Master Abstract Document

Canadian Student Health Research Forum (CSHRF) 2021

Hosted virtually by the University of Manitoba June 14 – 18, 2021

The design, synthesis, and application of new benzamides as antimicrobial molecules

Monalisa Abas1, Rachel Willim1, Tabitha E. Wood1

1The University of Winnipeg

Introduction: Antibiotic-resistant bacterial strains continue to rise, and new methods of antimicrobials are needed. A promising new target involves replication protein FtsZ. This protein, found only in prokaryotes, orchestrates the formation of a Z-ring. The Z-ring serves as the site of division, separating the singular bacterial cell into two identical daughter cells. This step is necessary in successful replication of bacteria.

The most promising reports of potent FtsZ inhibitors are benzamides. Upon benzamide binding to FtsZ, the cell elongates, and replication is completely inhibited. This project aims to investigates the design and synthesis of a library of benzamide compounds. These compounds will be tested against a range of different bacteria to assess their inhibitory properties.

The synthetic approach to benzamide synthesis may be achieved in many ways, however the Truce-Smiles rearrangement is of particular interest for its enhanced selectivity, reactivity and perfect atomeconomy. Additionally, the reaction achieves the formation of a new and highly desirable carbon-carbon bond.

Methods: Ullmann coupling utilizes a copper catalyst and is used in the formation of the aniline starting material. Microwave-assisted TCT peptide coupling is used in the formation of the aryl benzamide intermediate by combing the starting aniline and a corresponding acid. The final synthesis involves the Truce-Smiles rearrangement. The rearrangement is achieved by directed ortho lithiation, lithium halogen exchange, or deprotonation. The compounds are characterization by standard methods of chromatography and spectroscopy.

The compounds will be tested against a range of bacterial species. The broth microdilution method will be used to determine minimum inhibitory concentrations. Standard phase-contrast microscope techniques will be used to observe phenotypic changes upon benzamide binding.

Results: The Ullmann coupling is reproducible with the aniline and pyridine-derivative aniline compounds. Additionally, the microwave-assisted amide synthesis has successfully synthesized the nitro-derived benzamide compound intermediates that have been proposed.

Conclusion: The synthesis of the aniline starting material and nitro-derived benzamide compound intermediates have been successful thus far. These proposed methods of synthesis will continue to be used.

Trends of COVID-19 incidence in Manitoba and public health measures: March 2020 to March 2021

Laila Aboulatta1, Kaarina Kowalec1,2, Sherif Eltonsy1,3

1College of Pharmacy, Rady Faculty of Health Sciences, University of Manitoba, Canada 2Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Sweden 3The Children's Hospital Research Institute of Manitoba, Canada

Introduction: The increasing spread of SARS-CoV-2 has prompted much of Canada to take unprecedented measures. Currently, there is no timeline to demonstrate the effectiveness of the implemented mitigation measures in controlling the burden of COVID-19. The aim of this report was to examine the impact of the implemented public health measures on the incidence of COVID-19 in Manitoba.

Methods: Using the Manitoba Health, Senior and Active Living COVID-19 dataset, we examined the temporal trends of daily reported COVID-19 cases and the coinciding public health measures implemented from March 12, 2020 to March 1, 2021.

Results: We developed a model that demonstrates the trend of COVID-19 incidence before, during and after the implementation of different mitigation measures. On March 20, 2020, the Premier of Manitoba declared a state of emergency to limit the viral spread. From March 13 to April 17, 2020, several mitigation strategies were applied including physical distancing, limiting gatherings, wearing masks, closure of universities, non-urgent medical care restriction, and non- essential travel limitations. Due to these restrictions, the daily cases decreased by 97% from 33 (April 1) to 1 (May 1, 2020). Between May 4 and July 17, 2020, the reported cases stabilized, and some restrictions were lifted. However, during the second wave in the Fall 2020, the number of cases increased by ~176% from 58 on October 7 to 160 on October 14, 2020. Since October 19, Manitoba had three-digit daily cases for 3 consecutive months, and the number peaked during November 4-27, 2020 (ranging from 227-593). Additional restrictions implemented included limiting social contacts to household members, non-essential items could not be purchased in-store, closing of gyms and restaurants, with all of Manitoba placed in the highest response level (“critical”). Mitigation measures were extended through January 2021, and the number of cases dropped to 52 cases on March 1, 2021.

Conclusion: This report provides estimates that physical distancing in conjunction with other containment measures can reduce the COVID-19 burden. Future studies into the extent of the implementation of the restrictions are necessary.

Transplantation of young bone marrow stem cells can delay bone aging in mice

Lina Abu-Nada1,4, Younan Liu2, Faez Al hamed2, Simon Tran2, Guylaine Ferland3, Vahab Soleimani1, Faleh Tamimi2, Monzur Murshed1,2,4

1Faculty of Medicine, McGill University, Montreal 2Faculty of Dentistry, McGill University, Montreal 3Faculty of Medicine, Université de Montréal, Montreal 4Shriners Hospital for Children, Montreal, Qc, Canada

Background: Recent discoveries have shown that systemic manipulation such as parabiosis, blood exchange and young plasma transfer, can counteract many important symptoms of aging. This rejuvenation effect has been attributed to circulatory factors produced by several cells (haematopoietic or non-haematopoietic). However, the specific involvement of bone marrow (BM) or haematopoietic cells in producing these factors and their effects on bone remodeling is still unclear. Our objective is to develop a model of old mouse transplanted with young/old marrow cells through BM transplantation and assess the effects on the aging process, specifically in bone.

Method: An aged mouse model with young/old BM/haematopoietic system has been created. In particular, aged C57BL/6 mice (18-month old) were total-body irradiated to eliminate their endogenous BM, and then transplanted with BM cells from either young (2-month) or old (18-month old) mice. Flow cytometry using a transplanted cell-specific marker (green fluorescent protein) confirmed BM cell engraftment. After six months of transplantation, mice were euthanized, and their long bones and vertebrae were collected and analyzed using micro CT, mechanical testing and histology.

Results: A mouse model of “old mice transplanted with young/old BM cells” was successfully created with ~85% engraftment. Our results showed that rejuvenating the BM/hematopoietic system through young bone marrow transplantation reversed old age-associated low bone mass traits in mice. Specifically, young bone marrow transplantation improved bone trabecular microarchitecture both in tibiae and vertebrae of old mice and increased the number of osteoblasts and osteoclasts compared to old bone marrow transplantation.

Conclusion: Young bone marrow cells may represent a future therapeutic strategy for osteoporosis. The findings of this study could radically alter our understanding of aging, and direct further examination of the cell signaling process involved in age- related diseases.

Implementing SDM in interprofessional home care teams supporting caregivers of frail elderly facing housing decisions: a stepped wedge cluster randomised trial

E. Lionel Adisso1, Louis-Paul Rivest1, Monica Taljaard2, France Légaré1

1Laval University, Quebec City, Quebec 2University of Ottawa, Ontario, Canada

Introduction: One of the toughest decisions faced by cognitively impaired older adults with loss of autonomy (frail elderly) is whether to stay at home or move to a care facility. This study aims to evaluate the effect of the addition of a training programme in IP-SDM to the passive dissemination of a decision guide on the proportion of caregivers reporting an active role in the decision-making process about the housing of the frail elderly.

Methods: A stepped wedge cluster randomised trial involving nine Integrated (University) Health and Social Service Centers (CISSS/CIUSSS) in the province of Quebec was conducted. Participants were home care teams working in recruited CISSS/CIUSSS, and caregivers of frail elderly (≥ 65 years) who were receiving care from a home care team. Participating CIUSSS/CISSS were randomised to one of four intervention periods. Eligible caregivers had made a housing decision for the frail elderly. The intervention, targeted IP home care teams, consisted of a 1.5-hour online tutorial and a 3.5-hour skills- building workshop in IP SDM. The comparator was a decision guide distributed passively at the beginning of the project. The primary outcome was the proportion of caregivers reporting an active role in the decision-making process. We performed intention-to-treat multilevel analysis and used a generalized linear mixed model.

Results: Of the 339 caregivers, 167 were in the control periods and 172 in intervention periods. They were mostly female – 122 (73.1%) versus 117 (68.0%), more than 60 years old – 63.7 (IQR, 55.1 – 74.1) years versus 68.6 (IQR, 59.4 – 77.2) years, respectively in control and intervention periods. Caregivers had college or more level in 51% versus 46%. After adjusting for clustering, time effects, age, sex, and education attainment, the proportion of caregivers reporting an active role increased by 13.0% from control to intervention conditions even if our data did not suggest such difference significant (Proportion ratio = 1.13; 95%CI: 0.53-2.39; p=0.750).

Conclusion: In home care setting, the addition of a training program in IP-SDM targeting healthcare teams to the passive dissemination of a decision guide seemed not necessary to increase the proportion of caregivers reporting an active role in the decision-making process.

Addressing the Toxic Drug Supply Epidemic through a University-Community Partnered Intervention

Lauren Airth1-3, Nelly D. Oelke1, 4-6, Thomas Pool1, Rebekah Underhill1, Joan Bottorff1,7-9

1University of British Columbia – Okanagan 2Canadian Association of Nurses for the Environment 3Harm Reduction Nurses Association 4Co-Scientific Director, Rural Coordination Centre of British Columbia 5BC Ministry of Health, Primary and Community Care Research Advisory Committee 6Rural Health Research Network Advisory Committee 7Member of Research Advisory Council (UBCO representative) 8Member Advisory Panel to develop the RNAO Best Practice Guideline: Supporting Tobacco Interventions in Indigenous Pre- and Postnatal Women and their Families 9Member of CIHR College of Reviewers

Introduction: The impacts of COVID-19 are impeding substance use harm reduction measures and contributing to record numbers of toxic drug deaths. With the burgeoning attention on privilege and distribution of resources, it is critical that universities contribute to solutions for this epidemic. The aim of this study is to assess the feasibility of a university-community partnered substance use harm reduction intervention (UCPI).

Objectives: 1) To better understand the impact of the toxic drug supply on people who use drugs and service providers; 2) To develop a UCPI to enhance harm reduction approaches; 3) To evaluate the implementation and outcomes of a pilot UCPI.

Methods: Community stakeholders, people with lived experience, campus community members, and harm reduction experts contributed to the development of this UCPI. Using mixed methods, this study will employ the RE-AIM (Reach, Effectiveness, Adoption, Implementation, and Maintenance) framework to evaluate the implementation process and the intervention. Focus groups and interviews with participants will be recorded, transcribed, and analyzed, alongside art, for thematic analysis and will include descriptive statistics. Data will also be analyzed to examine patterns in service use and results of drug checking. Feedback will be sought from participants to validate the results.

Results: The UCPI began in December 2020 through the University of British Columbia’s Okanagan (UBCO) campus, Interior Health, and the BC Centre for Substance Use. UBCO students contribute to the program through communications, education development and delivery, and harm reduction service delivery. Harm reduction services include sterile supplies and drug-checking through spectrometry and test strips. The UCPI is delivered in four locations in three communities. Services have resulted in large-scale drug alerts regarding drugs with high potential for overdose, and clients report that this service has prevented overdoses. The UCPI has also facilitated rich learning experiences for students.

Conclusion: Although the formal execution of this study has yet to begin, the preliminary results are promising. The findings will provide an enhanced understanding of the impacts of the epidemic on people who use drugs and the service providers, and a model for UCPI to guide similar interventions in other communities to prevent toxic drug deaths.

Tools to visualize latently-infected CD4+ in lymphoid organs in vivo

Oluwaseun Ajibola1, Nnamdi Ikeogu1, Xinyun Liu1, Paul Lopez1, Roshan Parvarchian1, Alon Hershhorn2 and Thomas Murooka1

1University of Manitoba, Faculty of Health Sciences, Department of Immunology and Medical Microbiology and Infectious Diseases, Winnipeg, MB. 2University of Minnesota, Division of Infectious Diseases and International Medicine, Minneapolis, MN.

Introduction: Antiretroviral therapy (ART) has helped HIV become a manageable chronic infection by repressing viremia to undetectable levels in infected individuals. However, this treatment is not curative as treatment interruption causes viremia to rebound to pre-treatment levels due to viral latency, a state of reversible non-productive infection of individual cells established early during acute infection. These cells could act as a viral reservoir and hide in various anatomical sites posing a barrier to achieving a complete HIV cure. Therefore, the inability to completely eradicate these viral reservoirs remains a significant barrier towards achieving a cure. Hence, it is vital to fully understand these latent reservoirs’ cellular and molecular mechanisms to develop effective therapeutic measures.

Methods: One molecular tool that allows us to better understand HIV latency generation and maintenance is the fluorescent protein-based HIV reporter system, which can reliably track latently-infected cells over time. However, a significant limitation of previously published reporters is that they lack expression of critical viral proteins Nef and Env. To overcome these limitations, we generated a novel full-length, R5-tropic dual-fluorescent HIV reporter that encodes two different fluorescent markers: Nef-E2Crimson fusion protein under the control of the HIV-LTR and the EF1a-HTLV1 promoter driving ZsGreen1 expression (HIVNef-CRMZY). Infection of primary CD4+ T-cells with this reporter allowed us to discriminate productively infected (E2Crimson+ZsGreen1+) and latently-infected (E2Crimson-ZsGreen1+) cells visually. The lymph node (LN) is known to be one of the largest HIV reservoirs, but the distribution and migratory pattern of latently infected CD4+ T-cells here are currently unknown. Thus, using this reporter, we will uncover whether there are specific LN niches that help maintain this population in vivo using advanced microscopy approaches in humanized mice.

Significance: This study will be the first to visually characterize latently infected CD4+ T-cells in vivo. The localization, distribution and migration of these cells are novel, and their potential interaction with other cells may establish the role of cell-cell contacts in maintaining the HIV reservoir. My study will identify new therapeutic targets that will block latency generation and help reduce the HIV reservoir size, so life-long ART is no longer required.

Impact of semaphorin-3E deficiency on natural killer cells development

Abdulaziz Alamri1, Abdelilah Soussi-Gounni1 and Sam K P Kung1

1Department of Immunology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada

Natural killer (NK) cells are part of the innate immune system. They are characterized by their unique ability to kill virus- infected and tumor cells through direct cytotoxicity or cytokines/chemokines production that activates both the innate and adaptive immune systems. Semaphorin 3E (Sema-3E) is a secreted axon guidance protein that has emerged as a novel mediator of cell migration and proliferation, adhesion, differentiation, and angiogenesis in several organs upon its binding to cognate receptors. However, the role of Sema-3E in the regulation of NK cell function has not been investigated. Here, we demonstrate for the first time that Sema-3E deficiency diminishes NK cell count in vivo. However, NK cell maturation, defined by the expression of CD11b and CD27 as well as major NK cell receptors (such as NKP46, KLRG1, NKG2D, and Ly49 C/I/G/H) were not been altered spleen and bone marrow from Sema-3E-deficient mice. To further address the importance of Sema-3E in regulating NK cell function, we first compared the expression of CD107a and IFN-γ in wild-type (WT) and Sema-3E-deficient NK cells using the major histocompatibility complex (MHC) class I-negative mutant mouse lymphoma cell line (RMA-s) as target cells in vitro. We observed that Sema-3E-deficient NK cells exhibited less CD107a and IFN-γ expression than WT NK cells. Together, these results reveal a crucial role for Sema-3E deficiency in regulating NK cell development and function.

This work is supported by NSERC, CCSRI (to SKPK) and KSU (to AA).

Organ-specific alterations in level and expression pattern of von Willebrand factor in response to aging

Parnian Alavi1, Douglas Brown2, Radya Yousef Abdualla1, John Lewis2, Nadia Jahroudi1

1Department of Medicine, University of Alberta, Edmonton, Canada 2Department of Oncology, University of Alberta, Edmonton, Canada

Introduction: Von Willebrand factor (VWF) is a prothrombotic endothelial specific protein and unregulated elevated VWF levels present a risk factor for thrombus formation. Aging process has been associated with increased circulating levels of VWF, however the mechanism is unknown. Our objectives are 1) To explore the regulation of VWF expression during aging. 2) To determine the association of increased VWF expression with thrombogenicity during aging. 3) To determine the molecular mechanism of agerelated upregulation of VWF expression.

Methods: Elisa, Western blot, and RT-PCR analyses were used to determine circulating plasma, cellular protein, and mRNA levels of VWF in young and aged mice. Immunofluorescent analyses of major organs were performed to establish the vascular pattern of VWF and the presence of platelet aggregates. Cultured endothelial cells were used as an in vitro model of aging to explore the mechanism of increased VWF levels.

Results: Increased plasma levels of VWF were observed in aged mice. VWF mRNA and protein levels were increased in the endothelium of the brains, lungs, and livers, but not kidneys and hearts of aged mice. The distribution of VWF expression in target organs was altered from primarily large vessels in young, to include small vessels in the aged mice. Increased platelet aggregates formation in vessels of aged organs were concomitant with increased VWF expression, consistent with increased thrombogenicity. Prolonged maintenance of endothelial cells in culture, resulting in cell senescence, correlated with increased VWF mRNA levels. Increased VWF expression was specifically detected in senescent cell population. Aged mice treated with lipid nanoparticles (LPN) that targets p53 expressing senescent cells for destruction, exhibited a significant reduction in platelet aggregates formation in the brain vasculatures compared to control.

Conclusion: VWF levels and expression patterns are significantly increased in response to aging in an organ-specific manner, which is concomitant with increased platelets aggregate formation as a risk factor for age-associated thrombotic disorders. A potential mechanism of age-associated increase in VWF expression maybe through cell senescence. The ultimate goal is to design appropriate vascular bed specific targeted therapies to combat thrombogenic complications that occur with aging.

Environmental Enrichment Reduces Intermale Aggression and Alters Brain Activation Patterns in Group-housed Mice

Muhammad S. Aldhshan1, Tooru M. Mizuno1

1Dept. of Physiology and Pathophysiology, Rady Faculty of Health Sciences, University of Manitoba

Background: Aggression emanates in the brain through coordination between the subcortical structures such as the amygdala, the hypothalamus and the prefrontal cortex (PFC), where emotions are born and matured. Group-housing male mice under standard laboratory conditions triggers aggression to establish dominance which is a social imperative in social animals. Environmental enrichment (EE) is a housing protocol that improves laboratory rodents' welfare by providing complex stimulations that sponsor the display of species-typical behaviours. EE promotes various health benefits via enhanced expression of brain-derived neurotrophic factor (Bdnf), a gene encoded by multiple splice variants that exhibit brain region-specific expression and have distinct functions, including modulating aggression. Interestingly, enriched mice are considered less aggressive than mice housed under standard conditions, but the exact mechanism remains unknown.

Hypothesis: EE reduces aggression by altering Bdnf mRNA expression in a variant- and brain region-specific manner.

Design: 4-week-old male C57BL/6 mice were group-housed (5/cage) under standard or EE conditions for 6-8 weeks. EE mice were housed in large cages supplemented with a house, running wheels, igloos, wood logs, a tube maze and nesting materials. Aggressive interactions were visually evaluated throughout the experiment. Mice were euthanized, and brain tissues were collected for gene expression analysis.

Findings: The incidence of aggressive behaviour was significantly reduced in EE mice (17.1%) compared to control (51.4%, P < 0.005 by chi-squared test). EE caused a significant reduction in Fos, a marker of neuronal activation, mRNA levels in the PFC and the hypothalamus (P < 0.05 by Student's t-test, n = 8-9/group) without a significant change in the amygdala. mRNA levels of Bdnf variant I in the PFC and the hypothalamus were significantly reduced in EE mice compared to control (P < 0.01 or 0.05 by Studentʼs t-test, n = 8-9/group), while it was increased significantly in the amygdala of EE mice (P < 0.05 by Studentʼs t-test, n = 6-9/group). EE did not significantly alter Bdnf variants IIc, IV, and VI mRNA levels in all tissues.

Conclusion: EE diminishes intermale aggression by altering brain activation pattern and Bdnf mRNA expression in a variant- and brain region-specific manner.

Acknowledgment: NSERC and GETS

Role of Scleraxis in Perivascular Fibrosis

Danah AlHattab1, Michael Czubryt1, 2

1University of Manitoba 2St. Boniface Research Albrechtsen Centre

Perivascular fibrosis (PVF) is one of the most significant pathologies related to blood vessel dysfunction. PVF results in an impairment of vascular tone due to an increase in protein build up synthesis in the vessel wall, increasing vascular stiffness and reducing lumen diameter. PVF is both caused by and exacerbates numerous vascular diseases including diabetes, atherosclerosis and in particular, hypertension, which accounts for 13% of deaths globally (WHO World Health Report, 2002). Adverse patient outcomes include increased risk of morbidity and mortality related to stroke, ischemic heart disease and heart failure. Multiple studies have investigated various therapeutic approaches to interfere with PVF, but to date this condition remains untreated, thus novel approaches to target fibrosis development and progression are urgently required.

Our lab identified the transcription factor scleraxis as a novel master regulator of fibrotic signaling in the myocardium, showing scleraxis is sufficient to induce fibroblast to myofibroblast phenotype conversion, a critical step in the development of fibrosis in many tissue types, and directly up-regulates ECM genes in cardiac fibroblasts. In contrast, scleraxis loss attenuates cardiac ECM gene expression. Angiotensin II (AngII) was reported to induce vascular fibrosis via activation of the transcription factor Smad3 in aortic vascular smooth muscle cells. Our lab has shown that Smad3 physically interacts with scleraxis, and critically requires scleraxis to drive TGF/Smad fibrotic signaling. Our preliminary data has revealed that scleraxis is expressed in the arterial wall of vessels and scleraxis expression is elevated in high pressure versus low pressure vessels. We also found that AngII induces scleraxis expression. We thus hypothesize that scleraxis is sufficient and necessary to induce perivascular fibrosis.

My pressure myography data reveals an increased vascular stiffness and hypertrophic remodeling of the vessel wall of 3rd order mesenteric arteries in scleraxis overexpression of SMCcre mice. Also, it shows that scleraxis overexpression in vascular smooth muscle layer increase the thickness of the aortic vascular wall through histological sections. Thus, contributing to the progression of perivascular fibrosis.

Youth Substance Use: A Critical Analysis of Tensions Between Federal Policy Discourse and Frontline Service Provision in Ontario Abstract

Farihah Ali1

1York University

Introduction: Substance use – ranging from experimentation to problematic use and addiction – is most common among youth. Evidence shows that the earlier in life individuals begin to use substances, the higher the risk for addiction. Adolescence and young adulthood are life stages when behaviours and habits become established. They are also periods of social and developmental change as youth navigate through challenges. In order to respond to substance use issues in Ontario, the substance use service provider arena is guided by federal policies and offers a range of services intended to support youth experiencing substance use issues.

My dissertation had three objectives: 1) to assess the experience of frontline service providers to shed light on their perspectives on challenges faced by youth who use substances; 2) to evaluate representations of substance use among youth in a federal substance use strategy document that informs provincial level practice; and 3) to assess the policy implications of the tensions between dominant representations of substance use in policy documents and the lived experience of frontline service workers in the field of substance use.

Methods: To achieve these objectives, I conducted an online survey of Ontario service providers recruited from youth- oriented addiction substance use treatment organizations, I followed up with qualitative key informant interviews of a sub- sample of willing survey participants, and I assessed dominant representations of the problem of substance use using the critical policy approach of WPR (Bacchi, 1999), through an examination of the 2017 National Canadian Drugs and Substance Use Strategy (CDSS).

Findings: My findings revealed significant tensions between theory and practice. While frontline providers expressed the need for harm-reduction, non-pharmacological and prevention initiatives for youth, the National Strategy downplayed this need, as well as the significance of the social determinants of health (SDOH), while largely framing the behaviours of users of substance as falling under the jurisdiction of the criminal justice system.

Conclusion: I offer policy recommendations on how to reduce the identified gaps between dominant representations and practice and propose strategies, such as a national youth substance use strategy which integrates the recognition of the SDOH and harm reduction and prevention initiatives.

BCL2L13 Regulation of Non-Small Cell Lung Cancer Metastasis via Mitophagy and Anoikis

J. Alizadeh1, 2, 3, M. Mowat3, JH Ko1, S. Ghavami1, 2, 3

1Dept. of Human Anatomy and Cell Science, Max Rady College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada 2Children Hospital Research Institute of Manitoba, Biology of Breathing Theme, University of Manitoba, Winnipeg, MB, Canada 3Research Institute in Oncology and Hematology, CancerCare Manitoba, University of Manitoba, Winnipeg, MB, Canada

Introduction: Lung cancer is one of the most important causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) comprises ~80% of lung cancer cases. Mitophagy, a selective autophagy, refers to continuous mitochondrial housekeeping, ensuring recycling of damaged mitochondria. Our group has recently showed that general autophagy is possibly associated with NSCLC metastasis through regulation of epithelial to mesenchymal transition regulation. Also, one of the requirements of metastasis is acquiring a mesenchymal phenotype and resistance to detached induced apoptosis (anoikis) in tumor cells. Therefore, we continued our investigation on regulation of NSCLC metastasis focusing on mitophagy and anoikis. Several mediators are involved in mitophagy induction including BCL2L13, a member of the Bcl2 family. It has been shown that BCL2L13 is associated with tumor metastasis. In the present study, we have investigated the regulatory role of BCL2L13 in NSCLC metastasis via mitophagy and anoikis.

Methods: Human A549 and mouse LLC lung cancer cells were treated with transforming growth factor-beta1 (TGFβ1) to analyze mitophagy induction and subcellular localization of BCL2L13 using Immunoblotting, Immunocytochemistry (ICC) and Transmission Electron Microscopy (TEM). Next, BCL2L13 gene was inactivated in human A549 and mouse LLC lung cancer cells using specific shRNA against BCL2L13. BCL2L13 role in cellular phenotype and resistance to anoikis was evaluated by immunofluorescence microscopy, immunoblotting and flow cytometry.

Results: We showed that TGFβ1 induces mitophagy ([LC3β-II lipidation, p62 and TOMM20 degradation using immunoblotting for mitochondrial fraction]; [colocalization of LC3 puncta and MitoTracker, colocalization of TOMM20 and LAMP1 using ICC]; [mitochondrial structures in autophagosomes using TEM]) and the mitochondrial localization of BCL2L13. Furthermore, we showed that mesenchymal markers [Vimentin and N-cadherin] are upregulated in Bcl2l13-/- cells, indicating acquirement of a mesenchymal phenotype. Our flow cytometry results also showed that apoptosis is significantly reduced in BCL2L13-/- cells compared to wild-type cells indicating anoikis resistance in BCL2L13-/- cells.

Conclusion: Our results highlight a possible role for BCL2L13 in mitophagy and anoikis resistance in NSCLC cells. We will later address how BCL2L13 is involved in regulation of NSCLC phenotype and anoikis resistance and will also address it in NSCLC animal and human model.

Regulation of Airway Inflammation: Cathelicidin LL-37 selectively augments IL-17A/F-mediated inflammation

Anthony Altieri1, Hemshekhar Mahadevappa2, Dylan Lloyd2, and Neeloffer Mookherjee1,2,3

1Department of Immunology, University of Manitoba 2Manitoba Centre for Proteomics and Systems Biology, Winnipeg, MB 3Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Introduction: Asthma is a heterogeneous disease reflecting different pathophysiology. Inhaled allergen-mediated disease can be Th2-driven or Th2-low/Th17-driven. The Th2-low/Th17-driven disease is predominantly neutrophilic inflammation and associated with non-responsiveness to inhaled corticosteroids. There are currently no effective therapies for the control of such steroid-unresponsive, severe asthma. This disease is characterized by increased abundance of IL-17A/F and antimicrobial host defence peptide LL-37 in the lungs.

Objective: To characterize the interplay of IL-17A/F & LL-37 in airway inflammation and identify the molecular targets of IL- 17A/F and LL-37. These targets may represent pivotal checkpoints in airway inflammation that can be exploited to develop new therapeutic strategies.

Methods: Human Bronchial Epithelial Cells (HBEC-3KT) were stimulated with IL-17A/F (50 ng/mL). Cell lysates (n=5) were probed using high content aptamer-based proteomic array. Differential analysis was performed on normalized log2 protein expression values, along with Welch’s t-test (p<0.05) to identify differentially abundant proteins. Subsequently, the identified IL-17A/F-mediated molecular signature was independently validated using ELISA and western blots, with HBEC- 3KT and Primary Bronchial Epithelial Cells (PBEC) isolated from three individuals undergoing lung resection in vitro, in the presence/absence of LL-37 (0.25 uM).

Results: Proteomic profiling and subsequent independent validation in HBEC-3KT and PBEC determined that IL-17A/F increased the intracellular abundance of neutrophil-associated antimicrobial peptide (AMP) LCN2 & neutrophil-associated chemotactic factor GRO. In addition, LL-37 selectively augmented IL-17A/F-mediated inflammation in bronchial epithelial cells: LL-37 decreased IL-17A/F-mediated LCN2 production, whereas combinations of IL-17A/F + LL-37 enhanced the abundance of neutrophil-associated AMP Elafin, TH17-associated chemokine CCL20, and GRO. LL-37-mediated regulation of IL-17A/F-signature inflammatory mediators was associated with NF-kB activation.

Conclusion: Physiological concentrations of LL-37 selectively alter IL-17A/F-mediated inflammation in bronchial epithelial cells. LL-37 suppresses IL-17A/F-mediated LCN2 production while enhancing IL-17A/F-mediated Elafin, GRO and CCL20 production. Overall, this study demonstrates the role of LL-37 in augmenting IL-17A/F-mediated responses associated with severe airway inflammation that may be exploited to develop new therapies.

Electronic Dashboard to Monitor Student Clinical Experiences

Hollis Lai1, Doris Lunardon2, Melanie Chouinard3, Cody Surgin4, Nazila Ameli5

1Associate Professor, School of Dentistry, University of Alberta, Edmonton, Canada 2Associate Chair - Clinic Operations, School of Dentistry, University of Alberta, Edmonton, Canada 3Clinic Supervisor - Patient Services & Systems, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada 4Data specialist, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada 5Ph.D. student, School of Dentistry, University of Alberta, Edmonton, Canada

Introduction: Tracking mechanisms of clinical experience for students at dental schools provide feedback for patient assignment, student experience, and learning progression. A learning progression dashboard provides instructors and students with detailed information on their progress of learning, diversity of experiences, and clinical performance. The purpose of the present study is to evaluate the improvement gained from how an electronic dashboard using Axium software can be used to manage concerns regarding student clinical experience and their progress during clinical studies at the faculty of Dentistry, University of Alberta.

Methods: A secondary data analysis study was conducted after we designed and created an electronic dashboard using php and used dental students’ records from 2017-2021 (about 35000 student experience records per year) collected on the dashboard to determine their clinical experience and compare it between 2017-2018 and 2018-2019 academic year. Dashboard data contained what dental procedures a dental student required to do across all disciplines across time and the number of completed tasks of an individual. Each clinical discipline listed a different number of required procedures with a different number of attempts required. Using two time points, the end of January and the end of April, students’ experience were compared. Univariate statistics was used to describe changes across collected data. All analysis has been conducted in R and we used t-test for detecting the differences in the number and categories of codes by dental students and p=0.05 was determined as significant.

Results: The number of codes and the category of codes showed significant differences between the academic year 2017- 2018 and 2018-2019 for both third year and fourth year dental students after one term and two terms. (p< 0.05) The number of recorded codes was 8597 (26%) greater in the year 2018-2019 compared to the previous year which indicates the efficiency of dashboard in improving the clinical experience of dental students.

Conclusion: Connecting recent technologies with assessment strategies that support education and learning, educators can continuously improve the learning analytics field by designing information management methods such as dashboards in support of better learning outcomes.

Effects of S100A9 on macrophage cellular reprogramming in the context of sepsis

Andy An1, Beverlie Baquir1, Arjun Baghela1, Olga Pena1, Robert EW Hancock1

1Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, BC, Canada

Introduction: Survivors of sepsis, a life-threatening dysregulated immune response to infection, experience sustained immunosuppression, increasing the risk of secondary infections and death. One hypothesized contributor of immunosuppression early and later in sepsis is cellular reprogramming (CR), in which macrophages become “immunoparalyzed” after repeated stimulations, unable to respond appropriately to pathogens (e.g., cannot produce TNFα). Our lab developed a CR gene signature that can predict sepsis development in patients. In this signature is S100A9, which codes for a calcium-binding inflammatory protein upregulated in septic monocytes. Thus, we hypothesized that adding S100A9 might induce CR in macrophages and removing S100A9 might prevent CR or shorten its duration.

Methods: CR was induced in the macrophage cell line THP-1 by two stimulations of lipopolysaccharide (LPS) and cytokine production was profiled using ELISA. This was compared to cytokine production from non-CR inflammatory macrophages (one stimulation of LPS), stimulation with recombinant human S100A9 (rhS100A9), or stimulation with LPS in the presence of tasquinimod, an S100A9 inhibitor. In addition, CRISPR-Cas9 technology was used to generate induced pluripotent stem cell-derived macrophages with a frameshift mutation in the S100A9 gene. CR was similarly induced in these mutant macrophages and cytokine expression was profiled longitudinally.

Results: TNFα production in THP-1 macrophages stimulated with rhS100A9 was similar to that of CR macrophages, suggesting exogenous S100A9 could induce CR. Inducing CR in THP-1 macrophages in the presence of tasquinimod also showed altered production of TNFα, IL-8, IL-10, and CXCL-10 compared to both CR and non-CR macrophages. Production of TNFα and IL-10 after CR induction was the same in S100A9 mutant macrophages compared to wild-type. In addition, there was partial reversal of the CR phenotype five days after CR induction for both mutant and wild-type macrophages.

Conclusions: S100A9 was able to induce CR in THP-1 macrophages. Using an S100A9 inhibitor affected CR induction but did not prevent CR in these macrophages as cytokine profiles were also dissimilar to non-CR macrophages. Mutant S100A9 macrophages were indistinguishable from wild-type macrophages in both TNFα and IL-10 production and CR duration. These results suggest that S100A9 may be a player but not a key regulator in CR.

iPSCs display a unique set of MHC I-associated peptides shared by cancer cells

Anca Apavaloaei1, Marie-Pierre Hardy1, Jean-Philippe Laverdure1, Gregory Ehx1, Chantal Durette1, Joel Lanoix1, Mathieu Courcelles1, Dev Kapil Chauhan2, Sebastien Lemieux1, Mick Bhatia2, Pierre Thibault1, Claude Perreault1

1Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Canada 2McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Canada

Introduction: The development of cancer immunotherapeutics is limited by the lack of well-defined tumor-specific antigens (TSAs). Growing evidence indicates that cancer cells and pluripotent stem cells, i.e. embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), share unknown TSAs that could be immunotherapeutic targets. Indeed, cancer cells and iPSCs show a significant transcriptomic overlap and, when used as prophylactic vaccines, iPSCs prevent tumor growth in mouse models of lung, breast, and skin cancer. This study aimed to identify the nature of pluripotency-associated TSAs shared between human iPSCs and cancer cells.

Methods: We used a proteogenomic approach combining RNA-sequencing and mass spectrometry to study the MHC I- immunopeptidome specific to three human iPSC samples. iPSCs were also analyzed after a 72h treatment with IFN-γ to boost MHC I levels and increase our chance for pluripotency-associated MHC I-associated peptide (paMAP) discovery.

Results: We identified 46 paMAPs which were absent from the RNA-seq of healthy somatic tissues and adult stem cells, but 40 of them were expressed in multiple cancer types from The Cancer Genome Atlas (TCGA). paMAPs were non-mutated, and 50% of them derived from ostensibly non-coding genomic regions (e.g., introns, intergenic regions) transcribed in pluripotent stem cells. Six of the paMAPs were previously identified as TSAs using tumor samples, indicating that novel paMAP-coding sequences have a high potential to generate shared TSAs between patients across multiple cancer types. Bioinformatic analyses revealed that hypomethylation and aberrant signaling events regulate paMAP expression across TCGA cancers. However, even though at least two of the paMAPs are immunogenic, paMAP presentation did not confer a survival advantage in patients from TCGA cohorts. Indeed, the number of paMAPs was correlated with immune evasion, which may explain their preferential expression in advanced cancers and their lack of spontaneous immunogenicity, reinforcing the importance of TSA-based vaccinations.

Conclusion: Our study reveals that the immunopeptidome of iPSCs reflects their pluripotency state and provides new insights into the molecular underpinnings of stemness in cancer. The antigens shared by iPSCs and tumors represent attractive targets for immunotherapy of poorly differentiated cancers.

A Neurochemical Atlas of the Brain

Jessica Archibald1,2, Erin L. MacMillan4,5, John L.K. Kramer1,3,6,7

1International Collaboration on Repair Discoveries (ICORD), University of British Columbia, Vancouver 2Department of Experimental Medicine, Faculty of Medicine University of British Columbia; 3Department of Anesthesiology, Pharmacology and Therapeutics, Faculty of Medicine, University of British 4Department of Radiology, Faculty of Medicine, University of British Columbia; 5 Philips Health Canada 6Djavad Mowafaghian Center for Brain Health (DMCH); 7Hughill Center.

Introduction: Stated by the 2020-21 Departmental Health Canada Plan, the opioid epidemic is listed as one of the four priority areas to strengthen the health-care system. Following the implementation of COVID-19 measures, there has been a 74% increase in opioid-related deaths1. A strategic research priority is improving access to evidence-based treatments. Central to evidence-based treatments are advanced neurophysiological techniques. These are used to examine neurological conditions including neurodegenerative (e.g., Alzheimer's), psychiatric conditions (e.g., schizophrenia) and chronic pain (e.g., back pain). These approaches focus on function in terms of electrophysiology (i.e., EEG, MEG)2–4 and blood flow (i.e., fMRI, ASL)5–8. Measurement of neurochemicals via H-MRS (Proton-magnetic resonance spectroscopy), is a technique that provides a unique opportunity to evaluate neurochemical levels, colloquially termed The Virtual Biopsy.

Aim: To determine if clinical pain is associated with significant changes in neurochemistry. As the primary excitatory and inhibitory neurotransmitters, respectively, glutamate and GABA9 are integrally involved in brain function. Preliminary findings show an 8.3% increase in glutamate occurs in the cingulate cortex when experimental pain first appears. The trend for glutamate to increase during is notably higher in females10. Based on this, one may ask: Can we predict pain sensitivity based on a neurochemical atlas?

Methods: I am mapping neurochemicals in the cingulate gyrus in healthy participants to determine ‘normal’ ranges of neurochemicals, distribution and variability. This map will be applied in populations with clinical pain. I hypothesize that patients will show higher glutamate, and lower GABA levels, as well as changes in variability and distribution. Given that, there are significant differences in how males and females perceive pain11–14, I hypothesize there will be differences in their neurochemical patterns.

Results/Feasibility: Patient data will be collected in Switzerland under the supervision of Dr. Schweinhardt (Friedman Award 2021). Alongside publications, central to the project is a deliverable that aims to democratize the study of brain health through the creation of an open-source tool for analysis of MRS data.

Significance: The gap between physiological knowledge and specialized measurement techniques has led to a lack of diagnostic and treatment measures, my research aims to tackle this problem.

Rhythmic ENG activity induced by 4-aminopyridine in the decerebrate rat

Katrina E. Armstrong1, Kristine Cowley1, Katinka Stecina1, Larry M. Jordan1, Tabrez J. Siddiqui1, Michael F. Jackson2

1Department of Physiology and Pathophysiology, University of Manitoba 2Department of Pharmacology and Therapeutics, University of Manitoba

Introduction: 4-aminopyridine (4-AP), a non-selective blocker of voltage-gated potassium channels, is a drug currently used clinically to treat demyelinating disorders such as SCI. Classical research into the components of the locomotor central pattern generator (CPG) has used 4-AP in spinalized cat models to facilitate synaptic transmission and potentiate the effects of drug induced rhythmic activity. Previous application of 4-AP in the acute decerebrate spinal cat and the anaesthetized spinal rat elucidated two salient features on rhythmic activity: high-frequency synchronous activity in flexor and extensor nerves, and once initiated, can be recorded uninterruptedly for several hours. To this day, it is still unknown how 4-AP produces synchronous activity in functionally different nerves. The aims of this study are to investigate the rhythmic activity induced by 4-AP in a decerebrate rat model and to examine the potential synchronization of neuronal activity induced by 4-AP.

Methods: Experiments were performed in decerebrated (un-anaesthetized) female rats (Tph2-cre, n=4) while recording flexor (common peroneal) and extensor (tibial) nerve activity as well as arterial blood pressure. 4-AP was administered intra- arterially (I.A) by titrating the dose to determine the lowest amount of drug needed to induce rhythmic activity. The frequency and coordination of the rhythmic activity produced was analyzed using SCRC software.

Results: Rhythmic activity in all ENG nerves was observed 20 minutes after I.A. injection of 4-AP (0.1-0.5 mg/kg) and continued to be active for more than an hour. We attempted to perturb nerve activity by peripheral nerve or midbrain electrical stimulation. The frequency of all ENG activity was much higher than normal fictive locomotion induced by midbrain stimulation. Most of the rhythmic activity recorded was synchronous in all nerves based on preliminary analysis. The frequency of alternation and other basic electroneurogram parameters describing coordination of left-right and flexor- extensor activity will be compared in more detail.

Conclusion: Administration of 4-AP in the decerebrate rat produced results similar to the acute decerebrate spinal cat, in terms of robust rhythmic hindlimb activity. This drug could be useful to study the components of the CPG that may produce the synchronization of functionally different nerves.

Factors Affecting Women's Participation in the Health Workforce in Fragile and Conflict- Affected Countries: A Scoping review

Basnama Ayaz1

1Lawrence S. Bloomberg Faculty of Nursing, University of Toronto

Introduction: The full participation of women as healthcare providers is critical to achieving universal health coverage and Sustainable Development Goals by 2030. However, women in the Fragile and Conflict-Affected States (FCASs) not only experience the common gender biases and inequities that prohibit women's participation in the healthcare workforce in many countries but face heightened risk of violence from political conflicts and war. A scoping review to examine the impact of gender on women's participation and career trajectories in the health workforce in FCASs identified factors affecting women's participation in the health workforce.

Methods: Following Arksey and O'Malley's scoping review framework, we searched published literature from five health sciences databases and grey literature. Two reviewers independently screened the title and abstract, followed by a full-text review for shortlisted sources against the set criteria. Based on the World Bank's harmonized lists for 2018 and 2019, 36 countries categorized as FCASs were included in the literature search.

Results: In 10 FCASs, women constituted up to 46% of the health workforce. Women comprised up to 57% of physicians and 29% of nurses. Men occupy leadership positions in both nursing and medicine. Constraints for women included professional hierarchies, gendered sociocultural norms, and security conditions. Women in the health workforce in FCASs struggled to function at their full capacity due to poor remuneration, lack of career advancement opportunities, and gender-sensitive policies. Donors and implementing partners narrowly focused on programs that served women's reproductive health rather than a comprehensive approach to women's health.

Conclusion: The review identified multiple challenges and constraints facing efforts to create gender equity in the health workforce, particularly for women in FCASs. Expanding female nurses' and physicians' capacity is critical to provide gender-sensitive care for women beyond the reproductive health in the FCASs.

Rationale, Design and Baseline Characteristics for the Strategies to OPpose SUGARS with Non- nutritive sweeteners Or Water (STOP Sugars NOW) trial

Sabrina Ayoub-Charette1,2, Néma McGlynn1,2, Tauseef Ahmad Khan1,2, Sonia Blanco Mejia1,2, Laura Chiavaroli1,2, Meaghan Kavanagh1,2, Maxine Seider1,2, Danielle Lee1,2, Amna Ahmed1,2, Rachel Asbury2,3, Madeline Erlich2,3, Vasanti Malik1,4, Richard Bazinet1, Anthony J Hanley1, Cyril WC Kendall1,2,5, Lawrence A Leiter1,2,6-8, Elena M Comelli1,9, John L Sievenpiper1,2,6-8

1Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, 2Toronto 3D Knowledge Synthesis and Clinical Trials Unit, Clinical Nutrition and Risk Factor Modification Centre, St. Michael’s Hospital, Toronto, Ontario 3School of Nutrition, Faculty of Community Services, Ryerson University, Toronto, Ontario 4Department of Nutrition, Harvard T.H. Chan School of Public Health; 5College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan 6Division of Endocrinology and Metabolism, Department of Medicine, St. Michael’s Hospital, Toronto, Ontario 7Department of Medicine, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario 8Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario 9Brian Lawson Centre for Child Nutrition, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario

Introduction: Health authorities recommend reducing added or free sugars to ≤5-10% energy. Much attention has focussed of the reduction of SSBs with the recommendation that SSBs be replaced with unsweetened healthy alternatives such as water but not non-nutritive sweetened beverages (NSBs). There are concerns that non-nutritive sweeteners do not have the intended benefits and may induce glucose intolerance through changes in the gut microbiome. Whether NSBs have benefits similar to water in their intended substitution for SSBs is unclear.

Methods: To address this question, we have undertaken the STOP Sugars NOW trial (NCT03543644), a pragmatic “head-to-head” crossover randomized controlled trial of the effect of NSBs (the intended substitution) versus water (the standard of care) as a replacement strategy for SSBs on glucose tolerance and gut microbiome diversity. We recruited overweight or obese participants with a high waist circumference who regularly consume ≥1 SSBs/day. Each participant underwent a ≥2-week run-in period followed by three 4- week treatment phases in random order (usual SSBs, equivalent NSBs, or water) with each phase separated by a ≥4-week washout. The two primary outcomes are change in glucose tolerance and gut microbiome beta- diversity. Adherence to the interventions will be assessed by objective biomarkers of added sugars (13C/12C isotopic ratio in serum fatty acids and urinary fructose and sucrose) and non-nutritive sweeteners (urinary sucralose and acesulfame-potassium).

Results: The trial started on June 1st, 2018 with the first participant undergoing randomization on August 1st, 2019 and the last participant finishing on October 15th, 2020. We screened 1,086individuals, out of which 80 were randomized. Baseline characteristics showed a mean age of 41.8±12.9y, BMI of 33.8±6.7kg/m2, waist circumference of 108.7±13.4cm, and mean SSBs intake of 2 SSBs/day.

Significance: The results of this trial will directly inform public health guidance on the use of NSBs in sugars reduction strategies.

Prenatal cannabis exposure: levels of maternal-fetal transmission and effects on fetal brain development in rats following inhaled delivery

Samantha L. Baglot1, Catherine Hume1, Gavin N. Petrie1, Robert J. Aukema1, and Matthew N. Hill1

1Department of Cell Biology and Anatomy, University of Calgary, Calgary, AB, Canada

Introduction: Cannabis use during pregnancy is as high as 10-20%. Human and animal studies of prenatal cannabis exposure (PCE) have shown growth retardation and alterations in neurobehavioral trajectories. However, little is actually known about maternal-fetal transmission of Δ⁹-tetrahydrocannabinol (THC, major psychoactive component of cannabis) or the mechanism through which THC alters neurodevelopment. Animal models allow for examination of such mechanistic questions, as well as control over the timing and amount of exposure. THC acts on the endocannabinoid system (eCB), which is heavily interconnected with the immune system. Both systems are highly involved in brain development. Thus, THC may exert its effects on neurodevelopment through direct modulation of the eCB system and/or indirect modulation of the immune system. Our studies perform translational work utilizing the most common route of cannabis consumption in humans – inhalation – during pregnancy in rats to examine the effects on maternal-fetal transmission of THC and fetal brain and immune system development.

Methods: Pregnant rats were exposed to cannabis via inhalation (10% THC; 15-min session) either acutely (on gestational day [G] 21) or chronically from G10-21 or G1-21. Dams and their fetuses were collected across gestational timepoints to examine: 1) THC and metabolites levels in maternal blood, placenta, and fetal brain, 2) fetal brain eCB levels, 3) fetal brain eCB and immune-related gene expression, and 4) fetal brain cytokine (immune signaling molecule) levels.

Results: We show highly correlated levels of THC and metabolites in maternal blood, placenta, and fetal brain. Interestingly, chronic exposure of THC during pregnancy produced altered metabolism compared to acute exposure in dams and their fetuses, suggesting metabolic tolerance to chronic cannabis exposure. Furthermore, our results indicate that ~30% of circulating maternal THC reaches the fetal brain following inhalation. We found no impact of PCE on whole fetal brain eCB levels. Analysis of gene expression and cytokine levels are ongoing.

Conclusion: These results highlight the importance of an animal model of inhaled THC delivery. Further, this research is important to establish placental ability to breakdown cannabis and determine the level and timeframe of fetal exposure, as well as the effects of PCE on fetal neurodevelopment.

Invariant natural killer T cells in HIV infection: An optimized method to assess cellular functionality

Allison L Balasko1, Julie LaJoie1,2, Keith R Fowke1,2,3,4

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg MB 2Department of Medical Microbiology, University of Nairobi, Nairobi Kenya 3Department of Community Health Sciences, University of Manitoba, Winnipeg MB 4Partners for Health and Development in Africa

Background: Invariant Natural Killer T (iNKT) cells are innate lymphocyte cells bridging the innate and adaptive immune systems as one of the first responders critical in combatting viral infection. Our lab has shown that iNKT cell exhaustion and dysfunction are increased in HIV infection. To further characterize this cellular dysfunction, both multi-hour cytokine and multi-day proliferation assays must be optimized. Traditionally, in vitro iNKT stimulation has been achieved by α-GalCer, a CD1d-restricted lipid antigen used to activate iNKT cells. However, it has been shown that in HIV infection, CD1d is down- regulated, potentially affecting our ability to study iNKT cell function properly in HIV-positive samples. However, we have shown the NIH tetramer loaded with PBS-57 (α-GalCer analogue), which is normally used to stain iNKTs for identification via flow cytometry, can double as a stimulant of iNKT cells if allowed time to incubate with iNKTs. This stimulates iNKT cells independently of CD1d-restricted antigen presentation.

Hypothesis: We hypothesize the tetramer stimulates iNKTs via the PBS-57 α-GalCer analogue cell binding, providing a reliable stimulation independent of CD1d-specific antigen presentation.

Methods: In HIV-negative donors, we have optimized multi-hour cytokine and multi-day proliferation assays assessing iNKT functionality and exhaustion through either α-GalCer or PBS-57-tetramer stimulation.

Results: iNKTs have been successfully activated with 40.2% IFN-γ production after 10hr α-GalCer stimulation, versus 58.2% via the PBS-57-tetramer. A 13-day proliferation time course showed an average iNKT fold-expansion of 8.4, 107.1, 256.0 and 814.9 by α-GalCer stimulation on day 6, 8, 10 and 13 respectively, versus 10.1, 136.4, 209.9 and 937.5 by PBS-57- tetramer.

Significance: As CD1d is downregulated in HIV infection as an evasion strategy to suppress iNKT activation, the study of a CD1d-independent method for iNKT stimulation is relevant when assessing HIV-positive samples, as well as designing therapeutic adjuvants for use in HIV-positive individuals. Future goals are now to apply these iNKT stimulation techniques to assess cellular functionality and exhaustion in HIV infection.

You are what your great-grandparents eat: glyphosate exposure results in decreased locomotor activity and altered anxiety-like behaviour in male mice that persist three generations after exposure

Jacqueline A. Barnett1, Maya Bandy1, Andrea Verdugo Meza1, Jiayu Ye1, Natasha Haskey1, Miranda M. Hart,1 Julien Gibon1, and Deanna L. Gibson1,2

1University of British Columbia Okanagan Campus, Biology Department, Kelowna, BC 2University of British Columbia Okanagan Campus, Faculty of Medicine, Kelowna, BC

Background: Agricultural practices, including desiccation and the development of glyphosate-resistant crops, have led to increased glyphosate levels within our food. While cytotoxic effects of glyphosate and glyphosate-based herbicides have been reported, their potential impact on gut commensal bacteria and behaviour has yielded conflicting results due to dose discrepancies. Glyphosate prevents the synthesis of aromatic amino acids by inhibiting the Shikimate pathway, which, besides killing weeds, can inhibit bacterial growth, promoting bacterial dysbiosis. Additionally, aromatic amino acids are important precursors for many neurotransmitters. We hypothesized that exposure to physiologically relevant levels of glyphosate leads to dysbiosis, resulting in behaviour changes that persist across generations in rodents.

Methods: We examined the acceptable daily intake currently set by the environmental protection agency (EPA) (1.75mg/kg body weight/day.) Parental generation animals were exposed to glyphosate during breeding and pregnancy. F1, F2 and F3 generations of animals were not directly exposed to glyphosate. At 16 weeks of age, offspring were subjected to open field behavioural testing.

Results: Male C57Bl/6 mice whose great-grandparents were exposed to EPA level glyphosate during pregnancy exhibited decreased locomotor activity and increased anxiety-like behaviour compared to both female mice receiving the same treatment and animals whose great-grandparents were not exposed to glyphosate. Overall, male mice whose great- grandparents were exposed to glyphosate travel less distance and travel at a lower body velocity than control mice and females exposed to the same treatment. Moreover, male mice exhibit increased anxiety-like behaviour, including increased immobility (freezing) and decreased exploratory behaviour. Additionally, animals whose great-grandparents were exposed to glyphosate exhibited clinical symptoms of gastrointestinal disease, including weight loss and diarrhea.

Conclusion: Glyphosate exposure may influence behaviour by reducing important neurotransmitter precursors and promoting microbial dysbiosis within the gut. Moreover, these changes appear to persist across generations without direct glyphosate exposure to offspring. These results suggest that exposure to glyphosate, at doses previously deemed safe by the EPA, may have implications for neurodevelopment, behaviour and gastrointestinal health across generations. Characterization of the microbiome within these animals and quantification of neuromodulatory peptide levels within their serum is underway. Additional behaviour testing is needed to characterize anxious behaviours more thoroughly.

Investigating Host-Pathogen dynamics of Mycobacterium Paratuberculosis utilizing an Enteroid Model System

Grace M. Baruta1, Hong Zhang1, Laurie Alston1, Simon A. Hirota1

1Department of Physiology & Pharmacology, University of Calgary

Background: Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease (JD) in ruminants. Following infection, JD may present as enteritis, leading to nutritional deficiencies and wasting, often causing premature culling of livestock. Beyond veterinary medicine, several mycobacterium species, including MAP, have been implicated in human infection in immunocompromised individuals. While MAP has also been incriminated in causing Crohn’s disease in humans (a claim that has yet to be substantiated), there are confirmed cases of MAP infection in humans. Given the economic burden associated with JD, considerable efforts have sought to understand MAP infection dynamics. However, these processes have not been completely characterized, hindering our ability to generate novel anti-infective agents. While the current paradigm suggests that MAP travels to the small intestine, where it gains entry through the epithelium, the exact cellular tropism and the mechanism(s) of entry are not well defined. Therefore, we have developed an ex vivo enteroid-based system to visualize invasion of MAP using a GFP-expressing MAP strain. With this, we sought to test the hypothesis that MAP invasion occurs via M cells through receptor-mediated transcytosis.

Aims: 1) Characterize experimental system and visualize MAP invasion 2) Determine cellular tropism for MAP 3) Uncover mechanisms underlying MAP invasion

Methods: Enteroids (2D and 3D) were generated and M cell differentiation induced by the addition of RANKL. Confluent ileal monolayers were exposed to GFP-expressing MAP strain (K10 pWES4). Confocal microscopy was performed, and measured barrier function via transepithelial electrical resistance (TEER).

Results: We generated 3-D enteroids and confluent enteroid-derived monolayers with functional M cells. MAP was detected mainly within M cells. Furthermore, alterations in TEER following MAP exposure in monolayers cultured with RANKL suggest the existence of a novel mechanism by which MAP disrupts the barrier to invade the mucosa.

Conclusion: Our results suggest that MAP translocates across the epithelium predominantly via M cells. This newly optimized approach provides an experimental system that will enable us to better characterize M cell-MAP interactions, with the hopes of identifying new therapeutic targets to prevent the spread of MAP and reduce economic impact of JD.

Functional Characterization of the Interaction Between Adenylyl Cyclase Isoform 6 and the G protein G alpha S

Anjali Y. Bhagirath, Vikram Bhatia, Manoj Reddy Medapati, Nisha Singh, Martha Hinton, Prashen Chelikani, Shyamala Dakshinamurti

1Departments of Pediatrics and Physiology, University of Manitoba 2Biology of Breathing, Children's Hospital Research Institute of Manitoba (CHRIM) 3Department of Oral Biology, University of Manitoba, Winnipeg

Stimulation of Adenylyl cyclases (ACs) in response to G-protein Gαs catalyzes the production of cyclic AMP (cAMP), a key second messenger that regulates diverse physiological responses. There are 10 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. We have previously shown that under hypoxic conditions, AC6 is amenable to post translational modification by nitrosylation at cysteine residues. Nitrosylation was also found to affect AC reactivity. In the absence of crystallographic data, to identify the nitrosylable cysteines, we previously modeled the interaction between AC6 and Gαs and identified key cysteine residues predicted to be involved in the interaction. In this study, we confirmed that our previously identified residues in AC6 and Gαs are in fact important for the activation of AC6. Using functional assays, we have shown that Cys1004 in AC6 (subunit C2) and Cys237 in Gαs present at the AC-Gαs interface are important for the activation of AC6. We further provide preliminary evidence to show that mutating Cys 1004 in the second catalytic domain of AC6 makes it amenable to inhibition by Gi which possibly accounts for its decreased functional activity.

Improving Peritoneal Dialysis Catheter Success – Lessons Learned from the Clinical Improvement Team

Tiffany Blair PhD Candidate, Rod Stryker MD FRCPC, Nicolette Sinclair MD FRCPC, Paul Babyn MD FRCPC,

Vincent Framework Team* College of Medicine, University of Saskatchewan, Canada

Introduction: It is important to identify best practice, implement solutions to ameliorate cause-specific peritoneal dialysis (PD) technique failure, and optimize clinical practice. Extending technique survival on PD remains a major challenge to optimizing outcomes for patients while increasing utilization. Failure rates are associated with significant burden and hardship to the patient, and an overall increase in cost to the health system. Current methods of PD catheter insertion have resulted in high failure rates in patients1o and 2o failure rate: 2016 31/62 (50.0%); 2017 23/71 (32.4%); 2018 11/50 (22.0%); 2019 8/54 (14.8%); 2020 5/57 (8.8%).

Objectives:  To identify prognostic factors which make significant contributions to improved health status for patients who choose peritoneal dialysis as a treatment modality  To improve access to PD, and design a culturally sensitive model of care  To demonstrate cost saving through avoidance of hemodialysis, improved uptake of PD, reduced operating room time, and reduction of primary/secondary PD catheter failure.

Methods: The interdisciplinary clinical improvement team (CIT) participated in the Canadian Patient Safety Institute Measuring and Monitoring for Patient Safety Framework (MMSF) national collaborative. The team was challenged to improve the quality and safety of patient care with clinical pathways, overcome traditional ‘department’ boundaries, and better capture and understand the patient and family care experience. The CIT oversees the development, monitoring, and continuous improvement of clinical pathways drawing upon evidence based best practice.

Results: Through focused efforts, the CIT has reduced PD catheter failure rate by from 31/62 (50.0%) in 2016, to 5/57 (8.8%), representing a 41.2% reduction. Process improvements implemented were standard workflow and PD assessment, exit site marking for interventional radiology only, standard room set up, patient safety questionnaire, quarterly review of metrics and reporting, ongoing case reviews, and lead time reduction (time to referral, assessment, PD insertion, and PD train).

Conclusion: The MMSF served to translate real time data so it is useful to take action, gap analysis for process improvement, identifying strengths and weaknesses, and promoting a culture of safety and continuous improvement.

Using social media discourse to inform health care: Developing qualitative methodology to analyze Reddit data

Lisa Blundell1, Maria Mathews2

1Memorial University of Newfoundland 2Western University

Introduction: Patients use social media to learn about their disease and to receive peer support. Data from social media sites are often publicly accessible and could be used by researchers to better understand patients’ concerns and experiences. Large amounts of data are readily available, but few methods have been developed to collect and analyze such data. Our objective was to determine how to retrieve and qualitatively analyze data from Reddit, a social news network and discussion website.

Methods: We reviewed methodology of published social media research to understand research considerations. We used R, an open-source software environment for statistical computing and the ‘RedditExtractoR’ package to retrieve data. We implemented our methods using subreddit ‘r/ostomy’ to observe how 3500+ people discussed food while having an intestinal ostomy due to bowel disease. We extracted post titles, content, and comments containing chosen keywords related to food. We cleaned and reformatted the data, and imported the data into Nvivo, data analysis software. Using Nvivo, we catalogued usernames and recorded demographic characteristics from information within their posts and comments.

Results: Our final data file was organized and in an easy-to-read format that allowed us to qualitatively analyze the data. We conducted a thematic analysis by independently coding responses and highlighting themes to produce a coding template and using Nvivo software. Through our analysis, we were able to identify common patient concerns such as worries about nutrient absorption, food restrictions and avoidance, and symptom management. Users also discussed the difficulties of having an illness and the value r/ostomy provided by creating an opportunity to talk to others with lived experience of having an ostomy. Reddit contained rich data of good quality and because Reddit users use pseudonyms to communicate with one another, posts appeared disinhibited, potentially attributed to the use of pseudonyms on the Reddit platform.

Conclusion: The utilization of Reddit data in health care research can provide a candid view of people’s health and health care experiences. Our findings indicate that Reddit is a rich data source and studies using these data have the potential to inform health care services and triangulate research findings.

Studies of depression and anxiety using Reddit as a data source: Scoping review

Nick Boettcher1

1Department of Community Health Sciences, Cumming School of Medicine, University of Calgary

Introduction: Interest in studying depression and anxiety using publicly available social media data has grown considerably in the last decade. The discussion platform Reddit has become a popular social media data source in this area of study, in part because of the unique ways in which the platform is facilitative of research. The objective of this review is to understand the scope and nature of research using Reddit as a primary data source for studying depression and anxiety.

Methods: A scoping review was conducted following the Arksey and O’Malley framework. Academic databases searched include MEDLINE/PubMed, EMBASE, CINAHL, PsycINFO, PsycARTICLES, Scopus, ScienceDirect, IEEE Xplore, and ACM database. Inclusion criteria were developed using the Participants/Concept/Context framework outlined by the Joanna Briggs Institute Scoping Review Methodology Group. Eligible studies featured a methodological focus on analyzing depression and/or anxiety using naturalistic written expressions from Reddit users as the primary data source.

Results: 54 Studies were eligible for review. Key methodological features included a comparatively larger focus on depression versus anxiety, an even split of original and premade datasets, a common analytic focus on predictive classification of mental health states, and practical implications which often recommended new methods of professionally- driven monitoring and outreach for Reddit users.

Conclusion: Studies of depression and anxiety using Reddit data are currently driven by a prevailing methodology favoring a technical, solution-based orientation. Researchers interested in advancing this research area will benefit from further consideration of conceptual issues surrounding interpretation of data from Reddit using the medical model of mental health. Further efforts are also needed to locate accountability and autonomy within practice implications suggesting new forms of engagement with Reddit users. Through illuminating the scope and nature of the research landscape using Reddit data to study depression and anxiety, this review contributes to advancing understandings at the intersection of social media, research practice, and public mental health.

Developmental follow-up practices for children with congenital heart defects: A national environmental scan

Marie-Eve Bolduc1, Janet Rennick2,3,4, Isabelle Gagnon1, Marie Brossard-Racine1,4,5, Annette Majnemer1,4,5

1School of Physical and Occupational Therapy, McGill University, Montreal, Canada 2Department of Nursing, The Montreal Children’s Hospital, McGill University Health Centre 3Ingram School of Nursing, McGill University, Montreal, Canada 4Department of Pediatrics, McGill University, Montreal, Canada 5Department of Neurology and Neurosurgery, McGill University, Montreal, Canada

Introduction: Developmental surveillance, screening and evaluation are central to the early identification of developmental impairments in children at-risk. However, data is lacking on the current developmental follow-up practices of children with congenital heart defects (CHD) post open-heart surgery as well as barriers to optimal practices across Canada. Therefore, the objective of this study was to describe current Canadian developmental follow-up practices and to explore barriers to optimal developmental follow-up.

Methods: This is a cross-sectional survey involving health professionals in the developmental follow-up of children and adolescents with CHD in all eight Canadian hospitals that provide openheart surgery to infants. Telephone interviews were conducted. The telephone survey collected both qualitative and quantitative data and was divided into three sections: 1) descriptive information, 2) current follow-up practices and 3) feasibility and acceptability of current guidelines and barriers to developmental follow-up.

Results: Although most children benefit from developmental surveillance at every medical visit, only half of the tertiary care centers that perform open-heart surgery in Canada have a systematic follow-up program that includes standardized evaluation, and developmental screening at specific timepoints during childhood. When available, these programs are only accessible to a subset of children with CHD requiring open-heart surgery who are deemed higher risk. The large majority of surveyed Canadian institutions (7/8) describe current practices as suboptimal and would like to develop a more systematic developmental follow-up program or expand an existing one. They described the lack of human and/or financial resources and limited dedicated time as important barriers to offering optimal developmental surveillance, screening, and evaluation. Furthermore, the lack of knowledge of existing intervention services and the presence of a waitlist further limits timely access to intervention services.

Conclusion: Only a subset of children with the most complex congenital heart defects requiring open-heart surgery benefit from a systematic developmental follow-up program that includes surveillance, screening and evaluation in Canada, as recommended in guidelines by the American Heart Association. Hence, current surveillance practices may fail to promptly identify children and adolescents who experience developmental challenges. It is essential to develop policy recommendations to optimize developmental follow-up practices in Canada for high-risk populations.

Medical Predictors of Social-Emotional and Adaptive Functioning in School-Age Pediatric Intestinal Failure

Bianca C. Bondi1,2,3, Anna Gold1,2, Christina Belza1,4, Glenda Courtney-Martin1,4,5, Stephanie So1,4,6, Yaron Avitzur1,4,7, Paul W. Wales1,4,5,8

1Group for Improvement of Intestinal Function and Treatment (GIFT), The Hospital for Sick Children 2Department of Psychology, The Hospital for Sick Children 3Department of Psychology, York University 4University of Toronto 5Research Institute, The Hospital for Sick Children 6Department of Rehabilitation Services, The Hospital for Sick Children 7Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children 8Division of General and Thoracic Surgery, The Hospital for Sick Children

Introduction: Long-term survival in pediatric intestinal failure (IF) has improved over recent decades due to medical/surgical advances and the development of multi-disciplinary rehabilitation programs. Focus has shifted towards identifying neurodevelopmental morbidities and promoting optimal quality of life; however, there has been very little exploration into social emotional and adaptive functioning.

Methods: Children (4-18 years) in our IF rehabilitation program underwent neuropsychological assessments (2012-2019) which included caregiver- and teacher-reported social-emotional (BRIEF/BRIEF-2, BASC-2/BASC-3) and adaptive (ABAS- II/ABAS-3, SIB-R, Vineland-II, BASC- 2/BASC-3) functioning questionnaires. Results were compared to age-matched norms (mean=100; SD=15) using one-sample z-tests. Medical variables showing ≥3 significant correlations with social-emotional and adaptive functioning outcomes were included in linear regression analyses to explore medical factors independently associated with psychological outcomes.

Results: Overall, 74 children (55% males) underwent assessments (median assessment age: 6.5 years, IQR:6.1-8.1). Most children (72%) were premature (median gestational age [GA]: 34 weeks, IQR:29-37; median birth weight [BW]: 2.1kg, IQR:1.1-2.6). The most frequent IF etiology was necrotizing enterocolitis (NEC; 32%). Although caregiver- and teacher-report indicated broadly average-range mean scores, given the standard deviations, the group performed significantly poorer than age-matched norms on executive functioning, internalizing emotional problems (i.e., anxiety, depression, somatization), and adaptive functioning (Figure). GA, NEC diagnosis, duration of first year hospitalization, and total ambulatory clinic appointments correlated with ≥3 social-emotional and adaptive functioning outcomes while BW, small and large bowel length, total septic episodes, and total parenteral nutrition days did not. In the regression, adjusted for GA, NEC diagnosis predicted internalizing emotional problems, while the duration of first year hospitalization and total ambulatory clinic appointments were both nonsignificant.

Conclusions: Relative to population norms, the group displayed executive dysfunctions, emotional issues, and poor adaptive functioning. NEC diagnosis and GA, rather than IF related factors, posed heightened risk for emotional issues. Future research should explore demographic risk factors to further inform clinical screening and targeted intervention.

Figure. Social-Emotional and Adaptive Functioning in School-Age Pediatric Intestinal Failure Compared to Age-Matched Population Norms

Longitudinal alterations in aeurovascular coupling accompany L-DOPA induced dyskinesia development

Samuel Booth1, Dali Zhang1, Ji Hyun Ko1

1University of Manitoba

Introduction: Levodopa is a highly effective treatment for Parkinson’s disease (PD), but comes with the risk of the patient developing a motor complication known as Levodopa induced dyskinesia (LID). Previously, dissociation in cerebral blood flow and metabolism in the basal ganglia have shown to be associated with dyskinesia. However, we do not know if this dissociation precedes dyskinesia onset, which would indicate it is a relevant underlying mechanism.

Methods: 20 Sprague Dawley rats underwent unilaterial lesion of the substantia nigra with 6-OHDA was performed to induce hemiparkinsonian symptoms. Animals received 2 mg/kg L-DOPA per day which induces dyskinesia symptoms in a portion of animals within the experimental timeframe. Dyskinesia symptoms were assessed after L-DOPA injection in each animal on days 1, 11, and 21 by abnormal involuntary movements test. Cerebral metabolism and blood volume were measured by imaging with Fluorodeoxyglucose positron emission tomography (FDG-PET) and dynamic susceptibility contrast (DSC) MRI respectively. Animals were scanned on day 1 and day 22, both off L-DOPA and after L-DOPA treatment.

Results: Of the 20 animals that were treated with L-DOPA, 7 animals developed L-DOPA induced dyskinesia symptoms (LID group) while 13 animals remained free from dyskinesia symptoms. In the non-LID animals L-DOPA treatment significantly reduced FDG uptake ON L-DOPA from the first dose (lesioned side p = 0.009, t=3.148; non-lesioned side p=0.031, t=2.502). After 22 days of L-DOPA treatment, levodopa induced striatal hypometabolism was not as great (lesioned side p = 0.07, t=2.029; non-lesioned side p=0.097, t=1.829). In contrast, L-DOPA failed to result in striatal hypometabolism in the LID animals, either at baseline or after 22 days of L-DOPA treatment. In the LID animals only, striatal blood volume was significantly increased ON-L-DOPA after 22 days of chronic treatment, which was not observed at baseline (lesioned hemisphere, p=0.042, t = -2.569; non-lesioned hemisphere, p=0.028, t = -2.872).

Conclusions: non-LID animals were distinguished from LID animals by L-DOPA dependent reductions in striatal metabolism at the first dose of L-DOPA. Increases in cerebral blood volume distinguished LID animals from non-LID, however this increase developed later and was not present from the first dose.

Exploring new targeted therapies for SHH medulloblastoma

Stephanie Borlase1, Tamra Ogilvie1

1University of Manitoba

Medulloblastoma is the most common malignant primary pediatric brain tumor and is currently divided into 4 molecular subgroups that exhibit different genomic alterations, gene expression profiles and response to treatment. The 4 subgroups are WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. SHH medulloblastoma is characterized by activation of the SHH pathway and is comprised of very high-risk groups of both children and infants. Personalized therapies for SHH medulloblastoma are lacking, as SHH pathway antagonists are not predicted to work in younger patients with tumors harboring mutations in downstream SHH pathway genes. Novel, targeted therapies that have the potential to reduce toxicity and improve survival are urgently needed. Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Our previous studies have identified novel roles for the CD271/p75 neurotrophin receptor and the MEK/ERK signaling pathway in contributing to SHH medulloblastoma growth and tumor progression. CD271+ cells exhibit up-regulated MEK/ERK signaling and targeting this population with the MEK inhibitor selumetinib reduced endogenous CD271 levels, stem/progenitor cell proliferation, survival and migration in vitro as well as tumor growth in vivo. Together, our data provided the first evidence that the MEK/ERK pathway is a therapeutic target in human SHH medulloblastoma. The “best in class” MEK1/2 inhibitor trametinib is blood brain barrier penetrant and is currently undergoing clinical testing in other pediatric brain tumours. However, trametinib has not been assessed in SHH medulloblastoma. A major objective of my MSc project is to evaluate the effect of trametinib on SHH medulloblastoma cell properties both in vitro and in vivo. My preliminary data demonstrates that trametinib treatment has a more significant effect on MEK inhibition as well as tumorsphere size, stem/progenitor cell proliferation and survival in vitro. These effects are observed in the lower nM range relative to the uM range for selumetinib. Our new findings reveal novel roles for trametinib in targeting CD271+ SHH medulloblastoma cells, and warrant further investigation of this potent MEK1/2 inhibitor in our preclinical animal models.

“It’s A Big Difference between Having an Opinion on something and Actively Doing It:” Physician and Nurse Practitioner Non-Participation in Medical Assistance in Dying

Janine Brown1

1University of Saskatchewan, College of Medicine Health Sciences Program; University of Regina, Faculty of Nursing

Introduction: There are numerous considerations and challenges when developing safe and sustainable Medical Assistance in Dying (MAID) programs in Canada within Bill C-14’s parameters. Healthcare practitioners (HCPs) who are willing to participate in the formal MAID processes of patient assessment and MAID provision are pivotal to ensuring patients' access to MAID care. Few HCPs participate in MAID assessments and provisions, and this research explored the factors that influenced this.

Methods: Using an interpretive description methodology and considering Social Contract Theory and the Ruggiero Approach to Moral Dilemmas and Decision-Making, 35 physicians and nurse practitioners who identified as non-participators in formal MAID processes were interviewed. Data included participant interviews, field notes, and reflective content. Demographic and contextual data was also collected to account for the personal and practice context of the participant. With the support of NVivo12, the data was analyzed using reflexive thematic analysis. Initial patterns of meaning were returned to participants for member checking before interpretation and theming and an expert panel analysis check.

Results: Broad representation in the demographic and contextual data was noted. As HCPs consider the consequences of participation and their moral ideas and obligations in a changing social contract, different non-participation thresholds were identified. These thresholds were influenced by numerous endogenous (originating within the HCP) and exogenous (originating external to the HCP) factors. This culminated in the Model of Factors Influencing Non-Participation in Formal MAID process. The model illustrates the complex, fluid, and interrelated factors that contemporaneously influenced HCPs’ reconciliation of the endogenous factors and the intentional contemplation of the exogenous factors. As these factors interact and HCP’s personal and professional circumstances change, the individual participation thresholds may also evolve.

Conclusion: Considering HCPs within their personal, patient, practice, and community contexts are vital to understanding non-participation in ethically complex care. Non-participation MAID includes both conscientious objection to MAID and non-participation in MAID for reasons other than conscience. The resultant practice-focused suggestions will support HCPs as they build safe and satisfying practices within their moral space while supporting the social contract of care by facilitating patient’s access to legally available EOL care.

Mechano-arrhythmogenicity is Enhanced in Late Repolarisation during Ischemia and Driven by a TRPA1-, Calcium-, and Reactive Oxygen Species-dependent Mechanism

Breanne A. Cameron1, T. Alexander Quinn1,2

1Department of Physiology & Biophysics, Dalhousie University, Halifax, NS 2Department of Biomedical Engineering, Dalhousie University, Halifax, NS

Introduction: Cardiac dyskinesis during ischemia results in arrhythmias via mechanically-induced changes in electrophysiology (‘mechano-arrhythmogenicity’). While cellular mechanisms of mechano-arrhythmogenicity are unknown, ischemic alterations in voltage-Ca2+ dynamics may create a vulnerable period (VP) for mechanoarrhythmogenicity during late repolarisation.

Methods: Rabbit LV myocytes were paced at 1Hz and rapidly stretched (10-18% increase in sarcomere length over ~110ms) during diastole or the VP in control (CTL) or simulated ischemic (SI) conditions. Drugs were used to buffer Ca2+ (BAPTA), stabilise ryanodine receptors (dantrolene), block mechano-sensitive TRPA1 (HC-030031) or KATP channels (glibenclamide), or to chelate (NAC), block (DPI), or increase (fluorophore photoexcitation) reactive oxygen species (ROS) production. Voltage-Ca2+ dynamics were simultaneously monitored by dual fluorescence imaging with a single camera-optical splitter system to assess the VP (= Ca2+ transient - action potential duration). Diastolic Ca2+ was measured with ratiometric imaging.

