knowledge of the target. This research aims to investigate vastness of the FGT College of Medicine, death in RMS cell lines. microbiome by characterizing known or unknown microbial species that may contribute to the varied Methods: MTT assay and Nicoletti flow cytometry analysis were used to measure cell death and apoptosis Diseases Research Center, University of Manitoba, susceptibility to sexually transmitted infections in females. in SJCRH30 (RH30) human RMS cells and the non-transformed mouse myoblast cell line C2C12, following Public Health Agency of Methods: Cervical vaginal lavage (CVL) samples were collected from women with varying HIV status Winnipeg, MB TMZ treatment (100 μM). Canada, Winnipeg, MB; (positive, negative, control, “resistant”). Total nucleic acids (TNA) were extracted using NucliSense Results: We monitored autophagy using transmission electron microcopy (autophagosome and EasyMag system. Unbiased high-throughput sequencing (UHTS) was conducted on all samples and the autophagolysosome formation) and immunoblotting (LC3 lipidation, Atg15-12 conjugation, p62 3The Bioinformatics Core, derived amplicons were subjected to sequencing using Illumina MiSeq V2 500 cycle kit. Illumina degradation). The effect of bafilomycin A1 on TMZ-induced cytotoxicity was also evaluated. We showed National Microbiology sequencing reads were uploaded to bioinformatics pipelines (KRAKEN and One Codex) to process that TMZ decreased the viability of RMS cells in a dose/time-dependent manner and induced Laboratory, Public Health taxonomical classification for all microbial species against existing microbial databases. The relative accumulation of sub-G1 cell population, representing apoptotic cells. Interestingly, TMZ induced Agency of Canada abundance and species diversity from each sample in every category were also examined. apoptosis by 16-fold in the RH30 cells, but only increased apoptosis by 63% in C2C12 cells. In RH30 cells, Results: Successful UHTS amplification of total nucleic acid samples revealed varying band sizes from 200 TMZ decreased the expression of antiapoptotic proteins Bcl-XL and Mcl-1, and increased the expression bp to 1200 bp. The number of classified host reads were varied from sample to sample. All host reads of the death gene Nix. Moreover, we showed that TMZ altered biochemical markers of autophagy, such were excluded from further microbial profiling. Data analysis with KRAKEN (primary pipeline) and One as LC3 lipidation, Atg5-12 conjugation, and p62 degradation, and induced morphological evidence Codex (confirmatory pipeline) displayed charts and tables of detected microbial species in each of four autophagy, including accumulation of autophagosomes and autophagolysosome, determined by microbial categories and their relative abundance in the examined samples. KRAKEN showed fluctuating transmission electron. Finally, treatment of RMS cells with the autophagy flux inhibitor (Bafilomycin A1, numbers of classified reads after host reads were filtered. However, discrepancies in the results provided 4 and 6 nM) had no significant effect TMZ-induced cell death in either cell line. by the two pipelines exist due to their alignment and referencing strategies. Conclusion: Our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both Conclusion: NGS and metagenomics can resolve the complexity of the FGT microbiome. Using KRAKEN RH30 and C2C12 cells; however, cell death induction by TMZ does not appear to be dependent on and One Codex bioinformatics pipeline with UHTS approach, it is possible to profile each microbial Bafilomycin A1 inhibited processes, such as autophagosome-lysosome fusion or autolysosome species for further downstream investigation. It is adaptable for any metagenomics studies in which acidification. non-specific microbial identification and profiling is required. The Role of Microglial PARP-1 in Alzheimer’s Disease Understanding the Ecological Relations Between Marginalization and Acute Psychiatric Abbas Rezaeian Mehrabadi, Jin Hee Kim, Gary Odero, Lory Tessler, Tiina Kauppinen Care Sebastian Rios, Christopher M. Perlman Department of Introduction: Alzheimer’s disease (AD) is a main cause of dementia. AD pathology includes synaptic and Pharmacology and neuronal loss, Aβ accumulation, neuroinflammation and compromised BBB integrity. Microglia, the brain School of Public Health Introduction: Socio-environmental factors such as discrimination, social exclusion, isolation, and other immune cells have been suggested to play a key role in neuroinflammation acting as the drivers. While Therapeutics, University and Health Systems, forms of social disadvantage have been associated with increased risk of mental illness and utilization of microglial pro-inflammatory and neurotoxic release activity can damage brain cells, microglial ability to mental health services. of Manitoba; release trophic factors, phagocytose and prune synapses can also support brain health. We have identified University of Waterloo, Methods: Bivariate analysis and logistic regression models are employed to understand the relationships Neuroscience Research poly(ADP-ribose) polymerase-1 (PARP-1) as a key regulator of microglia. Here we tested the hypothesis Waterloo, ON between inpatient mental health data and a Census-based index that measures marginalization in that microglial PARP-1-mediated pro-inflammatory functions can directly disrupt BBB integrity (in vitro) Program, Kleysen Institute Ontario. Two datasets are linked via a common geographic unit, the Forward Sortation Area. Thus, the and also promote other AD pathological events accelerating disease progression (in vivo). Ontario Marginalization Index (ONMarg), which geographically measures four domains: material for Advance Medicine, Methods: The effect of primary murine microglia cultures and their release activity after Aβ stimulation deprivation, ethnic concentration, residential instability, and dependency is linked to the Ontario Mental Winnipeg, MB was assessed on cells forming BBB; astrocytes and brain endothelial cells. Astroglial activation was Health Reporting System (OMHRS), which contains information about all the inpatient psychiatry analyzed by GFAP expression. Endothelial integrity was assessed by quantitating expression of tight admissions in Ontario. Service use, socio-demographics, clinical characteristics, diagnoses, and algorithms junction proteins (TJPs) and determining paracellular permeability. To study whether chronic pro- that identify a person’s strengths, needs, and risks in various areas (i.e., behaviour, social, financial, inflammatory microglial activation accelerates AD progression in 3xTg-AD mice, we performed functioning, vocational, and clinical) are evaluated against the different dimensions of the ONMarg. hippocampus-injection with microglia-directed lentiviral construct of mutant PARP-1 (LV-Iba1-mPARP-1- Results: The majority of inpatient mental health services recipients live in neighbourhoods with high RFP), which transduces constitutively active PARP-1 expression in microglia. The effect of mPARP-1 vs. levels of social deprivation, ethnic concentration, residential instability and dependency scores. There is control lentivirus injected animals was assessed 2-3 months later at 4-5 months of age by behavioral tests a clear positive relationship between the type of mental health diagnosis and the degree of and immunohistochemistry-staining. neighbourhood marginalization among recipients of inpatient mental health services. This relationship is Results: Aβ-stimulated primary murine microglia released NO and TNFα, triggered activation of also true for social and service use characteristics, as well as mental health symptoms. These results co-cultured astocytes and reduced TJP expression in endothelial cells in a PARP-1 dependent manner. confirm that socio-environmental factors play an important role in mental illness and highlight a new Both NO and TNFα stimulation of endothelial cells reduced their expression of TJPs (ZO-1 and Occludin) way to study these factors using publicly available data. and increased paracellular (FITC-dextran, IRDye) permeability. Microglial pro-inflammatory activation in Conclusion: Identifying and understanding the context of where a person lives can influence policy and the hippocampus of 3xTg-AD mice accelerated AD progression. Mice injected with LV-Iba1-mPARP-1-RFP help ensure that services and programs are available to those who need it, and reduce inequities through had memory deficits in NOR test already at 4 months of age, whereas AD mice injected with the control appropriate targeted care. LV showed signs of memory deficits after 5 months of age. Immunostaining of the post-mortem 3xTg-AD mice brains revealed that LV-Iba1-mPARP-1-RFP promoted pathological aspect of AD. Conclusion: Our in vitro and in vivo data demonstrates that microglial PARP-1 mediated pro-inflammatory responses can directly drive AD pathology/progression. Frailty, Resilience, and Mortality of Old Men: Manitoba Follow-Up Study A CRISPR-Cas9 Approach Followed by Whole Transcriptome Shotgun Sequencing to Elizabeth Sachs Identify the Transcriptional Regulatory Role of MEIS1 in Restless Legs Syndrome Faezeh Sarayloo1, Alanna Grant2, Helene Catoire2, Daniel Rocheforr, Patrick A. Dion2, 3, Guy A. Rouleau2, 3 University of Manitoba, Introduction: Frailty, a vulnerable state often experienced with aging, increases the risk of various health Community Health outcomes such as hospitalization, falls, and institutionalization. Most notably, the experience of frailty 1Department of Human Restless legs syndrome (RLS), the most common movement disorder in the elderly in western populations, can be attenuated. Therefore the operationalization of frailty has implications for health care and public Sciences, Rady Faculty of Genetics, McGill is characterized by an irresistible desire to move the legs and abnormal sensations in the lower limbs. A policy. That said, the literature has not yet reached universal agreement on the definition or successful GWAS on RLS identified common variants associated with this disorder located in intron 8 of 2 Health Sciences, operationalization of frailty. Current perspectives view frailty as an accumulation of phenotypic University; Montreal MEIS1 gene and the intergenic region between MAP2K5 and SKOR1. These results were also replicated by Winnipeg, MB components, as an accumulation of deficits, or as a resultant score from self-rated frailty scales. Neurological Institute, other independent studies. Our group subsequently showed that MEIS1 risk haplotype is associated with Furthermore, the relationships between frailty, resilience, and mortality have not yet been fully explored. McGill University; decreased mRNA and protein expression of this gene in RLS patients’ LCL and thalamus samples. We also Objective: To fill these gaps, the present this study will: 1) examine the possibility of recovering from a examined the SKOR1 mRNA expression in patient cells with the MEIS1 risk haplotype and observed a 3Department of frail state, thereby exhibiting evidence in favour of resilience to frailty; and 2) determine whether self- significant decrease in its expression, suggesting a regulatory role of MEIS1 on SKOR1 expression. To rated frailty is a predictor of mortality. Neurology and further confirm this regulatory role, a luciferase reporter assay followed by electro mobility shift assay Methods: The Manitoba Follow-up Study (MFUS) is an ongoing 69-year prospective cohort study that Neurosurgery, McGill showed a direct interaction between MEIS1 and SKOR1 promoter. Given the fact that MEIS1 has a examines health and well-being in a sample of World War II pilots and aircrew. The MFUS successful aging transcriptional regulatory function, we hypothesis that this gene has an indirect regulatory role in RLS University, Montreal, QC questionanaires included frailty, resilience, and mortality in 2015 and 2016. Secondary analysis of this pathways. To further investigate that, we are overexpressing MEIS1 and also using CRISPR-Cas9 to data will be used to examine the research questions at hand. knockout this gene, separately in different human cell lines (HEK293 and SK-N-SH cells, a neuroblastoma Significance: Knowing whether and how resilience relates to frailty will influence provision and planning cell line). Whole transcriptome of these sets of cells as well as wild type cells with normal endogenous of health care programs and services. Assessing the relationship between self-rated frailty and mortality expression are extracted followed by RNA-Seq experiment. We believe that differential expression will serve to more clearly define and operationalize the concept of frailty. analysis of RNA-Seq data will lead to identify genes that are in fact regulated by these transcription factors, some of which might be novel genes with direct roles in RLS pathological pathways and ultimately with more understanding of RLS molecular mechanisms we could move forward to give these individuals the rest they deserve. Mimicking Classroom and Outdoor Settings to Understand Motor-Cognitive Task Coordination in Young Children Shikha Saxena

McGill University, Objective: Coordinating performance on concurrent motor and cognitive tasks entails simultaneous highly relevant to clinicians who provide functional or dual task training to improve task coordination in Montreal, QC interaction between the two processes, for example walking and talking at the same time. Motor- children. Motor-cognitive task coordination ability might differ for different movement skills. Therefore, cognitive task coordination is highly relevant to everyday activities, but is not well understood in young task coordination during fine and gross motor functions should be assessed and treated separately. children. The purpose of this study was to investigate the effects of age on motor-cognitive task coordination in typically developing children aged 5-8 years using task paradigms similar to classroom and outdoor settings. Methods: This cross-sectional study included 200 typically developing children aged 5-8 years, assessed for task coordination ability under two paradigms. Task coordination in outdoor settings for young children generally involves walking while talking, and in classroom setting task coordination is required to write/draw while listening to teacher and answering questions. The combination of these tasks was mimicked in a lab setting. The paradigm chosen to mimic outdoor settings was walking while performing an audio-verbal working memory task; and the one that mimicked classroom settings was drawing while performing the same audio-verbal working memory task. Children performed the tasks separately (single task) and concurrently (dual task). Task coordination was quantified by measuring the performances for walking (number of steps), drawing (number of errors) and working memory (number of errors) under single and dual task conditions. Results: No age differences in the performance costs of either motor or cognitive tasks in any paradigm (p>0.05) were found. However, the children’s performance deteriorated under dual task conditions in both motor and cognitive tasks, and for both paradigms. Coordinating a working memory task while drawing led to a significant deterioration in drawing performance, whereas, the same cognitive load did not result in any significant difference in walking performance. The deterioration in working memory task performance was comparable whether walking or drawing. Conclusions: Coordinating cognitive tasks (listening, answering, memorizing) during writing or drawing can be more demanding than coordinating tasks while walking in young children. These findings are Medication Errors Committed by Nursing Students: Allocating Responsibility and Bayesian Evolutionary Analysis by Sampling Trees Applied to Extended Sequencing of Accountability Measles Virus for Molecular Epidemiology of Outbreaks. Catharine Schiller Helene Schulz1, Joanne Hiebert2, Alberto Severini1, 2

University of Northern Introduction: Medication errors committed by nursing students during clinical placements represent, in 1Dept. of Medical Background: Endemic circulation of measles virus in the Americas has been interrupted since 2002 and British Columbia, general, a vastly under-researched phenomenon. Research that currently exists about student-committed Microbiology, University all cases result from importation from endemic areas. Ongoing elimination efforts have contributed to medication errors focuses on the factors that lead up to the occurrence of the error. There is no research the decreasing diversity of measles virus genome, making it impossible to distinguish independent Vancouver, BC of Manitoba; 2National available that pertains to the events that take place after the error has already occurred. In particular, the importations from local transmissions when relying on traditional WHO-recommended genotyping allocation of responsibility and accountability for the error amongst the many potential individual and Microbiology Laboratory, targets. Therefore a higher-resolution genotyping or whole genome sequencing approach is needed. organizational contributors has not yet been explored. Students tend to perceive that they entirely Public Health Agency of Objectives: 1) calculate the rate of change and determine the relatedness of cases in outbreaks, 2) shoulder the blame when a medication error occurs and that system factors and other individuals who analyze the sequences for hypervariable regions for development of high-resolution genotyping test. Canada, Winnipeg, MB may have contributed to the error will not be adequately considered. Methods: Whole genome sequencing by Sanger and next-generation sequencing of a large Canadian Methods: In this study, the researcher used a mixed methods exploratory design within a critical realism and a large Brazilian outbreak followed by Bayesian Evolutionary Analysis by Sampling Trees (BEAST). philosophical framework to explore the phenomenon of student-committed medication errors. Through Results: Based on whole genome sequencing, the 1000nt M/F non-coding region (MF-NCR) accounts for data analysis, the ways in which responsibility and accountability for such errors are allocated, and the most of the variability seen in outbreak-associated viruses. BEAST revealed a clock rate of 3x10-3 and factors that influence these allocation decisions, will be explored. The semi-structured interviews will be 7x10-4 changes/nucleotides/year for the MF-NCR and whole genome, respectively. Based on the node analyzed using interpretive description. Quantitative data will be analyzed using descriptive statistics. ages, the main outbreak is distinguished from other unrelated importations. Results: Data collection (both qualitative and quantitative) was completed in January 2017. A total of 33 Conclusion: BEAST analysis of whole genome sequencing and extended genotyping of measles virus will actual interviews were completed although some of the participants held dual roles and therefore were empower epidemiological investigation as global measles elimination is approached, and the diversity of asked questions pertaining to all self-identified roles. This resulted in 9 student nurse interviews, 16 the measles genome decreases. clinical instructor interviews, 8 preceptor interviews, 8 educational institution leader interviews, 7 healthcare facility administrator interviews, and 1 regional representative interview. Data analysis is in the very beginning stages at this time. Work Integration Situations of Syrian Refugees in Canada Bmal1 Regulates the Rhythmic Expression of UCP2 in Pancreatic β Cells and Contributes Sonja Senthanar, Ellen MacEachen to the Control of Daily Cycles of Insulin Secretion and Glucose Tolerance Nivedita Seshadri, Michelle E. Jonasson, Tianna Flett, Kristin Hunt, Bo Xiang, Vernon Dolinsky, Christine A. Doucette School of Public Health Introduction: Work is an integral part of our lives, providing us with the income necessary for housing, and Health Systems, food, transportation, as well as social networks to help integrate into the community. For refugees, University of Manitoba, Background: Type 2 Diabetes (T2D) results from an inability of the pancreatic beta cell to secrete however, labour market barriers including non-recognition of foreign credentials, lack of language University of Waterloo, Winnipeg, MB sufficient insulin to compensate for insulin resistance. Upregulation of uncoupling protein 2 (UCP2) proficiency, and racial/ethnic discrimination, impedes work resulting in low-skilled, low-waged, precarious impairs glucose stimulated insulin secretion (GSIS) and is linked to β cell dysfunction associated with Waterloo, ON employment positions. Refugee women are particularly vulnerable to these positions resulting in adverse obesity and T2D. To understand the physiological function of UCP2 in the beta cell, we recently physical and mental health conditions. The aim of this research is to describe the work integration demonstrated that UCP2 expression over 24 hours is rhythmic and inversely regulates GSIS capacity and situations of Syrian refugees and explore barriers and facilitators to employment. Syrian refugees were glucose tolerance over 24 hours in mice. We now aim to explore the biological processes that drive chosen as they represent a unique opportunity for study – with a recent influx of roughly 30,000 into rhythmic UCP2 expression in beta cells Canada. Hypothesis: We hypothesize that the circadian clock drives the rhythmic expression of UCP2 and Methods: A qualitative research design guided by feminist grounded theory will be employed. This ultimately controls daily cycles of GSIS capacity and glucose tolerance. approach focuses on women’s subjective experiences and interpretations as a source of knowledge while Methods: Here, we used siRNA to knock down expression of the core circadian clock gene, Bmal1 in a examining micro- and macro-level influences, allowing us to explore gender, work, and health in a well-studied pancreatic clonal beta cell line (MIN6). These cells were synchronized and used to examine systematic way. Syrian refugees and key informants will be recruited in Fall 2017 through our community UCP2 expression patterns and GSIS capacities over 24 hours. partnerships with ShamRose Refugee Support Centre, Reception House Waterloo, and The Working Results: We observed dynamic Bmal1 expression in synchronized MIN6 cells with elevated expression at Centre – not-for-profit organizations in the Kitchener-Waterloo region with ranging services available to 4 hour when compared to 16 hours. We also observed that Bmal1 knockdown impaired GSIS at 4 hours refugees. We will conduct one-time, in-depth interviews either in-person or over the phone. Interviews compared to 16 hours. This suppression was associated with elevated Ucp2 mRNA at the same 4 hour will be audio recorded, transcribed verbatim, and entered into NVivo qualitative data software. Data will time point. We further demonstrated that upregulated Ucp2 mRNA associated with reduced glucose- be distilled into codes and codes linked and compared within and across data to develop key themes. induced ATP production, indicative of reduced mitochondrial uncoupling. Implications: This research is the first of its kind, to the best of our knowledge, that utilizes a qualitative Conclusions: Our study suggests that the circadian clock contributes to the regulation of rhythmic Ucp2 design guided by feminist grounded theory to explore interactions of work with power relations, familial mRNA expression, which in turn controls the temporal capacity of GSIS over the course of the day/night role, host-related policies, services, and norms, culture-related practices and norms, and labour force cycle and ultimately time-of-day glucose tolerance. Future studies will include the creation of a beta conditions. It will highlight the intricate interplay of work as a social determinant of health thus informing cell-specific knockout mouse model to determine if this finding is translatable to the in vivo state. development of interventions and guiding policy to improve and strengthen the health and well-being of all incoming refugee women. Mevalonate Cascade Inhibition Increases Cholesterol Pathway Inependent The Immune Activation Status of the Female Reproductive Tract Could Be Influenced by Temozolomide-Induced Apoptosis in Glioblastoma Cells Environmental Factors Via Epigenetic Mechanisms S. Shojaei1, J. Alizadeh1, N Koleini2, J. Thliveris1, E. Kardami1,2, S. Hombach-Klonisch1, T. Klonisch1, S. Ghavami1 Abu Bakar Siddik1, Vanessa Poliquin2, T. Blake Ball1, 3, Ruey-Chyi Su1, 3

