20th Annual McGill Biomedical Graduate Conference

Experimental Medicine Graduate Students’ Society

Published online: 21 October 2020

Cardiovascular and Respiratory Systems

The Role of Memory Gamma Delta T Cells in Hypertension and Vascular Damage

Kevin Comeau¹, Pierre Paradis¹, and Ernesto L. Schiffrin^1,2
¹Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research;
²Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Quebec, Canada
Corresponding Author: Kevin Comeau, email kevin.comeau@mail.mcgill.ca

Abstract

Background: We recently demonstrated that γδ T cells participate in the pathogenesis of hypertension. Evidence also suggests that memory T cells develop during an initial hypertensive episode, sensitizing mice to develop hypertension to further mild hypertensive challenges. However, whether memory γδ T cells develop and play a role in hypertension remains unknown. We hypothesize that memory γδ T cells develop after an initial exposure to a hypertensive stimulus, and that they respond to further mild hypertensive insult.
Methods: 10-12-week-old C56BL/6J mice were exposed or not to a hypertensive challenge (490 ng/kg/min angiotensin II (Ang II), SC) for two weeks, followed by a two-week washout period, and then infused with a subpressor dose of Ang II (140 ng/kg/min Ang II, SC) for two weeks. Blood pressure was measured via telemetry and memory γδ T cells were analyzed by flow cytometry.
Results: Mice exposed to the first hypertensive challenge had a systolic blood pressure ~30 mm Hg higher than the sham group after the subpressor hypertensive challenge. After 14-days of Ang II infusion, effector memory γδ T cells increased 1.4-fold in the spleen, 3-fold in the mesenteric artery perivascular adipose tissue (MA PVAT), and 1.8-fold in the mesenteric lymph nodes. Resident memory γδ T cells increased 2.4-fold in the MA PVAT, 1.8-fold in the bone marrow, and appeared in the Aortic PVAT.
Conclusion: An initial exposure to a hypertensive stimulus sensitizes mice to develop hypertension to a subsequent subpressor hypertensive challenge and results in the development of memory γδ T cells.

Secretome derived from amniotic stromal mesenchymal stem cell spheroids as a cell-free therapy for cardiac repair

Ida Derish^1,2, Kashif Khan^1,2, Renzo Cecere^1,3
¹ Research Institute at the McGill University Health Centre, Montreal, QC
² Division of Experimental Surgery, Department of Medicine, McGill University, Montreal, QC
³ Division of Cardiac Surgery, Department of Medicine, McGill University, Montreal, QC
Corresponding Author: Ida Derish, email ida.derish@mail.mcgill.ca

Abstract

Myocardial infarction (MI) occurs when one of the cardiac coronary arteries is obstructed and the resulting ischemia induces necrosis and maladaptive remodeling. Cardiovascular cells are unable to efficiently proliferate after MI, with no observable regeneration mechanism. Consequently, patients develop heart failure which is a leading cause of death worldwide with no curative therapies available. Over the past twenty years, stem cells have been utilized to regenerate cardiac tissue. However, the low engraftment of injected cells to the site of injury and their lack of differentiation into cardiomyocytes resulted in limited cardiac function improvements. A novel cell-free strategy that bypasses these obstacles utilizes the stem cell secretome. The latter contains soluble factors involved in cell proliferation, angiogenesis and anti-apoptosis. Moreover, stem cell spheroids (3D cultures) have been shown to be more efficacious compared to 2D cultures for tissue repair therapies via i) their greater cardiogenic potential and ii) higher concentration of regenerative factors within the secretome. Our project assesses the regenerative potential of the secretome derived from spheroids made of amniotic stromal mesenchymal stem cells (ASMCs) in vitro and investigates its mechanisms of repair through angiogenesis induction in human cardiac microvascular endothelial cells (HCECs). Neovessel formation was analyzed in vitro via tube formation, proliferation and cell migration assays. Use of 3D cultured ASMC-derived secretome increased metabolic activity and proliferation of HCECs compared to 2D cultured cell secretome. In sum, this study identifies a novel therapy for applications in cardiovascular repair, to increase survival and recuperate heart function after MI damage.