Results: The VP was longer in SI than CTL (146±7 vs 54±8ms; n=50 cells, N=6 rabbits; p<0.0001). Mechanoarrhythmogenicity during the VP was greater in SI compared to CTL (7 vs 1% of stretches induced arrhythmias; n=50, N=6; p<0.005) but was similar in diastole. Arrhythmias during the VP were more complex than in diastole (100 vs 69% had sustained activity; n=50, N=6; p<0.05). In the VP, arrhythmia incidence was reduced by BAPTA (2%; p<0.05), HC-030031 (1%; p<0.005), NAC (1%; p<0.005), or DPI (2%; p<0.05), while dantrolene had no effect. Fluorophore photoexcitation caused an increase during both the VP and diastole (29 and 14%, n=42, N=4; p<0.05). Glibenclamide reduced the size of the VP (109±6ms; p<0.0001), with an associated decrease in arrhythmia incidence (2%, n=50, N=6; p<0.05). SI increased diastolic Ca2+ (9±1%, n=25, N=5; p<0.0001), which was not prevented by NAC or HC-030031.

Conclusion: Mechano-arrhythmogenicity in ischemia is enhanced during the VP and involves TRPA1, Ca2+, and ROS, representing potential anti-arrhythmic targets.

Rehabilitation dogs for walking and balance training in children living with cerebral palsy: a pilot study

Valerie Caron1, Alison Oates1, Sarah Donkers1

1University of Saskatchewan

Introduction: Cerebral Palsy (CP) limits gross motor function in children and directly impacts their balance and walking ability. Animal-assisted interventions (AAIs) are a unique approach to combining animals with therapeutic initiatives. Dog- assisted rehabilitation increases psychosocial wellbeing, walking speed, and community ambulation. However, there is a lack of research exploring the use of a highly-trained rehabilitation dog paired with walking and balance training for children with CP. This research aims to co-create, pilot, and evaluate an AAI for walking and balance in children with CP.

Methods: This study is a 3-phase community-engaged mixed methods project. Phase 1 Intervention design: identifying critical components through literature search and stakeholder engagement, guiding the intervention design. Phase 2 Intervention pilot: a wait-control design will be used. Following baseline, participants wait 2 months before receiving the intervention allowing children to act as their own control. This accounts for the high intra-individual variability of gait and CP severity in this population. Phase 3 Intervention evaluation: qualitative methods will be used to explore enjoyment, motivation, and perceived benefit from both child and parent perspectives.

Results: A systematic review (Phase 1) to explore how walking and balance interventions for children with CP target the various domains of the postural control system as outlined by the Systems Framework for Postural Control was completed Jan 2021. Results highlighted the need to include cognitive processing, verticality, and reactive postural control within our intervention design. Stakeholder engagement further emphasizes the need to tailor interventions based on individual needs, goals, and the clinically-determined postural control target areas. We are currently in Phase 2, but are on hold due to COVID restrictions. Phase 2 will report on the effect of the AAI on neuromuscular and temporal-spatial parameters (EMG and kinematic data analyses). Phase 3 will theme participants’ perceived experiences to identify meaningful and impactful characteristics of the AAI.

Conclusion: This project represents the logical development of a complex AAI to address an unmet need. Combining quantitative and qualitative approaches and engaging stakeholders in the development will support a comprehensive exploration and help identify the critical components for a subsequent full scale efficacy trial.

The impact of early childhood traumatic brain injury on language outcomes

Carly A. Cermak1,2, Shannon E. Scratch1,2,4, Nick P. Reed1,2,5, Deryk S. Beal1,2,3

1Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, Ontario, Canada 2Rehabilitation Sciences Institute, University of Toronto, Toronto, Ontario, Canada 3Department of Speech-Language Pathology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada 4Department of Pediatrics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada 5Department of Occupational Science and Occupational Therapy, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada

Introduction: A traumatic brain injury (TBI) occurring in early childhood is detrimental to development. Despite this vulnerability imposed by early childhood TBI, little is known about language outcomes after injury. Understanding language outcomes across all stages of recovery is an essential first step towards improving TBI management, which ultimately impacts later scholastic and social success.

Methods: Four separate but related studies were completed: a retrospective chart review, a scoping literature review, and two systematic reviews with meta-analyses. Effect sizes were used to examine the magnitude of difference in language performance between children with TBI in comparison to control participants or normative data (in non-controlled studies).

Results: Effect sizes were medium in receptive language and small in expressive language in the short-term stage of early childhood TBI; impact of injury on expressive language may be an underestimate due to sample size discrepancies between language areas. Impairments in language tasks requiring verbal responses were evident in the long-term stage of early childhood TBI, with the largest effect sizes in severe TBI.

Conclusion: Language areas impacted in the short-term stage of early childhood TBI may continue to show persistent impairment in the long-term stage of early childhood TBI. These findings provide insight into TBI management, particularly as it relates to individualizing rehabilitation shortly after injury. Investigation into the vulnerability of expressive language tasks is warranted, particularly exploration of neural correlates in relation to performance on verbal tasks.

Focal Point of the Pandemic: Investigating the Equity Dimensions of Long-Term Care Policymaking during the First Wave of COVID-19 in Canada

Elias Chaccour1, Hamza Al-Shammaa1, Sean A. Hillier1

1Faculty of Health, York University

Introduction: The COVID-19 pandemic laid bare to the Canadian public the well-documented social, economic, and health inequities which residents and workers of LTC homes face. However, given the widespread issues documented through the pandemic response, it is clear policymakers failed to account for inequities while developing their policy response to LTC outbreaks. This study analyzes whether the pan-Canadian governmental policy response to the COVID-19 outbreaks in LTC homes accounted for health equity-related negative impacts on LTC residents.

Methods: Data was collected through comprehensive searches of policy announcements and media related to COVID-19 and long-term care using the following two electronic databases: Factiva and Google for an advanced search of federal and provincial government websites. The results were thematically analyzed to produce five policy streams: government administrative actions, guidelines, restriction on visitations, staffing regulation and government investment. An assessment of these policies’ equity implications on LTC residents was conducted using an adaptation of the Health Equity Impact Assessment tool (HEIA).

Results: We identified three major health equity issues that affect LTC residents: unique health and service needs, socioeconomic disparities, and limited socio-cultural connections. The HEIA analysis showed that LTC policies did not account for the negative impact of health inequities and exposed the uneven impact of the pandemic on residents of LTC facilities, which affected their safety, health, and access to services and resources. We conclude that this rushed policymaking process is intended to patch-up the downfalls of decades of systematic underfunding and under-regulation of LTC and cannot fully account for LTC residents’ equity needs.

Conclusion: This crisis shows the need for addressing inequities that affect LTC residents and the need for Canadian health policymakers to adopt a people-centred equity-based approach. We have recommended mitigation approaches to adjust COVID-19 responses in ways that mitigate the inequity-related negative impacts of pandemic policies on LTC residents.

Sympathetic nervous system and integration from V3 neurons in spinal cord injury

Camila Chacon1, Dr. Jeremy Chopek1

1University of Manitoba

There are roughly 85,556 people living with spinal cord injury (SCI) in Canada faced with many sedentary-related health complications in addition to paralysis. After injury, descending supraspinal fibres are severed, and spinal motoneurons and interneurons' synaptic connections are disrupted, resulting in lost sensory-motor function and a high rate of incidence in experiencing autonomic dysfunction. The leading cause of mortality and morbidity in individuals living with SCI is the secondary complication of cardiovascular dysfunction. The sympathetic nervous system is compromised in SCI because the lesion eliminates descending input from the from the autonomic command centre in the brainstem to sympathetic preganglionic neurons (SPNs) in the spinal cord. SPNs provide sympathetic outflow to the periphery and are found in intermediate lamina and central autonomic nuclei of T1-L2 spinal cord. SPN innervation at T1-T5 levels reaches the heart and regulates sympathetic activation. Epidural stimulation has been found to restore voluntary movements below injury site after SCI and can also improve autonomic function even though electrodes are placed over the lumbar cord and far from thoracic neurons innervating the heart. Why and how this occurs is unknown. We hypothesize that networks of neurons in lumbar spinal cord termed central pattern generators (CPGs) that are responsible for rhythmic locomotion also communicate with thoracic SPNs during movements to ensure metabolic support via long ascending propriospinal neurons. Recently, we have identified a potential target propriospinal interneuron based on their ability to relay ascending and descending motor commands within the spinal cord and their rhythmic activation during locomotion. We have also demonstrated synaptic contacts from these interneurons in lumbar regions make synaptic contacts directly on SPNs throughout the thoracic spinal cord. We are currently investigating the complexity of these synaptic contacts by examining the distribution of synaptic contacts on SPNs in the entire thoracic spinal cord and from what level of the lumbar spinal cord these contacts arise from. This knowledge will provide novel insight into their pattern of input onto thoracic SPNs, and optimal placement of epidural stimulators to activate lumbar motor networks and sympathetic neurons via propriospinal pathways to ameliorate SCI individuals' autonomic function.

Developing an aptamer-base tool targeting SARS-CoV-2 S protein for COVID-19 diagnostics and therapeutics use

Rose Chan1, Darwyn Kobasa2

1Department of Medical Microbiology, University of Manitoba, Winnipeg 2Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays are technologically demanding and time consuming. Development of aptamers, nucleic acid based biomolecules that specifically bind to viral protein targets, could be an approach that is more rapid than other options like monoclonal antibodies and RT- qPCR, increasing the flexibility to respond to the current and future pandemics. The objective of this study is to design aptamer candidates that binds to SARS-CoV-2 spike (S) protein, the viral surface receptor protein, which makes it a good target for diagnostic assays. The viral S protein that we will produce via a baculovirus AB vector system will be used to screen and select suitable candidates from the aptamer library that consists of up to 1015 randomized nucleic acid sequences that are 30 to 50 nucleotides long. Selection of S specific aptamers will be performed by successive cycles of systematic evolution of ligands by exponential enrichment (SELEX) resulting in aptamer candidates with high affinity to the target. The candidates will be analyzed via site-directed mutagenesis to determine the binding motif of the aptamers and binding affinity will be measured. Further optimization such as truncation and chemical modification will be applied to increase the candidate’s synthesis efficiency, binding affinity, stability, and resistance against environmental stress and nuclease degradation. In the future, colorimetric reporter add-ons can further enhance its diagnostic potential to meet the needs for field-deployable testing. The COVID-19 pandemic has highlighted the need for developing both diagnostic and therapeutic tools that can be used to rapidly respond to emerging diseases. To explore the therapeutic potential of the aptamer candidates, they will also be tested in vitro against a recombinant vesicular stomatitis pseudovirus (VSV) expressing SARS-CoV-2 S protein to examine its antiviral activity by their ability to neutralize viral infectivity. Since there is still no approved antiviral therapeutics for the disease, we proposed that the aptamer candidates could fill both diagnostic and therapeutic gaps. The aim of the study is to ultimately develop aptamer-based tools that have high antiviral potential and high sensitivity and specificity to detect SARS-CoV-2 in in vitro, and clinical samples.

Regulation of GLS1 Expression by Scleraxis in Cardiac Fibroblasts

Sikta Chattopadhyaya1,2, Raghu S. Nagalingam1,2, Pavit Narhan1, D. Allison Ledingham1, Michael P. Czubryt1,2

1Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre 2Department of Physiology and Pathophysiology, Rady Faculty of Health Sciences, University of Manitoba

Cardiac fibrosis occurs due to enhanced production of extracellular matrix proteins, particularly fibrillar collagens, impairing cardiac function and leading to heart failure and death. Fibrosis is a very high energy intensive process, yet it is unclear how the metabolic requirements are met. Glutaminolysis, which involves conversion of glutamine to glutamate by glutaminase (GLS), and conversion of glutamate to alpha-ketoglutarate to fuel oxidative metabolism via the tricarboxylic acid cycle, has been reported as a mechanism for meeting the energy demand in pulmonary and liver fibrosis, however its contribution in cardiac fibrosis has not been elucidated. Although the role of transcription factor Scleraxis has been established in the phenotypic conversion of cardiac fibroblasts to fibrotic myofibroblasts by our lab, Scleraxis’ role in glutaminolysis remains unelucidated. Our results here show Scleraxis expression was increased by 5 fold and GLS1 by 4 fold when comparing freshly isolated rat cardiac fibroblasts to fibrogenic myofibroblasts. While Scleraxis overexpression increased GLS1 expression 20- fold, its knockout in cardiac fibroblasts attenuated GLS1 expression by 90%. TGF-beta, one of the primary inducers of cardiac fibrosis, induces expression of Scleraxis. TGF-beta function is highly dependent on Scleraxis for many of its downstream effects. TGF-beta treatment of wild type mouse activated cardiac fibroblasts doubled the expression of GLS1, but failed to induce GLS1 expression in Scleraxis knockout cells. Via luciferase assay we found that there is a dose-dependent transactivation of the human GLS1 promoter by Scleraxis in NIH3T3 fibroblasts. While Scleraxis transactivated the hGLS1 promoter by about 2-fold, mutation of an E-Box Scleraxis putative binding site caused 70% attenuation of promoter transactivation. These findings suggest that Scleraxis is sufficient and required to regulate GLS1 expression to facilitate increased energy metabolism during cardiac fibroblast to myofibroblast conversion.

Genome-Wide Analyses Highlight the Role of Dyslipidemia, Inflammation, Calcification, and Adiposity in Aortic Stenosis

Hao Yu Chen1,2, Mark Lathrop3,4, James C. Engert1,2,4, and George Thanassoulis1,2, MD on behalf of the Therapeutic targets for AoRtic stenosis using GEneTics (TARGET) Consortium

1Division of Experimental Medicine, McGill University, Montreal, Canada 2Preventive and Genomic Cardiology, McGill University Health Centre and Research Institute, Montreal, Canada 3McGill University and Genome Quebec Innovation Centre, Montreal, Canada 4Department of Human Genetics, McGill University, Montreal, Canada

Introduction: No medical treatment is approved for aortic stenosis (AS), the leading form of incident valvular heart disease. Identifying genes that cause AS would further the development of new medical therapies.

Methods: In 10 European-ancestry cohorts totalling 653,867 participants (13,765 cases), we performed a genome-wide meta-analysis for AS (11,591,806 variants). We constructed a weighted polygenic risk score (PRS) using independent, genome-wide significant (p≤5×10-8) variants from the meta-analysis and assessed its association with AS among 257,231 White British participants of the UK Biobank (2,213 cases). To identify cross-phenotype associations, we examined 58 biomarkers, physiological measurements, and diseases, and performed Mendelian randomization for six modifiable traits associated with several variants. We also conducted gene- and expression-based analyses to identify additional genes.

Results: Eighteen independent variants at 16 loci were associated with AS at genome-wide significance; among these were 10 variants at 9 loci, including CELSR2-SORT1, HMGB1, and ARHGEF26, not previously reported for AS at genome-wide significance. The PRS was strongly associated with AS (odds ratio per SD, 1.38; 95% CI, 1.33 to 1.44; p=4.6×10-57) and improved the discriminative power for AS of a model containing cardiovascular risk factors (area under the curve, 0.71; 95% CI, 0.70 to 0.72; pdifference=2.0×10-11). In the phenome-wide association study, we observed 98 significant associations, including for height (8 variants); triglycerides, albumin, and coronary artery disease (6 variants each); and apolipoprotein B and C-reactive protein (5 variants each). Mendelian randomization indicated genetically-elevated levels of low-density lipoprotein cholesterol, apolipoprotein B, lipoprotein(a), and body mass index causally increased the odds of AS. Whole gene analysis identified 95 gene regions associated with AS, including LDLR (p=2.3×10-10). Expression-based analysis predicted differential expression of 42 genes in AS cases in at least 1 of 4 tissues examined, including increased IL6R expression in the blood (p=3.1×10-6) and increased LPA expression in the liver (p=1.5×10-7). We also predicted differential expression of co-regulated genes, including expression of miR-219, miR-491, miR-19a/b and miR-21 gene targets.

Conclusion: We identified novel genetic contributors which confirmed the involvement of calcification, altered lipid metabolism, adiposity, and inflammation in AS. Their potential as therapeutic targets warrants further investigation.

Altered central and peripheral glutathione in Alzheimer Disease and Mild Cognitive Impairment: a meta-analysis

Jinghan Jenny Chen1,2, Mathura Thiyagarajah1,2, Jianmeng Song3, Nathan Herrmann2, Krista Lanctôt1,2

1Department of Pharmacology, University of Toronto 2Sunnybrook Research Institute 3Institute of Medical Science, University of Toronto

Background: Increasing evidence implicates oxidative stress (OS) in Alzheimer Disease (AD) and Mild Cognitive Impairment (MCI). Depletion of the brain antioxidant glutathione (GSH) may be important in OS-mediated neurodegeneration, though post-mortem brain GSH changes in AD have been inconclusive. Recent in vivo measurements of brain and blood GSH may shed light on GSH changes earlier in the disease.

Aim: To quantitatively review in vivo GSH in AD and MCI compared to healthy controls (HC) using meta-analyses.

Method: Studies published before June 2020 with in vivo measurements of brain or blood GSH in MCI or AD with a HC group were identified using Medline, PsychInfo, and Embase. Standardized mean differences (SMD) and 95% confidence intervals (CI) were calculated for outcomes using random effects models. Outcome measures included brain GSH (Meshcher-Garwood Point Resolved Spectroscopy (MEGA-PRESS) versus non-MEGA-PRESS) in AD and MCI, and blood GSH (intracellular versus extracellular) in AD and MCI. The Q statistic and Egger’s test were used to assess heterogeneity and risk of publication bias respectively.

Results: For brain GSH, 4 AD (AD=135, HC=223) and 4 MCI (MCI=213, HC=211) studies were included. For blood GSH, 25 AD (ADD=1050, HCl=1031) and 7 MCI (MCI=434, HC=408) studies were included. Brain GSH did not differ in AD or MCI compared to HC; however, the subgroup of studies using MEGA-PRESS reported lower brain GSH in AD (SMD [95%CI] -1.45 [-1.83, -1.06], p<0.001) and MCI (-1.15 [-1.71, -0.59], z=4.0, p<0.001). Blood GSH was lower in AD (-1.10 [-1.58, -0.62], z=4.46, p<0.001). In a subgroup analysis, intracellular GSH was lower in MCI (-0.66 [-1.11, -0.21], p=0.025). Significant heterogeneity was observed in all analyses and supported the use of random effect models. Egger’s test indicated risk of publication bias in MCI brain GSH literature.

Conclusion: In vivo measures of GSH in AD and MCI had significant heterogeneity. Blood analyses suggested intracellular GSH decreases may be prominent in early disease stages with both intra- and extracellular decreases in later stages. Brain GSH may be decreased in AD and MCI but heterogeneity and potential bias indicate the need for measurement standardization and replication.

Rare Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

*Owen J. Chen1,2, Ester Castellsagué3,4,5, Mohamed Moustafa-Kamal1,2, Javad Nadaf6, Barbara Rivera3,4, Somayyeh Fahiminiya7, Yilin Wang1, 2, Caterina Pacifico1, Isabelle Gamache1, Lai Jiang8,9, Jian Carrot-Zhang3,7, Leora Witkowski3,4, Albert M. Berghuis2,10, Stefan Schoenberger11, Dominik Schneider12, Susanne Bens13, Reiner Siebert13, Colin J. R. Stewart14, Ziguo Zhang15, William C. Chao16, Celia M.T. Greenwood8,9,17, David Barford15, Marc Tischkowitz18, Jacek Majewski3,7, , William D. Foulkes3,4,20,21, Jose G. Teodoro1,2,19.

1Goodman Cancer Research Centre, McGill University, Montréal, Québec, Canada 2Department of Biochemistry, McGill University, Montréal, Québec, Canada 3Department of Human Genetics, McGill University, Montréal, Québec, Canada 4Department of Medical Genetics, The Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montréal, Québec, 5Translational Research Laboratory, Catalan Institute of Oncology, Bellvitge Institute for Biomedical Research, L’Hospitalet de Llobregat, Barcelona, Spain 6McGill University and Génome Québec Innovation Centre, Montréal, Québec, 7Research Institute of the McGill University Health Centre, Montréal, Québec 8 Lady Davis Institute, Jewish General Hospital, Montréal, Québec 9Department of Epidemiology, Biostatistics & Occupational Health, McGill University, Montréal, Québec 10Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, Québec 11Pediatric Hematology and Oncology, Children's Hospital, University of Bonn, Bonn, Germany 12Clinic of Pediatrics, Municipal Hospital Dortmund, Dortmund, Germany 13Institute of Human Genetics, University of Ulm & Ulm University Hospital, Ulm, Germany 14Department of Histopathology, King Edward Memorial Hospital, and School for Women’s and Infants’ Health, University of Western Australia, Perth, Australia 15Institute of Cancer Research, London, United Kingdom 16Faculty of Health Sciences, University of Macau, Macau SAR, China 17Departments of Oncology and Human Genetics, McGill University, Montréal, Québec 18Department of Medical Genetics, University of Cambridge, Cambridge, United Kingdom 19Department of Microbiology and Immunology, Montréal, Québec 20Program in Cancer Genetics, Department of Oncology and Human Genetics, McGill University, Montréal, Québec 21Department of Medical Genetics and Cancer Research Program, Research Institute of the McGill University Health Centre, Montréal, Québec

Background: CDC20 is a co-activator protein of the anaphase promoting complex (APC/C), an E3 ubiquitin ligase that targets mitotic substrates for degradation to allow for mitotic transit and cell division. Mitotic progression is regulated by mitotic checkpoint complex (MCC) proteins during the spindle assembly checkpoint (SAC), an important fail-safe that protects cells from aberrant chromosomal segregation. Previous studies have demonstrated that different variants of CDC20 have pathological implications, including aneuploidy and tumorigenesis. We have identified two distinct heterozygous missense mutations in the CDC20 gene from two families with familial ovarian germ cell tumours (fOGCTs). fOGCTs are very rare, suggesting that an underlying genetic link exists in these two families. Here, we characterize these patient CDC20 mutations and propose a mechanism by which these variants compromise the SAC.

Methods: CDC20 mutants were cloned into expression constructs and transfected into HeLa cells. The SAC was engaged in mitosis by the addition of the drug nocodazole to cells. Mitotic cells were harvested for co-immunoprecipitation (co-IP) experiments to assess for MCC protein interaction changes. HeLa cells and patient-derived skin fibroblasts were subjected to real-time microscopy to assess for differences in mitotic progression when the SAC is engaged. Finally, we derived a mouse model carrying a patient CDC20 mutation for further characterization.

Results: Co-IP experiments showed reduced mutant CDC20 interaction with BUBR1 (an MCC protein) during mitosis. Real- time microscopy showed that cells expressing the CDC20 mutant constructs underwent more mitotic slippage when the SAC was engaged compared to the WT. Reduced mitotic transit time was also observed in similar miscroscopy experiments performed on patient skin fibroblasts and mouse embryonic fibroblasts derived from our mouse model. In our mouse model, we observed that the Cdc20 variant cooperates with the E mu-myc onco-transgene in the lymphomagenesis in mice.

Conclusions: Our studies of the two CDC20 mutations found in fOGCT patients showed that these variants compromise the SAC via impaired interaction with the BUBR1 protein in the MCC. Our mouse model indeed suggested an oncogenic predisposition role in CDC20. Altogether, our data demonstrate a likely link between these CDC20 variants and tumorigenesis in our fOGCT patients.

Using an implantable optical sensor to monitor spinal cord oxygenation and hemodynamics after acute spinal cord injury

Amanda Cheung1, Lorna Tu1, Neda Manouchehri1, Kyoung-Tae Kim1,2, Kitty So1, Megan Webster1, Shera Fisk1, Seth Tigchelaar1, Sara S. Dalkilic1, Eric C. Sayre1, Femke Streijger1, Andrew Macnab3, Brian K. Kwon1,4, Babak Shadgan1,4

1International Collaboration on Repair Discoveries, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada 2Department of Neurosurgery, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, South Korea 3Department of Pediatrics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada 4Department of Orthopaedics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

Introduction: Current clinical guidelines for the treatment of acute spinal cord injury (SCI) patients recommend that mean arterial pressure (MAP) be augmented for 7 days post-injury to increase spinal cord perfusion and potentially improve neurologic function. However, it is difficult for clinicians to hemodynamically manage acute SCI patients without real-time physiologic information about the effect of MAP augmentation within the injured cord. In this study, we investigated the feasibility of a customized optical sensor, based on near-infrared spectroscopy (NIRS), to non-invasively monitor spinal cord oxygenation and hemodynamics during the first 7 days post-injury in a porcine model of acute SCI.

Methods: Six Yucatan mini-pigs received a weight-drop T10 contusion-compression injury. A multiwavelength NIRS system with a custom-made optical sensor was placed directly onto the dura at T9. Using NIRS, the spinal cord tissue oxygenation (Hbdiff) and concentrations of oxygenated (O2Hb), deoxygenated, and total hemoglobin (THb) were monitored before and after SCI. To validate the NIRS measures, an invasive intraparenchymal (IP) combined PO2/blood flow (SCBF) sensor was inserted into the spinal cord adjacent to the NIRS sensor at T11. Episodes of MAP alterations and hypoxia were performed acutely after injury, 2 days, and 7 days post-injury to simulate the hemodynamic changes SCI patients experience postinjury.

Results: The NIRS sensor demonstrated the ability to maintain physiologic measurements over the 7-day post-SCI period. Non-invasive NIRS monitoring identified changes in spinal cord hemodynamics and oxygenation levels during the MAP alterations and hypoxia. Changes of THb followed similar patterns of perfusion changes measured by the IP SCBF sensor, and changes of Hbdiff and O2Hb showed significant correlations with oxygenation changes measured by IP PO2 (p<0.0001).

Conclusion: Our novel NIRS sensor is feasible as a non-invasive technique to monitor real-time changes in spinal cord oxygenation and hemodynamics 7 days post-injury. Further refinement of the NIRS sensor would allow a clinically applicable device spine surgeons could place on the dura at the time of surgical decompression to monitor spinal cord tissue hemodynamics post-injury.

Isolated Lymphoid Follicles are a Microbiota-Regulated Niche for Intestinal Macrophages

Pailin Chiaranunt, Kyle Burrows, Louis Ngai, Eric Cao, Elen Liang, Anthony Wong, Abdul Momen, Slava Epelman, Arthur Mortha

Department of Immunology, University of Toronto, Toronto, ON, Canada Toronto General Hospital Research Institute, University Health Network (UHN), Toronto, ON, Canada

Introduction: Gut-resident macrophages (MPs), comprising of either fetal precursor-derived or adult monocyte-derived subsets, are critical in mounting anti-microbial host defense along the gastrointestinal tract. Colony stimulating factor 2 (CSF2), a myeloid growth factor abundantly secreted by tissue-resident innate lymphoid cells (ILC3s), regulates the development of distinct MP subsets to confer innate intestinal immunity in a microbiota-dependent manner. Recent data implicate both bacterial and non-bacterial (e.g. the protist Tritrichomonas musculis) members of the microbiota as drivers of ILC3 CSF2 production. Here, we examine the interplay between microbial signals, such as adenosine triphosphate (ATP), and ILC3-derived CSF2 within microanatomic niches of the gut in shaping MP heterogeneity to confer innate immunity against enteric pathogens.

Methods: The microanatomic localization of intestinal MP populations was determined using a combination of immunofluorescence imaging and microsurgical techniques. To determine the role of ILC3s and CSF2 on MP development, we performed adoptive transfers of purified ILC3s, either proficient or deficient in CSF2 production, into lymphopenic mice. Luminal ATP concentrations and intestinal MP turnover rates were assessed in mice constituted with varying microbiota compositions. Wild-type and CSF2-deficient mice were infected with the enteric pathogen Citrobacter rodentium to determine the impact of the microbiota-ATP-ILC3-CSF2-MP axis on inducing host defense.

Results: Immunofluorescence imaging and quantification of MPs across the colon revealed isolated lymphoid follicles (ILFs), ILC3-rich lymphoid aggregates that develop postnatally, as a microanatomic niche for CSF2-dependent MP development. CSF2-producing ILC3s were sufficient to restore the impaired monocyte-derived MP development in lymphopenic mice. Mice with more diversified microbiota contained higher levels of luminal ATP and exhibited increased replacement of MPs by monocytes, correlating with increased ILC3 CSF2 production. CSF2-deficient mice had reduced monocyte-derived MPs and displayed greater susceptibility to C. rodentium infection.

Conclusion: Our data demonstrate that microbiota-derived ATP activates CSF2 production in ILF-residing ILC3s to shape MP heterogeneity by inducing monocyte-derived MP development. Disruption of this microbiota-ILC3-MP axis results in an imbalanced MP composition and impaired host defense against enteric infections. Altogether, our data emphasize the importance of a diverse microbiota in driving a location-specific innate immune cell network that is critical for the maintenance of anti-bacterial host defense.

Population pharmacokinetic model-informed dosing of vancomycin in neonates

Erin Chung1,2, Winnie Seto1,2,3,4

1Department of Pharmacy, The Hospital for Sick Children, Toronto, ON, Canada 2Graduate Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada 3Child Health Evaluative Services, The SickKids Research Institute, Toronto, ON, Canada 4Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, ON, Canada

Background: Neonatal sepsis is an invasive bloodstream infection occurring in newborns, commonly caused by coagulase- negative staphylococci (CoNS) in the neonatal intensive care unit (NICU). Vancomycin is the drug of choice to treat CoNS sepsis, yet dosing remains a challenge in neonates due to significant pharmacokinetic variability. Therefore, we aimed to develop a population pharmacokinetic (popPK) model for vancomycin in neonates in order to derive more optimal initial vancomycin dosing to improve target attainment.

Methods: This was a single-centre, retrospective study that included neonates admitted to NICU receiving intravenous vancomycin. PopPK analyses were conducted using nonlinear mixed effects model (NONMEM). The final popPK model was then used to simulate dosing using Monte Carlo simulations. Therapeutic ranges used in the analyses included area-under- the-concentration-time-curve of 400-600 mg*h/L or trough concentration of 10-15 mg/L for central nervous system (CNS) infections or 5-12 mg/L for other infections.

Results: Out of 201 vancomycin courses, current initial vancomycin dosing resulted in 67-80% of vancomycin trough concentrations outside target range for CNS infections (10-15 mg/L) or 29-43% outside target range for other infections (5- 12 mg/L). A one-compartment model best described the observed data from 181 neonates with 296 detectable vancomycin concentrations. The mean clearance was 0.11±0.03 L/kg/h and volume of distribution (V) was 1.02±0.08 L/kg. Bodyweight, postmenstrual age (PMA), and serum creatinine (SCr) were significant covariates associated with clearance (p<0.001) and bodyweight was a significant covariate associated with V (p=0.009). Based on simulations with the popPK model, a new dosing guideline for vancomycin in neonates was derived with stratification by PMA and SCr, which improved probability of target attainment.

Conclusion: Neonates are at risk of not achieving target vancomycin concentrations, which increase their risk of ineffective therapy or nephrotoxicity. We suggest implementing a popPK model-informed vancomycin dosing guideline in neonates with therapeutic drug monitoring to improve target attainment.

Impact of early antiretroviral therapy on tissue resident myeloid cells in the liver and lung of SIV- infected rhesus macaques

Julien Clain1, Henintsoa Rabezanahary1, Gina Racine1, Ghita Benmadid-Laktout1, Ouafa Zghidi-Abouzid1, Jérôme Estaquier1

1Centre de Recherche du CHU de Québec, Université Laval, Québec, Qc, Canada.

Introduction: Whereas we demonstrated that early antiretroviral therapy (ART) efficiently prevents infection of monocytes in the blood, spleen and intestine of SIVtreated rhesus macaques (RMs) [1], little is known so far about the role of TRMs, and whether these cells may represent VRs in SIV-infected RMs after early ART.

Methods: RMs were infected with SIVmac251 and treated at day 4 with a cocktail of antiretroviral drugs. Cells from liver and lung were mechanically isolated. The phenotype of TRMs was analyzed by flow cytometry using specific antibodies including antibodies against CD14, CD16, CD44, TIM-4, CD117, CD206, MERKT, and LYVE (these markers were previously defined in mice). The levels of viral DNA and RNA were quantified by qPCR for each tissue. In situ hybridization was used to detect vRNA in tissues.

Results: Our results revealed that myeloid cells from liver and lung of SIV-infected RMs expressed mostly CD44, CD117, CD206 and LYVE markers, but represent a small proportion of liver and lung cells. Concomitantly, our data revealed that liver and lung of non-treated SIV-infected RMs both contain viral RNA and DNA that are positively correlated with the viremia. Furthermore, treated-RMs have no viral RNA and DNA both in the liver and lung.

Conclusion: Herein, we characterized the phenotypes of long-lived TRMs that colonize lung and liver of SIV-infected RMs. We also showed that early ART efficiently prevents early viral seeding both in the liver and lung. These results highlight the crucial importance of early treatment by decreasing anatomical VRs.

Providing Crisis Care in a Pandemic – A Virtual-based Crisis Stabilization Unit

Avery P. Clavio1, Katrina E. Pullia2, James M. Bolton1

1University of Manitoba 2Winnipeg Regional Health Authority

Introduction: The COVID-19 pandemic has given rise to challenges in the provision of mental health crisis care as public health restrictions have altered how these services are able to be provided.1 In response to the decreased accessibility of in-person mental health care and the potential increase in mental health needs of patients secondary to the pandemic, the Crisis Response Center (CRC) in Winnipeg, Manitoba, has rapidly virtualized the delivery of full-spectrum crisis care to individuals requiring urgent mental health services. The goal of this study was to describe the Virtual Crisis Stabilization Unit (vCSU), present patient demographics and outcomes, and comment on the feasibility and effectiveness of virtual models of care.

Methods: Winnipeg operates a 16-bed stepped-care unit for voluntary patients in crisis that do not require admission to the hospital. The vCSU was developed in March 2020 as an adjunct to the in-person CSU for safety reasons in the COVID- 19 pandemic. Patients could be referred after in-person presentations to the crisis centre/emergency department, or virtually after calling the crisis line. Individuals in the vCSU had access to all of the same resources as those admitted to the in-person CSU unit (access to coping skills-based classes, medication reminders, 24/7 access to crisis support). Patient demographics, presenting complaints, and outcome data were collected through discharge surveys for individuals admitted to the vCSU during a 6-month period.

Results: From March to August 2020 a total of 95 patients were admitted to the vCSU. Of these, thirty-one individuals (32.6%) had their vCSU intake either by phone or videoconference, therefore remaining at home for the entirety of their care. Seventy-six (39.2%) individuals had suicidal/self-harm behaviour at presentation, twenty-one (22.7%) reported depression and anxiety, seven (3.6%) had symptoms of mania or psychosis. Primary reasons for referral were risk/symptom monitoring and problem-solving/recovery planning, both occurring in sixty-nine patients (47.6%). Six individuals (6.6%) required psychiatric involvement; no individuals were admitted to hospital. Eighty-five individuals (90.42%) responded positively or neutrally about their vCSU experience.

Conclusion: A virtual crisis ward is a feasible and effective mechanism for providing access to care and connection to resources for patients experiencing crisis.

Targeted Metabolomic Profiling and Prediction of Cardiovascular Events: A Prospective Study of Patients with Psoriatic Arthritis and Psoriasis

Keith Colaço1-3, Ker-Ai Lee4, Shadi Akhtari1,3, Raz Winer5, Paul Welsh6, Naveed Sattar6, Iain B. McInnes6, Vinod Chandran2,3, Paula Harvey1,3, Richard J. Cook4, Dafna D. Gladman2,3, Vincent Piguet1,3, Lihi Eder1,3

1Women’s College Hospital, Toronto, Canada 2Schroeder Arthritis Institute, University Health Network, Toronto, Canada 3University of Toronto, Toronto, Canada 4University of Waterloo, Waterloo, Canada 5Rambam Medical Center, Haifa, Israel 6University of Glasgow, Glasgow, United Kingdom

Introduction: Psoriasis and psoriatic arthritis, collectively termed psoriatic disease (PsD), are chronic inflammatory diseases associated with increased cardiovascular (CV) risk. Metabolites comprise biomarkers that may add predictive value over traditional CV risk factors. We aimed to identify metabolites associated with CV events (CVEs) and to determine whether they could improve CV risk prediction beyond traditional CV risk factors.

Methods: Patients from a longitudinal PsD cohort without a prior history of CVEs were included. A targeted nuclear magnetic resonance (NMR) metabolomics platform was used to quantify 64 metabolite measures including lipoprotein subclasses, fatty acids and amino acids. The study outcome included CVEs occurring within 10 years of biomarker assessment. The associations of each metabolite with incident CVEs were analyzed separately using multivariate Cox proportional hazards regression models. Variable selection was then performed using the proportional sub-distribution hazards regression model adjusted for age and sex. The added predictive value of the selected metabolites to improve risk prediction beyond traditional CV risk factors was assessed using the area under the receiver operator characteristic curve (AUC).

Results: 977 patients with PsD, followed between 2002 and 2019, were analyzed (mean age 49.1 ± 12.6 years, 45.1% female). During a mean follow-up of 7.1 years, 70 (7.2%) patients developed incident CVEs. In Cox regression models adjusted for CV risk factors, alanine, tyrosine, degree of unsaturation, high-density lipoprotein (HDL) cholesterol, and medium and large HDL particles were significantly associated with decreased CV risk. Glycoprotein acetyls, apolipoprotein B, remnant cholesterol, very low-density lipoprotein (VLDL) cholesterol, and very small VLDL particles were associated with an increased CV risk. Thirteen metabolites were selected based on the algorithm. The age- and sex-adjusted expanded model (base model + 13 metabolites) significantly improved prediction of CVEs beyond the base model (only age and sex) with an AUC of 79.9 vs. 72.6, respectively (p=0.02).

Conclusion: Using NMR metabolomics, we identified a variety of metabolites associated with a lower and higher risk of developing CVEs in patients with PsD. Further study of their underlying association with CVEs is needed to clarify the clinical utility of these biomarkers to guide CV risk assessment in this population.

Diffusion Tensor MRI Reveals Myocardial Fibre Structure post Cell-Based Therapy in Myocardial Infarction

Moses Cook1, Wahiba Dhahri2, Graham A. Wright1,3,4, Michael A. Laflamme2, Nilesh R. Ghugre1,3,4

1Department of Medical Biophysics, University of Toronto 2McEwen Centre for Regenerative Medicine, University Health Network 3Schulich Heart Research Program, Sunnybrook Health Sciences Centre 4Physical Sciences Platform, Sunnybrook Research Institute.

Introduction: Cardiac regenerative therapies using pluripotent stem cell-derived cardiomyocytes (PSC-CM) (the graft) have shown early promise in repairing scar tissue post-myocardial infarction (MI). However, in vivo response, and therapeutic efficacy remain less understood. Magnetic Resonance Imaging (MRI) is non-invasive and the gold standard for assessing cardiac function, structure and viability. Our objective was to employ a 3D high-resolution Diffusion-Tensor (DT)-MRI (a technique capable of quantifying myofibril alignment) protocol to localize and characterize structural changes in the infarct and cellular graft regions after PSC-CM transplantation in an ex vivo guinea pig model of MI.

Methods: Guinea pig hearts were cryoablated to induce infarction and infarct regions were injected either with PSC-CMs (n=4), vehicle (n=2), or healthy control (n=2) on Week 2 post-MI. Hearts were harvested on Week 5 and perfusion fixed. Ex vivo MRI was performed on a 7T Bruker system. We utilized 3D DT-MRI (resolution=0.3x0.3x0.3mm, b-value 700s/mm2, 30 diffusion directions) to quantify myocardial structure parameters. Contrast enhancement (CE, resolution=0.2x0.2x1mm) was used to distinguish infarcted from healthy tissue via accumulation of gadolinium-DTPA injected prior to sacrifice. DT- MRI parameters were measured in infarct, graft and remote myocardium.

Results: PSC-CM engraftment was in agreement between CE-MRI and histology. The mean diffusivity of the graft region was not significantly different than that of the healthy control (1016μm2s-1+/-148 vs. 1009μm2s1+/207 respectively, p=0.41) and was improved compared to infarct (1770μm2s-1+/-208, p<0.0002). This suggests the DT-MRI measures within graft tissue were indicative of greater structural organization compared to the infarcted territory, attributed to the presence of PSC-CM graft.

Conclusion: DT-MRI can help evaluate the degree of structural remodelling after MI and how host-graft structural integration can potentially impact functional recovery. Future studies can incorporate in vivo MRI sequences to perform serial response to therapy.

URI-1 Levels are Elevated in Response to Hepatic Lipid Droplet Formation in Fatty Livers of db/db and C57BL/6 Mice Fed Trans-10,Cis-12 Conjugated Linoleic Acid and in Oleic Acid Treated H4IIE Rat Hepatoma Cells

Luis Cordero-Monroy, Peter Zahradka and Carla G. Taylor

University of Manitoba

Diets containing trans-10,cis-12 conjugated linoleic acid (t10,c12 CLA) cause fat mass loss in mice but also coincide with hepatic lipid accumulation and fatty liver through an unknown mechanism. Unconventional prefoldin RPB5 interactor-1 (URI-1) belongs to the prefoldin family of proteins that have been shown to coordinate nutrient availability by transcriptional regulation of genes involved in glucose and lipid metabolism. We hypothesized that an elevation of hepatic URI-1, induced by t10,c12 CLA, increases fatty acid uptake and accumulation through the modulation of lipid metabolism proteins leading to fatty liver. This study was designed to investigate the association of hepatic URI-1 with lipid metabolism proteins, lipid droplets and triglyceride (TG) accumulation in genetically obese db/db mice and lean C57BL/6 mice fed either a control (CTL) or a t10,c12 CLA (0.4% w/w) diet for 4 weeks. H4IIE rat hepatoma cells treated with 100 μM oleic acid (OA) were used to model hepatic lipid droplet accumulation in relation to URI-1 levels. The t10,c12 CLA diet resulted in increased liver:body weight ratios in lean and db/db mice and a 5-fold increase in hepatic lipid concentration in lean mice. BODIPY staining of liver sections revealed that the t10,c12 CLA diet increased hepatic lipid droplet numbers and size in lean mice. Levels of lipid metabolism proteins, specifically fatty acid binding protein 1, fatty acid translocase, and fatty acid synthase, were elevated with t10,c12 CLA feeding in both genotypes. Importantly, hepatic URI-1 levels were elevated in db/db and lean mice fed t10,c12 CLA, and were strongly correlated with liver:body weight ratios and hepatic TG concentration. Time course studies with H4IIE cells demonstrated that maximal lipid droplet formation and the highest levels of URI-1 occurred 24 hours after OA treatment. These results reveal a potential relationship between hepatic URI-1 levels and hepatic triglyceride accumulation. These results warrant further investigation into the causative role of URI-1 in propagating hepatic lipid droplet formation and fatty liver disease.

Intervention Preference Survey for People with Early Psychosis using Cannabis

Stephanie Coronado-Montoya1,2, Amal Abdel-Baki1,2, José Côté1,2, Simon Dubreucq1,2, Benedikt Fischer3,4, Navdeep Kaur2, Tania Lecomte1, Sophie L’Heureux5, Clairélaine Ouellet-Plamondon1,2, Marc-André Roy5.6, Didier Jutras-Aswad1,2

1Université de Montréal, Montréal, Canada 2Research Centre, Centre Hospitalier de l’Université de Montréal, Montréal, Canada 3Centre for Addiction and Mental Health, Toronto, Canada 4University of Auckland, Auckland, New Zealand 5Laval University, Québec, Canada 6Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Montréal, Canada

Introduction: Cannabis use is higher among people with psychosis than the general population and is often associated with an adverse course for psychotic disorders. Cannabis use in people with psychosis is associated with higher incidence of cannabis use disorder (CUD), poorer psychosocial functioning, more severe psychiatric symptoms, and increased suicidal ideation. The scarce literature available on cannabis interventions for people with psychosis has primarily focused on CUD treatments. A recent review on preventive cannabis interventions for people with psychosis without CUD found only five eligible studies, no intervention emerging as more efficacious. Evidence-based practice guidelines suggest that patient preference should guide intervention selection, even more so when there is no evidence clearly demonstrating one intervention to be superior to others. Additionally, addressing the concepts of preference and acceptability with patients for preventive interventions can lead to better adherence and better patient engagement in interventions. However, patient preferences for cannabis-related interventions, and specifically among young people with psychosis, have been understudied. The aim of the present study is to explore preferences of young adults with first episode psychosis (FEP) who use cannabis in relation to key characteristics of interventions to prevent cannabis-related harms, using a cross-sectional quantitative survey.

Methods / Results: The survey will be administered across Canada to 165 youth with FEP who 1) use cannabis but do not have a CUD or 2) have a CUD but are not seeking to treat their CUD. We will collect information on participants' sociodemographic characteristics and clinical history; we will then ask them to choose between different intervention or booster session scenarios that vary according to a set of characteristics (e.g., duration, frequency, etc.). To do this, we will use Discrete Choice Experiment methodology (DCE). Data analyses of the DCE section will be conducted using three Mixed Rank-ordered Logit models to identify the variables that most strongly predict the participants' choice of interventions.

Conclusion: The findings of the survey will highlight preferred characteristics of cannabis preventive interventions according to a patient perspective, which can inform future development and tailoring of preventive interventions offered to young adults with psychosis who use cannabis.

Non-Lactobacillus dominant vaginal microbiome induces inflammation and epithelial barrier disruption that increase HIV acquisition risk

Marina Costa Fujishima1, Alana Lamont3, Kenzie Birse3, Paul Lopez1, Christina Farr-Zuend3, Alicia Berard3, Max Abou3, Atta Yazdanpanah1, Oluwaseun Ajibola1, Adam Burgener2,3 and Thomas Murooka1,2

1University of Manitoba, Rady Faculty of Health Sciences, Department of Immunology 2University of Manitoba, Department of Medical Microbiology and Infectious Disease 3National HIV and Retrovirology Labs, JC Wilt Infectious Disease Research Centre, Public Health Agency of Canada, Winnipeg, Canada.

Introduction: The mucosal surface of a healthy female genital tract (FGT) is comprised of physicochemical, immunological and microbial components that serve as a rapid, first line of defense against infections. Alterations in any of these components have been associated with higher HIV acquisition risk. Analysis from >700 women from the CAPRISA004 cohort show that women with a non-Lactobacillus dominant vaginal microbiome were at significantly higher risk of sexual HIV acqusition, and that this strongly correlated with vaginal epithelial barrier disruption, inflammation and neutrophil accumulation. However, how FGT barrier function is impacted by changes in the vaginal microbiota, and a mechanistic understanding of mucosal neutrophils in this process, remains unclear. Here, we utilized microscopy and proteomic approaches to better define the interplay between vaginal microbial species, epithelial barrier function and neutrophil activation in vivo.

Methods: Balb/c mice were intravaginally inoculated with either PBS, L. crispatus (CT-1), Mobiluncus mulieris (CT-4) or Gardnerella vaginalis (CT-3/4). Cervicovaginal lavage (CVL) was collected on day 0, 2, 4, and 7 to confirm bacteria colonization through 16S rRNA sequencing and assess protein expression profile through mass spectrometry. Vaginal tissues were collected for immunohistochemical analysis. To determine neutrophils contribution to barrier disruption, barrier integrity was assessed in the presence or absence of neutrophils by intravaginally inoculating mice with lucifer yellow (0.45Da) and harvesting tissues to measure dye penetration into the epithelium by immunohistochemistry.

Results: Immunohistochemistry analysis demonstrate a larger neutrophil influx within the vaginal epithelium and the submucosa of mice inoculated with M. mulieris and G. vaginalis but not L. crispatus. High levels of inflammatory cytokines and neutrophil-related factors were also identified by proteomic analysis of collected CVL samples. Additionally, permeability assay revealed a significant loss of barrier integrity in mice inoculated with M. mulieris and G. vaginalis. However, upon depletion of neutrophils in vivo, vaginal barrier integrity was restored even in the presence of these non- optimal bacteria.

Conclusion: Our study demonstrates that the presence of non-optimal bacteria species in the FGT results in genital inflammation, high neutrophil activation and increases barrier breakdown. Excitingly, we show that neutrophils response to these non-optimal bacteria species directly impacts FGT barrier function in vivo and ongoing work will determine whether these changes in barrier function can directly enhance HIV acquisition.

Addressing Communication Barriers in Clinical Encounters between Healthcare Providers and Pregnant People with Obesity

Rachel Dadouch1, 2, Rohan D’Souza1, 2, Janet Parsons1,3

1Institute of Medical Science, University of Toronto 2Maternal-Fetal Medicine, Mount Sinai Hospital 3Li Ka Shing Knowledge Institute, St. Michael's Hospital

Introduction: Obesity in pregnancy may elevate risk of health challenges. Moreover, there are accommodations specific to larger physical size. Communication barriers impede management of these considerations. Healthcare providers (HCPs) and users perceive and experience obesity differently: their willingness to talk about weight, terminology and risk perception. These differences coupled with weight stigma permeate clinical encounters, compromising patient health; a critical effect for individuals with high Body Mass Indices (BMI) who have cited fear of healthcare settings. There is a dearth of weight- related communication strategies. We propose narrative inquiry to deepen our understanding of the obesity-inpregnancy clinical encounter before bridging these narratives via Brokered Dialogue.

Methods: This qualitative study employed a narrative approach. We conducted in-depth, individual interviews informed by stigma theory and prior research findings, to determine perceptions of care. Pregnant people with a BMI >30kg/m2 were purposively sampled from an antenatal clinic at a tertiary-care centre, as were clinicians including family physicians, obstetricians, nurses, anaesthesiologists, midwives and dieticians. Data collection and analysis were iterative and transcripts were coded inductively for themes and patterns using narrative analysis.

Results: Data collection and analysis are still underway. HCPs discussed communication challenges or lack thereof when equipment modifications substituted weight conversations. Certain situations entail deciding between avoiding mention of weight to minimize stigma versus adequately addressing weight for responsible care. HCPs wondered about patients’ awareness of the reasons behind getting specialized care. Patients shared past weight-related experiences, which included stigma or discrimination in the healthcare setting and otherwise. Communication was more positive in the tertiary-care obstetric setting, apart from incidents such as HCPs’ difficulty with fetal monitoring due to excess weight. Feelings of being misunderstood by society were shared. All participants valued constructive communication and discussed the impact of their own weight stories on how they approach each other in this clinical encounter.

Conclusion: Detailed accounts of weight-related conversations in an obstetric setting were explored, which demonstrated communication divides. Findings point to strategies to improve weight-related discussions, and further inform future research employing a film-based intervention, Brokered Dialogue, which mediates in-person tensions surrounding weight discussions, and helps bridge communicative divides.

The role of TGF-β1 in the early cerebellar circuit formation

Azadeh Dalvand1, Hassan Marzban1

1University of Manitoba

Many cerebellar neurological disorders are characterized by structural changes in neuronal connectivity. Recent experiments in our lab have shown that the foundation of the cerebellar connectivity is assembled by trigeminal afferent pioneer axons, however, the cellular and molecular mechanisms underlying the early cerebellar connectivity establishment is not clear. This study aims to determine how cytokines such as TGF-β superfamily regulate the complex cerebellar circuit formation that emerges from homogenous cell populations to provide insight into the cause of conditions such as autism spectrum disorder. In this study we have focused on the TGF-β1 and its downstream signaling pathways which are activated during early cerebellar development. Our data show a significant amount of active TGF-β1 in the mouse embryonic cerebellar homogenates at E9 and E10 that experience a downward trend with lowest amount at E13. This may suggest a role in attracting primary projection of early pioneer axons to the cerebellar nuclei neurons by activating different molecules such as Neural Cadherins, Bcatenin which are key players in neural cell migration, LC3 I/II and P62 in autophagy pathway that promote migratory phenotype in the cells. Our study is indicating a novel role for TGF-β1 and downstream signaling pathways that may regulate trigeminal derived pioneer axons to target neurons during early cerebellar circuit formation.

A novel anti-inflammatory role links the CARS2 locus to protection from coronary artery disease

Anh-Thu Dang1, 2, Sébastien Soubeyrand1, Paulina Lau1, Ruth McPherson1, 3

1Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, Canada; 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada 3Department of Medicine, Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, Canada.

Introduction: Coronary artery disease (CAD), caused by the plaque buildup in the artery walls affecting the blood supply to the heart, is the leading cause of mortality worldwide. A locus tagged by rs61969072 (T/G) was identified by a large genome-wide association study (GWAS) for CAD with protection conferred by the common allele (T). Expression quantitative trait loci (eQTL) analysis demonstrated increased CARS2 mRNA abundance in carriers of the protective allele in various human tissues suggesting CARS2 as a candidate causal gene. CARS2 encodes a mitochondrial cysteinyl-tRNA synthetase and is highly expressed in monocytes. Here we have explored the mechanisms by which CARS2 confers a protective effect on CAD risk.

Methods: We investigated the role of CARS2 in THP-1 monocytes. Monocytes were differentiated to macrophages and polarized to proinflammatory (M1) and anti-inflammatory (M2) macrophages. We profiled macrophages transfected with siRNA targeting CARS2 using a microarray and measured cytokine levels in cell supernatant using the Luminex Multiplex assay. Functional enrichment analysis was performed using Ingenuity Pathway Analysis to identify pathways associated with CARS2 knockdown. To recreate vessel wall conditions, macrophages, and smooth muscle cells (SMC) were maintained in co-culture.

Results: CARS2 gene expression was found to decrease in M1 and increase in M2 macrophages. Gene expression profiling revealed an increase in pro-inflammatory markers in response to CARS2 siRNA knockdown in macrophages and this was accompanied by an increased abundance of inflammatory cytokines in the cell supernatant. Functional enrichment analysis of impacted transcripts identified the anti-inflammatory IL-10 signalling pathway. Western blot analysis of CARS2 knockdown macrophages showed reduced STAT3 phosphorylation in response to IL-10, indicating a role for CARS2 in anti-inflammatory signalling. In co-culture experiments, we demonstrate that CARS2 knockdown in macrophages acts to alter SMC phenotype by decreasing expression of contractile genes, ACTA2 and TAGLN and increasing expression of inflammatory genes including IL1B and CXCL1.

Conclusion: These data highlight a novel anti-inflammatory novel role for CARS2 in human macrophages and smooth muscle cells that may underlie the protective effect of a common GWAS identified variant.

PulseNet International: Assessment of Whole Genome Sequencing Implementation Capacity and Challenges for Global Foodborne Disease Surveillance

Taylor Davedow1,2, Heather Carleton3, Kristy Kubota4, Daniel Palm5, Peter Gerner-Smidt3 and Celine Nadon1,2

1Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada 2Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Canada 3Centers for Disease Control and Prevention, United States 4Association of Public Health Laboratories, United States 5European Centre for Disease Prevention and Control, Stockholm, Sweden

Introduction: PulseNet International (PNI) is a global network of 88 countries and their regional and national public health laboratories who work together to track foodborne disease. The vision of PNI is to implement globally standardized surveillance using whole genome sequencing (WGS) for real-time identification and subtyping of foodborne pathogens. Several countries in North America and Europe have implemented WGS and have experienced significant benefits in disease mitigation. To broaden the routine use of WGS around the world, challenges and barriers must be overcome.

Purpose: To determine what challenges and barriers countries are encountering in their attempts to implement WGS and identify how PNI can provide support in order to improve and become a better integrated system overall. Methods: A qualitative, 22-question survey was designed to capture the experiences of countries in the implementation of WGS. The survey was administered online via the survey function of Google Forms (Alphabet, Inc., USA), to 55 PNI participating laboratories in Latin America and the Caribbean, Africa, the Middle East, and Asia-Pacific through their regional coordinators. Multiple responses per country were accepted if the survey was submitted by different participating laboratories. Data was coded and analyzed using Microsoft Excel (Microsoft, USA).

Results: Thirty-one (56%) different countries completed the survey and data from all 38 responses from Africa (45%), Asia- Pacific (16%), Latin America and the Caribbean (21%), and the Middle East (18%) were analyzed. One third of members do not use WGS, and only 8% reported using WGS for routine, real-time surveillance. The main barriers for WGS implementation were lack of funding (82%), gaps in expertise (59%), and training (56%). Members identified that an ideal system for global surveillance would be an all-in-one software that is free, accessible, standardized and validated.

Conclusions: The results of this survey highlighted the minimal use of WGS for foodborne disease surveillance. Although funding remains a major barrier to WGS, critical gaps in expertise and availability of tools must be overcome. The future priorities of PNI should include coordinating funding and training opportunities, and identifying solutions for a globally standardized surveillance platform to accelerate implementation of WGS and to create accessible solutions for all network members.

Whole Genome Sequencing Reveals High Level of Genetic Diversity in Antifungal Resistant Candida glabrata Isolates in Canada

Domenica G. De Luca1, Tanis C. Dingle2, Philippe Dufresne3, Jeff Fuller4, Greg German5, David Haldane6, Linda Hoang7, Lei Jiao8, Julianne Kus9, Kathy Malejczyk10, Caroline Sheitoyan-Pesant11, Markus Stein12, Morag Graham1, Gary Van Domselaar1, Amrita Bharat1

1National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB 2Alberta Precision Laboratories – Public Health Laboratory, Edmonton, AB 3Laboratoire de santé publique du Québec, Sainte-Anne-de-Bellevue, QC 4Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON 5Health PEI, Charlottetown, PEI 6QEII Health Science Centre, Halifax, NS 7BC Centre for Disease Control, Vancouver, BC 8Eastern Health, St. John’s, NL 9Public Health Ontario, Toronto, ON 10Roy Romanow Provincial Laboratory, Regina, SK 11Centre hospitalier universitaire Dr-Georges-L.-Dumont, Moncton, NB 12Diagnostic Services of Manitoba, Winnipeg, MB

Introduction: Candida glabrata is a major human fungal pathogen that can exhibit reduced susceptibility towards antifungal drugs. Here, whole genome sequencing (WGS) was utilized to elucidate the genomic epidemiology of antifungal resistant C. glabrata isolates.

Methods: The ten provincial public health laboratories in Canada provided 142 C. glabrata isolates. Resistant isolates from invasive infections were prioritized. Of 142 isolates, 80 (56%) were resistant to at least one antifungal drug (fluconazole, micafungin, amphotericin B) and 110 (77%) were collected from sterile sites. Sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit and sequenced on the NextSeq 550 platform. The Single Nucleotide Variant Phylogenomics pipeline version 1.1 was used to generate a maximum likelihood phylogenomic tree based on single nucleotide variants in the core genome. CBS138 was used as the reference strain for this phylogeny.

Results: The phylogenomic tree represented 68% of the C. glabrata core genome. Of 142 isolates, 127 (89%) grouped into one of 16 genetically related clusters. The five largest clusters accounted for 61% of the isolates (cluster 3, 15.5%; cluster 7, 10.6%; cluster 11, 10.6%; cluster 13, 8.5%; cluster 16, 15.5%). Few clusters grouped based on province of isolation, with the exception of cluster 9, which consisted of isolates exclusively from the same province in Central Canada. Some clusters grouped according to site of isolation, for example clusters 2, 5, 8, 9, and 10 were isolated from sterile body sites with the majority from bloodstream infections. Resistant isolates were present throughout the tree and all clusters contained at least one resistant isolate. Cluster 14 (n = 6) consisted entirely of isolates that were resistant to fluconazole. Cluster 16 (n = 22) had the largest number of resistant isolates with 15 resistant to fluconazole, three resistant to micafungin and one resistant to amphotericin B.

Conclusion: WGS revealed that there was a high level of genetic diversity in antifungal resistant C. glabrata isolates circulating in Canada. Only two branches were enriched for antifungal resistant isolates, suggesting that, in most cases, antifungal resistance may arise spontaneously due to selective pressure during treatment rather than dissemination of resistant clones.

Novel co-option of PDGFRA in Histone H3.3 G34-mutant interneuron progenitors promotes gliomagenesis

Shriya Deshmukh1*, Carol C.L. Chen2*, Selin Jessa3,4*, Djihad Hadjadj2*, Véronique Lisi2, Augusto Faria Andrade2, Damien Faury5, Steven Hébert2,4, Nicolas De Jay2,4, Michele Zeinieh2, Mathieu Blanchette6, Claudia L. Kleinman2,4#, and Nada Jabado1,2,5##

1Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Canada 2Department of Human Genetics, McGill University, Montreal, Canada 3Quantitative Life Sciences, McGill University, Montreal, Canada 4Lady Davis Research Institute, Jewish General Hospital, Montreal, Canada 5Department of Pediatrics, McGill University, and The Research Institute of the McGill University Health Centre, Montreal, Canada 6School of Computer Science, McGill University, Montreal, Canada *Equal contribution #Corresponding authors

Histone H3.3 glycine 34 to arginine/valine (G34R/V) driver mutations occur in lethal high-grade gliomas in the temporal- parietal cortex of adolescents and young adults. The striking location and age specificity suggests a developmental context permissive to G34R/V-mediated epigenetic perturbation and gliomagenesis. Assembling the largest cohort of these rare tumours to date, we show that 50% of G34R/V tumours (n=95) carry activating mutations in the PDGFRA oncogene. Although PDGFRA is a prototypical glial marker and G34R/V tumours are classified as gliomas, comparison of transcriptomic signatures to reference single-cell developmental forebrain atlases reveals that G34R/V tumours surprisingly resemble interneuron progenitors. G34R/V tumours express transcription factors such as GSX2 normally restricted to interneuron progenitor cells, while repressing expression of mature neuronal genes through G34R/V-mediated H3K27me3 deposition. Notably, PDGFRA and GSX2 are located immediately adjacent in the linear genome. By Hi-C chromosome conformation capture, we identify a chromatin loop connecting PDGFRA to active GSX2 enhancer elements in G34R/V tumours, suggesting that their lineage-of-origin facilitates PDGFRA co-option to promote aberrant overexpression and mutation. Expanded astroglial compartments in PDGFRA-mutant tumours suggest that mutant-PDGFRA potentiates gliomagenesis. In contrast, we show with CRISPR/Cas9-edited glioma cell lines and xenograft models that G34R/V mutations become dispensable for tumour maintenance. Together, our results suggest that G34R/V gliomas arise in GSX2-expressing interneuron progenitors, where G34R/V mutations modulate the epigenome to prevent terminal differentiation and support a progenitor state that is permissive to PDGFRA co-option and gliomagenesis. These findings underscore the importance of lineage and cell-type context in neoplastic processes, and suggest PDGFRA signaling as a novel potentially targetable pathway in lethal G34R/V high-grade gliomas.

Excitatory spinal interneuron circuit modules revealed through task-specific subpopulation recruitments

Dylan Deska-Gauthier1, Dr. Han Zhang1, Dr. Joanna Borowska1, Dr. Ying Zhang1

1Dalhousie University, Faculty of Medicine, Department of Medical Neuroscience

Animals exhibit a wide range of sensorimotor behaviours that emerge from the coordinated activities of neuronal circuits in the spinal cord. While spinal interneurons (INs) are known to play an essential role in establishing precise temporal patterns of muscle contraction, the principles governing recruitment of INs across different behaviours remain elusive. Here, we combine computational models to systematically analyze task-specific c-fos expression within the cardinal V3 IN population in the mouse spinal cord. Our analysis reveals a topographic arrangement of V3 INs into functionally distinct modular domains. Furthermore, we uncover molecularly discrete Nr3b3+, Onecut2+, Olig3+, Pou2f2+ and Prox1+ V3 IN subpopulation clusters that display unique neurogenesis timings, form unique connectivities, and most notably, differentially assemble within distinct V3 modular domains. Thus, our current work indicates developmentally and genetically discrete V3 IN subpopulations as the building blocks of topographically clustered spinal circuits engaged in different sensorimotor tasks.

LRRTM1 and LRRTM2 regulate synapse development and plasticity through distinct roles in developing and mature hippocampal circuits

Shreya H. Dhume1,2*, Steven A. Connor3,4, Fergil Mills5, Parisa Karimi Tari3,4, Sarah HM Au-Yeung3, Benyamin Karimi1,2, Shinichiro Oku3, Reiko T. Roppongi1,2, Hiroshi Kawabe6,7,8, Shernaz X. Bamji5, Yu Tian Wang9, Michael F. Jackson1,10, Nils Brose6, Ann Marie Craig3 and Tabrez J. Siddiqui1,2,11,12

1Neuroscience Research Program, Kleysen Institute for Advanced Medicine, Health Sciences Centre, Winnipeg, MB 2Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, MB 3Department of Psychiatry and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC 4Department of Biology, York University, Toronto, ON 5Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia 6Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany 7Division of Pathogenetic Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 1-5-6 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan 8Department of Gerontology, Laboratory of Molecular Life Science, Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe, 2-2 Minatojima-minamimachi Chuo-ku, Kobe 650-0047, Japan 9Division of Neurology, Department of Medicine and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC 10Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB 11The Children's Hospital Research Institute of Manitoba, Winnipeg, MB 12Program in Biomedical Engineering, University of Manitoba

Neurons in the brain communicate at specialized junctions known as synapses, which are comprised of pre- and post- synaptic compartments. Synapses are highly plastic and undergo activity dependent changes in synaptic strength, a phenomenon required for learning, memory, other aspects of cognition and behaviour. A class of cell-surface adhesion proteins known as synapse organizers mediates the development of synaptic connections. Leucine rich repeat transmembrane neuronal proteins (LRRTM) are postsynaptic cell adhesion molecules that promote excitatory synapse development through binding to their presynaptic receptors, neurexins. To study the role of LRRTM1 and LRRTM2 in vivo, we did a temporal knockout study of both Lrrtm1 and Lrrtm2 where we deleted both proteins in developing brain and adult brain. We studied synapse development and plasticity in hippocampal neuronal circuits where we found that these proteins maintain morphology and plasticity in both developing and developed circuit.

LRRTM1 and 2 are not required for these roles in dentate gyrus of hippocampus. LRRTM1 and 2 also regulate endurance of fear memory, a memory encoded by dorsal hippocampus in both the circuits. Thus, our study shows that LRRTM1 and 2 regulate excitatory synapse development and plasticity in dorsal hippocampus of both developing and mature brain circuits.

Predicting Individual Gut Microbiome Responses to Resistant Starch

Peter A. Dobranowski1, 2, Hongye Qin1, Krystal Walker1, James Butcher1, Kendra Hodgkinson1

1University of Ottawa, Ottawa, ON, Canada 2Ottawa Institute of Systems Biology

Introduction: Resistant starch (RS), present in potatoes, corn, and commercial supplements, evades digestion and reaches the colon relatively intact. A guild of specialized microbes can degrade and ferment RS into organic acids, including the health-promoting metabolite, butyrate. Previous clinical trials have shown that RS fermentation (i.e. butyrate production) depends on the RS treatment and the individual’s microbiome. So far, no method has been developed to predict if an individual will benefit from RS.

Methods: The novel in vitro assay, RapidAIM, enables us to predict how an individual’s microbiome responds to treatments, such as RS. We incubated fecal slurry obtained from 15 pediatric inflammatory bowel disease patients with a panel of 9 RS that vary by physicochemical property and botanical source. After 18 hours of incubation, we measured changes in supernatant pH (organic acids) and bacterial relative abundances using 16S rRNA gene sequencing.

Results: Individuals’ microbiomes show remarkable variability with which RS they degrade and ferment. However, two tiers of responses (high and low) emerged using k-means clustering. pH changes correlated significantly with butyrate-producing bacteria relative abundances, suggesting that changes in pH are in part mediated by the activity of butyrate-producing bacteria. Lastly, the relative abundances of previously-reported RS-active bacteria are significantly enriched in high responders compared to low responders.

Conclusion: RapidAIM enables us to interrogate mechanisms by which an individuals’ microbiome degrades and ferments RS. The relative abundance of known RS-active bacteria may serve as a biomarker to predict responses to RS. Future work will validate butyrate production in vitro and in vivo using longitudinal multi-omic microbiome analyses following a clinical trial.

Assessment of Bladder Function After Spinal Cord Injury Using a Novel Pressure Measurement Device

Adam Doelman1, Steve Majerus2, Margot Damaser2, Brian K. Kwon1, 3

1University of British Columbia 2Cleveland Clinic, Cleveland, OH, USA 3Vancouver Spine Surgery Institute, Vancouver, BC, Canada

Introduction: Without question, bladder dysfunction remains one of the leading causes of morbidity after spinal cord injury (SCI). Our overall objective is to develop a wireless sensor that can be implanted into the bladder to provide continuous bladder pressure monitoring in individuals living with SCI. In order to facilitate the transition from “bench-to-bedside” we will be testing the UroMonitor in our previously established minipig model of SCI. In this pre-clinical study, we are comparing bladder pressures recorded from the “gold-standard” urodynamics catheters to the UroMonitor during urodynamic (UDS) evaluation before and after spinal cord injury in adult minipigs.

Methods: In the present study we evaluated 20-30kg adult female Yucatan minipigs before and after a 20cm contusive- compression SCI at the T10 level. Animals were first anesthetized and a UroMonitor was inserted transurethrally into the bladder by a certified veterinary technician. After insertion, a dual-channel intravessicle catheter was inserted into the bladder to simultaneously record bladder pressure and provide retrograde filling during UDS.

Results: UroMonitor insertion was accomplished either “free-hand” or using our silicone insertion tool. Once inserted, we found the UroMonitor reliably reproduced bladder pressure patterns with a high degree of correlation in all of the UDS experiments performed. Pearson correlation coefficients between UDS and UroMonitor were between 0.59-0.87 (p<0.001). Further, similar values were recorded between the UDS catheter and UroMonitor when assessing baseline pressure, non- voiding contraction pressure, threshold pressure to void and peak voiding contraction pressure. At this time, there does not appear to be an effect of the UroMonitor on cystometric voiding capacity during UDS.

Conclusion: Based on the preliminary results, we’ve demonstrated we can successfully insert the UroMonitor into the bladder lumen with a minimal degree of difficulty and have developed techniques to extract the device using an externalized suture. We conclude that the UroMonitor is able to accurately measure bladder pressure during UDS in intact and SCI minipigs with sub-cmH20 level of accuracy. Future directions for this project will include more frequent UDS evaluation in up to 10 animals as well as the assessment of ambulatory bladder function to simulate a more “physiological” fill-empty cycle.

A comparison of copper tolerance determination methods with Pseudomonas aeruginosa and the effects of copper on growth medium Ph

Ali Doucet1, Michael R. Mulvey1,2, George Golding1,2, and Denice C. Bay1

1University of Manitoba, Department of Medical Microbiology and Infectious Diseases, Winnipeg, Manitoba, Canada 2Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba, Canada

Introduction: Copper is an important antimicrobial metal that inhibits the growth of both Gram-negative and Gram-positive bacterial species. However, its efficacy is threatened by emerging copper tolerance mechanisms in opportunistic pathogens like Pseudomonas aeruginosa. Presently, there is a lack of experimental consistency in measuring copper tolerance. As a result, there is no consensus for breakpoints for copper, making the comparison of results between studies difficult. These issues include the acidification of media due to the copper salts, copper precipitation at high phosphate concentrations, and limited copper bioavailability in growth media. Here, we examine these issues by comparing P. aeruginosa strains to two commonly used copper salts (CuSO4 and CuCl2) using copper susceptibility testing and copper exposure methods.

Results: Visual precipitation of copper was seen in all minimal media we tested, but copper precipitation at high concentrations was not observed in rich medium Mueller Hinton broth (MHB), when compared to lysogeny broth (LB). The growth media with added CuSO45H2O (0-8 mg/mL) or CuCl2 2H2O(0-5.46 mg/mL) showed increasing media acidity with increasing copper concentrations. To confirm that copper susceptibility was due to its pH acidification, we compared P. aeruginosa strains growth in MHB containing CuSO4 and in MHB adjusted to its acidic pH values at the same copper concentration. Minimum inhibitory copper concentration values for each strain corresponded to its acidic pH-adjusted medium, revealing that growth inhibition was likely due to pH reductions. To overcome this, we performed P. aeruginosa time course copper exposure experiments with and without CuSO4 in sterile water at concentrations that did not reduce pH and assessed their metabolic activity with an ATP-based luminescence assay.

Conclusions: This exposure method minimizes copper acidity effects, improving the accuracy of copper tolerant concentration measurements, making it useful for copper tolerance determination in future studies.

Single and combined effects of NOD2 and Mincle in the innate immune recognition of live and dead Mycobacterium tuberculosis

Jean-Yves Dubé1,2,3, Fiona McIntosh2,3, Juan G. Zarruk4, Samuel David4, Jérôme Nigou5, Marcel A. Behr1,2,3,6

1Department of Microbiology and Immunology, McGill University. 2Infectious diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre. 3McGill International TB Centre. 4Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre. 5Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier, Toulouse, France. 6Department of Medicine, McGill University Health Centre, Montréal, Canada.

Mycobacterium tuberculosis (Mtb), the primary cause of tuberculosis (TB), was the leading killer due to a single infectious agent in the pre-COVID era. However, mycobacteria have also been useful to humanity as immunotherapeutic reagents: bacille Calmette-Guérin (BCG) prevents TB in children and is an effective treatment for bladder cancer in adults. Complete Freund’s adjuvant (CFA, dead Mtb in mineral oil) is the dirty little secret of immunologists, used to elicit cell-mediated immunity in various animal models of immunization and autoimmunity. To understand innate immune recognition of mycobacteria, we have investigated the contributions of the pattern recognition receptors (PRRs) Mincle and NOD2 in mouse models of immunization and infection. Mincle and NOD2 respond to the mycobacteria-specific microbe associated molecular patterns (MAMPs) trehalose dimycolate (TDM) and N-glycolyl muramyl dipeptide (MDP), respectively. Using genetic knockouts and synthetic MAMP chemical complementation, we demonstrated the essentiality of TDM plus MDP for the adjuvant effect of CFA. With low doses of these MAMPs in combination, we achieved adjuvant activity in both an ovalbumin model of cell-mediated immunity and experimental autoimmune encephalomyelitis (EAE). During live Mtb infection in a low-dose aerosolization model, Mincle and NOD2 played early and late roles: essential for timely mounting of the lymphocyte response and protective from premature death after months of infection, respectively. Data with double knockout of both Mincle and Nod2 suggest NOD2 plays a dominant role in resistance to Mtb infection. However, single and double knockouts of PRRs all result in late reductions in survival. Thus, while PRRs are powerful tools to elicit immunity, they are individually (and in small combinations) mostly dispensable for the host during infection, allowing for the genetic diversity in PRRs that we observe in humans.

Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells

Angela Duong1, Jean-Martin Beaulieu1, Liliana Attisano1, Ana C. Andreazza1

1University of Toronto

Introduction: Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues.

Methods: To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages.

Results: Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs.

Conclusion: We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

The Dietary Fatty Acids Palmitate and Oleate Differentially Impact BeWo Trophoblast Lipid Processing

Zachary JW Easton1,2, Ousseynou Sarr1, Lin Zhao1, and Timothy RH Regnault1,2,3,4,5

1Department of Physiology and Pharmacology 2Collaborative Graduate Specialization in Developmental Biology 3Department of Obstetrics and Gynaecology, University of Western Ontario 4Children’s Health Research Institute 5Lawson Research Institute, London, ON, Canada

Background: Maternal gestational obesity is associated with altered placental lipid processing highlighted by placental steatosis and reduced mitochondrial beta-oxidation. However, maternal dietary fat consumption has recently been demonstrated to modulate placental function independent of maternal body composition. As the fatty acids (FA) palmitate (PA) and oleate (OA) are the most abundant fats in Western culture diets and their levels in maternal circulation are elevated with obesity, these fats themselves may be important in regulating placental lipid processing. The goal of this project was to characterize lipid metabolic function in PA and OA exposed BeWo trophoblast cells and further elucidate the independent impacts of dietary FA on the placenta.