1Department of Human Introduction: Glioblastoma (GB), the most common and aggressive brain tumor, has a low survival of 1University of Manitoba, Background: Sexually transmitted infections (STI) have a substantial impact on the Canadian health. The Anatomy and Cell Science, about 15 months after diagnosis and chemotherapy with Temozolomide (TMZ), one of the most Medical Microbiology & innate immunity at the FGT is highly efficient at fending off invading pathogen. However, changes in commonly used treatments for this cancer. Statins, mevalonate (MEV) cascade inhibitors, are best known immune activation due to changes in mucosal milieu or host hormones could greatly compromise University of Manitoba; for their cholesterol (CH) lowering effect but have recently attracted attention because of their beneficial Infectious Diseases; immune defence and affect cellular susceptibility to infections. These environmental factors and 2St. Boniface Research effects on cancer patients in the presence of other chemotherapy agents. The mevalonate cascade 2University of Manitoba, hormones are known to affect cellular gene expression profile by altering cellular epigenetic programming. Centre, University of provides farnesyl (FPP) and geranylgeranyl pyrophosphate (GGPP) for small Rho GTPase activation via Department of Obstetrics Here, we hypothesize that changes in cervicovaginal H2O2, lactic acid and butyric acid levels will have prenylation process. In the current investigation, we study if simvastatin (Simva) (Blood Brain Barrier direct impact on cellular epigenome, protein acetylation resulting in changes of the pro- and anti- Manitoba, Winnipeg, & Gynecology; 3National permeable statin) sensitizes human GB cells to TMZ-induced apoptosis. inflammatory cytokine/chemokine profiles. Canada Material and methods: U251 and U87 were pretreated without (control) or with low, non-toxic doses of HIV & Retrovirus Objectives: To test the hypothesis, levels of H2O2, lactic acid, butyric acid and cytokine/chemokine in the Simva. (1 and 2.5μM) and/or MEV (5μM), CH (5μM), FPP (15μM), GGPP (15μM) (both are sources for Laboratories, National cervicovagianl fluid (CVF) samples will be determined and related to the cellular activities of histone prenylation) for 4h before treatment with TMZ (100μM) for 48-96h. MTT assay and propidium iodide flow deacetylase (HDAC) and acetyltransferase (HAT) in the matched cervicovagianl mononuclear cells (CMC) Laboratory for HIV cytometry were used to evaluate cell viability and apoptosis. We used immunoblotting and transmission samples. electron microscopy to investigate autophagy upon combination of Simva and TMZ treatment in GB cells. Immunology, Methods: Informed consent will be sought from healthy Manitoban women who visit the clinic for a Results: Our results showed that co-treatment with TMZ and Simva induces a significant increase (P<0.01) Winnipeg, MB routine OB/Gyn Pap test. A questionnaire collecting information on menstrual cycle, sexual history, of TMZ-induced apoptosis in both U87 and U251 cells. The increase in apoptosis occurs in parallel to an contraceptive, STIs, weight and height will be administered. CVF will be collected with a Weck-Cel sponge increase in autophagy (LC3-β lipidation, decrease in p62, and accumulation of double-membrane in the cervical os prior to the use of cytobrushes and scrapers to collect CMC. CVF samples are stored at vacuoles) in Simva/TMZ treated cells compared to Simva and TMZ alone. Interestingly, the increase of -80°C until analysis. Measurements of pH, H2O2, lactic acid, butyric acid and cytokine/chemokine will be TMZ-induced apoptosis in Simva co-treated cells was not dependent on mevalonate cascade and performed using commercial kits and services. Nuclear protein lysate will be isolated and tested for HDAC addition of MEV, CH, FPP and GGPP had no significant (P>0.5) effect to reverse it. and HAT enzymatic activities. Conclusion: Our results show that combination of Simva and TMZ provoked a significant increase in TMZ- Significance: This study will provide insights on the role of the dynamic environment in affecting the induced apoptosis in U87 and U251 cells which is not dependent on the classic cholesterol metabolism epigenetic programming to shape the immunologic environment at the FGT. Such knowledge will pathway. establish the basis for mechanistic studies to understand how factors at cervicovaginal mucosae boost or impair the innate immune memory against STIs. The Use of SSRIs and SNRIs During Pregnancy and the Risk of Autism: A Population Investigating the Role of BNIP3 in Regulating Cell Proliferation and Transcriptional Based Retrospective Cohort Study Utilizing Linkable Administrative Data Control of IGF2BP1 Deepa Singal1, Dan Chateau2, Laurence Katz3, Chelsea Ruth4, Elizabeth Wall-Weiler1, Ana- Hanlon-Dearman5, Marni Brownell6 Amandeep Singh, Spencer B. Gibson, Elizabeth S. Henson

1 Department of Community Introduction: There is worldwide debate over the risk of autism spectrum disorders (ASD) in children University of Manitoba; Introduction: Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) is a BH3 only pro-apoptotic member Health Sciences, University of exposed to in-utero selective serotonin reuptake inhibitors (SSRIs)/selective serotonin norepinephrine of Bcl-2 family of cell death regulating proteins. BNIP3 function in the cell is dependent on its localization. Manitoba; 2Department of Research Institute in reuptake inhibitors (SNRIs) versus children born to mothers with untreated maternal depression during Under normal oxygen conditions, BNIP3 is present at low levels in the nucleus. Under low oxygen Community Health Sciences, pregnancy, making the management of depression during pregnancy a significant clinical challenge. Oncology and Research Scientist, Manitoba conditions, BNIP3 is upregulated and localizes to cytoplasm where it associates with mitochondria and Centre for Health Policy, Previous studies are significantly limited by inability to account for confounding by indication and Hematology, induces cell death. However, if BNIP3 is overexpressed in the nucleus, it represses the transcription of disease severity. We utilize population based administrative data and sophisticated analyses to delineate University of Manitoba; Winnipeg, MB certain genes associated with cell death and thus inhibits cell death. Observations in BNIP3 knockout 3Department of Psychiatry, the effects of untreated maternal depression versus the effects of antidepressant exposure on the onset mice brain showed increased number of cells in different parts of the brain. Moreover, RNA sequencing Section Head, Child and of ASD by investigating whether children of mothers with mood/anxiety disorder during pregnancy had of astrocytes showed three-fold increase in expression of Insulin-like growth factor 2 mRNA binding Adolescent Mental Psychiatry, an increased risk of ASD when exposed in-utero to SSRIs/SNRIs versus children born to mothers with protein 1 (IGF2BP1). IGF2BP1 is a fetal oncogene that is normally only expressed during fetal development 4 University of Manitoba; Section mood/anxiety disorder during pregnancy not treated with antidepressants. but increased expression of this protein is seen in adults with various cancers. IGF2BP1 promotes cell of Neonatology, Department of Methods: This study included all mother-newborn pairs in Manitoba (born from 1996 to 2009, with proliferation, growth, adhesion and migration- hence could play a vital role in tumor growth and Paediatrics and Child Health, follow-up till 2014) where the mother had a diagnosis of mood/anxiety disorder 90 days prior to Research Scientist, Manitoba metastasis. conception until delivery. From this cohort we identified children whose mothers had at least two Centre for Health Policy, Methods: A number of assays will be used to measure proliferation differences between wildtype and University of Manitoba; prescriptions for an SSRI or SNRI during pregnancy (n=857), and children of mother’s who did not have a BNIP3 knockout mouse embryonic fibroblasts (MEFs). RTCA (real time cellular analysis) measures increase 5Department of Paediatrics and prescription for any antidepressant during pregnancy (n=5830). Confounders perceived as clinically and in cell index over real time. Cytation 5 is a high throughput cell imaging reader that can be used to Child Health, Section Head, statistically relevant using a high-dimensional propensity score model macro were adjusted for using quantify changes in cell number over several days. A cell staining dye, CFSE, will also be used. Differences Developmental Paediatrics, inverse probability of treatment weights. Poission regression and Cox Proportional Hazard Ratios models in IGF2BP1 levels will be quantified by western blotting. Gene knockdown via RNA interference will be University of Manitoba; were used to estimate risk of ASD in offspring. employed to study effects of IGF2BP1. 6Department of Community Results: Use of SSRIs/SNRIs during pregnancy was not significantly associated with an increased risk of Results: RTCA and Cytation 5 assays have shown BNIP3 knockout MEFs have significantly higher Health Sciences, Manitoba ASD; HR 0.78 (95% CI 0.34 to 1.77), RR 0.73 (95% CI 0.33 to 1.61). proliferation than wildtype MEFs. Further analysis has to be performed to confirm the results. Centre for Health Policy, Max Conclusions: When taking into account underlying indication and disease severity, taking SSRIs/SNRIs Rady College of Medicine, Rady Conclusion: Nuclear BNIP3 appears to play a role in suppressing cell proliferation. Further confirmation during pregnancy does not increase risk of ASD in children of women who have prenatal mood/anxiety Faculty of Health Sciences, of these findings can define a new function for nuclear BNIP3. University of Manitoba, disorder. Winnipeg, MB The Effect of Timing of Oseltamivir Chemoprophylaxis in Controlling Influenza A H3N2 Hepatic JAK2 Protects Against Atherosclerosis Through Circulating IGF-1 and Influenza B Outbreaks in Long Term Care Facilities in Manitoba, Canada, 2014-2015: Tharini Sivasubramaniyam1, 2, Stephanie Schroer2, Angela Li1, 2, Sally Shi1, 2, Rickvinder Besla1, 2, Jara Brunt1, 2, Joshua Lopes1, A Retrospective Cohort Study Michelle Bendeck1, Clinton Robbins1, 2, Minna Woo1, 2 Davinder Singh1, Depeng Jiang1, Paul Van Caeseele1, 2, Carla Loeppky1, 3 1University of Toronto; Introduction: Fatty liver is an emerging independent risk factor for atherosclerosis. However, the 1University of Manitoba; Introduction: Influenza is a major cause of morbidity and mortality in long term care (LTC) facilities. The 2University Health mechanisms by which fatty liver promotes atherosclerosis are difficult to delineate due to its common association with other metabolic abnormalities such as insulin resistance and diabetes. The JAK-STAT 2Cadham Provincial Infectious Diseases Society of America recommends the use of oseltamivir for chemoprophylaxis during Network, Toronto, ON outbreaks, but the evidence supporting its use in this setting is not strong. As well, the impact of timing pathway is a major signalling pathway downstream of cytokines and growth factors. We previously Laboratory; 3Manitoba -/- of chemoprophylaxis in this setting has not been thoroughly evaluated. This study will examine the effect showed that deletion of JAK2 in hepatocytes (L-JAK2 ) of mice led to profound steatosis spontaneously. Health, Seniors and Active of the timing of administration of oseltamivir chemoprophylaxis for the control of influenza A H3N2 and Interestingly, those defects commonly associated with fatty liver including systemic insulin resistance and glucose intolerance were absent in these mice. Thus we asked whether fatty liver in the absence of Living, Winnipeg, MB influenza B outbreaks among residents in LTC facilities in Manitoba, Canada during the 2014-2015 influenza season, after controlling for other institutional factors. other commonly associated risk factors affected atherogenesis. -/- Methods: A retrospective cohort study will be conducted of all LTC facility influenza A H3N2 outbreaks Methods: In order to assess the role of hepatic JAK2 in atherogenesis, we bred L-JAK2 mice into 2 -/- (n=94) and influenza B outbreaks (n=11) in Manitoba during the 2014-2015 influenza season. Since the atherosclerosis-prone models, apolipoprotein E knockout mice (APOE ) and low-density lipoprotein -/- -/- -/- -/- -/- outcomes are not all independent, a hierarchical logistic regression analysis will be conducted. An empty receptor knockout mice (LDLR ), generating L-JAK2 APOE and L-JAK2 LDLR mice, respectively. -/- -/- model will be used to determine the intra-class correlation. The independent variables will be included L-JAK2+/+APOE and L-JAK2+/+LDLR littermates were used as controls. Starting at 6 weeks of age, in the model as institutional level variables and individually modeled. A stepwise forward modelling mice were placed on an atherogenic diet for 12 weeks after which metabolic parameters, hepatic strategy will be used to determine the best multiple variable main effects model, including both steatosis and atherosclerotic plaque burden were assessed. -/- -/- -/- statistically and clinically significant variables. Continuous variables will be assessed for linearity and Results: L-JAK2 in both atherosclerosis-prone APOE and LDLR models showed similarly profound model variables will be assessed for co-linearity. The final main effects model will be assessed for fatty liver without glucose intolerance or insulin resistance. Interestingly, these mice developed over a significant interactions between the time to oseltamivir prophylaxis and other main effects variables. All 2-fold increase in plaque burden compared to controls as assessed by Oil-Red-O staining of the descending analyses will be two-tailed and conducted at an alpha level (α) of 0.05. aorta and the plaques in the aortic arch appeared more advanced with increased macrophage content Conclusion: This will be the largest examination of the use of oseltamivir for chemoprophylaxis for and decreased smooth muscle cell content. We had previously shown that JAK2 was required for growth influenza A H3N2 in LTC facilities, and the first to examine the effect of timing of oseltamivir hormone signaling in liver resulting in low circulating insulin-like growth factor-1 (IGF-1) and hypothesized -/- -/- chemoprophylaxis for the control of influenza B outbreaks in LTC facilities. It will provide the strongest this to play a causal role in the increased atherosclerosis. To test this hypothesis, L-JAK2 APOE mice piece of evidence for or against the use of oseltamivir chemoprophylaxis in this setting and will help were infused with IGF-1 analog or vehicle while on an atherogenic diet for 12wks. Our data show that inform infection prevention and control policy in LTC facilities in North America, Europe and possibly restoring circulating IGF-1 attenuates atherosclerotic plaque burden in hepatic JAK2-deficient mice. other regions. Conclusion: Our study identifies an essential role of hepatic JAK2 and its regulation of IGF-1 as an important mechanistic link between fatty liver and atherosclerosis. Indigenous Culture and Language as a Source of Mental Wellness and Resiliency Continuous Renal Replacement Therapy Is Associated with Acute Myocardial Injury in J. Erynne Sjoblom Critically Ill Patients Marat Slessarev1, 3, Fabio Salerno2, Ian Ball1, Christopher Ellis3, Christopher McIntyre2, 3 University of Manitoba, Introduction: The oppression of Indigenous peoples in Canada through historical and on-going Winnipeg, MB processes of colonization has resulted in social inequities that are at the root of mental health problems 1Divisions of Critical Care Introduction: Intermittent renal replacement therapy is associated with dialysis-induced acute myocardial at both the individual and community level. The majority of research with Canadian Indigenous Medicine; 2Department of injury, in both chronic and acute treatment. Continuous Renal Replacement Therapy (CRRT) is often populations has focused on prevalence and individual-level predictors of mental health problems; favoured in critically ill patients with acute kidney injury (AKI), hypotension or shock. Lower ultrafiltration 3 however, research in recent years has been increasingly concerned with the importance of community- Nephrology; Department rates, characteristic of CRRT, reduce systemic hemodynamic stress and may be cardio-protective. We level factors, especially with respect to addressing the impacts of colonization. Nevertheless, few studies of Medicine, and assessed the impact of CRRT on the development of acute segmental myocardial injury, in critically ill have explored, from the perspectives of community members, how community-level factors protect and Department of Medical patients requiring dialysis for AKI. promote mental wellness. Thus, the objective of this qualitative study was to explore Indigenous peoples’ Hypothesis: CRRT will not induce cardiac injury (i.e. will be cardio-protective) in critically ill patients with Biophysics;4Schulich understandings of how community environments protect mental wellness and how these environments AKI. can be strengthened and supported in a way that acknowledges the realities of communities. School of Medicine & Methods: We used 2D-echo and speckle tracking analysis software (EchoPAC, GE Healthcare) to measure Methods: This study utilized a community-based participatory research approach, respecting First Dentistry, Western global and segmental left-ventricular myocardial longitudinal strain in 12 critically ill patients presenting Nations principles of Ownership, Control, Access and Possession. Moreover, the study was framed by with AKI. Measurements were made at baseline immediately prior to and 4, 8 and 24 hours after initiation University, London, ON Indigenous methodological research design principles, supported by modified grounded theory for of CRRT. analysis and data organization. A combination of purposive and snowball sampling was utilized to recruit Results: Measurements were completed in 11 patients. 10/11 patients developed new regional wall a total of 17 participants from a First Nation Tribal Council and community located within the Tribal motion abnormalities, with 8 developing these as early as 4 hours after CRRT start. The number of Council area (Manitoba). Interviews were conducted using a conversational approach to facilitate affected segments varied from 1 to 11 (out of 12). Of 11 patients, 7 (58%) died in the ICU, with 5 of those participant and researcher co-creation of knowledge. dying within 2 days of CRRT initiation. Results: Indigenous culture arose as an important source of mental wellness and resiliency, particularly Discussion and Conclusions: Our results show that CRRT is associated with new regional myocardial in relation to how traditional teachings, ceremony, and spirituality facilitate the creation of meaning, injury. This injury was associated with high mortality. These results are concerning with respect to the enabling reflection on self, relationships, and history, healing from trauma, and promoting unity, respect, presumed safety of CRRT (compared to other dialysis modalities) in critically ill patients. Further direct and a sense of shared purpose. Indigenous language arose as an integral source of community-level comparison of CRRT with other dialysis modalities is warranted to assess the potential for relative wellness, counteracting the disconnections that have resulted from colonization by bolstering community cardio-protection. unity and identity, by strengthening emotional connections between family and community members, and by preserving cultural teachings and worldviews that are inherent in the language. Conclusions: Mental health promotion and policy with Indigenous communities should consider incorporating activities that promote and protect Indigenous culture and language as an important means of collective healing from the impacts of colonization. The Influence of Altering Anionic Phospholipid Membrane Composition on Cationic The Living Stories of Chronic Kidney Disease for First Nations People Substrate Specific Multidrug Efflux Pump Function in Escherichia Coli Mary Smith Carmine J. Slipski, Denice C. Bay University of Victoria, Aims: Further the cultural consciousness, reclaim ancestral traditions and good health, express the voices University of Manitoba Introduction: Multidrug resistant bacteria are a world-wide threat to human health, and the problem is Victoria, BC of those who have suffered through colonialism, identify and address systemic barriers, increase knowledge that furthers cultural safety, further Indigenous methodology as a way of healing Medical Microbiology and exacerbated with the selective pressures placed on bacteria by the constant wide-spread over use of antiseptic compounds in clinical, industrial, and household applications. Low level exposure to these Background: Within a First Nations background, I questioned mainstream western methodologies and Infectious Diseases, antiseptics can lead to reduced membrane permeability and decrease the efficacy of other antimicrobials, the lack of consistency with ways of Indigenous being and knowing. I searched for a deeper connection Winnipeg, MB such as antibiotics. A major mechanism for antiseptic resistance in bacteria are efflux proteins, which to the earth, nature and dreams. encompass many different families with varying substrate specificity. Our focus is on cationic substrate Ontology and Epistemology: When the sweet grass is smelled the beautiful scent can only bring positive specific efflux pumps, and the role anionic phospholipids in bacterial membranes have on the folding thoughts that cast away the dark lonely images. Together the birch bark and the sweet grass, a sacred and function of these transporters. There is a gap in knowledge of what role phospholipid-protein medicine, symbolize all earnest and good things and healthful ways of living. The porcupine quills interactions have in the function of efflux pumps that can expel these antiseptics. Knowledge gained in represent the spirit animals and the clans that create a purpose and direction through our living within this research could be used to develop novel efflux pump inhibitors, find new lipid targets for drug our relations. The four points of the blackened flower quills signify the four directions further reinforcing susceptibility, and to help guide the development of new antimicrobials. the interconnectedness and guidance through the land and mother earth. Methods: Efflux pumps EmrE (SMR family), MdtK (MATE family), and MdfA (MFS family) will be PCR Relational Axiology: Heartfelt relations find healing like the sweet grass that weaves us together and the amplified from Escherichia coli strain BW25113 and cloned into the plasmid vector pMS119-EH. The vector grandfather teachings of wisdom, love, respect, bravery, honesty, humility, and truth. At its crux is the containing each efflux pump will be transformed into various E.coli mutant strains containing specific value of respect, where all dialogues between people and nature are respected, linked and deciphered phospholipid synthesis pathway gene deletions, or transporter gene deletions in order to eliminate as through the virtues inherent within the grandfather teachings. The meaning of truth extends beyond to many confounding factors as possible, and to test the effect of phospholipid composition in the embrace the land and everything on it as a living entity. An ethical methodology requires congruence membrane on efflux topology and function. Active sites of each efflux pump will be verified by site- with the ontological, epistemological and axiological uniqueness of Indigenous peoples within their directed mutagenesis. Phospholipid content of each E.coli strain will be confirmed by TLC. The efflux contextual experiences. activity of each pump type in the various strains will be tested against varying concentrations of Methods: Dreams and sharing circles – unfold the living stories - beyond that of being lived but continues commonly used cationic antimicrobials utilizing high throughput assays to determine if efflux topology through the past, into the present and beyond, is not linear but transforms in relation to all the living and and function are affected by varying membrane phospholipid compositions. non-living immersed within the cosmos. The living story transpires from the dreams and sharing circles Expected results: From previous research, it is expected that anionic phospholipids will have the greatest to express the methodology rooted within an Indigenous way of being. The living stories of persons are impact on cationic substrate efflux function and topology, and that reduction in the expression of rich dialogues interconnecting culture, history, health and beyond. zwitterionic phosphatidylethanolamine (PE) will result in greater expression of anionic phospholipids. Expected conclusions: Anionic phospholipids have a large impact on the insertion, folding, and topology of cationic efflux proteins, and provide promising targets to improve the efficacy of cationic antiseptics. Apoptotic Oxidized Phosphatidylcholines (OxPCs) Are Produced During Renal Ischemia Computational Approaches in Epigenetic Drug Discovery Reperfusion Injury Emese E. Somogyvari, Selim G. Akl, Louise M. Winn Zahra Solati, Andrea Edel, Karmin O, Yue Shang, Amir Ravandi Queen’s University, Introduction: Epigenetics describes the regulation of genomic functions leading to heritable changes in University of Manitoba, Introduction: Renal ischemia/reperfusion (I/R) injury is the leading cause of acute kidney injury and one Kingston, ON gene expression that are outside of the genetic sequence. The misregulation of epigenetic modifications, such as DNA methylation, has been shown to lead to an increased incidence in several diseases including Winnipeg MB of the major factors affecting patient survival after renal transplantation. One of the mechanisms is through the generation of reactive oxygen species and the release of active mediators resulting in cancer. The development of drugs treating disorders of the epigenome has been gaining interest in structural damage to the kidney. Oxidized phosphatidylcholines (OxPCs) are produced in cardiac I/R as recent years due to their potential to reverse epigenetic modifications. However, knowledge about the certain types of OxPCs, including POVPC, PGPC and PONPC, can mediate I/R injury through apoptotic underlying mechanisms of the epigenome still remains limited and the development of specific drugs pathways. The aim of this study was to identify these apoptotic OxPCs in renal I/R. has shown to be difficult. Additionally, there is an increased amount of methylation and matched gene Methods: Kidney ischemia was induced in eight male Sprague-Dawley rats (250-300 g) by clamping the expression data, yet there exist few tools for their integrative analysis. In this regard, a machine learning left renal pedicle for 45 min followed by reperfusion for either 6h or 24h. As control, sham-operated rats approach supplemented by transcriptional regulatory networks and differential methylation analysis is were sacrificed at corresponding time points. Kidneys were harvested in ice cold potassium phosphate proposed to identify potential drug targets involved in epigenetic diseases. buffer. In order to extract phospholipids from renal tissue, a portion of each kidney was weighed and Methods: A dataset of gene expression and matching methylation data was collected for normal and subjected to lipid extraction. OxPCs were identified and quantitated using liquid chromatography cancer samples taken from patients with kidney renal clear cell carcinoma (KIRC). A dataset of transcription coupled to electrospray ionization tandem mass spectrometry using an internal standard. factor (TF) information was also collected and used as labels in classification. Multi-label support vector Result: Fifty six distinct OxPCs were identified in rat kidney which included fragmented OxPCs, terminal machines (SVM) was used to predict TF-gene interactions from which two networks were created: one of furans, isoprostanes and long-chain products. Among apoptotic fragmented OxPCs, PONPC was the most normal samples and the other of cancer samples. Important subnetworks can be isolated by integrating abundant compound after both 6h and 24h reperfusion (0.25±0.09 and 0.57±0.02ng/mg, respectively). differential methylation analysis results and by comparing the two networks. PONPC concentrations were even greater after 24h compared to 6h of I/R (p=0.014). Following 6h of Results: Analysis is ongoing, however preliminary results have led to the isolation of a subnetwork reperfusion, among the apoptotic fragmented OxPCs studied, only PONPC in the I/R group was including various genes pertaining to transcription, histones, and a histone deacetylase isoform. significantly elevated relative to the sham group (0.25±0.09and 0.15±0.10ng/mg, p<0.05). However, after Conclusion: Preliminary results look promising. A successful model will give insight on epigenetic 24h reperfusion, POVPC, PGPC and PONPC were all significantly greater in the I/R group compared to the mechanisms affecting KIRC and potential drug targets, and could be applied to other cancers and sham group (p<0.05). epigenetic diseases. Conclusion: We have shown for the first time that OxPC molecules increase after I/R injury in rat kidney. In addition, the levels of apoptotic fragmented OxPCs increased with increased time of reperfusion. Therefore, the attenuation of their activity would be considered as a potential therapeutic approach to reduce acute kidney injury. The Many Faces of E4-ORF3 Protein in Cellular Reprogramming by Human Adenovirus Do Spouses Bereaved by Suicide Experience Worse Mental Health? A Population-Based Andrea Soriano, Jasmine Frost, Leandro Crisostomo, Richard Jung, Sandi Radko, Peter Pelka Longitudinal Investigation of Bereavement Associated Mental Disorders Rae Spiwak, Brenda Elias, Jitender Sareen, Laurence Y. Katz, Mariette Chartier, James M Bolton University of Manitoba, Human adenovirus E1A is the first gene product expressed during viral infection. E1A is involved in Winnipeg, MB remodeling of the cellular environment by altering cellular pathways as a preliminary step for efficient University of Manitoba, Objective: The following study utilizes a longitudinal population based sample of spouses bereaved by viral replication. E1A targets cellular hub proteins, that is, proteins involved in multiple regulatory Winnipeg, MB suicide and selected sudden death cohorts to determine if rates of physician-diagnosed mental disorders pathways, in order to alter intracellular environment. Interestingly, recent data has shown that E1A also differ for individuals bereaved by suicide, sudden natural death (SND), and sudden unintentional injury binds to the viral protein E4-ORF3. E4-ORF3 is one of seven proteins encoded by the Early region 4 (E4) (UI). gene, which are required for successful viral replication. E4-ORF3 is a 14 kDa protein expressed early in Methods: Married individuals whose spouse died between 1998 and 2008 by suicide (n=365) were infection, it is involved in suppression of the interferon response, stimulation of viral RNA splicing and compared to spouses bereaved by SND (n=1000) and UI (n=270) over the same time period. Inverse inactivation of tumor suppressors. E4-ORF3 forms a scaffold network in the nucleus through self- probability treatment weights were calculated using age, sex and income to ensure groups were polymerization and simultaneously binds and inactivates multiple tumor suppressors such as p53, PML, comparable. Generalized estimating equations were used with a poison distribution to calculate relative TRIM24 and MRN. In addition, E4-ORF3 prevents p53 DNA-binding on p53 target promoters, by forming rates comparing mental disorders 5 years before and after spousal death. A group by time interaction a nuclear structure that induces heterochromatin formation at these promoters. Recently we found that term was used to determine if bereavement groups had significantly different rate changes for disorders Nek9, a cellular serine/threonine kinase is recruited to the nuclear structures formed by E4-ORF3 in from the pre to post periods. dependent manner. Co-localization of Nek9 with E4-ORF3 suggested that Nek9 may play a role in Results: Spouses bereaved by suicide had the greatest proportion of depression post bereavement E4-ORF3 mediated silencing of cellular genes that would negatively impact virus growth. We observed (50.96%), as compared to SND (33.70%) and UI (38.52%) spouses. A significant interaction term (P=0.047) that Nek9 was found to block activation of p53-regulated gene GADD45A, which is an important player was found signifying that the rate change in depression in the pre to post period was significantly in stress response and p53-mediated cell cycle arrest. In addition, we observed that E4-ORF3 is critical for different between suicide and UI bereaved spouses. Suicide bereaved spouses also had increased rates of efficient viral growth, as mutations in E4-ORF3 lead to a significant inhibition of virus growth. These any mental disorder both pre (ARR=1.34, 95% CI=1.02-1.76, P=0.036) and post death (ARR=1.22, 95% results demonstrate that E4-ORF3 mediates the inactivation of tumor suppressors in order to overcome CI=1.01- 1.47, P=0.035) when compared to UI spouses. Post-bereavement, adjusted models found that cellular blocks to viral replication. Recently, we observed that E1A co-localizes to E4-ORF3 tracks in the suicide bereaved spouses had greater rates of depression only when compared to spouses bereaved by nucleus, indicating that E1A may be recruiting E4-ORF3, or vice versa. This interaction was verified as a SND (ARR=1.37, 95% CI=1.12-1.69, P=0.002). No differences were found in rates of mental disorders direct interaction through a GST-pulldown assay. A viral protein to viral protein interaction has never between UI and SND groups. been previously found in HAdV and this is important as it can give us insights into the mechanisms used Conclusion: Sudden spousal bereavement is a time of vulnerability for depression. Significant differences by E1A to alter cellular environment. in rates of depression were found between UI and suicide bereaved spouses. Suicide bereaved spouses have greater rates of depression and any mental disorder in the period before bereavement. Prospective studies are needed to examine the role of caregiving, the deceased’s pre-death health, and the role of guilt and stigma on bereavement related health to inform targeted interventions. The Role of Oxidized Phosphatidylcholine (OxPC) In Rat Cardiomyocyte Cell Death Human Polyclonal Antibodies Produced in Transchromosomal Bovine Prevent Lethal Aleksandra Stamenkovic1, 2, Kimberley A. O’Hara1, 2, David C. Nelson1, 2, Grant N. Pierce1, 2, Amir Ravandi1, 3 Zika Virus Infection and Testicular Atrophy in Mice Derek R. Stein1, Joseph W. Golden3, Bryan D. Griffin1, 2, Bryce M. Warner1, 2, Charlene Ranadheera1, Leanne Scharikow1, Angela 1Institute of Introduction: Phosphatidylcholine (PC) represents the most abundant class of phospholipids in Sloan1, Kathy L. Frost1, Darwyn Kobasa1, 2, Stephanie A. Booth1, 2, Matthew Josleyn3, John Ballantyne4, Eddie Sullivan5, Jin-an Cardiovascular Sciences, biological membranes. PC is highly susceptible to oxidation leading to a large production of OxPCs Jiao5, Hua Wu5, Zhongde Wang6, Jay W. Hooper3, David Safronetz1, 2 during simulated myocardial ischemia/reperfusion (I/R) injury. Two fragmented OxPCs: POVPC St. Boniface Hospital; (1-palmitoyl-2-(5’-oxo-valeroyl)-sn-glycero-3-phosphocholine) and PONPC (1-palmitoyl-2-(9’-oxo- 2Department of nonanoyl)-sn-glycero-3-phosphocholine), were the most abundant OxPC’s generated during I/R injury. 1Zoonotic Diseases and Zika virus (ZIKV) is rapidly spreading throughout the America’s and is associated with significant fetal complications, most notably microcephaly. Due to the ongoing pandemic, rapid development and Physiology and There was also a correlation between the concentration of POVPC and PONPC and cardiomyocyte cell Special Pathogens, National death. However, the mechanism for the cardiomyocyte cell death induced by OxPCs still remains treatment using polyclonal antibodies for pregnant women at risk of ZIKV related complications could be Pathophysiology, Microbiology Laboratory, unknown. Ferroptosis, an iron dependent form of cell death induced by accumulation of lipid oxidation a safe alternative to vaccination. We found that large quantities of human polyclonal antibodies could be Public Health Agency of 3Department of Internal products, is a possible mechanism of OxPC action. Therefore, the main aim of our study was to investigate rapidly produced in transchromosomal bovines (TcB) and successfully used to protect mice from lethal 2 Medicine, University of this process in detail. Canada; Department of ZIKV infection. Additionally, antibody treatment eliminated ZIKV induced pathology in immunologically Methods: Adult rat ventricular cardiomyocytes were isolated and treated with 1-palmitoyl-2-oxovaleroyl- Medical Microbiology, privileged sites such as the brain and testes and protected against testicular atrophy. These data indicate Manitoba, Winnipeg, MB sn-glycero-3-phosphocholine (POVPC), at concentrations of 0.1μM, 1μM, 5μM, and 10μM. Following a 1 h University of Manitoba, that rapid development and deployment of human polyclonal antibodies could be a viable countermeasure treatment, Live/DeadTM assays were performed, cells were counted and the data presented as the against ZIKV. Winnipeg, MB; 3Virology percentage of live vs. total cells. When present, ferrostatin-1, an inhibitor of ferroptosis, was added together with selected OxPCs. To investigate the protective role of ferrostatin-1 in I/R, cells were exposed Division, United States to 1 h ischemia followed by 1 h of reperfusion, and ferrostatin-1 was added at the time of reperfusion. Army Medical Research Results: POVPC was cardiotoxic under cell perfusion conditions. When added with POVPC, ferrostatin-1 Institute of Infectious blocked the cardiotoxic effects of POVPC on its own (p<0.001). Ischemia and I/R challenge caused a Diseases, Fort Detrick, MD; significant decrease in cell viability. However, ferrostatin-1 had a protective role when added at the time 4Aldevron, Fargo, ND; 5SAB of reperfusion. Conclusion: Oxidized phospholipids are potent mediators of cardiomyocyte cell death under control Biotherapeutics, Sioux Falls, conditions and in ischemia/reperfusion injury. The mechanism involves the ferroptotic pathway. These SD; 6Department of Animal, preliminary results are promising and may lead to a new therapeutic approach to prevent cell loss during Dairy and Veterinary I/R injury. Sciences, Utah State University, Logan, UT Difference-Makers in Human Affective Distress: Perspectives on Causation and Recovery Gained from Qualitative Inquiry Into Lived Experience Thomas Lieven Steur