Development and Validation of Supportive Bioreactors for Cardiovascular Tissues

Meera Kanagalingham^1,2, Anthony J. Deegan^1,2, Jake E. Barralet^1,2,3
¹Department of Surgery, Faculty of Medicine, McGill University, Montreal, QC;
²McGill University Health Center, Montreal, QC;
³Division of Orthopaedics, Research Institute of McGill University Health Center, Montreal, QC
Corresponding Author: Meera Kanagalingham, email meera.kanagalingham@mail.mcgill.ca

Abstract

Cardiovascular disease remains a major cause of death worldwide; however, cardiac tissue engineering offers potential with numerous developments, such as biomaterials that can repair and restore function to infarcted hearts. One hurdle that continuously hinders progress is the lack of angio-inductive processes that would enable long-term tissue survival. With that, our lab has previously reported research in this area. This project, therefore, intends to utilize this experience to 1) build and validate an electrical stimulation bioreactor, 2) refine and validate a perfusion bioreactor, and 3) combine the use of both to improve the duration of potential viability of ex vivo venosomes. To do this, our in-house-built electrical bioreactor was first validated using fresh rodent hearts by assessing contractile function. Next, a refined perfusion bioreactor was validated by demonstrating prolonged viability and contractility in rodent hearts. An in vivo-produced venosome surgically implanted for 4 weeks was then perfused for 7 days in vitro. Tissue viability was assessed via immunohistochemical protocols. Rodent hearts were first shown to contract under electrical stimulation for a few hours post surgical removal. Following perfusion, however, rodent hearts were shown to contract under electrical stimulation for up to 24 hours post surgical removal. After perfusing the venosome in vitro, significant viability was observed compared to a static equivalent. Both bioreactors developed and validated throughout this project demonstrate the potential of such technology for improved tissue viability. Additionally, the growth and sustainability of a viable venosome demonstrates our ability to produce and maintain a potentially applicable vascular network.

Vγ6+ γδ T CELLS PLAY A ROLE IN ANGIOTENSIN II-INDUCED HYPERTENSION

Ahmad Mahmoud¹, Antoine Caillon¹, Pierre Paradis¹ and Ernesto L. Schiffrin^1,2
¹Vascular and Hypertension Research Unit, Lady Davis Institute for Medical Research
²Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montréal, Québec, Canada.
Corresponding Author: Ahmad Mahmoud, email ahmad.mahmoud2@mail.mcgill.ca

Abstract

Background: We demonstrated that a small subpopulation of T cells expressing the γδ T-cell receptor (TCR) plays a key role in hypertension. γδ T cells can be subdivided according to the TCR vγδariant (V) subtype. A subpopulation of lungs and skin γδ T cells that are Vγ6+ and produce interleukin (IL)-17A was shown to respond promptly to pneumococcal infection and skin inflammation. However, γδ T cell Vγ subtypes involved in hypertension are still unknown.
Methods: Eleven- to 13-week-old C57BL/6J male mice were infused or not with angiotensin (Ang) II (490 ng/kg/min, SC) for 14 days. The γδ T cell Vγ subtypes were profiled and markers of activation of Vγ6+ γδ T cells were determined by flow cytometry.
Results: In spleen and mesenteric lymph nodes (MLNs) the most abundant γδ T cells Vγ were Vγ1/2+ and Vγ4+ followed by Vγ6+,Vγ5+ and Vγ7+. In thoracic aortic (TA) perivascular adipose tissue (PVAT), the most abundant γδ T cell Vγ was Vγ6+ followed by Vγ4+, Vγ1/2+, Vγ5+ and Vγ7+. In mesenteric artery (MA) PVAT, the most abundant γδ T cell Vγ subtype was Vγ6+ followed by Vγ4+, Vγ7+, Vγ5+ and Vγ1/2+. Ang II increased the frequency of Vγ6+ γδ T cells in the spleen and TA PVAT, whereas it only tended to increase them in MA PVAT.
Conclusion: Vγ6+ γδ T cells were the most abundant Vγ subtype in PVAT. Targeting a γδ T cell variant subtype could be a therapeutic approach to reduce inflammation in hypertension.