Methods: BeWo trophoblast cells were cultured in F12K media supplemented with 100 μM PA, OA or a 1:1 combination of PA+OA (P/O) conjugated 2:1 to Bovine Serum Albumin (BSA, 0.3%) for 72H. Chloroform-methanol extraction was utilized to collect cellular lipids and FA profiles of the extracted lipids were determined using gas chromatography-flame ionization detection. Thin layer chromatography-flame ionization detection was additionally utilized to examine the relative abundance of neutral lipid species. Additional BeWo trophoblast cultures were collected for transcriptomic analysis via Clariom S mRNA microarray. Transcripts with ±1.3 fold-change were considered differentially expressed and functional pathways analysis was examined using WikiPathways.

Results: OA-treated BeWo trophoblasts displayed an increased abundance of triglyceride (TG) species. Dietary-FA treatment altered BeWo trophoblast FA profiles and we specifically observed that PA treatment increased while OA and P/O treatment decreased the C16:1n7/C16:0 FA ratio (measure of SCD-1 Δ9-desaturase activity). Transcriptomic analysis highlighted that elevated FA levels were sufficient to alter expression of genes involved in lipid metabolic pathways (including SCD1, ACSL5, ACCα, ACCβ and ACADVL).

Conclusion: Overall, these results demonstrate that elevated levels of PA and OA differentially impact placental lipid metabolic processes and further highlights the importance of maternal dietary fat composition in modulating placental function. As recent reports indicate that many lean individuals also consume high fat diets, this study may provide insight into how maternal dietary fats modulate placental function not only in obese pregnancies, but also in lean pregnancies impacted by poor maternal diet.

Is flaxseed equivalent and/or synergistic with ACE inhibition in the prevention of chemotherapy- induced cardiotoxicity?

Eekhoudt CR1, Varghese S1, Cheung D1, Austria JA1, Thliveris J2, Aukema HM3, Ravandi A1, Pierce GN1, Singal PK1, Jassal DS1,4,5

1Department of Physiology and Pathophysiology, University of Manitoba 2Institute of Cardiovascular Sciences, Albrectsen Research Centre, Manitoba 3Department of Internal Medicine, University of Manitoba 4Department of Human Anatomy and Cell Science, University of Manitoba

Background: While Doxorubicin (DOX) and Trastuzumab (TRZ) are two common anti-cancer agents used to treat breast cancer, their use is associated with adverse cardiotoxic side effects. Recent studies have evaluated the role of pharmaceuticals, including perindopril (PER), and nutraceuticals, including flaxseed (FLX), in the prevention of DOX+TRZ mediated cardiotoxicity. However, little is known on whether FLX is equivalent and/or synergistic to PER in the prevention of chemotherapy induced cardiotoxicity.

Method: A total of 60 wild-type C57Bl/6 female mice were randomized to either regular chow (RC) or FLX-supplemented diets for a total of 6 weeks. On weeks 4, 5, and 6, mice were further randomized to receive an intraperitoneal injection of: i) 0.9% saline; ii) DOX (8mg/kg/wk); iii) TRZ (3mg/kg/wk); or iv) DOX+TRZ to create a chronic in vivo murine model of chemotherapy induced cardiotoxicity. Additionally, mice received PER (3mg/kg/daily) daily via oral gavage. Weekly echocardiography and hemodynamic parameters were evaluated followed by histological analysis at the end of week 6.

Results: In mice treated with RC+DOX+TRZ, left ventricular end diastolic diameter (LVEDD) increased from 2.8±0.2 mm at baseline to 4.3±0.2 mm by week 6. Prophylactic administration of either PER or FLX alone partially prevented adverse LV remodelling with LVEDD values of 3.4±0.3 mm and 3.5±0.2 mm, respectively (P<0.05). Similarly, the left ventricular ejection fraction (LVEF) in mice treated with RC+DOX+TRZ decreased from 75±2% at baseline to 37±3% at week 6. Prophylactic treatment with either PER or FLX alone partially attenuated LV systolic dysfunction with LVEF values of 63±2% and 62±2%, respectively (P<0.05). Prophylactic treatment with the combination of PER+FLX, however, was not synergistic at preventing adverse LV remodelling. Histological analyses confirmed significant disruption of myofibrils, and loss of sarcomere integrity in the RC+DOX+TRZ treated mice. Prophylactic administration of FLX or FLX+PER was markedly cardioprotective in preserving myofibril integrity at week 6 in mice receiving the combination therapy of DOX+TRZ.

Conclusion: In a chronic model of DOX+TRZ induced cardiotoxicity, although FLX was equivalent to PER in the prevention of adverse LV remodelling, the combination of FLX and PER was not synergistic.

Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML

Grace Egan1, 2, G.E Thomas1, Parasvi S. Patel1, Jordan Chin1, Aaron Botham1, Rose Hurren1, Neil MacLean1, Razq Hakem1, Aaron D. Schimmer1

1Princess Margaret Cancer Centre, Ontario Cancer Institute, Toronto, ON, Canada 2Division of Hematology/Oncology, The Hospital for Sick Children, Toronto, ON, Canada

Relapse of acute myeloid leukemia (AML) is the main cause of mortality in this disease. Leukemia stem cells (LSCs) contribute to relapse however the mechanisms underpinning chemoresistance are not well understood. We explored the DNA damage response in AML LSCs. We discovered that expression of DNA repair pathway genes were upregulated in functionally defined LSC vs bulk AML cells (n= 227, GSE76008), NES = 1.74, FDR = 0.057, and undifferentiated (n= 7) vs committed AML samples (n = 4, PMCC cohort), NES = 2.041, FDR = 0.000. Next, we investigated the expression of DNA repair genes and response to DNA damage in FACs sorted stem and bulk fractions of 8227 cells. 8227 cells are primary AML cells that maintain a hierarchical organization with stem cells in the CD34+CD38- fraction. Compared to bulk cells, LSCs had increased expression of homologous recombination and non-homologous-end-joining repair genes. Sorted 8227 cells were treated with daunorubicin and DNA damage and repair were evaluated by measuring intensity of 53BP1, RAD51 and γH2AX by fluorescent microscopy and quantified using image J. Compared to bulk cells, 8227 LSCs demonstrated increased DNA damage repair with increased 53BP1 and RAD51 expression and decreased γH2AX. We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. To understand how stemness impacts DNA repair, we selectively over-expressed HK2 in the nucleus 2 of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence. Overexpressing nuclear HK2 increased stem cell function as shown by clonogenic growth assays and mouse marrow engraftment. We treated these cells with daunorubicin and measured DNA repair. Over- expressing nuclear HK2, increased 53BP1 and RAD51 expression and decreased γH2AX, similar to the LSC phenotype. As measured by the COMET assay, these cells also had decreased levels of double strand DNA breaks. Over-expression of nuclear HK2 also conferred resistance to daunorubicin as measured by clonogenic growth assays.

In summary, LSCs have increased levels of DNA repair genes and increased rates of DNA repair after chemotherapy exposure. The accelerated repair seen in LSCs may contribute to chemoresistance and subsequent disease relapse.

Gaining an insight on the genomic evolution of Sudan virus serially passaged in guinea pigs

Karla Emeterio, Wenjun Zhu, Shihua He, Logan Banadyga, and Michael Drebot

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba 2National Centre for Foreign Animal Disease, Canadian Food Inspection Agency 3Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada

Sudan virus (SUDV) is a filovirus classified as a Risk Group 4 pathogen and linked to human cases of severe hemorrhagic fever in Sudan and Uganda with an average case fatality rate of about 50%. The lack of licensed prophylactis and therapeutics to treat SUDV disease remains a concern. Initial evaluation of filovirus countermeasures is primarily conducted in small rodents, such as mice, guinea pigs, and hamsters; however, filoviruses are largely apathogenic in immunocompetent rodents. Adaptation is achieved through serial passaging of the virus in the host animal, during which the virus acquires genomic mutations that contribute to virulence and lethality. A single guinea pig animal model currently exists for SUDV, for which repeated passaging of the virus resulted in a guinea pig-adapted virus carrying 16 nucleotide changes, 6 of which resulted in an amino acid change. In our study, we sought to discern which adaptive mutations may be relevant in SUDV pathogenesis in guinea pigs by looking at changes in the viral genome throughout the adaptation series. By deep sequencing the virus genome obtained from each passage, we were able to track the appearance and changes in frequencies of each mutation over time with high precision. Our results revealed that SUDV acquired multiple transient mutations throughout passaging, with the majority of mutations appearing within the untranslated regions of the genome. Three of the formerly identified adaptive mutations in viral proteins (VP) 40, 30 and 24 were also found to be present in the starting virus and increased in frequency over time. Most adaptive mutations appeared in earlier passages and reached a frequency of 99% or higher near the end of the adaptation series, with the exception of the P204L mutation in VP24, which reached a frequency of 100% by passage 7. Nonetheless, this high frequency of the P204L mutation was not sustained throughout remaining passages. Interestingly, we also identified a novel mutation, L203P, in the VP40 gene. Overall, our results provide insight into virus evolution that occurs during host adaptation, and they highlight mutations that may play a significant role in SUDV pathogenesis.

Coordinated Changes of Sialylated Glycans Control B-cell Responses in the Germinal Centre

Jhon R. Enterina1, Susmita Sarkar2, Laura Streith1, Matthew S. Macauley1,2

1Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada 2Department of Chemistry, University of Alberta, Edmonton, AB, Canada

Introduction: Germinal centres (GC) are a crucial component for an effective humoral immunity against invading pathogens. It is the site where B-cells undergo iterative clonal expansion and diversification of their immunoglobulin genes following an infection. Following somatic hypermutation, high affinity clones arising from the GC are selected and then differentiate into either memory B-cells and antibody-secreting plasma cells. In the GC, B-cells are routinely identified through the distinct changes on their surface carbohydrates. One striking modification relates to the monosaccharide sialic acid. In mice, this change is mediated through downregulation of an enzyme called CMAH. As a consequence, GC B-cells lose the preferred ligand for CD22, a member of the sialic acid-binding immunoglobulin-type lectins (Siglecs) and a co-inhibitory receptor of the B-cell antigen receptor. Since CD22 regulates B-cell function, we hypothesize that this specific carbohydrate remodeling on B-cells control the GC reaction by modulating the activity of CD22.

Methods: To test our hypothesis, we developed a transgenic mouse model that can conditionally express CMAH on B-cells under the control of Cre-recombinase. We crossed this model with Mb1Cre and AidCre mouse lines to selectively express CMAH in all mature and GC B-cells, respectively. Bone marrow chimera mice models were generated from these models. We then immunized these chimera mice with a T-dependent antigen, chicken ovalbumin, to track the effect of constitutive CMAH expression in B-cell responses in the GC.

Results: We identified that constitutive expression of CMAH in B-cells restricts the formation and/or maintenance of GC B- cells following immunization. More significantly, we determined that this observed defect is dependent on CD22, highlighting that coordinated loss of preferred ligands for CD22 on B cells plays a critical function in the GC. Additionally, mice lacking CD22 expression or with sustained CMAH expression in the GC mounted poor antibody responses, which we have pinpointed to decreased proliferation and increased apoptotic cell death in the GC B-cell compartment.

Conclusion: Overall, our study uncovers the crucial role of carbohydrate remodeling and CD22 in B-cell competitiveness in the GC.

High fat diet-induced inflammation stimulates adiposity-promoting neurons to drive obesity

Lisa Z. Fang1, Victoria Linehan1, Maria Licursi1, Michiru Hirasawa1

1Division of Biomedical Sciences, Faculty of Medicine, Memorial University

Background: Currently over 25% of adults in Canada are obese, posing an increased risk of type 2 diabetes, cardiovascular disease, and cancer. This is in part due to the overconsumption of high-fat diet (HFD). HFD causes a chronic low-grade inflammation of the hypothalamus, a brain region that controls body weight. Hypothalamic inflammation ultimately promotes weight gain, however, the mechanism linking inflammation to weight gain remains unclear. We previously found that melanin-concentrating hormone (MCH) neurons, a hypothalamic cell group known to promote adiposity, are activated by chronic HFD consumption. Thus, we tested the hypothesis that MCH neurons mediate the hypothalamic inflammation- induced weight gain.

Methods: Male rats and mice were fed either a standard low-fat rodent chow or HFD for up to 14 weeks. After the feeding period, their brain tissues were collected to perform in vitro electrophysiology where the activity of MCH neurons was recorded in real time. Immunofluorescence staining was performed to identify the localization of receptors.

Results: Prostaglandin E2 (PGE2) is an inflammatory mediator involved in peripheral inflammation during obesity and may also be induced in the brain. Thus, we asked whether PGE2 could be responsible for HFD-induced activation of MCH neurons. Indeed, PGE2 activated MCH neurons from chow-fed animals, mimicking the HFD effect. Both the HFD and PGE2 effect was blocked by the PGE2 EP2 receptor (EP2R) antagonist, indicating that HFD activates MCH neurons through PGE2- EP2R signaling. Using immunofluorescence staining, we confirmed that EP2R are localized on MCH neurons. To test the functional role of PGE2-EP2R signaling on weight gain, we generated mice that lacked EP2R on MCH neurons (MCHEP2R KO). MCH neurons of MCHEP2R KO mice were not activated by HFD, confirming the role of EP2R in activating these cells. Further, MCHEP2R KO mice fed 14 weeks of HFD were largely protected from diet-induced obesity, adiposity, and hepatic steatosis.

Conclusion: PGE2-EP2R signaling in MCH neurons is necessary for HFD-induced weight gain and fat accrual. This explains how the inflammatory milieu in the brain can lead to excess weight gain during HFD. Moreover, this mechanism could serve as a novel therapeutic target for diet-induced obesity in the future.

Predicting Drug Potency: A Simple Model with Quantum Chemistry Calculation to Score Potency of Small PARP Inhibitors

Yuhua Fang1, Geoffrey Tranmer1

1University of Manitoba

Introduction: Drug design in silico is an indispensable tool for modern drug design and development. In silico drug design includes traditional computer-aided drug design using software such as Schrodinger’s Maestro, and machine learning-based drug-protein affinity prediction and drug generation. So far, both methods lack accuracy in potency prediction. In silico drug design is difficult for researchers without computer science backgrounds. Poly(ADP-ribose) polymerase-1 (PARP1) is involved in DNA-repair pathways using nicotinamide adenine dinucleotide (NAD) as the substrate. Small molecule PARP-1 inhibitors (PARPi) that are nicotinamide analogs bind to the nicotinamide (NI) binding site. These inhibitors are consistent in docking pose and pattern. As they are fundamental for PARPi development, an efficient and reliable method to predict potency is benefiting by screening candidates virtually.

Methods: Crystal structures of the PARP-1-inhibitors complexes were obtained from Protein Data Bank and analyzed using PyMol. Compounds were synthesized or purchased and their potency was tested using a fluorescence-based PARP-1 enzymatic assay. Literature-reported potency were obtained from PubChem. Electrostatic potential surfaces were calculated by Gaussian 09W at Hartree–Fock level using the 3-21G basis set. With electrostatic potential, we introduced a scoring model based on Coulomb's Law.

Results: We introduced a scoring method standing on Columb’s Law, compounds’ electrostatic potential as well as ligand- protein interactions revealed by crystal structures. We compared our scores, along with experimental and literature- reported potency and corresponding docking scores from Maestro. The results show that our scores were more representative of the small molecules’ experimental and literature-reported potency inside the NI site compared to Maestro’s docking scores.

Conclusion: This simple scoring function based on the electrostatic potential of compounds can predict small inhibitor’s potency inside the NI site. The experiment also provides us an alternative method to predict drug potency in silico that combines chemical calculation and protein analysis. Moreover, it indicates that different ligand-protein interactions may have distinct weights when influencing the ligand-protein affinity, which needs to be considered more carefully in the future in silico prediction.

Systematic Analysis of the Breast Cancer Secretome to Decipher miRNA Tumor Biology

Riley Feser1, Sujit Maiti1, Braydon Nault1, Vincent C Chen1 and Mousumi Majumder1

1Brandon University

Introduction: One in eight Canadian women are diagnosed with breast cancer in their lifetime. However, early detection can save 99% of patients. Circulating small RNAs, known as microRNA (miRNA/miR), show aberrant expressions in tumors compared to normal breast tissues and promote aggressive breast cancer and could serve as potential biomarkers. We have identified miR526b and miR655 as oncogenic and tumor promoting miRNAs. We recently showed that cell-free conditioned media (referred to as the secretome) from miR526b/miR655-overexpressing tumor cells modify the tumor microenvironment, promoting breast cancer phenotypes. A substantial amount of the human proteome consists of secretory proteins. Thus, a miRNA-high cell secretome analysis will identify the novel secretory proteins regulating the cancer phenotypes promoted by miR526b/miR655 cell secretions.

Methods: Cell-free conditioned media was collected from the stable miRNA overexpressed breast cancer cells (MCF7- miR526b and MCF7-miR655) and empty vector transfected MCF7-Mock cells. Cell secretomes were analysed using nano- high-performance liquid chromatography-mass spectrometry. The mass spectrometry data was analyzed to identify up and downregulated proteins. A systematic bioinformatic analysis was completed using various databases to validate miRNA- high secretome expression potentially controlling previously established breast cancer phenotypes and their potential as blood-based breast cancer biomarkers.

Results: 1535 up and down regulated miRNA secretory proteins coded by 442 genes were identified. Our secretome threshold of >1.5/<-1.5 log2 fold change and >0.3 -log10 p-value reduced our initial secretions to 136 protein IDs coding for 39 genes. Protein-coding genes absent from the breast cancer proteome were disregarded, and a classical signal peptide analysis was performed. This found 25 classically secreted proteins coded by six genes TXNDC12, MYL6B, SFN, FN1, PSMB6, and PRDX4.

Conclusion: In silico validation of identified markers functions in breast cancer will decipher the mechanisms of miRNA regulating the tumor microenvironment. Presence of proteins in cell secretome also suggests that identified secretory protein marker might also serve as cancer biomarker. The projects end goal is to identify and validate a blood biomarker for early breast cancer detection. In the future, this knowledge can be translated towards the development of blood test kit for early breast cancer detection.

Humoral responses to the measles, mumps and rubella vaccine are slightly impaired in Leigh Syndrome French Canadian patients

Adrien Fois1,2, Anne-Marie Boucher-Lafleur3, Julie Thompson Legault4, Christian Renaud2, Charles Morin5, Christine Des Rosiers4, Lise Coderre1, LSFC Consortium6, Catherine Laprise3, Sylvie Lesage1,2

1Immunology-oncology Section, Maisonneuve-Rosemont Hospital Research Center, Montréal, Québec 2Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal, Québec 3Centre intersectoriel en santé durable, Université du Québec à Chicoutimi, Saguenay, Québec 4Research Center, Montreal Heart Institute, Montréal, Québec 5Hôpital de Chicoutimi, Chicoutimi, Québec

Leigh Syndrome French Canadian (LSFC) is a rare autosomal recessive metabolic disorder characterized by severe lactic acidosis crises and early mortality. LSFC patients carry mutations in the LRPPRC gene which lead to defects in the respiratory chain complexes and mitochondrial dysfunction. Mitochondrial respiration modulates cellular metabolic activity, which impacts many cell types including immune cell differentiation and function. Hence, we postulated that, in addition to neurological and metabolic disorders, LSFC patients may show impaired immune activity. To gain insight into the effectiveness of the immune response in LSFC patients, we examined the quality of the response to the measles, mumps and rubella (MMR) vaccine in LSFC patients by measuring antibody levels to MMR in the plasma. In a cohort of LSFC patients, the response to the MMR vaccine in LSFC patients was variable, with some individuals showing a complete response, while others had partial or ineffective responses. The results suggest that the mutations in the LRPPRC gene present in LSFC patients may affect the immune response to some vaccines. Vaccine recall responses in this fragile population should be considered to ensure full protection against pathogens.

Investigating the Mumps outbreaks in Canada through Whole Genome Sequencing

Jasmine Rae Frost1, Elizabeth McLachlan2, Joanne Hiebert2, Alberto Severini1,2

1Dept. of Medical Microbiology, University of Manitoba; 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB

Despite the fact that the Mumps vaccine has been in use in Canada for over three decades, the mumps virus (MuV) has remained endemic. Recent years have seen an increase in mumps outbreaks in vaccinated populations, particularly in young adults. These outbreaks have been almost exclusively comprised of genotype G infections. It is unclear what is driving these outbreaks, but some possible explanations are it could be due to a vaccine escape, or waning immunity. Our objective is to determine if the genotype G strains in these outbreaks are endemic in Canada, or due to multiple importations, as well as determine if there are factors (such as vaccination status) that are driving these outbreaks. Through the use of whole genome sequencing of Canadian outbreaks, combined with epidemiological data such as age, sex, vaccination status and geographical location we aim to identify if there is a vaccine escape MuV circulating in Canada. This will also allow us to identify if there are certain groups who may be more at risk for future outbreaks. We are focusing on the Manitoba 2016- 2018 outbreak (3000 cases) and the Newfoundland/Nova Scotia 2018 outbreak (150 cases). Using a novel whole genome sequencing method for mumps, we have sequenced 260 full genomes directly from clinical samples. Based on the phylogenetic and Bayesian analysis of these genomes, we see that the provincial outbreaks diverged separately from one another. This suggests that these outbreaks occurred independently, and were not the result of endemic transmission within Canada. Future work will continue focusing on determining if the genotype G strains are endemic in Canada, and work will be done on pairing the sequencing data to the epidemiological data, to identify factors that are driving the outbreaks. Overall this project will generate data that will be used to inform future vaccination and outbreak policy surrounding mumps. It will also identify characteristics of the mumps outbreaks that will allow us to combat future outbreaks more efficiently.

Double Dissociation of Two Pedunculopontine Neuronal Populations in Prepulse Inhibition of Startle and Reward

Niveen Fulcher1, Erin Azzopardi2, Cleusa De Oliveira2, Steven R. Laviolette1,2, Susanne Schmid1,2

1Neuroscience Graduate Program, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON

Introduction: The pedunculopontine tegmental nucleus (PPTg) is a midbrain structure involved in many innate functions, such as arousal, reward association, and sensorimotor gating. Although commonly referred to as a cholinergic structure, it also contains glutamatergic and GABAergic neurons. A longstanding assumption was that cholinergic projections to the reticular formation mediate prepulse inhibition (PPI) of the startle response. PPI deficits accompany many neuropsychiatric illnesses, such as schizophrenia, autism spectrum disorder (ASD), and Tourette’s Syndrome, yet underlying mechanisms are unclear. Additionally, literature suggests that cholinergic PPTg projections to the VTA mediate opioid-induced dopamine activation via VTA M5 muscarinic receptors. While chronic lesions of the PPTg have disrupted PPI and blocked conditioned place preference (CPP; i.e. reward association), selective inhibition of PPTg cholinergic neurons does not alter PPI. In this study, we aim to discern the involvement of PPTg cholinergic and glutamatergic neuron populations in reward association versus sensorimotor gating via PPI of startle testing.

Methods: We intracranially delivered either cholinergic- or glutamatergic-neuron specific inhibitory DREADDs (designer receptors exclusively activated by designer drugs) bilaterally into the rat PPTg. DREADDs are derived from engineered G- protein-coupled receptors (GPCRs) and are a widely used chemogenetic technique that allows for transitory inhibition/activation of specific cell types following administration of clozapine-N-oxide (CNO). Animals were tested for startle, PPI, and morphine-induced CPP after receiving a systemic injection of DREADD activator, CNO, or saline (control). Extensive validation and analyses were included to confirm DREADD efficacy and spread via immunofluorescence combined with fluorescence in situ hybridization (FISH).

Results: This study shows that DREADD inhibition of cholinergic PPTg neurons does not disrupt PPI, but impedes the acquisition of morphine-induced CPP. In contrast, transient inhibition of glutamatergic PPTg neurons significantly disrupts PPI, but has no effect on the acquisition of morphine-induced CPP.

Conclusions: Our results highlight, for the first time, a double dissociation between the role of PPTg glutamatergic and cholinergic neuron populations in reward association versus sensorimotor gating. These data further corroborate the evidence that cholinergic PPTg neurons do not mediate PPI, suggesting that instead glutamatergic PPTg neurons mediate PPI.

Determining health coverage eligibility for Ontario-born babies of people with precarious immigration status

Monica Gagnon1

1University of Toronto

Introduction: Across Canada, an estimated half-million people are living without legal immigration status. While many are long-term residents of Canada, they lack citizenship rights. However, their children, if born on Canadian soil, are lawful citizens of Canada. In Canada, healthcare coverage is a right of citizenship that is administered provincially. Current Ontario policy charges hospital staff with determining whether newborns are eligible for coverage through the Ontario Health Insurance Plan (OHIP), yet judgements appear arbitrary. OHIP is sometimes denied to these newborns. My doctoral research critically analyzes the discourses impacting OHIP eligibility decisions. My research objectives are to: (1) characterize the relevant dominant discourses in Canada; (2) examine the impact of these discourses on OHIP eligibility decisions in Ontario hospitals.

Methods: In this qualitative research study, I employ critical discourse analysis (CDA) as an analytical strategy, investigating the ideologies embedded in different discourses. My dataset includes interviews, policy and advocacy documents, and news media. My analysis is guided by the conceptual framework of bordering practices, which is used to understand how people deemed non-members are excluded from access to services or other forms of belonging within a society.

Results: Preliminary results indicate that dominant exclusionary discourses such as birth tourism impact OHIP eligibility decisions in Ontario hospitals, leading to restrictive decisions in hospital settings that inhibit access to OHIP for newborns. This in turn inhibits access to care for these children, placing an undue logistical, financial and health burden on families.

Conclusion: This research examines how people in Canada think, talk about, practice, and ultimately determine who is “deserving” of rights such as healthcare. The research is particularly important in Canada, where healthcare coverage is a valued aspect of the national identity, yet where the right to healthcare and the rights of people living without status are contested.

Siramesine and Lapatinib induce synergistic cell death in prostate cancer cells

Emily A. Garcia1,2, Elizabeth S. Henson2, Spencer B. Gibson1,2

1Research Institute of Oncology and Hematology, CancerCare Manitoba, Winnipeg, Manitoba, Canada 2Department of Biochemistry and Medical Ghossenetics, University of Manitoba, Winnipeg, Manitoba, Canada

Introduction: Prostate cancer is the most common cancer affecting men often resulting in aggressive development with poor prognosis. Novel therapeutic strategies include combinational treatments aiming to reduce the negative effects of chemotherapy, such as drug resistance. The use of lysosomotropic agents offers a new possibility since they disrupt lysosomal membranes and can trigger a series of events leading to cell death. In addition, combining lysosome disrupting agents with targeted inhibitors can induce cell death in different cancer types. Therefore, we hypothesized that lysosomotropic agents alone or in combination with tyrosine kinase inhibitors will induce cell death in prostate cancer cells.

Methods: siramesine and desipramine were tested in PC3, DU145, and LnCaP cells. To determine whether lysosome membrane permeabilization occurred after treatment, cells were stained with Lysotracker. To detect reactive oxygen species levels, cells were stained with dihydroethidium and Mitosox, respectively. Changes in mitochondria membrane potential were measured with tetramethylrhodamine. Lastly, cells were stained with C11 Bodipy to detect lipid peroxidation. All experiments were analyzed by flow cytometry and were repeated in combination with lapatinib. To determine whether reactive oxygen species were responsible for siramesine-induced cell death, we treated cells with the reactive oxygen species scavenger alpha-tocopherol.

Results: Siramesine was most effective drug in inducing cell death. Lysosome membrane permeabilization increased only slightly after treatment but we found reactive oxygen species levels significantly increased. These effects were accompanied by a decrease in mitochondrial membrane potential and an increase in lipid peroxidation levels. When alpha-tocopherol was added before treatment, cell death decreased. Furthermore, the combination of siramesine and lapatinib induced synergistic cell death in all cancer cell lines tested.

Conclusion: Our results suggest siramesine alone and in combination with lapatinib were significantly effective at inducing cell death in all prostate cancer cell lines tested. These effects were more prominent in PC3 cells and therefore, hold the potential to treat advanced prostate cancer.

High containment pathogen infection in Brown Adipose Tissue

Lauren Garnett1,2, Bryan D Griffin1, Kaylie N Tran1, Bryce, Anders Leung1, Darwyn Kobasa1, Jim Strong1,3 M Warner1

1Department of Medical Microbiology and Infectious Diseases, Faculty of Health Sciences, University of Manitoba, 2Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Canada, 3Department of Paediatrics and Child Health, College of Medicine, Faculty of Health Sciences, University of Manitoba

Ebolavirus (EBOV) is a highly lethal zoonotic infection from the filoviridae family, with a fatality rate up to 90% in humans. The reservoir host species of EBOV is currently unknown; however, it is suspected to be African fruit bats as Marburgvirus, from the same virus family, has been isolated from this species. In addition to filoviruses, bats are a well-recognized reservoir for numerous other pathogens including rabies, henipaviruses and coronaviruses. Some of these pathogens have been found to persist within the adipose tissue of the host bats and be reactivated under certain conditions. A main stimulant of this reactivation, which could aid in cross species transmission, is temperature and climate change. For these pathogens, this has been currently attributed to brown adipose tissues metabolic capacity and distinct ability for burning energy to produce heat. However, it is unknown whether EBOV infects or persists within adipose tissue or if metabolic changes such as temperature are leading to altered infectious shedding and potential spillover events. We hypothesize that EBOV persists within the brown adipose tissue depots and that altering the metabolism of these cells can lead to re-emergence from persistence causing increased infection and viral shedding. This is important as EBOV is a highly fatal pathogen that has been identified as an agent of biodefence concern with pandemic potential. Preventing cross species transmission and emergence of new zoonotic viruses such as EBOV relies on understanding determinants for spillover risk. Therefore, identifying reservoir tissues in host species and understanding factors contributing risk of zoonotic spillover events is critical.

Rectal microbiota diversity in Kenyan men who have sex with men (MSM) is inversely associated with frequency of receptive anal sex, independent of HIV status

Henok GEBREBRHAN1, Cheli KAMBARAN1, Aida SIVRO1,2,3, Wendy ADHIAMBO4, Naomi SIELE4, Michael G. BECKER5, Jie LI5, Sandra CHOI1, Ruth S. MWATELAH1, N. Vincent REYES5, Maureen AKOLO4, Peter NJOGU4, François CHOLETTE1,5, John HO5, John KIM5, Shelley W. PETERSON6, Irene MARTIN6, Paul SANDSTROM1,5, Supriya D. MEHTA7, Robert R. LORWAY8, T. Blake BALL1,5, Joshua KIMANI1,4, Paul J. MCLAREN1,5, Hezhao JI1,5, Lyle R. MCKINNON1,2,4,5

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba 2Centre for the AIDS Programme of Research in South Africa (CAPRISA) 3Department of Medical Microbiology, University of KwaZulu-Natal, Durban, South Africa 4University of Nairobi Institute of Tropical and Infectious Diseases, University of Nairobi, Nairobi, Kenya 5JC Wilt Infectious Disease Research Centre National Microbiology Laboratory, 6Public Health Agency of Canada, Winnipeg, Manitoba, 7Canada School of Public Health, University of Illinois at Chicago, Chicago, Illinois, USA. 8Institute for Global Public Health (IGPH), University of Manitoba, Winnipeg, Manitoba, Canada.

Objective: Both HIV infection and identifying as MSM have been linked to altered rectal microbiota composition, but few studies have studied sexual behavioural associations with rectal microbiota within MSM. In addition, most rectal microbiota studies in MSM have been limited geographically to Europe and North America, and replication of findings in lower- and middle-income countries is lacking.

Methods: We enrolled MSM from Nairobi, Kenya, and determined their HIV/STI status. Rectal specimens were obtained for 16s rRNA sequencing of the rectal microbiota, and sexual behaviour was characterized using a standardized questionnaire. Microbiome differences were modelled using non-parametric statistics, Bray-Curtis ecological distance metrics, and analyses of differential taxa abundance. Multivariable linear regression was used to model HIV status and recent sexual activity as predictors of alpha diversity, controlling for a range of covariates.

Results: Alpha diversity was consistently lower in Kenyan HIV-infected MSM (n = 80), including those on antiretroviral therapy (ART) compared to HIV uninfected MSM. A statistical trend was observed for clustering of HIV status by Prevotella or Bacteroides dominance (p=0.13). Several taxa were enriched in HIV+ men including Roseburia, Lachnospira, Streptococcus, and Granulicatella. Receptive anal sex with several types of sexual partners (paying, regular, casual) was associated with lower Chao1 and Simpson diversity, independent of HIV status, while HIV infection was associated lower Chao1 (p=0.030) but not Simpson diversity (p=0.49).

Conclusions: Both HIV infection and sexual behaviour-related were associated with rectal microflora alpha diversity, in particular richness, but not Prevotella spp. dominance, in Kenyan MSM. Associations were more robust for sexual behaviour.

The metabolic pattern that makes trefoil factor family member 2 (Tff2) KO protected from high- fat diet-induced obesity

Abdelaziz Ghanemi1,2, Aicha Melouane1,2, Octave Mucunguzi1,2, Mayumi Yoshioka1, Jonny St-Amand1,2

1Functional Genomics Laboratory, CREMI, Québec Genome Center, CHUL-CHU de Québec Research Center, Québec, Québec G1V 4G2, Canada 2Department of Molecular Medicine, Faculty of Medicine, Laval University, Québec, Québec G1V 0A6, Canada

Introduction: Trefoil factor family member 2 (TFF2) is a small gut peptide. We have previously shown that Tff2 knock out (KO) mice are protected from high-fat (HF) diet-induced obesity (De Giorgio et al., 2013a). Thus, exploring Tff2 KO-related pathways of mice at the genomic, proteinic and biochemical levels would allow us to elucidate the processes behind this protection from obesity.

Methods: To explore the metabolic and energetic effects related to Tff2 deficiency, we used sampled blood from the previous study to measure levels of free fatty acids, glucose, glycerol and triglycerides in serum. Expression levels of selected genes and proteins related to energy metabolism in the skeletal muscle, liver and adipose tissue were also studied.

Results: Following the 12-wk challenging of Tff2 KO and WT mice with both HF and low-fat diet, Tff2 KO mice had lower levels of serum glucose, triglycerides and glycerol. Importantly, western blotting and Q_RT-PCR revealed that the expression levels of selected genes and proteins are toward less fat storage and increased energy expenditure by enhancing lipid and glucose utilization via oxidative phosphorylation.

Conclusion: We mapped a part of the metabolic and biochemical pathways of lipids and glucose involving the adipose tissue, liver, skeletal muscle and sympathetic nervous system that protect Tff2 KO mice from the HF dietinduced obesity. Our data highlight Tff2-related pathways as potential targets for obesity therapies.

Secondary Prevention of Anal Cancer among Men living with HIV: Acceptability and Suitability of Anal Pap Cytology and HPV DNA Type Testing

Jennifer L Gillis1, Janet M Raboud1, Aisha Lofters2,3, Olli Saarela1, Ann N Burchell1,2,4

1Dalla Lana School of Public Health, University of Toronto, Ontario Canada 2Department of Family and Community Medicine, Faculty of Medicine, University of Toronto, Ontario, Canada 3Women’s College Research Institute, Women’s College Hospital, Ontario, Canada 4MAP Centre for Urban Health Solutions, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Unity Health Toronto, Ontario, Canada

The risk of anal cancer is magnitudes higher among men living HIV than the general population. To contribute to an evidence-base in support of anal cancer screening guidelines, this dissertation evaluates the acceptability of screening among men living with HIV and sets the foundation for work examining the suitability of anal cytology and HPV DNA type testing for screening this population.

A sample of 1677 men living with HIV enrolled in the Ontario HIV Treatment Network Cohort study completed a questionnaire module on HPV, its associated diseases, and their prevention in 2016-2017. Three objectives examined acceptability of anal cancer screening by 1) quantifying prevalence of screening, 2) examining the role of past screening on beliefs and willingness to be screened, and 3) evaluating facilitators to screening, HPV knowledge and self-perceived risk for anal cancer. In this sample, disparities in anal cancer screening were observed, wherein men from some racialized groups and heterosexual men were less likely to have discussed screening or to have been screened. The vast majority (90%) of men, however, were willing to undergo screening in the future. Positive beliefs regarding screening were associated with higher willingness. Behavioural intention does not ensure uptake, however, and factors such as health literacy and perceived susceptibility can influence men’s participation. Half of the study sample were unaware of HPV and the majority (73%) felt they had no or low chance of getting anal cancer. Specific risk-focused knowledge was associated with higher perceptions of risk. Findings will inform community-led health literacy efforts to improve participation in anal cancer screening.

The final objective provides methodological guidance for the analysis of screening studies when verification of screening results is not possible for all participants. This simulation study assessed the utility of statistical methods to account for verification bias that arises in these scenarios. Findings suggest multiple imputation may be most suitable for handling complex missing data scenarios common to screening studies. This work will inform the analysis of data from a forthcoming anal cancer screening study to determine the best algorithm by which to screen for anal cancer among men living with HIV.

Impact of differential gene expression in inflammatory pathways on HIV susceptibility

Shanelle N Gingras1,2, Jeffrey Tuff2, Naima Jahan1, Slim SA Karim3, Quarraisha A Karim3, Lenine J Libenberg3, Jo-Ann S Passmore3,4, Lyle R McKinnon1,3, Paul J McLaren1,2

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada 2JC Wilt Infectious Diseases Research Centre, Public Health Agency of Canada, Winnipeg, Canada 3Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa 4University of Cape Town, Cape Town, South Africa

Introduction: The Centre for the AIDS Programme of Research in South Africa (CAPRISA)-004 trial assessed the efficacy of a 1% tenofovir gel in preventing sexual transmission of HIV. Tenofovir gel reduced transmission by 54% overall; however, the efficacy was ablated in women with genital inflammation. Systemic and mucosal inflammation increase HIV susceptibility through recruitment of HIV target cell to the site of exposure, causing mucosal barrier damage and increased availability of HIV susceptible cells. Many extrinsic factors that contribute to inflammation have been well studied (e.g. age, condom use, STIs). However, the contribution of intrinsic host genetic factors has not been adequately explored. Studies of immune function in healthy populations have shown large contribution of genetic variability to gene expression in inflammatory cell populations, many of which have been linked to chronic diseases. Similar studies in the context of HIV have yet to be done.

Hypotheses: Monocytes from individuals exhibiting inflammatory cytokine profiles at the female genital tract differentially express genes within known inflammatory pathways. This differential gene expression is regulated by genetic variants whose frequency differs in individuals that do and do not exhibit inflammatory phenotypes. Such variants will also influence an individual’s risk of HIV acquisition.

Methods: Monocytes isolated from peripheral blood mononuclear cells from 60 participants in the CAPRISA-004 trial have been allocated to three stimulation conditions (LPS, IFN-b, unstimulated) and RNA has been isolated for RNA Sequencing. We have approximately 80% power to detect genes differentially expressed 2.5-fold between groups, considering two phenotypes; mucosal inflammation (N=17 high inflammation; 43 low inflammation) and HIV acquisition (N=28 seroconverters; 32 HIV-). Significantly differentially expressed genes will be investigated for genetic regulation using an expression quantitative trait locus (eQTL) framework comparing genome-wide genotype data to gene expression level. Significantly associated eQTLs will be tested for association with HIV acquisition in CAPRISA-004 and an independent cohort.

Significance: This study will increase the representation of African populations in genomic research, while determining the genetic contribution to inflammation and thereby HIV susceptibility. We expect that host genetics is an important driver of inflammation. Targeting these variants with pharmaceutics can reduce future HIV infections.

Characterization of MERS-CoV monoclonal antibodies as antibody-based therapeutics

Nathan Glowach, Teresa Cabral, Darwyn Kobasa

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba; 2Public Health Agency of Canada, Winnipeg

Middle-Eastern Respiratory Syndrome Coronavirus emerged in Saudi Arabia in 2012 and has become endemic in regions of the Middle East. Although the rate of transmission in humans is relatively low, the high mortality rate (~35%) is concerning. Further, there are no licensed vaccines or therapeutics to treat individuals infected with the virus. In a natural viral infection, the humoral immune response produces neutralizing antibodies that bind to the virus and prevent it from entering host cells. Researchers have been successful in developing neutralizing monoclonal antibodies that can be used to treat MERS- CoV infection. However, the receptor binding domain (RBD) of the spike protein (which is the primary target of antibody- based therapeutics) is under high selective pressure, resulting in mutations in the RBD-dipeptidyl peptidase 4 (DPP4) receptor interface. This lowers the efficacy of these treatments. Our approach is to fully characterize a set of six neutralizing monoclonal antibodies targeting the RBD of the MERS-CoV spike protein, which were purified from murine hybridoma cell lines. The neutralizing activity of the antibodies will be confirmed using a standard virus neutralization assay. We will also perform an evaluation of the cross-neutralization activity of the antibodies with other pathogenic coronaviruses (i.e. SARS- CoV, SARS-CoV-2). Additionally, we aim to identify specific amino acid residues which are essential to antibody binding and neutralization through epitope mapping. Lastly, the binding affinity of the antibodies will be assessed using bio-layer interferometry (BLI) and surface plasmon resonance (SPR) technologies. Downstream, we intend to humanize our antibodies and evaluate their therapeutic potential using a transgenic mouse model that can be infected with MERS-CoV. The full characterization of novel neutralizing antibodies is essential for the discovery of more efficacious therapeutics to complement those previously published for MERS-CoV infection, thereby reducing the currently high mortality rate of this deadly virus.

Biochemical Characterization and Inhibition of SARS-CoV-2 Polymerase

Calvin Gordon1#, Egor Tchesnokov1#, Emma Woolner1, Dana Kocinkova1, Jason Perry2, Joy Feng2, Danielle Porter2, Matthias Götte1,3

1 Department of Medical Immunology and Microbiology, University of Alberta, Edmonton, Canada 2Gilead Sciences, Inc. Foster City, CA, USA 3Li Ka Shing Institute of Virology at University of Alberta, Edmonton, Canada #CG and ET contributed equally to this work

Introduction: Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID- 19, has brought the coronavirus family to the global public health forefront. The RNA-dependant RNA polymerase (RdRp) is a crucial component of the SARS-CoV-2 replication machinery, directing genome replication and transcription. RdRp is the target for the nucleotide analogue prodrug remdesivir (RDV), i.e the only approved antiviral small molecule drug for the treatment of COVID-19 in the US and Canada.

Methods: We utilized the viral protease and co-expressed the non-structural proteins nsp7, nsp8, and nsp12 in insect cells to yield an active SARS-CoV-2 RdRp complex. RNA synthesis was monitored on short model primer/template substrates mimicking a random elongation complex.

Results: Kinetic analysis of the RdRp revealed that the triphosphate form of RDV (RDV-TP) is incorporated more efficiently than its natural counterpart ATP. Furthermore, the incorporation of RDV-TP at position i resulted in RNA synthesis inhibition following the incorporation of 3 additional nucleotides (i+3), suggesting that a plausible mechanism of action is “delayed chain termination”. Modelling studies point to serine 861 as the residue responsible for a clash with the inhibitor at i+3. The 1’-CN moiety of the incorporated RDV-TP comes into close proximity with the side chain of S861. Mutagenesis of S861 to a glycine eliminates delayed chain termination. The result of this study reveals the structural requirements for delayed chain- termination and suggest that mutations at position 861 can reduce susceptibility to RDV. Finally, a second mechanism of RDV inhibition along with a mechanism of resistance was determined as a result of RDV-TP incorporated into the template- strand.

Conclusion: RDV inhibits the SARS-CoV-2 RdRp via two independent mechanisms, delayed chain termination in primer- strand synthesis and template-dependent inhibition. Moreover, characterizing already identified in vitro RDV resistance mutants provided the link between biological and biochemical antiviral assays.

Cell lines to model LAG3 inhibition of T cell activation

Colin G Graydon1, Shifa Mohideen1, Jason Kindrachuk1, Keith R Fowke1,2,3,4

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba 2Department of Community Health Sciences, University of Manitoba 3Department of Medical Microbiology, University of Nairobi 4Partners for Health and Development in Africa

Introduction: LAG3 is an immune checkpoint, a negative co-receptor upregulated during chronic activation of lymphocytes. This upregulation occurs during autoimmunity, chronic infection and cancer, during which LAG3 regulates the immune response. LAG3 blocking antibodies are currently in clinical trials to manipulate this role. However, despite its importance, LAG3 is poorly understood relative to other immune checkpoints. While other immune checkpoints have conserved inhibitory sequences in their cytoplasmic domains, LAG3 has none that are known. Therefore, LAG3’s mechanism of inhibition is a black box. We hypothesize that LAG3 inhibits early TCR signaling events.

Methods: Through lentiviral transduction we created Jurkat T cells that express either full-length LAG3 (LAG3+), a LAG3 mutant lacking the cytoplasmic domain (LAG3ΔCY), or do not express LAG3 (LAG3-). Confirmation of LAG3 expression was done by flow cytometry. We activated these cells using Staphylococcal enterotoxin E or D (SEE or SED) coated Raji B cells. IL-2 is measured by ELISA and phosphorylation events are measured using a kinomics array combined with phospho-flow cytometry.

Results: LAG3 was expressed on a majority of LAG3+ cells, but expression was low on LAG3ΔCY cells. Interestingly, activation with SEE restored LAG3 expression to regular LAG3+ levels. Confirming the function of LAG3, IL-2 production after SEE or SED activation was reduced by half on LAG3+ cells, but not on LAG3ΔCY cells, when compared to LAG3- cells. Changes in kinase activity was also observed between cells expressing full-length LAG3 and those expressing mutant LAG3 or no LAG3.

Conclusion: The created model demonstrates LAG3 inhibitory function and demonstrates the inhibition of kinases involved in T cell receptor signaling. In the absence of cellular activation, LAG3 surface expression is dependent on its cytoplasmic domain.

Annual Trends in Prevalence and Incidence of Hepatitis-C Virus Infection in Manitoba between 1998 and 2018

Sai Krishna Gudi1, Sherif Eltonsy1, Joseph Delaney1,2, Carla Osiow3,4, Silvia Alessi- Severini1

1College of Pharmacy, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada 2Department of Epidemiology, University of Washington, Seattle, WA, USA 3National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada 4Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada

Background: Hepatitis-C virus (HCV) infection is a major cause of liver-related morbidity and mortality worldwide. Epidemiological data of HCV infection in the Canadian province of Manitoba are limited.

Objective: To determine trends in annual prevalence and incidence of HCV infection in Manitoba between 1998 and 2018. Methods: A population-based retrospective study was conducted using data from the Manitoba Centre for Health Policy (MCHP) repository. Using the test results provided by the Cadham provincial laboratory (CPL), individuals in Manitoba with any diagnosis (acute and chronic) of HCV infection (based on positive HCV RNA) were identified. Demographic characteristics of the population were described. Annual prevalence and incidence rates (crude and standardized) were calculated for the overall population and stratified by sex, regional health authority (RHA), residence area (urban vs. rural), and income quintile.

Results: A total of 8,721 HCV cases were diagnosed between 1998 to 2018 in Manitoba with a mean age of 40.5 years ± 13.6 (SD). The overall crude HCV incidence and prevalence were estimated as 0.03% and 0.37% during the study period, respectively. No significant change was observed in the standardized HCV incidence rate (per 100,000) during the study period (54.3 in 1998 and 54.8 in 2018). The standardized HCV prevalence (per 100,000) increased from 52.5 in 1998 to 655.2 in 2018. In all study years, HCV prevalence based upon sex, RHA, region, and income was observed to be higher in males (747.8 per 100,000), Winnipeg RHA (792.1 per 100,000), urban region (782.8 per 100,000), and low-income quintiles (1481.7 per 100,000).

Conclusions: Although incidence rates of HCV infection in Manitoba appeared to have initially declined, rates showed an upward trend by the end of the study period (1998-2018). Prevalence increased steadily. Further studies will look at HCV medication utilization in the population of Manitoba.

CORM-2 preconditioning ameliorates apoptosis of OPCs via mitochondrial-derived vesicles quality control

Ying Guo, Teng Guan, Sida Sun, Chunting Jin, Guohui Zhang, Jiming Kong

1Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, MB, Canada 2Department of Forensic Medicine, Hebei North University, Zhangjiakou, Hebei, China

CORM-2 preconditioning ameliorates apoptosis of OPCs via mitochondrial-derived vesicles quality control Ying Guo1,2, Teng Guan1, Sida Sun2, Chunting Jin2, Guohui Zhang2, Jiming Kong1 1Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, MB, Canada. 2Department of Forensic Medicine, Hebei North University, Zhangjiakou, Hebei, China. Introduction: Carbon monoxide (CO) has been thought to be neurotoxic to white matter which could induce delayed neurologic sequelae. However, recent studies have also proved that Carbon monoxide releasing molecule-2 (CORM-2), a donor for carbon monoxide, prevents axon demyelination and oligodendrocyte precursor cells (OPCs) apoptosis, but the mechanisms are still obscure. Mitochondrial-derived vesicles (MDVs) are recently discovered as earlier Mitochondrial quality control (MQC) to oxidative stress. Here, we investigated the role of MDVs in CORM-2 precondition and the protective mechanism. Methods: Mitochondria were observed by live-cell imaging, immunofluorescent microscopy, super-resolution structured illumination microscopy, and electron microscopy in Chinese hamster ovary (CHO) and primary cultured OPCs. Mitochondrial function was assessed by Mito Tracker Red and JC-1 test. Sodium dithionite (Na2S2O4) with EBSS for 3 hours was applied to mimic the Oxygen and Glucose Deprivation (OGD) condition for CHO cells and 500μM CORM-2 for 6 hours as severe oxidative stress for OPCs. ROS generation in mitochondria and whole-cell was evaluated separately by Mito SOX and DCFDA assay. Cell proliferation and viability were quantified by the WST test. Results: CORM-2 could stimulate MDV formation in both Chinese hamster ovary (CHO) cells and OPCs without interfering with mitochondrial homeostasis. However, inhibition of lysosome activity disturbed MDV quality control indicated by condensed mitochondria. Further studies have shown that preconditioning with CORM-2 prevented OPCs apoptosis under the toxicity condition of CORM-2 and decrease the nuclear translocation of AIF (P < 0.0001). Conclusion: Our study provides direct evidence that preconditioning with CORM-2 promotes MDV formation without interfering with mitochondrial morphology and function which can ameliorate severe oxidative damage to OPCs.

Real-time neural feedback of mesoscale cortical GCAMP6 signals for training mice

Pankaj K. Gupta1, Timothy H. Murphy1

1University of British Columbia

Introduction: Mice can learn to control specific neuronal ensembles using sensory (eg. auditory) cues (Clancy et al. 2014) or even artificial optogenetic stimulation (Prsa et al. 2017). In the present work, we measure mesoscale cortical activity with GCaMP6s and provide graded auditory feedback (within ~100 ms after GCaMP fluorescence) based on changes in dorsal- cortical activation within specified regions of interest (ROI)s with a specified rule.

Methods: We define a compact, low-cost optical brain-machine-interface (BMI) capable of image acquisition, processing, and conducting closed-loop auditory feedback and rewards, using a Raspberry Pi. The changes in fluorescence activity (ΔF/F) are calculated based on a running baseline (eg. 5 sec.). Two ROIs (R1, R2) on the dorsal cortical map were selected as targets. We started with a rule of ‘R1-R2’ (ΔF/F of R1 minus ΔF/F of R2) where the activity of R1 relative to R2 was mapped to frequency of the audio feedback and if it were to cross a set threshold, a water drop reward is generated. To investigate learning in this context, water-deprived tetO-GCaMP6s mice (N=8) were trained for 30-minutes every day on the system for several days, with a task to increase audio frequency leading to reward.

Results: We found that mice could modulate activity in the rule-specific target ROIs to get an increasing number of rewards over days. Analysis of the reward-triggered ΔF/F over time indicated that mice progressively learned to activate the cortical ROI to a greater extent.

Conclusion: We developed an open-source system for closed-loop feedback that can be added to experimental scenarios for brain activity training and could be possibly effective in inducing neuroplasticity.

Employment and Accommodation Needs in Individuals with Traumatic Brain Injury: A Pilot Study

Sara Hanafy1, Angela Colantonio1, Sarah Munce2, Sally Lindsay3

1Rehabilitation Sciences Institute, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada 2Toronto Rehabilitation Institute, University Health Network, Toronto, Ontario, Canada 3Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, Ontario, Canada

Introduction: Traumatic brain injury (TBI) is a leading cause of disability worldwide. Sex and gender influence employment in TBI. A large facilitator to employment in TBI is having workplace accommodation, however in many cases accommodations are unavailable or may not fit the needs of the individual. Further, it is unknown how the Coronavirus Disease 2019 (COVID-19) pandemic is impacting employment and accommodations for persons with TBI. This study aims to investigate sex and gender specific workplace accommodations in persons with TBI, while considering the impact of COVID- 19 on transitioning to work and on mental health in adults with TBI.

Methods: The proposed research is a pilot study with an observational cross-sectional design. Sixty adults with TBI, including men, women and gender diverse people within the age range of 18-65 years inclusive, will be recruited. An online survey will be self-administered through Research Electronic Data Capture. The survey includes questions on demographics (e.g., sex, gender, age, ethnicity, injury severity, mechanism of injury); questions from the Canadian Survey on Disability 2017 on employment status, requirements and unmet needs for workplace accommodations; and questions from Statistics Canada on the impact of COVID-19 on work status.

Results: Data collection is in progress. Planned analyses include multinomial logistic and multivariable linear regression analyses to evaluate the relationships between the predictor (i.e., sex, gender) and main outcome variables (i.e., the number and type of accommodations needed, change in employment status and mental health due to COVID-19). Descriptive statistics, between-group comparisons for sex and gender, and sex-specific and gender-specific stratification will be completed to understand emerging trends.

Conclusion: Sex and gender influences in TBI can serve to inform rehabilitation professionals, employers and persons with TBI, to enable sex- and gender-sensitive interventions for community participation practices. Findings from this study will contribute to the body of evidence on sex- and gender-specific workplace accommodations, while bridging the knowledge gap of how to improve transition to work in persons with TBI. Results will also further the understanding of the specific needs of men, women and gender-diverse persons with a disability during community participation post-discharge, including during unprecedented times.

Development of an Educational Virtual Reality Simulation to Reduce Medication Administration Errors: Preliminary Results from a Needs Assessment

William Harding1

1University of British Columbia

Background: There is a pressing need for high-quality simulations that are effective, cost-efficient, and scalable to accelerate health professional training due to predicted health professional shortages and evolving clinical practice guidelines. Virtual reality has been proposed as a potential solution to provide affordable simulation experiences in nursing. While virtual reality simulation research has been increasing over the past 20 years, recent reviews have suggested that there is a limited body of evidence rigorously examining their effectiveness in nursing. It is critical that virtual reality simulations are developed to target behaviors that have the greatest impact on measurable clinical outcomes to support rigorous testing of their educational effectiveness.

Methods: Following the INACSL Standards of Best Practice for Simulation Design, a needs assessment was conducted to guide the development of an educational virtual reality simulation. Health professionals involved in a Medication Safety Team from Health Authority in British Columbia were recruited. Data were collected using semi-structured interviews and supplemented with reflective field notes. Data were analyzed using content analysis and presented at a subsequent meeting alongside technical simulation design considerations to support the simulation development and validate the findings.

Results: Preliminary findings suggest that the simulation should target specific behaviours related to safe medication administration practices. This includes training nurses to dismiss distractions while preparing medications, verify concentrations of ‘high-risk’ medications, and strictly follow independent double-check protocols. To promote these desired behaviours, the development of dialogue and decision choices within the simulation were proposed. Data also suggested that the simulation should also orientate new graduate nurses to their workplace and workflow, identify and address gaps in knowledge and skills, provide individual feedback, and produce reports that clinical nurse educators can use to better support new graduate nurses.

Conclusion: Educational virtual reality simulations designed to minimize medication administration errors should provide nurses with opportunities to consolidate their medication administration competencies by replicating realistic clinical practice situations and promoting safe medication administration behaviours.

Gene expression profiles of interstitial lung disease: a systematic review and meta-analysis

Daniel He1,2, Sabina A. Guler3, Casey P. Shannon2, Scott J. Tebbutt1,2, Christopher J. Ryerson1

1Department of Medicine, University of British Columbia 2PROOF Centre, Vancouver, BC 3University of Bern

Introduction: Fibrotic interstitial lung diseases (ILDs) are a group of >200 diseases characterized by excessive deposition of extracellular matrix in the lung interstitium, resulting in decreased oxygen uptake into the blood. Numerous studies have generated gene expression profiles of individual ILD subtypes through the use of high-throughput ‘transcriptomics’ technologies that measure the entire genome, and have identified numerous molecular changes that may be representative of the pathophysiology of specific ILD subtypes. However, a consensus on the aberrant molecular processes that differ between ILD subtypes has yet to be determined; transcriptomics studies have been frequently limited by small sample sizes due to the high cost.

Methods: To address this gap in knowledge, we have screened 5,337 publications and identified 33 relevant studies to conduct our meta-analysis. We extracted microarray and RNA-seq data from the Gene Expression Omnibus (GEO), and integrated data using the Multivariate INTegration (MINT) framework.

Results: A majority of the studies focused on the ILD subtype idiopathic pulmonary fibrosis (IPF). Using the data from these studies, we used MINT to develop a classification model of IPF that had an AUC of 0.940 on our training datasets (n=17) and an AUC of 0.9541 on our test dataset. Within the IPF model, 12 genes were identified as explaining the most variance between IPF and control subjects, most of which are associated with fibrosis (e.g. COL3A1, COL17A1). Interestingly, we identified 2 genes (MYRF and ADRB2) that have not been previously implicated in the disease mechanisms of any ILD subtypes.

Conclusion: The preliminary results from this systematic review and meta-analysis confirm previous fibrotic processes of IPF, but also highlight new mechanisms of disease that may have been overlooked in single studies.

Composite Adverse Outcomes in Obstetric Studies: A Systematic Review

Dylan Herman1,2, Kar Yee Lor4, Abdul Qadree5, Daphne Horn6, Rohan D’Souza2,3

1Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. 2Division of Maternal and Fetal Medicine, Department of Obstetrics and Gynaecology, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Room 3-908, Toronto, Ontario M5G 1X5, Canada. 3Lunenfeld-Tanebaum Research Institute, Mount Sinai Hospital, Toronto,Ontario, Canada. 4Faculty of Medicine, University of Aberdeen, Aberdeen, Scotland, UK. 5Department of Chemical and Physical Sciences, University of Toronto, Toronto, Ontario, Canada. 6Centre for Addiction and Mental Health (CAMH), Toronto, Ontario, Canada.

Introduction: Composite outcomes are increasingly being used in obstetric trials. The aim of this systematic review is to critically appraise the use of composite outcomes in obstetric RCTs with an intention of identifying limitations and providing potential solutions for future research.

Methods: The study protocol was prospectively registered. Medline, Embase, Cochrane Databases and www. clinicaltrials.gov were searched for randomized controlled trials (RCTs) published in English between 1999 and 2019, using search terms related to pregnancy and composite outcomes. Study eligibility criteria: RCTs involving an obstetric condition that reported on a composite outcome. Study appraisal and synthesis methods: Screening and data extraction were performed in duplicate, and a descriptive synthesis and critical appraisal of composite obstetric outcomes, is presented.

Results: Of the 4170 results screened, we identified 156 RCTs, reporting on 181 composite outcomes. Of these, 158 composite outcomes related to general morbidity and mortality, either exclusively maternal (n=20), fetal-neonatal [perinatal] (n=116) or maternal and perinatal (n=22) were included in the final analysis. Obstetric composite outcomes included between two and 16 components. Components that comprised these composite outcomes were often dissimilar in terms of severity and frequency of occurrence, unlikely to have similar relative risk reductions and sometimes unrelated to the study’s primary objective – important pre-requisites to consider while constructing composite outcomes. In addition, composite adverse obstetric outcomes often do not incorporate the perspectives of pregnant persons, embrace a holistic view of health or consider outcomes related to both members of the mother-fetus dyad.

Conclusions: Composite outcomes are being increasingly used as primary outcomes in obstetric RCTs, based on which study conclusions are drawn and clinical recommendations made. However, there is a lack of consistency with regard to what components should be included within a composite adverse obstetric outcome and how these components should be measured. The use of novel research methods such as concept mapping may be able to address some of the limitations with the development of composite adverse obstetric outcomes, to inform future research.

Insights from Oxygenation-Sensitive Cardiovascular Magnetic Resonance Imaging: A Novel Non- Invasive, Needle-Free Assessment of Coronary Vascular Function

Elizabeth Hillier1,2, Matthias G. Friedrich1,3

1Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada 2Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 3Departments of Medicine and Diagnostic Radiology, McGill University, Montreal, QC, Canada

Introduction: Cardiovascular magnetic resonance imaging (CMR) is the current gold standard imaging modality for the quantitative assessment of left ventricular function, with recent advances such as tissue characterization allowing for a non- invasive, “virtual biopsy” and assessment of myocardial tissue based on the different magnetic properties of tissue. However, one of the main limitations against the widespread adoption of CMR is its dependence on extrinsic agents. Oxygenation-Sensitive Cardiac Magnetic Resonance Imaging (OS-CMR), and its utilisation of the inherent magnetic field properties of deoxyhemoglobin, has overcome these limitations and revolutionized the potential of CMR. OS-CMR delivers a needle-free, non-invasive imaging test. OS-CMR is able to harness the inherent differences in magnetic susceptibility between oxy- and deoxyhemoglobin into an MRI signal intensity. Used in combination with validated, vasoactive breathing maneuvers, OS-CMR can dynamically evaluate the myocardial oxygenation reserve in cardiovascular disease states.

Methods: Vasoactive breathing maneuvers consisting of a 60s period of paced hyperventilation, inducing coronary vasoconstriction, followed by a voluntary maximal breath-hold, inducing coronary vasodilation, were utilized in combination with OS-CMR to investigate Coronary Artery Disease (CAD) (n=18), women presenting with symptoms of Ischemia with No Obstructive Coronary Artery disease (INOCA) (n=20), and Heart Failure (HF) (n=20) patient populations.

Results: A composite marker derived from OS-CMR with breathing maneuvers has shown a reduced oxygenation, function, and heart rate response in areas downstream of an obstructive coronary stenosis. Women presenting with ischemia and found to have no obstructive coronary artery stenosis were found to have a heterogenous myocardial oxygenation response characterized by regional areas of impaired oxygenation. Heart failure patients were found to have a globally impaired oxygenation reserve in both the heart and brain when compared to age-controlled healthy volunteers.

Conclusion: Impaired endothelial dysfunction, reflected in a reduced myocardial oxygenation reserve, may have important clinical and therapeutic consequences in macrovascular and microvascular disease states. Adding only four minutes to a regular CMR scan, breathing maneuvers in combination with OS-CMR images appear as a safe and efficient diagnostic approach for a onestop, comprehensive, and simultaneous assessment of coronary vascular function and associated ventricular function.

Role of Machine Learning in Ultrasonic Characteristcs of Bone Like-Porous Material

Mohammad Hodaei1, Pooneh Maghoul1

1University of Manitoba

Ultrasonic characteristics of human cancellous bone using Artificial Neural Network (ANN) and solving the inverse problem is considered. The human cancellous bone is simulated using a two-infinite length porous slab in which the medium pores are filled with a relatively low and high viscous fluid such as air and bone marrow respectively. The ultrasonic wave propagation in the porous slab is modeled based on Biot’s theory modified by JKD model to consider the effect of tortuosity, especially in the clinically relevant ultrasound frequency range. The discrepancy of the analytical theory and ANN’s results are minimized to calculate inversely the required parameters of the medium which are so sensitive to transmission and reflection. The inverted parameters are: viscous characteristic length, tortuosity, porosity, Poisson ratio, and Young modulus of the porous slab. It is discovered here that estimating Young modulus and Poisson’s ratio of the medium are more reliable on longitudinal waves alone regardless of the viscosity of the medium while considering both longitudinal and transverse waves to estimate viscous characteristic length, tortuosity, and porosity for medium filled with a relatively high viscous fluid is so essential.

Towards a Genome-wide Fingerprint of Antibiotic Resistance Determinants in the Cystic Fibrosis Pathogen Burkholderia cenocepacia K56-2

Andrew M Hogan1, Silvia T Cardona1

1Department of Microbiology, University of Manitoba

Introduction: Patients with cystic fibrosis experience re-occurring polymicrobial pulmonary infections that are a leading cause of mortality. Among the infecting bacteria, Burkholderia cenocepacia causes a rapidly progressing form of necrotizing pneumonia and bacteremia known as “cepacia syndrome”, with few treatment options due to high intrinsic antibiotic resistance. The application of next-generation sequencing has powered recent genome-wide explorations revealing both the contributions of cryptic resistance mechanisms and routes to overcome them. Here, we lay the foundation for such an exploration into the epidemic clinical isolate B. cenocepacia K56-2 with an in-depth survey into its resistance arsenal.

Methods: Microplate dilution series were used to determine growth dose-response curves for a panel of 87 antimicrobials from over 30 diverse classes, including clinically relevant antibiotics. The sequenced K56-2 genome was used for bioinformatic searches using BLAST and the Comprehensive Antibiotic Resistance Database (CARD).

Results: Despite many antimicrobials not causing full growth inhibition, we observed important differences and similarities in and among structural classes. For example, tetracyclines and quinolones were up to two orders of magnitude more potent than aminoglycosides and cationic peptides. Within the cephalosporins, while ceftazidime by itself has poor activity, the related siderophore conjugate, cefiderocol, possessed an MIC that was 512-fold lower. Cross-reference to CARD shows K56-2 putatively possesses over 250 unique known resistance mechanism-encoding genes, including extended-spectrum β-lactamases, carbapenemases, broad-spectrum efflux pumps, and outer membrane modification systems. Furthermore, we have constructed and validated a barcoded transposon mutagenesis scheme to quantitatively profile genomic contributions to antimicrobial resistance, which will be the focus of future work.

Conclusions: To our knowledge, this is the most diverse antimicrobial dose-response screening in B. cenocepacia. Many of the trends in activity are explained by the presence of known resistance mechanisms. We expect these studies to yield valuable insight into novel therapeutic avenues for treating infections caused by B. cenocepacia and related bacteria.

Blocking CSPGs receptors enhances spinal neuronal replacement by transplanted human directly reprogrammed neural precursor cell and improves locomotor recovery after spinal cord injury

Seyed Mojtaba Hosseini1, Arsalan Alizadeh1, Narjes Shahsavani1, Jeremy Chopek1, Jan-Eric Ahlfors2, Soheila Karimi- Abdolrezaee1,3

1Department of Physiology and Pathophysiology, Spinal Cord Research Centre, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada 2New World Laboratories, Laval, Quebec, Canada. 3Children Hospital Research Institute of Manitoba, Winnipeg, Manitoba, Canada

Traumatic spinal cord injury (SCI) is a leading cause of permanent disabilities in young adults. Neurological impairments after SCI are substantially attributed to the progressive degeneration of spinal cord neurons. To date, effective replacement of damaged neurons and functional restoration of spinal cord circuitry remain challenging in preclinical studies of SCI. Thus, development of targeted regenerative therapies are critically needed. Transplantation of neural precursor cells (NPCs) offers a promising approach for regenerating new neurons. However, our preclinical studies indicate that long-term survival of engrafted NPCs and their differentiation into functionally integrated neurons is severely limited in the hostile milieu of SCI. We previously demonstrated that injury-induced upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) impedes survival and migration of engrafted NPCs after SCI. Our in vitro investigations revealed CSPGs directly inhibit NPCs by signaling through two protein tyrosine phosphatase receptors, LAR and PTP-σ. Here, we have developed a novel strategy to block the inhibitory effects of CSPG signaling on transplanted human directly reprogrammed NPCs (drNPCs) in a preclinical model of compressive/contusive SCI in rats. We have pharmacologically blocked LAR and PTP-σ in drNPCs by two functionally inhibiting peptides, ILP and ISP, respectively. Our in vitro studies confirmed CSPGs potently suppress survival, growth and proliferation of human drNPCs, which can be reversed by co-inhibiting LAR and PTP-σ. Inhibition of CSPG receptors also promoted differentiation of drNPCs into spinal interneurons and motoneurons, and supported their maturation, dendritic complexity, synapse formation and physiological activity. In rat SCI, co-inhibition of LAR and PTP-σ by systemic delivery of ILP/ISP remarkably promoted long-term survival, transplant volume and biodistribution of human drNPCs in the injured spinal cord, and enhanced their neuronal differentiation, maturation and synaptogenesis. Importantly, combined application of drNPCs with ILP/ISP treatment synergistically improved locomotor recovery after SCI. Mechanistically, transcriptomics analysis of human drNPC progenies in the injured spinal cord uncovered that CSPGs impede neuronal differentiation, at least in part, through inactivation of Wnt signaling, which was further validated in our in vitro studies. Altogether, we have identified a new targeted, translationally feasible strategy with the potential to optimize neuronal replacement and functional recovery after SCI.

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates anti- tumor immune responses

Fan Huang1, 2, Christophe Gonçalves1, Margarita Bartish1, 2, Wilson H. Miller, Jr.1, 2, Sonia V. del Rincón1, 2

1Lady Davis Institute, Jewish General Hospital, Montréal, Canada 2Division of Experimental Medicine, McGill University, Montréal, Canada

Introduction: Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance, however, the mechanisms are not completely understood and thus therapeutically unexploited.

Methods: To understand the role of the MNK1/2-mediated phosphorylation of eIF4E in melanoma phenotype switching and anti-tumor immunity, we generated an immunecompetent mouse model where melanoma can be induced and eIF4E cannot be phosphorylated.

Results: Using mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phosphoeIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and MDSCs, and increased CD8+ T-cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable anti-tumor immunity, was detected in mice administered a MNK1/2 inhibitor and anti-PD-1 therapy.

Conclusion: Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a new strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

Novel mechanisms related to DHA’s atheroprotective effects in endothelial cells: eNOS activity is regulated by DHA via p38MAPK and MSK

Shiqi Huang, Carla Taylor, Peter Zahradka

University of Manitoba; Canadian Centre for Agri-Food Research in Health and Medicine (CCARM)

Novel mechanisms related to DHA’s atheroprotective effects in endothelial cells: eNOS activity is regulated by DHA via p38MAPK and MSK. Shiqi Huang1,2, Carla Taylor1,2, Peter Zahradka1,2 1 University of Manitoba 2 Canadian Centre for Agri- Food Research in Health and Medicine (CCARM) Introduction: Our laboratory previously reported that docosahexaenoic acid (DHA) activates p38 mitogen-activated protein kinase (MAPK) differently in growing and quiescent human endothelial cells, which represent the atherogenic and healthy states in vivo, respectively. Endothelial nitric oxide synthase (eNOS) activity differs between these two states. Since eNOS can be regulated by p38MAPK and mitogen-stimulated kinase (MSK) is a p38MAPK substrate involved in cell proliferation and inflammation, we hypothesized that DHA’s atheroprotective actions require eNOS activation via the p38MAPK/MSK pathway. Thus, our objective was to investigate the role of p38MAPK/MSK in the eNOS response to DHA and determine whether the proposed pathway is growth state-sensitive. Methods: EA.hy926 cells were cultured on Matrigel-coated plates to sub-confluent and quiescent states and treated with DHA ± SB202190 or SB747651A, inhibitors of p38MAPK and MSK, respectively. eNOS activation was quantified by Western blot detection of Ser1177 phosphorylation. Results: eNOS activation by DHA in EA.hy926 cells was concentration-, time-, and growth state-dependent. p38MAPK inhibition suppressed eNOS activity in sub-confluent cells and increased eNOS activity in quiescent cells, while MSK inhibition suppressed eNOS activity in both growth states. eNOS activity remained suppressed with DHA treatment under MSK inhibition and showed no dose- or growth state-dependent effects. In contrast, when p38MAPK was inhibited, high dose DHA activated eNOS in sub-confluent cells, but dose-dependently decreased the elevated eNOS activity of quiescent cells. Conclusions: eNOS activity in endothelial cells is modulated by DHA via p38MAPK and MSK. The growth state-dependent regulation of p38MAPK and eNOS by DHA provides novel insight into the molecular mechanisms of DHA’s atheroprotective actions in relation to health status.

Evaluating the potential of FDA approved and natural product compounds as novel antivirals against seasonal influenza virus

Marnie Hustins, Robert Vendramelli, Darwyn Kobasa

University of Manitoba and Public Health Agency of Canada

Seasonal influenza viruses cause approximately 250,000-500,000 deaths globally every year, making them a significant burden on the healthcare system. Currently, the best intervention against seasonal influenza is the annual vaccine, which provides protection against Influenza Type A subtypes H1N1, H3N2, and Type B viruses, all of which are endemic in humans. However, vaccine strains must be decided one year in advance of the season, as the dominant seasonal strains may change year to year, which can lead to reduction in vaccine efficacy. Typically the first line of post-exposure defense for influenza infection is antivirals such as Oseltamivir and Zanamivir. However, influenza quickly gains resistance against antivirals, limiting their value for sustained use during outbreaks. By utilizing ApexBio’s DiscoveryProbe pre-aliquoted drug panels against a seasonal strain of H1N1 influenza (A/Canada/RV733/2007), we aimed to discover novel antivirals for use during seasonal influenza outbreaks. In vitro screening of ApexBio’s FDA-approved drug panel identified 2 drugs that provided protection against RV733 H1N1 with limited cytotoxicity in MDCK cells, as determined by XTT assay, while ApexBio’s Natural Product library panel identified 12 compounds that provided protection with limited cytotoxicity. We have also determined the concentration at which these 14 compounds are maximally effective against RV733, and future directions include determining the mechanism of action of these compounds. These novel antivirals could provide new possibilities for treatment of influenza infections, particularly to treat complicated or severe cases of infection or to provide additional options for the management of outbreaks and pandemics, such as the 2009 Swine Flu Pandemic.

PSYCHOSOCIAL STRESS, SOCIAL SUPPORT, AND ALLOSTATIC LOAD IN CANADIAN FIREFIGHTERS: WHAT DOES THE DATA SAY?

Somkene Igboanugo1, Phil Bigelow1, John Mielke1

1School of Public Health and Health Systems, University of Waterloo.

Introduction : Psychosocial factors are recognized as a major source of stress in the workplace, and chronic exposure to such stressors has been connected with increased risk for chronic diseases. Given their importance to public safety, we wondered whether the duty-related psychosocial stressors experienced by firefighters could affect their long-term health and well-being.

In designing our study, we used the “allostatic load” model, which describes how exposure to stressors becomes biologically embedded in a manner that can affect chronic disease development. We aimed to improve understanding of the psychosocial stress encountered by firefighters, how these experiences may become biologically embedded (i.e., their allostatic load [AL]), and whether the relationship between stress and its embedding may be moderated by social support.

Methods: Participants were 63 active firefighters from the Waterloo Fire Service. Workplace psychosocial stress was measured using the Sources of Occupational Stress Scale, while Social Support Scale for Firefighters measured social support. AL was measured using an 8-item index including anthropometric data, heart rate variability, and hair cortisol based on predefined clinical threshold values. Analytical models were applied to determine the relationships between perceived stress, key measures of their general health, and social support.

Results: A substantial proportion (27%) of firefighters reported high workplace stress, and a sample mean of 2.68 ± 1.34 was recorded for the AL. Participants’ age (r = .25) and length of service (r = .38) had a significant positive association with perceived stress. The multiple regression analysis showed a positive association between perceived stress and AL in our sample following adjustments for potential confounders; however, the association was non-significant. The social support measure showed a significant inverse relationship with perceived stress (r = -.385); no significant moderating effect of social support was observed between stress and AL.

Conclusion: Although we did not observe a significant association between perceived psychosocial stress and AL in our sample, our findings highlight the progressive increase in stress level with time on the job and as firefighters age. High levels of social support were observed to reduce stress levels, thus, emphasizing its importance in improving the well-being of firefighters.