University of Manitoba, Introduction: The biomedical conceptualization of “depression” as an objective disease entity stems from literature which finds physicians eschewing diagnostic checklists and algorithms, and respectfully th Winnipeg, MB a 17 century biological science paradigm and is an inappropriately positivist idea which discounts negotiating treatment for depression with individuals while attending to functioning, social context, individual agency, disregards social context, fails to accommodate inter-subjectivity and process, and treatment preferences, and potential for stigma. The effectiveness of an integrated mental health system medicalizes suffering. Qualitative inquiry into the perspectives of people with lived experience can shed with an array of services which respects individual agency and choice is upheld. light on how individuals conceptualize the experience, including their views on fit or lack of fit with the dominant psychiatric discourse of biomedical individualism; what ‘difference-makers’ or risk factors are identified by participants as contributory to the genesis of the emotional distress; and what ‘difference- makers’ are perceived as helpful to recovery. Methods: Adult participants were recruited who self-identified as having experienced emotional distress, in adulthood, for a period of two weeks or longer; were treated with (and possibly still were taking) prescription antidepressant medication for this distress; and described themselves as having achieved a degree of recovery. In-depth interviews were audio-recorded, transcribed verbatim, coded and analyzed using thematic analysis methodology. Findings: Data clustered around three main overlapping ‘umbrella’ theme categories: multifactorial conceptual understandings; context pertinent to the experience of distress and recovery; and trajectories of recovery from acute distress to negotiation of ambivalence toward treatment, enlisting of supports, and reclaiming of agency. A variety of factors were named in both the genesis of and recovery from depression; difference-makers which were felt to be essential for recovery included development of trusting relationships with primary care physicians and/or psychiatrists, counselling, and self-help organizations and community agencies. Significance: Theoretical orientation is relevant to understanding and recovery in depression. Physicians appear to be practicing more than the dominant biomedical discourse surrounding depression would imply, as human factors such as trust, empathy, optimism, acknowledgement of contexts, and acknowledgement of agency and even of resistance were recurring themes. Findings are consistent with Development and Validation of a New Pediatric Resuscitation and Trauma Outcome (PRESTO) Score Using the U.S. National Trauma Data Bank Etienne St-Louis1, 2, Robert Baird1, 2, Tarek Razek1, James A. Hanley3

1Department of General Background: The worldwide burden of pediatric traumatic injuries is very significant and a large low-resource settings. Indeed, it is composed of “low-tech” variables that can be reliably collected even Surgery, McGill University proportion of childhood injury deaths occur in Low and Middle Income Countries. Trauma registry data in austere environments. Further research is necessary to allow in situ validation using prospectively can help improve the care of injured pediatric patients; however, there is a need for a pediatric trauma collected LMIC data. Health Centre; outcomes benchmarking tool that is adapted for low-resource settings. Inadequate LMIC trauma data 2Department of Pediatric availability precludes development and validation such a score using “local” data. Therefore, we aim to Surgery, McGill University apply literature-informed and consensus-based limitations to North-American data from the U.S. National Trauma Data Bank {NTDB). Health Centre; Methods: The National Trauma Data Bank was used to analyze data on all pediatric trauma patients aged 3Department of 0-14 years from 2007-2012. A sample including all cases and 10% of non-cases was used for analysis. Epidemiology, Demographic, physiologic, anatomic and outcome variables were collected. The primary outcome was Biostatistics and in-hospital death. Multiple imputation was performed to account for the missing data. Stepwise logistic regression was performed to identify independent predictors of the primary outcome in these patients. Occupational Health, The Pediatric Resuscitation and Trauma Outcome (PRESTO) model was created using above-mentioned McGill University LMIC limitations to include only “low tech” variables. It was submitted to 10-fold cross-validation, compared to the full model and to the gold-standard TRISS model as well using ROC analysis, using Delong’s test. Results: PRESTO consists of age category, low blood pressure, AVPU status, need for supplemental oxygen, pulse, oxygen saturation, and an interaction term for age and pulse. The resulting model has an AUC of 0.98 compared to the AUC of 0.99 for the full model and AUC of 0.89 for TRISS. The sensitivity and specificity of PRESTO, using a threshold value of 0.129, are 95.7% and 94.0%, respectively. Although Delong’s test for two correlated ROC curves indicates a significant difference in AUC between PRESTO and the full model, PRESTO satisfies the requirements of a low-resource setting and is inherently adapted to children. Conclusion: We believe PRESTO is the best tool for pediatric trauma outcomes benchmarking in Characterization of a Novel OTX2-Driven Self-Renewal Program in Medulloblastoma M. Stromecki1, N. Tatari1, L. Morrison1, R. Kaur1, G. Palidwor2, C. Porter2, Patryk Skowron4, M. Wölfl3, T. Milde4, M. Taylor5, T. Werbowetski-Ogilvie1

1Regenerative Medicine Objectives: Medulloblastoma (MB) is a highly heterogeneous primary malignant pediatric brain cancer Our results offer critical insights into the molecular drivers of these most aggressive MB tumors and Program, Department of that is frequently accompanied by metastatic dissemination and poor long-term prognosis. The most provide an informed framework to pursue novel targeted therapies aimed at differentiating MB tumors aggressive tumors are refractory to conventional chemotherapy and radiation, namely due to the cells. Biochemistry and Medical treatment-resistant stem cells. Our lab previously discovered that the gene orthodenticle homeobox 2 Genetics, University of (OTX2), which is amplified or overexpressed in the majority of aggressive MBs, is a central regulator of Manitoba, Winnipeg, MB; stem cell function or self-renewal in these tumors. However, the molecular mechanisms by which OTX2 regulates these functions are still unknown. 2Ottawa Hospital Methods: Here, we employed complementary bioinformatics approaches to characterize the OTX2 Research Institute, regulatory network, including global gene expression profiling and chromatin immunoprecipitation Ottawa, ON; 3University of (ChIP) sequencing (ChIP-seq). Group 3 and 4 MB patient samples were evaluated for correlations between expression of axon guidance pathway genes, OTX2 and survival by a 2-sided Wilcox-sum test as well as a Wuerzburg, Würzburg, Pearson correlation coefficient. Gain and loss of function studies were performed. 4 Germany; German Results: Global gene expression analysis revealed that 3614 transcripts were differentially expressed Cancer Research Center, (P<0.05; ± 2-fold) following OTX2 knockdown. Of these, transcripts associated with axon guidance Heidelberg, Germany; signaling represented the top dysregulated networks. ChIP-seq identified putative OTX2 DNA binding sites and revealed statistically significant associations between OTX2 and these axon guidance pathway 5 Arthur and Sonia Labatt genes. Semaphorin signaling was the most overrepresented pathway across all datasets including, Group Brain Tumour Research 3 and 4 MB patient samples, with expression of all pathway genes being upregulated following OTX2 KD. Centre and Program in Moreover, SEMA4D expression was identified as a novel prognostic biomarker. Functional validation studies demonstrated that increased levels of semaphorin pathway molecules are associated with a Developmental and Stem decrease in selfrenewal. Downstream pathways known to mediate the effects of semaphorin signaling Cell Biology, The Hospital such as, RHOA, were also differentially expressed following OTX2 KD. Furthermore, RHOA pathway for Sick Children, inhibitors demonstrated a partial rescue in sphere formation following OTX2 KD. Conclusions: Group 3 and 4 MB have the worst prognosis, yet we know the least about these subtypes. Toronto, ON Altering Underlying Processes of Vascular Calcification Is Sufficient in Reducing Vessel Stiffening in Experimental CKD Bruno A. Svajger1, Kimberly J. Laverty1, Cynthia M. Pruss1, Emilie C. Ward1, Martin P. Petkovich1, Rachel M. Holden2, Michael A. Adams1