Arsenic is a unique modulator of macrophage polarization toward pro-atherogenic phenotypes

Kiran Makhani¹, Nicolas De Jay², Coralie Charpy⁴, Luis Fernando Negro Silva¹, Maryse Lemaire⁴, Claudia L. Kleinman², and Koren K. Mann^1,3
¹Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada;
²Department of Human Genetics, McGill University, Montreal, Quebec, Canada;
³Department of Oncology, McGill University, Montreal, Quebec, Canada;
⁴Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
Corresponding Author: Kiran Makhani, email kiran.makhani@mail.mcgill.ca

Abstract

Arsenic toxicity is a global health concern and is correlated with adverse cardiovascular outcomes, including atherosclerosis. Atherosclerotic plaque formation is a complex process with macrophages being major players in both its initiation and progression. Macrophages accumulate lipids and modulate the microenvironment through secreting cytokines and recruiting other immune cells to the site. There is phenotypic heterogeneity of macrophages residing in plaques, the most prevalent of which are broadly characterized as M1 (pro-inflammatory) and M2 (anti-inflammatory) subtypes in response to microenvironmental cues. Here, we hypothesized that arsenic exerts proatherogenic effects by skewing the relative abundance of pro- and anti-inflammatory macrophages towards pro-inflammatory M1 macrophages. To test this hypothesis, we cultured bone marrow derived macrophages from C57BL/6 mice in vitro and differentiated them into M1 (IFNγ-induced) or M2 (IL-4-induced) phenotypes in the presence or absence of 50 ppb arsenic over 48 hours. Gene expression was assessed by RNA sequencing. Principal Component Analysis showed that unpolarized (M0), M1, and M2 macrophages could easily be distinguished. Surprisingly, arsenic did not result in a separate population of “arsenic-exposed macrophages”, but rather altered different gene expression within each subtype. Within M2 macrophages, many chemokines were altered by arsenic. In particular, CCL17 and CCL22 mRNA and secreted CCL17 and CCL22 protein levels were decreased in the presence of arsenic. These anti-inflammatory chemokines recruit Tregs leading to the plaque resolution. We will correlate arsenic exposures with decreased CCL17/22 to decreased Tregs and increased plaque size in vivo.

All-in-one continuously monitoring device for patients’ vitals

Zanib Nafees¹
¹McGill University Health Centre, Montreal, QC
Corresponding Author: Zanib Nafees, email zanib.nafees@mail.mcgill.ca

Abstract

Background: Continuously monitoring patient vital signs is important in the intensive care unit as unexpected incidents can occur any time. Patient parameters such as heart rate and rhythm, respiratory rate, blood pressure, blood-oxygen saturation, and other vitals are some of the common patient vitals that are needed to be monitored continuously. My aim is to investigate and validate one such physiological monitoring device named VTLAB in an ICU adult population at the McGill University Health Care (MUHC) center. VTLAB is a cuffless and calibration-free personalized monitoring device which continuously measures all vital signs including 1) arterial blood pressure, 2) oxygen saturation, 3) respiratory rate, 4) body temperature, and 5) heart rate. I hypothesize that patients recovering from undergoing cardiac surgery would be able to wear VTLAB and similar readings would be collected to validate VTLAB wearable with other conventional devices.
Method: Patient and setting: This observational study will be conducted in the general medical- surgical/trauma ICU at the MUHC Glen hospital. A convenience sample of 50 adult patients, that underwent cardiac surgery (age > 18) will be recruited for this study. Participating patients will wear VTLAB healthcare smartwatch for a recurring 24-h period.
Result/Conclusion: By comparing the VTLAB wearable with regular devices used in the ICU, I will be able to validate the VTLAB wearable on its accuracy and precision in monitoring patients’ vitals. By adapting a more user-friendly and all-in-one compact device, we will be simplifying the needs of not only the patients but making patient monitoring easier for the caregivers.