A hydride transfer complex reprograms NAD metabolism preventing senescence and allowing primary cell transformation

Sebastian Igelmann1,2, Frederic Lessard1, Ana Fernandez-Ruiz2, Jacob Bouchard1, Oro Uchenunu3, Marie-Camille Rowell2, Stephane Lopes-Paciencia2, David Papadopoli3, Aurelien Fouillen1,4, Katia Julissa Ponce1,4, Genevieve Huot1, Lian Mignacca1, Mehdi Benfdil1, Paloma Kalegari1, Haytham M. Wahba1, Jan Pencik5, Nhung Vuong2, Jordan Quenneville6, Veronique Bourdeau1, Laura Hulea7, Etienne Gagnon6, Lukas Kenner5, Richard Moriggl8, Antonio Nanci1,4, Michael N. Pollak3, James G. Omichinski1, Ivan Topisirovic3, and Gerardo Ferbeyre1,2

1Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, QC, Canada 2CRCHUM Research Center, Montréal, QC Canada. 3Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, QC, Canada. 4Faculty of Dental Medicine, Université de Montréal, Montréal, QC, Canada. 5Department of Pathology, Medical University of Vienna, Vienna, Austria. 6Institut de recherche en immunologie et en cancerologie (IRIC), Université de Montréal, Montréal, QC Canada. 7Maisonneuve-Rosemont Hospital Research Centre, Departement de Medicine, Université de Montréal, Montreal, QC, Canada. 8Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.

Introduction: Cancer is one of the leading causes of death in Canada thus making the understanding of initiation processes of cancer a central point of research. Important aspects of cancer initiation include the bypass of tumor suppressor mechanisms as well are changes in the metabolic requirements of precancerous cells. One barrier for tumor progression that is circumvented in cancer cells is cellular senescence. Cellular senescence is characterized by a stable cell cycle arrest and can be triggered by various oncogenic events such as constant activation of the oncogene H-RAS. Other key aspects of senescent cells include the decrease in important regulators of redox homeostasis namely NAD+ and NADPH.

Hypothesis: In cancerous cells, the level of NAD+ and NADPH are significantly higher than in senescent cells thus raising the question of how cancer can increase NAD+ and NADPH to bypass senescence.

Results: Here, we report a novel multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+ and restores the levels of NAD+ and NADPH during the cellular transformation process. This hydride transfer complex, coined HTC, is formed by Malate Dehydrogenase 1, Malic Enzyme 1 and cytosolic fraction of Pyruvate Carboxylase. HTC enzymes are found as phase-separated bodies in the cytosol of cancer cells and can be assembled in vitro from purified proteins. These enzymes are repressed in senescent cells but restored by p53 tumor suppressor inactivation. Furthermore, HTC enzymes are highly expressed in vivo in mouse and human prostate cancer models and their inactivation triggers senescence. Using proximity ligation assay to visualize HTC enzymatic complexes on tissue samples, we were able to correlate aggressiveness of tumor assessed by KI67 staining with the amount of HTC complex foci in prostate cancer. Finally, exogenous expression of HTC bypasses the senescence response and cooperates with oncogenic RAS to transform primary cells.

Conclusion: In global, we provide evidence for a new multi-enzymatic complex, with de novo functions that reprogram metabolism and prevent cellular senescence. Inhibition of the formation of the HTC complex might allow targeting specifically the de novo function of this complex with fewer effects on normal enzyme function.

Precise quantitation of PTEN by immuno-MRM: a tool to resolve the PTEN biomarker controversy in many cancers

Sahar Ibrahim1,2, Mark Basik2,3,4, Gerald Batist2,3,4, René P. Zahed2,5, Christoph H. Borchers2,4

1Division of Experimental Medicine, McGill University. 2Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University. 3Segal Cancer Center, Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University. 4Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montréal, Québec, Canada.

Introduction: The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR-signaling and is commonly found downregulated in many cancers such as breast cancer (BC) due to several transcriptional and post-transcriptional mechanisms. Conflicting immunohistochemistry (IHC) and western blot (WB) data have sparked a controversy about PTEN’s role as a prognostic and predictive biomarker in these cancers. This has impeded the precision required to correlate minor PTEN-expression changes in tumors to clinical data.

Methods: Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (iMRM)] enables the development of highly sensitive and specific assays for low- abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. We developed and validated a fully-standardized, highly sensitive, robust iMRM assay for PTEN quantification that includes an 11-min micro-flow LC-MRM analysis on a triple-quadrupole mass spectrometer as well as a two-point internal calibration (2-PIC) strategy. We apply PTEN iMRM to samples from metastatic colorectal cancer and BC patients as well as BC-derived patient-derived xenografts PDXs treated with paclitaxel to be able to evaluate whether there is a clinically significant correlation between PTEN protein Sahar Ibrahim Abstract CSHRF concentration and cancer patient’s prognosis as well as response to different therapeutics including the new targeted ones and the conventional chemotherapeutics,

Results: Our PTEN iMRM assay using the 2-PIC strategy enables precise quantitation of PTEN concentrations in cell lines, fresh frozen- and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 μg of extracted protein, with high inter-and intra-day precision (CV 6.3%). PTEN levels in BC PDX samples that were determined by iMRM correlate well with semi-quantitative IHC and WB data produced under standardized conditions. iMRM, however, allowed precise PTEN quantitation -- even in samples that were deemed to be PTEN-negative by IHC or WB -- while requiring substantially less tumor tissue than WB.

Conclusion: The PTEN iMRM assay provides a much-needed tool for the study of PTEN to be able to validate its use as a prognostic and predictive biomarker in many cancers.

Digital health equity in online sexually transmitted and blood-borne infection testing in British Columbia

Ihoghosa Iyamu1,2,, Mark Gilbert1,2

1School of Population and Public Health, University of British Columbia (UBC), Vancouver, BC 2British Columbia Centre for Disease Control, Vancouver, BC, Canada

Background: A plethora of digital health interventions have recently been implemented to foster more efficient access to health services, especially for sexually transmitted and blood-borne infections (STBBI). A key challenge has been making sure that these interventions help to reduce health inequities, rather than reinforcing existing, or creating new inequities. Yet, many digital health interventions have not properly considered health equity in their design and development. Using GetCheckedOnline.com (GCO), a BC based online STBBI testing service targeted at people who experience marginalization, we aim to use a health equity framing to examine how features of GCO’s design differentially affect access, use and experiences of attrition (i.e. exit from GCO without completing service) among users.

Methods: Using a mixed-methods sequential explanatory study, we will first analyze clickstream data (data tracking users’ website navigation behaviors) linked with routinely collected program data. We will describe attrition patterns among various GCO users, and identify webpages and design features along the service cascade with high risk of attrition. We will assess the association between users’ equity-related sociodemographic characteristics (age, gender, sexual orientation), website navigation behaviors (e.g. device used, time spent on website), and attrition, using logistic regression with generalized estimating equations. Using findings from this analysis, we will purposively sample individuals from sociodemographic groups most at risk for attrition. Using key informant interviews, we will seek to understand design and implementation-related factors and processes that shape the differential experience of attrition from GCO. The data will be analyzed using inductive techniques based on grounded theory, to develop a theoretical understanding of these factors and processes.

Results: We hypothesize that there will be differential access and use of online STBBI services through GCO. Findings will help identify groups most at risk for attrition, and contribute to our understanding of design and implementation-related factors that may be addressed to equitably improve outcomes on GCO.

Conclusions: Emergent theoretical frameworks on digital health equity have lacked sufficient detail on the contribution of design and implementation related factors to equity of outcomes. Our study will contribute much needed information on possible design considerations to improve equity in other STBBI digital interventions.

Screening virus-host dependency factors via functional genomics in silico and in vitro for drug targeting

Rubendren Jamilchelvan1,2, Riley H Tough1,2, Xia Liu3, Eric Enns3, Paul J McLaren1,2

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 2National HIV and Retrovirology Laboratory at the JC Wilt Infectious Diseases Research Centre, National Microbiology Labs, Public Health Agency of Canada, 3Bioinformatics Group, National Microbiology Labs, Public Health Agency of Canada

Introduction: Viruses are obligate intracellular pathogens that require many host cell components to establish and maintain infection. Multiple genome-wide knockout/knockdown studies have identified host proteins that are essential for viral replication. These host dependency factors (HDF), are good candidates to develop novel antivirals, but defining which may make good targets and which may lead to drug toxicity is challenging. Genes in the human body interact with each other in signalling cascades or pathways. When a gene is inactivated, the other remaining genes in the pathway may compensate for the loss-of-function and the biological process may continue without negative health outcomes. In this case, the inactivated gene can be considered to be a non-essential gene. Intersecting genes found to be essential for viral replication but non-essential for host function may open new avenues for development of therapies.

Methods: We performed a comprehensive literature review to identify genome-wide studies of viral HDFs for multiple viruses. We identified more than 25 studies covering HIV, Hepatitis C, Hepatitis D, SARS-CoV-2, SARS-CoV, Ebola, Influenza A, Zika, Dengue and West Nile viral models. These HDFs will be intersected with the genome aggregation database (gnomAD) using an in-house bioinformatic pipeline, the genome non-essentiality and loss-of-function identifier (gNALI). The gnomAD resource contains >125,000 human exome and >15,000 whole-genome sequences from healthy individuals with specific information on genes harbouring loss-of-function polymorphisms. The output of gNALI is a list of HDFs that are essential to viral replication and non-essential for host survival.

Results: A total of 3177 HDFs were confirmed for all these viruses, with 312 genes implicated in more than 1 virus. Lists will be processed using gNALI which can process 800 HDFs within 70 minutes. HDFs harbouring homozygous loss-of-function polymorphisms in gnomAD, i.e., those that are non-essential to the host, will be assessed by bioinformatic platforms such as DAVIDv6.8 and REACTOME to predict the pathways they are involved in. Results observed in silico, will be confirmed via in vitro methods in future analyses.

Conclusion: Better understanding of the interaction of viruses with non-essential host genes can aid in the development of new anti-viral drugs that target host proteins.

Needles in a Haystack: Finding Inborn Errors of Immunity in Blastomycosis

Paul Jankowski1, 2, Emma Lee2, John Embil3, Yoav Keynan1, Paul McLaren1, 2

1Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba 2JC Wilt Infectious Diseases Research Centre, Public Health Agency of Canada 3Department of Medicine, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada

Introduction: Blastomyces dermatitidis is a thermally dimorphic fungus typically found in wet organic-rich soil and wooded areas in Manitoba and northwestern Ontario. B. dermatitidis infections occur when its spores are inhaled causing blastomycosis. In immunocompetent individuals, blastomycosis severity ranges from mild, self-limiting illness to severe life- threatening disease. This observed variability may be explained by the presence of inborn errors of immunity (IEI). IEI are monogenic germline mutations in genes of the immune system that can affect an individual’s susceptibility to certain infectious agents and their risk of developing severe disease. In a systematic literature scan, we have identified 35 IEI- causing genes in mycobacteria and dimorphic fungi. However, there is limited information on their impact on blastomycosis. Discovery of IEI involved in blastomycosis could offer a genetic explanation for the observed disease variation in immunocompetent individuals.

Methods: Whole blood samples were collected from 18 individuals previously diagnosed and treated for blastomycosis and 10 household controls with similar risk factors. DNA was extracted and whole exome libraries were prepared and sequenced on an Illumina MiSeq. Whole exome sequences were aligned to the reference human genome (build 38) and genetic variant calling was performed using the Genome Analysis Toolkit.

The genetic variants will undergo quality control to remove low-quality variants and those likely due to sequencing errors, reducing the possibility of false positives. Each variant in the refined variant callset will be annotated to its corresponding gene locus. The impact of the variants on protein structure and function will then be assessed by in silico prediction and database searches to identify potentially pathogenic variants. These will be used for downstream association analysis by logistic regression modelling to find enrichment of genetic variants in blastomycosis cases compared to household controls, focusing on previously reported IEI-causing genes. Burden tests will be used to increase power by combining variants within the same gene to test its association with blastomycosis.

Significance: Discovery of IEI could uncover immunological pathways needed to control infection. A better understanding of the immune system components involved in blastomycosis could inform novel therapeutics and diagnostic tools that could improve clinical outcomes.

Nevirapine-induced leukopenia in mice is associated with increase in corticosterone

Alison Jee1, Samantha Sernoskie1, Jack Uetrecht1

1University of Toronto

Introduction: Nevirapine is an antiretroviral used in the treatment of HIV infection. Its use is limited due to the risk of idiosyncratic drug reactions (IDRs) such as idiosyncratic drug-induced liver injury and skin reactions. Multiple lines of evidence including HLA associations suggest that IDRs involve an adaptive immune response. However, an adaptive immune response requires a prior innate immune response, likely due to reactive metabolite formation, which is unlikely to be idiosyncratic or symptomatic. We performed the present study to characterize early changes in inflammatory mediators and immune cells in mice caused by nevirapine that precede an IDR.

Methods: Male C57BL/6 mice were administered nevirapine by oral gavage once daily and sacrificed at two endpoints: three hours after the first dose (3h) and three hours after the third dose (3d+3h). Protein levels of inflammatory mediators in serum and organ homogenates were measured by ELISA. Leukocyte subsets in blood, spleen, and inguinal lymph nodes (iLN) were enumerated by flow cytometry. Corticosterone levels were measured by competitive ELISA.

Results: Treatment with nevirapine resulted in no change or decreases in serum, spleen, and liver levels of interleukin-6, serum amyloid A 1, and CXCL1 at 3h. Nevirapine treatment also decreased spleen and iLN weights, and decreased cell counts of most leukocyte subsets in blood, spleen, and iLN at 3d+3h. To explain this apparently immunosuppressive effect, we assayed corticosterone levels, which were increased in serum, spleen, and liver at 3h.

Conclusion: These data show that nevirapine causes decreases in some inflammatory mediators and multiple leukocyte subsets after three days of treatment, which may be explained by the systemic increase in corticosterone observed at 3h. This may represent a means of immunosuppression to prevent the immune system from unnecessarily attacking host cells that may be modified by reactive metabolites. Indeed, leukopenia has been observed clinically with multiple drugs that cause different IDRs, so this may be an important feature of drugs that cause IDRs. Further studies are required to understand the mechanism by which corticosterone is increased. Understanding the mechanisms of IDRs is necessary for their prevention and treatment, leading to the safer use of medications.

Impact of direct-acting antiviral treatment on mortality related to extrahepatic manifestations: findings from a large population-based cohort in British Columbia, Canada

Dahn Jeong1,2, Stanley Wong2, Mohammad Ehsanul Karim1,3, Naveed Z. Janjua*1,2 for The BC Hepatitis Testers Cohort Team

1School of Population and Public Health, University of British Columbia, 2206 E Mall, Vancouver, BC 2British Columbia Centre for Disease Control, 655 West 12th Avenue, Vancouver, British Columbia 3Centre for Health Evaluation and Outcome Sciences, 588-1081 Burrard St, St Paul’s Hospital, Vancouver, BC

Introduction: In addition to liver-related mortality, chronic hepatitis C virus (HCV) infection is associated with mortality due to extrahepatic manifestations (EHM). Direct-acting antivirals (DAA) are highly effective therapy for HCV and have been associated with reducing liverrelated mortality. Evidence of its impact on EHM-related mortality is lacking. This study assessed the impact of DAA treatment on reducing EHM-related mortality using large population-based linked administrative data.

Methods: British Columbia (BC) Hepatitis Testers Cohort includes ~1.3 million people tested for HCV since 1990. Among individuals with a chronic HCV infection, those who received at least one DAA treatment were matched to those who never received treatment. We assessed EHM-related mortality including deaths due to diabetes, cardiovascular, cerebrovascular, renal diseases, rheumatoid arthritis and neurocognitive disorders. Individuals were followed from index date to the earliest of 1) EHM-related death 2) other death or 3) end of study (2019/12/31). To assess the impact of DAA treatment and sustained virologic response (SVR), we compared three groups of individuals: treated & SVR, treated & no-SVR, and untreated. We computed EHM-related mortality rates and used inverse probability treatment weighting (IPTW) in multivariable Fine-Gray subdistributional hazards model.

Results: Study population included 10,254 individuals who were treated & SVR, 440 individuals who were treated & no-SVR and 10,694 untreated individuals. EHM-related mortality rates were: 5.86/1,000 PYFU (95% confidence interval [CI] 5.00- 6.87) in treated & SVR; 25.32/1,000 PYFU (95% CI 16.67-38.45) in treated & no-SVR; 24.61/1,000 PYFU (95% CI 22.78-26.59) in untreated. In the IPTW-weighted multivariable model, the treated & SVR group had the greatest reduction in EHM mortality (adjusted hazard ratio [aHR] 0.15, 95% CI 0.13-0.17), followed by the treated & no-SVR group (aHR 0.44, 95% CI 0.32-0.62) compared to the untreated group. Older age and history of hypertension, cardiovascular diseases, cirrhosis, diabetes, end-stage renal disease, hepatocellular carcinoma, statin use and co-infection with HIV or HBV were all associated with higher EHM- related deaths.

Conclusion: The virologic cure of HCV following DAA treatment was associated with a significant reduction in EHM-related mortality. This highlights the need for providing timely diagnosis and treatment for people living with HCV to reduce EHM- related mortality.

GCM1 and ASCL2 Cooperate to Regulate Development and Function of the Extravillous Trophoblast Lineage in Human Trophoblast Stem Cells

Mariyan J. Jeyarajah1, Gargi J. Bhattad1, and Stephen J. Renaud1,2,3

1Department of Anatomy and Cell Biology, Western University 2Children’s Health Research Institute 3Lawson Health Research Institute, London, ON, Canada

Introduction: Placental maldevelopment is a leading cause of sickness and death among mothers and babies and thus, understanding placental development is clinically important. The human placenta is comprised of trophoblast stem (TS) cells that give rise to various sublineages including the extravillous trophoblast (EVT) lineage. EVTs are critical for invading maternal arteries and widening them to promote adequate blood flow to the baby. Insufficient EVT development can result in several serious obstetrical complications that can jeopardize the health of the fetus. Glial cells-missing 1 (GCM1) is a transcription factor that is crucial for proper placentation in mice, and is thought to be important for the formation of another human placental sublineage, syncytiotrophoblast. However, the role of GCM1 in human TS cell differentiation, particularly in the regulation of EVT development, is still unknown.

Methods: Immunohistochemistry was used to determine localization of GCM1 in 6-week human placenta. Lenti-shRNA mediated knockdown of GCM1 was performed in TS cells and RNA-sequencing was used to identify differential gene expression between control and GCM1-knockdown TS cells. Matrigel-coated transwell assays were performed to determine EVT invasiveness. Chromatin immunoprecipitation (ChIP) was utilized to determine GCM1 binding to DNA.

Results: Immunohistochemistry in human placentas showed expression of GCM1 in EVTs and syncytiotrophoblast. Lenti- shRNA was effective in knocking down GCM1 expression in TS cells by 77% (P<0.05). RNA-sequencing revealed differential expression of 1128 genes (fold-change2, FDR<0.05) between control and GCM1-knockdown EVTs. GCM1-knockdown EVTs clustered closely with undifferentiated TS cells. EVT-specific markers HLA-G, MMP2, and ITGA1 were downregulated by 73%, 80%, and 47%, respectively in GCM1-deficient cells (P<0.05). GCM1-knockdown EVTs showed 53% decreased invasiveness (P<0.05). Additionally, we detected a 62% decreased expression of ASCL2, a master regulator of rodent and human placentation, in GCM1-deficient EVTs (P<0.05). In silico analysis of the ASCL2 promoter revealed a putative GCM1 binding site. Using ChIP, we confirmed that GCM1 binds to this site. Knockdown of ASCL2 in TS cells resulted in poor EVT development and function.

Conclusion: GCM1 is highly expressed in EVTs and binds upstream of ASCL2 to regulate development and function of EVTs. This research will reveal fundamental mechanisms governing EVT development.

Identification of Core Immune-Related Genes in the Alcoholic Liver Disease

Yao Jiang1, Chuyi Zeng1, Yongcan Guo2, Hualin Tao3

1The Affiliated Hospital of Southwest Medical University 2Traditional Chinese Medicine Hospital Affiliated to Southwest Medical University 3The Affiliated Hospital of Southwest Medical University

Alcoholic liver disease (ALD) is a class of liver diseases caused by long-term drinking and its’ initial manifestation is fatty lesions. Nevertheless, it has not been elucidated whether immune-related genes (IRGs) play an important role in the ALD at an early stage. Therefore, this study aims to explore IRGs, which play an essential role in the early ALD, via constructing mice and cellular model of the ALD. First, bioinformatics analysis were performed, including differential analyses, functional enrichment analysis, construction of protein-protein interaction (PPI) network, as well as identification of core genes. Second, we constructed a mouse model of the early ALD, then we used hematoxylin & eosin (HE) staining and oil red O staining of liver tissues to examine this animal model. Third, human L02 hepatocytes were cultured and CCK-8 assay was used to determine optimal alcohol concentration and stimulus time for hepatocytes. Additionally, alcoholic fatty liver cell model was established in vitro. Next, TG kit and oil red O staining for hepatocytes were used to detect whether alcoholic fatty liver cell model formed. Subsequently, real-time fluorescent quantitative PCR (RT-qPCR) was applied to detect the expression levels of core genes among mice and hepatocytes from experimental and control groups, respectively. Ultimately, immunohistochemistry (IHC) method was employed to detect the proteins expression and localization of core genes. Based on bioinformatics analysis, CXCL1 and CXCL6 genes were considered to be core genes. And CCK-8 assay confirmed that 200 mM and 300 mM ethanol were suitable concentrations and 36 h was optimal treatment time. Also, RT- qPCR revealed that CXCL1 and CXCL6 genes were significantly up-regulated in mice alcoholic fatty liver tissues and human alcoholic fatty liver cells. Meanwhile, IHC staining indicated that CXCL1 and CXCL6 proteins were over-expressed in the experimental groups. Bioinformatics analysis revealed that CXCL1 and CXCL6 genes, considered to be core genes of the ALD, might play an important role in the formation of ALD at an early stage. In addition, CXCL1 and CXCL6 mRNAs and proteins were over-expressed in the early ALD model, which may be potential targets of the diagnosis and treatment for ALD.

Deconvolution of bulk gene expression profiles to characterize the tumour immune landscape of early onset breast cancer

Yong Won Jin1, Pingzhao Hu1, 2, 3, 4

1University of Manitoba 2George and Fay Yee Centre for Healthcare Innovation 3Research Institute in Oncology and Hematology, CancerCare Manitoba 4University of Toronto

Introduction: Young age at diagnosis (age < 40) is considered to be an independent factor for poor clinical outcomes in breast cancer patients. Patterns of tumor infiltrating lymphocytes (TILs) may provide insight into the underlying biology behind this disparity, which is yet to be discovered. Deconvolution algorithms can be used to characterize tumor infiltrating lymphocytes given the gene expression profile (GEP) of the bulk tumor tissue sample. Novel models of deconvolution based on deep learning framework will be used to extract distinct patterns of TILs in early onset breast cancer (age at diagnosis < 40) that are significantly associated with clinical outcomes and other molecular signatures.

Methods: Pseudo-bulk GEP data were simulated by leveraging single cell RNA-Sequencing (scRNA-seq) data from immune and breast cancer cells with known cell type compositions. Pseudo-bulk GEPs were used to optimize an artificial intelligence (AI) model with deep quartile regression to provide estimates for cell fractions given bulk GEPs. Then, the TIL landscape of early onset breast cancer was characterized, which were associated with clinical outcomes.

Results: Preliminary analyses revealed that clinical outcomes of early onset breast cancer patients were more significantly affected by the abundance of CD8+ cytotoxic T cells, but to a lesser extent in the old patients. However, due to several inconsistencies in estimates between existing methods, we constructed a model of deconvolution optimized under the deep learning framework in attempt to produce accurate predictions of cell type composition from bulk GEPs and better characterize the TIL fractions in breast cancer for clinical outcome analysis.

Conclusion: The canonical survival disparity of early onset breast cancer patients was observed but the TIL landscape identified by current deconvolution algorithms do not give consistent results. Novel models of deconvolution built on state- of-the-art deep learning frameworks leveraging scRNA-seq data have potential to produce robust estimates of cell type proportions to better characterize the TIL landscapes from bulk tissue GEPs.

Nanoscaled materials for triggered release of anti-scar agents for expedited scarless skin wound healing

Farinaz Jonidi Shariatzadeh1, Sarvesh Logsetty2 and Song Liu3*

1Biomedical Engineering, Faculty of Engineering, University of Manitoba 2Departments of Surgery and Psychiatry, Rady Faculty of Health Sciences, University of Manitoba 3Department of Biosystems Engineering, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

Scar not only affects appearance but also the functionality of tissue. Annually, 11 million people suffer from severe burn wounds many of whom have post-burn scarring. Biomolecules incorporated into a wound dressing can impact cell behavior and scar formation. Kynurenine (Kyn) is a tryptophan metabolite that upregulates MMP-1 and -3 expression and leads to scar-free healing. This small molecule has shown promising results as a cream-based anti-scar treatment. However, challenges remain, such as lack of effective diffusion to the wound site, and daily removal of the wound dressing, which causes a second injury. Additionally, the untriggered release of anti-scar drugs increases a temporary collagen-based scaffold's degradation rate and hamper wound healing.

We proposed incorporating responsive nanoparticles (NP) based on polyethylene glycol (PEG)- polypropylene sulfide (PPS) co-polymer into wound dressings as carriers for on-demand delivery of KyA to overcome these mentioned shortages. An increase in ROS (Reactive Oxygen Species) concentration inside the cell is one reason for scar formation. The smart NPs target the scar formation stage and release the drugs in the presence of ROS. The PEG-co-PPS undergoes an oxidative transition in ROS presence, which allows the on-demand release of the payload. It is hypothesized the NP enters the cell through the lipid diffusion pathway and forms pores in the endosome membrane and in the cytosol where ROS concentration is high, and releases the drug; otherwise, these NPs leave the cell through exocytosis.

PEG-co-PPS was synthesized through three steps. The obtained co-polymer was used to fabricate NPs through the double emulsion technique. The fabrication yield of NPs was 80%, and the drug's loading efficiency was 97%. The NPs with the size of 200 ±5 nm. H2O2 was used as a quantifiable source of ROS. The NPs showed reliable responsive release (92% within 72 hr) in the presence of H2O2 while less than 6% of the drug was released without H2O2. The release behavior is based on the zero-order model. All results demonstrated that the fabricated responsive NPs is a promising choice for on-demand delivery of anti-scar drug for scar-free wound healing. Further in vitro tests and gene expression experiments are required to prove the hypothesis.

19-Hydroxyeicosatetraenoic acid causes bronchodilation in murine tracheal smooth muscle

Ramandeep Kaur1, Jignesh Vaghasiya1, Anurag Sikarwar1, Shana Kahnamoui1, Andrew J. Halayko1, Christopher D. Pascoe1

1University of Manitoba

Introduction: Asthma exacerbations, triggered by excessive inflammation and airway narrowing, causes significant morbidity and mortality in Canadians. β-agonists are first line therapeutics that promote bronchodilation but can be rendered ineffective through desensitization of the β-adrenergic receptor. This necessitates the identification of alternative pathways to promote bronchodilation. Lipid mediators, called oxylipins, play an important role in coordinating a diverse array of lung cellular functions. Hydroxyeicosatetraenoic acids (HETEs) are a class of oxylipin produced in abundance in the airways with important roles in regulating cellular function in other organ systems. Albeit abundant, information about their role in coordinating airway smooth muscle (ASM) cell contractility in the context of asthma is scarce. Here, we hypothesize that 19-HETE causes relaxation of ASM in response to asthmatic stimuli triggering bronchoconstriction.

Methods: Media supernatant from primary cultured ASM cells (non-smokers, no lung disease, n=3) were analyzed for 19- HETE production by HPLC-MS/MS targeted lipidomics. Trachea without epithelium were collected from 8 weeks old BALB/c female mice (n=3). Trachea (2mm) were mounted in wire myograph chambers containing Kreb's buffer. Trachea were pre- contracted with 1µM methacholine (MCh) and cumulative dose-response curves generated with isoproterenol (10pM to 3.2µM) and 19-HETE (1nM to 10µM). A three-parameter dose response curve was constructed to determine LogEC50 for each compound. Data presented as mean ± SD.

Results: Confluent, serum deprived ASM cells produce 5.47±1.6 ng/mL of media of 19-HETE. 19-HETE causes a rapid reduction in MCh induced force, with 1µM 19-HETE causing 56.34±7.89% drop in force. This is an equivalent bronchodilation to 3.89nM of isoproterenol. Maximal relaxation was 71.06±12.61%, in response to 10µM 19-HETE. The LogEC50 dose for 19-HETE was -6.77, compared to -8.78 for isoproterenol.

Conclusion: Our results reveal that 19-HETE is produced by ASM cells and causes profound bronchodilation in murine trachea contracted with MCh. This suggests a potential role for 19-HETE in regulating ASM contractility and may present an opportunity for developing a novel bronchodilator for asthma. The molecular mechanism for this bronchodilation is currently unknown, but may involved prostacyclin receptor signalling. More research is required to understand if 19-HETEs bronchodilator effect is intact in asthma patients.

Building Blocks: Establishing Baselines for Pediatric Brain-Computer Interfaces

Dion Kelly1, Ephrem Zewdie1, Adam Kirton1

1Alberta Children's Hospital Research Institute, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada

Introduction: Children with severe neurological disabilities, unable to move or speak, may be intellectually normal and cognitively aware. They are literally trapped inside a body that does not work. For adults in such a “locked-in” state, brain- computer interfaces (BCIs) can restore their ability to communicate and interact with their environment by non-invasively acquiring, analyzing and translating brain signals into commands using electroencephalography (EEG). However, pediatric studies are limited. We have therefore established a novel clinical pediatric BCI program for children with disabilities. In order to gauge competency in pediatric patients, we aimed here to establish the ability of typically developing children to use EEG-based BCI systems.

Methods: Twenty-two neurotypical children ages 6 to 16 (mean age 11.36; 14 female) completed tasks in five different EEG- based BCI paradigms over two sessions. Sensorimotor rhythms (SMRs) and P300 event-related potentials, evoked by two- and three-tactor vibrotactile (VTP2, VTP3) stimuli, auditory stimuli, and visual flashing stimuli, were assessed for BCI control. Performance was assessed by accuracy scores for each paradigm. Potential factors affecting performance were evaluated.

Results: Our results demonstrate that children can effectively operate a P300-based BCI with accuracy scores depending on the paradigm. Children performed the best on the visual P300 paradigm, where highest mean accuracies were achieved at medium frequency visual stimulation of 8 flashes per row/column (94%; 95% CI = 0.87, 1.01), although significant differences in performance were only observed below 4 flashes. Age affected performance on the auditory P300 paradigm (r2 = 0.156, p = 0.028), while sex differences were observed in the SMR and VTP2 paradigms (p = 0.006). Only two participants were able to achieve accuracy scores greater than threshold (61%) for communication in the SMR paradigm (mean = 52.8%; 95% CI = 0.49, 0.56). One participant dropped out following the auditory paradigm, reporting extreme fatigue.

Conclusion: Many children can competently operate advanced BCI systems with accuracy scores comparable to established thresholds necessary for control. Performance likely varies by paradigm, algorithm, age and sex. This work provides the foundational knowledge necessary for the development of BCI paradigms and screening programs for children with severe neurological disabilities.

Immune checkpoint blockade augments changes within oncolytic virus-induced cancer MHC-I peptidome creating novel antitumor CD8 T cell reactivities

Youra Kim1, Prathyusha Konda2, Patrick Murphy3, Joao Paolo4, Stefan Stevanovic5, Steven Gygi4, and Shashi Gujar1,2,6

1Department of Pathology, Dalhousie University, Halifax, NS, Canada 2Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada 3Department of Biology, University of Prince Edward Island, Charlottetown, PEI, Canada 4Department of Cell Biology, Harvard Medical School, Boston, MA, United States 5Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany 6Department of Biology, Dalhousie University, Halifax, NS, Canada

Introduction: Immunotherapies aim to (re)educate the immune system to recognize and eliminate cancer cells, resulting in antitumor immunity that provides a highly specific and long-lasting protection. Cancer immunotherapy approaches based on oncolytic viruses (OVs) or immune checkpoint blockade (ICB) have shown promise in clinical settings and are being recognized for their capacity to re-instate CD8 T cell attack on cancers. Such re-instatement of antitumor CD8 T cell responses depends on the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for CD8 T cell-mediated tumor eradication.

Methods: Tumor-bearing mice were treated with OV and/or ICB. Tumors were processed for immuno-precipitation using anti-MHC-I antibodies. MHC-I peptides were identified and quantitated by mass spectrometry analysis, and tested for their CD8 T cell response-stimulating activity.

Results: Oncolytic reovirus monotherapy up- and down-regulates the expression of MHC-I peptides in a cancer type and oncolysis susceptibility-dependent manner. In comparison to either monotherapies, reovirus+ICB combination results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs). Most importantly, reovirus+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells.

Conclusion: These findings support the biological and therapeutic implications for therapy-induced changes to the MHC-I peptidome following combinatorial treatment with two emerging immunotherapies – OV and ICB. The elucidation of therapy-driven DEMHCPs provides an insight on the alterations to tumors in response to therapy as well as identifies immunogenic peptides that can be exploited for the development of next-generation cancer immunotherapies.

An investigation into an anomalous pyramidal cell type: atypical morphology and functional ramifications in the hippocampus

Adrienne Kinman1, Madeline Elder1, Sarah Erwin1, Mark S. Cembrowski1

1University of British Columbia

Introduction: This research investigates an anomalous pyramidal cell type, henceforth called “deep cells” in the subiculum of the hippocampus. Deep cells are structurally unique in that they completely lack radial oblique dendrites. This work will use in-vivo calcium imaging to elucidate how this remarkable change in structure effects neuronal function in the subiculum and its possible role during behaviour.

Methods: Our lab has constructed a transgenic cre line to access this deep cell type. I use this mouse line to image deep cells during spatial navigation and novel object tasks. During these experiments, I use wire-free 1-photon miniscopes to image the dorsal subiculum while mice undergo the o-maze and open field with two local objects, repeated over several timepoints.

Results: We found that the unique structure of deep cells drastically changed their neuronal activity and highlighted a robust and time sensitive correlation with novelty behaviors. Data from this project show that deep cells act on very slow timescales and have robust, sustained activity that appear to respond to encounters with novel, local objects. Deep cells show large increases in activity after interaction with a novel object on day one and this activity is substantially reduced by day four. Intriguingly, this cellular signature is still reduced 100 days after initial introduction and this cellular novelty phenotype can be resurrected with the introduction of another novel object. In comparison to control cells, deep cells show novel object- centered activity and no classic spatial phenotypes expected from pyramidal cells in the subiculum.

Conclusion: This data leaves us with intriguing evidence of a seemingly spatially uninvolved, novelty driven and morphologically distinct cell type previously undescribed in the subiculum. Future work will be integral to identifying the role of this cell in memory processes/dysfunction and the role of this novel cell type in other brain regions.

Retrospective Determination of Plant Toxin Crude Purification Methods via Proteomic Fingerprinting of Seed Accessory Proteins

Matthew Klassen1, Cindi Corbett1

1Bioforensic Assay Development & Diagnostics (BADD), National Microbiology Laboratory

Type II Ribosome-Inhibiting Proteins (RIPs) remain a significant biothreat challenge warranting continual innovation of advanced bioforensic capabilities. Abrin toxin is obtained from the seeds of Abrus precatorius, and bears identical mechanism of action and toxicity as ricin toxin. There are multiple methods available by which a person or group may produce a crude toxin sample, and molecular-based source attribution can link evidence to a perpetrator and assist in directing a criminal investigation. Here, a comprehensive bench-to-bioinformatics process has been created to retrospectively identify crude purification methods based on proteomic fingerprinting of non-toxic seed accessory proteins. Six different crude protocols were carried out on Abrus precatorius seeds, trypsin-digested, and shotgun proteomic data collected. Principal component analysis (PCA) revealed unique method-wise clustering of samples, and sparse partial-least squares discriminant analysis (sPLS-DA) classified and predicted all 6 separate crude methods. Furthermore, accurate intra- method discrimination of high- and low-quality reagent variations of two protein precipitation methods was achieved. 100% accuracy of retrospective prediction was maintained even when testing independently processed and randomized samples.

Comparing magnetic particle imaging (MPI) to MRI for the detection of breast cancer brain metastasis

Natasha N. Knier1, 2, and Paula J. Foster1, 2

1Department of Medical Biophysics, Western University, London, Ontario, Canada 2Robarts Research Institute, London, Ontario, Canada

Introduction: Magnetic particle imaging (MPI) is an emerging modality that detects superparamagnetic iron-oxide nanoparticles (SPIONs), with sensitivity and resolution depending heavily on SPION tracer properties. Synomag-D has been explored as a sensitive MPI tracer, yet no studies exist using this tracer to track cancer cells. Here, we use Synomag-D for MPI of breast cancer cells in the brain and compare to cells labeled with micron sized iron particles (MPIO) for MRI.

Methods: Human brain metastatic breast cancer cells (231BR) were labeled with Synomag-D and NOD/SCID/ILIIrg−/− (NSG) mice (n=6) were injected with 2.5x105 cells intracardially (IC). MPI of the mouse brain was performed on Day 0 and 7 and compared to MRI from a prior study with mice injected IC with 2.5x105 MPIO-labeled 231BR cells. A calibration line was generated from samples of Synomag-D to determine iron content for a given MPI signal.

Results: On Day 0, MPI signal was detected in 3/6 mouse brains and iron quantified (M=0.08ug). Signal was also seen in the lung/liver region since cells injected IC distribute throughout the body. We have shown that strong MPI signal impedes detection in nearby regions with low signal. Day 7 MPI revealed lung/liver signal reduced allowing brain signal detection in 5/6 mice. Brain signal and iron content decreased (M=0.012ug), as many cancer cells die and clear by day 7. In day 0 MRI, MPIO-labeled cells appear in the brain as signal voids, however, while void volume can be calculated, cell number cannot.

Conclusion: This is the first study to show that 231BR cells can be labeled with Synomag-D and detected in the mouse brain with MPI. Calculating iron content indicated detection of 16,000 cells on average in the brain with labeling of ~5pg of iron/cell. Employing MPI for experimental cancer cell tracking will allow for the detection and quantification of the arrest, clearance, and retention of cancer cells in vivo.

Low-dose ASA reduces HIV target cells at the female genital tract without inhibiting viral recall immune responses

Monika M Kowatsch1, Julius Oyugi1,2, Lucy W Mwangi2, Natasha Hollett1, Maureen Akolo3, John Mungai3, Joshua Kimani1,2,3, Julie Lajoie1,2, Keith R Fowke1,2,3

1University of Manitoba 2University of Nairobi 3Partners for Health and Development in Africa

Background: Globally 1.7 million new HIV infections occurred in 2019, therefore, new prevention methods are needed. Inflammation is a risk factor for HIV acquisition as it attracts HIV target cells to the female genital tract (FGT). Our lab conducted a study aimed at reducing HIV target cells at the FGT using safe, affordable, and globally available anti- inflammatory drug: acetylsalicylic acid (ASA/Aspirin). We found ASA decreased the proportion of HIV target cells (CD4+CDCR5+Tcells) at the FGT by 35%. However, this decrease in inflammation must not hamper the immune response to other infectious agents.

Hypothesis: We expect ASA to decrease HIV target cells without adversely effecting the immune response to recall antigens. Methods: Women from Nairobi, Kenya took low dose ASA (81mg) daily for 6 weeks. Blood was drawn prior to ASA and following 6 weeks daily ASA. Peripheral blood mononuclear cells (PBMCs) were isolated, frozen, and shipped to Winnipeg, Canada where they were stimulated with either 2µg/mL CEF (Cytomegalovirus, Epstein Barr, Influenza) or 8µg/mL HPV (Human Papilloma Virus) peptide pools. Stimulations were for 12 hours for cytokine detection or 7 days for proliferation.

Results: Following 6 weeks ASA there was an increase in the pro-apoptotic receptor CD95 in unstimulated CD8+Tcells (p=0.011) with increased IFNγ (p=0.006) and IL-2 (p=0.040), in CD4+Tcells and TNFα in CD4+ and CD8+Tcells (p=0.031, p=0.024 respectively) following HPV peptide stimulation. There was no change with CEF peptide stimulation and no impact of either stimulation on proliferative ability.

Conclusion: We show that the immune response to recall antigens is not impaired by 6 weeks of ASA treatment and may be boosted with HPV peptide stimulation. Our observation that ASA decreases HIV target cells at the FGT without adversely altering the ability of immune cells to respond to recall antigens supports ASA’s further assessment as a new HIV prevention tool.

Yeast-based high-throughput screening reveals differential biological effects of anthracycline- based cancer drugs

Divya Kriti1, Uche J. Ogbede1, Rutuja Pattanshetti1, Guri Giaever1, Corey Nislow1

1The University of British Columbia

Introduction: Anthracyclines are widely-used, effective anticancer treatments. Their use in different types of cancers has dramatically improved overall cancer survival statistics. However, despite the fact that anthracyclines differ in their molecular actions, they are often used interchangeably in the clinic. An additional concern with treatment is that patients often develop drug resistance. In this study, we identify variants that confer resistance to different anthracyclines and use these drug-resistant strains to characterize their mechanism of action.

Methods: An agar-based drug screen was employed to isolate spontaneous, doxorubicin-resistant strains (IC100 lethal dose) in wild-type haploid BY4741 and heterozygous diploid ssl2/SSL2 strains. Following nine days of drug exposure, 91 resistant isolates (30 haploid and 61 diploid strains) were identified and confirmed. These strains were screened using high-resolution growth curve analysis for their response to seven different anthracyclines: doxorubicin, daunorubicin, epirubicin, idarubicin, amrubicin, valrubicin, and etoposide. The genomic DNA from all 91 strains was isolated and sequenced to identify common and private variants that may contribute to anthracycline resistance.

Results: Growth parameters including doubling time, percentage inhibition, adaptation and maximum growth were determined for each strain. Whole-genome sequencing of all 91 strains allowed for the identification of the resistance- conferring variants. Using the MutantHuntWGS tool for variant analysis and characterization, 139 private variants and 4 common variants (present in all isolates) were found for the haploid strains. 155 private and 3 common variants were found for the heterozygous diploid ssl2/SSL2 strains. We are developing a pipeline that can correlate each strain genotype with its drug response phenotype, to be able to correlate all observations in yeast to individual patient variation and response to treatment.

Conclusion: Anthracyclines play an undisputed crucial role in cancer treatment. The risks associated with chronic administration of first-generation anthracyclines, daunorubicin and doxorubicin, along with its analogs, have stimulated an intensive effort to elucidate their mechanism of action. This study presents a systematic approach to identify variants of pharmacogenomic importance that alter efficacy in anthracycline drugs and elucidate the underlying mechanism. Analysis of such clinically actionable variants and the mechanistic differences between anthracyclines will lead to better treatment decisions and dosing guidelines.

Induction of autophagy mitigates TDP-43 pathology and translational repression of neurofilament mRNAs in mouse models of ALS/FTD

Sunny Kumar1, Daniel Phaneuf1, Pierre Cordeau Jr.1, Hejer Boutej1, Jasna Kriz1 & Jean-Pierre Julien1

1Department of Psychiatry and Neuroscience, CERVO Brain Research Centre, Université Laval, Quebec City, Canada.

Introduction: TAR DNA-binding protein-43 (TDP-43) is a nuclear DNA/RNA binding protein that plays key roles in transcriptional and translational regulation. TDP-43 cytoplasmic mislocalization and aggregation, called TDP-43 proteinopathy, is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The TDP-43 proteinopathy is associated with loss of TDP-43 nuclear function and gain of toxic function by protein aggregates contributing to neurodegenerative changes in ALS/FTD. So far, there is no effective therapy available for these neurodegenerative diseases.

Methods: Here using biochemical, immunohistological, and Ribotrap methods, we investigated the impact of TDP-43 proteinopathy on translational profile of neurons and tested the therapeutic effects of a novel semi-synthetic analog of withaferin-A (IMS-088), an inhibitor of inflammation pathway.

Results: Oral administration of IMS-088 reduced TDP-43 proteinopathy likely through a mechanism involving induction of autophagy. Moreover, IMS-088 treatment improved cognitive function and reduced neuroinflammation. Using the Ribotrap method for immunoprecipitation of neuronal ribosomes followed by mass spectroscopy analysis of newly synthesized peptides, we found that TDP-43 proteinopathy in one-year-old TDP-43A315T Tg mice caused a severe translational dysregulation of several mRNAs including translational suppression of neurofilament mRNAs. Oral administration of IMS- 088 rescued the translational defects associated with TDP-43 proteinopathy and restored synthesis of neurofilament proteins which are crucial for axon integrity and synaptic function.

Conclusion: Our study revealed translational dysregulation as a new gain of toxic function in TDP-43 proteinopathy, a phenomenon which can be rescued by the withaferin-A analog IMS-088 acting as an inhibitor of inflammation and inducer of autophagy.

A Systematic Review of Antiepileptic Drug Exposure during Pregnancy and Neonatal Birth Weight Outcomes and Interim Meta-analysis

Alekhya Lavu1, Christine Vaccaro1, Silvia Alessi-Severini1, Walid Shouman1, Sherif Eltonsy1,2

1College of Pharmacy, University of Manitoba, Canada 2The Children's Hospital Research Institute of Manitoba, Canada

Introduction: The prevalence of epilepsy in pregnant women is estimated at 0.3- 1%. Antiepileptic drug (AED) exposure in- utero has been associated with various adverse health outcomes in neonates including adverse birth weight outcomes.

Objectives: To systematically summarize the evidence on the association between AED exposure in pregnancy and adverse birthweight outcomes. Including Small for gestational age (SGA), low birth weight (LBW), birth weight (BW) as primary, and birth length, head circumference, and cephalization index as secondary outcomes. Methods: MEDLINE, EMBASE, Cochrane Library, Scopus, CINAHL, IPA, and Global Health databases were searched. Trials and observational studies were included. We used random-effects and fixed-effects models for meta-analysis based on the heterogeneity level.

Results: Out of 15720 studies identified by our search strategy, 4279 were excluded after deduplication. After the title, abstract, and full-text review we included a total of 60 studies in the analysis. We identified 21 studies on SGA. Of those studies, 3 studies with the heterogeneity of I2 =17% (p=0.30) compared women exposed to AED to women unexposed to AED and found a significant increased risk of SGA, RR=1.37 (95% CI; 1.29, 1.46), in exposed compared to unexposed women. Six studies reported SGA in exposed women with epilepsy vs unexposed women with epilepsy with higher statistical heterogeneity I2= 66% (p=0.01). The meta-analysis showed an increased risk of SGA but did not reach statistical significance (RR 1.30 (95% CI; 0.98, 1.73)). Since both exposed and unexposed groups have epilepsy, the results show a drug-disease risk separation for SGA. The sensitivity analysis showed comparable results.

Conclusion: This review demonstrates that women taking AED during pregnancy have a significant increased risk of SGA when compared to unexposed women. Additional studies are needed to investigate the disease drug separation. Further, the review will report additional birth weight/growth outcomes in infants exposed to AED in-utero.

In-Progress: Optimization of Flow Cytometry Panel to Assess Natural Killer Function and Activation in Populations from Nairobi Kenya

Toby Le1#, Monika M. Kowatsch1#, Julie Lajoie1,3 and Keith R. Fowke1,2,3,4

1Medical Microbiology and Infectious Diseases 2Community Health Science University of Manitoba 3Medical Microbiology University of Nairobi 4Partner for Health and Development in Africa #equal contribution

Background: Depot medroxyprogesterone acetate (DMPA) is the most popular form of injectable contraceptive in Sub- Saharan Africa, yet some epidemiological studies have associated it with increased risk for HIV acquisition. Previously, our lab found female Kenyan sex workers using DMPA, had higher levels of HIV target cells in their genital tract. Natural killer (NK) cells are innate immune cells that have been shown to exhibit decreased activity in exposure to progesterone. It remains unclear how DMPA, a progestin contraceptive, affects the activation and function of NK cells. The goal of this study is to optimize a flow cytometry panel to study blood NK cells.

Methods: The NK panel will measure marker expression of NK activation (CD38, NKp46, NKp30, KIR2DL5, KIR3DL1, NKG2a), NK memory (NKG2C, CD57) and migration (α4β7). Panel optimization will occur using blood samples collected from healthy Winnipeg donors. The optimization process is as follows: (1) optimum voltages setup, (2) full stain assessment, (3) antibody titration, (4) voltage titration, (5) plate titration, (6) fresh vs frozen stain analysis, and (7) stimulated/unstimulated assessment. Once optimized the panel will be used on DMPA-case and control samples collected from women in Nairobi, Kenya.

Results: The optimal voltage for each fluorescent marker was determined to detect lymphocytes at 2.5-3 the brightness of the machine’s background. For our full stain assessment, we discovered high levels of fluorescence spillover into the PE- Texas channel which we initially used for distinguishing/dead cells. To address this issue, we moved the detection of live/dead cells onto another detection channel (BV510). For our antibody titration step, we titrated all our markers thereby improving staining efficiency. Next, we titrated the voltages for several markers (KIR3DL1 and KIR2DL5) to increase the resolution of their detection. Finally, a panel titration determined the optimal volume for panel staining was 75 uL. Next, we will optimize this panel comparing blood cells in fresh vs frozen and stimulated vs unstimulated conditions.

Conclusion: Using this panel, we can assess NK cell activation using flow cytometry. We will use frozen blood samples collected from women in Nairobi, Kenya to measure how DMPA impacts function and activation of NK cells.

Dual Cannabinoid Receptor Agonist, CB13, Prevents Atrial Electrical Remodeling

Danielle Lee1,2, Michael Murninkas3, Yoram Etzion3, Hope D. Anderson1,2

1College of Pharmacy, Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada 2Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Research Centre, Winnipeg, Canada 3Department of Physiology and Cell Biology and Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Introduction: Atrial fibrillation (AF) leads to rate-dependent changes, which progressively increase the risk for recurrence and persistence of the arrhythmia (collectively defined as atrial remodelling). Atrial effective refractory period (AERP) shortening and conduction velocity prolongation are hallmarks of atrial remodelling markedly promoting AF substrate. The molecular mechanisms underlying atrial remodelling are not fully elucidated and therapeutic strategies to reduce AF burden by preventing atrial remodelling are not yet available. Cannabinoid receptor (CBR) ligands exert cardioprotective effects. We hypothesized that CB13, a peripherally restricted dual CBR agonist with limited brain penetration, would reduce atrial remodelling in both ex vivo tachypaced rodent hearts and in vitro atrial cardiomyocytes.

Methods: Sprague Dawley (SD) rat hearts were excised and hung on a Langendorff tachypacing set-up and atrial cardiomyocytes were isolated from 1-3 day old SD rat pups where Angiotensin II was utilized to provoke atrial remodeling. Hanging hearts and atrial cardiomyocytes were supplemented with CB13 or without (control). Effects of CB13 were assessed through electrophysiological (EP) parameters (AERP and conduction time) by analysis of high resolution electrogram recordings. Protein expression and molecular signaling pathways (AMPK, Cx43, PGC1a, LKB1, CAMKK2) were assessed through conventional western blotting. Hypertrophic markers (myocyte surface area) and mitochondrial function were also assessed.

Results: EP recordings showed significant reduction in AERP in control hearts pre- and post-tachypacing. CB13-treatment resulted in preservation of AERP compared to control. Protein expression of PGC1α was significantly increased by CB13 compared to controls. Upregulation of mitochondrial biogenesis may be related to increased phosphorylation of AMPKα compared to control. Furthermore, gap junction Cx43 was significantly downregulated in tachypaced controls vs. non-paced controls. Dual CBR agonist treatment did not alter CBR1 or CBR2 protein expression in atrial tissue.

Conclusion: CB13 exposure prevents AERP reduction and gap junction downregulation probably through mechanisms involving mitochondrial biogenesis. Thus, by inhibiting atrial remodeling CB13 and the subsequent activation of CBRs may be a viable treatment strategy for AF.

Anxiety and migraine as determinants of inability to complete caloric testing

Erika Lee1, Dr. Brian Blakley1

1University of Manitoba

Background: We have observed that many patients who are unable to complete caloric testing (CT) carry a high clinical suspicion of anxiety and/or migraine. CT tests vestibular function but also induces anxiety, nausea and discomfort. Anxiety is difficult to measure objectively but there is a known relationship between anxiety and vestibular symptoms. This study explores the possibility that inability to complete caloric testing is an objective finding that can be clinically useful in detecting anxiety or migraine in dizzy patients.

Methods: Responses to self-reported anxiety and migraine for 380 patients were recorded. Primary outcome was test completed or not completed due to nausea, vomiting. Secondary variables were age, unilateral weakness (%) and sum of caloric tests (d/s).

Results: Fisher’s exact test results indicated that reported anxiety was statistically significantly more common in patients who reported anxiety prior to testing (2-sided, p=0.023), whereas neither reported migraine (2-sided p=0.713) nor gender (p=0.098) were significantly associated with test incompletion. Cramer’s phi was only 0.087, suggesting a small effect size.

Conclusions: Clinicians should be aware that inability to complete CT may be related to anxiety although a causal relationship is yet to be proven.

The p66ShcA Adaptor Protein as a Contextual Metastasis Promoter

Kyle Lewis1,2, Alex Kiepas1,3, Jesse Hudson1,2, Peter M. Siegel1,3, Josie Ursini-Siegel1,2

1McGill University 2Lady Davis Institute for Medical Research 3Goodman Cancer Research Centre

Introduction: The p66Shca protein is one of three isoforms and the only one that produces reactive oxygen species. It does so in response to various stress stimuli, when it becomes phosphorylated on serine 36 allowing it to translocate to the mitochondria and transfer electrons from cytochrome C to molecular oxygen. In the context of breast cancer, p66ShcA is variably expressed in tumors and studies have yielded conflicting evidence that categorize it as either a positive or negative prognostic marker. We sought to better understand the role of p66ShcA in breast cancer progression and metastasis.

Methods: Explanted lung metastatic 4T1 cells had upregulated expression of p66ShcA in comparison to the parental cell line. Expression was abrogated using Crispr-Cas9 genomic editing and lines re-expressing wildtype p66ShcA (p66WT) or a mutant (p66S36A) unable to localize to the mitochondria were established to be examined alongside the knockout. Metastasis was examined by quantification of lung burden following primary tumor engraftment and resection or after injection of cells into the vasculature. Cell motility was examined using transwell assays and microscopy. Cells were seeded in non-adherent dishes to observe anoikis and circulating tumor cells were isolated for colony formation assays. Finally, lung colonization was visualized using fluorescently labeled cells and metastases were examined by IHC.

Results: We found that expression of p66WT was necessary for full metastatic potential from the primary tumor, but either p66WT or p66S36A, representing cytoplasmic p66ShcA, rescued metastatic ability from the vasculature. Cytoplasmic p66ShcA increased motility through interactions with focal adhesions and improved colonization and proliferation in lung tissue. An intact serine 36 was found to be necessary for enhanced resistance to anoikis and survival in the vasculature following intravasation.

Conclusion: These findings establish roles for p66ShcA in promoting the metastatic fitness of breast cancer in a manner that relies on both mitochondrial and non-mitochondrial pools of the protein.

Tracking the Genetic Diversity and Antigenic Variations of SARS-CoV-2: the UK perspective

Katherine Li, Hezhao Ji

Department of Medical Microbiology and Infectious Diseases, University of Manitoba National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Centre, Public Health Agency of Canada

Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.1.7 was discovered in November 2020 when infection rates in Kent, the United Kingdom, persisted despite increased national restrictions. B.1.1.7 transmission soon exploded, sparking a massive surge in UK infection rates and spreading world-wide. Genome-wide studies are urgently needed to identify the mutation patterns and their consequences on viral-host interactions in order to understand how variant lineages emerge. By studying how genetic diversity and antigenic variation contribute to SARS-CoV- 2 evolution, we may better understand and even predict future variants' emergence.

Methods: Three groups of complete SARS-CoV-2 genomes from England were gathered from GISAID based on specimen collection dates surrounding the first UK SARS-CoV-2 case, preceding B.1.1.7 emergence, and following B.1.1.7 establishment (groups A, B, and C, respectively). Sequences were aligned using MAFFT, and mutations were identified using BioEdit. The functional consequences of the identified mutations will be determined by 1) their linkages to varied immune epitopes in viral proteins predicted using IEDB; 2) their potential alterations to the corresponding proteins' 3D structure using SWISS-MODEL. Positive selection and recombination were detected with Datamonkey. Inter-group comparisons were then conducted to explore how mutations across the viral genome and the resulting immune epitope changes found before/after the emergence of B.1.1.7 influence SARS-CoV-2 evolution.

Results: Preliminary results showed a large increase in the number of mutations from group A to groups B and C. Non- synonymous mutations were more prevalent than synonymous mutations in groups B and C, and group C displayed the largest number of mutations present at population frequencies >10%. Analyses of the spike protein revealed no evidence of recombination in any group, however, the collective alignment of all groups showed evidence of both positive/diversifying and negative/purifying selection at nine sites each within the protein.

Conclusion: This study helps determine how/why SARS-CoV-2 has diversified and evolved within the UK population, which may inform the development of effective strategies towards the containment of the B.1.1.7 lineage and other SARS-CoV-2 variants of concern. Further investigations on how the identified mutations contribute to antigenic variation and protein function are underway.

Macrophage origins, dynamics and functions in the mouse diaphragm

Qian Li1, Salyan Bhattarai1, Feng Liang1, Eva Kaufmann1, Junying Ding2 and Basil J Petrof1

1Meakins-Christie Laboratories and Respiratory Division, McGill University, Montreal, Quebec 2Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China

Background & Objectives: Macrophages (MPs) play a key role in tissue homeostasis, immune surveillance, inflammation, and tissue repair. In broad terms the steady-state MP population in tissues can be derived from 2 major sources: post-natal adult bone marrow (BM) origin blood monocytes and pre-natal BM-independent progenitors seeded in the tissue during embryogenesis. The objectives of this study were: 1) To determine how BM-dependent versus BM-independent MPs contribute to the steady-state MP population in the diaphragm (DIA); and 2) To ascertain the responses of the different MP subsets to acute and chronic DIA muscle injury.

Methods & Results: To distinguish between BM-dependent and -independent MPs, chimeric mice were first generated by transplanting BM into irradiated recipient mice with a different CD45 allele (CD45.1 and CD45.2 MPs detected by flow cytometry). The DIA of recipient mice was shielded during irradiation to protect the resident MP population. At steady-state 8 wks later, ~45% of DIA MPs were BM-independent. To further address this issue, parabiosis was performed by surgically joining the blood circulations of CD45.1 wild-type (WT) and CD45.2 CCR2-/- mice. In CCR2-/- mice the percentage of CD45.1 MPs in the DIA was significantly lower than the corresponding percentage of CD45.1 blood monocytes, thus indicating once again a substantial BM (monocyte)-independent population. To explore the embryonic origin of BM-independent MPs in the DIA, the CX3CR1CreER–YFP:R26Td lineage tracing system was used. Pregnant mice received tamoxifen at days E7.5, E10.5 or E13.5, with subsequent analysis at day E18.5. The kinetics of labeled CX3CR1+ cells in the DIA suggested that the majority of these MPs originated from the aorta-gonad mesonephros. At birth most MPs (~90%) were CX3CR1+/CCR2- and this population then declined progressively to reach a plateau in adulthood. To determine the response to acute injury, the DIA was surgically exposed and treated with the myonecrotic agent cardiotoxin. An acute increase in MPs occurred primarily due to BMdependent MPs (>90%). Differences in local proliferation (Ki67, BrdU) of BM-independent MPs did not appear to be a major contributor. After recovery from acute DIA damage, the pre-injury steady-state ratio of BM-dependent to BM- independent MPs was restored. Finally, to assess the response to chronic DIA injury, muscular dystrophy (mdx) mice were used as recipients for transplanted CX3CR1gfp/- BM. At steady-state 8 wks later, the percentage of MPs in the DIA expressing pro-inflammatory (IL-1β, NOS2) as well as anti-inflammatory (CD206, IL-10) markers was significantly higher in BM-dependent (gfp+) compared to BM-independent (gfp-) MPs.

Conclusions: These findings indicate the existence of a major population of steady-state MPs within the adult DIA that is independent of BM-derived monocytes under homeostatic conditions. In addition, our results suggest that muscle MPs of different origins are likely to have different functions particularly in the setting of muscle injury.

Nutrient sensing in the dorsal vagal complex regulates hepatic lipid and glucose metabolism in rats

Rosa J.W. Li1,2, Battsetseg Batchuluun2, Song-Yang Zhang2, Mona A. Abraham1,2, Beini Wang1,2, Yu-Mi Lim2,3, Jessica T.Y. Yue4 & Tony K.T. Lam1,2,5,6

1Department of Physiology, University of Toronto, Toronto, Canada 2Toronto General Hospital Research Institute, UHN, Toronto, Canada 3Medical Research Institute, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4Department of Physiology, University of Alberta, Edmonton, Canada 5Department of Medicine, University of Toronto, Toronto, Canada 6Banting and Best Diabetes Centre, University of Toronto, Toronto, Canada

Introduction: Increased hepatic secretion of very-low density lipoprotein (VLDL) and unsuppressed glucose production contributes to dyslipidemia and hyperglycemia. The physiological and pathological mechanisms underlying lipid and glucose homeostasis remain to be further investigated. While hypothalamic regulation of lipid and glucose homeostasis is emerging in rodents, it remains unknown whether dorsal vagal complex (DVC) of the brain, which is similarly positioned beside a circumventricular organ to detect circulating factors and regulates energy homeostasis, can sense nutrients to regulate hepatic nutrient metabolism.

Methods: We infused oleic acid or glucose into DVC of healthy and high fat (HF)-fed rats and evaluated for any changes in lipid and glucose metabolism. Using lipoprotein lipase inhibitors to block VLDL clearance, we measured plasma triglyceride accumulation as a function of time to determine hepatic VLDL secretion rate. Using pancreatic basal-insulin euglycemic clamps with the tracer-dilution methodology, we evaluated the rate of glucose uptake and production.

Results: DVC oleic acid infusion suppressed hepatic secretion of VLDL. Inhibition of DVC long-chain fatty acyl-CoA synthetase (ACSL) disrupted this hypolipidemic effect, and disturbed lipid homeostasis during intravenous lipid infusions. DVC infusion of glucose suppressed hepatic glucose production. In contrast to hypothalamic mechanisms, this was independent of lactate metabolism as inhibition of DVC lactate dehydrogenase failed to disrupt DVC glucose sensing, and neither could DVC lactate infusion recapitulate the effects of glucose in lowering glucose production. Next, we tested DVC nutrient sensing in a short- term HF model characterized by increased hepatic VLDL secretion and hepatic insulin resistance to examine the pathological relevance of DVC nutrient sensing. The observed effects of both DVC oleic acid and glucose on hepatic nutrient release were abolished. Farnesoid X receptor (FXR) is a lipid- and glucose-regulating receptor activated by HF-feeding. Inhibition of DVC FXR in HF-fed rats rescued DVC oleic acid but not glucose sensing.

Conclusion: In summary, we report, for the first time to our knowledge, that DVC senses fatty acids and glucose to lower hepatic VLDL secretion and glucose production, respectively, in healthy but not HF-fed rats. Impairments in DVC nutrient sensing may lead to disruption of lipid and glucose homeostasis in obesity and diabetes.

SiO2 active retinoic acid response by aggravating ferroptosis in silicosis

Xingjie Li1,2, Ning Ma1,2, Hongyu Jiang1,2, Zongde Zhang1,2

1Inflammation & Allergic Diseases Research Unit, Affifiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China 2The School of Basic MedicalSciences, Southwest MedicalUniversity, Luzhou, Sichuan 646000, China

Lung parenchymal and phagocytic cell damage caused by inhalation of silica and silicon crystals contribute to silicosis. Retinoic acid is an enssential factor in the development of lung organs, it regulates transcriptional activity by forming heterodimer with RARs and RXRs and acting on RARE (retinoic acid response element) in the target gene promoter region. We hypothesize retinoic acid pathway is involved in the process of silicosis. We identified up-regulated RARa signaling in silicosis model induced by SiO2 in mice. Further, SiO2 active RARa pathway by modulate ferroptosis. Activation of lipid reactive oxygen reaction by SiO2 results in the induction of ferroptosis, whereas inhibition of this process may protect organisms from silicosis.

The leucine zipper EF-hand containing transmembrane protein-1 EF-hand is a tripartite calcium, temperature, and pH sensor

Qi-Tong Lin1, Peter Basile Stathopulos1

1Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario

Introduction: Mitochondrial calcium (Ca2+) is integral to vital ATP production required for life however excessive Ca2+ leads to cell death. Both processes are crucial to normal cellular function and thus, mitochondrial Ca2+ homeostasis is tightly regulated. Leucine zipper EF-hand containing transmembrane protein 1(LETM1) is an essential inner-mitochondrial membrane protein that regulates mitochondrial Ca2+ levels through the exchange of Ca2+ for protons(H+). Haploinsufficiency of LETM1 is associated with Wolf-Hirschhorn syndrome, a rare chromosomal disorder characterized by microcephaly, growth retardation, and early onset of epileptic seizures. LETM1 deficiency in yeast causes mitochondrial swelling and is lethal under non-fermentable conditions, and in mouse studies, homozygous LETM1 deletion is embryonically lethal within 6.5 days, indicating a fundamental importance of LETM1 function is conserved from lower eukaryotes to higher vertebrates. The matrix residing carboxyl (C)-terminal domain contains a highly conserved sequence identifiable Ca2+ binding EF-hand motif (EF1). Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca2+ flux, highlighting the requirement of EF1 for LETM1 function.

Methods: To understand the mechanistic role of this EF-hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation.

Results: Our data show that EF1 exhibits α-helical secondary structure that is augmented in the presence of Ca2+. Unexpectedly, EF1 features a weak but specific, apparent Ca2+-binding affinity, consistent with the canonical Ca2+ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4-fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF-hand properties, Ca2+ binding increases the exposure of hydrophobic regions, typical of EF-hands; however, this Ca2+-induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF-hand within LETM1 is unreactive to Ca2+ either in isolation or tandem with EF1.

Conclusion: Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca2+ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.

Development and validation of a dynamic, microfluidic culture model for assessing blood-brain barrier function in health and disease

Stacey Line1, Donald W. Miller1

1Department of Pharmacology and Therapeutics, University of Manitoba

Background: The application of microfluidic technology to various cellular barrier models, provides a more realistic setting where both fluid flow and cell-cell contact can be replicated under both physiological and pathophysiological conditions. Objectives: The objectives of the present study were to establish a dynamic micro-fluidic (DMF) brain microvessel endothelial cell culture model to more closely mimic the barrier properties of the cerebral micorovasculature that forms the blood-brain barrier (BBB). Once established, the DMF culture model was used to examine endothelial cell permeability and gene expression in response to different tumor cell microenvironments.

Methods: Human brain microvessel endothelial cell line (hCMEC/d3) were seeded (5,000 cells/µL) into the vascular compartment of Mimetas 3-lane microfluidic units under monoculture conditions and in the presence of various glioblastoma cell lines in the brain compartment. Barrier function was studied using fluorescent markers of either paracellular diffusion (fluorescein, fluorescein labeled dextran, IRdye 800) or transcellular transport (doxorubicin, rhodamine 800). Quantitative differences in expression of BBB specific genes in the DMF and traditional TranswellTM models was determined using RT-qPCR. Molecular and functional changes occurring in the DMF BBB culture model were compared to traditional 2-D TranswellTM hCMEC/d3 model.

Results: Flux of all the permeability markers was significantly reduced in confluent hCMEC/d3 within the DMF system. Permeability coefficients for fluorescein labeled dextran (FDX70K), IRdye 800, and Rhodamine 800 in the standard 2-D hCMEC/d3 monolayers were (2.64148E-06, 1.12863E-05, 1.02377E-06 Papp/s) compared to (4.209E-08, 8.600E-08, and 1.361E-08 Papp/s) for these same markers in the DMF model. Examination of the RNA expression profile for BBB specific molecular markers confirmed the presence of essential markers associated with endothelial cells of the BBB. Glut-1, P-gp, and Ve-Cadherin were significantly upregulated in the DMF studies compared to Transwell monocultures. Studies examining the effects of tumor cells on permeability of DMF and traditional Transwell culture models are ongoing.

Conclusions: The DMF culture model using hCMEC/d3 appear to provide a more accurate representations of BBB conditions occurring in vivo and can be used to study the effects of the tumor microenvironment on BBB function.

The role and mechanism of the IncARSA /PTEN/Akt/NF-κB/ IncARSA positive feedback regulatory loop in chemotherapy resistance of liver cancer

Yaling Li1

1Southwest Medical University

As routine first-line therapy, chemotherapy has been used to treat many cancers. However, the efficacy of chemotherapy for liver cancer is not satisfactory. Furthermore, there is no efficient therapeutic strategy for unresectable and post-surgery recurrent liver cancer. Therefore, elucidating the molecular mechanisms underlying liver cancer chemo-resistance, providing targets for enhancing liver cancer chemotherapeutic sensitivity, and identifying the liver cancer individuals sensitive to chemotherapy would have important clinical significances. In our previous study, we found that lncARSR enhanced liver cancer resistance to doxorubicin via inhibiting PTEN and activating Akt signaling pathway. Further study revealed that serum lncARSR expression indicated the sensitivity to doxorubicin, and Akt upregulated lncARSR via activating NF-κB signaling pathway. These data suggested that lncARSR/PTEN/Akt/NF-κB/lncARSR positive feedback regulatory loop exists in liver cancer chemo-resistance. In this study, we will further measure the expression or activation of lncARSR, PTEN, Akt, and NF-κB in liver cancer tissues and serum, analyze their correlation with doxorubicin sensitivity, explore the action mechanisms and roles of lncARSR/PTEN/Akt/NF-κB/lncARSR positive feedback regulatory loop in liver cancer chemo- resistance, and provide potential biomarkers for indicating liver cancer chemotherapeutic sensitivity and targets for enhancing chemotherapeutic efficacy.

Interpretable and extendable deep learning models for identifying cancer biomarkers from multimodal imaging and genomic data

Qian Liu1, Pingzhao Hu1

University of Manitoba1

Medical imaging (such as magnetic resonance imaging) has been used to extract biomarkers for cancer diagnosis. Traditionally, the imaging features need to be preprocessed subjectively by experienced radiologists. It has been suggested that these handcrafted imaging features, such as textures, are normally shallow and low-ordered. Deep learning is a type of machine learning methods that are composed of multiple data processing layers to learn representations of data with multiple levels of abstraction. Comparing with the handcrafted imaging features, the high-order deep imaging features are extracted automatically from raw image data through prespecified deep learning models. These models are usually treated as black boxes and, hence, it is difficult for us to interpret their biological and/or clinical meanings. For deep learning models to be readily adopted by clinical practices, they should be interpretable without sacrificing prediction accuracy. I hypothesize that there are significant associations between breast cancer-specific deep imaging phenotypes with the underlying genetic mechanisms. The overarching goal of this project is to propose interpretable and extendable deep learning algorithms for modeling multimodal breast cancer medical imaging and genomic data to identify clinically relevant biomarkers. There are three research aims: 1: To develop new interpretable deep learning models for identifying image surrogates, and systematically assessing the robustness of radiogenomic association findings. 2: To develop an integrating framework for effectively utilizing multiple data sources (including multiple both imaging and genomic sources), which can be extended in the future to incorporate wider data modalities. 3: To impute deep image features for the public genomic data sets without medical images using the pretrained radiogenomic association models and associate the predicted images features to the clinical outcomes. This research will help characterize useful information across multimodal cancer patient data. I expect to identify novel genomic signatures that are associated with deep imaging features. Furthermore, I expect that the deep learning methods can achieve a better performance than traditional methods. Such information might support improving medical imaging as a potential non-invasive examination to probe cancer molecular status, then help support clinical decisions and ultimately improve cancer patient’s care.

Characterization of sector RP-associated rhodopsin missense mutations in transgenic Xenopus laevis rod photoreceptors

Aaron D. Loewen1, Beatrice M. Tam1, Ross T. Scharbach1, Colette N. Chiu1, Orson L. Moritz1

1Department of Ophthalmology & Visual Sciences, The University of British Columbia, Vancouver, BC

Introduction: 15% of non-syndromic retinitis pigmentosa (RP) cases belong to a subclass called sector RP. Sector RP is defined by the localization of the disease phenotype to certain quadrants of the retina, usually the inferior. Mutations in RHODOPSIN (RHO) are the primary cause of sector RP. While these mutations have been identified in patients, their molecular consequences are largely unknown. We screened over 10 sector RP-associated RHO mutations in our Xenopus laevis model for retinal degeneration (RD) and protein localization as well as investigated the effects of changing protein stability via dark rearing on select variants of interest.

Methods: We generated transgenic X. laevis by injecting wildtype (WT) oocytes with a mixture of sperm nuclei and linearized plasmids containing a human RHO transgene (either WT or containing missense mutations) under control of a X. laevis rod opsin promotor. Tadpoles were raised in either cyclic light or constant dark for 14 days before euthanization. Tadpole eyes were enucleated and were immunolabelled using anti-mammalian rhodopsin antibodies for confocal microscopy or analysed for rhodopsin levels using a dot blot assay.

Results: Rhodopsin localization for most mutations were not significantly different from WT; however, some variants of rhodopsin were retained in the inner segment more than WT. We observed mitigated RD in the dark-reared condition for animals expressing the L31Q or T58R RHO transgenes compared to cyclic light conditions. However, D190G RHO-expressing animals failed to display mitigated RD in the dark-reared condition.

Conclusion: Dissimilarity in localization patterns between RHO variants suggests that the mechanisms of RP pathogenicity will also be variable. Similar to previously described models of sector RP, two of these mutations (L31Q and T58R) result in RD that is mitigated when animals are reared in complete darkness. However, RD caused by D190G RHO was not alleviated by dark rearing, suggesting that the origin of the sector RP phenotype in D190G RHO patients may be novel. This variability suggests that the term sector RP is an umbrella term for a disease that looks similar but likely has multiple underlying pathogenic pathways. Further screening of these mutations may unveil novel therapeutic targets.

Thymic B Cell Isotype Switching Contributes to Central T Cell Tolerance and Limits Autoimmune Diabetes

Félix Lombard-Vadnais1,2, Geneviève Chabot-Roy2, Javier Di Noia1,3,4, Sylvie Lesage2,3

1McGill University 2Centre de Recherche de l’Hôpital Maisonneuve-Rosemont 3Université de Montréal 4Institut de Recherches Clinique de Montréal

Type 1 diabetes (T1D) is an autoimmune disease mediated by autoreactive T cells. T cells are generated in the thymus, where autoreactive T cells are generally eliminated by interaction with various cell types presenting self-antigens, a process known as clonal deletion. In addition to dendritic cells and epithelial cells, thymic B cells also participate in this process. Interestingly, thymic B cells can undergo immunoglobulin isotype switching, which may confer them unique properties for inducing clonal deletion. Using a T cell transgenic mouse model of T1D, we demonstrate that isotype switching facilitates the clonal deletion of autoreactive T cells in the thymus. In non-transgenic mice, we find that isotype switching favors cellular interactions between thymic B cells and developing thymocytes. Furthermore, we show that inhibition of isotype switching in NOD B cells promotes the accumulation of diabetogenic T cells in the pancreatic lymph nodes and, consequently, accelerates T1D. Together, this work highlights the importance of B cell isotype switching in mediating central T cell tolerance and limiting the development of autoimmune diabetes. Understanding how isotype switching in the thymus modulates the T cell repertoire could lead to new therapies against various autoimmune diseases.

Protective effect of vitamin B in regulating Th17/Treg balance through DNA methylation in PM2.5 induced acute lung injury

Yingyu Luo1, Jun Bai1, Qingbi Zhang1

1School of Public Health, Southwest Medical University

Protective effect of vitamin B in regulating Th17/Treg balance through DNA methylation in PM2.5 induced acute lung injury Yingyu Luo1, Jun Bai1, Qingbi Zhang1 1School of public health, Southwest medical university Introduction: DNA methylation plays a momentous role in the imbalance of Th17/Treg which is the one of functions of PM2.5-induced acute lung injury, and rencent studys reported DNA methylation can be mediated by Vitamin B supplement. To make clarification of the protective effect of vitamin B complex on PM2.5-induced acute lung injury by adjusting methylation level, we established an animal study of Vitamin B supplement with a focus on the change of Th17/Treg balance in PM2.5 exposure rats. Methods: 64 SD rats were divided into eight groups: negative control (NC), low(0.4mg/ml), medium(2.0mg/ml) and high dose(10.0mg/ml) PM2.5 instillation groups, vitamin B protection+low, medium and high dose PM2.5 instillation groups and 5-Aza intervention+PM2.5 instillation group. Then we detected and compared the related cytokine levels, RORγt gene and protein levels, RORγt gene DNA methylation levels Results: The levels of IL-17, IL-6, TGF-β in BALF of low, medium and high dose PM2.5 groups were higher than those of control group; the levels of VitB protection+PM2.5 exposure groups and 5- Aza+PM2.5 intervention group were lower than those of the same dose PM2.5 single exposure groups (P<0.05). Compared with the same dose of PM2.5 alone exposure group, the RORγt gene and protein in the lung tissue of vitamin B intervention rats were decreased (P<0.05). The methylation level of RORγt gene in the VitB+PM2.5 intervention groups were higher than that in the PM2.5 alone exposure groups and 5-Aza group at the same dose (P<0.05). Conclusion: Vitamin B complex can reduce the acute lung injury caused by PM2.5 exposure by reversing the abnormal DNA methylation of RORγt gene regulated by Th17/Treg balance, which may play a role of protective effect on the body.