1Department of Introduction: Chronic kidney disease (CKD) is the progressive long-term decline in renal function that VC development may influence subsequent cardiovascular complications. That is, by altering an Biomedical and Molecular leads to hyperphosphatemia and imbalances in hormonal regulators of mineral homeostasis (FGF23, underlying process of VC, it may be possible to reduce the subsequent CVD burden despite not attaining PTH). Over half of CKD mortality is resultant of cardiovascular disease (CVD). Vascular calcification (VC) full inhibition of VC. Sciences, Queen’s and subsequent vessel stiffening is important to CVD development in CKD. We sought to examine the University; 2Department importance of a VC process, extracellular matrix degradation by matrix metalloproteinases (MMPs), on of Medicine, Queen’s CVD development. To do so we supplemented rats in an experimental model of CKD with an MMP-inhibitor. University, Kingston, ON Methods: CKD was induced in adult male Sprague-Dawley rats (n=27) using a high adenine diet (0.25% adenine). Rats were taken off dietary adenine upon attaining sufficient renal disease (creatinine >400μM) and separated into 3 groups: normal phosphate (0.5% dietary phosphate, NP-CKD, n=8), high phosphate (1% dietary phosphate, HP-CKD, n=11), or high phosphate with doxycycline (30 mg/kg BID, HP-DXY, n=8). Doxycycline is an MMP-inhibitor. After 12 days of treatment, hemodynamic changes (including pulse- wave velocity, PWV) were measured and rats sacrificed. Results: High phosphate treatment significantly elevated circulating phosphate and PTH compared to normal phosphate (p<0.0001 and p<0.05, respectively). VC was present in 75% of HP-CKD compared to 25% of both NP-CKD and HP-DXY. HP-CKD aortic phosphate and calcium content was significantly elevated compared to NP-CKD (p<0.01 and p<0.05, respectively). VC development was mitigated in HP-DXY with no significant differences compared to NP-CKD. HP-DXY rats had similar PWV to NP-CKD, with both markedly reduced compared to HP-CKD (HP-DXY vs HP-CKD p=0.0592, NP-CKD vs HP-CKD p=0.0852). Conclusion: MMP-inhibition sufficiently mitigated VC and subsequent vessel stiffening in experimental CKD despite the presence of hyperphosphatemia. Although some VC in HP-DXY occurred, the expected vessel stiffening was absent. These findings suggest that the manner, and not necessarily magnitude, of Diet Quality and Mortality Risk in Canada: A Population-Based Study Using Linked Exploring the Intersubjectivity Between the Couple, (the Person with Dementia and their Survey and Health Administrative Data Family Member), Living with Dementia in the Community Christopher A. Tait1, 2, Mary R. L’Abbé3, Peter M. Smith1, 4, Laura C. Rosella1, 2, 5 Barbara Tallman

1Dalla Lana School of Background: Diet quality indices are increasingly being used in epidemiologic studies. However, few University of Manitoba, This research explores the experience of living with dementia from the perspectives of the person with Public Health, University studies have investigated whether adherence to a specific set of dietary guidelines is longitudinally Winnipeg, MB dementia and their family member (couple) within the context of their everyday activities and relational associated with decreased risk of mortality. The objective of this study is to investigate the association life story. Symbolic interactionism, as the theoretical framework, directs the interpretation of 2 of Toronto; Institute for between diet quality, as assessed by the validated Canadian adaptation of the Healthy Eating Index (HEI), intersubjectivity as the way self is shaped within the couple’s interactions. A focused ethnographic Clinical Evaluative and the risk of all-cause mortality. Importantly, this is the first study to evaluate the presumed efficacy of approach captures the “emic” or the insider’s perspective of the couple through a triangulation of Sciences; 3Department of the HEI as a public health intervention strategy, using a population-based sample of Canadian adults. methods including individual and joint interviews, participant observation and photo-elicitation. This Methods: We used data from Ontario adult respondents to the 2004 Canadian Community Health Survey, poster presentation will describe several case studies of different couples. The poster will include selected Nutritional Sciences, linked to health administrative data at the Institute for Clinical Evaluative Sciences (n = 5,539). Adherence excerpts illustrating how couples integrate their past, present and anticipated future understandings to Faculty of Medicine, to the HEI was analyzed with a 24-hour dietary recall, where higher scores are congruent with a healthier their current circumstances. The following are three examples of some of the excerpt that will be used to University of Toronto; diet. Mortality status was ascertained through the Registered Persons Database, and tracked up to 11.9 describe the intersubjectivity of the couple: This first excerpt describes the husband with dementia’s years from baseline. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% understanding of his current relational dynamics with his wife; “And of course as you may gather shortly, 4Institute for Work & confidence intervals (CIs) for all-cause mortality, comparing those in the highest to the lowest quartiles we’re both independent minds and the agreements and understanding sometimes were not always in Health 5 Public Health of HEI scores. Given obesity’s potential role as a mediator of this association, we explored the effects of place.” The second excerpt is the wife of a husband with dementia describing changes in a meaningful Ontario, Toronto, ON removing body mass index (BMI) from the final model. We also conducted analyses stratified by BMI, to activity; “And then the other day I put on Lawrence Welk and he said, who’s he, he didn’t want to watch assess whether the association between HEI and mortality persisted across weight categories. it, so that’s it, changes, things change.” The third excerpt is the awareness expressed by the husband with Results: After multivariable adjustment, high HEI adherence was significantly associated with a 35% dementia of how their current circumstances might be reflected in their future: “Because I can’t visualize reduction in mortality risk [HR = 0.65, 95% CI = 0.48-0.89]. Significantly decreased mortality risk was anything positive about living in a demented state for another five or ten years.” Thematic analysis of the highest among Canadian males [HR = 0.58, 95% CI = 0.35-0.95]. Additional adjustment for BMI did not intersubjectivity of the couple suggests that couples’ make efforts to overcome their misunderstanding result in changes to the multivariable adjusted hazard ratios. However, stratified results revealed that diet and make connections with each other through the interactions that occur within their everyday quality was a weaker and less significant predictor of mortality at high levels of BMI. activities. The intersubjective perspective increases an understanding of impact of the relational Conclusions: This is the first study to provide epidemiological evidence that adherence to the dietary experience of living with dementia. This knowledge is foundational for developing clinical and policy recommendations of the HEI may decrease the long-term risk of mortality in the Canadian population. interventions that benefit the quality of life of those living with the experience of dementia. Our analyses highlight the importance of prospective observational studies to evaluate the utility of diet quality as a discriminating measure in predicting mortality risk in a population-based sample. Identifying Military and Veteran Family Cultural Competencies for Healthcare Providers Molecular Mechanism by Which Prominent Human Gut Symbiont Bacteroides Ovatus Linna Tam-Seto1, Heidi Cramm1, 2, Terry Krupa1, Patricia Lingley-Pottie3, 4, Heather Stuart5, Alice Aiken6 Utilizes Mixed-Linkage β-Glucan, a Major Health-Promoting Dietary Glycan Kazune Tamura1, Glyn R. Hemsworth2, Guillaume Dejean1, Constance M. E. Bahr3, Theresa E. Rogers3, Nicholas A. Pudlo3, Karthik 1School of Rehabilitation, Introduction: The National Defence and Canadian Forces Ombudsman’s Special Report, On the Homefront: Urs3, Eric C. Martens3, Gideon J. Davies2, Nicole M. Koropatkin3, Harry Brumer1 Faculty of Health Sciences, Assessing the Well-being of Canada’s Military Families in the New Millennium, describes how the military lifestyle can affect the lives of Canadian military and Veteran families (MVF). Frequent relocation can 1 Queen’s University, present challenges to the stability, mental health and well-being of MVF compounded by the stressors of Michael Smith Introduction: Gastrointestinal health is a cornerstone of our wellbeing and the composition and Kingston, ON 2Canadian accessing and navigating a new healthcare system. Unlike their American counterparts, MVF in Canada Laboratories, University of physiology of the gut microbiota is heavily influenced by the influx of complex glycans (commonly known as “dietary fibre”) from our diet. Mixed-linkage β-glucan (MLG) from cereals, in particular, has been Institute for Military and access the civilian health care system. The need for MVF cultural competency is being discussed in the British Columbia, American healthcare literature; however, little is known about the health care experiences of Canadian linked to a wide range of health benefits, including reduction of blood cholesterol levels and mitigating Veteran Health Research; Vancouver, BC; 2York MVF. This has also been identified as a priority by Canadian healthcare associations. Therefore, there is metabolic syndrome. Members of the Bacteroidetes, a dominant phylum in the human gut, possess an 3Department of urgency to conduct research to answer the question: “What are the cultural competencies required of Structural Biology arsenal of polysaccharide utilization loci (PUL) to target a wide range of complex plant polysaccharides. Methods: We have identified a PUL in B. ovatus that is transcriptionally upregulated in the presence of, Psychiatry, Faculty of healthcare providers when working with military and Veteran families?” Laboratory, University of Methods: The first phase of this multi-phase mixed methods study is aimed at developing a cultural and necessary for growth on, barley β(1,3)/β(1,4)-glucan, a representative MLG. Through combined Medicine, Dalhousie York, York, UK; 3University competency model and involved in-depth, one-on-one qualitative interviews. Participants were asked to biochemical, enzymatic, biophysical and structural characterization, we have illuminated key aspects of 4 University; Strongest describe and reflect on specific positive and negative health care interactions. Framework analysis was of Michigan Medical the molecular mechanism by which B. ovatus utilizes MLG. Families Institute, Halifax, used to identify cultural competencies from the perspective of the service user. School, Ann Arbor, MI; Results: Two outer membrane glycan-binding proteins facilitate capture and retention of the polysaccharide at the cell surface. Subsequently, a specific and efficient mixed-linkage endo-glucanase NS 5Centre for Health Results: Seventeen interviews were conducted including one with a military family, a Veteran and 15 spouses/partners of active members. Families identified the need to increase awareness among from GH family 16 localized on the cell surface cleaves MLG into two main breakdown products, which Services and Policy healthcare providers on the unique lifestyle and stressors experienced by military families. Lifestyle have been found to activate transcription of the PUL. These products are then imported to the periplasm Research, Faculty of factors such as increased mobility directly affect the abilities of MVF to access continuous healthcare. by a TonB-dependent transporter where they are reduced to monosaccharides by the action of two exo- glucosidases from GH family 3. Health Sciences, Queen’s Stressors including deployment and extended absences due to training exercises influence the types of healthcare supports MVF are seeking. There are also significant systemic factors that influence their Conclusion: Together, these data allow us to outline a pathway by which these key PUL gene products University, Kingston, ON healthcare experiences. act in concert to efficiently capture and metabolize dietary β-glucan in the competitive distal gut 6Faculty of Health Conclusion: The results of this study will be integrated with a second phase of the study that will capture environment. Professions, Dalhousie the healthcare provider perspectives on critical cultural competencies and how they came to develop them. Ultimately, this cultural competency model will broadly inform healthcare providers when working University, Halifax, NS with Canadian MVF. This study will also contribute to the growing body of knowledge on the healthcare experiences of Canadian MVF. Mechanism of Pannexin Channel Mechanosensitivity Novel Chemoprotective Role of the CTRP8-RXFP1 System in Glioblastoma Shubhamsingh Tanwar, Natalie Lavine, Michael F. Jackson Thatchawan Thanasupawat1, Aleksandra Glowgowska1, Jerry Krcek1, 2, Jason Beiko2, Marshall Pitz3, Sabine Hombach-Klonisch1, 4, Thomas Klonisch1, 2, 5 University of Manitoba; Ischemic stroke driven cytotoxic edema expedites Pannexin-1 (Panx1) channel activation, facilitating Kleysen Institute for chemotaxis-guided neuronal injury and death. Mechanisms underlying osmotic cell swelling-induced 1Department of Human Glioblastoma (GB) is the most common and highly aggressive form of primary brain cancer which is Panx1 regulation are poorly understood. The objective here is to establish and evaluate the contribution Advanced Medicine, Anatomy and Cell Science; usually fatal. The GB invasive phenotype and the development of treatment resistance are major clinical of F-actin and microtubules to swelling-induced augmentation of Panx1 channel activity in HEK 293 cells challenges. The underlying mechanisms remain poorly understood. We identified the presence of relaxin 2 Health Sciences Centre, stably expressing mouse panx1 (flag-mPanx1). In cells voltage-clamped at -60 mV, application of Department of Surgery; family peptide receptor1 (RXFP1) in GB and discovered C1q-tumor necrosis factor-related peptide8 Winnipeg, MB extracellular solution (ECS) with depreciating osmolarity induced a corresponding progressive 3Department of Internal (CTRP8) as a novel RXFP1 ligand in human GB cells. Here we demonstrate a novel mechanistic link magnification in Panx1 mediated currents. The effects of cytochalasin-D (CytD; F-actin depolymerizing medicine; 4Department of between the CTRP8-RXFP1 receptor-ligand system and DNA protective mechanisms in human GB. In drug) and nocodazole (NDZL; tubulin depolymerizing drug) were evaluated using immunofluorescence primary and established GB cells, CTRP8, like relaxin (RLN2) used as positive control, were able to activate Obstetrics, Gynecology & imaging and whole cell patch clamp recording. CytD not only inhibited the escalation in Panx1 channel RXFP1. This resulted in activated STAT3 signaling and protection from DNA damage caused by the response during osmotic cell swelling but also abated the basal channel activity [767 pA (control; n=7), Reproductive Medicine; chemotherapeutic drug temozolomide (TMZ), the drug of choice for GB. TMZ chemoresistance is a 319 pA (treated; n=8)] indicating that F-acting contributes to cell swelling induced mechanosensitive as 5Department of Medical common occurrence and a major cause for fatal outcome. Base excision repair (BER) pathway is an well as basal channel regulation. Tubulin disruption showed poor inhibition of channel activity important DNA repair pathway of TMZ induced DNA base alkylation. Failure or insufficient BER can result Microbiology & Infectious augmentation during osmotic stress-induced cell swelling. Transiently transfected HEK 293T cells in fatal double strand DNA (dsDNA) breaks which are detected by the marker γH2AX. Upon TMZ expressing Panx1 truncations at C-term (shown to interact with F-actin) further attest the decline in basal Diseases, College of treatment, CTRP8 mediated activation of RXFP1 was able to protect human patient GB cells from TMZ and stress-induced Panx1 mediated currents. To conclude, F-actin plays an important role in the Medicine, Faculty of induced DNA damage and enhanced GB survival. We found that activation of RXFP1 activated the STAT3 augmentation of Panx1 channel activity during osmotic stress-induced cell swelling. Health Sciences, signaling pathway and this caused the RXFP1- and STAT3-dependent up-regulation of the key BER member N-methylpurine-DNA glycosylase (MPG) catalyzing the first step of BER. RXFP1 increased both University of Manitoba, MPG protein content and MPG activity. Activated RXFP1 also increased the production of STAT3 targeted Winnipeg, MB anti-apoptotic factors Bcl-2 and Bcl-XL. Thus, we identified a novel CTRP8-RXFP1-STAT3 signaling pathway as a new protective mechanism against TMZ-induced DNA damage in patient GB cells. Our results indicate a novel role for the CTRP8-RXFP1 ligand-receptor system in STAT3-dependent mitigation of TMZ- induced DNA damage and increased GB survival and identify RXFP1 as a new tumor-promoting G protein coupled receptor in GB. Semaphorin 3E Protects Against Chlamydial Infection by Regulating Dendritic Cell Multiplexed Imaged-Based Screening Identifies Human Chromosome Instability Genes Functions Laura L. Thompson1, 2, Allison Baergen1, Zelda Lichtensztejn2, Kirk J. McManus1, 2 Rony Thomas, Shuhe Wang, Ying Peng, Sai Quio and Xi Yang 1Department of Introduction: Chromosome instability (CIN) is an abnormal phenotype frequently observed in cancer Department of Biochemistry and Medical that is characterized by an increase in the rate at which chromosomes or chromosomal fragments are Introduction: Chlamydia is an intracellular bacterial pathogen that causes multiple human diseases, gained or lost. CIN is associated with highly aggressive, multi-drug resistant cancers, disease recurrence, Immunology, University of including pneumonia and sexually transmitted diseases. The incidence of chlamydial infections is arising Genetics, University of and poor patient prognosis. Despite this, the majority of human CIN genes have yet to be elucidated, Manitoba, Winnipeg, MB significantly in Canada over the past 15 years. Until now, no vaccine is available for human chlamydial Manitoba; 2Research highlighting the need for studies aimed at identifying the defective genes that cause CIN. diseases. The main impedance for an effective vaccine development is the incomplete understanding of Institute in Oncology and Methods: In this study we developed and employed two image-based approaches capable of detecting protective immunity and immunopathology related to chlamydial infection. Semaphorin 3E (Sema3E), a CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. First, nuclear areas Hematology, CancerCare guidance molecule for neurons, has emerged as an essential mediator involved in cell migration, were quantified following silencing, where changes relative to controls reflect the DNA content changes proliferation and angiogenesis, although its role in infectious disease is not investigated. Manitoba, Winnipeg, MB that are characteristic of CIN. The second approach monitored micronucleus (MN) formation where Objective: We sought to understand the role ofSema3E in host defense against chlamydial lung infection increases in the number of micronuclei are indicative of DNA damage or mitotic defects that underlie CIN. using an established mouse model. Traditional cytogenetic approaches (i.e. mitotic chromosome spread analyses) were employed to validate Methods: Sema 3E-­‐KO mice and wild-­‐type (WT) mice were infected with Chlamydia muridarum (Cm) and a subset of putative CIN genes identified by the screen. Fluorescent microscopy techniques were utilized monitored for body weight changes, bacterial load, cytokine profile and pathological changes in the to investigate the aberrant mechanisms that drive CIN following gene silencing. lungs at day 7 and day 14 post infection. Lungs and Spleen from both groups of mice were isolated, Results: A multiplexed, high-content screen of 164 candidate CIN genes was performed in two unrelated, cultured and concentration of various cytokines in the supernatant was measured by ELISA. The karyotypically-stable cell lines. Collectively, 148 putative human CIN genes were identified. The most intracellular cytokine production by T cells and Dendritic cells were analyzed by flow cytometry. promising putative CIN genes including SKP1 were prioritized for subsequent validation based on the Results: We found that the Sema3E‐KO mice showed more bacterial burden and pathology upon number of assays that identified the gene and the strength of the CIN phenotype. Following gene chlamydial challenge when compared with the WT mice. Spleen and lung cells isolated from Sema3E‐KO silencing, data collected through Western blotting and assessment of mitotic chromosome spreads for mice produced significantly lower levels of IFN‐γ and IL‐17 than WT mice. Intracellular cytokine analyses numerical and structural chromosomal changes, supported the validation of SKP1 as a CIN gene. The also showed marked reduction of IFN‐γ and IL‐17 producing CD4+ and CD8+ T cells in lungs of Sema3E‐ increases in centrosomal aberrations and replication stress observed following silencing may represent KO mice compared to WT mice at day 7 and day14 post infection. Moreover, the surface phenotype and important contributors to CIN in SKP1-silenced cells. cytokine production of DC is altered in Sema3E‐ KO mice. Conclusion: These findings indicate that this screening approach is capable of detecting phenotypic Conclusion: The data demonstrate that Sema3E is a critical molecule, mediating host defense against changes associated with CIN, and can be utilized to uncover novel human CIN genes such as SKP1. chlamydial lung infection by modulating dendritic cell functions. This study provides cutting‐edge Identification and characterization of CIN genes will provide critical insights into CIN and oncogenesis, as findings to elucidate the role and mechanism of Semaphorin 3E in infections, specifically chlamydial well as identify potential targets that could be exploited in novel, precision medicine approaches for infection, which may have implications for developing prophylactic and therapeutic approaches. superior cancer treatment. Fidelity of Replication for the Measles Virus Polymerase Investigating the Value of a Tailored Knowledge Synthesis Approach for Rare Diseases: R. Thorington1, H. Schulz1, M. Carpenter1, 2, A. Severini1, 2 Focus Groups with Various Stakeholders Kylie Tingley1, Beth Potter1, Doug Coyle1, Pranesh Chakraborty2, Ian Graham1, 3, Kumanan Wilson1, 3 1Dept. of Medical In the year 2000, the World Health Organization began its efforts to eradicate the measles virus (MeV) Microbiology, University that lead to both a drastic decrease in the number of cases of measles globally and a decrease in the 1University of Ottawa; Objective: The objective of this study is to engage with stakeholders (i.e., patients/families, physicians, variability of the MeV genome, which has shown a remarkable stability for an RNA virus. While this of Manitoba; 2National 2Children’s Hospital of and policy-advisors) to better understand their perspectives on evaluating and synthesizing treatment apparent stability may be due to selective pressure, we also hypothesize that there is a high-fidelity effectiveness evidence for rare diseases. 3 Microbiology Laboratory, mechanism in the genome replication by the MeV RNA-dependent RNA polymerase (RdRp) complex. Eastern Ontario; Ottawa Methods: We conducted focus group interviews by telephone to elicit opinions on evaluating evidence Public Health Agency of Replication of MeV requires three proteins; the nucleoprotein, the phosphoprotein, and the large protein. Hospital Research for rare diseases from various stakeholder groups. A purposive sampling approach was used to ensure The large protein is the RdRp and it is responsible for all enzymatic functions related to transcription and Canada, Winnipeg, MB Institute, Ottawa, ON sufficient diversity and contrasting opinions. Using a semi-structured interview guide, participants were replication. The nucleoprotein’s role is to protect the genome and act as a guide for the RdRp, while the asked to describe their general perspectives on the challenges in rare disease research, their processes phosphoprotein acts as a bridge between the other two proteins. for evaluating and synthesizing evidence, and their thoughts on outcomes used in rare disease research. In order to examine the lack of variability in the MeV genome, we established an in vitro RNA synthesis Each interview was recorded and transcribed by a member of the study team. Transcripts were analyzed assay using the MeV RdRp, which can be used to shed more light on the molecular mechanism of MeV using interpretive description to identify key concepts and better understand stakeholder perspectives. replication and to determine the cause of the virus’s genetic stability. To accomplish this we have taken Preliminary Results: To date (February 2017), we have conducted two focus group interviews, with two approaches, one using endogenous replicative proteins isolated using a cell lysate. The other is the metabolic physicians (n=6) and policy advisors (n=3). Participants described many limitations of clinical use of recombinant proteins, which involves cloning the three proteins into the SUMOstar vector and trial evidence for rare diseases, including insufficient length of follow-up, lack of clinically meaningful then expressing them in a baculovirus system. Two approaches are being taken in order to account for outcomes, lack of standardized health outcome measures for rare disease populations, and poor external the many different variables that affect the success of the assay. Functionality of the assay and rate of validity (i.e., full clinical spectrum of disease not captured). Participants from both groups also expressed 32 incorporation for the RdRp will be monitored using P tagged nucleotides. With the establishment of the concerns about bias in industry-funded research. Participants in both groups suggested that if a in vitro synthesis assays, we will examine the fidelity of the MeV using the single completed hit model. treatment is transformative or there are no alternative treatments available, research studies do not need The large ratio between the number of primers and polymerases in this model allows for the analysis of to be as methodologically rigorous; however, some participants noted that this is rarely the case, any amplicons for correct nucleotide incorporation under the assumption that any primer will only have especially since supportive care can be used as a comparator. encountered one polymerase. Conclusion: The next step for this project is to conduct focus groups with other stakeholders, including patients/families. Data collected as part of these interviews will be used to shape the development of an evidence framework for evaluating and synthesizing treatment effectiveness evidence for rare diseases. The Role of Parkin in Mitigating Oxidative Stress in Parkinson’s Disease Evaluation of Mucosal Inflammatory Cytokine/Chemokine Response to Novel 12-PCS Jacqueline M Tokarew1, Daniel El Kodsi1, Julianna T Tomlinson2, 3, Michael G Schlossmacher1, 2, 3 Vaccine and Traditional Full Gag/Env Vaccine. Nikki Toledo1, Hongzhao Li1, Nancy Schultz-Darken2, Maria J. Alonso3, James B. Whitney4, Francis A. Plummer1 ,5, Ma Luo1, 5 1Cellular and Molecular Introduction: The causes of Parkinson’s Disease (PD) are complex however a subset of early-onset PD is Medicine, Faculty of associated with autosomal recessive mutations in several proteins including Parkin. No specific role for 1University of Manitoba, Background: Mucosal inflammatory responses influence susceptibility to HIV infection. We are conducting Parkin has been validated but it is believed that mitochondrial dysfunction is causing death of Medicine, University of Winnipeg, MB; 2Wisconsin a nonhuman primate study to compare efficacy of a vaccine targeting the 12 protease cleavage sites with dopaminergic cells that carry these mutations. Our lab has recently observed that the oxidation state of a vaccine targeting full Gag and Env. Evaluation of inflammatory responses to different vaccine modalities Ottawa; 2Brain and Mind National Primate Research Parkin is increased in older (>40 yrs old) vs. younger human brains (<20 yrs old) and led us to propose at the site of infection is very important. Research Institute, that Parkin’s high number of oxidation-sensitive cysteines enables it to migitate OS and prevent PD. Center, Madison, WI; Objective: Evaluate mucosal inflammatory responses in macaques vaccinated with rVSVgag/env or Methods: To test the ability of Parkin to mitigate OS, recombinant Parkin and various mutant and control University of Ottawa; 3University of Santiago de rVSVpcs/NANOpcs. proteins were expressed in either E. coli or human cell lines. Parkin’s reactivity towards H2O2 was measure Methods: Mauritian Cynomolgus macaques were immunized with rVSVgag/env, rVSVpcs/NANOpcs, or 3Program of Compostela, Spain; in situ using a chemiluminescence assay. Parkin’s ability to reduce intracellular reactive oxygen species rVSV vector. Weekly cervicovaginal lavage (CVL) and nasal swabs were collected for the multiplex protein Neurosciences, Ottawa (ROS) in vitro was measured using dichlorodihydrofluorescein-diacetate (DCFH-DA) in HEK293 and 4Harvard Medical School, assay (Bio-Plex 200 system). Bio-Plex magnetic beads coupled to primary antibodies in combination with SH-SY5Y cells treated with H2O2. The ability of Parkin to alter the cellular redox state was determined by Hospital Research Cambridge, MA; 5National biotinylated detection antibodies and PE-labeled streptavidin were used to measure cytokines/ measuring reduced and oxidized levels of glutathione before and after OS. Institute, Ottawa, ON Microbiology Laboratory, chemokines in the CVL samples. The cytokine panel consists of 15 (pro- or anti-) inflammatory cytokines/ Results: Parkin is a stronger thiol-dependent antioxidant than glutathione and DJ-1 (another autosomal chemokines to evaluate the inflammatory responses. recessive PD-linked protein), yet is weaker than catalase. These results are consistent with our DCFH-DA Public Health Agency of Results/Discussion: We observed lower levels of IL-1beta, IL-6, IFNγamma, and RANTES from the rVSVpcs/ measurements, in that Parkin is unable to reduce intracellular ROS when cells are treated with H2O2. Canada, Winnipeg, MB NANOpcs group at baseline and/or the week after immunization, while IL8, IL-17alpha and IP-10 were Overexpression of Parkin alters the cellular redox state. Parkin overexpression decreased the reduced lower in comparison only after the first boost. By one week after the first boost, most of the pro- form of glutathione but inhibited oxidation of glutathione under OS. inflammatory cytokines and chemoattractants either had the same or even lower levels for the rVSVpcs/ Conclusion: Although Parkin is unable to significantly reduce ROS in cells undergoing OS, it affects the NANOpcs group compared to the rVSVgag/env vaccine group. This result provides insight on the cellular redox state both under basal and OS conditions by altering glutathione levels. Given the potential of proposed rVSVpcs/NANOpcs vaccine to confer a more site-restricted inflammatory response, importance of glutathione redox state in facilitating numerous cellular functions, Parkin-mediated avoiding unnecessary and excess immune activation. alteration of the cellular redox state may prime cells with neuroprotective mechanisms. Conclusions: Levels of inflammatory cytokines/chemokines play an important role in HIV-1 susceptibility and disease progression. These preliminary data using a custom multiplex protein assay showed a potentially more focused immune response that could avoid excessive inflammatory response and localization of HIV-1 target cells. Characterization of the Autoantibody Response in Experimental Cervical Spinal Cord Stem Cell Engineering Toward the Construction of Brain Organoids for Use in Pre-Clinical Injury Screening of Drugs Against Alzheimer’s Disease Antigona Ulndreaj1, 2, Apostolia Tzekou1, Ahad M. Siddiqui1, Rachel Dragas1, 2, Emina E. Torlakovic5, Michael G. Fehlings1, 2, 3, 4 Roza Vaez Ghaemi, Ileana Co, Vikramaditya G. Yadav