P2X7 receptor knockout attenuates angiotensin II-induced hypertension and small artery injury

Brandon Shokoples¹, Pierre Paradis¹, Ernesto Schiffrin^1,2
¹Vascular and Hypertension Research Unit, Lady Davis Institute for Medical Research
²Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Quebec, Canada
Corresponding Author: Brandon Shokoples, email brandon.shokoples@mail.mcgill.ca

Abstract

Introduction: Inflammasome activation by binding of ATP released from damaged cells to the purinergic receptor P2X7 (P2RX7) could play a role in hypertension and vascular injury through release of interleukin (IL)-1β and immune cell activation. P2rx7 knockout (P2rx7–/–) prevented blood pressure (BP) elevation and renal damage in salt-dependent hypertensive models including Dahl-salt sensitive rats and deoxycorticosterone acetate-salt treated mice. However, it is unknown whether P2RX7 plays a role in angiotensin (Ang) II-induced BP elevation and vascular damage.
Methods: Ten to 12-week-old male C57BL/6J male wild-type (WT) and P2rx7–/– mice were sham-treated or infused with Ang II (1000 ng/kg/min) for 14 days. BP was determined by telemetry. Mesenteric artery function and remodeling was determined using pressurized myography. P2RX7 expression in immune cells was determined by flow cytometry. IL-1β secretion from bone marrow-derived macrophages (BMDM) or dendritic cells (BMDC) isolated from WT and P2rx7–/– mice was assessed by ELISA.
Results: P2RX7 knockout abrogated lipopolysaccharide- and ATP-induced IL-1β release from BMDMs and BMDCs. In WT mice, Ang II increased P2RX7 expression ubiquitously across B cells, T cells and myeloid cells in the spleen and bone marrow. P2rx7–/– mice display a reduced pulse pressure throughout Ang II infusion, accompanied by a significantly attenuated systolic BP compared to WT mice. Ang II-infusion induced small artery remodeling and endothelial dysfunction in WT mice, which was blunted in P2rx7–/– mice.
Conclusion: Ang II-induced hypertension influences P2RX7 expression on immune cells and P2RX7 knockout attenuates Ang II-induced hypertension development.

The Role of Sex Hormones on Plaque Instability in Men and Women with Severe Carotid Atherosclerosis

Diana Di Iorio^1,2, Karina Gasbarrino¹, Huaien Zheng¹, Stella Daskalopoulou¹, Terence Hébert³
¹Vascular Health Unit, Department of Medicine, McGill University, Research Institute of McGill University,
²Department of Physiology, McGill University,
³Department of Pharmacology and Therapeutics, McGill University
Corresponding Author: Diana Di Iorio, email diana.diiorio2@mail.mcgill.ca

Abstract

Background: Carotid atherosclerotic plaques can be stable or unstable, the latter more likely to rupture resulting in strokes. The causes of plaque instability are unknown. Men tend to have more unstable plaques than women, yet women have increased mortality rates post-stroke. Sex hormones influence the vasculature differently in men and women; until menopause estrogen offers women protection against cardiovascular disease. We hypothesize that sex hormones and their receptors affect plaque instability, where unstable plaques show higher levels of receptor expression.
Methods: Using liquid chromatography mass spectrometry, we measured circulating levels of testosterone, estradiol, androstenedione, and dehydroepiandrosterone (DHEA) in the blood of patients undergoing carotid endarterectomy. We classified plaques into 4 groups: women stable/unstable and men stable/unstable. We performed immunohistochemistry on plaques to quantify mean percent area stained for estrogen receptor-alpha (ER-a), estrogen receptor-beta (ER-b), G protein-coupled estrogen receptor (GPER), and androgen receptor (AR). qRT-PCR was performed to quantify receptor gene expression in plaques, as well as in peripheral blood monocytes obtained from a subset of our patient population.
Results: No significant differences in circulating testosterone, androstenedione, and DHEA levels were noted between patient groups (N=20 per group). Men had significantly greater staining for ER-a, ER-b, and AR in unstable vs stable plaques (p<0.05). ER-b and AR were not detected in patient-isolated monocytes, and no significant differences in ER-a and GPER expression were found in monocytes.
Discussion: Our preliminary findings indicate a possible association between sex hormone receptor expression levels and plaque instability. Our work may lead to hormone-specific therapies aimed at reverting unstable to stable plaques.