How do we Know What to Say and When to Say it? User-Informed Development of a Diabetes Prevention Text Messaging Intervention

Megan M MacPherson1, Sean R Locke2, Mary E Jung1

1University of British Columbia 2Brock University

Introduction: Prediabetes affects 1 in 5 Canadians and increases risk of developing type 2 diabetes by up to 70%. Interventions involving diet and physical activity (PA) remain a cornerstone of diabetes prevention; however, long-term adherence to such behaviours is low. Short messaging services (SMS) can improve adherence following behaviour change interventions, but information pertaining to content/delivery characteristics (message timing/frequency) is lacking. This project, situated within Phase-I of the Multiphase Optimization Strategy (MOST) framework, aims to develop a theory- based, user-informed bank of SMS messages for use following the Small Steps for Big Changes (SSBC) diabetes prevention program. MOST is a three-phase framework (I-Preparation; II-Optimization; III-Evaluation) aiming to develop interventions that are effective, efficient, and scalable; attributes which are key in effective translation into practice.

Methods: This work followed 3 broad steps: I) Development: A conceptual model informed by the Behaviour Change Wheel (BCW) was developed to establish links between SMS messages and underlying theoretical mechanisms. Qualitative research profiling SSBC alumni was used to identify barriers/facilitators to maintaining diet and PA. Barriers/facilitators were then linked to intervention functions (e.g., education, persuasion, enablement) and behaviour change techniques (BCT). An SMS bank was written to map onto this conceptual model (e.g., barrier=guilt -> intervention function=persuasion -> BCT=reframing -> message=“Don’t think of a slip as harmful; they are inevitable. How we react to slips and recommit to change is more important”). II) Evaluation: Stakeholders (SSBC coaches and alumni) evaluated message content using a validated framework on message clarity, utility, and relevance. Participant feedback was summarized, and messages were refined accordingly. III) Feasibility: The SSBC RCT demonstrated that messages from a coach led to increased logged PA 3-7 days following a message. A feasibility study using the above-mentioned messages is underway to determine participant preferences of delivery characteristics (message amount and timing) which will be further tested in MOST Phase-II.

Conclusion: Current literature underreports SMS development. Understanding how to optimally intervene via SMS requires rigorous development and stakeholder engagement. Subsequent MOST phases will build on this research towards an effective, economical, and scalable SMS intervention to improve long term behaviour change adherence following SSBC.

Development and Characterization of Novel Dendritic Cell (DC) Targeting Vaccine Against Human Immunodeficiency Virus (HIV)-1 Envelope Conserved Elements (CEs)

Mona Mahmoudi1, Zhujuin Ao1, Titus Olukitibi Abiola1, Gary Kobinger2, Xiao-Jian Yao1*

1Laboratory of Molecular Human Retrovirology, Department of Medical Microbiology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada 2Centre de Recherche en Infectiologie de l'Université Laval/Centre Hospitalier de l'Université Laval (CHUL), Québec, QC, Canada

Development of the human immunodeficiency virus type-1 (HIV-1) vaccine is an effective and powerful prevention method of the halting pandemic of the acquired immunodeficiency syndrome (AIDS). Dendritic cell (DC)-based HIV immunotherapeutic vaccine is very promising at optimizing the HIV-specific immune responses. Since the Ebola virus glycoprotein (EboGP) has strong DC-targeting ability, we hypothesized that the infusion of the highly conserved elements (CE) of HIV envelope glycoprotein including the Membrane-Proximal External Region (MPER), with the DC-targeting domains of EboGP (EboGPΔM), can direct these epitopes to the DCs/Macrophages and more efficiently elicit immunes responses in the host.

It is known that the mucin-like domain of Ebola GP is highly glycosylated, less conserved and dispensable for EBOV infection. In this study, we have replaced the mucin-like domain of Ebola GP with 9 arranged highly conserved elements (9CE) or MPER of HIV envelop glycoprotein to generate plasmids encoding EboGPΔM-9CE, EboGPΔM-MPER and/or HIV Env(M) transgenes. To investigate whether these fusion proteins are able to efficiently enter DCs/Macrophages, we co-transfected 293T cells with HIV vector (ΔRI/ΔE/Gluc), HIV packaging plasmid (Δ8.2), and EboGPΔM- 9CE and/or EboGPΔM-MPER plasmids to generate virus-like particles (VLPs) and used to infect human monocytes and Macrophages. Our results have shown that EboGPΔM- 9CE- and/or EboGPΔM-MPER- VLPs can efficiently target the a human monocytic cell line (THP-1) and monocytes derived macrophages (MDMs). Also, we investigated the entry ability of EboGPΔM-9CE-and/or EboGPΔM- MPER and/or HIV Env(M)VLPs in in vivo study in BALB/C mice to evaluate the potential T cell- and B cell-mediated immune responses of the VLPs based vaccines.

The results have demonstrated that fusion of EboGP with 9CE or MPER can be successfully expressed in the cells and also can incorporated into pseudotyped VLPs. However, the production of EBOGPΔM-MPER incorporation to VLPs is much less than the production of EBOGPΔM-9CE and HIV Env(M). Moreover, the EboGPΔM- 9CE- and/or EboGPΔM-MPER-VLPs can efficiently target THP-1 and MDMs. This strategy in the animal model(s) demonstrated that both EboGPΔM-MPER- and EboGPΔM-9CE-VLPs induced more robust responses at 76 days post-vaccination in comparison to HIV-Env-VLPs. However, EboGP-MPER fusions pseudotyped VLPs induced significantly higher anti-HIV MPER antibodies than native HIV Env VLPs in mice.

Exploring the Intended Behaviours of Youth Towards Providing Social Support to a Peer with a Concussion

Kylie Mallory1,2, Katherine Wilson3, Kiera DiLeonardo2, Andrea Hickling2,3 & Nick Reed1,2,3

1Rehabilitation Sciences Institute, Faculty of Medicine, University of Toronto, Toronto, Canada; 2Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, Canada; 3Department of Occupational Science and Occupational Therapy, Faculty of Medicine, University of Toronto, Toronto, Canada

Introduction: Youth experience longer recovery times after a concussion and a greater number of symptoms compared to younger children. Sustaining a concussion can lead to isolation and reduced social participation. Youth are strongly influenced by their social networks, and social support is sought out during concussion recovery. Despite this, the intended behaviours of youth towards supporting their peers after they sustain a concussion have yet to be explored. Therefore, the objectives of this study were (1) to identify the intended behaviours of youth towards providing social support to a peer with a concussion; and (2) to examine if demographic factors and concussion knowledge contribute to these intended behaviours.

Methods: A novel survey informed by the Theory of Planned Behaviour was completed by 200 youth (M=15.3 years, SD=1.52 years). The survey included demographic and concussion knowledge questions that were comprised of checkbox, yes/no, open-ended and true/false responses. Questions related to social support were comprised of 4-point Likert scales. Data analysis included descriptive statistics, Wilcoxon Rank Sum Tests and Spearman Rank Correlation Coefficients.

Results: Over 30% of youth reported intentions to engage in behaviours that endorsed isolating or discrediting their peers recovering from a concussion. Females (W=2411, p<0.0005), older youth (rs=0.411, p<0.0005) and having higher concussion knowledge (rs=0.219, p=0.002) were associated with more favourable intentions to provide social support. Participating in high-risk sports (hockey, soccer, football and/or basketball) resulted in significantly less favourable intentions to provide social support (W=6721, p<0.0005). Unexpectedly, a personal history of concussion or knowing someone with a history of concussion had no significant effect on intention to provide social support.

Conclusions: This study provides the first exploration of the intended behaviours of youth towards supporting their peers after they sustain a concussion. The findings identify factors that contribute to the intended behaviours of youth towards providing social support to their peers such as age, concussion knowledge and high-risk sport participation. As not all youth intend to provide social support, these findings have the potential to inform targeted education initiatives aimed at enhancing concussion knowledge and creating more supportive environments for concussion recovery in youth.

Combination of an Alginate-Based Islet Encapsulation Device with a Prevascularization Strategy to Enable Immunosuppression-Free Subcutaneous Islet Transplantation

Braulio A. Marfil-Garza1, Rena L. Pawlick1, Joshua Hefler1, Nidheesh Dadheech1, A.M. James Shapiro1,2

1Department of Surgery, University of Alberta, Edmonton, Canada 2Clinical Islet Transplant Program, University of Alberta, Edmonton, Canada

Introduction: Cellular encapsulation represents a promising alternative to enable immunosuppression-free islet transplantation (IT). Achieving this could allow widespread use of IT in many patients with type 1 diabetes. Its efficient implementation requires that implantation sites other than the intraportal circulation (i.e., the clinical standard) be explored. The subcutaneous space (SC), although promising, is a very hypoxic environment. We developed a strategy for subcutaneous IT that harnesses the foreign body response to promote and increase vascularization of the SC by implanting a Nylon catheter at this site before transplant. Following catheter removal, islets infused into this prevascularized pocket allow diabetes reversal (device-less approach - DL). This approach, however, is not immune protecting. Conversely, the recently-developed thread-reinforced alginate fiber for islet encapsulation (TRAFFIC) allows long-term diabetes reversal without immunosuppression in immunocompetent murine models. However, it has only been tested in the intraperitoneal (IP) cavity, which complicates clinical translation. Our objective is to assess the effectiveness of a DL + TRAFFIC approach to enable immunosuppression-free subcutaneous IT.

Methods: C57BL/6 mice will have catheters implanted into the SC of the abdomen 6 weeks before IT. On the day of transplant, TRAFFIC devices with 500 allogeneic islet equivalents (BALB/c donors) will be implanted. Controls include: TRAFFIC + direct SC implantation (no prevascularization), and subcapsular kidney transplantation (no encapsulation, quality control). Glucose monitoring thrice a week will extend to 180 days. Glucose tolerance tests will be done at 90-days post- transplant to assess metabolic capacity of the grafts.

Results: We have observed long-term diabetes reversal in our model of allogeneic IT using our TRAFFIC + DL approach, with normoglycemia maintained for 180 days without any immunosuppression. In contrast, TRAFFIC devices implanted directly in the SC did not correct hyperglycemia and islets implanted under the kidney capsule reversed hyperglycemia, but were rejected within 21 days.

Conclusion: Our TRAFFIC + DL approach supports long-term immunosuppression-free diabetes reversal and normoglycemia following IT. These findings are promising due to its potential for clinical translation. We are currently extending our experience with xenogeneic models (pig and human islets) to assess the potency and efficacy of our combined approach.

TNF-mediated production of MMP9 and MMP13 is suppressed by antimicrobial host defence

Courtney Marshall, Anthony Altieri, Hemshekhar Mahadevappa, Neeloffer Mookherjee

Manitoba Centre for Proteomics and Systems Biology, Department of Immunology, University of Manitoba

Introduction: Asthma is a heterogenous disease characterized by chronic inflammation in the lung. Tumor necrosis factor (TNF) is a critical mediator of airway inflammation and is associated with increased severity in chronic respiratory diseases. Recent studies have demonstrated that the human antimicrobial cationic host defence peptide LL-37, which is released from neutrophils during inflammation in the lungs, can regulate lung tissue remodeling. Therefore, in this study we examined the effect of LL-37 on TNF-induced metalloproteinases (MMP) which are critical effectors of lung remodeling.

Objective: To profile TNF-induced proteins, and examine effects of LL-37 on MMP production, in human bronchial epithelial cells. Methods: Human Bronchial Epithelial Cells (HBEC-3KT) were stimulated with TNF (20 ng/mL). Cell lysates (n=5) were probed using high content aptamer-based proteomic array. Differential analysis was performed on normalized log2 protein expression values, along with Welch’s t-test (p<0.05) to identify differentially abundant proteins. The proteins identified were independently validated by ELISA in HBEC-3KT, and in primary bronchial epithelial cells (PBEC) obtained from resected lung tissue (in submerged and Air-liquid interface cultures). Related signaling pathways were examined using Src pharmacological inhibitors, Dasatinib and SRC1 (1uM and 5uM). TNF-induced MMP abundance were further examined using ELISA, in the presence and absence of physiological concentration of LL-37 (0.25 uM), in HBEC-3KT.

Results: Proteomic profiling and independent validation demonstrated that TNF increased airway remodeling factors MMP9 and MMP13 in both cell lysates and supernatants, in HBEC and PBEC. Pharmacological inhibition of Src suppressed TNF- induced MMPs, showing the involvement of these pathways. Further, LL-37 significantly decreased the abundance of TNF- induced MMP9 and MMP13 in HBEC.

Conclusion: Physiological concentrations of LL-37 suppress TNF-induced airway remodeling factors MMP9 and MMP13 in bronchial epithelial cells. This study characterizes TNF-induced proteins that can be further examined as drug targets for respiratory inflammatory disease.

Characterization of adaptive metabolic heterogeneity in patient-derived glioblastoma cells

Emma Martell1, Tanveer Sharif1

1University of Manitoba

Introduction: Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults. The current standard of care for GBM patients consists of maximal surgical resection followed by radiation and/or adjuvant chemotherapy. Despite this, disease relapse and the development of highly aggressive and therapy-resistant recurrent tumors remains almost inevitable, with the average time of relapse occurring only 7-9 months after treatment. Because of this, only 5% of Canadians diagnosed with GBM will survive longer than 5 years. The problem with GBM tumors is that they are very heterogeneous, which means there exists different populations of tumor cells that display unique behaviours and energy requirements. This represents a major hurdle for finding effective treatments for GBM patients, as these populations respond differently to various therapeutic interventions. It is unclear how the metabolic makeup of different GBM populations plays a role in therapy response and the development of recurrent tumors. Hypothesis: Heterogeneous GBM tumor populations may display unique mechanisms of metabolic adaptation that regulates therapy response and the development of recurrent tumors.

Methods: This study utilizes heterogeneous GBM cells isolated from excised patient tumors as well as primary and recurrent tumor samples from matched patients. These patient-derived GBM cells are maintained in specialized serum-free 3D neurosphere culture conditions which has been established to conserve the phenotypic characteristics of GBM tumors. Transcriptomic, biochemical, and functional methods were used to characterize the metabolic phenotypes of various patient-derived GBM cells.

Results: Transcriptomic profiling revealed that heterogeneous patient-derived GBM tumor populations displayed unique metabolic profiles. Moreover, these distinct tumor populations differentially rewire their metabolism to adapt to various therapeutic interventions. The robust metabolic plasticity of GBM cells is further exemplified by the observation that the metabolic profiles of tumor samples taken from patient tumors at disease recurrence post-treatment differs from significantly samples taken from the same primary patient tumor prior to treatment.

Conclusion: Altogether, the findings from this project unveil novel insights regarding the regulation of metabolic adaptation in heterogeneous GBM tumors. This information may help improve patient outcomes by identifying potential metabolic targets to aid in the development of new combination therapies.

Cellular Responses of Neurons and Astroglia to Enterovirus Infection

Alan McGreevy1,2,3, Anna Majer1 and Timothy Booth1,2

1Viral Diseases Division, National Microbiology Laboratory, Winnipeg, Canada 2Department of Medical Microbiology and Infectious Diseases, University of Manitoba 3Department of Biology, University of Winnipeg

Introduction: Enterovirus infections cause half the clinical cases of aseptic meningitis in children. Enteroviruses, single- stranded positive-sense RNA viruses of the family Picornaviridae, are associated with illnesses including hand, foot and mouth disease, myocarditis, encephalitis and acute flaccid paralysis. Outbreaks of Enterovirus D68 in 2014, 2016 and 2018 were associated with acute flaccid myelitis and these new strains can replicate in human neuroblastoma cell culture. Echovirus 6 has also shown increased neurovirulence since 2016. Our lack of understanding of enterovirus pathogenesis is a significant barrier to developing effective therapeutics. My research investigates the host cellular response to neurotropic enteroviruses to understand mechanisms of pathogenicity in the central nervous system, which may identify potential therapy targets.

Methods: Human SVG astroglia and SK-N-SH neuroblastoma cell lines were inoculated with Echovirus 6 and Echovirus 11 to screen for viral replication and cytopathic effects. Supernatants were titrated using RT-qPCR and TCID50 assays to determine viral growth kinetics. Pilot study RNA sequencing was performed on SK-N-SH neuroblastoma cells infected with Echovirus 11 at MOI 0.1, using QIAGEN RNeasy extraction, Illumina TruSeq library prep and sequenced on the Illumina Hi- Seq platform.

Results: Echovirus 6 causes cytopathic effect in SVG astroglial cells but not neuronal SK-N-SH cells, while Echovirus 11 is cytopathic in both cell lines. SVG astroglial time courses at MOIs of 0.1 or 1 showed each virus reached 2×10^10 viral genomes per mL supernatant by 2 dpi. Three commercial kits for RNA extraction and three mRNA sequencing library prep kits were compared for RNA integrity and yield. Preliminary RNA sequencing data analysis is underway.

Conclusions: By examining target cell susceptibility and host cell responses to enterovirus infection, and confirming the significance of host proteins in knockdown cell lines, this work will elucidate mechanisms that modulate neurovirulence and identify potential targets for future therapeutic research.

Bisphenol A-Induced Increase of Hypothalamic miR-708-5p Levels May Contribute to Its Obesogenic Effects

Emma K. McIlwraith1, Calvin Lieu1, Denise D. Belsham1,2

1Department of Physiology, Faculty of Medicine, University of Toronto 2Departments of Medicine and Obstetrics and Gynecology, Faculty of Medicine, University of Toronto

Introduction: Bisphenol A (BPA) is a widely used endocrine disrupting chemical linked to obesity and has been termed an “obesogen”. BPA promotes weight gain by binding hormone receptors and directly increasing fat mass. It is beginning to be understood that BPA also acts in the hypothalamus, the brain region that centrally controls weight by modifying appetite and energy expenditure. In peripheral tissues, BPA alters levels of microRNAs (miRNA), which post-transcriptionally regulate gene expression, and are increasingly under investigation for therapeutic use. We therefore hypothesized that BPA alters specific hypothalamic miRNAs that underlie its obesity-promoting effects.

Methods: The murine adult mHypoA-59 cell line was treated with 100 μM BPA for 4 and 24 hours and the RNA was analyzed on the Affymetrix Genechip miRNA 4.0 array. Predicted miRNA targets were identified using miRwalk 2.0 and their mRNA was quantified using RT-qPCR.

Results: Of the over 3,000 murine miRNAs tested on the array, the levels of only 49 miRNAs were affected by BPA. One miRNA in particular, miR-708-5p, was upregulated by 28.8-fold and was therefore selected for further study. Overexpression of miR-708-5p with 25 nM of a mimic resulted in a 1.6-fold increase in neuropeptide Y (Npy), a potent appetite-stimulating peptide. Npy is not a predicted target of miR-708-5p, as such the miRNA may target a gene that represses Npy, ultimately causing an increase in Npy. Upon evaluation of the predicted targets of miR-708-5p using miRwalk 2.0, we identified two candidates: neuronatin (NNAT) and OCT-1. NNAT is a proteolipid linked to body weight regulation. To identify if NNAT regulates Npy expression in hypothalamic cells, NNAT was knocked down using a siRNA and overexpressed using a constitutively active plasmid. The second candidate, OCT-1, is a transcription factor that represses Npy gene expression. To establish if BPA and miR-708-5p reduce OCT-1 binding to the Npy regulatory region, we are conducting chromatin immunoprecipitation (ChIP) analysis.

Conclusions: BPA alters hypothalamic miRNA levels that may in turn mediate its obesogenic effects by elevating expression of appetite-stimulating genes. Understanding the specific mechanisms by which BPA promotes weight gain will reveal methods of minimizing its effects.

Spatial Analysis of Discarded Needles and Dropboxes in Calgary, Canada

Abreham Mekonnen1 ,Em M. Pijl2

1University of Lethbridge 2University of Manitoba

Introduction: Concomitant with the rise in the number of people who inject drugs has been an increase in unsafely discarded needles and injection debris. While the scholarly literature indicates that both supervised consumption services (SCS) and needle distribution programs reduce needle debris on the street, the public news media often report otherwise, suggesting that improperly discarded needles are a significant concern around these services.

Methods: Using Geographic Information Systems (GIS) software ArcGIS (Esri, 2020), we analyzed citywide geospatial data pertaining to needle debris, identifying its spatial distribution, tracking mitigation programs, identifying hotspot locations, and relating the distribution pattern to the presence of needle dropboxes.

Results: A total of 74,900 discarded needles were collected from 4,470 discrete locations (i.e., 4,470 retrievals) between January 2019 and September 2020. Needle debris was most dense in two central neighbourhoods: Beltline and Downtown Commercial Core. Analysis showed that 10% (n=7,667) of collected needles were within 300 meters of the SCS, 16.8% (n=12,608) were within a 500-meter buffer from the facility, and 43% (n=32,472) within 1-kilometre buffer from the facility. The city's central neighbourhoods contributed to 83% of all needle discards, which account for 73% of discrete locations. Additionally, 51% of discarded needles were collected from the Beltline (40%) and Downtown Commercial Core (11%) neighbourhoods, and these neighbourhoods accounted for 85% of clusters and 42 of 80 hotspots. Needle dropboxes were located in only six of the 19 neighbours with hotspots. There was a positive correlation between overdoses reported and the presence of needle debris. The onset of the COVID-19 pandemic restrictions was linked to a spike in the number of collected discards.

Discussion: Central neighbourhoods had high rates of improperly discarded needles and a lack of needle dropboxes. A causal relationship between needle debris and the area services (SCS, homeless shelters) cannot be definitively surmised as there is a lack of data prior to these programs starting. Conclusion: Needle debris is a complex social, environmental and public health issue that requires a multifaceted approach. GIS mapping is a powerful tool that can locate hotspots so that resources can be deployed.

The role of Hippo signaling in stromal-epithelial interactions in acinar-to-ductal metaplasia and pancreatic cancer initiation

Julia Messina-Pacheco1, Alex Gregorieff1

1Department of Pathology, McGill University, Montreal, QC, Canada

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths with a 5-year survival rate of approximately 7%. Current models suggest that PDAC may originate from acinar cell trans-differentiation into ductal-like cells. Acinar-to-ductal metaplasia (ADM) is triggered by chronic pancreatitis and/or mutations in K-Ras and may further develop into pancreatic intraepithelial neoplasia (PanIN). The progression to PDAC is commonly associated with a dense fibrotic stroma, excessive extracellular matrix (ECM) production, stromal cell proliferation and myofibroblast activation. Cancer-associated fibroblasts (CAFs) may contribute to cancer progression by further increasing ECM deposition and remodeling. The Hippo transcriptional effectors YAP1/TAZ have been identified as essential drivers of ADM and YAP1 is a tension-stimulated CAF activator that promotes ECM remodelling and stiffening, thereby generating a mechanosensory feed-forward loop that fosters a permissive microenvironment for pancreatic cancer progression.

Hypothesis: We hypothesize that the Hippo pathway may coordinate fibroinflammatory signals emanating from the stromal compartment during regenerative responses to acinar cell injury in the pancreas and progression towards PDAC.

Methods and Results: In order to unveil the differential gene expression profile of ADM, we performed a high-plex, spatial analysis of over 1800 RNA probes in normal acini, ADM and PDAC areas of FFPE human primary tumour tissue using NanoString’s GeoMx Digital Spatial Profiling (DSP) technology. Bioinformatic analysis revealed functional enrichment of multiple pathways in ADM, including K-Ras signaling, ECM organization and epithelial-to-mesenchymal transition. Further studies into the role of Yap in ADM will be conducted by immunofluorescence staining with ductal, stromal and fibroblast markers on human PDAC tissue. To study the role of Hippo signaling in stromal cells, we conditionally deleted Yap/Taz or Lats1/2 in Collagen1a2-producing cells in a murine model of caerulein-induced pancreatitis. The efficiency of Cre activation in pancreatic stromal cells was monitored by tamoxifen induction of Col1a2Cre-ERT; LSL-Tom mice. Lineage tracing experiments will be completed to verify the contribution of Col1a2-expressing cells to the stromal reaction.

Conclusion: Further insights into the mutational and expression gene profile of ADM, its regulatory network and signaling pathways may allow for the discovery of sensitive and specific early diagnostic biomarkers, as well as effective strategies to prevent pancreatic cancer development.

Integrating Patient Values into Clinical Trials Using Composite Endpoints: A Case Study Using the Control of Hypertension in Pregnancy Study

Rebecca K. Metcalfe1,2, Mark Harrison2,3; Joel Singer1,2; Laura A. Magee4; and Nick Bansback1,2,5

1School of Population and Public Health, University of British Columbia, Vancouver, Canada 2Centre for Health Evaluation & Outcome Sciences, Vancouver, Canada 3Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada 4Department of Women’s Health, King’s College London, London, United Kingdom 5BC SUPPORT Unit, Vancouver, Canada

Objective: Clinical trials in cardiovascular medicine routinely use composite endpoints (CEs) to capture the breadth of an intervention’s effects and improve statistical efficiency. Unfortunately, CEs can be difficult to interpret, especially when the direction of treatment effects varies by composite component or the clinical importance of the components is unequal. Using an existing trial comparing two pregnancy hypertension management strategies, this study explored how integrating patient values might change the interpretation of trial results.

Design and Method: A previous study explored patient preferences for management of pregnancy hypertension. The study used validated preference elicitation techniques to quantify the relative value of seven potential composite components identified in qualitative work. Latent profile analysis of overall preferences found three preference profiles; each gave different weight to potential components (see table). We integrated these weights for each profile with actual event data from the Control of Hypertension in Pregnancy Study (CHIPS) trial. Each component was scored 1 or 0 if it occurred or did not occur, respectively. The binary component score was then multiplied by the component weight and summed by participant to create a weighted CE score. This process was repeated for overall and preference profile weights. Mean CE values between treatment arms were compared using T-tests.

Results: Similar to the primary findings in the CHIPS trial, analyses using overall (and Profile 1) preference weights found no difference between the two treatment approaches. However, when data were analyzed using Profiles 2 and 3 weights, ‘tight’ control was statistically significantly better for individuals who prioritized avoiding early delivery and ‘less-tight’ control was significantly better for individuals who prioritized minimizing medication.

Conclusions: Although the original CHIPS trial found no difference in its primary composite outcome, our results suggest that integrating patient values can change the interpretation of the CHIPS trial results. Depending on patient preference profiles, a single trial may support different treatment recommendations. While the approach has important lessons for shared decision-making, it poses some challenges for implementing in trial designs, notably determining recruitment targets for adequate statistical power for each preference profile.

HCAR1 Nuclear Location Bias Drives Cancer Malignancy by Multiple Routes

Mohammad Ali Mohammad Nezhady1,2, Gael Cagnone2, Sylvain Chemtob1,2

1Programmes en Biologie Moléculaire, Faculté de Médecine, Université de Montréal, Montreal, QC, Canada 2Centre de Recherche du CHU Sainte-Justine, Montreal, QC H3T 1C5, Canada

Introduction: GPCR are virtually involved in all physiological processes. HCAR1, as a GPCR, is endogenously activated by lactate and has been shown to promote cancer malignancy via higher level of glycolysis due to Warburg effect. It has a high expression level in many cancers and negatively correlates with patient’s prognosis. However, its mechanism of action is ill- understood.

On the other hand, nuclear localization of several GPCRs have been described albeit its unusual feature for them. Additionally, it has been shown nuclear GPCRs can perform functions distinct from their cell surface counterparts in vivo. Here we demonstrate HCAR1 has a nuclear localization and this localization pattern promotes cancer malignancy by multiple routes.

Methods and Results: We determined HCAR1 nuclear localization pattern by cell fractionation, immunofluorescence confocal imaging and TEM. Site-directed mutagenesis showed ICL3 and phosphorylation of C-terminal domains are required for nuclear localization. We also demonstrated that this localization is ligand independent and there is a pool of nuclear HCAR1 (N-HCAR1) in the cells. We show N-HCAR1 induces intra-nuclear signaling through Gαi and Gßγ by WB and ELISA leading to increased phosphorylation of nuclear AKT and ERK resulting in increased cancer cell survival and proliferation. Our ChIP-sequencing data shows N-HCAR1 binds to the genes regulating cell migration and we prove N-HCAR1 promotes migration in cellulo. We identified N-HCAR1 interactome by Bio-ID coupled with mass spectrometry and found, it interacts with proteins involved in translation and DNA damage repair and our experimental data demonstrates that specifically the N-HCAR1 promotes both of those process in cellulo. Additionally, we identified the transcriptomic signature of N-HCAR1 and showed it regulated a larger gene network than its plasma membrane counterpart. Concordantly, our in vivo tumor xenografts and tail vein injections prove that tumors without N-HCAR1 have smaller size and tumor mass and lower metastatic rate as well.

Conclusion: Here we show an unusual localization of a GPCR in the nucleus and provide evidence that N-HCAR1 specifically promotes various hallmarks of cancer malignancy. The effect of N-HCAR1 is validate in vivo in tumor xenografts as well. Understanding these mechanisms can provide targets and cues for therapeutic developments.

Investigating the Role of LAG3 in HIV-1 Susceptibility

Shifa Mohideen, Colin Graydon, Thomas Murooka, Keith R. Fowke

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 2Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya 3Partners for Health and Development in Africa, Nairobi, Kenya 4Department of Community Health Sciences, University of Manitoba, Winnipeg 5Department of Immunology, University of Manitoba

Background: Upon HIV infection, T cell exhaustion is provoked resulting in the failure to produce appropriate effector cytokines. Such inhibition is mediated by the overexpression of surface [negative] co-receptors called immune checkpoints (IC). LAG-3, an IC, is enriched on CD4+HIV-infected T-cells, where it may pre-dispose cells to HIV infection thus contributing to susceptibility. This study seeks to clarify the role of LAG-3 as a potential co-receptor for viral entry in vitro.

Expected Results: An HIV-1 infection assay will test HIV susceptibility by comparing % of LAG-3+ and LAG-3- CD4+ T cell lines infected with single-replication HIV-GFP by flow cytometry. As LAG-3 is homologous and structurally similar to CD4 co-receptor, sharing the common ligand MHC-II, we hypothesize more LAG3+ cells are infected than LAG3- cells.

Conclusion: Understanding LAG-3’s role in HIV infection may provide valuable insight on the efficacy of a LAG-3 blockade in alleviating susceptibility and ultimately, inform a functional cure for HIV.

The gut mycobiome in pediatric multiple sclerosis: establishing a bioinformatics pipeline

N. Mok1, N. Knox2, D.L. Arnold3, A. Bar-Or4, C.N. Bernstein1, J. Forbes5, M. Graham2, C. Bonner2, J. Hart6, R.A. Marrie1, J. O’mahony7, E.A. Yeh5, F. Zhu8, G. Van Domselaar2, B. Banwell9, E. Waubant6, H. Tremlett8;

1University of Manitoba, MB/Canada, 2Public Health Agency of Canada, MB/Canada, 3McGill University, QC/Canada, 4University of Pennsylvania, PA/United States of America, 5University of Toronto, ON/Canada, 6University of California San Francisco, CA/United States of America, 7The Hospital for Sick Children, ON/Canada, 8University of British Columbia, BC/Canada, 9Children’s Hospital of Philadelphia, PA/United States of America

Background: Studies examining the role of the microbiota in multiple sclerosis (MS) often focus on the gut bacteria; few have considered a potential role of gut mycobiota. Methods for evaluating gut mycobiota are lacking and require systematic evaluation of sequencing protocols, reference databases, and bioinformatics pipelines to properly investigate possible gut mycobiome influences on MS.

Objectives: We set out to evaluate the performance of different sequencing conditions and analytical approaches for characterizing the gut mycobiome in a cohort of healthy individuals and cases with monophasic acquired demyelinating syndrome (mono ADS) or pediatric-onset MS.

Methods: We first assessed a mock-community control of 19 defined fungal organisms. We then assessed 201 stool samples obtained from our cohort of 52 healthy individuals, 49 individuals with mono ADS, and 46 participants with pediatric-onset MS. The fungal internal transcribed spacer 2 (ITS2) region was sequenced using the Illumina MiSeq platform. Varying concentrations of PhiX control library spike-in were tested to address low-complexity amplicon sequencing. Generated sequences were characterized by the UNITE database—a curated collection of eukaryotic ITS sequences—in conjunction with three fungal sequence analysis pipelines: LotuS, mothur, and PIPITS.

Results: Taxa identified in our mock-community differed across sequencing conditions but were similar between technical replicates. LotuS correctly classified 7 taxa each at species and at genus level; 5 were unclassified. Mothur correctly identified 5 species-level taxa and 11 at genus-level; 3 were unclassified. Lastly, PIPITS correctly identified only 3 species- level and 12 genus-level taxa, while 4 were unclassified. We successfully generated sequence data for 112 of 147 (76%) individuals (70 females; 42 males). Of the tested sequencing conditions, a spike-in of 50% PhiX produced the highest-quality reads.

Conclusions: The LotuS pipeline best identified fungal taxa in our mock-community, with optimal resolution to species level. Sequencing read quality was optimal when 50% PhiX was used for sequencing ITS2 amplicon libraries of stool samples. Establishment of this validated sequencing pipeline, confirmed using a mock-community with known fungal identities, will aid characterization of gut mycobiomes for our cohort of individuals with/without pediatric-onset MS.

Fibro-adipogenic progenitors and bioactive lipids interaction, a key partnership for muscle regeneration

Molina Thomas1,2, Fabre Paul1,2, Deprez Alyson1,2, Dumont Nicolas1,3

1CHU Sainte-Justine Research Center. Montreal, QC, Canada 2Department of pharmacology and physiology, Faculty of Medicine, Université de Montréal. Montreal, QC, Canada. 3School of rehabilitation, Faculty of Medicine, Université de Montréal. Montreal, QC, Canada

Introduction: Representing 40% of the body mass, the skeletal muscle is the most prevalent human tissue, that plays a vital role in daily activities. Due to its repeated use, muscle is subjected to frequent injuries. Consequently, it possesses a remarkable regenerative capacity involving the activation and fusion of muscle stem cells (MuSC) to form myotubes. Growing evidence shows that fibro-adipogenic progenitors (FAPs), a population of stem cells responsible for extracellular matrix production, are also important in muscle reconstruction by sustaining MuSC proliferation. As their name suggests, these mesenchymal cells are able to differentiate either in adipocytes or in fibroblasts orchestrating time dependent processes allowing the muscle to fully recover. Despite their role in muscle homeostasis, FAPs have been described to be broadly implicated in muscular diseases such as Duchenne muscular dystrophy (DMD) by promoting fibrosis accumulation. In order to better comprehend the mechanisms regulating the activity of FAPs during muscle regeneration, we focused on bioactive lipids, which are derived from omega-3 and -6 fatty acids, and are known to regulate inflammation and stem cell function in muscle. However, the role of these lipids on FAPs is still unknown.

Methods: We treated FAPs with different bioactive lipids and investigated several parameters. Furthermore, we are treating mdx mice (a well-known model of DMD) with these compounds to determine their impact on fibrosis, myogenesis, and muscle function.

Results: We have shown that FAPs express several bioactive lipids receptors as well as their synthesis enzymes. Our in vitro co-culture experiments revealed that FAPs are a main source of bioactive lipids that act as a paracrine factor to stimulate MuSC proliferation. Moreover, bioactive lipids also directly affect FAPs behavior in an autocrine manner to regulate their proliferation. RNAseq analysis are currently underway to determine their mechanism of action.

Conclusion and perspectives: This project has the potential to highlight a new mechanism implicated in muscle regeneration and provide an alternative for the current treatment for DMD.

Combined treatment of human islets with glucagon-like peptide-1 receptor agonist and interleukin-1 receptor antagonist protects β-cells in type 2 diabetes and islet transplantation

Mukta Moni1, Yoo Jin Park1, Dr. Lucy Marzban1

1University of Manitoba

Background: Type 2 diabetes (T2D) is characterized by peripheral insulin resistance and progressive β-cell death which leads to hyperglycemia. Formation of islet amyloid by aggregation of the β-cell hormone, islet amyloid polypeptide (IAPP), in patients with T2D, cultured and transplanted islets, contributes to β-cell death in T2D and poor long-term survival of transplanted islet grafts in T1D. Treatment with glucagon-like peptide-1 receptor agonists (GLP-1Ra) such as exenatide, or interleukin-1 receptor antagonists such as anakinra enhances β-cell survival.

Hypothesis: In this study, we tested whether combined therapy with exenatide and anakinra has synergistic effects on improving islet survival by reducing amyloid formation and β-cell apoptosis.

Methods: Isolated pancreatic human islets (purity >90%) were cultured free-floating in CMRL containing 11.1 mmol/l glucose (to form amyloid) without or with exenatide (10 nmol/l) and anakinra (10 µg/ml) or both. Culture medium was changed every 2 days. Paraffin-embedded islet sections were double-immunostained for insulin and TUNEL or thioflavin S. Micrographs were captured using an inverted fluorescence Zeiss microscope.

Results: Treatment with either exenatide or anakinra reduced β-cell apoptosis in human islets during culture (non- treated=7.4±0.9%, exenatide=3.6±0.5%, anakinra=3.9±0.7%; p<0.05). Combined treatment with exenatide and anakinra further reduced β-cell apoptosis as compared to treatment with exenatide or anakinra alone (exenatide+ anakinra=2.1±0.3%; p<0.05). Reduced β-cell death closely correlated with lower number of amyloid positive islets during 7- day culture (control=0.7±0.2%, non-treated=12±4%, exenatide=5±1%, anakinra=4±0.8%, exenatide+anakinra=2±0.5%; p<0.05).

Conclusion: Treatment with GLP-1Ra (exenatide) and IL-1RA (anakinra) reduces amyloid formation and improves β-cell survival in human islets during ex-vivo culture. Combined treatment with exenatide and anakinra has additive effects on protecting islet β-cells from amyloid toxicity, therefore may provide an effective strategy to enhance survival of β-cells in conditions associated with islet amyloid formation such as T2D and islet grafts in T1D.

Unravelling interactions between genetics, nutrition, and sex using experimental models of early- onset type 2 diabetes

Taylor S. Morriseau1,2, Kristin L. Hunt1,3, Cuilan Nian4, Vernon W. Dolinsky1,2, Francis Lynn4, Christine A. Doucette1,2,3

1Diabetes Research Envisioned and Accomplished in Manitoba (DREAM) Theme of the Children's Hospital Research Institute of Manitoba (CHRIM), Winnipeg, MB 2Departments of Pharmacology and Therapeutics, University of Manitoba 3Physiology and Pathophysiology, University of Manitoba, Winnipeg, MB, 4BC Children’s Hospital Research Institute, Vancouver, BC

Background: Manitoban Indigenous youth, particularly female adolescents, experience the highest rates of early-onset type 2 diabetes (T2D) in Canada. 40% of these youth harbour a genetic variant in the HNF-1α gene (HNF-1αG319S). In G319S carriers, clinical evidence implicates pancreatic β-cell dysfunction compounded by modern dietary stress. We hypothesize that the G319S variant alters β-cell insulin secretion differentially by sex and by dietary intake.

Methods: CRISPR/Cas9 was used to knock-in the G>A.955 substitution into MIN6 β-cells (G319S-MIN6) and C57/BL6 mice (G/S and S/S) compared to control (G/G). To determine the impact of the G319S variant on β-cell function, oxygen consumption rates and insulin secretion were measured in basal and glucose-stimulated MIN6 β-cells. To assess whole- body physiology, body weight, insulin sensitivity, and ex vivo insulin secretion in pancreatic islets from male and female mice were assessed at 6-months-of-age.

Results: In female mice, glucose-stimulated insulin secretion (GSIS) in G/S islets was reduced 3.5-fold relative to G/G mice, but this impairment was not observed in G319S-MIN6 cells or in islets from male mice. In these male-derived models, a consistent reduction in basal insulin secretion (BIS) was observed (>2.8-fold). The suppression of BIS may be driven by a 1.5- fold elevation in fatty-acid β-oxidation as measured in G319S-MIN6 relative to control. At the whole-body level, suppressed BIS in male G/S mice translated into reduced fasting plasma insulin (3.4-fold) and elevated blood ketones (2.6-fold), with no changes in body weight or insulin sensitivity.

Conclusion: In a female G/S mouse model, the impairment in glucose-stimulated insulin secretion may contribute to accelerated diabetes onset under modern dietary stress. This is being assessed by placing G319S-expressing mice on a “Western” high-carbohydrate diet. In male-derived models, the G319S variant shifts β-cell metabolism resulting in elevated fatty-acid oxidation and suppressed BIS, possibly triggering ketogenesis and reducing systemic glucose utilization. To better align G319S-expressing mice with their apparent metabolic dependencies, studies evaluating the use of a high-fat, low- carbohydrate diet (one that resembles the content of traditional food sources) are currently underway.

Uncovering the physiological roles of gut hormone receptor signalling in intestinal-triglyceride secretion

Nadya M. Morrow1,2, My-Anh Nguyen1, Natasha A. Trzaskalski1,2, Antonio A. Hanson1,2, Evgenia Fadzeyeva1,2, Erin E. Mulvihill1,2

1University of Ottawa Heart Institute 2Department of Biochemistry, Microbiology and Immunology, Ottawa, Ontario, Canada

Introduction: The small intestine receives hormonal, immunological, and neuronal inputs to ensure the proper delivery of dietary triglycerides (TG) to fuel the body. Patients with Type 2 diabetes and metabolic disease secrete more TG than healthy individuals following the same meal, contributing to increased risk of cardiovascular disease. The changes in the regulation of TG secretion during metabolic disease are poorly understood. Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are meal-stimulated, gut-derived hormones that potentiate glucose-stimulated insulin secretion linking food ingestion with utilization. Additionally, GLP-1R signalling reduces intestinal-TG secretion in healthy individuals and patients with diabetes, and Glp1r-/- mice display increased intestinal-TG secretion rates compared to wild- type controls. However, we do not understand the factor(s) driving this increase in the absence of GLP-1R signalling. Since the sister peptide, GLP-2, and GIP are also meal-stimulated gut peptides that increase intestinal-TG secretion, we aimed to uncover their contribution to dysregulated lipoprotein secretion. We also aimed to identify the receptor-expressing cells and signaling circuits in the gut responsible.

Methods: We challenged GLPDKO (Glp1r-/-Glp2r-/-), DIRKO (Glp1r-/-Gipr-/-), and wild-type mice to an oil gavage and collected blood samples up to 4 hours post-gavage to measure intestinal-TG secretion. We also used RNAscope in situ hybridization to visualize Gipr mRNA in jejunal tissue sections.

Results: Intestinal-TG secretion was not significantly different between male or female DIRKO mice compared to wild-type controls when fed chow or a high-fat diet. By contrast, chow-fed male GLPDKO mice secreted less TG, and female GLPDKO mice secreted more intestinal TGs than wild-type controls. High-fat feeding reversed this trend in male mice: GLPDKO mice secreted more intestinal TGs than wild-type mice. These results suggest that GIPR signalling is responsible for the increased intestinal-TG secretion observed in Glp1r-/- mice. Additionally, we have observed distinct, non-epithelial Gipr-expressing cells in the villus microenvironment, rich in immune, vascular, and neuronal cells.

Conclusion: In conclusion, GIPR signalling appears to play a sex-specific role in intestinal-TG secretion and diet-induced obesity in male mice. Identifying the local GIPR signalling engaged upon nutrient ingestion is required to uncover potential therapeutic strategies that target the pro-atherogenic gut-heart axis.

Identifying the molecular mechanism for the pain caused by lionfish venom

Stephanie Mouchbahani-Constance1, Albena Davidova1, Hugues Petitjean1, Reza Sharif-Naeini1

1Department of Physiology and Cell Information Systems Group, McGill University, Montreal, Quebec, Canada

Introduction The lionfish (Pterois volitans) is a venomous invasive species found in the Caribbean and Northwestern Atlantic. It poses a growing health problem due to painful stings that it delivers, for which no treatment or antidote exists. Our prior work provided the first insight into the venom’s cellular target – a subset of pain-sensing neurons, termed nonpeptidergic nociceptors. The aims of my current project are to 1) isolate the algogenic toxin in lionfish venom and identify its target and 2) identify how predators of the lionfish evolved a resistance to the painful venom.

Methods We use High Pressure Liquid Chromatography (HPLC) to fractionate the venom and screen fractions for activity on human nociceptors derived from induced pluripotent stem cell (iPSC) using a high throughput screening system (FLIPR). The active fraction will be subject to mass spectrometry in order to identify the sequence of the algogenic toxin and use calcium imaging and electrophysiology to identify the toxin’s receptor. For aim 2, we will clone the gene encoding the toxin’s receptor from predators of the lionfish (Spotted moray eel, Gymnothorax moringa) and evaluate its sensitivity to the venom’s algogenic toxin using calcium imaging and electrophysiology. Finally, I will align the sequences of the human and eel receptors to identify divergent domains, and generate chimera of this receptor to demonstrate whether insensitivity to lionfish venom can be conveyed to the mouse or human receptor by simply swapping domains with those from the moray eel’s sequence.

Results Calcium imaging results using thapsigargin show that the venom acts on an ion channel to specifically activate non- peptidergic nociceptors. Furthermore, activation of these cells by the venom elicits the release of ATP to activate neighbouring cells, causing amplification of the pain signal. We confirmed this channel’s identity using pharmacological blockers and siRNA molecules as well as multiple cell types (mouse dorsal root ganglion (DRG) neurons, human DRG neurons and human iPSC-derived nociceptors).

Conclusion These results will 1) identify novel modulators of the pain pathway, 2) identify novel therapeutic targets in the treatment of lionfish envenomations and 3) advance our knowledge on the acquisition of venom resistance through evolution.

Sex-specific role of fibrillin-1 in adipose tissue development and function

M.L. Muthu1*, K. Tiedemann2,3*, S. Komarova1,2,3#, D.P. Reinhardt1,2#

1Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, Canada 2Faculty of Dentistry, McGill University, Montreal, Canada 3Shriners Hospital for Children, Montreal, Canada * Co-first authors, # Co-senior authors

Introduction: Fibrillin-1 is a 350 kDa large extracellular glycoprotein present throughout the body. Mutations in fibrillin-1 cause a wide spectrum of type I fibrillinopathies, including Marfan syndrome (MFS). Individuals with MFS are typically lipodystrophic, however a subgroup is also characterized by a high body mass index. Therefore, our current study aims to address the hypothesis that fibrillin-1 deficiency in MFS interferes with normal regulation of adipogenesis, resulting in abnormal fat metabolism.

Methods: To determine the role of fibrillin-1 in adipose tissue homeostasis in vivo we employed two MFS mouse models - Fbn1mgR/mgR and Fbn1C1039G/+. Fbn1mgR/mgR are hypomorphic mice that produce reduced amounts (25%) of normal fibrillin-1. Fbn1C1039G/+ carry a missense mutation in fibrillin-1 (C1039G). To elucidate the adipogenic mechanisms in vitro we used bone marrow derived mesenchymal stem cells (MSCs) isolated from wild type mice, Fbn1mgR/mgR mice, Fbn1C1039G/+ mice.

Results: Fibrillin-1 was expressed in both mouse and human white adipose tissues surrounding the mature adipocytes. As expected Fbn1mgR/mgR mice displayed a significant reduction in fibrillin-1 levels whereas no change was observed in Fbn1C1039G/+ mice. Male Fbn1mgR/mgR mice displayed a significantly higher weight of inguinal white adipose tissue (WAT) and brown adipose tissue (BAT), compared to littermate controls (LC) while Fbn1C1039G/+ showed trends of increase. Elevated levels of plasma insulin, cholesterol, triglycerides, HDL and insulin resistance was observed in the male Fbn1mgR/mgR mice. Fibrillin-1 reduction in male mice promoted adipogenic gene expression markers and led to hypertrophic expansion of mature adipocytes compared to LC. In vitro, MSCs isolated from wild-type mice were treated with an adipogenic cocktail with or without recombinant fibrillin-1 C-terminal (rFBN1-C) fragment. rFBN1-C significantly reduced adipocyte differentiation and specifically the adipogenic commitment in the MSCs by suppressing the insulin signaling pathway and adipogenic gene expression. An increased number of mature adipocytes and adipogenic gene expression was also observed when MSCs were differentiated from both male and female Fbn1mgR/mgR mice.

Conclusion: Overall, our study shows that the altered adipose tissue metabolism observed in MFS mice depends on the type of fibrillin-1 deficiency and the biological sex, establishing a critical role of fibrillin-1 in adipose tissue development.

Characterization of cytokine receptor expression in the female genital tract

Ruth Mwatelah, Florence Mutua, Aida Sivro, Cheli Kambaran, Sandra Kiazyk, Nzioki King’ola, Sammy Wambua, Peter Gichangi, Marissa Becker, Sharmistha K. Mishra, Lyle R. McKinnon.

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada. 2JC Wilt Infectious Diseases Research Centre, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. 3Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu-Natal, Durban, South Africa 4International Centre for Reproductive Health, Mombasa, Kenya 5Pwani University, Kilifi, Kenya 6Centre for Global Public Health, University of Manitoba, Winnipeg, Manitoba, Canada 7Department of Medicine, University of Toronto, Toronto, Ontario, Canada 8Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.

Background: Cytokines expression levels in the female genital tract (FGT) are influenced by external factors like sexually transmitted infections, commensal microbiota, and intravaginal practices. Increased genital cytokines have been associated with risk of HIV acquisition, but few studies have focused on expression of the cytokine receptors through which they signal.

Objective: This study aims to quantify expression levels of a wide range of cytokine receptors and Toll-like receptors, and correlate their expression to microbiome, cytokine expression, sexually transmitted infection and behavior associated with sex work among adolescent and young women (ADGYW) from Mombasa, Kenya.

Methods: We collected cervicovaginal samples using a SoftcupTM from 70 sexually active ADGYW, a subset of whom engage in transactional sex and/or sex work. RNA was extracted from the Softcup pellets, and gene expression measured using the Quant studio™ 12K Flex system, A panel of 55 genes were assessed, consisting of cytokine receptors, toll-like receptors and genes representing major immune cell subsets. Microbiome and sexually transmitted infections (STI) were measured by sequencing the V3-V4 region of 16s rRNA and qPCR respectively. Correlation of receptors, microbiome and behavioral variables was carried out using R studio and SPSS.

Results: The prevalence of HIV infection, dysbiosis and any STI was 17.1%, 54.23% and 34.3% respectively. Vaginal microbiota were classified as Lactobacillus or non-Lactobacillus dominant, and alpha diversity was positively correlated with CCR6, CXCR3 and CXCR7 (p<0.05). Positive correlations were observed between markers of immune cells (CD4, CD45, CD69 and CD14) and several cytokine receptors (IL8Rb, IL1R1, IL1R2, IL1RAP, IFNR1, IFNR2, IL6R, TNFRSF1 and TNFRSF1, all p< 0.05). Recent sexual intercourse correlated positively with cytokine receptor expression (IL6R, IL8Rb, TNFRSF1, TNFRSF1, IFNR1 and IL1R2, all p<0.05).

Conclusion: When there is evidence of immune cell presence, there is more cytokine receptor expression suggesting the cells may increase the receptors, and/or the receptors may be expressed mainly on immune cells. Effects of cytokines are likely to be more dramatic if receptor expression is high allowing completion of signaling loop in HIV infection.

Role of microglial PARP-1-centered signalling in promoting the synaptotoxic actions of amyloid- beta oligomers (AβOs)

Olha Myhalatyuk, Albert Yeung, Tiina M. Kauppinen, Michael F. Jackson

1Department of Pharmacology & Therapeutics, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 2Kleysen Institute for Advanced Medicine, Health Sciences Centre, Winnipeg, Manitoba, Canada

Introduction: Alzheimer’s disease (AD) is a neurodegenerative disorder characterised by memory loss and cognitive decline due to Aβ accumulation. AβOs are potent synaptotoxins, that disrupt glutamatergic synaptic transmission and plasticity, notably NMDAR-dependent long-term potentiation (LTP). AβOs also initiate pathological pro-inflammatory responses in microglia, however the extent to which these contribute to impaired synaptic transmission and plasticity is poorly understood. Our past work shows that microglial PARP-1 is a central signalling hub that drives inflammation through NF-κB. Moreover, our recent work shows that sustained microglial PARP-1 activation requires Ca2+ feedback as a result of downstream Ca2+ permeable transient receptor potential melastatin-2 channel (TRPM2) activation. My study aims to examine whether microglial pathological responses promoted by PARP-1/TRPM2 signalling cascade leads to impaired synaptic plasticity and cognitive decline.

Methods and Results: To validate the role of microglial PARP-1 signalling in driving synaptotoxic AβO responses, we created microglia-targeted conditional PARP-1 knockout (PARP-1cKO) mice using Cre-lox recombination system. I derived acute hippocampal slices from PARP-1cKO and WT mice and recorded field excitatory post-synaptic potentials (fEPSPs). Once the baseline amplitude was established, LTP was induced by high-frequency stimulation at 100Hz. The different treatments (Ctrl vs. AβO) and microglial PARP-1 expression status (WT vs. PARP-1cKO) did not affect the baseline amplitude, but AβO- induced LTP inhibition seen in WT slices, was prevented in PARP-1cKO slices. To investigate the role of TRPM2 channels I used 4-6 weeks old WT mice and utilized clotrimazole to disrupt the signalling feedback loop. As with PARP-1cKO, treatment with clotrimazole did not affect the baseline amplitude in hippocampal slices but prevented the synaptotoxic effects of AβOs on LTP.

Conclusion: My findings demonstrate that synaptotoxic effects of AβOs are primarily mediated by PARP-1 signalling hub in microglia. However, PARP-1 inhibition might not be optimal approach to halt microglial pathological cascades as PARP-1 is expressed by all cell types and involved in variety of physiological functions. The pharmacological targeting of TRPM2 channels with abundant microglial expression might be more suitable option/approach. Future experiments will establish whether disruption of microglial PARP-1/TRPM2 protects against cognitive decline in a mouse model of AD.

Strain Profiles of Creutzfeldt-Jakob Disease (CJD) in Canada

Jennifer Myskiw1,2, Lise Lamoureux2, Michael Coulthart3, Stephanie Booth1,2

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg 2Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg 3Canadian CJD Surveillance System, Public Health Agency of Canada, Ottawa

Introduction: Prion diseases are rare invariably fatal neurodegenerative disorders affecting humans and animals. These diseases are associated with a structural change of a naturally occurring prion protein found within the brain to a misfolded pathogenic form. Chronic Wasting Disease (CWD) is a prion disease that has reached epidemic proportions within North America’s deer and elk populations. With the rapid spread of this disease, there are concerns over possible transmission to humans who consume CWD infected meat. If CWD were to cross the species barrier to humans it would likely exist as a novel strain of human prion disease (broadly referred to as Creutzfeldt-Jakob disease or CJD). However, it is impossible to know what human CWD would present with or whether it would differ from other CJD cases. Additionally, there are currently no convenient laboratory methods to study CJD strain differences experimentally. For these reasons, we have applied novel technologies in CJD surveillance to analyse strain diversity of CJD in Canada.

Methods: Samples from 32 CJD confirmed patients who resided in Alberta or Saskatchewan at their time of death have been categorized into venison consumers and non-venison consumers. Analysis of the samples include temperature and protease resistance profiles, glycoform ratio analysis, and real time quaking-induced conversion seeding profiles. Atypical cases identified have been inoculated into deer mice to observe clinical characteristics of disease.

Results: Using the techniques previously described, we found CJD cases of the same type to have remarkably similar molecular profiles. Subsequently, outliers were easily identified. We report two CJD cases with atypical properties as well as two cases of a rare subtype of CJD called variably protease-sensitive prionopathies or VPSPr. We hypothesize the two CJD cases with atypical properties represent novel strains of disease.

Conclusions/Next Steps: By characterizing Canadian CJD strains we intend to develop a baseline through which atypical cases of CJD, such as those that may arise from CWD exposure, could be readily identified in the future. Future steps include analyzing clinical characteristics of disease of the atypical CJD types once all mice succumb to prion infection.

Reliability of the Body Language Coding Scale (BLCS) for children with Autism Spectrum Disorder (ASD)

Ilana D Naiman1,2, Kelly P. Arbour-Nicitopoulos1, Jessica Brian2, and F Virginia Wright2

1University of Toronto 2Bloorview Research Institute

Background: Autism Spectrum Disorder (ASD) affects 1/66 children in Canada and is characterized by intense, repetitive, or restricted interests, and deficits in social communication including challenges using body language. As part of my doctoral thesis, the Body Language Coding Scale (BLCS) was developed to interpret body language of children with ASD during physical activity (PA). The BLCS is an observational scale that divides body language cues into Positive (n=10), Negative (n=7) and Neutral (n=2) categories. PA was the chosen focus as children with ASD often do not meet the recommended PA guidelines therefore do not gain the associated benefits. One common barrier expressed by parents of children with ASD is that their child was excluded from activities because the instructors did not have an understanding about their communication. The rationale for the development of the BLCS was that if instructors could better understand body language of children with ASD, they would be able to provide more inclusive PA environments.

Methods: Twenty-six videos (30-45minutes) of children with ASD completing the Ignite Challenge, a 13-item advanced gross motor skills assessment were viewed. Participants were 6-12 years, and able to follow complex 3-step instructions. Four raters (UofT MScPT students) were paired together. Each pair rated 12-14 videos using the BLCS and then re-rated 6-7. Intraclass correlation coefficients were used to assess inter- and intra-rater reliability.

Results: Intra-rater reliability was excellent for Positive (0.91) and Negative (0.94) categories and good for Neutral (0.84) category. Inter-rater reliability was good for Positive (0.89) category and poor for Negative (0.39) and Neutral (0.00) categories.

Conclusions: Feedback from raters resulted in seven changes to the measure including the removal of Neutral category and rewording of various cues. These changes are significant enough to require subsequent reliability work (currently ongoing with the assessment of validity and feasibility). The next step is implementing the BLCS into PA programs to measure engagement of children with ASD when taught by instructors who use it. The BLCS is the first measure to quantify body language and with successful results has the potential to positively impact the way children with ASD engage in PA.

Disparities in multimorbidity and mortality among people living with and without HIV across British Columbia’s health regions: A population-based cohort study

Ni Gusti Ayu Nanditha1,2 Grace Zheng2, Hiwot M. Tafessu1, Taylor McLinden1, Andreea Bratu1, Jacek Kopec3,4, Robert S. Hogg1,3, Julio S. G. Montaner1,2, Viviane D. Lima1,2

1British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada 2Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, Canada 3School of Population and Public Health, University of British Columbia, Vancouver, BC, Canada 4Arthritis Research Canada, Richmond, BC, Canada 5Faculty of Health Sciences, Simon Fraser University, Burnaby, BC, Canada

Objectives: Longer survival has increased the likelihood of antiretroviral-treated people living with HIV (PLWH) developing age-associated comorbidities. We compared the burden of multimorbidity and all-cause mortality across HIV status in British Columbia (BC), and assessed the longitudinal effect of multimorbidity on all-cause mortality among PLWH.

Methods: Antiretroviral-treated PLWH aged ≥19 years and 1:4 age-sex-matched HIV-negative individuals from a population- based cohort were followed for ≥1 year during 2001-2012. Diagnoses of seven age-associated comorbidities were identified from provincial administrative databases and grouped into 0, 1, 2 and ≥3 comorbidities. Multimorbidity prevalence and age- standardized mortality rates (ASMRs) in both populations were stratified by BC’s health regions. Marginal structural models were used to estimate the effect of multimorbidity on mortality among PLWH, adjusted for time-varying confounders affected by prior multimorbidity.

Results: Among 8,031 PLWH and 32,124 HIV-negative individuals, 25% versus 11% developed multimorbidity, and 23.53 deaths/1000 person-years (95% confidence interval [95%CI]: 22.02-25.13) versus 3.04 (2.81-3.29) were observed, respectively. PLWH in Northern region had the highest ASMR, but those in South Vancouver Island experienced the greatest difference in mortality compared to HIV-negative individuals. Among PLWH, compared to those with zero comorbidities, adjusted hazard ratios for those with 1, 2 and ≥3 comorbidities were 3.36 (95%CI: 2.86-3.95), 6.92 (5.75-8.33), and 12.87 (10.45-15.85), respectively.

Conclusion: PLWH across BC’s health regions experience excess multimorbidity and associated mortality. We highlight health disparities which are key when planning the distribution of healthcare resources across BC, and provide evidence for improved HIV care models integrating prevention and management of chronic diseases.

IRE1α is Essential for Podocyte Proteostasis and Mitochondrial Health

J.R. Navarro-Betancourt1, J. Papillon1, J. Guillemette1, T. Iwawaki2, C.F. Chung1, and A.V. Cybulsky1

1DepartmentofMedicine, McGill University 2DepartmentofLifeScience, Kanazawa Medical University

Introduction: Glomerular epithelial cell (GEC)/podocyte proteostasis is disrupted in glomerular diseases, leading to endoplasmic reticulum (ER) stress. To maintain proteostasis, the ER activates the unfolded protein response (UPR), which includes upregulation of chaperones and clearance of misfolded proteins through autophagy. Inositol requiring enzyme-1α (IRE1α), a protein kinase and RNase, resides in the ER membrane and transduces the UPR. This study characterizes the mechanisms by which IRE1α regulates proteostasis in GECs.

Methods: Mice with podocyte-specific deletion of IRE1α (IRE1α KO) were produced by breeding IRE1α flox/flox mice with mice expressing podocin-Cre recombinase. Nephrosis was induced with an injection of adriamycin (ADR). GECs were cultured from glomeruli of IRE1α flox/flox mice and IRE1α was deleted by transduction of Cre recombinase. Oxygen consumption rate (OCR) was quantified with the Seahorse mitochondrial stress test. Mitochondria were examined by fluorescence microscopy and flow cytometry using MitoTracker dyes.

Results: Podocyte-specific IRE1α KO mice had greater ADR-induced albuminuria over a 4 week period, compared to control littermates. ADR increased the expression of ER chaperones in glomeruli of control mice, but this upregulation was impaired in IRE1α KO mice. Autophagy induction was blunted in ADR-treated IRE1α KO animals, evidenced by reduced LC3-II and increased p62 levels, compared to treated controls. Electron microscopy showed prominent swelling of the ER and striking mitochondrial injury in podocytes of ADR-treated IRE1α KO mice. In cultured GECs incubated with tunicamycin (TM) to induce ER protein misfolding, deletion of IRE1α or chemical inhibition of the IRE1α RNase with 4μ8C attenuated upregulation of ER chaperones and autophagic flux (formation of LC3-II), compared to control. LC3 gene transcription and total LC3 protein levels were enhanced in TM-treated GECs, in an IRE1α-dependent manner. Compared to control, IRE1α KO GECs showed decreased maximal and ATP-linked OCR. Mitochondrial membrane potential was lower in IRE1α KO GECs than in control, while total mitochondrial mass was similar in both groups. Inhibition of IRE1α signaling increased ER stress- induced apoptosis.

Conclusion: Stress-induced chaperone production, autophagy and mitochondrial health are compromised by deletion of IRE1α. The IRE1α pathway plays an important cytoprotective role in glomerular disease associated with podocyte injury and ER stress.

Evaluating the Impacts of Reduced SKP2 Expression on Chromosome Instability in Colorectal Cancer

Nicole Neudorf1,2, and Kirk J. McManus1,2

1Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB 2Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, MB

INTRODUCTION: In 2020, colorectal cancer (CRC) was the third most commonly diagnosed and second most lethal cancer in Canada. This is partly attributed to ~50% of all new diagnoses occurring at late stages (III or IV), when the 5-year survival is as low as 8%. Accordingly, understanding the mechanisms driving cancer development and progression is essential to develop novel treatment strategies to improve the lives and outcomes of those living with CRC. Chromosome instability (CIN; ongoing changes in chromosome complements) occurs in ~85% of CRCs and is proposed to be a driving force in cancer pathogenesis, however, the abnormal genes and cellular pathways causing CIN remain largely unknown. Preliminary data from the McManus laboratory suggest that reduced expression of the gene SKP2 induces CIN, but the underlying mechanism remains to be determined. Accordingly, we now seek to investigate the impact reduced SKP2 expression has on CIN in a CRC context.

METHODS: SiRNA-based silencing and quantitative microscopy approaches were employed to assess the impact reduced SKP2 expression has on CIN in three karyotypically stable cell lines, namely HCT116 (CRC), 1CT (non-malignant colonic epithelial) and A1309 (1CT derivative). Single cell quantitative imaging microscopy approaches were used to determine the impact diminished SKP2 expression has on CIN associated phenotypes relative to a non-targeting control (siControl). These phenotypes include changes in nuclear areas and micronucleus formation, which are associated with large-scale DNA changes (e.g., ploidy) and chromosome mis-segregation, respectively.

PRELIMINARY RESULTS: To date, nuclear area distributions and micronucleus formation have been evaluated in all cell lines following SKP2 silencing. In HCT116, reduced SKP2 expression corresponded with statistically significant increases in nuclear area distributions relative to siControl. In 1CT, SKP2 silencing induced significant increases in nuclear areas, as well as trending increases in micronucleus formation, however these increases were not deemed statistically significant. In A1309, reduced SKP2 expression induced significant increases in nuclear area distributions and micronucleus formation relative to siControl.

CONCLUSIONS: Preliminary findings show that reduced SKP2 expression induced CIN phenotypes in many colonic cellular conditions and are in agreement with SKP2 being a novel CIN gene with potential pathogenic implications in CRC.

Hypoxia Controls Tumour Associated Macrophage Phenotype in Pancreatic Ductal Adenocarcinoma

Luke A. Neufeld1,2, Rahul S. Shinde3, Tracy L. McGaha1,2

1University of Toronto, Toronto, ON, Canada 2Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada 3Wistar Institute Cancer Center, Philadelphia, PA, USA

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and has the lowest 5-year survival rate of the common cancers. PDAC is resistant to most therapies, including immunotherapy, yet paradoxically often exhibits a significant immune infiltrate. Interestingly, hypoxia and tumour associated macrophage (TAM) presence in PDAC correlate with reduced survival. Since macrophage immune-suppressive function can be potentiated by hypoxia, we hypothesized that intratumoural hypoxia drives TAM phenotype, which contributes to PDAC progression and immune evasion.

Methods: We used tumour conditioned medium (TCM) and hypoxic culture to examine the role of low oxygen tension in controlling macrophage phenotype in the context of PDAC. We analyzed cells in vitro by qPCR and flow cytometry for TAM markers, as well as assessing their immune-suppressive capacity by coculture with T cells. In vivo, we used the hypoxic tracer pimonidazole (PIMO) to compare TAM in areas of differing oxygen tension in an orthotopic, surgical model of murine PDAC. We also examined the specific role of Hypoxia Inducible Factor 1-alpha (HIF1α) in vitro and in vivo using HIF1αflox LysMCre mice that specifically lack HIF1α in macrophages.

Results: Hypoxia significantly enhanced the expression of TAM-like markers in macrophages cultured with TCM. Interestingly, TCM alone was a stronger driver of HIF1α protein accumulation than culture in hypoxia (0.2% O2). Macrophages conditioned with TCM in both normoxia and hypoxia significantly inhibited T cell proliferation and effector molecule production in a coculture assay. In vivo, TAMs exhibited increased exposure to hypoxia relative to other immune cell types and hypoxic TAMs showed an altered phenotype that was dependent on macrophage-intrinsic HIF1α. Importantly, macrophage-specific deletion of HIF1α reduced TAM numbers and altered their phenotype, resulting in reduced tumour burden compared to controls.

Conclusion: Hypoxia is a critical factor driving the TAM phenotype in PDAC. Moreover, this unique, hypoxic phenotype is dependent upon macrophage-intrinsic HIF1α signalling promoting immune suppression and tumour growth.

Immunoregulatory mechanisms in the female genital tract: the roles of IL-2, c-maf and regulatory T cells

Faisal Nuhu1, Janilyn Arsenio2, Thomas Murooka3, Lyle R. McKinnon1,4

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Canada 2Manitoba Center for Proteomics and System Biology, University of Manitoba, Winnipeg, Canada 3Department of Immunology, University of Manitoba, Winnipeg, Canada 4Centre for the AIDS Programme Research in South Africa (CAPRISA), Durban, South Africa

Introduction: Inflammation is central to the initiation of an effective host immune response. However, if dysregulated, it can compromise the integrity of the mucosal barrier and increase HIV transmission risk. Understanding immunological and molecular mechanisms at mucosal sites may represent a novel HIV prevention strategy. In contrast to many inflammatory cytokines, homeostatic regulators such as IL-2, IL-15, and GM-CSF were associated with protection from HIV acquisition in South African women enrolled in CAPRISA-004 trial. We hypothesized that IL-2 decreases inflammation in the FGT by increasing the frequency and/or function of regulatory T cells (Tregs), thereby decreasing the risk of HIV acquisition. Also, a transcription factor called c-maf which has emerged to play a major role immune cells regulation may play a central role in the mechanism by which Tregs limits inflammation at the mucosal site.

Methods: We modelled the immunological effects of low dose IL-2 administration in the humanized bone marrow, liver and thymus (BLT) mouse model. BLT mice (n=6) with >50% human CD45+ reconstitution (defined by flow cytometry) were used. The mice were randomized into 3 groups: The first group of mice were dosed 8 times with 1000IU IL-2 vaginally (n=2), the second group were also dosed 8 times with 1000IU IL-2 intraperitoneally (n=2) and the last group (control group (n=2)) dosed with PBS. The mice were euthanized at the end of the experiment to obtain the tissues. Tregs were defined by Live CD3+CD4+CD25+CD127-. Again, in this preliminary study we compared the expression of c-maf in PBMCs and ectocervical biopsies using flow cytometry.

Results: Mice dosed with IL-2 via the vaginal and intraperitoneal routes had a higher frequency of Tregs of CD4+ T cells in the FGT (8.5% and 7.06% respectively) as compared to 6.62% in the control group. c-Maf was detected in cervical biopsies (5 of 6) with minimal detection in PBMCs. This further support that this transcription factor plays important roles that may be restricted to mucosal tissues.

Conclusion: These preliminary data reveal an interesting association between vaginally administered IL-2 and female genital tract Treg frequency, providing proof of principle for expanded experiments to determine how IL-2 impacts the female genital milieu more broadly, including modulation of HIV susceptibility. Significance: This study may shed light on strategies for reducing inflammation and HIV risk at a mucosal level.

Dissecting the Role of the Phosphatidylinositol 3-Kinase (PI3K) Signal Transduction Pathway in B Cell Regulatory Functions and Humoral Immune Responses

Folayemi Olayinka-Adefemi1, Chukwunonso Onyilagha2, Nipun Jayachandran3, Sen Hou1, Ping Jia1, Jude Uzonna1, Aaron Marshall1

1University of Manitoba 2National Centre for Foreign Animal Disease, Canadian Food Inspection Agency 3University of Pennsylvania

Introduction: The PI 3-kinase delta (PI3Kδ) isoform protein is essential for B cell activation. Trypanosoma parasites engage in several mechanisms to manipulate and evade host B cell and antibody responses critical for optimum immunity. We sought to determine the impact of PI3Kδ in immunity to Trypanosoma congolense infection.

Methods: Intraperitoneal (IP) Infection of mice with 1000 parasites of TC13, Immunophenotyping of cells using Flow cytometry, IP treatment of WT mice with the PI3Kδ-inhibitor : Idelalisib, Antibody and Cytokine analysis using Mesoscale ELISA.

Result: PI3KδD910A mutant mice with a genetic loss of the protein show a surprisingly enhanced control of parasitemia in early infection (7-9 days), when compared with wild-type (WT) C57BL/6 mice. Drug treatment with Idelalisib, an FDA approved drug to acutely inhibit PI3Kδ, during infection in WT mice led to improved early control of parasitemia, consistent with results from genetically deficient mice. Both mutant mice and Idelalisib-treated mice showed a delay in polyclonal B cell activation and CD80/86 expression. The analysis of cytokine in the blood and peritoneal cavity showed higher IFN and lower IL-10 levels in the drug-treated group at early time points, indicating a more proinflammatory environment. Innate B1 cells that fail to develop in the PI3Kδ genetic mutants are identified as the major IL-10 producing cells in the peritoneal cavity in early infection, and B1 cell production of IL-10 was significantly impaired following PI3Kδ-inhibitor treatment of WT mice. We generated of a novel mouse model PI3KδGOF/B that harbours a B cell specific gain-of-function (GOF) mutation in the PI3Kδ protein and in early infection. They showed a phenotype that is in contrast with the PI3KδD910A mutant and Idelalisib-treated groups in early infection. Results further showed that despite PI3Kδ’s impact on early parasite control in the different PI3Kδ mouse groups, ultimate mice survival was presumably hinged on the PI3Kδ-mediated generation of humoral immunity, in the form of parasite-specific antibodies which is severely compromised in the PI3KδD910A mutant mice when compared to PI3Kδ-inhibitor treated or PI3KδGOF/B mice.

Conclusion: PI3Kδ inhibition impacts both regulatory B cell functions, affecting the early innate response, and generation of critical protective antibodies.

Development and Characterization of a Novel M2e-Based DC-Targeting Universal Vaccine Approach against Influenza Virus

Titus Olukitibi1,2, Zhujun Ao1,2, Hiva Azizi4, Mona Mahmoudi1,2, Darwyn Kobasa3, Kevin Coombs2, Gary Kobinger4, and Xiaojian Yao1,2

1Laboratory of Molecular Human Retrovirology, 2Department of Medical Microbiology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada. 3Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada 4Centre de Recherche en Infectiologie de l'Université Laval/Centre Hospitalier de l'Université Laval (CHUL), Québec, QC, Canada

Introduction: The deadly Influenza virus responsible for approximately 650,000 death annually vaccines can be stopped with a universal vaccine. Also, enhancing the Dendritic cells (DCs) using Ebola glycoprotein (EboGP) could induce stronger immune responses. We, therefore, hypothesize that the fusion of influenza hemagglutinin stalk (HAcs), and ectodomain matrix protein (M2e) with DC-targeting domains of EboGP (EboGPΔM) will efficiently deliver influenza HA, M2e or both to DCs and induce more robust immune responses against influenza virus strains.

Method: We fused the EboGPΔM with HAcs, M2e or both to generate an EboGPΔM-HAcsM2e or EboGPΔM-tM2e and incorporated into VLPs or rVSV. The DCs/macrophages were infected with the VLPs to evaluate their DC-targeting ability in vitro by measuring the Gaussian luciferase produced by the VLP in the supernatant. Furthermore, we immunized BALB/C mice with rVSV-EboGPΔM-HAcsM2e, EboGPΔM-tM2e or PBS intramuscularly and investigated the immune responses in the serum and nasal wash of the mice using ELISA. We also challenged the immunized mice with A/PR8/1934 or H3N2 intranasally to evaluate the protective potential of the candidate vaccines.

Results: Our results demonstrated that the fusion of EboGPΔM with HAcs, M2e or both targets the epitopes to DCs/macrophages. Furthermore, ELISA results revealed significantly high levels of IgG and IgA anti-M2 and anti-HA in the serum and nasal wash of the immunized mice. In vivo infection of the immunized mice demonstrated the protection efficiency of EboGPΔM-HAcsM2e and EboGPΔM-tM2e. We will additionally check for protection against H5N1.

Conclusion: This study has provided strong evidence for developing a novel and efficient M2e-based DC- targeting universal influenza vaccine approach.