1Division of Genetics and Introduction: Autoimmune diseases affect 5-7% of the world’s population, most of which present with University of British Introduction: The discovery of anti-neurodegeneration drugs has been stymied primarily by the absence Development, Toronto circulating autoantibodies. Previous studies in a model of mid-thoracic spinal cord injury (SCI), have Columbia, Vancouver, BC of reliable pre-clinical drug screening platforms, particularly disease models for the identification of shown that autoantibodies against spinal cord antigens are pathogenic. The majority of clinical SCI cases promising lead compounds. Our goal is advancing the development and validation of a scalable Western Research occur at the cervical level, but it remains unknown whether cervical SCI results in the development of methodology to precisely construct three-dimensionally structured brain tissue exhibiting tau Institute, Krembil autoantibodies against the spinal cord. In this study, we asked whether autoantibodies are generated aggregation for use as a pre-clinical assay. (1) construction of the cerebroids, (2) tool development for Neuroscience Center, following cervical SCI and we characterized this response in the sub-acute (2 weeks post-injury), chronic subjecting the cerebroids to controlled bouts of force, (3) assessment of the pre-clinical utility of the (10 weeks post-injury) and late chronic (20 weeks post-injury) phase of the disease in rats. cerebroids via testing with known MT-stabilizing drugs. University Health Network Methods: Cervical SCI was induced in female Wistar rats with a 35g modified aneurysm clip which Methods: Assembly of the cerebroids proceeds in two stages. In the first stage, spherical aggregates 2Institute of Medical compressed the spinal cord for 60 sec at the C7-T1 level. Sham animals underwent the same surgical comprising cortical neurons, astrocytes and microglia are derived from induced pluripotent stem cells Science, Faculty of procedure, except for clip compression. (iPSCs). Separately, two-dimensional cultures of BBB endothelial cells are also be derived from iPSCs. In Results: We found increased levels of IgG and IgM antibody deposition within the lesioned spinal cord at the second stage, the differentiated BBB endothelial cells are re-suspended and transferred to a stirred- Medicine, University of 2 weeks, but not at later time points of this study. We further identified reactive astrocytes surrounding tank bioreactor along with the spherical neuronal aggregates, wherein tight control of the bioprocessing Toronto 3Department of the cavity at the injury epicenter, and neurons in the anterior horn as some of the cells where the conditions facilitates deposition of the suspended endothelial cells onto the aggregates. Neurosphere Surgery, University of antibodies were primarily located. Moreover, rats with SCI had higher levels of circulating autoantibodies formation are continually monitored visually throughout the differentiation process to verify against cultured astrocytes compared to sham animals. Peripherally, we observed a robust autoimmune Toronto 4University of developmental stages and the culturing conditions are optimized to achieve a defined cytoarchitecture activation in the spleen. IgG- and IgM- antibody producing cells (plasma cells) in the spleen were within the neurospheres. A two-dimensional co-culture of astrocytes and endothelial cells will initially be Toronto Spine Program, significantly expanded following SCI. Also, compared to controls, splenic cells from SCI rats had increased investigated to identify the culturing conditions that facilitate formation of perivascular appendages University of Toronto proliferation capacity and produced more antibodies when stimulated with injured spinal cord between the astrocytes and endothelial cells. Once the cerebroids are ready, they are traumatized and 5Department of homogenate. tau aggregation is quantified by the release of pro-inflammatory cytokine release. Conclusion: These findings demonstrate that the immune system is activated against components of the Results: Immunohistochemical staining of cortical neurons, astrocytes, neural progenitor cells, and Laboratory Hematology, lesioned spinal cord by generating autoantibodies which enter the spinal cord and target neurons and aggregated tau proteins validate the use of cerebroids as scalable human brain models. University of Toronto, astrocytes. However, this process does not persist beyond the sub-acute phase of injury. This is the first Conclusion: The proposed work combines concepts and insights from stem cell bioengineering, organoid Toronto, ON study to characterize the expansion of IgG and IgM autoimmune responses after cervical SCI. Future biomanufacturing, neuroscience and biomedical instrumentation. Our proposal to construct and research will investigate the implication of autoantibodies in the progression of cervical SCI. traumatize cerebroids in order to test drugs could eventually lead to the development of one of the first ever drugs to treat neurodegenerative disorders, which will directly benefit Canadians, and may prove to be a game changer for pharmaceutical testing and molecular medicine. Striatal Tyrosine Hydroxylase DNA Methylation Is Associated with Human Cocaine Characterizing the Urinary Peptidome of Early Type 1 Diabetes to Infer Protease Activity Dependence in the Kidneys Kathryn Vaillancourt2, Carl Ernst1, 3, Gang G. Chen1, Alexandre Bramoulle1, Jean Francois Theroux1, Aura Fiori1, Gilles Maussion1, Julie A.D. Van1, Ihor Batruch2, Eleftherios P. Diamandis2, 3, James W. Scholey1, 4, Ana Konvalinka1, 4 Erin Calipari4, Benoit Labonte4, Eric Nestler4,Deborah C. Mash5, Gustavo Urecki1, 2, 3 1Institute of Medical Introduction: Diabetes is the leading cause of kidney disease worldwide. Early functional and structural 1McGill Group for Suicide Background: Cocaine dependence is a chronic relapsing disorder with long-term negative consequences Science, University of changes in type 1 diabetes may arise from dysregulated protease activity and this activity is specific to certain proteases and protein substrates. Resultant peptides excreted into urine provide a footprint of Studies, Douglas Hospital and, to date, no pharmacological treatment. The development and trajectory of cocaine dependence Toronto, Toronto, ON; coincides with lasting behavioral and cellular changes, including dysregulated gene transcription, in intrarenal proteolysis. We aim to compare urinary peptidomes of adolescents with uncomplicated type 1 2 Research Center, Verdun, numerous brain areas. Recently, several studies have identified epigenetic marks, including histone post- Department of diabetes and infer changes in intrarenal protease activity. QC; 21ntegrated Program translational modifications, that are associated with the acquisition of compulsive drug seeking in animal Pathology and Laboratory Methods: We collected second-morning, midstream urines from 15 cases with type 1 diabetes and 15 age- and sex-matched controls from the observational arm of the Adolescent Diabetes Cardio-Renal in Neuroscience, McGill models, but little is known about the role of epigenetics in human cocaine dependence. We are Medicine, Mount Sinai particularly interested in DNA methylation as it represents a mitotically stable mark that has been shown Intervention Trial at SickKids Hospital in Toronto. Urines were subjected to 10kDa filter centrifugation to University; 3Department Hospital, New York, NY; to be altered by environmental cues. isolate naturally occurring peptides. Filtered peptides were fractionated by strong cation exchange liquid of Psychiatry, McGill Methods: We profiled the methylome of 25 dependent cocaine users and 25 controls used Reduced 3Department of chromatography and analyzed on Q-Exactive mass spectrometer. MaxQuant software was used for University, Montreal, QC; Representation Bisulfite Sequencing (RRBS) in two addiction-relevant regions of the striatum, the nucleus Laboratory Medicine and peptide/protein identification and label-free quantification. Peptide Extractor and Proteasix were used to accumbens (NAc) and caudate nucleus (CD). We validated multiple differentially methylated regions infer protease activity based on amino acid sequences of peptides. 4Icahn School of Pathobiology, University (DMRs) in each brain region and replicated a DMR in the tyrosine hydroxylase (TH) gene using bisulfite Results: Both groups were similar in most clinical characteristics, except for those relating to Medicine, Mount Sinai sequencing. Fluorescence activated cell sorting (FACS) allowed us to identify cell-type specific of Toronto; 4Division of hyperglycemia and diastolic blood pressure. The mean duration of diabetes for cases was 7.1 ± 2.7 years. Hospital, New York, NY; methylation changes, and we used an animal model of chronic cocaine seeking, transcriptional assays Nephrology, Toronto A total of 6349 peptides from over 750 proteins were quantified. We applied a stringent cut-off filter to identify and isolate peptides found in all samples, resulting in 163 peptides. Fifteen peptides were 5Department of and in vitro experiments to further investigate the impact of DNA methylation on this cocaine-related General Hospital, Toronto, genomic loci. significantly different between groups; however, only five remained significant after Benjamini-Hochberg Neurology, University of Results: We found numerous DMRs in both brain regions, including within exon 8/9 of TH that are more ON adjustment. These peptides included four uromodulin fragments and one clusterin fragment. Uromodulin Miami Miller School of methylated in the cocaine group. Methylation negatively correlates with TH expression in the CD of the peptides were situated between a known cleavage site by hepsin and its GPI-anchor inside the external hydrophobic patch, which is important in the polymerization of uromodulin. The clusterin peptide Medicine, Miami, FL cocaine group, but not in control CD. We replicated this effect in the NAc of an independent cohort (N=18 per group), and of chronically self-administering mice (N=8-10 per group). This hypermethylation originated from the alpha chain near the nuclear localization signal. Peptide intensities were strongly appears to be neuron-specific and impedes enhancer activity at this locus. correlated with glycated hemoglobin, HbA1c. Conclusions: Hypermethylation of TH is associated with chronic drug seeking behavior and may have Conclusions: Differences between cases with uncomplicated type 1 diabetes and healthy controls can be regulatory potential. Further research will uncover the functional relationship between epigenetic detected in the urinary peptidome before the onset of microalbuminuria. We identified endogenous dysregulation of TH and the time course and trajectory of chronic cocaine dependence. A Novel Role of Vaccinia Virus MRNA Capping Enzyme in Determining Virus Host Range Jessie Varga1, Yvon Deschambault2, Jingxin Cao1, 2 uromodulin and clusterin peptides, whose urinary excretion rates strongly correlated with HbA1c. 1University of Manitoba; Introduction: Vaccinia virus, the prototypic member of Poxviridae, encodes the immune modulator Further investigation into the interplay between uromodulin and hepsin in hyperglycemic conditions 2National Microbiology protein, E3L. The primary regulatory function of E3L has largely been attributed to the biochemical may shed light onto early kidney dysfunction. capability of binding double-stranded RNA (dsRNA), thereby inhibiting induced cellular responses. Laboratory, Winnipeg, MB However, mutational analysis of the E3L gene has revealed that the dsRNA binding capacity of the E3L protein is not essential for its biological function. Additionally, studies replacing the E3L gene with an ortholog from other poxviruses revealed that the ortholog from sheeppox virus, CPV034, was not able to rescue viral replication in cell culture, even though the protein retained the ability to bind dsRNA. Further analysis of viral propagation in various cell lines have demonstrated the E3L protein, and its orthologs, appear to be critical in determining the host range of poxviruses. We propose that CPV034 is not sufficient to restore E3L function due to the inability to interact with one or more vaccinia virus derived cofactors. Approach: Ethyl methanesulfonate (EMS) mutagenesis of a recombinant vaccinia virus, in which E3L was replaced with CPV034, uncovered two independent mutations capable of rescuing viral replication. These mutations occurred in the heterodimeric mRNA capping enzyme. These mutants were then used to create E3 knockout viruses. These viruses were analyzed for the ability to modulate host immune system pathways. Specifically, activation of protein kinase R and eIF2 were analyzed using Western blotting, and suppression of cytokines IL-6, TNF-α and IFN-β were analyzed using RT-qPCR. Conclusion: EMS mutagenesis of a recombinant vaccinia virus expressing CPV034 resulted in isolates that regained the ability to replicate in cell culture. Whole genome sequencing analysis revealed two independent mutations, one in the D1R gene (the result of a G801S substitution) and another in the D12L gene (the result of a S131N substitution). These proteins form the heterodimeric mRNA capping enzyme of vaccinia virus and are candidates for the cofactors between the host immune system and the viral antagonist, E3L protein. Comparative Resource Utilization and Workload in Patients with Pandemic H1N1 Sustained Peripheral Inflammation Triggers Central Anandamide Hydrolysis to Promote Pneumonia, Non-H1N1 Viral Pneumonia and Non-Viral ARDS Anxiety Gloria Vazquez-Grande, Anand Kumar Haley A. Vecchiarelli, Maria Morena, Kaitlyn Tan, Catherine Keenan, Martin Sticht, Kira Leitl, Winnie Ho, Min Qiao, Keith A. Sharkey, Matthew N. Hill Department of Medical Background: During the 2009-2010 H1N1 influenza pandemic anecdotal reports suggested an extremely Microbiology and Critical high resource utilization and workload. Given the certainty of future influenza pandemics, a rigorous Hotchkiss Brain Institute, Introduction: There is a high degree of comorbidity between chronic inflammatory conditions (e.g. assessment of resource utilization and workload impact of pandemic influenza relative to similar non- Care Medicine, University of Calgary, inflammatory bowel diseases, arthritis) and stress-associated neuropsychiatric disorders (e.g. anxiety, influenza respiratory illness could help inform future pandemic planning. depression); however, the mechanisms underlying these comorbidities are not fully elucidated. The University of Manitoba, Methods: Three cohorts of adult patients were identified from a high quality regional ICU database in the Calgary, AB endocannabinoid system, the system in the body that cannabis acts on, reduces anxiety and inflammation, Winnipeg MB 2009 peri-pandemic period 1) H1N1 confirmed (n=58) 2) other confirmed viral pneumonia (n=25) and 3) making it an ideal candidate to investigate the mechanisms of these comorbidities. Our aim was to adult respiratory distress syndrome (ARDS, n=46). Patient records were reviewed to identify epidemiological investigate if the endocannabinoid system is altered following peripheral inflammation (colitis), and to factors and use of laboratory tests, imaging studies, pharmacologic therapies and supportive interventions. determine the relevance of these changes for inflammation-induced anxiety, in a rodent model. The Therapeutic Intervention Scoring System (TISS-28) score was calculated for each patient for the first Methods: Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid to adult 10 days of admission, to determine overall resource utilization and workload. Comparisons were made male rats. Seven days after induction of colitis, levels of the endocannabinoids, anandamide (AEA) and using t-tests, Mann-Whitney test or rank sum test as appropriate. 2-arachidonylglycerol (2-AG), were measured in the amygdala, hippocampus, hypothalamus and medial Results: There were no significant differences between groups in terms of age (47.0 ± SD 18.3 yrs) and prefrontal cortex (brain regions that regulate stress and anxiety) using mass spectrometry. Anxiety like ICU length of stay (13.9 ± 11.8 days). Survival to hospital discharge was significantly higher in the H1N1 behaviors were assayed using the elevated plus maze. cohort than the ARDS cohort (90.0 vs 67.4%, p=.0112). Similar survival trend existed compared to the Results: AEA levels were reduced in the amygdala, hippocampus and medial prefrontal cortex. In the non-H1N1 cohort (72.0%, p=.0771). After Bonferroni correction, no differences in cumulative use of any amygdala, the decrease in AEA was accompanied by an increase in the activity of its metabolic enzyme, laboratory tests or imaging studies were found between groups. Among pharmacologic therapies, the fatty acid amide hydrolase (FAAH). In contrast, 2-AG levels were increased in the hippocampus and H1N1 cohort had significantly higher use of intravenous sedatives and narcotics; whereas, the number of medial prefrontal cortex. There was also an increase in anxiety levels, as measured by a decreased time antibiotic days was significantly higher in the non-H1N1 group. The average TISS-28 score was similar in spent in the open arm of the elevated plus maze. The colitis-induced increase in anxiety was reversed by all groups over the first 10 days of ICU admission. However, resource utilization with the ARDS cohort an acute intracerebroventricular administration of a FAAH inhibitor (PF- 4458945), and the ability of the declined more rapidly over the first 10 days of ICU admission than the other groups. FAAH inhibitor to be anxiolytic was correlated with the animal’s colon macroscopic tissue damage score. Conclusion: Although patients with 2009 pandemic influenza H1N1 exhibit increased utilization of Conclusion: Our findings reveal a role for AEA in the development of inflammation-induced anxiogenesis sedative and narcotic agents compared to other groups, their overall resource utilization and workload is and provide a molecular mechanism for the comorbidities of these conditions. Together our findings similar to other comparable patient groups. While preplanning may be required to ensure an adequate increase the understanding of the mechanisms behind anxiety behaviors in chronic inflammatory states, supply of these drugs in another pandemic situation, resource planning does not require a higher than and suggest that similar to stress-induced anxiety, inflammation-induced decreases in AEA signaling are anticipated workload for individual patients. likely relevant for the change in emotional behaviors associated with inflammatory states. BioID Proximity Labeling Identification of Androgen Receptor Signalling Networks in Predictors of Having First Child Taken Into Care At Birth: A Population-Based Castration Resistant Prostate Cancer Longitudinal Cohort Study Lauriane Vélot1, 2, Frédéric Pouliot1, 2, 3, Nicolas Bisson1, 2, 4 Elizabeth Wall-Wieler, Leslie L. Roos, Marni Brownell, Nathan C. Nickel, Dan Chateau