New role of PACE4 in familial hypercholesterolemia via stabilisation of the LDLR protein

Sepideh Mikaeeli^1,2, Roxanne Desjardins³, Edwidge Marcinkiewicz¹, Robert Day³, Nabil G. Seidah¹
¹Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, QC;
²Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC;
³Institut de Pharmacologie de Sherbrooke, Department of Surgery/Urology Division, Faculté de Médecine et des Sciences de la Santé, Sherbrooke, Qc.
Corresponding Author: Sepideh Mikaeeli, email sepideh.mikaeeli@mail.mcgill.ca

Abstract

PCSK9 is the 9th member of the mammalian proprotein convertases family and is implicated in Familial hypercholesterolemia (FH). The inhibition of PCSK9 resulted in a 15-20% reduction in the incidence of cardiovascular disorders (CVDs). Nonetheless, our understanding of the mechanism behind FH has remained largely incomplete to this day. PACE4 (PCSK6) is the 6th member of the convertase family and has important role in prostate cancer and blood pressure regulation. PACE4 is widely expressed in many tissues including liver, but its function therein is unknown. Our objective was to investigate the role of PACE4 in the liver. Our in-situ hybridization assay confirmed that PACE4 is highly expressed in mouse liver. Furthermore, mass-spectrometry and pull-down assays showed a direct interaction between secreted PCSK9 and PACE4 in HepG2 cells. Co-expression of PCSK9 and PACE4 reduced the ability of PCSK9 to enhance the degradation of the LDLR, possibly by inhibiting PCSK9 function on the LDLR. Interestingly, extracellular PACE4 not only increases the levels of LDLR but also improves its function as HepG2 cells exhibited higher LDL uptake, independently from PCSK9. These surprising results raise the possibility that PACE4 might be able to inhibit the function of PCSK9 indirectly via stabilizing the LDLR at the cell surface. In agreement, PACE4 KO mice exhibited a trend toward lower levels of total LDLR in liver. We are presently expanding our results and techniques to better understand this puzzling effect of PACE4 on LDLR.

Cellular and Molecular Biology

RAB-10 functions opposite of the AGEF-1/Arf GTPase/AP-1 pathway to regulate vesicle trafficking

Aida Sobhani^1,2, Olga Skorobogata^1,2, Ali Fazlollahi², Kimberley Gauthier^1,2, Christian E Rocheleau^1,2,3
¹Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.
²Research Institute of the McGill University Health Centre, Montreal, Canada.
³Division of Endocrinology and Metabolism, Department of Medicine, McGill University, Montreal, Canada.
Corresponding Author: Aida Sobhani, email aida.sobhani@mail.mcgill.ca

Abstract

Cell polarity is critical for the function of the many cell types. In C. elegans, basolateral localization of the LET-23 Epidermal Growth Factor Receptor (EGFR) in the vulval precursor cells (VPCs) is required for receiving the LIN-3 EGF-like inductive signal and thus for the development of the vulva. The EGFR signaling pathway is frequently over-activated in many human cancers. A class I/II Arf GTPase pathway regulated by AGEF-1, a homolog of the mammalian BIG1/2 Arf GEFs, antagonizes LET-23 EGFR signaling by promoting its apical trafficking. Loss of the AGEF-1/Arf GTPase/AP-1 pathway results in increased basolateral EGFR and over induction of vulva tissue. Thus, this pathway is required to maintain the proper levels of EGFR at the basolateral membrane to ensure proper vulva induction. We found that the RAB-10 GTPase has novel antagonistic interactions with the Arf GTPase pathway to regulate LET-23 EGFR trafficking and signaling. Hence, it is a positive regulator of EGFR signaling and basolateral localization.
The focus of my project is to find how RAB-10 interacts with the Arf GTPase pathway and which effectors and regulators function with RAB-10 to regulate LET-23 EGFR localization and signaling.
Both RAB-10 and the AGEF-1 and Arf GTPase pathway localize to Golgi and recycling endosomes to regulate polarized trafficking in epithelial cells, however, nothing is known about how their functions are coordinated. Characterizing RAB-10 and its role in promoting signaling is thus of great interest as it will identify novel regulators of EGFR and will determine how these trafficking pathways intersect.