Understanding the Origins of GBM: PDGFA & Aberrant Mitosis in Neural Progenitor Cells

Hiba Omairi1,2, Michael D. Blough1,2, Gregory J. Cairncross1,2

1The Clark H Smith Brain Tumour Centre, Calgary, Alberta, Canada 2Charbonneau Cancer Institute, Calgary, Alberta, Canada

Introduction: Glioblastoma (GBM) is a fatal brain cancer that has eluded major therapeutic advances. Although significant progress has been made in describing GBM’s molecular composition, treatments haven’t improved. Revolutionizing treatment may lie in understanding the origins of GBM, such that it may be prevented or intercepted at an early curable stage. Emerging evidence suggests that the mitogen Platelet-derived Growth Factor A (PDGFA) plays an important role in GBM initiation. To ascertain the earliest stages, we have developed a unique murine replica of GBM which allows the study of malignant transformation of neural progenitor cells (NPCs), the putative cell of origin of human GBM, by PDGFA. In this model, P53-null NPCs are cultured in stem cell growth factors: Fibroblast Growth Factor and Epidermal Growth Factor (EGF/FGF), or in PDGFA. Contrary to their counterparts, cells in PDGFA undergo a cancerous transformation and attain growth factor independence in about 100 days.

Methods: NPC cultures are established from the neurogenic subventricular zone (SVZ) of p53-null mice and supplemented with PDGFA or FG/EGF as controls. Preliminary data demonstrate early apoptosis, attenuated cell division and chromosome instability in cells cultured in PDGFA; we hypothesize that the malignant transformation of NPCs is an adaptation to PDGFA- induced mitotic stress. To test this hypothesis, High-resolution Immunofluorescent (IF) staining and live-cell imaging were used to study NPCs mitotic phenotypes and proliferative behaviours in PDGFA.

Results: live-cell imaging revealed that over 50% of cell divisions in PDGFA were visibly abnormal and had longer mitotic transit times. IF microscopy revealed abnormal nuclear phenotypes, including micronuclei, multinucleated cells, chromatin bridges and lagging chromosomes, additional features of mitotic stress in PDGFA. These findings prompted us to consider that PDGFA may alter centrosome function, a major mitotic regulator, leading to progressive genomic instability and transformation. We have recently documented a variety of centrosome anomalies in transforming and transformed NPCs, including supernumerary centrosomes, centrosome fragmentation and multipolar mitosis.

Conclusion: Our data indicate that PDGFA induces abnormal mitosis and centrosomal abnormalities in NPCs, suggesting that transformation occurs through cellular adaptation to mitotic stress. This work opens the doors to exploring early preventative or interceptive therapies of GBM.

PRE-HIV CD4+ T CELL PROFILES AS PREDICTORS OF HIV ACQUISITION AND DISEASE COURSE DURING THE FIRST YEAR OF HIV INFECTION

Tosin Omole1, Aida Sivro1,2, Naima Jahan1, Jai Lingappa3, Glenda Gray4,5, Lyle McKinnon1,2

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 2Centre for the AIDS Program of Research in South Africa (CAPRISA), University of Kwazulu-Natal, 3University of Washington, 4South African Medical Research Council, 5HIV Vaccine Trials Network (HVTN).

There is heterogeneity in the susceptibility to HIV infection among individuals. While some people are more susceptible to HIV infection, others are resistant to the disease despite multiple exposures to the virus. The frequency of target cells such as CD4+T cells is an important factor in determining HIV outcome. Productive HIV infection may depend on whether there are enough target cells for the virus to access. Previously, we quantified pre-HIV frequencies of α4β7+CD4+T cells – a gut- homing integrin – in women enrolled in the CAPRISA004 cohort and found that the women with higher frequencies of α4β7+ cells were more likely to acquire HIV and experienced faster disease progression. We, therefore, aim to investigate further the association of HIV target cells, including CD4+ phenotypes such as α4β7+, Th17 and regulatory T cells with the risk of HIV acquisition and disease progression in two independent cohorts that differ from CAPRISA004 by mode of HIV acquisition, sex, clade, and exposure to HIV prevention candidates. We hypothesize that higher pre-HIV frequencies of the HIV target cells will correlate with higher rates of HIV acquisition and disease progression, and this correlation will be modified by sex, clade, and exposure to HIV prevention intervention. In a blinded fashion, using flow cytometric methods, the frequency of the CD4 phenotypes is quantified in peripheral blood mononuclear cells from high-risk HIV-negative individuals enrolled in the Partner's PrEP and HVTN 503 cohorts. In a retrospective nested case-control manner, we will correlate the frequencies of CD4+ phenotypes with HIV outcomes. We sampled HIV cases as those who seroconverted during the studies and matched these to 3 or 4 controls based on study-specific criteria. We defined HIV progression as peak and setpoint viral load and CD4 count below 500 cells/mm3. We will use conditional logistic regression to estimate associations of HIV acquisition, Spearman rank correlation to measure associations with viral load, and Cox regression analysis to determine the time to CD4 decline. We hope that this present study will answer broader epidemiological questions regarding CD4+T cells' role in HIV pathogenesis and provide evidence in terms of which type of immune environment favours viral replication.

The effects of differential chronic locus coeruleus stimulation in a pre-tangle tau rat model

Tamunotonye Omoluabi1, Abhinaba Ghosh1, Sarah E. Torraville1, Kyron D. Power1, Camila Reinhardt1, Aida Maziar1, Qi Yuan1

1Memorial University of Newfoundland

Introduction: Braak and colleagues have proposed that the appearance of soluble hyperphosphorylated (pre-tangle) tau in the brainstem structure known as the locus coeruleus (LC), marks the beginning of the tau pathology that may eventually develop into AD. Although pre-tangle tau is prevalent in human brains at mid-age, the selective vulnerability of a subpopulation of people to developing AD is unknown. Braak et al. have shown that pre-tangle tau spreads from the LC to other brain regions, including memory-encoding structures such as the hippocampus. We proposed that LC neuronal activity would itself influence tau pathology and spread. LC neurons exhibit both phasic and tonic firing modes, which differentially modulate behaviour. We hypothesized that LC phasic activity would ameliorate tau pathology and restore cognitive function, whereas chronic LC tonic activity would augment tau pathology and cognitive impairment. Using optogenetics, we studied whether and how chronic phasic and tonic LC activation alters cognitive function in an LC pre- tangle tau model.

Methods: Viral vectors carrying a human pseudophosphorylated tau gene (AAV9-DIO-htauE14- EGFP) and an opsin channel (AAVdj-DIO-eChR2-mCherry) were infused bilaterally into the LC of 2-3-month-old Tyrosine Hydroxylase-Cre rats, mimicking the onset of tau pathology in humans. Control rats were infused with htauE14 and a reporter AAV without ChR2. Six-to- seven months after virus infusion, rats underwent optical cannula implantation. Rats then received either 10Hz phasic or 25Hz tonic LC activation for 20-30 minutes daily for six weeks. Two months following stimulation, rats underwent a battery of behavioral tests, followed by immunohistochemistry.

Results: Phasic LC activation improved spatial and olfactory discrimination, paralleled by higher LC fiber densities in both the piriform cortex and dentate gyrus. Chronic tonic LC activation impaired cognition and induced anxiety- and depression- like behavior.

Conclusion: Using optogenetics to regulate LC activity, our work suggests that promoting phasic LC neuronal activity may delay or prevent AD progression. In contrast, stress-inducing LC tonic activity may accelerate AD progression.

Identification of Cytoplasmic and Nuclear C-Terminal to LisH (CTLH) Complex Interactors in Mammalian Cells

Gabriel Onea1,2, Matthew ER Maitland1,2,3, Gilles Lajoie2,3, Caroline Schild-Poulter1,2

1Robarts Research Institute, Western University, London, ON, Canada 2Department of Biochemistry 3Don Rix Protein Identification Facility

Introduction: The CTLH complex is a tumour-suppressive E3 Really Interesting New Gene (RING) ubiquitin ligase that regulates many intracellular signaling pathways. It is comprised of 9 proteins that each contain conserved protein binding domains; of these proteins, Required for Meiotic Division 5 Homolog A (RMND5A) and Macrophage Erythroblast Attacher (MAEA) also contain RING domains required to transfer ubiquitin to specific protein substrates. Although its E3 ligase activity has been recently characterized, the endogenous subcellular localization of the complex and compartment-specific protein interaction network – which encompasses many potential E3 ligase substrates – has not been identified.

Methods: We first optimized the chemical fractionation method used to generate cytoplasmic and nuclear HeLa cell extracts. We then identified CTLH complex interactors by Affinity Purification Coupled to Mass Spectrometry (APMS) using Ran Binding Protein M (RanBPM) – the main CTLH complex scaffold protein – as bait in the cytoplasmic and nuclear extracts. Using the list of high-confidence interactors, we identified a number of potential novel functions of the complex by STRING enrichment analysis. Finally, we validated interactions between cytoplasmic and nuclear RanBPM with compartment- specific candidates in vitro and in situ by co-immunoprecipitation assay (CoIP) and Proximity Ligation Assay (PLA), respectively.

Results: All CTLH complex members were shown to interact in cytoplasmic and nuclear lysates. In addition, we identified 27 cytoplasmic, 155 nuclear, and 31 nucleocytoplasmic high-confidence RanBPM interactors by APMS. Subsequent STRING enrichment analysis revealed that the CTLH complex may be involved in many conserved Gene Ontology (GO) processes such as Ribonucleoprotein Complex Biogenesis and Chromatin Assembly. By CoIP, we validated the interaction between RanBPM and cytoplasmic Tankyrase1/2, as well as nuclear proteins HDAC2, MacroH2A1, and p14ARF. The interactions between RanBPM and Tankyrase 1/2 and RanBPM and MacroH2A1 were also visualized in situ by PLA, suggesting that these interactions may be physiologically relevant.

Conclusion: In summary, we identified a number of novel compartment-specific CTLH complex interactors using subcellular fractionation coupled to APMS. We then validated a subset of these interactors in vitro and in situ, thus confirming the high quality of our dataset and implicating the CTLH complex in a number of novel processes.

Understanding Crosstalk between Endoplasmic Reticulum Stress Sensors

Gideon Ong1, Wafa Kammouni1, Katarzyna Mnich4, Afshin Samali4, Susan E. Logue1,2,3

1Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, University of Manitoba 2Research Institute in Oncology and Hematology, CancerCare Manitoba 3Children’s Hospital Research Institute of Manitoba 4Apoptosis Research Centre, National University of Ireland Galway, Ireland

Introduction: The unfolded protein response (UPR) is a pro-survival response activated upon accumulation of unfolded proteins within the lumen of the Endoplasmic Reticulum (ER); a state known as ER stress. IRE1, PERK and ATF6 are three ER anchored stress sensors which monitor ER health. When activated by unfolded proteins, these stress sensors trigger signaling cascades which aim to reduce levels of unfolded proteins – collectively referred to as the UPR. Dysregulated activation of ER stress sensors, notably IRE1, has been reported in Triple Negative Breast Cancer (TNBC). Inhibition of IRE1 has been proposed as a novel treatment for TNBC, with IRE1 inhibitors being recently developed. To exploit IRE1 inhibitors in a safe manner, it is important to understand how they work and whether they impact the wider UPR signaling network. In this study, we ask if blocking IRE1 signaling influences the functionality of other ER stress sensors, in particular PERK.

Methods: Tumorigenic and non-tumorigenic cell lines were treated with a range of chemical or physiological inducers of ER stress in the presence or absence of IRE1 inhibitors. The impact of attenuating IRE1 signaling upon PERK signaling was assessed by immunoblotting and qPCR.

Results and Conclusion: IRE1 RNase inhibitors, in addition to blocking IRE1 signaling, also lowered expression of PERK and reduced its downstream signaling. IRE1 mediated regulation of PERK was observed in all cell lines tested and with diverse ER stress inducers indicating it is a conserved mechanism and not selective to one particular cell line or stimulus. Our results point to a previously unreported amplification mechanism through which IRE1 boosts PERK expression upon ER stress. We propose IRE1 RNase inhibitors, in addition to blocking IRE1 dependent signaling, also reduce PERK signaling effectively limiting two arms of the UPR.

The Role of Metabolic Enzymes in the Immunopathogenesis of Leishmaniasis

Somtochukwu Stella Onwah, Zhirong Mou, Ping Jia and Jude E Uzonna

1Department of Immunology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB

Introduction: Leishmaniasis is a spectral disease with clinical manifestations ranging from self-healing cutaneous ulcers to highly fatal visceral disease that affects the spleen, liver and bone marrow. Although there is currently no effective vaccine against human leishmaniasis, vaccination is possible because people who recover from natural infection are immune to reinfection. The lack of an effective vaccine is due to poor understanding of key immunodominant antigens and correlates of protective immunity in infected animals. We recently demonstrated that naturally processed peptide fragments, PEPCK335–351 and DLD63–79 derived from Leishmania glycosomal Phosphoenolpyruvate carboxykinase (PEPCK) and Dihydrolipoyl dehydrogenase (DLD) respectively, induced strong protective IFN--producing T cell response in mice infected with Leishmania major. Given that DLD and PECPCK are key metabolic enzymes of Leishmania, we hypothesize that they play a role in parasite virulence and modulation of host immune response.

Methods: We generated PEPCK and DLD gene-deficient (KO) and add-back (AB) strains of L. donovani and L. major respectively, using the CRISPR-Cas9 gene-editing tool technology. We confirmed the deletion of these genes and ABs by PCR and Western blot. We assessed the impact of this deficiency on parasite proliferation in axenic culture and bone marrow-derived macrophages in vitro and on the expression of costimulatory molecules on bone marrow-derived dendritic cells.

Results: Targeted deletion of PEPCK in L. donovani was confirmed on an agarose gel and western blots, showing the absence of the genes or their expression. Growth kinetics in axenic culture and macrophages show that this deficiency results in reduced proliferation in comparison to wild-type (WT) and AB parasites. CD11c+ cells infected with PEPCK deficient parasites displayed increased expression of costimulatory molecules compared to cells infected with WT and AB parasites.

Conclusion: These observations indicate that PEPCK is an important metabolic enzyme of Leishmania and its deficiency results in impaired parasite proliferation in axenic culture and infected macrophages as well as increased costimulatory molecule expression in infected dendritic cells. Further studies will assess the impact of PEPCK and DLD gene deficiency in disease pathogenesis in vivo by comparing lesion size, parasite burden and host immune response in mice infected with WT, KO and AB parasites.

The Effect of Different SUMO on HIV-1 Integrase Stability and Viral Replication

Chidi Onyejiegbu1

1University of Manitoba

The Human Immune Virus (HIV) invasion of humans since the discovery has continued, making it one of the deadliest global epidemics. A virion results from HIV integration and replication in a cell made possible by the interplay of viral enzyme integrase and cellular factors. Integrase is a karyophilic property that integrates the viral cDNA to the host genome for degenerative human immune system disease. Intense global research to inhibit the integration and progression of HIV in a host led to the discovery of Integrase Strand Transfer Inhibitors (INSTIs). Due to the resistance induced by this virus, the INSTIs have not maintained their standard. Therefore, it is necessary to research an alternative to the existing INSTIs using cellular and viral mechanistic approaches to restrict HIV replication without future resistance due to mutation. A Small Ubiquitin-like Modifier (SUMO) protein is present in all eukaryotic cells with different isoforms, regulating growth, development and cellular processes such as nuclear transport, signal transduction, transcription, apoptosis, etc. However, little is known about how SUMO has different isoforms, localization, sequence expressions, and an effect on viral integrase stability and replication. Unlike ubiquitin which targets proteins for degradation, SUMO was attached to integrase enzymatically by a post-translational modification process to replace integrase functions and determine the effect of different isoforms of SUMO on HIV-1 integrase stability and viral replication. We investigated the expression of HIV-1 integrase in the presence or absence of SUMO2 or SUMO3 by immunoblotting and Immunofluorescence assay while viral production was by ELISA kit and Luciferase assay. Our data showed that SUMO2 could induce HIV-1 integrase degradation while SUMO3 showed protection and have no significant effect on integrase degradation. Lastly, we over-expressed SUMO2/3, investigated the effect on viral production and expression, and our data showed that over-expression of SUMO2/3 in a cell could decrease and control viral production and expression with 86% significance. This investigation also revealed that SUMOylation could affect viral production at both the early and late stage of viral infectivity. In conclusion, this study has added to mechanisms of how HIV integrase could be controlled and inhibited in a cell.

A dyadic depression self-management intervention for adults with chronic physical diseases and concomitant depression and their caregivers: Study proposal for a mixed methods intervention adaptation and pilot randomized controlled trial

Lydia Ould Brahim1, Sylvie Lambert1,2, Nancy Feeley1, 3, Jane McCusker4, 2, Erica EM Moodie4, Christine Genest5

1Ingram School of Nursing, McGill University, Montreal, Quebec 2St. Mary’s Research Centre, Montreal, Canada 3Centre for Nursing Research & Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada 4Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada 5Faculty of Nursing Sciences, Université de Montreal, Montreal, Quebec

Background: Caregivers, unpaid family members or friends, provide over 70% of care for adults with chronic diseases. Having limited formal support, caregivers often experience high levels of burden and negative health impacts. Approximately 20% of adults with chronic diseases experience concomitant depression, which raises their risk of mortality and negatively affects their caregivers’ health. Including caregivers in interventions may positively impact use of the intervention and decrease attrition, optimizing intervention efficacy. Given healthcare resource constraints, providing a dyadic intervention (care recipient and caregiver participate) using acceptable and cost-effective modes of delivery, such as self-management, is a priority.

Objectives: In phase 1, the DIRECT-sc toolkit, an existing depression self-management intervention for adults with chronic conditions and co-occurring depression, will be adapted to include a prescribed caregiver role. In phase 2, a pilot RCT of the adapted dyadic intervention, DIRECT-support, will be conducted.

Methods: Overseen by a steering committee of experts, the phase 1 adaptation process will include consolidation of evidence from systematic reviews, relevant theory, and results of interviews with care recipient-caregiver dyads (n = 24) and healthcare professionals (HCPs) (n = 12). The preliminary version of DIRECT-support will be tested with 5 dyads and 5 HCPs (n=15) and its acceptability assessed using validated questionnaires and semi-structured interviews or focus groups with all participants. Phase 2, a multi-site, parallel-group pilot RCT, will be conducted with 26 dyads (n = 52), randomized to (a) DIRECT-support for 6-weeks or (b) a usual care control group. Data on feasibility (e.g., recruitment and attrition rates), acceptability (e.g., intervention use, satisfaction), and participant outcomes (primary outcome depression using the CES-D) will be collected using study logs and validated questionnaires. Semi-structured interviews will be conducted with a subset of participants to further explore acceptability.

Conclusion: With greater prevalence of chronic diseases, the demands on caregivers are rapidly increasing. An innovative dyadic approach holds promise as a source of support for individuals with chronic disease and their caregivers. This study will provide feasibility and acceptability data needed to elucidate the challenges and potential benefits of dyadic interventions in chronic disease care.

Assessment of Orotic Acid as a Superior Carrier for Lithium in the Management of Bipolar Disorder

Anthony G. Pacholko1, Lane K. Bekar1

1University of Saskatchewan

Introduction: Lithium carbonate (LiCO) is a mainstay treatment option for the control of bipolar disorder (BD) symptomatology. However, LiCO is not without its faults, specifically a narrow therapeutic index and common thyroid, parathyroid and renal complications. Lithium orotate (LiOr) is an alternative lithium compound that is suggested to have transport and uptake properties that may allow for markedly reduced dosages in treatment regimens, which could theoretically mitigate toxicity concerns and improve patient compliance.

Methods: As a first step in the exploration of LiOr as a replacement for traditional lithium compounds, we aimed to establish dose response curves for a range of LiOr and lithium chloride (LiCl) concentrations (0, 1, 5, 10 and 15 mg/kg for LiCl; 0, 1, 1.5, 2.5, 5, 10 and 15 mg/kg for LiOr; concentrations represent elemental lithium content) in wild-type mice using an amphetamine-induced hyperlocomotion (AIH) model, which features mania-mimetic locomotor characteristics known to be sensitive to blockade by lithium. Lithium was injected intraperitoneally (IP) 30 minutes prior to administration of 3 mg/kg of d-amphetamine (dA), also delivered IP. Animals were recorded for 120 minutes in an open field and scored for locomotion.

Results: Peak dA-induced hyperlocomotion was observed between 70-to-120 minutes post-injection; thus, LiOr/LiCl effects were contrasted in this window. Following a single treatment, the maximum effective dose was 2.5 mg/kg for LiOr and 10 mg/kg for LiCl. LiOr, but not LiCl, elicited a 100% blockade of dA-induced hyperlocomotion. Intriguingly, repeated once-daily administration of LiOr over 7 consecutive days prior to induction of AIH further reduced the required dose to 1.5 mg/kg, whereas no reductions in dosage requirements were noted for LiCl.

Conclusion: LiOr displays improved potency and efficacy relative to LiCl in the attenuation of AIH. Further reductions in the required dose of LiOr, but not LiCl, following repeated administration suggests that LiOr accumulates within the CNS to a greater degree over time. Future work will focus on mechanistic explorations of the LiOr transport mechanisms and pharmacokinetics potentially underlying this putative enhanced CNS entry/accumulation, as well as confirmation of enhanced therapeutic potential in a d-amphetamine withdrawal model known to display manic- and depressive-mimetic phenotypes.

EEG correlates of physical effort and reward processing during reinforcement learning

Dimitrios J. Palidis1,2,3 and Paul L. Gribble1,2,4,5

1The Brain and Mind Institute, Western University, London, Ontario, Canada 2Department of Psychology, Western University 3Graduate Program in Neuroscience, Schulich School of Medicine and Dentistry, Western University 4Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University 5Haskins Laboratories, New Haven, Connecticut

Introduction: Effort-based decision making is often modeled using subjective value, a function of reward discounted by effort. We asked whether EEG event-related potential (ERP) correlates of reinforcement processing are also modulated by physical effort. In particular, we measured the feedback-related negativity/reward positivity (FRN/RP), an ERP component related to learning from reward, loss, and punishment outcomes.

Methods: Human participants performed a task in which they were required to accurately produce target levels of muscle activation to receive rewards. Muscle activation was recorded with electromyography (EMG) during isometric knee extension. On a given trial, the target muscle activation required either low or high effort. The effort was determined probabilistically according to binary choices, such that the responses were associated with 20% and 80% probability of high effort. This contingency could only be learned through experience, and it reversed periodically. Binary reward feedback depended on accurately producing the target muscle activity.

Results: Participants adaptively avoided effort by switching responses more frequently after choices that resulted in hard effort. Feedback after participants’ choices that revealed the resulting effort requirement did not elicit modulation of the FRN/RP. However, neural responses to reinforcement outcome after effort production were increased by preceding physical effort. Source decomposition revealed separable ERP components. The FRN/RP was sensitive only to reward, whereas the reward × effort interaction was observed only in a late positive component that resembled the P300.

Conclusions: Contrary to economic models of choice, neural reinforcement signals did not respond to effort as a cost or an aversive outcome. However, effort retrospectively increased the neural responses to reward outcomes. This may relate to paradoxical behavioral findings whereby rewards requiring more effort to obtain can become more powerful reinforcers.

Toward targeted positron emission mammography and ultrasound-guided biopsy in high-risk women with breast cancer

Claire Keun Sun Park1,2, Jeffrey Bax2, Lori Gardi2, Eric Knull2,3, Aaron Fenster1,2

1Department of Medical Biophysics, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada 2Imaging Research Laboratories, Robarts Research Institute, London, ON, Canada 3School of Biomedical Engineering, Faculty of Engineering, Western University, London, ON, Canada

Introduction: Definitive diagnosis of breast cancer still requires a suspicious lesion to be sampled, most commonly with an image-guided biopsy. Positron emission mammography (PEM) is a breast-dedicated functional imaging modality, which combines high-resolution imaging components and pre-eminent fluorodeoxyglucose (FDG) radiotracers to image changes in metabolic activity, a hallmark of cancer growth. Advancements in PEM show potential to improve breast cancer detection, independent of breast tissue density, but anatomical context and realtime needle visualization are not available. To address these challenges, we have developed a guidance system integrating ultrasound (US) imaging and a biopsy intervention with highresolution PEM imaging. This work evaluates the accuracy of PEM-US-guided biopsy in breast phantoms under simulated PEM localization.

Methods: We developed a novel mechatronic system designed to operate with an advanced Radialis PEM system (Thunder Bay, Ontario) developed by our collaborators. The system contains a clinician-operated guidance arm and biopsy device with an integrated US transducer and core-needle biopsy gun focused on a remote-center-of-motion. Registration of the system to a machined, simulated PEM detector plate was performed to guide the needle relative to the simulated PEM image. Tissue-mimicking breast phantoms with targets in known positions were fabricated to simulate breast lesions. The needle was guided to a lesion, and its 3D positioning error and component in-plane and cross-plane errors were quantified in the PEM image. Mock biopsy procedures were performed (N=20) and the needle targeting error (NTE) as the shortest distance between the needle centerline and target lesion was quantified.

Results: The PEM-US-guided biopsy system was able to successfully sample 20 simulated breast lesions. The 3D positioning error was 0.85±0.30mm (N=20) with 0.64mm in-plane and 0.44mm cross-plane component errors. The NTE was 1.08±0.46mm (N=15) allowing for targets 1.32mm in radius to be sampled with 95% confidence.

Conclusion: We present a highly accurate PEM-US-guided breast biopsy system allowing for sampling of small early-stage and heterogeneous cancers presenting with multiple regions of genetic variability. This shows potential utility for improved image-guided tumour sampling in highrisk women with dense breasts. Future work will integrate the biopsy system with the Radialis PEM system toward clinical use of PEM-US-guided biopsy.

HIV-bearing DCs regulate T cell migration and cell-cell contact dynamics to enhance viral Spread

Wan Koh1, Paul Lopez1, Oluwaseun Ajibola1, Roshan Parvarchian1, Umar Mohammad1, Ryan Hnatiuk1, Jason Kindrachuk2 and Thomas Murooka1,2

1University of Manitoba, Rady Faculty of Health Sciences, Department of Immunology, Winnipeg 2University of Manitoba, Department of Medical Microbiology and Infectious Disease, Winnipeg, Canada

Introduction: HIV dissemination from the genital mucosal tract to the lymphoid organs is the first critical step towards systemic infection. We have previously shown that the trafficking of cellassociated HIV from the genital mucosa to lymphoid organs played a dominant role in viral spread early after sexual transmission in humanized mice. Here, we have extended these observations by addressing the role of migratory DCs in the capture, retention and transfer of HIV particles to susceptible T cells through trans-infection, a route of viral transmission that occurs through cellcell contact.

Methods and Results: Using primary monocyte-derived dencritic cells (DCs), we show that mature DCs captured HIV on the cell surface, mediated by Siglec-1 binding, and that captured virus rapidly formed dense clusters within a tetraspanin-rich area near the uropodia (or trailing edge) of migrating DCs. We show that chemotactic migration towards lymph node homing chemokines CCL19/21 and S1P were maintained by HIV-bearing DCs. Using a 3D collagen matrix model to visualize dynamic cell-cell interactions, CD4+ T cells engaged in prolonged contacts with HIV-bearing DCs that were dependent on gp120:CD4 and LFA-1:ICAM-1 adhesive contacts. Abrogating stable DC:T cell contacts significantly reduced HIV transmission. Finally, we identified distinct signaling pathways that are activated in T cells during co-culture with DCs bearing wildtype HIV, but not in HIVEnv, using a custom human kinomics array platform (containing >900 phospho-target motifs). Using functional network analysis, IPA and InnateDB, we observed mTOR, ERK and Ca2+ pathways are upregulated during DC:T cell contact in the presence of wildtype, but not Env-deficient HIV.

Significance: These data argue that T cells accumulate signals downstream of the CD4 molecule that impact their motility and function as they contact HIV-bearing DCs, some of which may increase virus infection. Together, we highlight an unexpected mechanism by which surface-bound HIV particles on DCs can function as adhesive receptors to contact and restrain motile T cells to facilitate efficient HIV spread within lymphoid tissues.

Investigating the role of condensin-mediated chromosome morphogenesis during mitosis

Alyssa Pastic1, Annahat Kochhar1, Damien D’Amours1

1University of Ottawa

Introduction: Effective transmission of genetic material during mitosis is vital to the survival of all living organisms. At the onset of mitosis, loosely organized chromatin is packaged into distinct chromosomes in a process is known as chromosome condensation. Mutations that impair this process cause genomic instability, one of the most powerful drivers of cancer. Chromosome condensation is regulated by a pentameric enzyme complex known as condensin, though exact mechanism used by this protein to compact chromatin is incompletely understood.

Methodology: Our team takes advantage of Saccharomyces cerevisiae (budding yeast) as a model organism to study chromosome morphogenesis. Many of the fundamental genes that control cell division in yeast, including condensin, have a significantly conserved equivalent in humans. The benefit of using yeast is that this organism allows for genetic deletions and manipulations to be performed in ways that would be extremely difficult in other organisms.

Results: Published work from our team has highlighted the importance of a short region of the condensin complex that is highly regulated by cell-cycle kinases to trigger chromosome condensation. This region is located specifically in the SMC4 subunit of condensin as a unique extension of the N-terminal end (SMC4-NT). The presence of this extension is conserved among all eukaryotes, and we have shown that its deletion from yeast causes lethality. Preliminary data reveals a novel role for the SMC4-NT in binding to DNA. We have identified at least two putative DNAbinding motifs and have shown that their removal causes severe growth defects and death in yeast.

Conclusion: Collectively, our findings suggest a role for the SMC4-NT in regulating the binding of condensin on chromosomes during cell division. Our findings will have a major positive impact on human health by revealing new genetic vulnerabilities that could be used for cancer treatment.

Panx1 channel activation by endoplasmic reticulum signalling- key to decipher the code of Alzheimer’s disease

Patil C.S*1,2,3, Lavine N.E1,2,3 & Jackson M.F1,2,3

1Kleysen Institute for Advanced Medicine, 2Department of Pharmacology & Therapeutics, 3University of Manitoba, Winnipeg, MB, Canada

Introduction: A crucial neurotoxic event associated with initial stages of Alzheimer’s disease is loss of Ca2+ homeostasis, especially within the endoplasmic reticulum (ER). Herein, amyloid-β oligomers (AβOs) provoke aberrant activation of Ca2+ permeable NMDA receptor (NMDARs) and sensitize Ca2+ release through ER. Altered ER Ca2+ release dynamics caused by AβOs is sensed by the stromal interacting molecule proteins (STIMs), which are known to activate surface expressed Ca2+ permeable channels through protein-protein interactions. Past studies have shown that overactivation of NMDARs activate pannexin (Panx1) channels. We now show that AβOs sensitizes Panx1 activation by NMDAR via ER resident STIM proteins.

Methods: Cultured hippocampal neurons from CD1, Panx1 WT and Panx1 KO mice were used for electrophysiological recordings. Recombinant expression system (HEK 293T cells) were used for molecular interaction studies between Panx1 and STIM.

Results: First we show that excitatory synaptic deficits induced by AβOs require Panx1. Moreover, Panx1 activation by NMDARs is significantly increased by AβO treatment and is notably reduced in STIM knockdown neurons. Therefore, to establish the mechanism of Panx1 activation by STIMs, we generated Panx1 mutants with deletions targeting intracellular N- and C-term domains. Electrophysiology and co-immunoprecipitation studies show that a domain within the N-term of Panx1 is required for activation by STIM. Further, NMDAR-dependent Panx1 activation was eliminated in cultured neurons expressing a Panx1 mutant unable to bind STIMs. Lastly, using an N-term recognising Panx1 antibody we validated our mechanism of Panx1 activation. Altogether our data confirms the importance of the Panx1-STIM interaction region in regulating activity of Panx1 channels. Future experiments are aimed to study the effect of inhibiting Panx1 functions in AβO treated neurons.

Conclusion: Ongoing work will allow us to gain important insights into the mechanisms by which AβOs disrupt the function of the ER and aggravate atypical activation of Panx1 channels. By investigating mechanism of Panx1-STIM interaction downstream of NMDAR, we hope to provide rationale for discovery of potential therapeutic targets for impeding detrimental effects of AβOs.

MultimodalPET-MR imaging of the selective activation of serotonergic neurons in living rodent brains

Jarrad Perron1, Bryan McIntosh1,2, Sidney Leggett1, Vanessa Palmer3, Sheryl Herrera3, Katrina Armstrong1, Larry Jordan1, Katinka Stecina1, Melanie Martin4

1University of Manitoba 2CancerCare Manitoba, University of Manitoba 3Cubresa Inc 4Univeristy of Winnipeg

The main advantage of hybrid PET-MR imaging systems is the ability to correlate anatomical with metabolic information directly. The bulk of commercially available PET-MR systems are quite large and expensive and mostly used on humans rather than for preclinical animal studies. This has led to a gap of knowledge in PET-MR imaging of small animal models used in preclinical research. Our work takes advantage of a new imaging system developed by Cubresa called 'NuPET'. This device is a MR-compatible PET scanner placed around the subject while they are within the toroidal bore of a MR scanner. With this equipment we are attempting to demonstrate the selective activation of serotonergic neurons in living rodent brains. To specify which neurons are to be activated, we use Designed Receptors Exclusively Activated by Designer Drug (DREADD) technology. These DREADDs are designer G-protein-coupled receptors. Neurons at the site of a stereotactic injection are transfected with a viral vector containing the proteins necessary to force expression of DREADDs in genetically modified rats. These may then be activated by administering the designer drug clozapine-N-oxide (CNO). This technique allows for precise spatiotemporal control of receptor signaling in vivo. Over two experiments (N=5, N=2) we have attempted to image the effect of DREADDs-mediated excitation of 5-HT neurons in rats. Voxel-based analysis of the data thus far show no confirmed statistically significant differences between rats given saline and those given CNO. Numerous methodological issues have been discovered within the experimental design, and are being addressed for a new trial of the technique and technology.

The authors wish to acknowledge funding from NSERC partnership grants, Mitacs, Cubresa, and Research Manitoba.

Transmission dynamics and phenotype-genotype resistance mapping of mycobacteria

Virginia Pichler1,2

1University of British Columbia 2British Columbia Centre for Disease Control

Introduction: Mycobacterial infections persist as one of the leading causes of death worldwide. There are an estimated 10 million new tuberculosis (TB) cases annually, 500,000 of which are drug resistant. Other similar infections caused by non- tuberculous mycobacteria (NTM) are on the rise globally, primarily manifesting as pulmonary disease. In the effort to control transmission, improve treatments and manage drug resistance, new strategies using bioinformatic tools are crucial to compliment and improve current epidemiological and culture-based methods. Leveraging -omics data can generate more comprehensive profiles of infections by directly detecting transmission, identifying circulating dominant clones and outbreak clusters and drug resistance markers.

Methods: 77 NTM and 70 TB clinical isolates collected in BC will undergo drug susceptibility testing (DST) and nucleic acid extraction for genomic and transcriptomic analyses. Sequence data will be used to perform phylogenetic analyses and determine transmission clusters and subsequently to generate resistance profiles based on SNP (single nucleotide polymorphism) mutations. DST results will be captured as minimum inhibitory concentrations (MICs), which will be overlaid on the resistance profiles to find correlations. Phenotypic expression will be further evaluated by subjecting each isolate to subinhibitory antibiotic concentrations and using the transcriptomes in a differential expression analysis to reveal gene regulation under stress. The differentially expressed genes and SNPs will serve as inputs for an association analysis to determine the variants responsible for regulation during drug exposure.

Expected Results: Through correlating MIC values and SNPs, phenotype-genotype inference may be improved by linking resistance to specific drug thresholds. Statistically significant correlations between resistance mutations and differentially expressed genes in the association analysis will be classified as expression quantitative loci (eQTLs), genetic variants directly responsible for expression during drug resistance.

Conclusions: The results generated will not only add specificity to the drug thresholds required to treat and infection based on strain type, but will provide new targets for future functional genetic studies. This integrated study will overall provide greater interpretation power and resolution for clinical mycobacterial infections. Transitioning completely to -omics-based methods will allow for more swift treatment and control practices in BC and beyond in Canadian and international contexts.

Decellularized Brain Tissue for the Study of Glioma

Alisha Poole1, Donna Senger1, Stephen Robbins1

1Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary

Introduction: Cancer research has primarily focused on specific genetic mutations within the cancer cells. However, it is apparent that the environment that surrounds the tumor influences tumor growth and progression. The environment of the brain is particularly unique, and presents many challenges to the treatment of glioma. Thus, we hypothesize that the brain tumor microenvironment, specifically the extracellular matrix, contributes to glioma progression and therapeutic resistance.

Methods: Brain tissue was cut into 1mm sections by vibratome and decellularized using a protocol adapted from standard decellularization literature. DNA content of the decellularized material was determined by nanodrop. Change in glycosaminoglycan content from non-decellularized to decellularized material was assessed using the Blycan Sulfated Glycosaminoglycan Assay Kit (Biocolor). Removal of cellular proteins was monitored using western blot. Difference in ECM from tissue to tissue was analyzed by employing TMT labeled LC-MS/MS. Human patient derived brain tumor initiating cell xenografts (BT147, BT73) were decellularized and GFP labeled U87 glioma cells seeded at a concentration of 10,000 cells per decellularized tissue section and visualized over 48 hours. Cell proliferation was analyzed using fluorescence and RT- PCR (Ki67, ERK1, PI3K) at the 48 hour point.

Results: Decellularized brain tissue was determined to contain less than 3% double stranded DNA, with more than 97% double stranded DNA having been removed. Both glycosaminoglycan assay and western blot showed an increase in ECM proteins and a decrease in cellular proteins in decellularized tissue. Mass spectrometry analysis of tissue to tissue ECM variation showed no significant difference between tissue samples. U87 cells seeded on BT73 decellularized tumor and contralateral hemisphere showed the highest fluorescence over the 48 hour period (10 fold and 4 fold change respectively), indicating that the cells adhere better to these ECM environments, allowing the cells to proliferate. Similarly, PCR results showed an increase in Ki67, ERK1, and PI3K in the cells seeded on BT73 tumor and contralateral hemisphere decellularized tissue, indicating that these ECM environments promote proliferation.

Conclusion: Decellularized models provide a better representation of the ECM environment and the opportunity to study the impact of the ECM environment on tumor progression and therapeutic resistance in vitro.

Metagenomic and metatranscriptomic profiling of gut microbiome functional activity

Molly Pratt1,2, Morag Graham1,2, Denice Bay1, Charles Bernstein3,4, Gary Van Domselaar1,2

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB 3Department of Internal Medicine, University of Manitoba, Winnipeg, MB 4University of Manitoba IBD Clinical and Research Centre, Winnipeg, MB

Recently, an imbalance in the gut microbial community (known as dysbiosis) has been described in a variety of chronic diseases such as Multiple Sclerosis (MS), Type II Diabetes Mellitus (T2DM), and Inflammatory Bowel Disease (IBD). Metatranscriptomics is an emerging technique for studying microbial communities, including the human gut microbiome. When combined with whole-genome analyses, metatranscriptomics can provide additional insights into the functional activity and metabolic environment of the microbiome. Thus, investigation of functional activity in dysbiotic environments may help to elucidate the etiologies of chronic diseases and to inform potential treatments. Although metatranscriptomics provides valuable complementary information to metagenomics, the methodologies used to collect, store, prepare, sequence, and analyze metatranscriptomic samples are much less developed relative to metagenomic samples. The objectives of this research are to develop a method for fecal sample collection and processing that will allow for accurate metatranscriptomic analysis of microbiome functional activity. Briefly, healthy fecal samples will be used to test the effect of different storage and extraction conditions on metatranscriptomic profiles. Stool is first collected in a controlled environment that simulates home collection protocols used in cohort studies. Prior to freezing and storage, the fresh stool is aliquoted into one of three preservation conditions. Microbial DNA and RNA from the samples will be extracted using several commercially available kits, and the comparative effect of both the preservant and the extraction kit on the metatranscriptomic profile of the sample will be observed from sequence data. Shotgun sequencing of nucleic acid libraries will be performed using Illumina NextSeq short read sequencing technology, producing high coverage sequence data containing millions of reads. Sequence data will be filtered, quality assessed, and finally analyzed using bioinformatic tools such as MetaPhlAn2 and HUMAnN2 for taxonomic and functional profiling, respectively. Tested conditions will be compared using differential abundance analysis. Developing a consistent method for metatranscriptomic data collection and analysis will allow for metatranscriptomics to be applied more widely—and consistently—in relevant disease research. Cohort design for microbiome studies will benefit from a sample collection and storage method that is amenable to functional profiling as well as metagenomic analysis.

miRNAs target TFF3 influence the epithelial-mesenchymal transformation in PTC

Xiao-Jing Qin1

1Hebei North University

The incidence of thyroid carcinoma (TC) accounts for about 3% of systemic malignancies. It ranks first among endocrine system malignancies and is also one of the fastest growing malignancies in recent years. The main reason for the growth rate is papillary thyroid carcinomas (PTC), accounting for about 80% of TC. MicroRNA (miRNA) is a type of non-coding single- stranded RNA molecule with a length of 18 to 22 nucleotides. The previous research of the research group found that TFF3 can promote the epithelial-mesenchymal transition（EMT）of TPC-1 cells by activating the MAPK signaling pathway and enhance the migration and invasion of TPC-1 cells. Based on this, this project further explores the miRNAs that regulate the expression of TFF3 and provides potential targets for the treatment of PTC. First, the bioinformatics website was used to predict the miRNAs that may regulate TFF3 are miR-7-5p and miR-143-3p, and verified by the dual luciferase reporter gene experiment. Real-time quantitative PCR (RT-qPCR) was used to analyze the expression levels of miR-7-5p and miR-143-3p in PTC. Then, using the TPC-1 cell line as the research object, miR-7-5p and miR-143-3p were overexpressed in TPC-1 cells by transfection of miR-7-5p mimic and miR-143-3p mimic. Cell growth curve, scratch test, and Transwell test were used to detect the effects of miR-7-5p and miR-143-3p on the proliferation, migration and invasion of TPC-1 cells. Finally, western blot and immunocytochemical staining were used to detect the effect of overexpression of miR-7-5p and miR-143-3p on the epithelial-mesenchymal transition (EMT) marker (E-cadherin, N-cadherin and Vimentin) of TPC-1 cells. The results showed: (1) The target miRNAs that may regulate TFF3 were selected by bioinformatics technology as miR-7-5p and miR- 143-3p. (2) The results of the dual luciferase reporter gene showed that the luciferase activity of the TFF3 vector carrying the wild-type 3'UTR region and the miR-7-5p/miR-143-3p co-transfected cells was respectively reduced to 0.5 times and 0.1 times of the control group; while the mutant 3'UTR vector lacking the target site was co-transfected with miR-7-5p/miR- 143-3p, and the relative fluorescence intensity did not change significantly.

Subgrouping Primary Synovial Sarcoma Based on Epigenomic Landscape

Alvin Qiu1,2, Edmund Su3, Qi Cao3, Marcus Wong3, Michelle Moksa3, Torsten O. Nielsen1,2,4, Martin Hirst2,3,5

1Vancouver Coastal Health Research Institute, Faculty of Medicine, University of British Columbia, B.C., Canada 2Interdisciplinary Oncology Program, Faculty of Medicine, University of British Columbia, B.C., Canada 3Department of Microbiology and Immunology, Michael Smith Laboratories, University of British Columbia, B.C., Canada 4Department of Pathology and Laboratory Medicine, University of British Columbia, B.C., Canada 5Canada’s Michael Smith Genome Sciences Centre, BC Cancer, B.C., Canada

Background: Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by a pathognomonic chromosomal translocation leading to the production of SS18-SSX, a fusion oncoprotein. Previous research shows that SS18-SSX associates with BAF, a chromatin remodelling complex, suggesting epigenetic mechanisms drive this cancer. We hypothesize that SS can be sub-grouped based on epigenomic state and that these subgroups relate to disease severity.

Methods: We profiled 31 cases of primary human SS using chromatin immunoprecipitation sequencing (ChIP-seq) for histone modifications and RNA-seq for transcriptomes. Publicly-available cell line data were obtained for comparison.

Results: Unsupervised hierarchical clustering of genome-wide histone ChIP-seq density for the transcriptionally active marks (H3K27ac, H3K4me1, H3K4me3) reveals two major SS sub-groups: Group 1 and Group 2. Enhancers (regions marked by H3K27ac) from Group 2 tumors show higher levels of BAF binding compared to Group 1 enhancers. Using published isogenic cell line models, Group 2 enhancers also show greater overlap with binding sites of non-oncogenic BAF complexes (lacking SS18-SSX) compared to oncogenic BAF complexes (containing SS18-SSX). Genes associated with Group 2 enhancers are expressed at lower levels in SS compared to other soft-tissue sarcomas, suggesting Group 2 tumors express SS signature genes at lower levels that Group 1 tumors. Treating SS cells with quisinostat, a histone deacetylase inhibitor, leads to cell death and increases the expression of Group 2 enhancer genes. Clinical data show that Group 2 tumors are lower grade tumors.

Conclusion: SS can be sub-grouped based on epigenomic state and sub-groups are associated with differences in BAF activity. Using a histone deacetylase inhibitor pushes cells into a phenotype that resembles the less aggressive sub-group, and therefore may be a therapeutic strategy in SS.

Influenza A Virus Utilizes PSMA2 for Downregulation of NRF2-Mediated Oxidative Stress Response

Mahamud-ur Rashid1, Kevin M. Coombs1

1University of Manitoba

Influenza A virus (IAV), an obligatory intracellular parasite, utilizes host cellular molecules for completing its replication cycle and suppresses immune responses. Proteasome subunit alpha type-2 (PSMA2) is a cellular protein that was highly expressed in IAV infected human lung epithelial cells (A549). It is a part of the 20S proteasome complex which degrades or recycles defective proteins and involves in proteolytic modification of many cellular key regulatory proteins. However, the role of PSMA2 in IAV replication is not well understood. In this study, knockdown (KD) of PSMA2 in human lung epithelial cells caused a significant reduction of progeny IAV in the supernatant, but virus protein expression was not affected inside the infected cells. This indicates, PSMA2 is a critical host factor for the IAV replication cycle. For better understanding, the mechanism, proteins dysregulated by PSMA2 KD, IAV infection in wild type cells, and IAV infection in PSMA2 KD cells, were identified by SomaScan (targeted 1322 proteins) and analyzed by IPA (Ingenuity Pathway Analysis) software. We found six cellular signaling pathways, including Phospholipase C signaling, Pak signaling, and NRF2- mediated oxidative stress response signaling were inhibited by IAV infection but significantly activated by PSMA2 KD. Further analysis of NRF2 mediated oxidative stress response signaling, we found IAV inhibits the accumulation of reactive oxygen species (ROS) inside the cells, but ROS level significantly increases during IAV infection in PSMA2 KD cells. However, IAV infection causes a significant reduction of NRF2 expression but remains relatively unaffected in PSMA2 KD cells during infection. Overall, this study indicates that IAV utilizes PSMA2 to scape viral clearance via NRF2 mediated cellular oxidative response, but further study is required to understand the mechanism clearly.

Identification of Novel Lipid Droplet Factors that Regulate Autophagy and Cholesterol Efflux in Macrophage Foam Cells

Sabrina Robichaud1,2, Garrett Fairman1,2, Viyashini Vijithakumar1,2, Esther Mak2, David Cook4, Alexander Pelletier1,3, Barbara Vanderhyden4, Daniel Figeys1,3, Mathieu Lavallée-Adam1,3, Kristin Baetz1,3 and Mireille Ouimet1,2

1Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Rd., Ottawa, ON 2University of Ottawa Heart Institute, 40 Ruskin St, Ottawa, ON, K1Y 4W7, Canada 3Ottawa Institute of Systems Biology, 451 Smyth Rd., Ottawa, ON, K1H 8M5, Canada 4Ottawa Hospital Research Institute

Introduction: Atherosclerosis is characterized by an accumulation of lipid-rich foam cells within the arteries. Autophagy is a constitutive process that maintains homeostasis by recycling cellular components. Macrophage autophagy is a highly anti- atherogenic process that helps maintain cellular homeostasis. In foam cells, autophagy was demonstrated to contribute to the degradation of lipid droplets (LDs) via a selective form of autophagy called lipophagy. Selective autophagy relies on tags such as ubiquitin and selectivity factors to label specific cargo for degradation. Yet, how LDs are targeted for autophagy remains poorly defined. Our study was aimed at identifying LD factors responsible for lipophagy in macrophage foam cells.

Methods: To identify lipophagy factors in macrophage foam cells in an unbiassed manner, we employed mass spectrometry to qualify the LD proteome. Using siRNA array in combination with high-content microscopy and cholesterol efflux screens, we assessed the functional role of these candidate lipophagy factors.

Results: We confirmed the presence of known LD-associated structural and metabolic proteins in the LD proteome. Additionally, we found the association of several proteins related to the ubiquitination machinery and autophagy, along with other novel factors that could regulate lipophagy. We observed that knocking down several of these genes, including Maplc3b and Tfeb significantly reduced cholesterol efflux, suggesting a role for these proteins in lipophagy-mediated LD catabolism. Furthermore, we identified OPTN and NBR1 as a novel cargo receptor for lipophagy.

Conclusion: Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Therapeutic targeting of these novel lipophagy factors may represent a means to enhance macrophage lipophagy to promote reverse cholesterol transport for the treatment of heart disease.

Transcriptome analysis reveals docosahexaenoic acid and α-linolenic acid affect cholesterol metabolism and migration of monocytes through similar and distinct gene pathways

Lisa Rodway, Samantha Pauls, Harold Aukema, Carla Taylor, Peter Zahradka

University of Manitoba, Canadian Centre for Agri-Food Research in Health and Medicine

Objectives: One of the earliest events in atherosclerotic plaque formation is the migration of monocytes to damaged blood vessels, and the accumulation of cholesterol in monocyte derived macrophages. The fish-oil derived omega-3 fatty acid docosahexaenoic acid (DHA) has been shown to inhibit this process. There is limited evidence suggesting α- linolenic acid (ALA) has a similar effect, however, ALA has not been directly compared to DHA. Therefore, the primary objective of this study was to compare the gene expression profiles of monocytes that have been exposed to either ALA or DHA and subsequently examine the effect of these fatty acids on monocyte cholesterol content and migration in a cell culture model.

Methods: Whole transcriptome analysis was performed on total mRNA isolated from a human THP-1 monocyte cell line treated with ALA, DHA or vehicle for 48 h. Candidate genes identified via fold change and Ingenuity Pathway Analysis were validated by qPCR. Assays to measure total cholesterol content and migration (in response to chemokine) were subsequently performed on monocytes treated with ALA or DHA to corroborate the predicted outcomes of our analysis.

Results: Transcriptome analysis identified a series of genes associated with cholesterol metabolism and migration to be altered by ALA and DHA treatment. The change in mRNA levels for candidate genes were validated by qPCR, with similar expression patterns as in transcriptome analysis. Based on these data, cholesterol content was predicted to be reduced by both fatty acids, ALA would increase migration and DHA would have no effect . Functional assays revealed that ALA and DHA decreased cholesterol content to a similar extent. Additionally, contrasting our predictions DHA significantly decreased migration, while ALA had no effect.

Conclusions: The results suggest ALA and DHA influence may affect monocyte migration through distinct gene pathways, while cholesterol metabolism may be regulated similarly. Furthermore, functional assays indicated that only DHA treatment reduced migration, while both fatty acids reduced cholesterol content. Due to the critical role of monocyte migration and cholesterol content in the pathophysiology of atherosclerosis, it may be concluded from this study both DHA and ALA may exert protective effects in different ways as they relate to individuals at risk for cardiovascular disease.

Chronic Stress Physically Spares Innate-Like Invariant T Cells but Perturbs Their Functional Fitness in a Cell-Autonomous, Glucocorticoid Receptor-Dependent Fashion

Patrick T. Rudak1, Joshua Choi1, S.M. Mansour Haeryfar1

1Department of Microbiology and Immunology, Western University

Introduction: The nervous system serves numerous critical roles in the regulation of immune responses. Consequently, psychological stress can result in immunosuppressive states that are conducive to the development of infection and cancer. Yet, whether psychological stress impacts the functions of innate-like invariant T cells including invariant natural killer T (iNKT) cells and mucosa-associated invariant T (MAIT) cells, which participate in early host defense against pathogens and tumors, has historically remained undefined.

Methods: We leveraged multiple well-established models of psychological stress in mice, including prolonged restraint stress, chronic variable stress, and repeated restraint stress. Afterwards, we injected mice intraperitoneally with agonists of iNKT cells or MAIT cells to evaluate their functional fitness in vivo.

Results: We demonstrate that stress abrogates TH1- and TH2-type cytokine production by iNKT cells in vivo, effects which are lost upon habituation to predictable, homotypic stressors. Instead, activated iNKT cells in stressed mice trigger an abnormal systemic inflammatory response characterized by striking levels of IL-10, IL-23, and IL-27. Pharmacological inhibitors, conditional knockout systems, flow cytometric analyses, and gene expression analyses revealed that these dysregulated responses are driven by iNKT cell-intrinsic glucocorticoid receptor (GR) signaling. Accordingly, iNKT cells upregulate the co-inhibitory molecule TIGIT in a cell-autonomous, GR-dependent manner, and blockade of TIGIT partially restores their functional capacity in stressed mice. Ultimately, iNKT cells from stressed mice exhibit a diminished ability to promote cytotoxicity against target cells and, in a GR-dependent fashion, fail to prevent pulmonary metastases of B16 melanoma. We also demonstrate that MAIT cells upregulate TIGIT and are incapable of mounting optimal TH1- and TH2- type cytokine responses during stress. Importantly, stress-induced suppression of invariant T cell responses is not simply due to their cellular death since we found human and mouse iNKT and MAIT cells to be unusually refractory to glucocorticoid-induced apoptosis.

Conclusion: Collectively, our findings reveal a novel mechanism of stress-induced immunosuppression involving innate-like invariant T cells. Since invariant T cells continue to be proposed as attractive targets for immunotherapy, our work implicates the stress response as a potential barrier for the efficacy of future iNKT or MAIT cell-based immunotherapies.

Evaluation of a taxon-specific reference database for pathogen detection in complex food matrices using a metagenomic approach

Jillian Rumore1,2, Matthew Walker2, Celine Nadon1,2, Aleisha Reimer2, Natalie Knox1,2

1Department of Medical Microbiology & Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada; 2National Microbiology Laboratory, Canadian Science Centre for Human and Animal Health, Public Health Agency of Canada, Winnipeg, Manitoba, Canada

Introduction: The accuracy of culture independent pathogen detection in foods using metagenomics is contingent on the quality and composition of the reference database, which is required to assign generated reads to a taxonomic classification. Sequence contamination and, lack of representation and taxonomic diversity in the reference database can lead to a high number of false positive (FP) classifications, potentially resulting in faulty conclusions that could have substantial consequences for food safety and regulatory decision-making. To improve classification accuracy, inclusion of microbial sequences from a diverse representation of taxonomies in universal reference databases are recommended. However, these databases require substantial computational resources, which may not be feasible for many laboratories. In this pilot study, our aim was to assess the performance of a foodborne pathogen (FBP)-specific reference database (taxon-specific) compared to the use of a universal reference database for detection of Listeria monocytogenes, a priority foodborne pathogen, in complex food matrices. To the authors’ knowledge, this is the first study to compare a taxon-specific and a universal reference database for the purpose of foodborne pathogen detection in complex food matrices.

Methods: Publicly available complete genomes for bacteria, viral, archaea and human, as well as contaminating sequences (available from UniVec and EMVEC databases) were used to construct the universal reference database. A subset of complete genomes representing priority enteric pathogens were used to build the FBP-specific database. To assess database performance, metagenomic-based pathogen detection of L. monocytogenes in artificially contaminated turkey deli meat and prepackaged spinach was applied. All reads classified as L. monocytogenes were investigated for accuracy.

Results: Removing host sequences from the datasets prior to taxonomic classification resulted in considerably fewer FP classifications; however, slightly more FP classifications were identified for prepackaged spinach samples compared to turkey deli meat samples when analyzed with the FBP-specific reference database. The number of true positive classifications was consistent across reference databases, however, considerably less computational resources were required for the FBP-specific reference database.

Conclusions: Comparable results were achieved for detection of L. monocytogenes in foods using a taxon-specific reference database when the appropriate quality control measures, classification tools, and analysis parameters were applied.

Reduced Expression of Ubiquitylation and Deubiquitylation Genes Induces Chromosome Instability that May Promote Colorectal Cancer Development

Kailee A. Rutherford1,2, Cindy Atayan2, Mirka Sliwowski2, Zelda Lichtensztejn2 and Kirk J. McManus1,2

1Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB 2Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, MB

OBJECTIVES: In 2020, colorectal cancer (CRC) accounted for approximately 27,000 new cancer diagnosis and took the lives of nearly 10,000 Canadians. To improve patient outcomes, more effective therapies are needed, which requires a greater understanding of the cellular events driving CRC pathogenesis. Chromosome instability (CIN; ongoing changes in chromosomal complements) is a driver of cancer development observed in approximately 85% of CRC cases, yet the causal genes (i.e. CIN genes) remain largely unknown. Recent findings from the McManus laboratory implicate aberrant ubiquitin regulation as a novel driver of CIN. Thus, we now seek to assess all genes encoding ubiquitylation and deubiquitylation proteins to determine their impacts on CIN.

METHODS: Transient siRNA silencing was employed to assess 94 deubiquitylation and 582 ubiquitylation genes for their impacts on CIN relative to a negative control (siControl) in two karyotypically stable colonic cell lines: HCT116 (malignant) and 1CT (non-malignant). Quantitative imaging microscopy was utilized to assess increased nuclear area heterogeneity and micronucleus formation, which are associated with large- and small-scale changes in DNA content, respectively. Genes were prioritized based on conservation of phenotypes and clinical relevance, which was determined by assessing genomic copy number and mRNA expression data from The Cancer Genome Atlas (TCGA). Complementary siRNA approaches were employed to evaluate gene silencing efficiency, CIN-associated phenotypes and changes in chromosome numbers.

PRELIMINARY RESULTS: To date, the siRNA screen of deubiquitylation and ubiquitylation genes identified 269 putative CIN genes in at least three of the four assay conditions. Subsequent patient-based analyses revealed 26 genes are heterozygously lost in at least 10% of CRC cases and diminished mRNA expression correlates with worse patient outcomes. Four genes (FBXL5, FBXO33, JOSD1, USP4) have been prioritized for further evaluation, with western blots, nuclear areas and micronucleus formation analyses identifying USP4 is a novel CIN gene in colonic contexts.

CONCLUSION: Preliminary findings identified USP4 as a novel CIN gene with clinical implications in CRC. Collectively, this work will expand our understanding of the pathogenic events driving CRC, which is crucial to ultimately develop more effective therapies to improve patient outcomes.

The feasibility of transcranial direct current stimulation (tDCS) as an adjunct to inpatient physiotherapy in pediatric acquired brain injury (ABI): Year 1 eligibility and recruitment

Jennifer Ryan1,2, Deryk Beal1,2, Darcy Fehlings1,2, Danielle Levac3, Virginia Wright1,2

1Holland Bloorview Kids Rehabilitation Hospital 2University of Toronto 3Northeastern University

Introduction: Children with moderate to severe ABI require intensive rehabilitation early in recovery to address motor deficits. Despite considerable improvement with inpatient physiotherapy (physio), motor recovery typically plateaus and deficits persist. Thus, adjuncts to physio are frequently explored to enhance motor recovery. tDCS modulates brain activity and enhances motor skill learning when coupled with motor skill practice but has not been studied in pediatric ABI. The primary objective of this study is to evaluate the feasibility of a ‘tDCS+physio’ protocol in an existing pediatric inpatient ABI rehabilitation program and determine if a randomized control trial (RCT) to evaluate its effectiveness is warranted.

Methods: This feasibility RCT randomizes 30 children (5-18 years) with moderate to severe ABI to receive active or sham tDCS paired with their existing inpatient physio at Holland Bloorview Kids Rehabilitation Hospital (i.e. tDCS+physio). Participants receive 20 minutes of either active or sham tDCS immediately prior to 4 physio sessions each week for 4 weeks (16 sessions). Feasibility is evaluated by tracking process, recruitment, and treatment indicators. Participants, therapists, assessors, and primary investigators are blinded to treatment allocation.

Results: Since January 2020, 152 children were admitted for inpatient ABI rehabilitation at Holland Bloorview and screened for study eligibility. Six children were eligible and three enrolled. Recurrent reasons for exclusion: age, tDCS contraindications, short admission duration, behaviour. Screening data and control charts outlining recruitment to date indicate that recruiting 30 children in a reasonable time frame is unlikely. Mitigating actions include adjusting eligibility criteria and adapting analysis plans based on three enrollment rates.

Conclusion: Results will inform whether tDCS is a practical adjunct to inpatient physio in pediatric ABI from tolerability and eligibility/recruitment perspectives. Gross motor outcomes will provide a basis for sample size calculations for a future effectiveness RCT if warranted. If effective, tDCS+physio could enhance motor outcomes in children with ABI.

Nanocomposite hydrogels for 3D bio-fabrication applications

Mahmoud A. Sakr1, Su Ryon Shin2, Sumi Siddiqua1, Keekyoung Kim3,4

1School of Engineering, The University of British Columbia, Kelowna, BC, Canada 2Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham Women's Hospital, Cambridge, Massachusetts, USA 3Department of Mechanical and Manufacturing Engineering, University of Calgary, Calgary, AB, 4Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB,

The principal building materials in biofabrication are generally hydrogels mixed with various types of cells, also known as bioinks. The bioink is a crucial component of the biofabrication process, as they must possess appropriate physical parameters required for the printing process and simultaneously offer an optimal microenvironment for cell survival, proliferation, migration, and biosynthetic activity. Tissue-engineered analogs that approximate the structural complexity and functionality of native tissues have significant potential for regenerative medicine. Nanocomposite hydrogels are a class of hybrid hydrogels that constitute nanomaterials as the dispersed phase within the crosslinked matrix of the hydrogel to reinforce certain properties that the hydrogel lacks. Nanomaterials have been frequently used to enhance the mechanical properties of hydrogels. Therefore, the major objectives of this research are to develop two different kinds of nanocomposite hydrogels using natural and synthetic clay nanoparticles (NPs) and an electrically conductive nanocomposite hydrogel for the fabrication of bone and cardiac tissues. The development of novel nanocomposite hydrogels with altered physical, biological, and electrical properties will provide an opportunity to investigate a variety of biofabrication applications. Both objectives include synthesizing the nanocomposite hydrogels. Characterization, comparison, and optimization of material properties of nanocomposites hydrogels for the use of bioinks. Testing the bio printability and biocompatibility of nanocomposite hydrogels and fabricate bone and cardiac tissues with the optimized bioink. Results from the research studies conclude that bentonite clay nanoparticles can enhance mesenchymal stem cells differentiation and enhance the mechanical properties of gelatin-based hydrogels. Swelling ratio and scanning electron microscope showed a change in the pore size of the nanocomposite hydrogel which plays a positive role in cell elongations and degradation rate of the hydrogel. Moreover, ornithine can be used as a source of ionic conductivity to produce electrically conductive hydrogels which can be used in cardiac tissue engineering. Cell encapsulation and electrical conductivity measurements showed an enhancement in cell proliferation and cell morphology. Both materials are naturally derived and biodegradable and support the elongation and differentiation of stem cells.

Role of Akyldihydroxyacetone phosphate synthase on host immune response and virulence of Leishmania major

Enitan Salako1, Zhirong Mou1, Ping Jia1, Stephen M Beverley2, Jude E Uzonna1

1Department of Immunology, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada. 2Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA.

Introduction: Leishmaniasis is a spectral disease with clinical manifestations ranging from mild self-healing skin ulcers to chronic mucocutaneous infection and severe systemic infection. While drugs are available for treating the disease, most are expensive or highly toxic. Interestingly, recovery from Leishmania infections leads to long-lasting protective immunity, suggesting that the disease can be prevented through vaccination. A key challenge is determining the antigens that could either be used as recombinant vaccine candidates or targeted for the generation of attenuated parasite to be used as live- attenuated vaccine. Alkyl-dihydroxyacetone phosphate synthase (ADS) is the critical enzyme involved in the biosynthesis of glycerol-containing ether lipids, which is required for the synthesis of glycosylphosphatidylinositol (GPI). GPI is important for anchoring lipophosphoglycan (LPG) and gp63, which are major virulence factors of the parasite, to the cell membrane. Deficiency of ADS synthesis leads to impaired synthesis of GPI-anchored molecules resulting in attenuated virulence. However, the impact of ADS deficiency on the immunopathogenesis of cutaneous leishmaniasis has not been studied.

Methods: The growth kinetics of ADS deficient (ADS KO) L. major parasite in axenic culture were compared to wild-type (WT) and Add-back (ADS-AB) parasites in axenic culture. Also, bone marrow-derived macrophages were infected with WT, ADS KO and Add-back parasites and infectivity and parasite proliferation were measured and compared at different times after infection by staining cytospin preparations with Giemsa stain and counting under a light microscope.

Results: Our preliminary study shows that ADS gene deficiency affects the growth kinetics of L. major in axenic culture. In addition, ADS deficient parasites showed lower macrophage infectivity in vitro compared to their wild-type (WT) controls.

Conclusion: Deficiency of ADS in Leishmania major affects parasite growth rate in axenic culture and infectivity in macrophages in-vitro, confirming the critical role of GPI-anchored in parasite proliferation and infectivity. Further studies will assess the impact of ADS gene deficiency in disease pathogenesis and host immune response in infected mice.

Taming the Cytokine Storm by dampening the NF-κβ butterfly effect: Repurposing Montelukast for the attenuation and prophylaxis of COVID-19 symptoms

Nitesh Sanghai1, Geoffrey K. Tranmer1,2

1College of Pharmacy, Rady Faculty of Health Science, University of Manitoba, Winnipeg, MB R3E 0T5, Canada. 2Department of Chemistry, Faculty of Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.

The two main complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are the development of acute respiratory distress syndrome (ARDS), as well as the development of complications from a hyperinflammatory cytokine profile (a cytokine storm). NF-κβ (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex involved in cytokine production and inflammation, with attenuation of NF-κβ associated with a reduction in cytokine production. Montelukast, an FDA and Health Canada approved asthma drug, has been shown to inhibit the signalling of NF-κβ, and other proinflammatory mediators, such as interleukin-6,8,10, TNF-α, MCP-1. Herein, we outline the scientific rational behind our hypothesis that repurposing Montelukast to target suppression of NF-κβ activation in COVID-19 positive patients will result in a corresponding reduction of proinflammatory mediators, thereby attenuating cytokine production, and dampening of the cytokine storm. Additionally, we postulate that a reduction in proinflammatory mediators by Montelukast will result in a mitigation of severe COVID-19 symptoms and serve as a therapeutic for SARS- CoV-2 infections. In this manner, we propose to target attenuation of COVID-19 symptoms, rather than the virus, as an effective therapeutic strategy to improve COVID-19 related clinical outcomes. In support of our hypothesis, we explore the latest clinical reports on COVID-19 and cytokine storm syndrome, as well as the clinical role of NF-κβ in COVID-19 pathogenesis, including results from the literature on Montelukast and its ability to modulate NF-κβ, reduce cytokine production, and mitigate RNA virus infections. Currently, as part of a larger team approach, we are preparing to initiate a clinical trial protocol for the study of the efficacy of montelukast in patients who are newly identified as COVID-19 positive, The COvid-19 Symptom MOntelukast Trial (COSMO), NCT04389411.

The role of humoral immunity in influenza-associated pulmonary aspergillosis

Nicole Sarden1, Bryan G. Yipp1

1Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary

Introduction: Respiratory viral infections continue to be a worldwide annual problem. H1N1, a particularly deadly strain of influenza, has been the predominant diseasecausing strain for the last decade, despite annual H1N1 vaccinations. Concurrent bacterial superinfections are commonly found in influenza-infected patients, however, a newly appreciated and concerning observation are H1N1-infected patients who develop invasive opportunistic infections with environmental fungi despite not been pharmacologically immunosuppressed. Indeed, the environmental fungi Aspergillus fumigatus is normally restricted to causing invasive lung disease in neutropenic or highly immunosuppressed patients. While there is ample research investigating bacterial superinfections, the biological basis of influenza-associated aspergillosis remains poorly understood. Since neutropenia represents a clinical risk factor for developing invasive aspergillosis, we hypothesize that H1N1 impairs neutrophil recruitment and/or function in the lung thereby increasing susceptibility to concurrent fungal infection.

Methods: Mice were infected intranasally with a sublethal dose of influenza virus and after 5 days infected with A. fumigatus. Clinical outcomes, mortality and pulmonary fungal burden were assessed. Innate and adaptive immune cells in the lungs were quantified by flow cytometry and neutrophil behavior was observed with lung intravital microscopy.

Results: H1N1 predisposes to invasive infection with A. fumigatus and worse clinical outcomes in a mouse model. Neutrophils and B cells are required for host defense against A. fumigatus. Absence of anti-Aspergillus protective antibodies during influenza infection have downstream effects in neutrophil function, which lead to worse outcomes in coinfected mice.

Conclusion: Humoral immunity is affected during influenza infection which may disturb anti-fungal immunity.

Determining the Underlying Mechanisms of Enhanced Viral Fitness for Widely Circulating SARS- CoV-2 Variants

James Saville1, Dhiraj Mannar1, Xing Zhu1, Wei Li2, Sriram Subramaniam1

1University of British Columbia 2University of Pittsburgh

The recently emerged B.1.1.7 (“UK”), B.1.351 (“South Africa”), P.1 (“Japan/Brazil”) and B.1.427/B.1.429 (“California”) variants of SARS-CoV-2 have all been designated by the American Centers for Disease Control and Prevention (CDC) as variants of concern (VoC). Initial genomic surveillance reports indicate that these variants confer higher infectivity and immune-system evasion. Common amongst these variants are mutations within the spike glycoprotein, which facilitates viral attachment and entry into human cells via its binding partner human angiotensin converting enzyme 2 (hACE2). These variants share multiple common mutations within the receptor binding domain (RBD) of the spike glycoprotein, which contains the interaction interface between spike protein and hACE2. Mutations within the RBD region are posited to increase viral fitness by increasing hACE2 binding affinity, or by decreasing the activity of neutralizing antibodies – the majority of which are directed against the RBD. We are engaged in efforts to systematically dissect the contributions of these VoC RBD mutations towards increasing hACE2 affinity and evading neutralizing antibodies. Our approach involves a combination of cryo-EM structural analyses and assays that measure hACE2 and neutralizing antibody binding. We have determined cryoEM structures of several RBD variants in complex with hACE2, illuminating the underlying inter-molecular interactions that facilitate the observed differences in hACE2 binding. Our studies suggest that the combination of these mutations is additive in their ability to increase hACE2 binding and that multiple single amino acid mutations are able to completely abolish binding of many neutralizing antibodies. We are also exploring the consequences of combining multiple VoC RBD mutations into a single spike protein to study the effects of potential, yet-to-emerge, combinatorial variants. These combinatorial mutations also emerge naturally, as evidenced by the discovery of variants that combine the mutations found in the B.1.1.7 (“UK”) with the E484K mutation, previously found only in the B.1.351 (“South Africa”) and P.1 (“Japan/Brazil”) variants. These results have implications for predicting the behaviour of future emerging variants and informing how second-generation vaccines may be improved to limit the spread of currently circulating variants.

Evaluating the impact reduced FBXO7 expression has on chromosome instability and cellular transformation in colorectal cancer

Michaela C. L. Palmer1,2, Tooba Razi3, Nicole M. Neudorf1,2, Ally C. Farrell1,2, Zelda Lichtensztejn2 and Kirk J. McManus1,2

1Department of Biochemistry and Medical Genetics, University of Manitoba, 2Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, MB, 3Faculty of Science, University of Manitoba

Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed and second most lethal cancer among Canadians. Therefore, it is imperative to enhance our understanding of the genes and mechanisms driving CRC development to ultimately improve patient outcomes. Chromosome instability (CIN), the increased rate of chromosome gains and losses, is an on-going and dynamic process posited to drive cellular transformation, an essential process for disease development. However, the aberrant genes and mechanisms underlying CIN are largely unknown. Preliminary data suggest FBXO7 is a CIN gene (i.e. decreased expression induces CIN) and is a clinically relevant as heterozygous FBXO7 (FBXO7+/-) loss is found in ~30% of CRCs. Importantly, FBXO7+/- loss and decreased mRNA expression are associated with worse overall survival in CRC patients. This project seeks to shed light on the early etiological events driving CRC development by determining the long-term impact FBXO7+/- loss has on CIN.

Methods: To evaluate the long-term effects diminished FBXO7 expression has on CIN, FBXO7+/- knockout models and a control (NT-Control) were generated using CRISPR/Cas9 in karyotypically stable, non-malignant colonic epithelial cells (A1309). FBXO7+/- models were assessed using three CIN assays, namely changes in nuclear area, micronucleus formation, and chromosome enumeration analyses every two weeks for ~2.5 months. Conceptually, changes in nuclear areas correspond to large-scale changes in DNA content, whereas micronuclei are associated with chromosome missegregation events. Mitotic chromosome spreads were generated to assess specific changes in chromosome numbers, while cellular transformation was assessed in colony formation and anchorage-independent growth assays.

Preliminary results: Two FBXO7+/- models were generated that expressed FBXO7 at ~40% of endogenous levels in western blot analyses relative to NT-Control. Long-term CIN assays revealed on-going and dynamic changes in CIN-associated phenotypes over ~2.5 months, including increases in cumulative nuclear area distributions, micronucleus frequencies and aberrant chromosome numbers (gains and/or losses). Interestingly, FBXO7+/- loss was associated with increased clonogenic and anchorage independent growth.

Conclusion: Our findings identify FBXO7 as a novel CIN gene that drives cellular transformation in A1309 cells and provides unprecedented insight on the impact reduced FBXO7 expression may have in the early etiological events contributing to CRC pathogenesis.

Identification of HIV escape mutations to a novel host genomic locus associated with control of HIV replication

Vanessa Schulz1,2, Rupert Capina2, Jeff Tuff2, Lara Lewis3, Joshua Kimani4, Lyle R McKinnon1,3,4, Ayesha Kharsany3, Paul J McLaren1,2

1Department of Medical Microbiology & Infectious Diseases, University of Manitoba 2National HIV and Retrovirology Lab at the JC Wilt Infectious Diseases Research Centre, National Microbiology Laboratories, Public Health Agency of Canada 3Centre for the AIDS Programme of Research in South Africa 4Department of Medical Microbiology, University of Nairobi

Restriction of HIV viral load within the infected population is key to ending the AIDS epidemic. Achieving this at the population level will likely require new strategies for limiting HIV replication. Some individuals, referred to as elite controllers, have genetic variants, particularly in the HLA region, that function to limit HIV replication. However, HIV can develop escape mutations to evade host pressure, counteracting their effect. A recent genome- wide association study (GWAS) of >3,800 of HIV+ individuals performed by our group identified a novel human genetic locus on chromosome 1 that associates with control of viral replication. A variant within this region, rs59784663(G), is associated with an average ~0.3 log10 reduction in HIV viral load (p=2.0x10-9) and is downstream from the protein coding gene chromodomain helicase DNA binding protein 1 like (CHD1L). However, despite the viral load decreasing effect of rs59784663, some individuals with the protective allele still show high viral loads. Given the high mutation rate and short generation time of HIV replication we hypothesize that selective mutation of the viral genome allows HIV to escape restriction by CHD1L. We will test this hypothesis by combining human and viral genomic data from ~1000 people living with HIV from Kenya and South Africa. Human genetic variants in the CHD1L region, including rs59784663, will be tested for association with amino acid variants identified across the HIV proteome in the same individuals. If CHD1L does confer some restriction on HIV, viral mutations associated with CHD1L variants are expected. This approach has the potential to reveal regions of conflict between the host and viral genomes, increasing our understanding of viral evolution and host control of HIV.