1Centre de Recherche sur Introduction: Prostate cancer (PC) is the most frequently diagnosed cancer in Canadian men and is the University of Manitoba, Background: This study links events and diagnoses in the two years before childbirth to identify women le Cancer; 2Division 3rd cause of cancer mortality. The Androgen Receptor (AR) is activated by androgens (e.g. testosterone), Winnipeg, MB at higher risk for having their first child taken into care by child protection services at birth. which leads to prostatic cell proliferation. The primary treatment for advanced PC is androgen‐deprivation Methods: Linkable administrative data was used to create a population-based cohort of women whose Oncologie, Centre de therapy, which is achieved via surgical or chemical castration. Nevertheless, most PC will become first child was born in Manitoba, Canada between January 1, 2002 and February 28th, 2012 and lived in Recherche du Centre castration‐resistant (CRPC). Although new anti‐androgens (e.g. Enzalutamide) were developed to the province at least two years before the birth of their first child (n = 53,565). A logistic regression model Hospitalier Universitaire improve patients’ survival, their efficacy is still very limited and most CRPC patients will die from the determined the adjusted odds ratios of having a child taken into care at birth. The model includes disease within a few years. We postulate that by gaining insights into AR signaling networks in the variables before the pregnancy, during the pregnancy, and at the birth of the child. (CHU) de Québec; context of CRPC, we could better direct patients towards the best‐suited therapy and propose new Results: Characteristics having the strongest association with a woman’s first child being taken into care 3Département de therapeutic targets. at birth were mother being in care at the birth of her child (AOR = 11.10), substance abuse in both before chirurgie, Université Laval; Methods: To this aim, we took advantage of an innovative proteomics approach, namely proximity‐ and during the pregnancy (AOR = 8.94), schizophrenia (AOR = 6.69) developmental disability (AOR = labeling (BioID), to characterize global AR signaling networks in hormone‐dependant LAPC4 cells. 6.45), and not having had any prenatal care (AOR = 5.47) The covariates included produced a strong 4Département de Biologie Results: We identified 45 AR‐associated proteins in non‐stimulated cells, 35 of which were not previously model (C-Statistic = 0.927). Moléculaire, Biochimie reported. Upon androgenic stimulation, the AR signaling network increased to 320proteins, including Interpretation: Most characteristics of women deemed to be at high risk for having their child taken into Médicale et Pathologie, 278 (253 were novel) that were restricted to androgen-stimulated cells. Enzalutamide treatment resulted care at birth are modifiable or could be mitigated with appropriate services. Greater efforts should be Université Laval, in a loss of 259 proteins from the network when compared to stimulated cells. As expected, this made to prevent a child to be taken into care at birth and allow for mother-child bonding in the critical reproduced quite faithfully the status of non‐stimulated LAPC4 cells. Interestingly, we identified 7 first year of life. Quebec City, QC proteins in the AR network specifically after Enzalutamide treatment. Conclusion: These are interesting targets that may be relevant for the acquisition or the prediction of Enzalutamide resistance. Hence, they could become in time alternative therapeutic targets for CRPC treatment, to be used when Enzalutamidefails. FGF-16 and Acute Doxorubicin Cardiotoxicity: An Early Target for Early Protection Effects of Qufeng Tongqiao Recipe on Microtubule Related Protein 1 Light Chain 3, Jie Wang, Peter A. Cattini Beclin-1 and AI in Hippocampal CA1 Area of Vascular Dementia Rats Raoqiong Wang1,Haixia Qian2,Xue Bai1 Department of Physiology Doxorubicin is a drug used widely in chemotherapy, however, cardiotoxicity that can lead to heart failure and Pathophysiology, is a major risk factor for cancer survivors. Strategies are needed to protect the heart. Fibroblast growth 1Southwest Medical Introduction: Based on the observation of microtubule associated protein 1 light chain 3 (MAP1-LC3), factor (FGF) 16 is a cardiac maintenance and protective factor. Evidence from Fgf-16 null mice indicates University of Manitoba, University, Chongqing Shi, autophagy protein Beclin-1, and Apoptosis Index (AI) in vascular dementia (VD) model rats, investigating that FGF-16 promotes cardiomyocytes proliferation and increases resistance to cardiac fibrosis and the protection mechanism of Qufengtongqiao Decoction on VD rats. 2 Winnipeg, MB hypertrophy in an angiotensin II-induced model of injury. FGF-16 supplementation also prevents an China; Chinese and Methods: The rats were randomly divided into sham operation group, model group, western medicine acute doxorubicin-induced decrease in left ventricle contractility in isolated mouse hearts. However, the Western Medicine group, Qufengtongqiao Decoction the high, medium, and low dose group by random table method. The effects of doxorubicin on endogenous FGF-16 levels and the effects of FGF-16 against doxorubicin- Hospital of Dazhou City, western medicine group was given nimodipine. The high, medium, low dose groups were given different induced cardiomyocyte damage have not been reported. Our hypothesis is that FGF-16 is an early target dosage of Qufengtongqiao Decoction respectively. Sham operation group and model group were given Sichuan Sheng, China of doxorubicin that may contribute to cardiotoxicity, and thus maintaining/increasing FGF-16 levels will normal saline . Used different methods to test the learning and memory abilities, the expression of offer protection against doxorubicin-induced cardiomyocyte damage. The level of FGF-16 expression in LC3,Beclin-1 protein and AI . 8 week-old Sprague-Dawley rat hearts was assessed after intraperitoneal injection of doxorubicin (15mg/ Results: Compared with the sham operation group, the platform quadrant dwell time of model group kg) as well as in neonatal rat cardiomyocytes after 1μM doxorubicin treatment. In both cases, FGF-16 RNA and each treatment group was shortened, and the frequency of crossing the virtual platform decreased, levels were significantly decreased within 6 hours of doxorubicin treatment, and evidence of a role for the the escape latency (EL) was prolonged, the difference was statistically significant (P<0.05); Compared cardiac transcription factor Csx/Nkx2.5 in this downregulation was also obtained. In addition, with the model group, platform quadrant dwell time of the western medicine group and the high, overexpression of FGF-16 increased resistance to doxorubicin-induced apoptosis and necrosis in isolated medium dose group was extended, and the frequency of crossing virtual platform increased, the EL was cardiomyocytes. This corresponded to an increase in efflux transporter Mdr1a expression and reduction shortened in the last three days, the difference was statistically significant (P<0.05). 3) Expression of LC3, in intracellular calcein and doxorubicin concentration, consistent with increased efflux activity. In Beclin-1 protein and AI: Compared with the sham operation group, the expression of LC3 ,Beclin-1 protein summary, cardiac production of FGF-16 appears to be an early target of acute doxorubicin cardiotoxicity, and AI in model group and each treatment group increased, the difference was statistically significant but increased FGF-16 levels offers protection against doxorubicin-induced cardiomyocyte cell death (P<0.05). Compared with the model group, the expression of LC3 ,Beclin-1 protein and AI in the western potentially by effects linked to efflux activity. Understanding the effect of FGF-16 on efflux drug medicine group, and the high, medium dose groups was significantly higher, the difference was transporter production and function may offer a further target for prevention in doxorubicin-induced statistically significant (P<0.05). 4) LC3,Beclin-1 protein and AI have a significant negative correlation. cardiotoxicity. Conclusion: Qufengtongqiao decoction can improve the learning and memory abilities, raise expression of LC3 and Beclin-1, and improve cell apoptosis with considerable dose-effect relationship. Sin Nombre Virus Infection in Deer Mice: Reactivation, Transmission, and Strategies for MRI-Guided Focused Ultrasound Gene Delivery to the Brain Using AAV6 and AAV1/2 Prevention Under Control of the Neuron-Specific Synapsin Promoter Bryce Warner, David Safronetz, Gary Kobinger Danielle Weber-Adrian1, 2, Joseph Silburt1, 2, Zeinab Noroozian1, 2, Kairavi Shah3, Alison Burgess4, Sebastian Kügler5, Kullervo Hynynen4, 6, Isabelle Aubert1, 2 1Department of Medical Introduction: Sin Nombre virus (SNV) is the primary cause of hantavirus cardiopulmonary syndrome (HCPS) in North America, a severe disease characterized by vascular leakage, cardiopulmonary edema, Microbiology, University 1Biological Sciences, Introduction: The blood brain barrier (BBB) prevents delivery of most therapeutics from accessing the and shock with a mortality rate close to 30%. To date, there have been greater than 100 confirmed cases 2 brain from the bloodstream, including adeno-associated virus (AAV) serotypes 6 and the mosaic 1/2. of Manitoba; National in Canada. Hantaviruses are carried by rodent reservoirs, with SNV being found in Peromyscus maniculatus Sunnybrook Research Instead of using surgical, intracranial delivery, MRI-guided focused ultrasound (MRIgFUS) application can Microbiology Laboratory, or deer mice. Infection in humans is caused by inhalation of aerosolized virus from contaminated rodent Institute; 2Laboratory transiently and locally disrupt the BBB, thereby permitting access to the brain after systemic injection of excreta. Currently, little is known about the dynamics of SNV infection in deer mice or what factors lead Public Health Agency of Medicine and AAV. To prevent transgene expression in non-target organs, the neuron-specific synapsin promoter was to increased shedding and transmission of the virus. It has been hypothesized that physiological changes Canada, Winnipeg, MB; Pathobiology, University used. such as heat shock responses or changes in sex hormone levels may influence viral replication and Methods: AAV6 and AAV1/2-synapsin expressing green fluorescent protein (GFP) was injected through a 3Laval University, Quebec shedding. The goal of this project is to determine how SNV is transmitted between deer mice, what of Toronto; 3Institute of tail vein catheter in C57Bl/6 mice and targeted to the striatum and hippocampus using MRIgFUS. The factors might be involved in increased viral replication and shedding, and to test strategies for prevention City, QC Medical Science, number of NeuN (neuronal marker)-positive, GFP-positive cells in the brain was compared between AAV of transmission such as vaccination of naive mice. University of Toronto; serotypes and quantified using unbiased stereology methods. Transduction of non-target organs Methods: For transmission experiments, three uninfected deer mice per cage will either be housed with including the liver, heart, and muscle was quantified using droplet digital PCR (ddPCR). an infected mouse (direct transmission) or in a soiled cage that previously housed an infected mouse 4Physical Sciences, Results: AAV6-synapsin-GFP resulted in a significantly higher percentage of NeuN-positive, GFP-positive (indirect transmission) for six weeks to determine the rate of transmission. Additionally, heat shock Sunnybrook Research cells in brain than AAV1/2-synapsin. The AAV6 serotype also showed four-fold lower levels of liver responses will be induced by oral gavage of paeoniflorin to assess their role in viral replication, shedding, Institute, Toronto, ON; transduction as compared to the AAV1/2 mosaic serotype. and transmission. The effect of testosterone levels on viral replication and shedding will be assessed in 5 Conclusion: AAV6-synapsin-GFP is a promising candidate for MRIgFUS-mediated gene delivery to the castrated male deer mice implanted with osmotic pumps filled with either testosterone or oil as a control. Neurology, University of brain. AAV6-synapsin-GFP demonstrated higher expression efficiency in the brain, and lower transduction Finally, deer mice vaccinated against SNV will be included in transmission experiments to assess the Göttingen, Göttingen, of the liver than AAV1/2-synapsin-GFP. potential of vaccination for prevention of transmission of the virus. Germany; 6Medical Results: Hsp70 expression can be induced in deer mice by oral gavage of 50mg/kg paeoniflorin and sustained administration of the drug has no adverse effects on the mice. Biophysics, University of Conclusion: We hope to shed light on how SNV is transmitted between deer mice, what factors play a role Toronto, Toronto, ON in facilitating viral replication and shedding, and how to potentially prevent transmission of the virus through vaccination. DLC1-Deficient Breast Cancer Cell Models for Therapeutic Targeting Drug Repurposing for the Treatment of Experimental Alport Syndrome Nicole A. Wilkinson, Brent J. Guppy, Kirk J. McManus, Michael R.A. Mowat Vanessa R. Williams1, Ana Konvalinka1, 2, Xuewen Song3, Nicholas Maksimowski1, Xiaohua Zhou2, Rohan John4, York Pei2, 3, James W. Scholey1, 2 Dept. of Biochemistry and Introduction: Breast cancer is the second most common cause of cancer-related deaths in Canadian Medical Genetics, women, primarily due to metastatic disease. DLC1 codes for a Rho-GTPase activating protein that has 1Institute of Medical Introduction: Alport Syndrome (AS) is a hereditary nephropathy caused by mutations in genes that both tumour and metastasis suppressor functions. Diminished DLC1 expression occurs in approximately University of Manitoba; Science, University of encode type IV collagen. It is characterized by early proteinuria and progressive injury eventually leading 50% of breast cancers making it an ideal candidate for therapeutic interventions that could conceivably to end-stage renal disease. Currently there are few effective therapies for AS. Therefore we applied a drug 2 Research Institute in target both primary and metastatic disease. In this study, we employ short hairpin (sh)RNA gene silencing Toronto; Department of repurposing strategy, utilizing data on drugs currently approved for use in humans, to identify a novel AS Oncology and and CRISPR/Cas9 gene editing approaches to generate two complementary DLC1-deficient breast cell Medicine, Division of treatment. models that will be employed in subsequent drug target screens. Hematology, CancerCare Nephrology, University Methods: 129/SvJ Col4a3 knockout (KO) and wild-type (WT) mice were studied at 4 and 7 weeks (n = 8 Methods & Results: To generate DLC1 knockdown cells, MCF7 breast adenocarcinoma cells were per group). Global gene expression profiling of RNA from renal cortical samples was performed with the Manitoba, Winnipeg, MB Health Network; transduced with four independent pGIPZ shDLC1 vectors, and stable clones were generated. Western Affymetrix Mouse Gene 2.0 ST Array. Significance Analysis of Microarrays (SAM) was used to identify blots were used to identify the two most efficient shRNAs. To generate DLC1 knockout cells, CRISPR/Cas9 3Division of Genomic differentially expressed genes. In silico drug repurposing with the Connectivity Map (CMAP) was used to strategies have been employed. First, MCF7 cells were transduced with a Cas9 expression cassette and Medicine, University identify drugs expected to effectively treat murine AS. Histological analyses of formalin-fixed mouse two clonal Cas9-expressing cell lines were subsequently selected for targeting with three independent kidney sections were performed. Plasma and 24-hour urine samples were collected. Polar kidney sections Health Network; DLC1 guide RNAs. Initial screens of 48 candidate clones were evaluated without any DLC1-deficient cells were snapfrozen and then used for Western blot analysis. being identified. To increase the probability of generating and isolating DLC1-deficient cells, lentiviral 4Departments of Results: Mice with AS developed a progressive rise in albuminuria, plasma creatinine, and blood urea vectors encoding guide strands, blue fluorescent protein and puromycin resistance are being employed Pathology and Laboratory nitrogen (BUN). These changes were associated with glomerular sclerosis and tubulointerstitial fibrosis. to allow for enhanced enrichment of edited cells. Once generated, these DLC1-deficient cellular models Medicine and SAM was used to generate a disease progression signature of 927 differentially expressed genes (FDR < will be phenotypically and biochemically characterized compared to control cells. 1%, > 2-fold difference), comparing 4- and 7-week-old KO versus WT mice. The progression signature was Conclusion: These validated DLC1-deficient cellular models will subsequently be employed in drug target Pathobiology, University used to query the CMAP. Vorinostat, a lysine deacetylase (KDAC) inhibitor, was the top drug predicted to screens to identify putative therapeutic targets of DLC1-deficient cells using synthetic lethal screens. Health Network, reverse the signature (p = 0.00032). KO mice were treated with vehicle or vorinostat daily from 4 to 7 Further validation in pre-clinical models may provide the basis for the development of novel breast Toronto, ON weeks of age. Vorinostat treatment induced hyperacetylation of kidney protein lysine residues. Treatment cancer therapeutics. tended to attenuate injury as indicated by improvement of albuminuria, plasma creatinine, BUN, and tubulointerstitial fibrosis. Vorinostat treatment was associated with decreased αSMA expression, and reduced expression of proinflammatory cytokines. Conclusion: In silico drug repurposing identified a novel therapeutic approach to AS. In vivo testing of the putative therapy showed that vorinostat exerts a renoprotective effect. Further studies will better define the mechanisms underlying the protective effect of this novel therapy and thoroughly investigate specific mechanisms of KDAC inhibition in AS. We Ask Because We Care: Feasibility and Acceptability of Sociodemographic Data Unravelling the Relationship Between Early and Late-Onset Preeclampsia. What Does the Collection in Urban Health Care Settings in Saskatoon Placental DNA Methylation Profile Reveal? Hazel Williams-Roberts1, Erin Beckwell2, Lara Murphy2, Mel Brockman2, Cordell Neudorf1, 2 S. L. Wilson 1, 2, K. Leavey 3, P. von Dadelszen 2, 4, B. Cox, 3, 5, W. P. Robinson1, 2