Interferon-γ amplifies airway smooth muscle-mediated CD4+ T cell recruitment by upregulating the secretion of C-X-C-motif chemokine receptor 3 ligands

Rui Sun¹, Joyce H. Jang¹, Anne-Marie Lauzon¹, James G. Martin¹
¹Meakins-Christie Laboratories and Research Institute of McGill University Health Centre, Montreal, QC, Canada
Corresponding Author: Rui Sun, email rui.sun4@mail.mcgill.ca

Abstract

Background: Airway smooth muscle is enlarged in asthma and the degree of which is a strong predictor for airflow limitation and disease severity. T cell infiltrates have been found within asthmatic human ASM, which potentially contributes to airway smooth muscle cell (ASMC) proliferation. In this study, we explored the capability of ASMC to recruit CD4+ T cells and characterized the signaling pathways involved.
Methods: The modified Boyden Chamber assay was adopted to study ASMC-mediated T cell recruitment in vitro. Activated healthy CD4+ T cells were placed in the upper chambers, and healthy ASMCs were seeded in the lower chamber. The amount of CD4+ T cells migrated to lower chamber over 24 hours was assessed. Treatments with chemokine receptor antagonists, siRNAs, or ligand neutralizing antibodies were employed to decipher the identity of the recruitment signal.
Results: Compared to the blank control, the presence of ASMCs in the lower wells enhanced CD4+ T cell chemotaxis. The chemotactic effect was inhibited by C-X-C motif chemokine receptor 3 (CXCR3) antagonist AMG487, as well as the neutralizing antibodies against its ligands CXCL10 and 11, but not CCR3 or CCR5 antagonists. ASMCs constitutively secreted CXCL10, and signaling through CD4+ T cell-derived interferon-γ (IFN-γ) induced STAT1 activation in ASMCs and subsequent upregulation of CXCR3 ligand secretion. Disruption of IFN-γ signaling reduced STAT1 activation, chemokine secretion and CD4+ T cell recruitment by ASMCs.
Conclusion: CXCR3 mediates CD4+ T cell recruitment to ASMCs. CD4+ T cells-derived IFN-γ amplifies the recruitment signals via upregulating chemokine secretion by ASMCs.

Apoptosis as a driver of Tissue Morphogenesis in the mouse Urogenital System

You Chi Tang¹, Khoren Ponsin², Magdalena Schindler¹, Anmar Khadra², Maxime Bouchard¹
¹Goodman Cancer Research Centre and Department of Biochmistry, McGill University.
²Department of Physiology, McGill University
Corresponding Author: Emily Tang, email you.tang@mail.mcgill.ca

Abstract

Apoptosis is a crucial and tightly regulated process during embryonic development for tissue morphogenesis. Apoptotic cell death has been shown not only to sculpt the tissue, but also to act as active driving forces to shape organisms during development. The examples of morphogenetic apoptosis have mainly been described in drosophila, but rarely shown in vertebrates.
Previously, or lab has described an exquisite system of apoptosis involved in early urogenital system (UGS) maturation, where the elimination of common nephric duct (CND) results in ureter-bladder connection. In this system, progressive CND elimination is shown to strictly depend on regulated apoptosis to bring the ureter in contact with the bladder. If this apoptotic rate is altered, diseases such as ureter obstruction and reflux will be resulted.
Recently, we observed that apoptotic cell removal in the CND is proceeded by non-professional phagocytosis by surrounding duct cells, and act as morphogenetic motor to shape the UGS by bringing the ureter closer to the bladder.
Using ex vivo tissue culture experiments and mathematical modeling calculations, we conclude that CND elimination is driven by a rapid phase of apoptotic cell volume decrease and phagocytosis. Whereas apoptotic body degradation within a non-professional phagocyte does not contribute as much to force generation during CND morphogenesis, and it proceeds at a much slower rate.
Together these results identify that apoptotic cell volume decrease and non-professional phagocytosis are drivers for apoptosis-mediated morphogenesis during embryonic mouse development.