Exploring the experiences of pelvic health physiotherapists in Canada: Preliminary results from a qualitative descriptive study

Stephanie Scodras1, Jacquie Ripat2, Euson Yeung1, Susan B. Jaglal1,3, Heather Colquhoun1, Nancy M. Salbach1,3

1University of Toronto 2University of Manitoba 3KITE-Toronto Rehabilitation Institute

Introduction: Pelvic health physiotherapy (PHPT) is considered an advanced and complex area of practice involving the assessment and treatment of people with pelvic health conditions. PHPT education is not standardized in Canada, which may lead to diverse experiences and challenges. Little is known about the experiences of pelvic health physiotherapists working in Canada. This study aims to explore the education and practice experiences of pelvic health physiotherapists in Canada.

Methods: Following a qualitative descriptive methodology, I conducted semi-structured interviews with 20 pelvic health physiotherapists (19 females, 1 male) with <1 to >10 years of PHPT experience, working in private practice (85%) and hospital (10%) settings in 6 provinces. Interviews were audio-recorded and transcribed verbatim. I analysed the qualitative data using a reflexive thematic analysis approach situated in an interpretivist paradigm.

Results: Six themes were identified. ‘The Right Fit’ represents physiotherapists’ satisfaction with filling a clinical need in society and the gratification from working with patients with pelvic health conditions. ‘Mutual Vulnerability’ represents the shared physical and emotional vulnerability that is experienced by the patient and provider due to the intimate nature of practice. ‘Therapeutic Alliance is Key’ captures the importance that physiotherapists place on the patient-provider relationship and the steps taken to communicate clearly and sensitively. ‘Breaking out of the Silo’ represents physiotherapists’ process of integrating the pelvic floor with the rest of the body and moving beyond treating physical components, often adopting a biopsychosocial approach to treat the whole person. ‘An Ongoing Learning Process’ reflects that learning does not have an endpoint and involves formal education, learning from experience, seeking advice and information from reputable sources, and sharing knowledge with others. Pelvic health physiotherapists feel ‘Responsibility and Accountability’ towards patients and the physiotherapy profession. These factors act as a flexible boundary that dictates how pelvic health physiotherapists can and should act but expands with the growth of the field.

Conclusions: PHPT is a rewarding and intimate practice that requires physiotherapists to adopt sensitive and holistic approaches to working with patients. Physiotherapists feel responsible to engage in ongoing learning to meet patient needs in this growing field.

Effect of HNF-1a G319S variant on liver function in the fasted and non-fasted states

Manuel Sebastian1

1Department of Physiology and Pathophysiology

BACKGROUND: Rates of youth-onset Type 2 diabetes (T2D) in the Anishininiiwuk (Oji-Cree) linguistic group of central Canada are among the highest in the world. A genetic variant in the hepatic nuclear factor-1a (HNF-1a) gene, known as HNF-1a G319S, strongly associates with T2D in this population. HNF-1a is known to play an important role in the liver and pancreas, however, it is unclear how the G319S variant influences its function. We hypothesize that the G319S variant promotes greater hepatic glucose production and hepatic triglyceride storage, which historically may have conferred an advantage during states of prolonged fasting and low dietary carbohydrate availability; however, interaction of this gene variant with colonization-driven changes in diet and lifestyle now promotes hyperglycemia and T2D development.

METHODS: CRISPR/Cas9 gene editing was used to knock-in the HNF-1a G319S G>A single nucleotide substitution into C57BL6 mice. Male and female G/G (wild type), G/S (heterozygous) and S/S (homozygous) mice were fed a standard chow diet, sacrificed at 3 months of age, and liver tissues are collected for gene expression, triglyceride content, and glycogen content measurements. Additionally, 3-month mice were fasted for 24 hours, and liver tissue collected for the same measurements outlined above. Blood glucose measurements were taken before and after fasting, and blood ketone measurements are taken after fasting.

RESULTS: In female G319S mice, a trend towards higher blood glucose was observed in the non-fasted state along with decreased liver triglyceride levels. In the fasted state, G319S female mice displayed a trend towards higher blood ketones, and lower blood glucose. G/S and S/S male mice also show decreased liver triglyceride and increased expression of genes involved in ketone synthesis. In the fasted state, male G319S mice showed a trend towards lower plasma insulin levels and decreased blood glucose levels.

CONCLUSIONS: Under the non-fasted state, G319S mice have elevated blood glucose levels, and under the fasted state, mice with the variant have higher ketones, and lower blood glucose. This indicates a greater shift in metabolism to utilization of ketone bodies when fasted, which is further validated by increased expression of ketogenic genes and decreased liver triglyceride levels.

Whole-genome sequencing of SARS-CoV-2 for Canada’s COVID-19 surveillance

Grace E. Seo1,2, Anna Majer2, Elsie Grudeski2, Rhiannon Huzarewich2, Russell Mandes2, Kirsten Biggar2, Darian Hole3, Philip Mabon3, Natalie Knox1,3, Gary van Domselaar1,3, Tim F. Booth1,2

1Department of Medical Microbiology & Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada 2Viral Diseases Division, National Microbiology Laboratory, Winnipeg, MB, Canada 3Bioinformatics Section, National Microbiology Laboratory, Winnipeg, MB, Canada

As of March 2021, the total number of Coronavirus Disease 2019 (COVID-19) cases have reached 12 million worldwide. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) — the etiologic agent of COVID-19 — has accumulated numerous mutations since the start of the pandemic. The most troubling mutations identified reside within the viral spike gene, the same region for which developed vaccines target. Some mutations have been found to increase viral transmissibility, disease severity, and decrease vaccine effectiveness. In order to monitor for the emergence and spread of SARS-CoV-2 variants carrying these concerning mutations, a genomic-based surveillance program is necessary. Like many other countries, genomic surveillance to monitor SARS-CoV-2 variants became of absolute importance in Canada to protect Canadians from emerging variants of concern and to ensure that current COVID-19 vaccines remain effective. The focus of my research is on developing whole genome sequencing (WGS) standards for a national SARS-CoV-2 genomic surveillance program, the first of its kind for viral pathogens in Canada. Using the developed sequencing standards, our lab at the National Microbiology Laboratory has been performing WGS of SARS-CoV-2 for outbreak investigations and variant detection since the start of the pandemic. To date, we have detected numerous variants of concern across Canada using the established genomic surveillance infrastructure. SARS-CoV-2 genomic surveillance has been used to inform public health measures in an effort to protect Canadians. Keywords: SARS-CoV-2 variants, whole-genome sequencing, genomic surveillance

Characterization of the early proinflammatory response to clozapine in rats: relevance for idiosyncratic drug-induced agranulocytosis

Samantha C. Sernoskie1, Alison Jee2, and Jack P. Uetrecht1,2

1Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada 2Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada

Introduction: Although clozapine is a highly efficacious anti-schizophrenic medication, it is infrequently prescribed due to the risk of idiosyncratic drug-induced agranulocytosis (IDIAG). The mechanism of IDIAG is unknown, but evidence suggests it involves an adaptive immune response against clozapine-modified proteins. Progression to IDIAG is rare, yet most patients experience a transient innate immune response early in treatment. Thus, using a rat model, we investigated the induction of this proinflammatory response, focusing on myeloperoxidase (an enzyme that bioactivates clozapine) and inflammasome involvement.

Methods: Female Sprague Dawley rats were administered clozapine (30 mg/kg, i.p.) and immune parameters were evaluated up to 24 hours. Whole blood was collected for differential blood counts. Plasma and protein homogenates (spleen, bone marrow, liver) were collected for inflammatory mediator measurement using ELISA. Immune cells were further profiled using flow cytometry. In some experiments, inhibitors of caspase-1 (VX-765) or myeloperoxidase (PF-1355) were co-administered.

Results: Blood counts showed increased neutrophils and decreased lymphocytes by 3 hours, with changes resolving by 24 hours. Using flow cytometry, a decrease in blood B cells was noted at 3 hours, as well as decreased blood and spleen T cell subsets. Increased neutrophils, monocytes, and natural killer cells were also observed in the spleen. This was accompanied by elevated proinflammatory mediators, including protein levels of IL-1β, CXCL1 (a neutrophil chemoattractant), and neutrophil elastase in the plasma, spleen, bone marrow, and liver, often by 1.5 hours. Interestingly, treatment with fluperlapine, a structural analogue of clozapine, did not cause neutrophilia or increase CXCL1. Clozapine-induced neutrophilia was moderately dampened by pre-treatment with a VX-765 and several immune changes in the blood and spleen were attenuated by pre-treatment with PF-1355.

Conclusion: Clozapine rapidly induced organ-specific immune changes, some of which may have been attenuated by inhibition of reactive metabolite formation (i.e., myeloperoxidase inhibition) or inhibition of inflammasome activation (i.e., caspase 1 inhibition). Ultimately, a better mechanistic understanding of the immune response to clozapine may reveal ways to prevent or treat IDIAG, enabling safer use of this highly effective medication in patients. This research was supported by CIHR, Mitacs, OGS, UofT and the Leslie Dan Faculty of Pharmacy.

Household Food Insecurity is associated with Depressive Symptoms in the Canadian Adult Population

Mojtaba Shafiee1, Hassan Vatanparast1,2, Bonnie Janzen3, Sara Serahati2, Pardis Keshavarz1, Parisa Jandaghi1, Punam Pahwa3,4

1College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada. 2School of Public Health, University of Saskatchewan, Saskatoon, Canada 3Department of Community Health and Epidemiology, University of Saskatchewan, Saskatoon, Canada 4Canadian Centre for Health and Safety in Agriculture, University of Saskatchewan, Saskatoon, Canada

Introduction: It is essential to identify factors associated with depression as it is a highly prevalent and disabling mental disorder. The aim of this study was to examine the association between depressive symptoms and household food security status among the Canadian adult population.

Methods: This is a cross-sectional study of the adult population in the five provinces and one territory (Northwest Territories) of Canada using data from the 2015-2016 Canadian Community Health Survey–Annual Component (n=19,118). Depressive symptoms were assessed using the 9-item Patient Health Questionnaire. Household food insecurity was measured using the Household Food Security Survey Module. A weighted logistic regression analysis with robust variance estimation technique was performed.

Results: Approximately 22% of the Canadian adult population reported mild-to-severe depressive symptoms, and 8.3% were from households classified as food insecure. Household food insecurity remained a significant predictor of mild-to-severe depressive symptoms even after adjustment for other known risk factors (ORajd: 2.87, 95% CI: 2.33-3.55, p<0.001). In the multivariable model, significant associations were also found with multimorbidity, lower household income, a history of illicit drug use, being a current smoker, being a widowed/divorced/separated, obesity, and being a non-drinker. Significant interactions also emerged between employment status and age (p=0.03), employment status and gender (p<0.001), and physical activity level and gender (p<0.001).

Conclusion: Household food insecurity is associated with depressive symptoms in Canadian adults. Additional longitudinal research is required to further elucidate the nature of this relationship.

Availability of neuregulin-1beta1 protects neurons in spinal cord injury and against glutamate toxicity through caspase dependent and independent mechanisms

Narjes Shahsavani, Arsalan Alizadeh, Hadeep Kataria, Soheila Karimi-Abdolrezaee

Department of Physiology and Pathophysiology, Regenerative Medicine Program, Spinal Cord Research Centre, Children’s Hospital Research Institute of Manitoba, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

Introduction: Spinal cord injury (SCI) causes sensorimotor and autonomic impairment that partly reflects extensive, permanent loss of neurons at the epicenter and penumbra of the injury. Strategies aimed at enhancing neuronal protection are critical to attenuate neurodegeneration and improve neurological recovery after SCI. In rat SCI, we previously discovered that tissue levels of neuregulin-1beta 1 (Nrg-1β1) is acutely and persistently downregulated in the injured spinal cord, and its decline contributes to the dysregulated, pro-inflammatory milieu of injury. We demonstrated restoring the declined levels of Nrg-1β1 fosters oligodendrogenesis and promotes an anti-inflammatory, pro-regenerative response in resident glia and infiltrating leukocytes, which culminates in improved recovery of function after SCI. Nrg-1β1 is well-known for its critical roles in the development, maintenance and physiology of neurons and glia in the developing and adult spinal cord. However, despite this pivotal role, Nrg-1β1 specific effects and mechanisms of action on neuronal injury remain largely unknown in SCI.

Methods: In the present study, we used a clinically-relevant model of compressive/contusive SCI in rats and assessed the effects of Nrg-1β1 treatment on neurons and oligodendrocytes in acute (3 days post injury) and subacute (7 days post injury) stages of SCI. In line with in vivo studies, we have studied the direct effects of Nrg-1β1 on neurons in vitro in a model of glutamate excitotoxicity in primary cortical neurons. Excess glutamate is a major inducer of cell death after SCI. Neurons were exposed to glutamate (10 µM) for 1 hour in culture. Then, they were treated with Nrg-1β1 (10 and 50 ng/ml) for 8 hours.

Results: we demonstrate Nrg-1β1 provides early neuroprotection through attenuation of reactive oxygen species, lipid peroxidation, necrosis and apoptosis in acute and subacute stages of SCI. Mechanistically, availability of Nrg-1β1 following glutamate challenge protects neurons from caspase-dependent and independent cell death that is mediated by modulation of mitochondria associated apoptotic cascades and MAP kinase and AKT signaling pathways.

Conclusion: Altogether, our work provides novel insights into the role and mechanisms of Nrg-1β1 in neuronal injury after SCI, and introduces its potential as a new neuroprotective target for this debilitating neurological condition.

Assessing the Cross Neutralization of mumps antibody between different genotypes

Saba Shaikh1, Jasmine Frost1, Elizabeth McLachlan2, Alberto Severini1, 2

1Department of Medical Microbiology and Infectious Disease, College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada 2Department of Viral Exanthemata and Sexually Transmitted Disease, National Microbiology Lab, Winnipeg, MB, Canada

Introduction: Several mumps outbreak have been reported with 24 mumps outbreaks across nine provinces across Canada between January 1, 2016 and July 31, 2018 with majority of the cases in adolescents and adults between the ages of 15 to 39 years. The outbreaks observed were due to genotype G whereas the vaccine strain is of genotype A. Mumps (MuV) is a paramyxovirus belonging to the Rubalavirus family, with a lipid envelope and a non-segmented, negative strand RNA genome of 15.3 kb. The virus encodes for seven different proteins including the two glycoprotein Hemagglutinin (HN) and the Fusion (F) responsible for attachment and fusion of the viral envelope to the target cell membrane. The glycoproteins are also an important target for the immune system to produce neutralizing antibodies against, which are believed to be protective against infection. Mumps infections in vaccinated individuals could be due to waning immunity or antigenic differences between genotype A and genotype G. The antigenic differences in the glycoproteins could result in reduced recognition of vaccine induced antibodies to the genotype G mumps virus leaving the population susceptible to mumps infection. Therefore, it is important to understand whether the vaccine induced antibodies are protective against genotype G infection.

Methods: Stable cell lines expressing the F and the HN protein from genotype A and genotype G will be created using lentivirus transduction in Mel-Juso cell line. Western blot paired with Fluorescent associated cell sorting (FACS) will be used to assess the success of transduction using high titer patient sera. Cross reactivity of antibodies between genotype will be assessed by western blot, immunofluorescence and FACS analysis using patient sera from unvaccinated individual, unvaccinated individual with history of genotype G as well as vaccinated individual with genotype G infection and vaccinated individual without genotype G infection. This will allow us to assess whether antibodies specific from genotype G infection can bind to the genotype A proteins and antibodies specific from genotype A infection can bind to the genotype G protein.

Conclusion: The project will help us identify whether mumps outbreak is due to antigenic differences in the HN and the F protein the two genotypes that results in reduced antibody recognition.

Basal Cell Adhesion Molecule: A Novel Human Cytotrophoblast Progenitor Marker

Matthew J. Shannon1, Barbara Castellana1, Jennet Baltayeva1, Jasmin Wächter1, Jenna Treissman1, Jenna Treissman1, 2, Hoa Le1, 2, Ji Soo Yoon2, 3, Francis Lynn2, 3, Alexander G. Beristain1, 2

1Department of Obstetrics and Gynaecology, University of British Columbia, BC Children's Hospital Research Institute

Introduction: Establishment of the human placenta is dependent on specialized placental cells called trophoblasts. Multiple trophoblast sub-types exist to facilitate placenta-related functions and maintain maternal and fetal heath. Unfortunately, little is known about the cellular and molecular processes controlling human trophoblast stem cell maintenance and differentiation. To address this, our laboratory has established a single-cell RNA sequencing dataset from first trimester placentas to identify the molecular programs important for trophoblast stem cell establishment, renewal, and differentiation.

Methods: Transcriptomic profiles from over 50,000 first trimester human placental cells were generated using the 10X Genomics Chromium platform or collected from the ArrayExpress data repository. Data normalization, clustering, and lineage trajectory analyses were performed. 3D human trophoblast organoids were then established and expanded from trophoblast stem cells for flow cytometry, quantitative PCR (qPCR), immunofluorescence (IF), and organoid differentiation assays.

Results: We found 8 distinct trophoblast states within the first trimester placenta, representing undifferentiated trophoblasts, called cytotrophoblast (CTB; 4 states), mature hormone-producing syncytiotrophoblast (SCT; 1 state) responsible for nutrient and gas exchange, as well as highly invasive extravillous trophoblasts (EVT; 3 states) that remodel maternal vasculature and dampen the maternal immune response against the placenta/fetus. The trophoblast stem cell markers TP63, YAP1, and CDX2 were upregulated within a putative cytotrophoblast progenitor (CTBp) population that had increased expression of the cell surface glycoprotein Basal Cell Adhesion Molecule (BCAM). Lineage trajectory analyses and qPCR of human trophoblast stem cells revealed that BCAM expression decreased with trophoblast differentiation into mature SCT and EVT. IF of first trimester placental villi and trophoblast organoid cultures validated BCAM expression is specific to CTBs. Finally, organoid establishment following flow sorting of BCAM+ and BCAM- CTB populations demonstrated that BCAM+ CTBs possess greater capacity for organoid formation and growth.

Conclusion: We have identified BCAM as a novel marker of first trimester human cytotrophoblast progenitor cells, providing improved methods for isolating and studying the mechanisms regulating human trophoblast stem cells, human trophoblast differentiation, and healthy placental development, all critical factors for ensuring a successful pregnancy.

“Making Sense of Food Allergies”: An integrated knowledge translation approach to mobilizing health research findings

Emily Shantz1 & Susan J. Elliott1

1Department of Geography & Environmental Management, University of Waterloo, Waterloo, ON

Introduction: Food allergy is a serious health condition characterized by onset of allergic reactions following ingestion of a food allergen. Severe reactions can lead to anaphylaxis and even death. A national study reported that 50% of Canadians are affected, either directly or indirectly (i.e. non-allergic, but prepares/consumes food in an allergen-controlled environment), by food allergies (Harrington et al., 2012). While many resources exist for those directly affected, a gap remains for those indirectly affected.

Methods: In 2015, the UK charity Sense About Science published 'Making Sense of Allergies' (Sense About Science, 2015). A Canadian version was developed focusing on food allergies. Current scientific research and Canadian guidelines were reviewed and synthesized. An integrated knowledge translation (iKT) approach was used in which knowledge users such as scientists, allergists, and patient support organizations (e.g., Food Allergy Canada) were involved throughout the research process, reviewed content, and contributed recommendations at key stages. Professional graphic design services were engaged to ensure the document was attractive, user-friendly, and accessible to a variety of audiences.

Results: 'Making Sense of Food Allergies', a publicly-available evidence-informed resource that provides information and is designed to dispel common myths about food allergies, was developed. Stakeholders identified a need to target content towards those indirectly affected, and advised on adapting scientific concepts and language for intended end-users. Topics include the science, prevention, diagnosis, and management of food allergies and allergic reactions. The document will undergo extensive public release in Spring 2021. To reach intended knowledge users, knowledge brokering activities will promote dissemination through partner networks and a targeted social media strategy will be employed.

Conclusions: This resource can be used by those directly or indirectly affected by food allergies, such as individuals with food allergies, their family and friends, community members, policymakers, and health professionals. Providing educational tools to the general public has the potential to contribute substantially to quality of life of allergic individuals and their families, ultimately leading to improved food allergy management policy and practice in Canada.

Assessing Neuropsychological Outcomes Amongst Youth with Persistent Post-Concussion Symptoms in Comparison to a Youth Anxiety Control Group

Elena Sheldrake1,2, Nick Reed1, Shannon E. Scratch1,2

1Rehabilitation Sciences Institute, University of Toronto 2Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital

Introduction: Pediatric concussion is a serious public health concern. Although most children recover quickly, approximately 30% of children experience prolonged symptoms for more than 4 weeks (persistent post-concussion symptoms, PPCS). While the symptoms are heterogeneous, PPCS are strongly correlated to difficulty in recovery, especially in behavioural and emotional domains, and it is challenging for clinicians to predict which youth are more likely to experience PPCS following concussion. Individuals with PPCS often report emotional changes following injury, including irritability and aggression, as well as mental health impacts such as anxiety and depression. This is extremely troubling in child and adolescent populations, who are at a vulnerable developmental age, and can have lasting impacts on many facets of their lives including academics and social relationships. This project objective is to investigate brain function through assessments for behavioural and emotional responses with PPCS and with anxiety to understand their psychological profiles and well-being

Methods: A participant group of youth diagnosed with PPCS will be compared to a control group of youth diagnosed with generalized anxiety disorder, both groups aged 12-18 years (age and sex matched controls,) with a sample size of 15-20 each, undergoing parallel neuropsychological and observational assessments. The neuropsychological test battery will measure PCSS characterization and symptom profile, emotional and behavioural responses, as well as anxiety characterization. Neuropsychological analysis will entail multivariate, partial regressive modelling using pipelines to examine differences in neuropsychological outcomes across concussion and control groups, controlling for important covariates (e.g. gender, IQ, age, socioeconomic status, concussion history, medical history, developmental history).

Results: Recruitment and data collection is to commence in summer 2021, and preliminary findings will be shared once completed.

Conclusion: Project outcomes will foster discussions within and beyond the research environment by targeting those who are at greatest risk for psychological morbidity to manage the aftermath of concussions and impacts on mental health and wellbeing, as well as clarify ambiguities between PPCS and mental health symptom interconnection through diagnostic assessments.

Trends of Utilization of Antiepileptic Drugs in Pregnancy in Manitoba, Canada A 20-year utilization study

Walid Shouman1, Joseph Delaney1, Alekhya Lavu1, Sherif Eltonsy1,2

1College of pharmacy Rady Faculty of Health Science, University of Manitoba 2The Children's Hospital Research Institute of Manitoba, Canada

Introduction: Studies in Canada and around the world have shown an increase in the utilization of antiepileptic drugs (AEDs) in the general population. Both epilepsy and AEDs could affect healthy pregnancies and lead to adverse neonatal outcomes.

Objective: Examine time-trends of utilization of AED therapies among pregnant women in Manitoba, Canada. Method: We conducted a population-based cohort study using administrative health databases from the province of Manitoba. Pregnancies of women living in Manitoba between 1995 and 2018 were included. Utilization was identified by Anatomical Therapeutic Chemical codes. Four groups of pregnant women were created based on AED exposure and epilepsy diagnosis. A woman was considered to have epilepsy if she has 1 or more medical claims or 1 or more hospitalization for epilepsy during the 5 years prior to delivery. Utilization trends of AEDs were examined using linear regression.

Results: Out of 273,492 pregnancies identified, 812 (0.3%) had epilepsy diagnosis and were exposed to AEDs, 963 (0.35%) had an epilepsy diagnosis and were unexposed, and 2742 (1%) were exposed to AEDs and did not have epilepsy diagnosis. Overall, the number of pregnancies exposed to AEDs increased significantly from 0.56% in 1997 to 2.21% in 2018 (p<0.0001). The percentage of pregnant women with epilepsy exposed to AEDs did not change significantly from 0.37% in 1997 to 0.36% in 2018 (p=0.2354). Whereas the percentage of pregnant woman without epilepsy exposed to AEDs increased significantly from 0.19% in 1997 to 1.85% in 2018 (p<0.0001). In the total cohort of pregnancies, 1439 (0.53%) were exposed during the whole pregnancy, 1369 (0.5%) were exposed in the first trimester, 63 (0.02%) were exposed in the second trimester, and 184 (0.07%) were exposed in the third trimester. Clonazepam was the most commonly used AED during the study period (1953 users, 0.71%), followed by gabapentin (785 users, 0.29%) and carbamazepine (449 users, 0.16%).

Conclusion: No major shifts in the use of AEDs were observed among women with epilepsy. Concerns about the increase use of AEDs driven by indications other than epilepsy require additional research on the safety of these agents and their indications to inform prescribers and policymakers.

Cervicovaginal microbial profiling using the cpn60 universal target reveals important associations with the mucosal immune milieu

Elinor Shvartsman1,2, Catia T Perciani3, Meika Richmond1,2, Justen NH Russell2,3, Sarah J Vancuren4, Janet E Hill4, KAVI-ICR5, Lyle R McKinnon1,2, Paul Sandstrom1,2, and Kelly S MacDonald1,2,3,6

1Department of Medical Microbiology and Infectious Disease, University of Manitoba, Winnipeg, Manitoba, Canada 2JC Wilt Infectious Diseases Research Centre, Winnipeg, Manitoba, Canada 3Department of Immunology, University of Toronto, Toronto, Ontario, Canada 4Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 5Kenyan AIDS Vaccine Initiative- Institute of Clinical Research (KAVI-ICR), University of Nairobi, Nairobi, Kenya 6Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Introduction: Bacterial vaginosis (BV) is associated with negative reproductive health outcomes, including increased risk to HIV, possibly by altering the mucosal immune milieu. Gardnerella bacteria are thought to play a role in the development of BV, however, the impact that the different Gardnerella species have on cervicovaginal immunity remains largely uncharacterized. Here, we used cpn60 microbial profiling to resolve the different Gardnerella subgroups, and examined the associations between cervicovaginal microbial communities and markers of inflammation, cell recruitment, and immune activation in a longitudinal cohort of reproductive age Kenyan women.

Methods: Cervicovaginal secretions and cervical cytobrush samples from participants enrolled in the longitudinal KAVI-VZV- 001 clinical trial were used in this ancillary study. Cervicovaginal secretions were used for microbial profiling using the cpn60 universal target and cytokine quantification using the MSD-UPLEX platform. Flow cytometry of cytobrush samples was used to measure the frequency of cellular markers of immune activation, integrins, or HIV receptor/co-receptor expression on cervical T-cells.

Results: We resolved ten cervicovaginal microbial communities, three of which were dominated by different Gardnerella subgroups, as well as one polymicrobial community. Gardnerella subgroup D was mostly associated with polymicrobial communities. Using Lactobacillus (non-iners) dominant microbial communities as reference, we found that Gardnerella dominated microbial communities and polymicrobial communities were associated with increased levels of the cytokines IL-1β, IFN-γ, TNF-α, and IL-17 (P<0.05). Similar associations were also found with IL-1α and IL-6 although those varied in significance. Divergent associations were discovered in Gardnerella dominated community associations with the chemokine IP-10.

Specifically, Gardnerella subgroup A dominance and polymicrobial communities were associated with reduced concentrations of IP-10 in both unadjusted and adjusted models (P<0.0001), whereas Gardnerella subgroup C dominance was associated with a relative increase in IP-10, although this did not reach statistical significance. Generally, these associations did not translate to significant differences in the relative frequencies of cellular markers of immune activation or CCR5 expression on T-cells.

Conclusion: We found important associations between microbial composition (focusing on Gardnerella diversity) and mucosal cytokines and chemokines. These findings may have important implications in elucidating the role different Gardnerella species play in BV and the BV-HIV link.

Suicidal thoughts and behaviors in early psychosis: what do we know and what do we need to know?

Roxanne Sicotte1,2, Srividya N. Iyer4,5*, Barnabé Kiepura2, Amal Abdel-Baki1,2,3*

*SNI and AAB are co-senior authors and contributed equally to this work. 1Research Center Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada 2Department of Psychiatry and Addiction, Faculty of Medicine, Montréal, Québec, Canada 3Clinique JAP, Centre hospitalier de l’Université de Montréal, Montréal, Québec, Canada 4Department of Psychiatry, McGill University, Montréal, Québec, Canada 5Prevention and Early Intervention Program for Psychosis (PEPP), Douglas Mental Health University Institute, Montréal, Québec, Canada

Introduction: People with psychotic disorders are at high risk for suicide, especially in the early stages of the illness. A better understanding of the evolution of suicidal thoughts and behaviors (STBs) and factors associated therewith could help to assess the suicidal risk of patients with first-episode psychosis (FEP) more accurately, and to address modifiable factors to mitigate their effect and thus reduce the risk of suicide.

Objectives: Describe the evolution of STBs in persons with FEP, identify factors associated with suicidal ideation, suicidal attempts and death by suicide in FEP patients and identify critical gaps that remained to be filled in this field of research.

Method: A systematic review was conducted according to PRISMA guidelines. Relevant studies were identified by searching PubMed, Medline, PsycINFO, Embase, EBM Reviews, and references lists of relevant articles. Screening of articles, data extraction and quality assessment were done independently by two reviewers.

Results: Of 3,177 references, 17 studies of 11 non-overlapping samples (N=14, 907) with varying lengths of follow-up (1- 41.7 years) were included. The prevalence of STBs decreased over follow-up, but up to 21.6% made at least one suicide attempt, 27% had suicidal ideation, and between 1% and 4.3% died by suicide during follow-up. Fifty-three factors were assessed across studies. Only male sex, depressive symptoms, and STBs occurring early during follow-up emerged as factors associated with subsequent STBs. Early intervention for psychosis decreased STBs in the first three years following receipt. Other factors were assessed in a single study, yielded conflicting results, or were not associated with STBs. Informed by our review, several factors that have been associated with STBs in the general population have not been studied in people with FEP and could help to better target those at risk.

Discussion: The high prevalence of STBs following onset of psychosis highlights the need for early detection and intervention in FEP and for ongoing assessment of suicidal risk throughout follow-up, with attention to identified risk factors. Overall, the literature base is limited albeit of acceptable quality. Thus, additional well-designed longitudinal studies including all factors that may influence both suicidal ideation and behaviors are needed.

Studying the effects of short-chain fatty acids on cervicovaginal epithelial function

Abu Bakar Siddik1, Vanessa Poliquin2, Jennifer Chan3, Robert John Lotocki2,4, Alon Altman2,4, Alberto Severini1,5,6, T. Blake Ball1,6, Ruey-Chyi Su1,6

1University of Manitoba, Medical Microbiology & Infectious Diseases 2University of Manitoba, Department of Obstetrics, Gynecology & Reproductive Sciences 3Women's Outpatient Dept, Women's Hospital 4CancerCare Manitoba 5Virial exanthemata and STD, National Microbiology Laboratories, Winnipeg, Manitoba 6JC Wilt Infectious Diseases Research Centre, Winnipeg, Manitoba

Short-chain fatty acids (SCFAs) are microbial derived metabolites produced in the gut. Besides being used as an energy source by colonocytes, SCFAs also enter the blood circulation. Recently, SCFAs have been shown to negatively regulate inflammation. However, most studies focused on the gut epithelium. Excessive immune activation has been associated with an increased risk of acquiring HPV and HIV-1 infection. As vaginal mucosa is the primary site of sexually transmitted infections, we were curious whether SCFAs were present at the vaginal mucosa and if SCFAs had a role in regulating the function and property of the vaginal epithelium, as the first line of defense against infection. This study assessed the levels of SCFAs and inflammatory cytokine/chemokine in cervicovaginal fluid (CVF) of healthy and HPV infected Manitoban female participants to determine the relationship between the two. In addition, this study examined the direct effects of SCFAs on epithelial barrier integrity.

CVF samples from healthy women (n=31) and HPV-infected women with low-grade squamous epithelial lesions (LSIL) (n=16) were analyzed for SCFAs and 18 inflammatory cytokine/chemokine using liquid-chromatography mass spectrometry and multiplex-protein assay, respectively. HPV screening was done by detecting HPV DNA in the vaginal swab and fluid samples.

The level of SCFAs in the healthy women's CVF was: acetate (0.16-2.8mM), butyrate (0.35-41.9μM), propionate (0.23- 67.0μM. There was no significant correlation between the 18 proinflammatory cytokines/chemokines that were examined and these SCFAs level. To our surprise, the levels of proinflammatory cytokine/chemokine were not significantly different between the healthy and HPV-infected women with LSIL. We further found that treating polarized epithelial cell lines derived from cervicovaginal mucosa with SCFAs resulted in decreased epithelial integrity, measured with transepithelial electrical resistance.

In summary, SCFAs may have no effects on inflammation at the vaginal mucosal but could alter the integrity of vaginal epithelium.

Thiopurines activate an antiviral unfolded protein response that blocks influenza A virus glycoprotein accumulation

Patrick Slaine1, Mariel Kleer2, Brett Duguay1, Eric Pringle1, Eileigh Kadijk1, Shan Ying1, Aruna Balgi3, Michel Roberge3, Craig McCormick1, Denys Khaperskyy1

1Dalhousie University 2University of Calgary 3University of British Columbia

Introduction: Influenza A viruses (IAVs) use host cell machinery to synthesize viral proteins. Most viral mRNAs are translated by free ribosomes in the cytoplasm, whereas mRNAs encoding the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA) are translated in the endoplasmic reticulum (ER) and traffic to the cell surface to participate in viral assembly and egress. I study two candidate antiviral thiopurines 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo) that impede viral glycoprotein synthesis by activating the cellular unfolded protein response (UPR) that responds to the accumulation of misfolded proteins in the ER. 6-TG and 6-TGo are currently FDA approve compounds that I am looking to re-purpose as an antiviral against IAV infection.

Methods: Human lung adenocarcinoma A549 cells were infected with IAV (A/Puerto Rico/08/1934 (H1N1)) and treated with 6-TG and 6-TGo to determine effects on viral protein synthesis and replication. Infectious virions were enumerated by plaque assay. Viral proteins were analyzed by immunoblot after collecting whole cell lysate from infected cells. Viral mRNA synthesis and genome replication were measured by RT-qPCR from total RNA harvested from infected and treated cells. UPR activation was analyzed by immunoblot and RT-qPCR. Host responses were manipulated with chemical inhibitors and by CRISPR knockout of the UPR sensor kinase PERK.

Results: Treating infected cells with 6-TG and 6-TGo reduced viral titers by ~100-fold. While these thiopurines did not affect viral mRNA synthesis and genomic replication, we observed a marked decrease in viral glycoprotein processing and accumulation that correlated with activation of the UPR. UPR modulation via chemical chaperones partially restored viral glycoprotein processing and accumulation. PERK knockout or chemical inhibition of PERK-dependent downstream responses restored synthesis of viral proteins but did not rescue viral titers, as the viral glycoproteins were not properly glycosylated.

Conclusions: We demonstrate for the first time that 6-TG and 6-TGo activate the UPR, and this response inhibits IAV replication. This provides a new conceptual framework for host targeted antivirals and identifies the UPR as a potential target for host targeted antivirals.

Establishing a prion infection model with neural progenitor derived cultures that overexpress cellular prion protein

Jessy Slota1,2 and Dr. Stephanie Booth1,2

1Department of Medical Microbiology and Infectious Diseases, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada 2Prion Diseases Section, National Microbiology Laboratory, Winnipeg, MB, Canada

Introduction: Prion diseases are fatal, progressive neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrPC) into a disease-associated conformation (PrPSc). Accumulation of PrPSc in the brain is associated with astrocytic gliosis concomitant with neuronal dysfunction and death, leading to degeneration of neuronal tissues and rapid neurocognitive decline. Structural differences between distinct PrPSc isolates are thought to encrypt the pathological features of the ensuing disease. However, the relationship between the biophysical and pathophysiological properties of prions remain poorly characterized due to a lack of available model systems.

Methods: Here we have attempted to establish a model of prion infection using human neural progenitor stem cells (ReN VM cells). ReN cells can be proliferated as neural progenitors and then differentiated into cultures of neuronal and glial cell types upon growth factor removal. Lentiviral vectors were used to overexpress different versions PrPC in this cell culture system, which was confirmed using western blotting. Following lentiviral transduction, immunofluorescence was used to confirm that the cells appropriately differentiate into neuronal cell types by staining for beta-III-tubulin (TUBB3) and glial cell types by staining for glial fibrillary acidic protein (GFAP). Mouse-PrPC overexpressing ReN cells were inoculated with Rocky Mountain Laboratory mouse-adapted scrapie (RML) and maintained for up to 4 weeks post infection. Real-time quaking induced conversion (RT-QuIC) was used to detect and quantify PrPSc that had replicated in the cultures.

Results: Lentiviral transduction followed by antibiotic selection resulted in human neural progenitor cell cultures that overexpress human, mouse and hamster versions of PrPC. Despite overexpression of PrPC, the neural progenitors still appropriately differentiated into neuronal and glial cell types. PrPSc was detected using RT-QuIC in neural progenitor derived cultures at multiple timepoints post infection and using multiple formats for the infection model.

Conclusions: PrPC overexpressing human neural progenitor cells were effectively obtained using lentiviral transduction. Efforts towards optimizing a novel model system of prion infection using these cells are still ongoing. If successful, this system could be used for studying prion species barriers, examining the effects of PrPC variants on prion replication and for characterizing human clinical disease isolates.

AMP-activated protein kinase requires myristoylation to regulate cellular metabolism

Tyler K.T. Smith1, Bruce E. Kemp2, Morgan D. Fullerton1

1Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada 2St. Vincent’s Institute of Medical Research and Department of Medicine, University of Melbourne, Fitzroy, Australia

Introduction: Cardiovascular disease is the leading cause of death worldwide. Once considered a lipid disorder alone, cardiovascular disease is increasingly recognized as a disorder of inflammation resulting from dysregulated metabolism. AMP-activated protein kinase (AMPK) is a conserved heterotrimeric energy sensor that broadly signals to pathways that support catabolism and oppose anabolism. For this reason, AMPK has been an attractive therapeutic target for cardiovascular diseases. However, given the multitude of co- and post-translational modifications of AMPK, its biological role in disease remains an important area of research. In this study, we investigate the biochemical implications of a co- translational myristoylation event on the AMPKβ subunit. Myristoylation is a covalent linkage of the fatty acid myristate to glycine residues, usually resulting in increased membrane association or regulation of protein activity.

Methods: Using mouse embryonic fibroblasts (MEFs) and mice that harbour a glycine-to-alanine knock-in (KI) mutation at the AMPK myristoylation site (G2A KI), we report here that myristoylation confers an additional layer of regulation to AMPK by preventing ubiquitination and degradation of AMPK subunits.

Results: Compared to WT cells, G2A KI MEFs had reduced AMPK subunit protein levels independent of changes in mRNA. Pharmacological inhibition of myristoylation using an N-myristoyltransferase inhibitor similarly decreased AMPK subunit protein levels in WT, but not G2A KI MEFs. This decrease in protein levels was not due to altered heterotrimer formation, as WT and G2A KI MEFs had the same subunit stoichiometries following endogenous immunoprecipitation. We traced the decrease in stability to elevated subunit ubiquitination using Tandem Ubiquitin Binding Entity (TUBE) pulldown experiments, although the specific subunit and residues affected are still unknown.

Conclusion: Ongoing and future work will focus on elucidating the machinery responsible for myristoylation-dependent AMPK ubiquitination. These are the beginnings of a project that will map how AMPK myristoylation affects immune cell metabolism in response to inflammatory stimuli. This will be further applied to atherosclerosis, where we will measure atherosclerotic plaque development in WT and G2A KI mice fed a western diet. Taken together, this work identifies a new AMPK protein stability-based role for myristoylation that may be important in an inflammatory disease such as atherosclerosis.

Modeling the Risk of Major Osteoporotic Fracture: A Comparison amongst Multi-State, Sub- Distributional and Cox Proportional Hazard Models

Shamsia Sobhan1, Shuman Yang1, 2, 3, Lin Yan1, William D. Leslie3, Lisa M. Lix1

1Department of Community Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada 2Department of Epidemiology and Biostatistics, School of Public Health, Jilin University, Changchun, Jilin, China 3Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada

Introduction: In many epidemiological follow-up studies, participants can experience multiple types of events. For example, in studies about major osteoporotic fracture (MOF), a common condition amongst older adults, interest lies in estimating the risk of fracture; however, death is a competing event that is often ignored. Conventional time-to-event models do not capture the dependence amongst health states (e.g., fracture and death), which could result in biased risk estimates. Multi- state (MS) models, an extension of survival models, account for this dependence, but are rarely used for estimating fracture risk. Our objective was to compare the conventional Cox proportional hazards (PH), sub-distributional hazard (SDH) competing risks, and illness-death MS models for estimating MOF risk.

Methods: Our study cohort included 61,393 individuals 50+ years from the Manitoba Bone Mineral Density Database. MOF (hip, wrist, humerus, spine) were identified from diagnosis codes in linked administrative data, as well as death. Risk estimates for demographic, comorbid, and clinical characteristics were produced using univariate PH, SDH and MS models. Proportionality and Markov assumptions were assessed for PH and MS models. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated.

Results: Overall, 9.6% of cohort members had an MOF, 2.5% died after an MOF, and 12.4% died without having an MOF. HRs for most risk factors were similar for the three models. However, there were substantial differences in the effect of age (ref: <70 years) on MOF risk for PH (HR70-79 years 2.04 [95% CI 1.93, 2.16]; HR80+ years 3.68 [95% CI 3.43, 3.94]), SDH (HR70-79 years 1.87 [95% CI 1.76, 1.98]; HR80+ years 2.77 [2.59, 2.97]) and MS (HR70-79 years 1.59 [95% CI 1.51, 1.68]; HR80+ years 2.81 [95% CI 2.63, 2.99]) models. The MS model also produced estimates of the effect of age on death after fracture (HR70-79 years 1.37 [95% CI 1.24, 1.52]; HR80+ years 3.61 [95% CI 3.25, 4.01]).

Conclusion: The MS model has potential benefits over the PH and SDH models to produce unbiased risk estimates while accounting for dependence amongst health states. Future research will investigate how the amount of bias varies with the magnitude of dependence amongst health states.

Implementing a Workplace Violence Reporting System for Nurses in a Healthcare Setting in Pakistan

Somani R.1, 3 , Muntaner C.1, 3 , Hillan E.1 , Velonis J. Alisa.2 Smith P.1 ,3, 4

1University of Toronto, Canada 2University of Illinois Chicago, USA 3Dalla Lana School of Public Health, University of Toronto, Canada 4Institute for Work & Health, Toronto

Introduction: Workplace violence (WPV) is a serious occupational problem in any society. The magnitude of WPV is high in hospitals, due to a stressful environment and the nature of the work. Nurses are prone to WPV as they work closely with patients and their family members. Implementing interventions to reduce WPV have remained challenging for healthcare organizations due to the under reporting of incidents of WPV. An easy and simple WPV reporting system within the hospital is the key to encouraging nurses to report incidents of violence.

Research Question: What is the process of utilizing an implementation science framework to establish a WPV reporting system for nurses in a healthcare setting in Karachi, Pakistan?

Methodology: This study will follow the implementation science approach. To achieve this purpose, the study will utilize a qualitative exploratory design for the data collection. This study will be guided by the Active Implementation Frameworks (AIFs), which is recommended by National Implementation Research Network (2005). Implementation stages include in this study are: (a) Exploration, (b) Installation, (c) Initial implementation, (d) Full Implementation. Online In-depth Interviews (IDIs) will be conducted with nurses and nursing supervisors during the exploration phase. The interventions that will be utilized in this study are: (a) The introduction of the Violence Incident Form (VIF), by Arnetz, 1998, and (b) Trainings (champions, nurses, supervisors). After the initial implementation IDIS will be conducted with nurses, nursing supervisors and hospital administrators.

Outcomes: The outcome measures in this study are (a) Acceptability: Perceived effectiveness of the implemented WPV reporting system. Stakeholders’ perception that the implemented intervention is agreeable (b) Adoption: Stakeholders’ willingness for adapting the intervention as a routine practice, (c) Sustainability: Key stakeholder’s perceptions of the sustainability of the implemented intervention

Conclusion: Interventions to reduce WPV will only be achieved if hospital management is aware of the severity of the issue and are involved in creating a violence-free environment for healthcare providers. Overall, a safe work environment encourages nurses to remain in the nursing profession and provide quality care to patients, which will lead to a positive impact on health within society.

Ketone Therapy as a Novel Approach for the Treatment of Heart Failure

Shubham Soni1,2, Nikole J. Byrne1,2, Shingo Takahara1,3, Mourad Ferdaoussi1, Rami A. Batran1,4, Ahmed M. Darwesh4, Jody L Levasseur1, Zaid H. Maayah1,2, John M. Seubert1,4, John R. Ussher1,4, Jason R.B. Dyck1,2

1Cardiovascular Research Centre, University of Alberta, Edmonton, Alberta, Canada 2Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada 3Division of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan 4Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

Introduction: The failing heart has metabolic defects which impairs energy production and contractile function. Recent studies show that the failing heart upregulates ketone metabolism and may rely on ketones as a fuel. Ketones, namely β- hydroxybutyrate (βOHB), are produced by the liver during low-carbohydrate conditions, such as fasting. While βOHB may be beneficial in heart failure (HF) because it is an energy-efficient substrate, it may also be of benefit by acting as a signaling molecule. However, whether chronic elevations in circulating ketones are beneficial remains unknown. Thus, regardless of the role that βOHB plays in HF development, elevating circulating βOHB levels beyond what is normally observed in HF may serve as a novel therapy to improve cardiovascular outcomes in patients with HF.

Methods: To determine whether elevating circulating ketones may improve cardiac function in HF, we utilized an inducible skeletal muscle-specific knockout of succinyl-CoA:3-ketoacid-CoA transferase (SCOT-KO), the rate limiting enzyme in ketone catabolism. We then subjected SCOT-KO and wildtype littermate mice to transverse aortic constriction (TAC) surgery to generate pressure overload-induced HF. Heart function (echocardiography), protein (immunoblot, immunohistochemistry) and gene expression (quantitative-PCR), and blood metabolites (βOHB, glucose) were analyzed.

Results: SCOT expression was significantly reduced in skeletal muscle of SCOT-KO compared to wildtype mice without changes in cardiac SCOT expression. Given that ketones are largely utilized in skeletal muscle, the SCOT-KO resulted in a 1.6-fold increase in fasted circulating βOHB compared to wildtype mice. Interestingly, this rise in circulating βOHB was associated with protection from TAC-induced cardiac remodelling (hypertrophy, fibrosis) and decline in systolic (49.8% vs 35.4% ejection fraction) and diastolic (E/A, E/E’) function in SCOT-KO compared to wildtype littermates. These improvements occurred without changes in cardiac ketolytic enzymes, suggesting metabolism-independent pathways. Further analysis revealed that SCOT-KO mouse hearts had lower inflammation (IL-6, IL-18, CX3CL1) and macrophage infiltration (galectin-3, F4/80).

Conclusion: Together, these data are the first to show that elevating circulating ketones, via a skeletal muscle SCOT-KO, prevents cardiac dysfunction, cardiac inflammation, and macrophage infiltration in mice with HF. Thus, elevating circulating ketones in HF shows promise as a novel therapeutic approach in the management and treatment of HF.

Chromatin dynamics and transcription factor networks driving hepatic fate in iPSC derived hepatocytes

Tabea L Stephan1, 2, Rebecca Cullum2, Sibyl Drissler1, 2, Stephen Duncan3, Pamela A Hoodless1, 2

1University of British Colubia 2BC Cancer Research Institute 3Medical University of South Carolina

Introduction: How the hepatocyte transcriptome is established during development, including how the underlying chromatin dynamics control cell identity and function is unclear. While several transcription factors (TFs) for aspects of liver development have been identified in mouse models, chromatin dynamics and regulating TFs in human cells are not well studied and the lack of consistency and functionality of iPSC derived mature hepatocytes suggest a knowledge gap. Here, we describe the changing epigenome during in vitro hepatocyte differentiation and identify missing links in the transcription factor network.

Methods: We are using in vitro differentiation of iPSCs to hepatic progenitors as a model of liver cell differentiation. By mapping post-translational histone tail modifications across the genome at several timepoints we developed a comprehensive map of enhancers and promoters and their activation status during hepatic cell differentiation. We are then using TF motif enrichment analysis to identify factors directing these dynamics. We further developed an inducible knockdown system to remove TFs of interest during hepatic differentiation. Currently, we are investigating the role of TBX3 on the specification of the hepatic chromatin.

Results: We found a highly dynamic epigenome with enhancers being activated and shutdown rapidly. We identified a small fraction of enhancers which are primed for activation in definitive endoderm and which are not shutdown but activated during further differentiation towards hepatocytes. This small group of driver enhancers appears crucial for hepatic cell fate as it includes enhancer for master regulators of hepatocyte cell fate as HNF4a and CEBPa. Our analysis suggests TBX3 as a potential regulator of these first hepatic-specific activated enhancers. Effectively specifying the hepatic lineage from other endodermal lineages through precise enhancer activation would add a new role to the importance of TBX3 during hepatic development.

Conclusion: A better understanding of human liver development and the underlying changes in the chromatin regulation will aid in the development of efficient methods to generate in vitro derived mature hepatocytes to potentially treat end- stage liver diseases. In addition, the experiments outlined here will also be of value for research into the regulation of chromatin during liver regeneration and oncogenesis.

Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci

Deepthi Sudarshan1, Nikita Avvakumov1, Marie-Eve Lalonde1, Nader Alerasool2, Karine Jacquet1

1St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec- Université Laval Research Center, Laval University Cancer Research Center, Quebec City, QC, Canada 2Donnelly Centre for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

Introduction: Chromosomal translocations are often drivers of cancer by producing gain-of-function fusion proteins. These events are particularly interesting in the case of genes coding for epigenetic factors. Recent genomic studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESS) and ossifying fibromyxoid tumors (OFMT) leading to an in-frame fusion of PHF1 (PCL1), a protein associated with the Polycomb repressive complex 2 (PRC2) complex, to four different subunits of the NuA4/TIP60 complex (EPC1/2, P400, MEAF6, BRD8). In contrast to the histone H4/H2A acetylation and H2A.Z loading activities of NuA4, PRC2 is linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27 (H3K27).

Method: We created isogenic cells expressing the EPC1-PHF1 fusion and fusion partners and performed biochemistry and functional genomics.

Results: In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks throughout the genome, linked to aberrant gene expression, in particular over an entire topologically associated domain that encompasses part of the HOXD cluster. Furthermore, we show that JAZF1, implicated with PRC2 components in the most frequent translocations in ESS, is a potent transcription activator that physically interacts with NuA4/TIP60.

Conclusion: Altogether, these results suggest that most chromosomal translocations linked to Endometrial Stromal Sarcomas employ the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes leading to mislocalization of histone marks and aberrant polycomb target gene expression.

Characterizing the Relationship Between SUMOylation and TDP-43 in the context of Amyotrophic Lateral Sclerosis

Terry R. Suk1-3, and Maxime W. C. Rousseaux1-4

1Department of Cellular and Molecular Medicine, University of Ottawa 2uOttawa Brain and Mind Research Institute 3Eric Poulin Center for Neuromuscular Disease 4Ottawa Institute for Systems Biology

Introduction: TDP-43 is an essential DNA/RNA binding protein at the center of Amyotrophic Lateral Sclerosis (ALS). Although mutations in TDP-43 are a primary cause of only around 4% of familial ALS cases, nearly all cases of ALS present with the nuclear to cytoplasmic mislocalization and aggregation of TDP-43. Therefore, identifying mechanisms regulating TDP-43 that may be disrupted in ALS is critical to identify novel therapeutic inroads broadly applicable for the majority of ALS patients. Few studies have suggested a link between TDP-43 and SUMOylation, an evolutionarily conserved and essential post translational modification that regulates a variety of protein functions. However, characterizing natively SUMOylated proteins in the central nervous system remain challenging due to limited tools to study this modification.

Methods: To characterize proteins natively SUMOylated in the mouse brain we generated a novel HA-Sumo2 knock-in mouse line. We immunoprecipitated SUMOylated proteins from mouse brain lysate of HA-Sumo1 and HA-Sumo2 mice and identified substrates differentially targeted in the mouse brain. In human cell lines, we used epitope-tag immunoprecipitation assays to characterize interactions with SUMO2.

Results: We identified Tdp-43 as a potential target of SUMOylation specifically by Sumo2 in the mouse brain. Co- immunofluorescence analysis revealed co-expression throughout the brain. Using cell-based assays we validated an interaction between TDP-43 and SUMO2 suggesting a potentially conserved and ubiquitous mechanism regulating TDP-43 function.

Conclusion: We show that there is an interaction between TDP-43 and SUMO2 providing a potential mechanism required for the normal regulation of TDP-43. We are currently exploring how inhibiting SUMOylation of TDP-43 through genetic mutations affects the normal functions of TDP-43 and how it may correlate with ALS pathology.

The Cellular Landscape of the Retrosplenial Cortex

Kaitlin Sullivan1, Adrienne Kinman1

1University of British Columbia

Introduction: The retrosplenial cortex (RSC) is a multimodal integration center participating in many higher-order cognitive. Broadly divided into the granular RSC and dysgranular RSC, these two vast subregions appear to differ based upon coarse anatomical definitions and connectivity differences. However, the organizational principles of these regions, which underlie such a multiplicity of functionality are not well defined. As such, we are left with a vague ‘black-box’ notion of how the RSC is capable of such computational feats. To uncover this gap in current knowledge, we employed a bottom-up approach, resolving previously unknown cell types in the RSC via spatial transcriptomics, and further mapping the connectivity of these cell types. This data provides a framework for deeper investigations into how this brain region functionally contributes to behaviour.

Methods: A combination of single cell RNA sequencing (scRNA-seq) and multiplexed fluorescent in situ hybridization (mFISH) was used to resolve the spatial location and unique transcriptomic profiles of previously unresolved cell types within the RSC. Higher-order properties of these cell types, such as cell body size and projection target were identified via fluorescent Nissl stain or retrograde AAV injections in concert with mFISH.

Results: The RSC is comprised of 11 different cell types organized in a laminar fashion with layer 5 containing multiple different cell types. These cell types have unique gene expression profiles, spatial location, morphology. Our dataset suggests a unique subset of cells in layer 5 may be the main subcortical projection cells for the entire brain region.

Conclusion: The data presented provides not only a resolved map of the cell types comprising the RSC, but further counters conventional thought regarding gene expression in the cortex. Despite a consensus in the literature that gene expression changes in the cortex are broad and graded, our dataset uncovers discrete and region-specific marker gene expression that opposes this canonical view.

Evaluation of viral sensitizing antibody-drug conjugate (ADC) and oncolytic virus combination regimen in novel murine models of HER2+ cancer

Zaid Taha1, Nouf Alluqmani1, Mathieu JF Crupi1, Rozanne Arulanandam2, Jean-Simon, Diallo1

1Cancer Therapeutics, Ottawa Hospital Research Institute; Department of Biochemistry, Microbiology, Immunology, University of Ottawa 2Cancer Therapeutics, Ottawa Hospital Research Institute

Introduction: We have demonstrated that small molecules such as microtubule destabilizers (MDAs) and other novel synthetic viral sensitizers (VSes) can selectively enhance oncolytic virus (OV) activity in cancer cells. We have recently published that antibody drug conjugates (ADC) can be harnessed as a cutting-edge strategy to enable targeting of these small molecules to improve the efficacy of OVs. The human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase situated upstream of a number of survival signaling pathways. HER2 overexpression is associated with a potent and characteristically aggressive neoplastic phenotype, accounting for 20%+ of breast cancer diagnoses. Current anti-HER2 therapies include the ADC T-DM1 (Ado- trastuzumab emtansine), consisting of anti-HER2 monoclonal antibody, trastuzumab, conjugated to a potent viral- sensitizing MDA. To date, existing mouse models for the evaluation of such HER2-targeted therapeutics are either transgenic in nature, immunocompromised, or immunologically incompatible with common murine cancer cell lines. As such, no directly relevant models currently exist for the preclinical evaluation of human HER2 targeted immunotherapies.

Methods: We have generated a novel immunocompetent mouse model of human HER2 (HER2T). This model have been assessed for responsiveness to our validated combination OV/ADC strategy, as well as compatibility with multiple other HER2-targeted therapeutics.

Results: By combining our OVs with the clinically approved T-DM1 in multiple in vitro and ex vivo HER2T models, we demonstrate that our combination strategy of OV/ADC yields mechanistic synergy and improved efficacy, compared to monotherapies. We further demonstrate that our OV/ADC strategy yields potent humoral anti-tumour responses. Furthermore, we have validated our HER2T model by demonstrating functional paratope binding against HER2T in the context of NK cells as well as T-lymphocytes. Importantly, we demonstrate that our HER2T model is not significantly more immunogenic compared to parental syngeneic tumour models.

Conclusions: In sum, the combination of a viral sensitizing ADC with oncolytic rhabdovirus VSV51 yields high efficacy in vitro and in vivo. By way of our novel immunocompetent HER2T model, we are able to profile the immune impacts of our HER2-targeted treatment to better understand the mechanisms of action and present a novel murine model for the evaluation of other HER2-targeted therapies.

Clostridioides difficile on retail meat samples. A possible source of human clinical infections?

Derek Tan1,2, Michael Mulvey1,2, George Zhanel1, Denice Bay1, Richard Reid-Smith3, George Golding1,2

1University of Manitoba 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada 3Center for Food-borne, Environmental and Zoonotic Infectious Disease, Public Health Agency of Canada, Guelph, ON, Canada.

Introduction: Clostridioides difficile, is a spore forming anaerobic bacteria most commonly known as a human nosocomial pathogen. However, C. difficile spores can be found dispersed throughout the environment and can asymptomatically colonize and/or infect animals. Previous studies have shown that C. difficile spores can be isolated from commercially available beef, veal, pork, vegetables and seafood. However, a definitive link has yet to been made between food contamination and hospitalized cases. This study aims to isolate C. difficile from retail meat samples and compare them to human clinical isolates.

Methods: Frozen retail pork, beef and veal samples were obtained from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) program. Defrosted samples were massaged in PBS. The rinse was plated on C. difficile moxalactam norfloxacin (CDMN) agar and added to CDMN enrichment broth before being incubated anaerobically. Post incubation, the enrichment broth was subject to an ethanol shock treatment, centrifuged, spread on brucella blood agar and incubated. Growth on agar plates were inspected visually with suspected C. difficile colonies confirmed by multiplex PCR. Toxigenic positive isolates were molecularly characterized by ribotyping and pulsed-field gel electrophoresis (PFGE). Antibiotic susceptibility was determined by E-test strips.

Results: Overall, 10 of 644 meat samples (1.55%) tested positive for toxigenic C. difficile. Of the 644 samples: no isolates were obtained from 28 beef samples, 4 isolates were obtained from 116 veal samples and 6 isolates were isolated from 500 pork samples. All 10 isolates were A/B toxigenic positive and 2 isolates are binary toxin positive. Molecular typing by ribotyping and PFGE revealed strain types commonly found in human clinical isolates (e.g. NAP1 (rt027), NAP4 (rtNS195), and NAP11 (rt106)). The 10 isolates from meat samples were all susceptible to vancomycin, metronidazole, tigecycline, rifampin, and clindamycin, whereas the one NAP1 isolate was resistant to moxifloxacin.

Conclusion: A low percentage of retail meats are contaminated by C. difficile spores. All ribotypes and NAP types of isolated C. difficile from retail meat have been identified in human clinical isolates. Future work includes the use of WGS to genetically compare retail meat isolates from human clinical isolates.

Investigating the Mechanistic Effects of Oral Aspirin on HIV Prevention

Morgan K. Taverner, Paul Lopez, Keith R. Fowke, Thomas T. Murooka

1University of Manitoba, Department of Medical Microbiology and Infectious Diseases; 2University of Manitoba, Department of Immunology

Introduction: When used as prescribed, pre-exposure prophylaxis with anti-retroviral drugs, or PrEP, is highly effective in preventing HIV infections. However, underlying inflammation can significantly lower the efficacy of PrEP. Preventative strategies must include ways to mitigate genital inflammation. Our recent human cohort study found daily, low-dose oral aspirin (also known as acetylsalicylic acid (ASA)) permeates the genital mucosa and directly affects mucosal immunity by reducing the number of HIV target cells (CD4+CCR5+ T cells) present in the female genital tract (FGT). The underlying cellular mechanism of this finding is unknown.

Methods: C57BL/6 mice will be used as a model to study how ASA modulates inflammation and immune cell distribution in the FGT. Female mice are dosed with daily high or low oral ASA, and blood and vaginal secretions will be assessed every two weeks for ASA levels, inflammatory cytokines, and HIV target cell numbers. After eight weeks, final analysis will include immunohistochemistry to visualize T cell localization and distribution as well as measuring the ASA levels in the homogenized FGT. A dye will be used to evaluate FGT epithelial barrier integrity and HIV penetration depth during ASA treatment and compare to controls. Humanized BLT mice, which are susceptible to vaginal HIV challenge, will be used to determine whether ASA treatment lowers HIV infection. Humanized mice will be given oral ASA or PBS for 8 weeks, then challenged intravaginally with HIV weekly for 8 weeks, as previously done. Analysis will focus on when mice become viremic and their peak viral loads and compare these parameters between ASA and control groups. Initial studies on oral ASA dosing and T cell characterization in the FGT will be presented.

Significance and Impact: This study will be the first to investigate whether ASA’s anti-inflammatory effects can reduce FGT inflammation and improve vaginal epithelial barrier function in vivo to better protect from HIV infection. This study will also directly evaluate whether oral ASA regimens can reduce or prevent vaginal HIV transmission in vivo, experiments that are not possible in humans. This research may provide an additional HIV prevention option.

Novel non-invasive therapy induces repair in an MS model – Foundations for clinical translation

Nataliya Tokarska1,2, Hannah E. Salapa2,3,4, Michael C. Levin1,2,3,4, Bogdan F. Popescu1, Sarah J. Donkers2,5* and Valerie M.K. Verge1,2*

1Anatomy, Physiology and Pharmacology Department, University of Saskatchewan 2Cameco MS Neuroscience Research Centre 3Office of the Saskatchewan Multiple Sclerosis Clinical Research Chair, University of Saskatchewan 4Neurology Division, Department of Medicine, University of Saskatchewan 5School of Rehabilitation Science, College of Medicine, University of Saskatchewan *Co-Senior Authors (NT co-supervised by both)

Introduction: Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by immune-mediated segmental demyelination and variable degrees of axonal/neuronal degeneration. Efficient repair of demyelinated lesions is a major challenge of MS. In MS, demyelination can be followed early on by efficient remyelination, supporting that endogenous repair mechanisms exist. Our lab focuses on therapies that enhance intrinsic repair mechanisms following injury. We find that acute intermittent hypoxia (AIH; intermittent periods of reduced oxygen), a non- invasive therapy, improves outcomes in spinal cord and peripheral nerve injured animals in a manner akin to electrical stimulation. But its potential for repair in MS is unknown. We hypothesized that AIH treatment would enhance repair of the demyelinated CNS and mitigate disease progression in the MOG35-55 experimental autoimmune encephalomyelitis (EAE) mouse model of MS.

Methods: EAE was induced by immunization with myelin oligodendrocyte glycoprotein. At near peak EAE disease (score=2.5), mice received either AIH or Normoxia (control) treatment once daily for 7d, with mice followed for an additional 7d post-treatment. Animals were examined daily for clinical score changes and spinal cord tissue was processed and analyzed quantitatively to assess impact of AIH treatment on multiple indices of repair, including degree of myelination, axon protective phenotype, oligodendrocyte precursor cell (OPC) recruitment, and immune response modulation.

Results: Analysis showed 7d daily AIH treatment significantly improved clinical scores when treatment was started at near peak EAE disease. AIH treatment, as compared to Normoxia, resulted in significantly more myelin basic protein and elevated levels of axon protective phosphorylated neurofilaments, node of Ranvier Caspr+ve paranode reorganization and OPC recruitment. There also was a quicker resolution of the inflammatory response and polarization of macrophages/microglia toward a pro-repair M2 phenotype.

Conclusions: AIH treatment has a remarkable impact on improving behavioural and pathological outcomes. Collectively, these findings support a role for AIH treatment as a non-invasive therapy to enhance CNS repair following demyelination in an MS preclinical model. Further investigation to confirm these results and provide additional insight into the mechanisms and benefits of AIH treatment will allow for translation into a pilot clinical study in MS patients.

Impact of African-specific Genetic Variants on CHD1L, PRKAB2, and FMO5 Expression in an HIV- positive Cell Model

Riley H. Tough1,2, David M. Tang2, Shanelle N. Gingras1,2, Jeff F. Tuff2, Catherine M. Card2, Paul J. McLaren1,2

1Department of Medical Microbiology and Infectious Diseases, University of Manitoba 2JC Wilt Infectious Diseases Research Center, Winnipeg, MB, Canada

Background: We conducted a genome-wide association study (GWAS) of HIV-1 viral load setpoint in 3,879 individuals of African ancestry and detected a novel association on chromosome 1. The region includes three protein-coding genes; CHD1L, PRKAB2, and FMO5, that have not been previously associated with HIV infection. The top associated variant in the region, rs59784663 (p<6.38x10-10, β=0.30), is only common in African populations, ranging from 5-13% allele frequency. Therefore, we sought to identify causal genetic variants in the chr1 region and how they might regulate CHD1L, PRKAB2, or FMO5.

Hypothesis: We hypothesize that the presence of specific noncoding genetic variants in the chr1 region will regulate expression of CHD1L, PRKAB2, or FMO5.

Methods: To investigate this, we filtered variants in the chr1 region that were in LD (R2 ≥ 0.4) to rs59784663 and annotated variants using Ensembl’s Variant Effect Predictor. Variants in the chr1 region were analyzed for predicted regulatory impact in blood tissues using Phenotype Informed Noncoding Element Scoring (PINES). We also performed genotyping of variants in the chr1 region in N=251 people living with HIV from Kenya using a custom TaqMan RT-qPCR detection assay and Sanger sequencing.

Results: The Variant Effect Predictor identified no variants that change the coding sequence and the remaining were mainly intronic (58%) and upstream gene variants (17%). The two most significant noncoding variants from PINES analysis were rs72997588 (p ≤ 0.00001) and rs72997590 (p≤0.0001). These variants overlap with regulatory regions of CHD1L, a gene involved with DNA repair and chromatin remodeling. To assess if these variants affect gene expression in HIV-relevant cell types, PBMCs from Kenyan individuals with all three genotypes at rs59784663 will be sorted into CD4+ T cell and monocytes fractions. Cell fractions will be stimulated with anti-CD3/CD28 or IFN-α, respectively, and expression will be measured using RNAseq (CD4 T cells), qPCR (monocytes) and western blot (both cell types). Ongoing work will assess whether rs72997588 and/or rs72997590 regulate CHD1L or related gene pathways in these cell types.

Conclusion: This study seeks to characterize a novel mechanism of control for HIV infection and address the lack of diversity in HIV host genomics.

High-throughput sequencing defines donor and recipient HLA B-cell epitope frequencies for prospective matching in transplantation

Jenny N. Tran1, Oliver P. Günther2, Karen R. Sherwood1, Ruth Sapir-Pichhadze3, Paul A. Keown1,4

1Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada 2Günther Analytics, Vancouver, Canada 3Department of Medicine, McGill University and MU-HRI, Montreal, Canada 4Department of Medicine, University of British Columbia, Vancouver, Canada

Introduction: Human leukocyte antigens (HLA) are the cardinal targets for transplant rejection, but the enormous heterogeneity of HLA genes makes donor-recipient matching challenging. To overcome this, we combined next-generation sequencing, structural biology, and computational medicine to define the sequence, conformation, and distribution of critical target HLA epitopes and develop an innovative strategy for epitope-based matching to improve clinical transplantation.

Methods: 1846 kidney patients and donors were genotyped at the 11 HLA genes using Next-Generation Sequencing. Genotypes were converted to epitopes using HLAMatchmaker. Simulations were performed for a base-model using identical-ABO and waitlist rank for matching, and an epitope-matching model additionally using epitope-mismatch scores. Simulations were run in R, using provincial waitlist sizes and annual donor rates.

Results: 361 alleles were identified (206 at class I genes, 155 at class II genes) and translated into 150 eplets (59 class I, 91 class II), resulting in a 59% reduction in complexity. While the majority of alleles occurred at low frequencies (>50% present in <1% of subjects), epitopes were more broadly and uniformly distributed (>75% present in >30% of subjects). All simulation models with epitope-matching reduced HLA mismatch scores. Compared with the national base-model, deliberate epitope- matching decreased the median mismatch score from 27.35 to 9.3 across all genes; for class II genes from 16.8 to 1; for DRB1/3/4/5 from 6 to 0; and for DQB1 from 6 to 0. Waitlist and donor size was an important factor in determining compatibility, where ≥~250 patients allowed for the majority of matches to have a DQB1 score 0. Except for all genes combined and class II for small waitlists, the probability of a mismatch of 10 or lower was >95% across all waitlist sizes and gene loci.

Conclusion: Epitope-matching dramatically improves donor-recipient compatibility and it is feasible in a waitlist of 250 patients. Our results offers an opportunity for a unique program of epitope-based matching to reduce organ rejection in BC and across Canada.

Liver-specific depletion of DPP4 reduces hepatic gluconeogenesis but does not improve disease progression or inflammation in a mouse model of NALFD

Natasha Trzaskalski1,2, Branka Vulesevic1, My- Anh Nguyen1, Natasha Jeraj1,2, Evgenia Fadzeyeva1,2, Nadya Morrow1,2, Antonio Hanson1,2, Ilka Lorenzen-Schmidt2, Conor O’Dwyer1,3, Morgan Fullerton1,3, Erin E. Mulvihill1,2

1University of Ottawa Heart Institute 2The University of Ottawa 3Ottawa Institute of Systems Biology

Introduction: As a manifestation of metabolic syndrome, approximately 70% of patients with T2D will develop non-alcoholic fatty liver disease (NAFLD), a chronic disease that ranges from simple steatosis to severe cirrhosis characterized by inflammation and fibrosis. Previous studies have demonstrated that circulating DPP4, an enzyme dysregulated in patients with T2D, has direct pro-inflammatory actions. Our recent work demonstrated that increases in circulating DPP4 in the setting of metabolic disease derives from hepatocytes. Additionally, genetic elimination of Dpp4 from hepatocytes reduced markers of inflammation in both liver and adipose. Therefore, this study aimed to evaluate the efficacy of complete genetic elimination of Dpp4 vs selectively targeting hepatic Dpp4 in liver disease progression.

Methods: One-year-old Dpp4+/+, Dpp4-/-, Dpp4hep-/- or Dpp4GFP (Control) mice were fed a high-fat, high-cholesterol diet for 24 weeks to induce features of NAFLD. Hyperinsulinemic-euglycemic clamp, the gold standard method to assess insulin sensitivity and glucose production, was performed to evaluate the bi-directional relationship between glucose regulation and liver disease progression. NanoString mRNA analysis was performed on the liver and isolated F4/80+ cells (macrophages) to evaluate immunological pathways' expression. Further, histological, biochemical and expression analyses were used to determine and stage disease progression and severity.

Results: Hyperinsulinemic-euglycemic clamp revealed significantly decreased hepatic gluconeogenesis in both Dpp4-/- and Dpp4hep-/- mice compared to controls. NanoString mRNA expression revealed elevations in curated immunological pathways in both liver tissue and isolated F4/80+ cells in Dpp4-/- mice but not Dpp4hep-/- mice. However, despite significant changes in glycemic control and inflammatory pathways, both biochemical analysis and examination of liver histology demonstrated no differences in lipid accumulation or fibrosis between Dpp4-/-, Dpp4hep-/- and Dpp4GFP mice.

Conclusions: For the first time, these data identify the molecular target of DPP4 inhibitor drugs to lower glucose is derived from hepatocytes. However, despite a significant effect on glucose, these data suggest that the elimination of hepatocyte- specific Dpp4 can not improve lipid accumulation and fibrosis in a NAFLD mouse model.

Evidence for a role of epithelial cells in experimental model of inflammatory bowel disease

Diane M. Tshikudi, Noor Eissa, Abdoulaye Diarra and Jean-Eric Ghia

Department of Immunology Internal Medicine, University of Manitoba,

Introduction: Inflammatory bowel disease (IBD) which includes ulcerative colitis and Crohn’s disease are a life-long, severe inflammatory disorder of the gastrointestinal tract which significantly affect the quality of life of an estimated 270000 Canadians. Increase amount of evidence suggests that dysregulation of gastrointestinal immune response and impairment of intestinal epithelial barrier integrity play a substantial role in IBD pathogenesis. Deletion of chromogranin A (CHGA), a pro-hormone secreted by intestinal epithelial enteroendocrine cells, up-regulates anti-inflammatory cytokines, and reduce the onset and severity of ulcerative colitis. However, the impact of CHGA deletion to the intestinal epithelial barrier integrity in the context of an experimental model of dextran sulfate sodium (DSS)-induced colitis remain unclear. In this study, we sought to investigate the cytokine profiles of intestinal epithelial cells (IEC) following CHGA knockout mice treatment with DSS.

Methods: IEC and total colonic cells were isolated from colons extracted from six wild-type and three CHGA knockout mice untreated (control) or treated with 5% DSS. Interleukin (IL)-25 and IL-33 which are cytokines secreted by IEC, were measure using enzyme-linked immunosorbent assay.

Results: Elevate secretion of IL-25 was measured in CHGA knockout IEC at baseline which was significantly reduced in IEC from CHGA knockout mice treated with DSS 5%. Contrastingly, non-significant changes in IL-25 were measured between IEC from WT mice untreated or treated with DSS. Similarly, in WT epithelial cells, upon DSS treatment, IL-33 was not secreted. Furthermore, IL-25 and IL-33 secretion were not modulated in total colonic cells.

Conclusion: This study demonstrates that both IL-25 and IL-33 are regulated in epithelial cells in the context of experimental colitis model.

Obstructive Sleep Apnea, Body Mass Index, and Cellular Adhesion Molecules

Peres BU*1, Hirsch Allen AJ2, Kendzerska T3, Shah A2,4, Fox N2, Laher I2, Jen R2,4, Sandford A5, van Eeden S5, Ayas N2,4,6, Almeida F1,6

1Department of Oral Health Sciences, Faculty of Dentistry, The University of British Columbia, Vancouver, Canada 2Department of Medicine, Faculty of Medicine, UBC 3Department of Medicine, Division of Respirology, University of Ottawa, Ottawa, Canada 4Leon Judah Blackmore Sleep Disorders Program, UBC 5Centre for Heart Lung Innovation, St. Paul’s Hospital, UBC 6Canadian Sleep and Circadian Network

Objectives: To investigate the relationship between obstructive sleep apnea (OSA) severity, body mass index (BMI), and circulating levels of inflammatory adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule- 1, and E-selectin).

Methods: A cross-sectional clinical cohort study was performed and consecutive adults referred to the University of British Columbia (UBC) Sleep Laboratory for a polysomnogram (PSG) for suspected OSA provided a morning blood sample. Fasting blood (15 ml) was collected in the morning after PSG and serum/plasma was stored in a -80C freezer. Milliplex MAP Human Cardiovascular Disease Panel 1 (HCVD1-67 AK) multiplex Luminex (EMD Millipore, Etobicoke, ON, Canada) was used to determine the levels of ICAM-1, VCAM-1, and E-selectin. All assays followed the manufacturer protocols and were tested in duplicate. The samples were processed in batches.

Results: 488 patients were studied; the majority were male (68%) with a mean age of 50 years, mean AHI of 22.7 events/h, and mean BMI of 32 kg/m2. The descriptive statistics suggest a clear relationship between levels of obesity (overweight to obese) and OSA severity, as well as a gradual increase in adhesion molecules levels, especially ICAM-1 and E-selectin. In multivariable linear regression models, all three adhesion molecules were significantly associated with BMI (E-selectin, p<.0001; ICAM-1, p=0.0007; VCAM-1, p=0.0003). However, only E-selectin was independently associated with AHI (p=0.02) after adjustment of clinically relevant confounding factors (age, sex, BMI, AHI, Statin usage, ethnicity, diabetes, history of cardiovascular disease, and smoking status). There was no significant interaction between AHI and BMI for E-selectin (p=.33).