1University of Introduction: Despite recognition that conditions of daily living affect health, health care organizations 1University of British Introduction: Preeclampsia (PE), a maternal hypertensive disorder, can be divided into early-onset PE Saskatchewan; have not instituted measures to consistently capture or act upon critical social history details. This limits Columbia, Medical (EOPE; diagnosis, <34 weeks), and late-onset PE (LOPE; diagnosis >=34 weeks); however, the relationship opportunities to provide equity-oriented care and develop more responsive community programs that between them remains unclear. Placental insufficiency is more common in EOPE, while maternal factors 2 2 Public Health mitigate the impact of social risks. The study assessed the feasibility and acceptability of sociodemographic Genetics; BC Children’s may be relatively more important in LOPE. Our objective was to use placental DNA methylation (DNAm) Observatory, Saskatoon data collection across three urban health care settings. It also explored local facilitators and barriers to Hospital Research, profiling to investigate the etiology of EOPE and LOPE, and their relationship to one another, with an Health Region, implementation. Vancouver, BC; 3University overarching goal to identify potential candidate biomarkers for PE. Methods: A multiple case study was used to evaluate implementation of sociodemographic data Methods: Placental DNA from a discovery cohort (24 pre-term controls, 22 term controls, 22 EOPE, 18 Saskatoon, SK of Toronto, Dept. of collection across sites. The study draws on multiple methods including individual and group interviews, LOPE) and validation cohort (9 pre-term controls, 9 term controls, 19 EOPE, 11 LOPE) were run on the chart reviews and surveys to understand the diverse participant perspectives and experiences. Cross case Physiology, Toronto, ON; Illumina HumanMethylation450 array. Data were normalized using functional normalization. Linear synthesis was done to identify similarities and differences across health contexts. 4University of British regression was used to model pathology and fetal sex to identify top differentially methylated (DM) sites Results: Analysis revealed that it was feasible to collect selected sociodemographic information although (FDR<0.05, Δβ>0.1). Columbia, Obstetrics and there was a gradient of comfort depending on the question and context in which it was asked. High item Results: In the discovery cohort, 1703 DM sites were identified in EOPE compared to pre-term controls non responses were observed for questions related to annual household income and year of arrival to Gynecology, Vancouver and 5 in LOPE compared to term controls. The 5 DM in LOPE were also DM in EOPE. There was a correlation Canada. Patient participants who felt that data collection was important appreciated how it could be BC; 5University of Toronto, between the DNAm changes observed in EOPE in the discovery cohort compared to the validation cohort (r=0.62, p<2.2e-16). Using a nominal p-value and change in DNAm in the same direction as the discovery used to improve care, trusted institutional motives for collection and had positive experiences with the Obstetrics and health care system. Common reasons for dissenting views included perceptions that it was inappropriate cohort, 539/1703 sites validated. Clustering on these sites in both cohorts reveals 3 distinct clusters, 1 to ask or irrelevant to the provision of care and concerns about the potential for misuse of the information. Gynaecology, Toronto, ON associated with placental pathology, another with pre-term birth/gestational age, and the other Preferences for mode of administration of questions varied depending on participant characteristics. consisting mostly of term and pre-term controls. Interestingly some LOPE cases clustered with EOPE in Structural, organizational, provider and patient factors influenced implementation across sites. the placental pathology cluster. These cases all had an earlier onset of PE than the LOPE cases that Conclusion: There is growing interest in upstream approaches to the delivery of care. The study clustered with the pre-term or control clusters. demonstrated feasibility and acceptability for sociodemographic data collection however more work is Conclusions: Widespread changes in EOPE are observed. DNAm profiles may be better at classifying needed to support wider implementation of some determinants across local health care settings. some cases of PE based on etiology rather than onset of PE. These validated hits are a starting point to look at potential biomarkers capable of predicting EOPE/placental insufficiency prior to the onset of clinical symptoms. Extracellular Matrix Surface Thickness Regulates Self-Assembly of Three-Dimensional Activation of TRPM7 by Naltriben Enhances Migration and Invasion of Glioblastoma Cells Placental Trophoblast Spheroids Raymond Wong, Ekaterina Turlova, Zhong-Ping Feng, James T Rutka, Hong-Shuo Sun Michael K. Wong1, Madeline A. Green1, Tom Ewart2, Ponnambalam R. Selvaganapathy3, Sandeep Raha1 University of Toronto, Glioblastoma (GBM) remains the most common and aggressive malignant brain tumor originating in the 1Department of Medical Introduction: The human placenta is pivotal for the growth and survival of the fetus during pregnancy. Toronto, ON central nervous system. Diagnosis is lethal with a median survival of <15 months. Aberrant TRPM7 expression has been linked to GBM cellular functions. Here, using the human GBM cell line U87, we Science, McMaster Many of the signature functions of this multifaceted organ are directed by interactions between its trophoblast cells and the extracellular matrix (ECM). Therefore, the incorporation of ECM is essential for further established TRPM7 as a potential therapeutic target by evaluating the TRPM7 potentiator, University, Hamilton, ON; generating in vitro models that better replicate the three-dimensional (3D) cellular organization found in naltriben, on GBM viability, migration, and invasiveness. Firstly, with the whole-cell patch-clamp 2Evik Diagnostics, Ottawa, human tissues. However, appropriate utilization of laboratory-derived ECM biomaterials in placental technique, we demonstrated that naltriben enhanced the endogenous TRPM7-like current in U87 cells. With Fura-2 Ca2+ imaging, we showed robust Ca2+ influx following naltriben application. U87 cell ON; 3Department of trophoblast research remains poorly understood. In this study, we investigated the effect of varying thicknesses of two ECM surfaces, collagen I and Matrigel, on BeWo placental trophoblast cell morphology, migration and invasion (assessed with scratch wound assays, Matrigel invasion experiments, and MMP-2 Mechanical Engineering, proliferation, and gene expression (differentiation, syncytial fusion, invasion/migration, and nutrient protein expression) were significantly enhanced with naltriben, but not viability and proliferation McMaster University, transport). (evaluated with MTT assays). With Western immunoblots, we also assessed the protein levels of p-Akt/t- Akt, and p-ERK1|2/t-ERK1|2. We found that, in U87, naltriben enhanced the MAPK/ERK signaling pathway, Hamilton, ON Methods: Two different thicknesses (50 and 250 micrometers) of collagen I or Matrigel were manually casted for BeWo cell culture for up to 7 and 21 days. Immunofluorescence and microscopy was used to but not the PI3k/Akt pathway. Therefore, potentiated TRPM7 activity contributes to the devastating assess cellular morphology and spheroid organization. Expression of genes related to proliferation, migratory and invasive characteristics of GBM. apoptosis, differentiation, syncytial fusion, invasion, and transport were quantified via real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results: Matrigel, but not collagen I, induced self-organization of BeWo trophoblasts into 3D spheroids that maintained for up to 21 days. Notably, it was revealed that a critical hydrogel surface thickness was required to induce spheroid formation. Trophoblast spheroids exhibited thickness-dependent increases proliferation and invasive gene expression profiles (e.g., MMP9) compared to two-dimensional cultures. Genes involved in syncytial fusion (e.g., CGA, CGB) exhibited thickness-dependent decreases in expression. Conclusions: The generation of 3D, self-assembling spheroid cultures through regulation of ECM surface type and thickness contributes to a deeper understanding of cell-ECM interactions in placental biology. In consideration of the increased usage of 3D bioprinting and dynamic model systems within the last several years, a proper understanding and integration of ECM biomaterials will be a crucial step towards generating truly relevant models for pregnancy research. Prognostic Value of CT Perfusion for Predicting Good Functional Outcome in Ischemic Human Milk Oligosaccharides Prevent Experimental Necrotizing Enterocolitis by Stroke Patients After Successful Intra-Arterial Therapy Inducing Mucus Production Eric Wright1, Christopher d’Esterre2, Enrico Fainardi3, Ting-Yim Lee1 Richard Y Wu1, 3, Bo Li1, Yuhki Koike1, Pekka Maattanen1, Agostino Pierro1, 2 Philip M Sherman1, 3

1Department of Medical Introduction: Results from the EXTEND-IA, ESCAPE and MRCLEAN trials revealed intra-arterial therapy 1Research Institute, Introduction: Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm Biophysics, University of (IAT) is superior to IV-tPA for patients with proximal occlusions. Successful recanalization is defined as a Hospital for Sick Children; infants. Breastfed babies have a lower risk of NEC than those who are formula-fed, but mechanisms 2b or 3 on the thrombolysis in cerebral ischemia (TICI) scale. However, in the trials mentioned above, underlying this protection remain unclear. The objectives of this study were to investigate the effects of Western Ontario, London, 2Division of General and ~17% of IAT-treated patients had TICI scores of 2b/3 but still suffered poor outcomes (mRS≥3). Post- human milk oligosaccharides (HMO) on the intestinal epithelium in vitro and in a mouse model of NEC. ON; 2Department of procedural CT Perfusion (CTP) could identify patients who may have poor outcomes due to incomplete Thoracic Surgery, Hospital Methods: Transwell-grown Caco-2 gut epithelial cells were incubated with HMO isolated from human Radiology, Foothills reperfusion. The study objective was to determine the association between reperfusion and outcome in for Sick Children; donors and then challenged with enterohemorrhagic E. coli (EHEC) O157:H7, strain CL56. Barrier function a group of IAT-treated stroke patients with TICI scores of 2b/3. We hypothesized that ~15% of patients will was measured by transepithelial electrical resistance (TER), dextran flux, and tight junction protein Medical Center, University 3Departments of have poor outcomes despite having TICI scores of 2b/3, and that assessing reperfusion in this subgroup expression and localization. Five-day-old mouse pups were orally gavaged formula with or without HMO of Calgary, Calgary, AB; Laboratory Medicine and will more accurately predict 3-month functional outcome than using TICI scores alone. (20 mg/ml) and then challenged with hypoxia (5% O2, 95% N2) and lipopolysaccharide (4 mg/kg/day). 3Neuroradiology Unit, Methods: Ischemic stroke patients treated with IAT received admission and 24hr CTP, post-procedural Pathobiology, Pediatrics Coded ileal sections obtained at day 9 of life were scored for severity of mucosal injury. Azienda Ospedaliera- DSA, and 3-month mRS evaluation. Patients were included if they had a post-procedural TICI score of 2b/3 and Surgery, University of Results: EHEC challenge decreased TER (26.6±2.3% of baseline), increased dextran flux (813.3±130 ng vs. and admission NIHSS≤20. Ischemic tissue volume was quantified on admission and 24hr CTP images 0±50 ng in controls) and induced actin remodelling, whereas these changes were ameliorated in HMO- Universitaria Careggi, Toronto, Toronto, ON using previously derived time-to-max thresholds. Reperfusion scores (SRep) were defined as the percent treated polarized epithelia. HMO-treated cells had less EHEC adherence and increased levels of protein Florence, Italy difference in volume from admission to 24hr. ROC analysis was used to find a threshold SRep for predicting disulfide isomerase (PDI) and mucin production (p<0.01, n=4-5). In a murine model of NEC, HMO reduced good outcome (mRS≤2). mucosal injury, increased mucin-secreting cells, and lowered the incidence of NEC (0/8 in HMO vs. 7/9 in Results: ~43% of TICI 2b/3 patients in our database had poor outcomes. Mean SRep for good (n=15) and sham treated pups; p<0.05). Chemical inhibition of PDI activity abrogated the protective effects of HMO poor (n=11) outcome groups were 84±6 and 67±10% respectively. The optimal SRep threshold of 78.5% both in vitro and in vivo. had a sensitivity and specificity of 0.87 and 0.55 respectively for separating good or poor outcomes. Using Conclusions: HMO protect barrier integrity in cells exposed to an enteric pathogen and prevent gut the SRep threshold resulted in correct outcome prediction in 73.1% of patients, as opposed to only 57.7% damage in a rodent model of NEC by inducing mucus production. Future studies will characterize of patient outcomes correctly classified by TICI 2b/3 alone. whether there are differences in effects between donor milk samples. Conclusion: This pilot study involving 26 of 98 patients in our database showed that our SRep threshold can better predict outcome in 15.4% of TICI 2b/3 patients. For further validation with a view to clinical translation in acute stroke imaging, we will finish analyzing all patients in our database and compare the predictive power of our SRep threshold against TICI scores. The Function of the AP-1 Transcription Factors c-Jun and JunB in CD30+ Lymphomas Using Next Generation Sequencing to Detect Clinically Relevant Oncogene Mutations in Zuoqiao Wu1, Jingxi Zhang1, Julinor T.C. Bacani2, Robert J. Ingham1 Lung Cancer Minqi Xu1, Shantanu Banerji2, 3, 4, Gefei Qing1, 5 1Department of Medical Introduction: Our lab focuses on the function of the c-Jun and JunB function in the CD30+ lymphomas: Microbiology and classical Hodgkin lymphoma (cHL) and anaplastic lymphoma kinasepositive, anaplastic large cell 1Department of Introduction: Modern care of patients with lung cancer requires rapid and accurate diagnosis leading to lymphoma (ALK+ ALCL). These AP-1 family transcription factors are highly expressed in these lymphomas, Immunology and Li Ka Pathology, University of personalized therapies for individual patients based on molecular characteristics of their tumour. and we wanted to examine whether they played a role in proliferation and apoptosis in these cells. Detecting mutations that predict response to drug quickly and accurately is an essential part of this 2 Shing Institute of Virology, Methods: To examine the function of c-Jun and JunB in cHL and ALK+ ALCL cells, we generated stable Manitoba; Department process. Next-generation sequencing (NGS) technologies provide an alternative approach for detecting University of Alberta; knockdown cell lines with lentiviral particles expressing c-Jun or JunB shRNAs. We then performed of Internal Medicine, mutated oncogenes in cancer. We hypothesize that NGS is equal if not superior to standard methods for growth curves, TUNEL, BrdU labelling, and other assays to examine the consequence of c-Jun and JunB 2Department of University of Manitoba; identifying targetable mutations in genes like EGFR and ALK in lung cancer. knockdown in these cells. We also examined whether c-Jun and JunB are required for tumour growth in Methods: DNA and RNA from 38 formalin-fixed paraffin embedded non-small cell lung cancer (NSCLC) Laboratory Medicine and 3Department of Medical vivo, by performing xenograft experiments in mice. samples archived in Diagnostic Services Manitoba were collected and analyzed using gene enrichment Pathology, University of Results: c-Jun and JunB knockdown resulted in reduced cell proliferation in cHL cell lines. In contrast, Oncology, CancerCare methods from Archer Diagnostics followed by sequencing on the Illumina MiSeq NGS machine. Targeted JunB knockdown, but not c-Jun knockdown, led to decreased cell proliferation in ALK+ ALCL. Neither Alberta, Edmonton, AB Manitoba; 4Research DNA sequencing to detect the EGFR mutation was performed on 19samples while targeted RNA c-Jun nor JunB knockdown induced significant apoptosis in all but one cHL or ALK+ ALCL cell line sequencing was applied to 20 samples to identify the ALK gene rearrangement. The NGS results were Institute of Hematology examined. Cell cycle analysis indicated that the growth defect was associated with reduced cells in S compared with and confirmed with current clinical standard molecular tests for EGFR (real‐time PCR) and phase and a prolonged G0/G1 phase. Furthermore, Ki-67 and Pyronin Y staining suggested that and Oncology; ALK(immunohistochemistry and FISH). Results: Three cases were positive for the EGFR L858R mutation knockdown cells may arrest in G0. Finally, the xenograft experiments demonstrated that mice injected 5Diagnostic Services and 2 other samples harbored the EML4-ALK fusion genes as determined by NGS. The concordance with cHL cells expressing c- Jun/JunB shRNA formed smaller tumours than mice injected with cells Manitoba, Health Sciences between NGS and real‐time PCR for EGFR mutation detection was 88.9%. Additionally, the NGS expressing control shRNA. methodology also provided profiles of other genes commonly mutated in NSCLC including KRAS and Conclusion: Our research demonstrates that JunB is critical for cHL and ALK+ ALCL proliferation, but the Centre, Winnipeg, MB TP53. The consistency for ALK fusion testing was 100% between NGS and FISH. function of c-Jun differs in these related lymphomas. Moreover, we show for the first time that these AP-1 Conclusion: This study provides support that NGS is a promising diagnostic tool for mutation detection family transcription factors can promote CD30-positive tumour growth in vivo. in NSCLC and holds strong potential for an alterative approach to identifying clinically relevant targets such as EGFR and ALK. Furthermore, NGS provides more information on cancer driven gene mutations than other traditional methods. The Investigation of a Multi-Serovar Salmonella Enterica Outbreak Using Whole Genome Contribution of Pannexin-1 Activation to Amyloid-Beta Induced Synaptotoxicity and Sequencing Neurotoxicity Christopher Yachison1, 2, Matthew Walker2, Lorelee Tschetter2, Aleisha Reimer2, the PulseNet Canada Steering Committee, Albert Yeung, Yufeng Xie, Natalie Lavine, Michael F. Jackson Celine Nadon1, 2 Department of Introduction: Calcium dysregulation induced by toxic soluble amyloid-beta oligomers (AβOs) is thought 1Department of Medical Introduction: Canada’s national bacterial foodborne pathogen surveillance program, PulseNet Canada has Pharmacology and to lead to impairment of synaptic function (synaptotoxicity) and eventual cell death (neurotoxicity) in Alzheimer’s disease (AD). Processes which contribute to the loss of calcium homeostasis can therefore Microbiology, University utilized pulsed-field gel electrophoresis (PFGE) as its gold-standard molecular subtyping technique for Therapeutics, University outbreak detection and investigation. Recently, whole genome sequencing (WGS) has positioned itself to exacerbate the disease process. Pannexin 1 (Panx1) is a large-pore non-selective ion channel broadly 2 of Manitoba; National revolutionize bacterial foodborne surveillance laboratories as this method provides the highest resolution of Manitoba; Kleysen enriched in the post-synaptic density of cortical and hippocampal neurons. The open state of Panx1 2+ Microbiology Laboratory, possible for determining pathogen relatedness, and the decreasing costs and improved bioinformatic Institute for Advanced allows the flux of particles up to 1 kDA, including Ca , ATP, and IL-1β. Given its ability to mediate inward Ca2+ currents, we hypothesize that pathological activation of Panx1 contributes to calcium dysregulation Winnipeg, MB pipelines have made this an increasingly feasible methodology for routine surveillance use. Medicine, Health Sciences Rationale and Objective: Past in-depth analyses of WGS for Salmonella enterica outbreak detection and in AD. Centre, Winnipeg, MB response has been limited to priority serotypes only; however, in order to be useful for surveillance WGS Methods: Naturally secreted AβOs collected from the media of 7PA2 CHO cells expressing the variant of interpretation guidelines must be available for all Salmonella serovars. This project aims to fill this gap human APP mutation V717F was used as a proven treatment method. Acutely prepared hippocampal through the analysis of a past multijurisdictional, multi-strain, multi-serovar outbreak of Salmonella in slices from Panx1 KO and WT mice were pre-treated with 7PA2 conditioned media (7PA2-CM) and CHO Canada associated with chia products. control media (CHO-CM). Changes in long-term potentiation (LTP) between groups was recorded by an Methods: A multi-serovar outbreak from 2014 was selected for analysis that included 63 human cases and established LTP protocol. Primary hippocampal neurons from Panx1 KO and WT mice were also chronically 40 food isolates from four serovars (Hartford, Newport, Oranienburg, and Saintpaul). All outbreak isolates, treated with 7PA2-CM. Cell death was assessed by LDH assay. Changes in neurite morphology and as well as a further 265 isolates from human cases of those four serovars that occurred during the same time synaptic markers was assessed via MAP2, synaptophysin and PSD-95 immunostaining. period as the outbreak were sequenced using the Illumina MiSeq platform. Single nucleotide variant Results: Panx1 WT slices treated with 7PA2-CM media showed impairment in LTP, as seen by a significant phylogenomic (SNVPhyl) and whole genome multilocus sequence typing (wgMLST) analyses (a custom decrease in fEPSP slope 60 minutes post-tetanus. However, 7PA2-CM treatment of Panx1 KO slices in-house pipeline and BioNumerics, Applied Maths software plugin, respectively) analyses were applied to showed no significant change in LTP compared to control treatment. In primary hippocampal cultures the WGS data. PFGE, wgMLST and SNVPhyl results were compared for congruence. treated with 7PA2-CM, cell death and extensive dendritic blebbing were observed by 5 days. Results and Conclusions: SNVPhyl and wgMLST differ significantly in their approaches for analyzing WGS Conclusion: Panx1 contributes to deficits in synaptic plasticity induced by AβOs. Subsequent experiments data; however, both methods reached the same conclusions and provided increased resolution to the will evaluate whether cultured neurons from Panx1 KO mice are protected from AβOs induced synapse outbreak investigation. Additional clusters of illness were identified through WGS analysis, and other loss and cell death. Mechanisms of Panx1 activation in response to AβOs in acute hippocampal slice and clusters identified by PNC from these four serovars during this time were further resolved. Lastly, while WGS primary cultured neurons will also be explored. provided increased resolution its use cannot minimize the importance of classical epidemiological work. Effects of Maternal Influenza Virus Infection on the Differentiation of Embryonic Stem Cells Into Cardiomyocytes and Fetal Heart Development: Identifying Possible Mechanisms Involved in Congenital Heart Anomalies Ali Zahedi Amiri, Kevin M. Coombs

University of Manitoba, Introduction: As the most common type of fetal malformations, congenital heart defects (CHDs) affect along with inhibition or/and dysregulation of underlying signaling pathways involved in fetal Winnipeg, MB various stages of life, ranging from fetal demise to asymptotic defect in adulthood. Numerous cases of cardiogenesis. Our results might be applicable for promoting fetal immunization and maternal miscarriage in the course of different trimesters of pregnancy or birth with CHDs suggest a significant vaccination during pregnancy through targeting influenza-modulated mechanisms of impaired and tangible relationship between influenza infection in pregnant women and such a sort of anomaly in cardiogenesis. offspring, which leads to the abnormal formation of the fetal heart. Nevertheless, the possible mechanism of cardiac abnormalities in the fetus affected by maternal influenza infection has not been identified so far. Methods: Human induced pluripotent stem cells (iPSCs) were infected by influenza A virus and the differentiation was induced one day after infection. Using Real-time PCR, we determined the viral replication kinetics at various post-infection time points. The expression of virus-specific proteins were analyzed by western blotting. On the 4th to 6th days post-induction, qPCR and flow cytometry were utilized in characterizing ISL1+ and Flt+/Lin- cells which represent phenotypes of cardiac progenitor cells, and for distinguishing GATA4+, CD31+ and Nkx2.5+ cells that indicate the myocardial differentiation and angiogenesis. Immunofluorescence was used to assess αMHC+ and αActinin+ cells. Annexin A5 affinity assay and Live/Dead cell viability assay was performed at each post-induction time point to measure the rate of apoptosis. To determine whether there are major alterations in signaling pathways involved in cardiogenesis and the differentiation of embryonic stem cells into cardiomyocytes like Wnt, Notch, and FGF signaling networks, western blot was applied for analyzing over-expression/dysregulation of key genes in cardiogenic signaling pathways along with the genes implicated in cardiogenesis, 4 days after culturing Flt+/Lin- cells that were supposed to be isolated by performing fluorescence activated cell sorting (FACS) on the sixth day after induction. Significance: We expect that influenza virus leads to congenital heart defect through disrupting the differentiation of embryonic stem cells into cardiomyocytes during the initial formation of the fetal heart Tudor-SN, a Novel Linker, Facilitates Chromatin Ralazation and Activates ATM Signaling L1CAM Inhibits Histone Deacetylase 2 (HDAC2) Under the Pathological Conditions of in DNA Damage Alzheimer’s Disease Chunyan Zhang, Xiao Fu, Xi Yang, Jie Yang H-L. Zhang1, C-L. Hu1, J-K. Hu1, W-J. Zhao1, M. Schachner1,2