Identification of the telomere protein-telomerase domain interactions that regulate telomerase recruitment to telomeres, and cell immortalization

Chu, T.-W^2,3, Lambert-Lanteigne, P.³, Young, A.^1,3, Autexier, C.^1,2,3
¹Department of Anatomy and Cell Biology ²Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, PQ H3A 2B2, Canada; ³Bloomfield Centre for Research in Aging, Lady Davis Institute, Jewish General Hospital, Montréal, PQ H3T 1E2, Canada
Corresponding Author: Adrian Young, email adrian.young@mail.mcgill.ca

Abstract

Background: The catalytic component of telomerase, telomerase reverse transcriptase (TERT), utilizes an intrinsic RNA component, hTR, to catalyze the reiterative addition of a short telomeric DNA sequence to the ends of chromosomes, through a process known as repeat addition processivity (RAP). Recent work from our laboratory has outlined evidence to suggest that this process may be enabled by the interactions between the telomere associated protein TPP1 with the TERT specific ‘insertion in fingers’ domain (IFD). Additional evidence to support this was provided by cryo-electron microscopy structures of Tetrahymena telomerase demonstrating interactions between the IFD and a TPP1 paralog p50 in addition to interactions between TPP1 and the telomerase essential N-terminal (TEN) domain. These structural studies also found previously unreported interactions between the TEN and IFD domains. Additionally, hypomorphic IFD mutations have been identified in patients suffering telomeropathies like Dyskeratosis congenita (DC) and aplastic anemia (AA). Our current understanding of telomerase suggests tight regulation in order to sustain cell proliferation in renewal tissues while preventing malignancies. We hypothesize that specific TEN-IFD and TPP1-IFD interactions are essential for telomerase function, telomere maintenance and cell immortalization.
Methods: We will identify the TEN-IFD and TPP1-IFD interactions that regulate telomerase activity through the analysis of hTERT-IFD mutations in conserved residues and telomeropathy associated hTERT-IFD mutations.
Conclusions: The unique IFD-TEN-TPP1 interface is an attractive target to gain greater insight into telomerase activity and may provide valuable information enabling new designs for telomerase-specific interventions.

Regulation of HSP70 mRNA translation in health and disease

Lokha R Alagar Boopathy^1,2, Suleima Jacob Tomas^1,2, Maria Vera Ugalde^1,2
¹Department of Biochemistry
²CRBS
Corresponding Author: Lokha R Alagar Boopathy, email lokha.alagarboopathy@mail.mcgill.ca

Abstract

Cell functionality relies on a group of molecules known as proteins. Proteins are synthesized as a linear polypeptide chain that requires proper folding into a functional 3-Dimensional structure. Proper shaping of the newly synthesized protein relies on Heat shock proteins (HSP) or molecular chaperones. HSPs cooperate to maintain the protein balance (proteostasis) inside the cell by refolding of misfolded protein, prevent protein aggregation, and eliminate damaged protein. Several cellular stress conditions threaten cell functionality as they cause proteins to lose their conformation (misfold). Cells tackle the protein damage by two means; (i) they stall the production of all proteins to minimize their incorrect folding and, (ii) they induce the production of HSPs, especially the inducible isoform of HSP70, in a process known as the heat shock response (HSR). The inducible HSP70 assist the misfolded proteins by either refolding them to prevent their harmful aggregation or by targeting them for degradation. Therefore, the upregulation of HSPs is a critical cellular process to preserve proteostasis and survival to stress conditions. However, an increase in the expression of HSP70 is a hallmark of cancers and is associated with inhibition of apoptosis, malignancy and resistance to chemotherapy. My project aims to define the molecular mechanisms that enable cells to upregulate the production of HSPs despite the shut-down in general protein synthesis. Investigating the translation of HSP70 mRNA would pave the way to find new targets to cancer treatment.