Conclusions: Although all three adhesion molecules were associated with BMI, only E-selectin was independently associated with OSA severity. E-selectin may represent a useful biomarker in OSA patients in terms of cardiovascular disease prediction. Futures studies are needed to determine the clinical significance of the relationship between E-selectin and OSA.

Acknowledgements: CIHR (Sleep Disordered Breathing Team Grant), VCHRI Scientist Award, BC Lung Association Operating Grant.

An Investigation of Novel Hepatitis B Virus Quantitative Serum Biomarkers in Hepatitis B-Hepatitis D Virus Co-Infected Patients Enrolled in the Canadian Hepatitis B Virus Network

Alicia Vachon1,2, Jacqueline Day2, Elizabeth Giles2, Sébastien Poulin3,4, Carla S. Coffin5, Sarah Haylock-Jacobs5, Gerald Y. Minuk1, Karen Doucette6, Alnoor Ramji7, Scott Fung8, Curtis Cooper9, Carla Osiowy1,2

1University of Manitoba, Winnipeg, Manitoba, Canada 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada 3Clinique L’Agora, Montréal, Québec, Canada 4Clinique I.D., St-Jérôme, Québec, Canada 5University of Calgary, Calgary, Alberta, Canada 6University of Alberta, Edmonton, Alberta, Canada 7University of British Columbia, Vancouver, British Columbia, Canada 8University of Toronto, Toronto, Ontario, Canada 9University of Ottawa, Ottawa, Ontario, Canada

Introduction: It is estimated that Hepatitis D virus (HDV) affects 5% of people living with chronic hepatitis B worldwide. HBV- HDV co-infection results in severe liver disease, with a rapid progression to cirrhosis and hepatocellular carcinoma. Novel HBV biomarkers, such as quantitative measures of HBV serum RNA, HBsAg (qHBsAg), and anti-HBc antibody (qAnti-HBc), and detection of Nucleic Acid-Related antigen (NRAg), consisting of PreS1 and core antigens, have shown utility in predicting infection progression and risk of HBV reactivation. However, these biomarkers have not been analyzed in the context of HDV co-infection.

Methods: Longitudinal serum samples (n=85) from 18 HDV antibody positive patients followed between 2010 and 2020 were retrospectively analyzed. Most patients (14/18) were HDV RNA positive. 50% of patients were treated with nucleos(t)ide analogue therapy, with one patient also treated with Hepcludex™. HBV serum RNA was extracted from serum samples and measured by 3’ RACE RT-PCR methods. Serological biomarkers were measured by standard immunoassay methods. Differences between groups were determined using Fisher’s exact test and Mann-Whitney U-test. Correlations between markers were analysed using nonparametric Spearman’s rank test. P-values below 0.05 were significant.

Results: Of the 18 patients investigated (13 males, median age 43 [26-70] years, 14 HBeAg negative) the majority were non- Canadian born (13/18). Low HBV serum RNA levels (<3 log copies/mL) were observed among most along with low to undetectable levels of HBV DNA (P <0.0001). HBeAg positive patients with HBV DNA levels >6 log IU/mL also had increased HBV serum RNA levels (mean 5.3 log copies/mL). Serum RNA correlated with qHBsAg (P <0.001) and qAnti-HBc levels (P=0.01). An association was noted between qHBsAg levels and elevated mean ALT (>1xULN; P <0.001). A lack of NRAg detection was observed in HDV RNA negative individuals having negligible (<40 IU/mL) qHBsAg levels, suggesting reduced cccDNA transcriptional activity. Positive NRAg was associated with qHBsAg (P <0.001) and elevated ALT (>1xULN; P=0.0213). Although Hepcludex™ successfully reduced HDV RNA levels, tested HBV biomarkers remained stable over time.

Conclusion: During HDV infection, novel HBV markers were observed to correlate with hepatitis. HBV biomarkers show potential for use in patient management of HDV co-infected patients.

Oxidized Phosphatidylcholines Mediate Airway Narrowing by Inducing Cytoplasmic Ca2+ Flux in Airway Smooth Muscle Cells

Jignesh M. Vaghasiya1,3, Divleen Mangat1,3, Christopher D. Pascoe1,3, Andrew J. Halayko1-3

1Departments of Physiology & Pathophysiology, University of Manitoba, Winnipeg, MB 2Internal Medicine, University of Manitoba, Winnipeg, MB 3Biology of Breathing Group, Children’s Hospital Research Institute of Manitoba, Winnipeg, MB

Rationale: Oxidative-stress associated with asthma pathobiology, oxidizes phospholipid in lung cell membranes and lining fluid. We reported that oxidized-phosphatidylcholines (OxPC) accumulates in the lungs of mice and humans after allergen challenge, and associates with the severity of airway hyperresponsiveness (AHR). This is, in part, due to direct effects on airway smooth muscle, as OxPC triggers an acute increase in intracellular Ca2+ ([Ca2+]i) in human airway smooth muscle cells (HASM). Here, we test the hypothesis that OxPC induces airway narrowing, and correlates with the activation of a unique repertoire of pathways that regulate intracellular Ca2+ in HASM.

Methods: The Murine-thin-cut-lung slices (TCLS) and video-microscopy was used to assess airway narrowing (n=3 mice). Changes in [Ca2+]i in HASM cells were measured by Fura-2 fluorescent microscopy (n=4 cell lines). Lumen size of airways in TCLS, and [Ca2+]i in HASM was assessed for 3 min in response to 80 μg/mL OxPC (oxidized-1-palmitoyl-2-arachidonoyl-sn- glycero-3-phosphocholine) or oxidation-resistant PSPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine). To identify Ca2+ channels activated by OxPC, cells were pre-treated with inhibitors: xestospongine (5μM, IP3 channel); ryanodine receptor (ryanodine-100μM; caffeine-25mM; 8-bromo-cADP-ribose-100μM,); Gd3+ (500μM, non-specific Ca2+-channels); or HC-030031 (50 μM, transient-receptor-potential-ankyrin 1 (TRPA1)). Similarly in TCLS, we evaluated the impact of ryanodine and TRPA1 channel inhibitors on OxPC-induced airway narrowing. Data were analyzed by one-way ANOVA or t- test.

Results: In HASM cells, OxPC induced a rapid rise to peak [Ca2+]i (143.0±14.48 nM), and secondary [Ca2+]i oscillations in 76% of cells. The Gd3+ or HC-030031 pre-treatment, didn’t affect peak [Ca2+]i, but [Ca2+]i oscillations were abrogated. Ryanodine receptor inhibition significantly reduced OxPC-induced [Ca2+]i flux. We confirmed that the upstream regulator of ryanodine receptors, CD38 was involved in this effect, as 8-bromo-cADP-ribose also prevented OxPC effects. In murine TCLS, OxPC caused 15% airway narrowing, which was abrogated by pre-treatment with ryanodine receptor or TRPA1 inhibitors.

Conclusion: OxPC mediates airway narrowing via direct effects on airway smooth muscle, as they trigger Ca2+ influx via TRPA1 channel, as well as intracellular Ca2+ release, chiefly via CD38-cADP-ribose- regulated ryanodine receptor in the sarcoplasmic reticulum. This implicates a role of OxPC, which accumulate in the airway of asthmatics, as a direct determinant of AHR.

Funding: Supported by CHRIM and the CIHR Canadian Respiratory Research Network. JMV holds a Research Manitoba- CHRIM Fellowship, AJH is supported by the Canada Research Chairs Program.

Exercise to Prevent Anthracycline-based Cardio-Toxicity (EXACT 2.0) in Women with Breast Cancer

Varghese SS, Eekhoudt CR, Cheung D, Barnes C, Johnston WJ, Bortoluzzi T, Bews H, McKenzie M, Mittal I, Ismail U, Chiekwe CJ, Keats MR, Nguyen T, Pitz M, Mulvagh S, Grandy SA, Jassal DS

1Department of Physiology and Pathophysiology, University of Manitoba; 2Institute of Cardiovascular Sciences, Albretchsen Research Center, Manitoba; 3Department of Internal Medicine, University of Manitoba; 4Dalhousie University and Nova Scotia Health Authority, Halifax

Background: While chemotherapy-induced cardiotoxicity is a major cause of morbidity and mortality in breast cancer (BC) survivors, pre-clinical models have shown that exercise can be cardioprotective in this setting. Translating this finding to the clinical arena, our EXACT 1.0 study confirmed the feasibility of a hospital-based exercise program in women with BC; however, travel to the facility was a major barrier for participation. In the present EXACT 2.0 study, we investigated the cardioprotective benefits of a 6-month home-based aerobic exercise program in BC patients receiving anthracycline (AC) chemotherapy.

Methods: Women with BC receiving AC chemotherapy were randomized to either standard of care (control) or standard of care + 6-month home-based aerobic exercise program (AEX). The exercise program consisted of 2 home-based sessions/week (20-45 minutes; 35-85% heart rate reserve intensity). Heart rate monitors were provided to all participants to monitor physical activity throughout the study. Transthoracic echocardiography was conducted at baseline and a 6- month follow-up to assess left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS).

Results: A total of 11 women (49 ±10 years old) with BC were recruited and randomized, 5 to control and 6 to AEX. AEX participants had an average 94% adherence to the program. Ten patients received Adriamycin and Cyclophosphamide for 8 weeks and 1 patient received Fluorouracil, Epirubicin, and Cyclophosphamide for 9 weeks; 5 patients received adjuvant radiation therapy. Five women had cardiovascular risk factors including hypertension (n=1), hyperlipidemia (n=1), smoking history (n=2), and family history of coronary artery disease (n=2). In the control group, the mean LVEF was 63±1% at baseline and 60±2% at 6-months (p=NS). In the AEX group, the mean LVEF was 63±2% at baseline and 59±7% at 6-months (p=NS). Similarly, in the control group, the mean GLS was -18.7±0.4% at baseline and -18.0±0.1% at 6-months (p=NS). In the AEX, the mean GLS was -18.8±0.5% at baseline and 18.5±2.5% at 6-months (p=NS).

Conclusion: These preliminary findings demonstrate AEX was feasible in women undergoing BC therapy but had a null effect on cardiovascular mechanics.

Modulation of Cardiac Immune Pathways using Acellular Matrix Biomaterial for Post-Infarct Repair

Vishnu Vasanthan, Darrell Belke, Guoqi Teng, Justin Deniset, Paul W.M. Fedak

1University of Calgary, Calgary, Alberta, Canada

Introduction: Epicardial Infarct Repair (EIR) is an emerging therapy to promote adaptive healing after myocardial infarction. Bioactive “patches” are applied over ischemic myocardium during surgical revascularization. We showed acellular porcine small intestinal submucosal extracellular matrix (SIS-ECM) improves post-infarct cardiac function in animals by altering cardiac fibroblasts to promote angiogenesis and attenuate fibrosis. The effect on post-infarct inflammation is less explored. Herein, we examine how SIS-ECM modulates cardiac immune response in a murine infarct model and investigate a fibroblast-mediated mechanism.

Methods: Mice received coronary ligation (infarct) followed by either SIS-ECM implantation (n=12) or sham (n=10). Myocardial flow cytometry at 7 days quantified proangiogenic immune cells. Two-photon microscopy imaged embedded SIS-ECM and fibroblasts in the myocardium. RNA-sequencing analysis assessed inflammatory transcriptional changes in cardiac fibroblasts seeded on active SIS-ECM or inactive control. To assess fibroblast secretome, mouse 3T3 cell line fibroblasts were seeded on SIS-ECM or inactive control and conditioned media was analyzed using multiplex (n=6/group). To elucidate mechanism, 3T3 cells were seeded on SIS-ECM or a version with less nuclear material (D-ECM) and media was analyzed by multiplex (n=10/group).

Results: Mice receiving SIS-ECM had higher 7-day flow cytometric counts of VEGFR1+ monocytes (44±8 vs 15±3; p<0.01; SIS-ECM vs Sham) and VEGFR1+ neutrophils (74±10 vs 26±4; p<0.01). Two-photon microscopy showed fibroblasts being recruited into SIS-ECM. RNA analysis showed increased fibroblast transcription favoring neutrophil and phagocyte recruitment in the SIS-ECM group (p<0.05). Multiplex showed increased 3T3 production of angiogenic factor VEGF (364±27 vs 30±3 pg/mL; p<0.01), neutrophil chemoattractant KC (827±42 vs 641±40 pg/mL; p<0.01) and monocyte chemoattractant IP-10 (53±3 vs 39±3; p<0.01). Compared to SIS-ECM, 3T3 fibroblasts seeded D-ECM decreased production of VEGF (1572±58 vs 144±6 pg/mL; p<0.01; SIS-ECM vs D-ECM), KC (6512±181 vs 4715±172 pg/mL; p<0.01) and IP-10 (51±3 vs 39±5; p=0.04).

Conclusion: SIS-ECM modulates post-infarct inflammation to increase proangiogenic immune cells, possibly through a fibroblast-mediated mechanism. EIR with active SIS-ECM in mice increased proangiogenic immune cell content. Fibroblasts were recruited to SIS-ECM on microscopy, highlighting an opportunity for modulation. SIS-ECM alters fibroblast activity in- vitro to potentiate recruitment of proangiogenic immune cells and that remnant nuclear material is key to this mechanism.

Clinical Red Flags that Warrant Workup for Monogenic Allergic Diseases

Maryam Vaseghi-Shanjani1, Kelsey Smith1, Rahnuma Sara1, Bhavi Modi1, Catherine Biggs1, Stuart Turvey1

1Department of Pediatrics, British Columbia Children's Hospital, The University of British Columbia, Vancouver, BC

Introduction: Primary Immunodeficiency diseases (PIDs) are genetic disorders that affect the function and development of the immune system. Although PIDs have traditionally been identified on the basis of enhanced susceptibility to infections, we now appreciate that the presence of allergic diseases can also serve as an important sign of an underlying PID. In light of this, recently the term primary atopic disorders (PADs) was coined to describe this group of heritable monogenic immune disorders that prominently present with allergic features. As such, it is important for clinicians to recognize that allergic diseases such as food allergy, atopic dermatitis, and allergic asthma may indicate an underlying PID, especially in patients who present with severe, early onset, refractory and coexisting allergic conditions. Clinically, distinguishing PADs from common polygenic allergic diseases may be challenging as both conditions share common clinical features; nonetheless, the disease-causing mechanisms may differ between the two, necessitating different management strategies and therapeutic interventions.

Identifying monogenic allergic disease through next-generation sequencing (NGS) can dramatically improve clinical outcomes through the use of precision-based therapy that target the patient’s underlying molecular defect. It is therefore imperative that clinicians recognize PADs to be able to provide informed therapeutic options and improve patient outcomes. Here, through a comprehensive literature review we identify clinical features and warning signs that warrant assessment for PADs and discuss the benefits of timely diagnosis and management of these conditions.

Methods: We performed a thorough literature review on the 32 PADs causing genes that have been identified and described in literature, in order to come up with a list of clinical warning signs that would warrant workup for PADs.

Results & Conclusion: Currently, we do not have sufficient evidence to create sensitive and specific diagnostic algorithms that can guide clinicians in identifying which patients should undergo sequencing for potential PADs. However, our comprehensive review shows that PADs can present with accompanying clinical conditions such as elevated IgE and eosinophils, autoimmune disorders, malignancies, short stature, connective tissue abnormalities and enhanced risk of infections. These clinical features should lower the clinicians’ threshold for considering further workup for PADs.

Chrna5 expression identifies a specialized subgroup of prefrontal cortex neurons with enhanced affinity to acetylcholine

Sridevi Venkatesan1, Tianhui Chen1, Shreejoy Tripathy1,2,4, Eric E Turner5, Evelyn K Lambe1,2,3

1Department of Physiology, University of Toronto 2Department of Psychiatry, University of Toronto 3Department of Obstetrics and Gynaecology, University of Toronto 4 Krembil Centre for Neuroinformatics, CAMH 5Department of Psychiatry & Behavioral Sciences, Center for Integrative Brain Research, Seattle Children’s Research Institute

Introduction: Cholinergic modulation of the prefrontal cortex is essential for attention. Layer 6 corticothalamic neurons are critical to this cholinergic attention circuitry and express nicotinic acetylcholine receptor subunits encoded by the Chrna5 gene. Disrupting Chrna5 impairs attention, concurrent with its role in the rapid onset of postsynaptic cholinergic responses and preventing desensitization. Heterogenous Chrna5 expression in layer 6 predicts the existence of a specialized neuronal population beyond existing genetic markers that would be hypersensitive to cholinergic activation, and thus vital during attention. We propose to identify and examine this neuronal population.

Methods: We used Chrna5-Cre mice to characterize the spatial distribution and cholinergic responses of labeled Chrna5- cre expressing layer 6 neurons (Chrna5+) using GCaMP6s Ca2+ imaging and patch-clamp electrophysiology in brain slices. We compared these to a well-characterized population of layer 6 corticothalamic neurons expressing Syt6-cre or Syt6-gfp (Syt6+).

Results: We found that Chrna5+ and Syt6+ neurons shared only 40% overlap in layer 6. These populations showed different affinities to exogenous acetylcholine application, with Chrna5+ cholinergic responses more resistant to the competitive nicotinic antagonist DHBE, and Syt6+ neurons more susceptible. Chrna5+ responses to optogenetic release of endogenous acetylcholine showed faster onset and greater peak current compared to neighboring unlabeled cells. Ongoing work is probing single-cell transcriptomic data to assay differential expression of partner nicotinic receptor subunit mRNAs as well as the ‘nicotinic-interactome’ of molecules involved in trafficking, scaffolding, and post-translational modification in layer 6 neurons. Overall, we identify Chrna5+ neurons as a group with strong cholinergic modulation, which were not well represented in previous labeling approaches. Thus Chrna5-Cre mice are a powerful tool for future in vivo studies of attention.

Impact of Specialized Proprioceptive and Aerobic Training on Proprioception and Postural Control in Seniors

Marie Julie Vermette1,2, Louis Bherer1,2,3, Benjamin Pageaux1,2, François Prince4, Julie Messier1,2

1École de kinésiologie et des sciences de l’activité physique, Université de Montréal 2Research Center of the University Institute of Geriatrics of Montréal, Montréal, Canada 3Research Center of the Montreal Heart Institute, Montréal, Canada 4Department of Surgery, Université de Montréal

Introduction: Falls are a major adversary to the quality of life in seniors. They have been attributed to both a decline in proprioceptive function as well as an inability to efficiently allocate attentional resources to balance in multi-task conditions. Previous findings have shown postural control is more attentionally demanding in sedentary seniors than in young adults, particularly in conditions having high proprioceptive demand. Improved motor functions following a specialized proprioceptive training in healthy and diseased populations have been documented. However, none have directly measured proprioceptive acuity nor the impact of a training intervention on tasks having high proprioceptive demand. Importantly, aerobic training, another avenue has been shown to improve executive functions (i.e., attentional resources) in older adults. A combination of both these trainings (proprioceptive specific and aerobic training) might therefore have a positive impact on proprioception and postural control in seniors.

Methods: We will use two dual-task paradigms in which a primary task (proprioceptive ankle position matching or postural stability limit task) and a secondary cognitive task (i.e., subtraction) are performed separately and concurrently before (pre- test) and after (post-test) a 12-week training intervention. The intervention group will be engaged either in a specialized proprioceptive (n=25) or aerobic (n=25) exercise program developed by kinesiologists (3 times/week, 40 min/session, 12 weeks). The non- exercise control group (n=20) will be instructed to maintain their normal physical activities for the 12- weeks.

Expected Results: We expect that specialized proprioceptive training will improve the proprioceptive processing and postural control of seniors. Such training-related improvements are also expected to be associated with a reduction in the attentional cost of both primary tasks (i.e., improve dual-task costs). Additionally, we expect that the aerobic training will improve the allocation of attentional resources towards proprioceptive processing and postural control.

Conclusion: Testing the effectiveness of specialized interventions on proprioception and postural control is crucial to understanding the mechanisms by which postural instabilities occur in seniors and to developing optimal training interventions to prevent falls.

Synthesis and Evaluation of Reversible Covalent-Binding Anti-Cancer Drugs

Billy Vuong1, Brian Hasinoff1, Geoffrey Tranmer1

1College of Pharmacy, Rady Faculty of Health Sciences, University of Manitoba

Introduction: Camptothecins are effective anti-cancer agents due to their ability to inhibit topoisomerase 1 (Top1), which changes the topology of DNA for processes such as DNA replication. Inhibition of Top1 in rapidly dividing cancer cells interferes with their ability to replicate, leading to DNA damage and programmed cell death. Covalent binding drugs offer advantages over non-covalent drugs through stronger interactions with their receptor, which can improve potency, selectivity, and duration of action. These can influence the pharmacodynamics of the drug, potentially requiring less frequent and lower dosing amounts. We hypothesize that we can use organic synthesis to generate drug candidates that can form an additional covalent interaction with the Top1 cleavage complex, generating potent anti-cancer agents with improved pharmacokinetic and pharmacodynamic properties over current Top1 inhibitors.

Methods: Drug candidates were synthesized and purified by flash chromatography, followed by characterization by NMR and mass spectroscopy. Cell viability assays were performed utilizing MTT assay against HCT116 (colorectal) and U251 (glioblastoma) cell lines after 72 hours of drug exposure. Annexin-V flow cytometry experiments were performed on K562 (leukemia) cell lines after 48 hours of drug exposure. Covalent binding experiments were performed using agarose gel- based cleavage/relaxation assay on Top1 nuclear extract and PBR322 DNA.

Results: We have synthesized several potent anti-cancer candidates with nanomolar half maximal inhibitory concentrations (IC50s) against several types of cancer cell lines determined by MTT assay. Annexin-V assay with flow cytometry of several of our drug candidates against K562 cells showed similar apoptosis and cell death scatter profiles to camptothecin and irinotecan metabolite SN-38. Cleavage assays show ability of drug candidates to form ternary complexes with Top1 and DNA, resulting in stalled cleavage complex. Salt reversal assay suggest potential of one of our drug candidates to resist salt induced reversal of ternary complex through formation of covalent bond with Top1.

Conclusion: We have generated potent drug candidates with nanomolar IC50’s. Our compounds induce apoptosis and cell death in cancer cell lines through formation of a ternary complex with Top1 and DNA. Salt reversal assays suggest one of our candidates can form an additional covalent interaction with the Top1 cleavage complex.

Nociceptor Neurons Control Pollution-exacerbated Asthma

Jo-Chiao Wang1, Theo Crosson1, Tuany Eichwald2, Katiane Roversi1, Maryam Ahmadi1, Ali Ahmadi1, Mohammad Balood1 and Sébastien Talbot1

1Département de pharmacologie et physiologie, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada 2Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brasil.

Introduction Half of the severe asthma patients suffer from uncontrolled exacerbations. Our work in neuro-immunology has shown that, in the context of asthma, vagal nociceptor neurons drive a feed-forward inflammatory loop with lung immune cells, and that silencing these neurons reverses allergic airway inflammation (AAI). Here, we aim to expend these findings to a clinically relevant model of pollution exacerbated asthma.

Methods Experimental allergen Ovalbumin (OVA) was co-exposed with fine particulate matter (FPM) to OVA-sensitized wild type or sensory neuron-ablated TRPV1-DTA mice. Cells in bronchoalveolar lavage fluid (BALF) and lung were immunophenotyped by flow cytometry. Gene expression in isolated alveolar macrophages was assessed by RT-qPCR.

Results We found that mice co-exposed to FPM and OVA show an aberrant bronchoalveolar lavage fluid immune profile characterized by a mixed infiltration of neutrophil and eosinophil as well as the expansion of lung γδ T cells. Along with these changes, we found that the expression and the release of the neurotrophic factor artemin was increased in FPM- stimulated alveolar macrophage. In addition, to these changes, we discovered that the genetic ablation of sensory neurons prevents the development of pollution exacerbation of asthma.

Conclusion In terms of inflammatory cell infiltration in BALF and lung γδ T cell expansion, FPM exacerbates OVA-induced AAI in a TRPV1+ sensory neuron-dependent fashion. In parallel, artemin is induced by the exposure of FPM, which support the existence of an artemin-related neuroimmune network mediating the pollution exacerbation of AAI.

Comparison of preparation conditions of Pluronic chrysophanol micelles

Mai Wang1,Yin-yue Wang1,Haihong Zhang1, Jin Wang1, Shu Wang1*

1Department of Pharmacy, Hebei North University, China *Corresponding author

Introduction: Chrysophanol is a kind of traditional Chinese medicine component with many pharmacological effects. Because of its low bioavailability, this study focused on comparing the preparation processes of chrysophanol micelles containing Pluronic and choosing the best preparation conditions.

Methods: Chrysophanol was coated with Pluronic as drug carrier and micelles were prepared by thin film hydration method. An ultraviolet spectrophotometer was established for the determination of drug content. The conditions of physical co- dissolution method, organic solvent high centrifugal speed, hydration ultrasonic time and rotary evaporation temperature were selected. The physical co-dissolution method took the dissolution time and absorbance as indexes to compare the stirring method, magnetic stirring method, water bath vibration method and ultrasonic method. In the selection of organic solvents, the encapsulation efficiency was used as the index to compare the anhydrous ethanol, chloroform, dichloromethane and acetone. The encapsulation efficiency was used as the index to compare the high centrifugal speed (4000, 6000, 8000, 10000 r·min-1). The hydration ultrasonic time (10, 20, 30, 40 min) was compared using the encapsulation efficiency as the index. The rotary evaporation temperature was compared at 20 ℃, 40 ℃ and 60℃ with the encapsulation efficiency as the index.

Results: In this study, chrysophanol had a good linear relationship in the range of 0.10-10.00μg·mL-1.Dissolution time: ultrasonic method >water bath shock method >magnetic stirring method >stirring method; Absorbancy: ultrasonic method >water bath shock method >magnetic stirring method >stirring method; Organic solvent: acetone>chloroform>dichloromethane>anhydrous ethanol; High centrifugal speed:4000>8000>6000>10000(r·min-1); Hydration ultrasonic time: 40>30>20>10(min); Rotary evaporation temperature: 60>40>20(℃).

Conclusion: This study provides a laboratory basis for the preparation of Pluronic-chrysophanol micelles and a reference for the selection of experimental conditions for Pluronic-chrysophanol micelles in the future.

Exploring the active ingredients of Astragalus propinquus Schischkin and Panax notoginseng against chronic kidney disease based on network pharmacology

Wang Xiaojia1, Li Jianchun1, Tan Ruizhi1, Wang Li1*

Introduction: To explore the potential biological mechanism of Huangqi Sanqi Mixture on chronic kidney disease based on network pharmacology. Astragalus and Panax Notoginseng Mixture is a compound preparation made under the guidance of TCM theories. It is composed of Astragalus, Panax Notoginseng, Achyranthes Bidentata, Laminaria and other medicines. The specific mechanism for improving CKD is unclear.

Method: Use the TCMSP database to screen the active ingredients of Huangqi Sanqi mixture based on oral bioavailability (OB) and drug-like properties (DL) (conditional parameters: OB ≥ 30 & DL ≥ 0.18), and compare each of them on the Swiss Target Prediction database. The effective ingredients are used for target prediction, so as to obtain the target of the drug. Through the mining of CKD target points, the two target point data sets are analyzed to find the intersection target point, and the corresponding component is used as the effective component. GO/KEGG enrichment analysis was performed to find the potential signal pathways of Huangqi Sanqi mixture in the treatment of chronic kidney disease.

Results: A total of 39 active ingredients were found, including 15 astragalus, 11 achyranthes, 6 kelp, 4 notoginseng, and 1 angelica. In the SWISS database, the target prediction of each active ingredient was carried out, and finally 582 targets of Huangqi Sanqi mixture were obtained. Use "CKD" as a keyword search in NCBI GEO database (condition parameter: gene expression profile microarray data, single set of samples is greater than 20), a total of 2 sets of chips are obtained; differential gene annotation is performed through R language "Limma" package to obtain differences Genes and obtain their co-expression profiles. Then the detected redundant and disorderly disease-related target information was eliminated, and finally 1546 disease-related targets were obtained. Take the target of Astragalus Notoginseng mixture and the common target of the two chips to make the intersection to construct the drug-target-disease molecular network, and obtain 98 common targets; further, by strengthening the screening criteria of the targets (conditional parameters) ：|logFC|> 1& p- vaule <0.05), 24 differential genes were obtained, namely HDAC5, CA12, CD81, MYLK, BCL2, BCAT2, PRKCD, ICMT, CDK4, PTPRF, FAAH, HSD11B2, CAPN1 , CSNK2A2, among which PRKCD and PTPRF are the most differentially expressed. The enrichment analysis results of GO/KEGG biological process show that the common target is closely related to factors such as cellular oxidative stress. And it is mainly enriched in the AKT signaling pathway.

Low Field Magnetic Stimulation Promotes Myelin Repair and Cognitive Recovery in Chronic Cuprizone Mouse Model

Zitong Wang*1, 7, Akanksha Baharani*1, Zelan Wei2, Davin Truong2, Xiaoying Bi3, Fei Wang4, Xin-Min Li7, Valerie M. K. Verge5,6†, Yanbo Zhang1, 6, 7†

*equal contribution 1Department of Psychiatry, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada. 2College of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada. 3Department of Neurology, Shanghai Changhai Hospital, the Second Military Medical University, Shanghai, China 4Nanjing Brain Hospital, Nanjing Medical University, Nanjing, China 5Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK 6Cameco MS Neuroscience Research Centre, College of Medicine, University of Saskatchewan, Saskatoon, SK 7Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.

Introduction: Multiple sclerosis (MS) is an inflammatory demyelinating disease featured with neuroinflammation, demyelination, and oligodendrocytes' loss. Cognitive impairment and depression are common neuropsychiatric symptoms in MS that are poorly managed with the present interventions. This study aimed to investigate the effects of low field magnetic stimulation (LFMS), a novel non-invasive neuromodulation technology, on cognitive impairment and depressive symptoms associated with MS using a mouse model of demyelination.

Methods: C57BL female mice were fed with a 0.2% cuprizone diet for twelve weeks to induce a chronic demyelinating model followed by four weeks of cuprizone withdrawal with either sham or LFMS treatment. After each treatment, mice were implemented with inhaled euthanasia followed by a series of behavioral tests, immunohistochemistry staining, and western blot test.

Results: Present results in the demyelination phase indicated a successful establishment of a chronic CPZ-induced toxic mouse model. The lower percentage of alternation and higher immobility on behavioral tests revealed impaired cognitive function and increased depression level. Reduced expression of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and glutathione-S-transferase (GST-π) indicated severe myelin damage and mature oligodendrocyte loss. During the myelin repair phase, increased percent of alternation, decreased immobility, and restored weight gain were observed in mice with LFMS treatment compared to the Sham group, therefore suggesting LFMS improved cognitive function, ameliorated the depression-like behavior, and promoted weight regain. Immunohistochemical and immunoblotting data showed enhanced MBP and MOG in mice's prefrontal cortex with LFMS treatment, supporting that myelin repair was promoted. LFMS also increased the protein expressions of mature oligodendrocyte biomarker GST-π. TGF-β and associated receptors' expression was elevated with LFMS treatment, implicating this pathway in the response. In the present study, we have shown, for the first time, the beneficial effects of LFMS on the chronic CPZ-induced demyelination animal model.

Conclusion: Results from the present study revealed LFMS to have neuroprotective effects, suggesting that LFMS has potential therapeutic value for treating cognitive impairment and depression related to demyelination disorders. In addition, the TGF-β pathway might explain the neuroprotective properties of LFMS.

Examining attitudes towards a sugar-sweetened beverage tax: A critical discourse analysis

Anne Waugh1, Kelsey Mann1, Andrea Bombak2, Patricia Thille3, Natalie Riediger1

1Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada 2Department of Sociology, University of New Brunswick, Fredericton, New Brunswick, Canada 3Department of Physical Therapy, University of Manitoba, Winnipeg, Manitoba, Canada

Introduction: Global concerns about “obesity” have been increasing, and as a result a sugar-sweetened beverage (SSB) tax has been proposed as a possible intervention in various Canadian jurisdictions. However, a SSB tax, if implemented, may have consequences for health equity. In particular, a SSB tax may create or exacerbate existing stigmas, including weight stigma, given the link between SSB and weight.

Objectives: We were looking to answer the following: How are SSB and SSB taxation discursively understood amongst white residents of River Heights, Winnipeg?

Methods: We performed a secondary data analysis of 18 qualitative interviews, including 3 men and 15 women. Interviews were collected in 2019, audio-recorded and transcribed. Participants were residents of River Heights, a neighborhood in Winnipeg, as well as being  18 years and English speaking. Critical Discourse Analysis methods were used, to direct our focus on power and dominance, and thus the potential impacts on equity. Analysis was informed by Critical Weight Studies, particularly utilizing the idea of weight stigma.

Results: Preliminary results show that participants are mostly supportive of a sugar-sweetened beverage tax. Specifically, participants were found to be utilising discourses of anti-“obesity”, such as explicit weight stigma, where fatness was considered to be “disgusting”, both when describing themselves, as well as others. In addition, personal responsibility framing for weight, also a part of weight stigma, was used to justify their support for a SSB tax.

Significance: As a SSB tax is an intervention with global public health support, it is important to explore and consider the potential negative unintended effects before, or whether, it is implemented. Our results highlight the prominence and harm of weight stigma in discourse related to a SSB tax.

The management of physical health for people with psychotic disorders by family physicians: a systematic review

Joshua C. Wiener1, Myanca Rodrigues1, Kelly K. Anderson1,2

1Department of Epidemiology and Biostatistics, Schulich School of Medicine & Dentistry, University of Western Ontario 2Department of Psychiatry, Schulich School of Medicine & Dentistry, University of Western Ontario

Introduction: Psychotic disorders are severe mental illnesses that affect 1% of the population over the lifecourse. People with psychotic disorders have a higher prevalence of several chronic physical health conditions, which are often a consequence of lifestyle factors and antipsychotic medications. Family physicians play a central role in the management of physical health conditions and in the prevention and treatment of physical illnesses, but providing primary care to patients with psychotic disorders may be more complex. This systematic review examined the evidence regarding the management of physical health conditions among people with psychotic disorders by family physicians.

Methods: A comprehensive literature search was conducted in three electronic databases for articles published in English up to January 2019. Studies were included if: (1) study design was observational; (2) population was patients receiving care from family physicians; (3) exposure group was patients with psychotic disorders; (4) comparison group was patients without psychotic disorders; and (5) outcome was management of physical health conditions (i.e., adherence to guidelines and recommendations). Study details, subject characteristics, methods, and results were extracted from each study and summarized in a qualitative manner. Risk of bias (RoB) for each study was assessed using the CLARITY tool.

Results: A total of 12 articles met inclusion criteria, including 9 retrospective cohort studies with low RoB and 3 case control studies with moderate RoB. Among patients with diabetes (5 studies), those with psychotic disorders received lower rates of guideline-recommended monitoring. Among patients with cardiovascular disease (2 studies), those with psychotic disorders received lower rates of guideline-recommended prescriptions. Among patients at risk for cardiovascular disease (3 studies), those with psychotic disorders received lower rates of guideline-recommended screening. Among patients at risk for cancer (2 studies), those with psychotic disorders received lower rates of guideline-recommended screening.

Conclusions: Physical health management is an ongoing concern for individuals living with psychotic disorders. These individuals appear to receive poorer care of their physical health by family physicians when compared to individuals without psychotic disorders.

Genetic variants affecting the expression of two Mendelian deafness genes are associated with age-related hearing loss

MacKenzie Wilke1, Britt Drögemöller1,2,3

1Department of Biochemistry and Medical Genetics, Rady Faculty of Health Sciences, University of Manitoba 2The Children’s Hospital Foundation of Manitoba 3Research Institute in Oncology and Hematology

Introduction: Age-related hearing impairment has a high burden worldwide. Hearing aids, which work by amplifying sound, are the only effective treatments available for hearing loss. Age-related hearing loss has been shown to be genetically heterogeneous and has genetic mechanisms which are largely unknown. In order to shed some light on the genetic etiology of this complex trait, a transcriptome-wide association study was performed.

Methods: Transcriptome-wide association studies (TWAS) were performed on two independent hearing loss cohorts (discovery ( n =330,759) and replication ( n =52,409)), to determine which hearing loss associated variants have an effect on gene expression levels across various tissues. Both hearing loss genome-wide association summary statistics and expression reference panels from the Genotype-Tissue Expression database were used as input for TWAS analyses performed with S-PrediXcan. Genes that were significantly associated with hearing loss in the discovery cohort were investigated in the replication cohort. The role of these genes in hearing loss was validated using inner ear gene expression databases, overrepresentation analyses and annotation platforms.

Results: TWAS analyses in the discovery cohort identified significant associations between hearing loss and thirty-five genes. Two of these genes were replicated in the independent replication cohort: TRIOBP (discovery cohort: P =4.6x10 -8 , zscore=0.67; replication cohort: P= 7.8x10 -8 , zscore=1.2) and ILDR1 (discovery cohort: P =2x10 -7 , zscore=-0.002; replication cohort: P= 4.4x10 -4 , zscore=-0.48). Further investigation of these genes revealed that both genes were enriched for abnormal hair cell functions, had high expression in inner and outer hair cells, and were associated with hearing loss phenotypes in both mice and humans.

Conclusion: To the best of our knowledge, this is the first study to perform a TWAS of hearing loss. This TWAS identified two genes, TRIOBP and ILDR1 , that were associated with age-related hearing loss in two independent hearing loss cohorts. Several lines of evidence have supported the role that these Mendelian deafness genes play in hearing loss and further research is needed as studies have shown that variants affecting the expression of Mendelian deafness genes play a role in non-Mendelian forms of hearing loss.

The NOD-like receptor protein NLRP6 regulates the colonic mucus layer during Tritrichomonas infection

Nathaniel Winsor1, Paul Lemire2, Elisabeth Foerster1, Samuel Killackey2, Ziqi Liu2, Heather Maughan3, Cathy Streutker4, Dana Philpott1, Stephen Girardin1,2

1University of Toronto, Department of Immunology 2University of Toronto, Department of Laboratory Medicine and Pathobiology 3Ronin Institute, Montclair NJ 4Saint Michael's Hospital, Toronto ON

Introduction: Enteric parasitic infection represents a major health burden in the developing world. Pathogen detection often involves members of the NOD-like receptor (NLR) family of proteins. However, the role of these proteins in parasitic infection is poorly characterized. A key step in the intestinal anti-parasitic response is the growth of the mucus layer. NLRP6 is the most common NLR protein in intestinal epithelial cells and it has been suggested that NLRP6 regulates both goblet cell abundance and mucus secretion.

Methods: We developed a novel parasitic infection model utilizing protist species of the family Tritrichomonas (Tm). Protists were harvested and FACS-purified from the cecum of infected mice and delivered by oral gavage into recipient animals.

Results: Upon infection, Nlrp6-/- mice possess a thinner mucus layer when compared to Nlrp6+/+ littermates. We excluded a role for the microbiome, as regardless of Tm infection, 16S rDNA sequencing in Nlrp6-/- and Nlrp6+/+ littermates did not reveal significant differences. In the colon, infected Nlrp6-/- mice have increased levels of tuft, goblet and proliferating cells. IL-18 is matured downstream of NLRP6 activation. However, littermate-controlled Tm infections in Il18-/- mice did not phenocopy the effects of NLRP6 deficiency, suggesting an IL-18 independent role for NLRP6 in the colon. Defects in the mucus layer result in chronic inflammation and can contribute to the development of colorectal cancer (CRC). To assess the functional impact of the thinner mucus layer in Tm+ Nlrp6-/- mice, we crossed infected animals onto the Apcmin/+ background, a genetic model for CRC, and determined that chronically infected Nlrp6-/- Apcmin/+ mice have a greater intestinal tumour burden than Apcmin/+ littermates.

Conclusion: NLRP6 regulates mucus secretion in response to parasitic infection. Future work aims to determine whether the NLRP6-mucus axis is dependent on Caspase-1/11 activation.

Defining the Epigenetic Signature of Tissue Reparative Cardiac Macrophages for Therapeutic Utility

Anthony Wong1,2,#, Shabana Vohra2,4,#, Slava Epelman1-4

1University of Toronto, Department of Immunology, Toronto ON 2Toronto General Hospital Research Institute, University Health Network (UHN) 3Peter Munk Cardiac Centre, UHN 4Ted Rogers Centre for Heart Research, Translational Biology and Engineering Program #These authors contributed equally

Introduction: Ischemic heart disease following myocardial infarction (MI) is a leading cause of death worldwide. Until recently, cardiac macrophages, the most abundant immune cell type in the heart, were considered a single cell type, leading to conflicting results for their role in tissue injury and repair. Using single-cell RNA-Seq, our group has resolved 3 distinct cardiac macrophage subsets: TIMD4+CCR2- (TIMD4+) macrophages that mediate tissue repair, TIMD4-CCR2+ (CCR2+) macrophages that exacerbate injury, and TIMD4-CCR2- macrophages involved in antigen-presentation. We have also extended these observations beyond the heart in the murine lung, liver, kidney, and brain. The major limitation preventing the therapeutic exploitation of tissue-reparative cardiac TIMD4+ macrophages from stem cells is that we do not know the epigenetic and transcriptional mechanisms that drive their lineage-specific development.

Methods: We performed single-cell ATAC-Seq (assay for transposase-accessible chromatin) on total macrophages as well as bulk ATAC- and RNA-Seq of each individual macrophage subset from the hearts and lungs of adult mice. Then, we employed bioinformatics techniques to determine the patterns of chromatin accessibility that underlie transcriptional differences between each subset and each organ.

Results: Bioinformatics analysis of single-cell ATAC-Seq of total macrophages from the heart and lung reveals the existence of distinct macrophage clusters that mirrors what we have previously observed at the level of transcription via single-cell RNA-Seq. This suggests that macrophages organize into clusters at the chromatin level that correlate with their distinct gene expression profiles. To take a closer look at these differences, we then analyzed the chromatin and transcription of each individually sorted macrophage subset in bulk, and contrasted chromatin accessibility between each subset within each organ to determine subset-specific differences, and between subsets across organs to determine tissue-specific differences. This allowed us to characterize the differential accessibility of key genes and identify the lineage-defining transcription factors that specifically determine the TIMD4+ cardiac macrophage cell fate, providing essential clues into how their phenotype is induced and maintained in a steady-state environment.

Conclusion: These findings provide a framework for understanding how cardiac macrophages acquire subset-specific phenotypes and inform future efforts to develop post-MI cell therapies.

Gastric acid and escape to the systemic circulation represent major bottlenecks to host colonization by Citrobacter rodentium

Sarah E. Woodward1,2, Stefanie L. Vogt2, Jorge Peña-Díaz1,2, Ryan A. Melnyk2, Antonio Serapio-Palacios2, Madeline Wang2, Laurel M. Neufeld1, Kelsey E. Huus1,2, Cara Haney1,2, B. Brett Finlay1,2,3

1Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada 2Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada 3Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

Introduction: Diarrheal disease remains a leading cause of death in children under the age of five. Citrobacter rodentium (CR) is a natural murine pathogen used to model human diarrheal disease caused by pathogenic Escherichia coli. Many factors influence how CR infection progresses, including the host immune response and the microbiota populating the GI tract, which can act as barriers to colonization. Studies monitoring fecal shedding during CR infection of mice indicate that tight ‘bottlenecks’ occur, which restrict pathogen population size.

Methods: To investigate the nature of bottleneck events during CR pathogenesis, we used a technique called Sequence Tag- based Analysis of Microbial Populations (STAMP) which involves high-throughput DNA sequencing of wild-type, genetically barcoded organisms. Here we use a barcoded library of equally fit CR to infect C57Bl/6 mice. By sectioning the GI tract and systemic organs at early and peak infection, we were able to measure population size at various tissue sites, and quantify bottleneck events.

Results: A founding population of only 10-130 cells exists at each GI site, despite an inoculum dose of 108 CFU. Even the cecal patch, a preferred niche of CR, has one of the smallest bottleneck sizes (~50 cells) yet one of the largest pathogen burdens (~1.5x10^8 CFU/g). Founding population size differs between mucosal and lumenal CR subpopulations. Most of the inoculum was lost almost immediately after gavage, implicating gastric pH as a major bottleneck to early infection. Neutralizing gastric pH dramatically increases the downstream founding population size of CR. Exit of the GI tract into the systemic circulation, and subsequent colonization of distal organs, represents another infectious bottleneck, with one single barcode accounting for 77-87% of all CR harvested from spleen samples.

Conclusion: The GI tract is a severely restrictive environment for invading CR. We identify potential barriers to CR colonization, which could potentially be manipulated to control disease course. Of significance is how neutralizing stomach pH dramatically increases the population size of invading CR in the upper GI tract, particularly as recent studies indicate a current overuse of gastric acid suppressants, which may be eliminating a crucial early barrier to enteric infection.

Rapid identification and antimicrobial resistance prediction of Mycobacterium tuberculosis directly from patient sputa by use of whole genome sequencing

Michelle Wuzinski1,2, Meenu Sharma1,2 , Hafid Soualhine1,2,

1University of Manitoba, Department of Medical Microbiology 2National Reference Centre for Mycobacteriology, National Microbiology Lab

Background: Tuberculosis is primarily a respiratory disease caused by the bacterium Mycobacterium tuberculosis (MTB), belonging to the M. tuberculosis complex. Global increases in drug resistant isolates highlight the need for rapid and high- resolution tests to identify and predict drug resistance of MTB. Current culture-based identification methods are slow and labour intensive. DNA extraction from sputum is a viable solution but it is challenging due to presence of other bacterial and human DNA. Rapid detection of MTB from sputa using whole genome sequencing (WGS) has the capability of reducing turn-round times, providing antimicrobial resistance prediction and identification in a single assay.

Aim: The aim of the project is to develop a method of mycobacterial DNA extraction directly from sputum for the purpose of identifying species and predicting antimicrobial resistance to first and second-line MTB drugs.

Methods: Extraction protocol is validated using TB-negative sputum spiked with Mycobacterium bovis BCG. Sputum is decontaminated using sodium hydroxide/N-acetylcysteine to liquefy and remove contaminants. Protocol is developed based on osmotic lysis of eukaryotic cells and DNAse I treatment for host-DNA depletion, and mycobacterial DNA extraction using a modified Qiagen UltraClean Microbial Kit protocol and subject to WGS. DNA will be evaluated using Illumina before testing with Oxford Nanopore. For bioinformatic analysis, Kraken is used to asses sequence contamination, BioHansel and Mykrobe Predictor are used for genotyping and speciation/drug resistance prediction, respectively.

Results: Illumina sequencing was performed to asses DNA quality. Of 12 currently analyzed samples that passed sequencing, 50% had correct speciation and drug susceptibility predictions. Kraken showed that human cells lysis combined with DNase I removed large amounts of host DNA. 4 samples failed to be correctly genotyped by BioHansel due to coverage issues. 3 samples failed resistance prediction, one produced a discrepant result while 2 others failed to detect a resistance-conferring mutation present in M.bovis BCG.

Conclusion: Of currently sequenced DNA, those that achieved ≥ 20% mapping to reference were successfully able to be genotyped and give correct drug resistance profiles. Osmotic lysis combined with DNase I treatment was able to remove human DNA however further work is in progress to remove non-mycobacterial bacteria DNA present in sputum samples.

MXene Quantum Dots for Immunomodulation and Prevention of Allograft Vasculopathy

Weiang Yan1, Alireza Rafieerad1, Keshav Narayan Alagarsamy1, Abhay D. Srivastava1, Rakesh C. Arora1, Sanjiv Dhingra1

1University of Manitoba

Allograft vasculopathy is an aggressive form of accelerated atherosclerosis that manifests uniquely in transplanted hearts, lungs and kidneys. Activated blood vessel endothelial cells (ECs) stimulate alloreactive CD4+ and CD8+ T-lymphocytes to result in sustained inflammation. MXenes, an emerging class of transition metal carbides, have recently been shown to have unique immunomodulatory properties that may be leveraged to treat allograft vasculopathy. In this study, we present the synthesis, characterization and application of novel zero-dimensional tantalum carbide MXene (Ta4C3Tx) quantum dots for immunomodulation. A facile hydrofluoric acid-free etching protocol was developed to synthesize zero-dimensional MXene quantum dots (MQDs) from bulky Ta4AlC3 MAX phase. The resultant MQDs are 3 to 30 nm in diameter and are surface modified with carboxyl, hydroxyl and amine functional groups for biological interactions. Using an in vitro co-culture system, we found that MQDs interact with activated human ECs to reduce activation and pro-inflammatory Th1 polarization of allogeneic CD4+ lymphocytes. Mechanistically, we showed that treatment with MQDs significantly decreased endothelial surface expression of the T-cell costimulatory molecule CD86. Furthermore, when applied in an in vivo rat model of allograft vasculopathy, treatment with MQDs reduced lymphocyte infiltration and preserved medial smooth muscle cell integrity within transplanted vessel segments. Taken together, these findings suggest that these novel MQDs have potential as an effective treatment to prevent allograft vasculopathy.

Targeting TET2 mutant clonal hematopoiesis of indeterminate potential

Yitong Yang1, Amit Subedi2, Steven Chan1,2

1Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada 2Princess Margaret Cancer Centre, Toronto, Ontario, Canada

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition that occurs in individuals carrying hematologic malignancy-associated somatic mutations. Ten-eleven-translocation 2 (TET2) plays an essential role in DNA demethylation and is recurrently mutated in CHIP carriers. Pathological loss-of-function mutations on TET2 are haplo-insufficient and are associated to elevated risk of cardiovascular diseases and hematologic neoplasms; however, currently no intervention for TET2 mutant CHIP is available.

Methods: to study the phenotypes of TET2 mutations in an isogenic in vitro model, I harvested bone marrow HSPCs from mice carrying wildtype (Tet2+/+) or catalytically inactive TET2 (Tet2-/-) and established an immortalized cell line system by overexpressing the transcription factor HOXB4 through retroviral transduction. Subsequently, I carried out an in vitro chemical library screen using 37 small-molecule inhibitors with well-characterized potency and selectivity on epigenetic modifying enzymes in HOXB4 cells.

Results: The screen demonstrates that DOT1L inhibitor SGC0946 selectively reduces the proliferative advantage and induces apoptosis in Tet2-/- HOXB4 cells. Tet2-/- HOXB4 cells also show higher sensitivity than Tet2+/+ cells to another DOT1L inhibitor EPZ5676. In a subsequent assay with untransformed bone marrow HSPCs, pharmacological DOT1L inhibition reduces clonogenic capacity of Lin- Tet2-/- bone marrow cells and causes differentiation of myeloid progenitors. Reduced proliferative advantage is also seen in Tet2-/- bone marrow HSPCs transduced with Dot1l shRNAs. To elucidate the mechanisms that underpin DOT1L-mediated synthetic lethality in TET2 mutant cells, I conducted RNA-seq on HOXB4 cells and noticed prominent upregulation of thrombopoietin receptor (MPL) in Tet2-/- cells compared to wildtype. Intriguingly, Mpl was significantly downregulated in Tet2-/- cells following SGC0946 treatment. Genetic knockdown of Mpl and inhibition of the downstream kinase JAK2 with ruxolitinib dramatically reduce the competitive advantage of Tet2-/- HOXB4 cells. In addition, phospho-STAT5 is also strongly diminished in Tet2-/- cells by DOT1L inhibition.

Conclusion: the results suggest that homozygous loss-of-function mutations on TET2 induce a higher dependency on DOT1L in HSPCs for their proliferation and survival, potentially through transcriptionally activating MPL. Inhibiting DOT1L or targeting MPL/JAK/STAT signaling can potentially eliminate TET2 mutant clones and terminate CHIP progression in TET2 mutation carriers, thereby preventing subsequent pathological transformations.

Community-driven genomics of Helicobacter pylori in Arctic Indigenous communities unravels global signals of circulating strains

Jeremiah Yarmie1, 2, Natalie Knox1, 2, Morag Graham1, 2, Janis Geary3, Kaisa Thorell4, Karen Goodman3

1University of Manitoba, Winnipeg, MB 2Public Health Agency of Canada, Winnipeg, MB 3CANHelp Working Group, University of Alberta, Edmonton, Alberta 4University of Gothenburg, Gothenburg, Västra Götaland

Background: Helicobacter pylori is a leading cause of stomach cancer worldwide. Prevalence is often a sign of inequity. Carriage of H. pylori and resulting gastric diseases are more common and severe in Northern Indigenous communities compared to Southern Canada. Helicobacter pylori is a public health concern in many of these communities.

Objectives: The primary goal of this project is to determine the phylogenetic structure of Arctic, Manitoban, and global H. pylori isolates based on genomic data. We wish to investigate if circulating H. pylori strains have unique genetic features, for example in key virulence factors such as the cag pathogenicity island, that may be contributing to increased prevalence and disease severity.

Methods: Helicobacter pylori isolated from gastric biopsies collected from various CANHelp research partners and Southern Manitoba communities were cultured, underwent DNA extraction, and sequenced by Illumina MiSeq short-read sequencing technology. Sequences underwent quality control, filtering, and adaptor trimming using fastp. Contaminating reads were classified based on taxonomic grouping using Kraken2 and removed using the KrakenTools script suite. Genomes were assembled using the de novo assembler tool SPAdes and were evaluated and assessed for quality using QUAST. Genome annotation was done using the Prokka tool and manually curated using a reference annotation. Genome phylogeny was determined using the k-mer based tool Mashtree.

Results: Isolates from Southern Manitoba classified according to H. pylori sequence types from across the world based on phylogenetic clustering. The branch lengths of these clusters suggest that they do not share a recent common ancestor. In contrast, Isolates from the Arcticclustered into four distinct sequence type clades: hspAfrica1, hspNEurope, hspEAsia, and hspIndigenousNAmerica. These clades have short branch lengths, implying they may share a recent common ancestor and that circulating strains in CANHelp communities are distinct. Of these four clades, only the hspIndigenousNAmerica isolates lack the cag pathogenicity island.

Conclusions: H. pylori from Southern Manitoba cluster with isolates from various world geographies. Arctic isolates clustered as tighter clades with African, European, Asian, and Indigenous American signal. Indigenous strains in the Arctic lack the cag pathogenicity island, while African, European, and Asian strains maintain the locus, and my contribute to increased disease severity and prevalence in the Arctic.

Early evolutionary history HPG axis genes related to reproduction

Tamanna Yasmin, Sara V. Good and Margaret F. Docker

1Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada 2Department of Biology, University of Winnipeg, Winnipeg, Manitoba, Canada

Lampreys, extant representatives of ancient jawless vertebrates, are a crucial model organism for understanding the early evolutionary history of vertebrate reproduction. A draft of the sea lamprey genome derived from male testes was completed in 2018, and in 2020, a chromosome-based assembly was published providing valuable insight into the early evolutionary history of vertebrates. Jawless fish are the first extant lineage to possess a pituitary and are models for the development of the hypothalamic-pituitary-gonadal (HPG) axis signaling. The HPG axis is the key regulator of vertebrate gonadogenesis, starting with the stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropins (GTH) in the brain. Two GTHs are found in gnathostomes, i.e., luteinizing hormone (LH) and follicle- stimulating hormone (FSH). These hormones are dimers composed of an α and β subunits, in which α subunits are encoded by the same gene, and β subunits are encoded by different but homologous genes. FSH and LH play distinct roles in vertebrate biology, signaling via their cognate receptors, FSHR and LHR, respectively. In contrast, lampreys have a single GTH which is composed of dimers encoded by the GTHβ and a more primitive, GTHA2 gene, and possess a single GTH receptor. Here, RNA-Seq analysis was performed on a total of 28 gonad samples of male and female sea lamprey to identify genes associated with gonad development. A reference-guided de novo assembly pipeline was designed to allow aligning reads to the reference genome without the aid of reference annotation in order to identify novel genes. The genes associated with lamprey HPG axis were identified from sea lamprey genome. A comparative genomic analysis was done to compare the change of these genes from lamprey to other model vertebrates, i.e., human, chicken, zebrafish, and mouse. This study was done to understand the origin of these genes in vertebrates and to get further insight on how conserved the role of these genes across later model organisms.

Detection of SARS-CoV-2 Viral Particles using Direct, Reagent-Free Electrochemical Sensing

Hanie Yousefi1‡, Alam Mahmud2‡, Dingran Chang1‡, Jagotamoy Das1, Surath Gomis2, Jenise B. Chen3, Hansen Wang1, Terek Been4, Lily Yip5, Eric Coomes6, Zhijie Li4, Samira Mubareka5, Allison McGeer7, Natasha Christie8,Scott Gray-Owen4,8, Alan Cochrane4, James M. Rini4,9, Edward H. Sargent2, Shana O. Kelley1, 3, 9,10*

1Leslie Dan Faculty of Pharmacy, 2The Edward S. Rogers Sr. Department of Electrical and Computer Engineering, 3Department of Chemistry 4Department of Molecular Genetics, 6Department of Medicine, 7Department of Microbiology, 8Combined Containment Level 3 Unit, 9Department of Biochemistry, 10Institute of Biomaterials and Biomedical Engineering

Introduction: The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. This report is the first virus-detecting assay that uses the kinetic response of a probe-virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detecting, within five minutes, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.

Methods: A new electrochemical sensing mechanism developed in our laboratories that studies molecular motion on electrode surfaces has been used to detect the whole viral particle. First, antibodies specific to spike protein of SARS-CoV- 2 virus was conjugated to DNA probes and redox labels. Second, the whole molecular probe (aka molecular pendulum) was immobilized on gold electrode surface. The electrochemical readout used was chronoamperometry (CA). The bound vs. unbound states of the probes to the viral particle was monitored via measuring the travel time of the probes to the surface (movement induced with CA)

Results: A handheld device consisted of the molecular assay described above, and potentiostat were developed that could detect SARS-CoV-2 infection in Covid-19 infected hamsters as well as in infected patients’ saliva samples. The test results are available in less than 5 minutes with high specificity, sensitivity and robustness.

Conclusion: This new generation of platform point of care diagnostic devices holds the potential to be used in many different disease monitoring such as respiratory infections and chronic disease.

Microglia-derived C1q is required and sufficient for myelination in central nervous system

Qiang Yu, Nan Zhang, Teng Guan, Jiming Kong

Department of Human Anatomy and Cell Science, University of Manitoba

Introduction: Myelin is formed by oligodendrocytes in the CNS. It provides insulation and nutritional support for its ensheathed axons and is essential for rapid and efficient conduction of impulses along the axons. Deregulation of myelinaton causes neurological disorders such as leukodystrophies and multiple sclerosis. However, mechanisms that govern CNS myelination have not been fully understood. In an effort to understand how myelination is initiated, we have found that the presence of activated microglia is required for oligodendrocytes to make contact with neuronal axons. We have further identified that the molecule complement component 1q (C1q) derived from microglia is required and sufficient to initiate myelination. The study provides a mechanism for initiation of myelination and may provide insights into treatment of demyelinating diseases.

Methods: Spinal cord tissue culture from E14 rat embryos and neural stem cell culture were performed. Immunoprecipitation (IP) was used to test the existence of C1q in both spinal cord culture group and stem cell culture group. Immunofluorescence staining was used to evaluate myelination conditions by using specific markers for myelin (PLP) and axons (pNF-H).

Results: Formation of myelin started to occur when cells from brain or spinal cord tissues were co-cultured for about 3 weeks. Neural stem cells were able to differentiate neurons, oligodendrocytes and astrocytes but not microglia. There was no myelination in co-culture of cells differentiated from neural stem cells. By contrast, addition of microglia or microglia- conditioned medium induced massive myelination in the neural stem cells culture group. To search for molecular factors from microglia, we focused on C1q and found that microglia-conditioned medium with C1q stripped abolished its myelination-inducing activity. IP results showed existence of C1q in spinal cord culture, which favored myelination. Supplementation of spinal cord culture medium or C1q alone remarkably rescued myelination in co-culture of cells derived from neural stem cells.

Conclusion: C1q is required and sufficient for CNS myelination in vitro, which may serve as the key regulator in myelination and become a novel target for further studies in this field. We are currently verifying the in vitro data by using conditional C1q knockout mice.

Construction and application of prevention and control scheme of respiratory infectious diseases in outpatients based on information technology

Huang Yuyan1, Peng Bo1, Guo Shengmin2, Zheng Silin2

1School of Nursing, Southwest Medical University 2Affiliated Hospital of Southwest Medical University

Respiratory infectious disease(RID) has become a major public health and healthcare issue due to its multiple transmission routes, high infectiveness, and wide range of susceptible populations. As SARS, Avian Influenza and COVID-9 have broken out in recent years, a medical service mode called “Internet+” is proposed by the NHSA (national healthcare security admission) and various guidelines to optimize the consultation process. As one of the designated hospitals for the treatment of COVID-9 in Sichuan. It’s our responsibility to practice it initiatively. During the epidemic, we have developed a plan for regular prevention and control and a plan for scattered outbreaks based on domestic and international experience, which contributes to guiding the establishment of IT-ased outpatient RID prevention and control programs. It’s also conducive to combining human and machine protection, improving cooperation mechanisms to improve the efficiency of epidemic prevention and control. (1)Pre-onsultation: based on the original intelligent outpatient clinic with time slots, we will promote the knowledge of RID to the people who visit the clinic. Primary prevention, shift forward the prevention and control gates, the health code will be checked at the entrance, and temperature will be measured by automatic infrared thermography. Patients need to scan the traceable place code, if there is a suspected case, the route map can be backtracked to screen the close contact group in time and reduce the cross-nfection rate inside and outside the hospital. (2)In the clinic: use intelligent outpatient in-ospital navigation to optimize the consultation process and reduce the risk of mass gathering. Secondary prevention, re-heck at the triage, conduct epidemiological survey, and broadcast health education knowledge through the Internet of Things such as LED screen. Tertiary prevention, specialist physician re-heck temperature, during the whole treatment, both of them will under strict protective measures. (3)Post-isit, build a cloud platform to achieve a seamless connection between hospital-ommunity-ome. The population will be fully taught good living habits to reduce the occurrence of respiratory infectious diseases through the internet and fiber optics,etc. During the implementation of this program, a survey of usage is conducted to assess the effectiveness.

Inhibition of the STING-mediated interferon response by novel oxidative posttranslational modification

Zamorano Natalia1,2, Fortin Audray1, Caron Elise1, Chartier Stéfany1 and Grandvaux Nathalie1,2

1CRCHUM, Montréal, Québec, CA. 2Université de Montréal, Québec, CA.

Accumulating evidence supports a role of reactive oxygen species (ROS) in the regulation of several pathways involved in virus-host interactions, particularly those leading to the induction of antiviral type I and III Interferons. Dysregulation of these pathways lead to autoimmune and inflammatory diseases. The redox regulation of virus-host interactions is well documented, but the underlying mechanisms of the redox regulation remain ill-defined. ROS are known to modify signaling protein structure and activity through reversible oxidative post-translational modifications of Cysteines (Cys ox-PTM). To unveil the Cys ox-PTMs that affect signaling proteins involved in the innate antiviral response, we performed a proteome wide identification of the Cys ox-PTMs induced by ROS in vivo using maleimide-based bioswitch labeling coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification allowed identification of Cys ox-PTMs in proteins involved in processes that impact the host-virus interaction, including protein translation, intracellular protein and RNA localization and immune response. Among these, we uncovered the oxidation of STING, the central adaptor of the innate immune type I interferon pathway induced upon detection of DNA viruses. We provide the first in vivo demonstration of reversible oxidation of Cys148 and Cys206 of STING. Proteomic analyses led us to establish a new model in which Cys148 oxidation is constitutive, while Cys206 oxidation is inducible by oxidative stress or by the natural ligand 2’3’-cGAMP. Using in silico molecular modeling and mutational analyses, we found that STING oxidation on Cys206 plays an inhibitory role to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This study establishes a direct mechanism by which ROS control the cGAS/STING-dependent innate immune response. It provides new ground for the design of therapies targeting STING relevant to viral infections, such as vaccination, but also to autoinflammatory disorders.

CELLULAR DYNAMICS OF IMMUNE EVASION DURING LEISHMANIA MAJOR INFECTION

Romaniya Zayats, Zhirong Mou, Atta Yazdanpanah, Wan Koh, Paul Lopez, Jude E. Uzonna and Thomas Murooka

University of Manitoba, Faculty of Health Sciences, Department of Immunology, Winnipeg, Manitoba, Canada

Introduction: There is a balance between the immune response and parasite escape mechanisms. This leads to an establishment of immunity to reinfection which only exists with the presence of the parasite in the host. Leishmaniasis is a neglected tropical disease with no cure or vaccination strategy. Leishmania major parasites elicit a strong T cell response, yet evade complete clearance and persistently infect a small pool of cells. This mechanism of immune evasion remains unclear.

Methods: A novel TCR transgenic mouse model, where T cells recognize the immunodominant Leishmania- glycosomal phosphoenolpyruvate carboxykinase (PEPCK) peptide on L. major parasites is combined with in vitro and in vivo 2-photon microscopy approaches to visualize the dynamics of anti-Leishmania CD4 T cell responses and to characterize mechanisms that restrain their effector functions.

Results: In vitro live-cell imaging studies demonstrate that monocyte-derived macrophage:T cell interaction dynamics were transient at steady-state, but prolonged upon antigen recognition. This activation leads to a production of high levels of IFN and can be significantly suppressed by PEPCK-specific Tregs in vitro, as compared to polyclonal Treg controls. Co- culture of PEPCK-specific CD4+ T cells, L. major-infected monocyte-derived macrophages, and Tregs shows that antigen activation leads to a substantial increase in IL-10 levels, while decreasing IL-12, TNF, and IL-2 production in the culture. Intravital microscopy studies characterizing PEPCK-specific CD4+ T cell migration dynamics and tissue localization within skin lesions directly in live mice show a significant recruitment of adoptively transferred effector T cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition. We are currently characterizing whether effector T cell responses are altered in healed lesions, where persistently-infected cells are readily observed. Collectively, our findings show for the first time that Leishmania-specific Tregs influence effector CD4+ T cell responses and this could be a mechanism that derives antigen persistence in L. major infection.

Significance: The goal of this study is to identify barriers that need to be overcome in order to achieve complete parasite clearance in infected individuals. This work will describe the dynamic aspects of concomitant immunity generation and provide insights into the role of antigen-recognition in Treg suppression of immune responses.

Immune-cell Signature Predicts Survival in Human Papillomavirus-associated Head and Neck Cancer

Peter YF Zeng1, Matthew J. Cecchini2, Anthony C. Nichols1

1Department of Otolaryngology - Head and Neck Surgery, University of Western Ontario, London, Ontario, Canada 2Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada

Background: Human papillomavirus-driven (HPVP+P) head and neck squamous cell cancers (HNSCCs) is the fastest rising cancer in North America. There is significant interest in treatment de-escalation for these patients given the generally favourable prognosis. However, 15-20% of patients recur after primary treatment, reflecting a need for improved risk- stratification tools. Thus, we aimed to develop a prognostic method for predicting survival of patients with newly diagnosed HPVP+ PHNSCC.

Methods: We performed RNA-sequencing (RNA-seq) on a cohort of HPVP+ PHNSCC tumours. The LASSO algorithm was used to derive two prognostic scores, UWO3 and UWO4, which were evaluated using four independent HPVP+ PHNSCC RNA-seq datasets and a tissue microarray dataset.

Results: Analysis of HPVP+ PHNSCC tumours revealed three distinct TME subtypes associated with patient survival: immune rich, immune desert, and mixed. The four gene immune score, UWO4, was able to stratify patients into the three subtypes and was stongly associated with disease-free survival in the discovery and two external RNA-seq cohorts. We further derived a reduced score UWO3 based on three genes that have antibodies used routinely in clinical pathology labs. The UWO3 score, assessed by immunohistochemistry in a tumor microarray of HPVP+P HNSCC patients, was predictive of disease-free survival. Finally, UWO4 was able to identify patients who respond to 30 Gy radiation in two treatment de-escalation cohorts.

Conclusions: The UWO4 and UWO3 scores have the potential to guide therapeutic decision-making in HPVP+P HNSCC. Further clinical validation through prospective trials are required to establish their use in guiding therapeutic intensity.

Estrogen promotes lymphatic contractility by suppressing hemorrhagic shock-induced endoplasmic reticulum stress

Jia-Yi Zhai1, An-Ling Kang1, Cai-Juan Li1, Hui-Bo Du1, Meng Zhao1

1Institute of Microcirculation, Hebei North University, Zhangjiakou, PR China.

Background: The lymphatic contractility dysfunction is associated with the deterioration of hemorrhagic shock (HS). Endoplasmic reticulum stress (ERS) involves in the HS-induced organ injury, while estrogen treatment alleviates HS-induced organ injury. Here, we sought to evaluate the therapeutic effect of estrogen on the HS-induced lymphatic contractility dysfunction and the underlying mechanism.

Methods: Wistar male rats were subjected to HS. During resuscitation, estrogen- and ERS-related drugs were administrated via subcutaneous injection. ERS marker GRP78 was detected using Western blot. The survival rate of HS rats was recorded. The lymphatic contractility and reactivity were detected by using dynamic visualization microvascular observation system and vascular tension and ion concentration measurement system. Primary lymphatic smooth muscle cells (LSMCs) were cultured under hypoxia and reoxygenation (H/R) condition. ERS agonist tunicamycin (TM) was added to verify the effect of ERS on LSMC contraction to noradrenaline (NE).

Results: The 17β-estradiol (E2) administration reduced the expression of GRP78 induced by HS in lymphatic tissues, suggesting the inhibitory role of E2 on ERS. Meanwhile, we found E2 and ERS inhibitor 4-PBA promoted the survival HS rats in the first 72 hours. The administrations of E2, estrogen receptor (ER)-α agonist propyl pyrazole triol (PPT), ER-β agonist diarylpropionitrile (DPN), GPR30-selective agonist G1, 4-PBA significantly enhanced the contractility of mesenteric lymphatic vessel following hemorrhagic shock in vivo and in vitro. ICI 182,780 (ERα and ERβ selective inhibitor) and G15 (GPR30 selective inhibitor) partly abolished the beneficial effects of E2 treatment on the lymphatic contractility, confirming the ER receptor-dependent effects of E2. Furthermore, ERS agonist XCT-790 abolished the beneficial effects of E2, PPT, DPN, and G1 on the lymphatic contractility. Furthermore, the administrations of E2, PPT, DPN, and G1 inhibited H/R- and TM-induced hypo-contractility, and suppressed TM-induced ERS marker expression in LSMCs.

Conclusion: E2 promoted the lymphatic contractility after hemorrhagic shock by the inhibition of estrogen receptor- dependent ERS.

Low-grade Serous Ovarian Cancers Modelled with Human Fallopian Tube Organoids and Single Cell Sequencing

Joyce Zhang1,2, Dawn Cochrane1, Kieran Campbell1, Minh Bui1, Germain Ho1, Genny Trigo1, Winnie Yang1, Cindy Shen1,Sorab Shah1, David Huntsman1,2

1Department of Molecular Oncology, BC Cancer Research Centre; 2Department of Pathology and Laboratory Medicine, University of British Columbia

Introduction: Ovarian cancers are the most common gynecologic malignancies. Low grade serous ovarian carcinoma (LGSOC) is a rare tumor, accounting for 5% of all ovarian cancer cases. Most of LGSOCs are characterized by high fatality rates over the long term, with only 10-20% of women surviving 10 years after diagnosis, due suboptimal response to current chemotherapies. Understanding the molecular events is crucial for developing more informed therapeutic options. LGSOC harbours a relatively stable genome, with common activating mutations in BRAF, KRAS and NRAS. Recently, NRAS mutation (Q61R) were found to co-exist with EIF1AX mutations (G8E) in LGOSC, and the two mutated proteins functionally cooperate. Low incidence of this disease means it is poorly understood, and the resulting lack of available models further limits the study of the underlying mechanisms. We therefore propose to utilize organoid cultures, which consist of 3D multicellular units that resemble in vitro a tissue or organ of body.

Method: To reflect genetic background and cell of origin of LGSOC, NRAS(Q61R) and eIF1a(G8E) mutant proteins were overexpressed via lentiviral transduction in primary normal human Fallopian tube tissues. After allowing organoids to establish, gene expression alterations were resolved with single-cell RNA sequencing (scRNA-seq) 2 weeks after transduction. Organoid cytology was assessed for signs of transformation. Patient-derived tumor organoids (PDTOs) were cultured to assess how well our LGSOCmodelling organoids (LMOs) recapitulate the histological features of patient tumours.

Results: LMOs showed cytologic signs of transformation (increased nuclear/cytoplasmic ratio, prominent nucleoli, cellular pleomorphism). Papillary structures, a major histologic characteristic of LGSOC were observed in LMOs. PDTOs showed similar cytological features and organization as LMOs. From scRNAseq, we identified genes up-regulated in double-mutant compared to single-mutant organoids such as CA125 and TACSTD2. CA125 is one of the earliest identified biomarkers for ovarian cancer and has remained to be a useful serum marker despite limited sensitivity and specificity; whereas TACSTD2 overexpression has been found to correlate with a chemo-resistant, aggressive malignant phenotype.

Conclusion: We established a novel model that largely recapitulated LGSOC cytology by introducing co-occurring mutations into Fallopian tube tissues. Genes upregulated in double mutants included wellcharacterized biomarker (CA125) and a potential biomarker or therapeutic target (TACSTD2). Our work will be crucial for developing more targeted treatment options.

The anti-apoptosis of Fos in ischemic stroke and its mechanism

Yuxuan Zhang1, Qiancheng Mu1, Long Gu2

1Department of Neurosurgery, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, China 2Department of Emergency Medicine, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China

Based on our early bioinformatics analysis of the brain tissue of rats with middle cerebral artery occlusion (MCAO) and peripheral blood data of ischemic stroke patients, we found that the Fos, immediate early genes are strongly associated with the prognosis of ischemic stroke. To further evaluate the potential function of Fos, we established oxygen-glucose deprivation models (OGD) in HT22 neurons. Real-time quantitative (Rt-qPCR) and Western blot (WB) results showed that the Fos level was significantly increased at 3H after OGD and returned to normal at 6H, which was consistent with the sequencing results. By comparing the Co-Immunoprecipitation (Co-IP)with Fos protein at 0H and 3H after OGD, we found that the lysosomal membrane protein ITFG2 significantly increased, and lentivirus knockout of Fos could significantly increase the apoptosis of neurons. We speculated that after ischemia and hypoxia, Fos might inrease the cell apoptosis by increasing the lysosome and increasing autophagy of neurons. In conclusion, Fos has an important relationship with the prognosis of ischemic stroke. This gene can be used as a biomarker to provide a potential biological target for improving neuronal apoptosis and neurofunctional prognosis after ischemic stroke.

Identification of Candidate Genes Associated with Charcot-Marie-Tooth Disease by Network and Pathway Analysis

Min Zhong1, Qing Luo1, Ting Ye1, XiDan Zhu1, JinBo Liu1

1Department of Laboratory Medicine, The Affiliated Hospital of Southwest Medical University

Charcot-Marie-Tooth Disease (CMT) is the most common clinical genetic disease of the peripheral nervous system. Although many studies have focused on elucidating the pathogenesis of CMT, few focuses on achieving a systematic analysis of biology to decode the underlying pathological molecular mechanisms and the mechanism of its disease remains to be elucidated. So our study may provide further useful insights into the molecular mechanisms of CMT based on a systematic bioinformatics analysis. In the current study, by reviewing the literatures deposited in PUBMED, we identifified 100 genes genetically related to CMT. Then, the functional features of the CMT-related genes were examined by R software and KOBAS, and the selected biological process crosstalk was visualized with the software Cytoscape. Moreover, CMT specifific molecular network analysis was conducted by the Molecular Complex Detection (MCODE) Algorithm. The biological function enrichment analysis suggested that myelin sheath, axon, peripheral nervous system, mitochondrial function, various metabolic processes, and autophagy played important roles in CMT development. Aminoacyl-tRNA biosynthesis, metabolic pathways, and vasopressin-regulated water reabsorption were signifificantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway network, suggesting that these pathways may play key roles in CMT occurrence and development. According to the crosstalk, the biological processes could be roughly divided into a correlative module and two separate modules. MCODE clusters showed that in top 3 clusters, 13 of CMT-related genes were included in the network and 30 candidate genes were discovered which might be potentially related to CMT. The study may help to update the new understanding of the pathogenesis of CMT and expand the potential genes of CMT for further exploration.