University of Mantioba, The DNA damage response (DDR) is a coordinated process used to overcome threats and to maintain 1Center for Neuroscience, Introduction: Amyloid-β peptide (Aβ) is the major toxic agent in the pathogenesis of Alzheimer’s disease Winnipeg, MB genomic stability. Herein, we identify Tudor staphylococcal nuclease (Tudor-SN) as a novel regulator of Shantou University (AD), but its clearance is not enough to reverse the cognitive decline as the cognitive capacities are the DDR. We first provide evidence that Tudor-SN has bona fide interactions with a number of DNA repair constrained by an epigenetic blockade of gene transcription mediated by histone deacetylase 2 (HDAC2). Medical College, Shantou, protiens. Our functional analysis indicates that the loss of Tudor-SN sensitizes cells to DNA damage and The combination of Aβ reduction and HDAC2 inhibition may provide a more effective strategy. The neural apoptosis. Upon DNA damage, PARP1 promotes the chromatin accumulation of Tudor-SN via poly(ADP- Guangdong, China; 2W. M. cell adhesion molecule L1 (L1) has been reported to reduce Aβ aggregation and modulates cognitive ribosyl)ation. As a result, Tudor-SN accelerates chromatin relaxation via association with both the ATP- Keck Center for functions. We here studied the effects of L1 on HDAC2 under the pathological conditions of AD. dependent chromatin remodeler SMARCA5 and the histone acetyltransferase GCN5. Double-strand break Methods: Western blot analysis and immunohistochemistry were conducted to evaluate L1 and HDAC2 Collaborative (DSB)-induced chromatin relaxation can cause auto-phosphorylation of ATM kinase and activate DNA alterations in AD patient and APP transgenic mice; Reverse transcription PCR (RT-PCR) and western blot repair signaling. Accordingly, we demonstrate that loss of Tudor-SN function significantly reduces the Neuroscience, analysis were performed to investigate the effect of recombinant L1 (rL1) on HDAC2 in cultured mice phosphorylation of ATM and a subset of its substrates. In addition, homologous recombination (HR) and Department of Cell cortical and hippocampal neurons. The frontal cortex from both APP transgenic and wild type mice were non-homologous end joining (NHEJ) assays further demonstrate that Tudor-SN plays an essential role in used to perform co-immunoprecipitation (Co-IP) assay to address the association between L1 and Biology and Neuroscience, DNA repair. Our findings reveal a novel function of Tudor-SN in regulating the DDR. As the overexpression HDAC2. L1 mutation (D201N) knock-in mice were used to evaluate HDAC2 level in the frontal cortex and of Tudor-SN has been observed in different tumors, it is highly possible that inhibiting Tudor-SN Rutgers University, hippocampus. expression could sensitize cancer cells to radiotherapy. Piscataway, NJ Results: In an AD patient compared with a normal person and APP mice compared with the wild type, L1 was increased and HDAC2 has no apparent change in the frontal cortex, whereas L1 and HDAC2 were both increased in hippocampus. In the cultured cortical and hippocampal neurons, mRNA and protein levels of HDAC2 were elevated in response to Aβ1-42 exposure, and were decreased after rL1 treatment. Physical associations between L1 and HDAC2 were observed in the extracts of frontal cortex of APP transgenic and wild type mice. HDAC2 level was increased in the frontal cortex of L1 mutation mice. Conclusions: This study is the first to show that L1 inhibits HDAC2 in AD, suggesting potential therapeutic application in treating neurodegenerative diseases. The Effect of Multimorbidity on Change in Health-Related Quality of Life for Hip and Making Neutralizing Monoclonal Antibodies Against Marburg Virus Knee Arthroplasty Zirui Zhang Lixia Zhang1, 2, Olawale Ayilara1, 3, Richard Sawatzky4, Eric Bohm5, Lisa M. Lix1, 2 University of Manitoba; Introduction: Marburg virus (MARV) is a hemorrhagic fever virus of the family Filoviridae, and causes 1George and Faye Yee Introduction: Multimorbidity is defined as the co-occurrence of two or more chronic diseases where one National Microbiology Marburg virus disease in humans and nonhuman primates, a form of viral hemorrhagic fever. What is more, there are currently no Food and Drug Administration-approved vaccines for the prevention of Centre for Healthcare disease is not necessarily more important than the others. Multimorbidity may affect health outcomes Laboratory, Winnipeg, MB such as quality of life (QOL) and physical functioning. This study examined the effect of multimorbidity MVD, and the virus is considered to be extremely dangerous with the mortality rate of up to 90%. In this Innovation, University of on change in QOL one year after total hip arthroplasty (THA) and total knee arthroplasty (TKA). case, the WHO rates it as a Risk Group 4 Pathogen. Hence, it is necessary to find some ways against this Manitoba; 2Department Methods: Study data were from the Winnipeg Regional Health Authority Joint Replacement Registry for pathogen before next outbreak. As Mucin-Like Domain “hides” some important epitopes in GP, we delete Mucin to try to get more and better neutralizing Antibody. of Community Health patients who underwent THA or TKA between 2009 and 2015. HRQOL was measured prior to surgery and within one year post-surgery using Oxford Hip and Knee (OX) and twelve-item Short Form Survey (SF-12) Methods: Producing a VSV-based vaccine expressing a mucin-deleted glycoprotein from Marburg virus Sciences, University of Physical and Mental Component Summary (PCS, MCS) scores. Multimorbidity was defined as 2+ Angola. Then, using both the WT VSV-MARVGP and VSV-MARVGPdeltaM produce monoclonal antibodies. Manitoba; 3Department conditions from a list of twelve self-reported chronic conditions. Multivariable regression models were After that, compare the fraction of monoclonals that are neutralizing as well as the neutralization efficiency between the two vaccines. of Statistics, University of used to test the effect of multimorbidity on change in QOL after adjusting for age, sex, body mass index (BMI) and preoperative QOL scores. Expected results: Some antibodies produced from WT VSV-MARVGP and VSV-MARVGPdeltaM will be Manitoba, Winnipeg, MB; Results: Among 5,243 included patients (2,231 THA; 3,012 TKA), 3,257 (62.1%) had multimorbidity. The neutralizing. However, vaccinating mice with VSV-MARVGPdeltaM will produce more and higher 4School of Nursing, Trinity most common chronic conditions were hypertension (49.5%) and secondary osteoarthritis (42.5%). The efficiency neutralizing antibodies than WT VSV-MARVGP. Western University, mean age was 66.6 (SD=10.3) and 57.8% were female. Obesity was common in both patient groups (THA Expected conclusion: A vaccine expressing the mucin-deleted glycoprotein of Marburg virus will yield more and better neutralizing monoclonal antibodies than a vaccine expressing the full glycoprotein. Langley, BC; 5Department 39.8%; TKA56.9%). Pre-operative mean QOL scores were: MCS 52.0 (SD 11.8), PCS 29.7 (SD 8.2), and OX 42.6 (SD 8.0). The estimated effect of multimorbidity on QOL change scores for THA were: MCS -1.3 (SD of Surgery, University of 0.4), PCS -4.5 (SD 0.4), and OX 2.2 (SD 0.4; positive change in OX indicates worse QOL). Similar estimates Manitoba, Winnipeg, MB were observed for TKA, and all estimates were statistically significant (p < 0.001). A higher number of chronic conditions was associated with worse QOL for both THA and TKA (p < 0.01). For example, the estimated effect of three conditions on QOL change scores for THA was: MCS -2.3 (SD 0.6), PCS -5.7 (SD 0.7) and OX 3.1 (SD 0.6). Conclusion: Multimorbidity has a negative impact on improvements in QOL after joint placement. Information about the impact of multimorbidity can provide guidance to health care providers about the management of patients with chronic diseases. A Meta-Analysis of the Effectiveness of The Stop Now and Plan Program (SNAP) and its Investigating the Effects of Amyloid-p GxxxG-Motif-Targeting Agents on AP42-Induced Implications for SNAP Kenora, Ontario Toxicity Hanxiao Zhao, Depeng Jiang Phoebe (Yifei) Zhong

Department of Introduction: The SNAP® (Stop Now And Plan) program serves children aged 6 to 11 years old with McGill University, The amyloid-P (Aβ) peptides of Alzheimer disease (AD) are suspected as the causative agents of the Community Health aggressive and disruptive behaviours and/or conduct problems, and their families. SNAP is an evidence- Montreal, QC disorder. These small 4 kDa peptides are produced from the amyloid precursor protein (APP) via secretase based, gender specific cognitive-behavioural multi-component family-focused model developed at the processing. Soluble oligomers of a specific form of the Aβ peptide composed of 42 amino acid residues Sciences, University of Child Development Institute (CDI), Toronto, Canada more than 30 years ago. SNAP was being implemented (Aβ42) are highly toxic, and are believed to be the principal effectors of synaptic dysfunction and Manitoba, Winnipeg, MB by many organizations in Canada, United States and Europe. At this time, a wide variety of organizations neuronal loss. Our laboratory is interested in elucidating the molecular mechanisms of AP generation and (e.g., community-based, children’s mental health organizations, and school-based services) are delivering toxicity. As part of this work, we previously identified the GxxxG interaction motif in the α-helical APP the SNAP Program with high integrity and fidelity (as noted a SNAP license is required). We propose to transmembrane sequence to play a significant role in Aβ peptide generation and aggregation. In a conduct a systematic review and meta-analysis to explore the long-term, and short term effectiveness of transgenic Drosophila melanogaster model that constitutively expressed Aβ42 solely in the eyes of the SNAP program. The results of the meta-analysis will be applied to the ongoing implementation of SNAP animals, we have previously demonstrated that 033-substituted Aβ42 G33I exhibited reduced eye in Kenora, Ontario. malformations compared to controls (Harmeier et al, 2009). We now hypothesize that exogenous agents Methods: We will conduct a systematic review of the literature to establish the current state of knowledge targeting the GxxxG motif of the AP region will modulate the occurrence and the toxic behavior of Aβ on effectiveness of SNAP program, program integrity and fidelity. We will assess the effect size of included peptides in D. melanogaster models. Our overall objective for this project is to determine the efficacy of articles on aggression and delinquency scales, then we will qualitatively synthesize the extracted data GxxxG-motif-targeting agents to prevent Aβ42-induced toxicity in novel D. melanogaster model systems. from all included articles, we will pool the results using meta-analysis. Next we will compare the results Aim I: To develop novel D. melanogaster models for the evaluation of Aβ42-induced toxicity. Using with findings of evaluation of SNAP Kenora. transgenic D. melanogaster, we will express human Aβ42 solely in either the eyes or neurons using specific Implications and discussion: The systematic review will provide the most comprehensive assessment of promoters with the UAS/Gal4 system. To further differentiate between the developmental and induced current research on SNAP program effectiveness. Comparison between overall effectiveness and SNAP toxic effects of Aβ42, we will also cross in a temperature sensitive Gal80 strain. Gal80 is a transcriptional program in Kenora can help us understand both process and impact of implementing manualized mental repressor of Gal4, which is in tum needed for the transcription of human Aβ42 in the transgenic animals. health intervention program and lay groundwork for future implementation of SNAP. This novel system will allow us to precisely control the expression of human Aβ42 in a temporal manner by simple temperature modifications. Preliminary results have indicated that in these transgenics, both eye- and neuron-specific human Aβ42 expression induced toxicity in a temperature sensitive manner, with neuron-specific Aβ42 expressing mutants showing the most pronounced reduction in locomotor activity and survival. Alternatively, animals with temperaturecontrolled neuron-specific Aβ42 expression exhibited no changes in eye morphology as determined by confocal microscopy, Intrinsic 4-1BB Signals are Indispensable for the Establishment of an Influenza-Specific Tissue-Resident Memory CD8 T Cell Population in the Lung Angela C. Zhou, Lisa E. Wagar, Michael E. Wortzman, Tania H. Watts while eye-specific Aβ42 expression induced during development caused significant eye degeneration. University of Toronto, The induction of long-lived heterotypic T cell protection against influenza virus remains elusive, despite Aβ42 protein levels in the animals, as quantified by Western blotting, inversely correlate with both Toronto, ON the conservation of T cell epitopes. T cell protection against influenza is critically dependent on lung- survival and locomotion data. Currently, we are in the process of developing novel imaging techniques resident memory T cells (Trm). Here we show that intranasal administration of 4-1BBL along with (e.g. live imaging) that will be used in conjunction with our current methods to further complement influenza nucleoprotein (NP) in a replication-defective adenovirus vector to influenza pre-immune mice future experiments. induces a remarkably stable circulating effector memory (Tem) CD8 T cell population characterized by Aim 2: To test the impact ofGxxxG-motif-targeting agents on Aβ42-induced toxicity. We intend to use the higher IL-7Rα expression than control boosted T cells, as well as the establishment of a substantial lung novel D. melanogaster model developed in Aim 1 to test the efficacy of selected agents in rescuing the parenchymal CD69+ CD8 Trm population including both CD103+ and CD103- cells. These T cell responses Aβ42-induced toxicity via food supplementation studies. We have previously generated data showing persist to greater than 200 days post-boost and protect against lethal influenza challenge in aged (year that the AP-oligomer interacting peptide (AlP) was able to rescue Aβ42-induced eye degeneration in D. old) mice. The expansion of the NP-specific CD8 Trm population during boosting is critically dependent melanogaster (Barucker et al. 20 15). Using the newly developed temperaturecontrolled D. melanogaster on local rather than systemic administration of Ag and 4-1BBL as well as on 4-1BB on the CD8 T cells. models, we will first expand on this study by verifying morphological changes in the eyes of the animals Moreover, mixed bone marrow chimera studies determined that endogenous 4-1BB is critical for the using confocal microscopy, as well as assess survival and neurodegeneration via behavioural and establishment of lung-resident CD8 memory T cells post-primary influenza infection, under conditions locomotion assays. Accordingly, other GxxxG-motif-targeting agents will be tested using this model in where lack of 4-1BB had no impact on the size of the number of effector cells in the lung at the peak of future experiments Our group was the first to demonstrate a critical role for the GxxxG motif in AP the response. These findings establish both endogenous and supraphysiological 4-1BBL as a critical generation as well as in the aggregation and toxic properties of Aβ42. The results of this study will enable regulator of lung-resident memory CD8 T cell formation during influenza infection. us to estimate the potential of GSMs and other GxxxG-motif-targeting agents to act as powerful modulators that effectively reduce the occurrence of toxic Aβ42 species. We anticipate that the highly beneficial antiamyloidogenic effects of the GxxxG-motif-targeting agents will be a powerful strategy for early intervention. Targeting TGF-Beta Pathway in Squamous Cell Carcinoma by CRISPR/Cas9-Mediated Laser Therapy for Pediatric Burn Scars Genome Editing J. Zuccaro1, 2, I. Muser1, M. Singh1, J. Yu1, C. Kelly1, J. Fish1 Shufeng Zhou2, Peter Siegel2, Anie Philip1, 2 1Department of Plastic Background: The most common complication experienced by burn survivors is the development of 1Division of Plastic Background: Skin cancers is the most common cancer. Cancer stem-like cells (CSCs) have been identified and Reconstructive hypertrophic scarring. Hypertrophic scars appear thicker than normal scars and are associated with symptoms including redness, stiffness, pain, and pruritus. Laser therapy has recently emerged as a novel Surgery, Department of in squamous cell carcinoma (SCC) and implicated in cancer progression and metastasis. Epithelial-to- Surgery, Hospital for Sick mesenchymal transition (EMT) is a highly conserved cellular process that is involved in normal tool to improve hypertrophic scars. More specifically, early research has demonstrated the success of 2 2 Surgery; Department of embryogenesis and tissue repair, but it also contributes to tumor metastasis. In the context of advanced Children; Institute of using Pulsed Dye laser therapy (PDL) and ablative fractional CO2 laser therapy (AFCL) to improve scar Medicine, McGill stage tumors, transforming growth factor-beta (TGF-β). a strong EMT inducer, can promote cancer cells Medical Science, symptoms. The purpose of this study was to evaluate the effect of using laser therapy to treat hypertrophic burn scars in pediatric patients when using each laser individually and in combination at the same University, Montreal, QC to undergo EMT to generate CSCs, enhance tumor invasion and metastasis. CD109 is a TGF-β co-receptor University of Toronto, that negatively regulates TGF-β function. However, the significance of CD109 in squamous cell carcinoma procedure. Toronto, ON and its potential to regulate cancer progression and metastasis remain to be explored. To investigate Methods: A retrospective review was carried out for all patients who received laser therapy for their burn CD109 overall influence in SCCs progression, CD109 was knocked out in the A431 cancer cell line using scars at our pediatric institution. Relevant demographic information and details related to the burn injury CRISPR/Cas9, a powerful genome editing tool. were collected. Information regarding the type(s) of laser used as well as treatment timing was also Methodology: We sorted SCC cells into CD109high, CD109medium and CD109low subpopulations by collected. The primary outcome for this study was the change in scar symptoms following treatment with fluorescence-activated cell sorting (FACs), then analysed the alterations in EMT and stem markers (slug, laser therapy and was evaluated using a widely accepted burn scar assessment tool known as the snail, Twist, fibronectin and Sox2) by western blot and immunofluorescence. Cellular migration and Vancouver Scar Scale (VSS). invasion were respectively assessed in vitro by wound healing assay and matrigel invasion assay, whereas Results: 125 patients were treated over a total of 289 laser sessions, with the majority of patients tumorigenicity was evaluated by spheroid formation and noble soft agar assays. We also knocked out receiving 2-3 treatments (61%). The mean age at treatment was 6.6 years with more than 50% of patients CD109 in A431 cells using CRISPR/Cas9 genome editing and then analyzed the same parameters as above under the age of five years at the first treatment. All patients were treated with either PDL (n=7), AFCL in parental vs A431-CD109 KO cells. (n=117), or both PDL and AFCL (n=165). Laser settings varied and were dependent on symptoms and Results: We showed that SCC cells express CD109 heterogeneously and cellular levels of CD1 09 are patient skin type. Before-after VSS scores significantly decreased from 7.37 (SD, 2.46) to 5.76 (SD, 2.29) inversely correlated to the expression of TGF-β receptors and EMT markers, as well as to sternness, after a single treatment (p<0.005). migration, invasion and tumorigenicity. Importantly, the CRISPR/Cas9-mediated knockout of CD109 Conclusions: The results obtained from this study support the use of laser therapy to improve caused profound morphologic and molecular changes indicative of enhanced EMT, sternness, increased hypertrophic burn scars in the pediatric population. Moreover, this is one of the few studies to address invasive and tumorigenic potential. how both PDL and AFCL can be safely combined at the same procedure. Ultimately, rigorous randomized Conclusion: Our findings implicate CD109 as an important regulator of EMT, migration, invasion and controlled trials are needed to confirm the effectiveness of using laser therapy to treat hypertrophic burn sternness in human sees. scars. The Association of Faculties of Medicine of Canada Institute of Cancer Research Dalhousie University Institute of Circulatory and Respiratory Health McGill University Institute of Gender and Health McMaster University Institute of Genetics 2017 Memorial University of Newfoundland Institute of Health Services and Policy Research SPONSORS Queen’s University Institute of Human Development, Child and Youth Health University of Alberta Institute of Infection and Immunity University of British Columbia Institute of Musculoskeletal Health and Arthritis University of Calgary Institute of Population and Public Health University of Manitoba Child Health Research Institute of Manitoba Université de Montréal Dean of Graduate Studies, University of Manitoba University of Ottawa Dean of Medicine, University of Manitoba University of Saskatchewan E.L. Drewry Memorial Trust University of Toronto Health Sciences Centre Foundation, Inc. Western University Health, Seniors and Active Living, Government of Manitoba Apotex Fermentation Inc. Manitoba Medical Service Foundation Inc. Children’s Hospital Foundation Inc. Physician Resource Coordination Office (PRCO), Government of Manitoba Canadian Institutes of Health Research (CIHR) Research Manitoba Institute of Aboriginal Peoples’ Health St. Boniface General Hospital Foundation, Inc. Institute of Aging S.W. Prowse Memorial Trust Vice-President (Research), The University of Manitoba