Differential Efficacy and Toxicity of Anti-HIV shRNAs expressed from Type 3 RNA Polymerase III Promoters on a Lentiviral Vector

Michelle J. Chen^1,2,3, Ryan P. Goguen^1,3, Camille M.G. Malard^1,3, Anne Gatignol^1,2,3, Robert J. Scarborough^1,3
¹Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC, Canada
²Department of Medicine, Division of Experimental Medicine, McGill University, Montréal, QC, Canada
³Department of Microbiology and Immunology, Montréal, McGill University, QC, Canada
Corresponding Author: Michelle Chen, email michelle.chen@mail.mcgill.ca

Abstract

Background: There is still no cure that can eliminate the human immunodeficiency type 1 virus (HIV) from the body. It may be possible to functionally cure HIV infection using antiviral RNA-interference based molecules such as short-hairpin (sh)RNAs. Autologous hematopoietic stem cells may be modified with these molecules ex vivo, then retransplanted into the patient to generate replication-resistant cells. In humans, shRNAs can be expressed from type 3 RNA Polymerase (Pol) III promoters for high transcriptional activity and defined transcriptional start and stop sites. The choice of promoter can influence transcriptional efficiency, in turn impacting shRNA potency and possibly cytotoxicity. Thus, questions remain about which combinations of shRNAs and promoters will effectively inhibit HIV replication without inducing cytotoxicity.
Methods: We systematically tested different promoter-shRNA constructs to assess their ability to inhibit viral replication and evaluated their cell toxicity. We cloned shRNAs targeting different parts of the HIV genome into lentiviral vectors containing one of the three human Pol III promoters: 7SK, U6, and H1. We then assessed the constructs for their ability to inhibit HIV replication and for their cytotoxicity.
Results: Our data suggest that U6- and 7SK-promoted shRNAs delay viral replication. While some of these constructs also showed toxicity, not all U6- and 7SK-promoted shRNAs demonstrated a negative impact. These findings suggest that the efficacy and toxicity of a shRNA depends on the promoter and on the shRNA sequence.
Conclusion: Our results may inform the combinations of RNA-based molecules with the highest potential for clinical use.

Using single-molecule fluorescence microscopy to uncover neuronal vulnerability to protein damage

Suleima Jacob-Tomas^1,2, Lokha R. Alagar Boopathy^1,2, Maria Vera Ugalde^1,2
¹Biochemistry
²Center de Recherche en Biologie Structurale
Corresponding Author: Suleima Jacob-Tomas, email suleima.jacob@mail.mcgill.ca

Abstract

Neurodegenerative disorders (NDs) are diverse age-related conditions also described as “conformational diseases”. The hallmark of NDs is the accumulation of disease-specific proteins as toxic misfolded aggregates in certain regions of the brain. Misfolded proteins can lead to the loss of protein homeostasis (proteostasis) which causes neuronal dysfunction and death. A potential therapeutic strategy for NDs is to prevent the accumulation of misfolded proteins through the activation of the heat shock response (HSR). The HSR maintains proteostasis through the upregulation of heat shock proteins (HSPs), molecular chaperones that recognize misfolded proteins, and either re-fold them to their functional conformations and/or target them for degradation. However, how to manipulate the expression of HSPs to obtain a therapeutic effect in neurons remains unclear. The regulation of the HSR in neurons is more complex than what we have learnt from culturing non-neuronal cells. Our preliminary data suggest that primary hippocampal neurons induce less HSP70 mRNA levels as compared to non-neuronal cells in response to heat stress. In this study we investigate the induction of HSP70 in primary hippocampal neurons using single-molecule fluorescence in situ hybridization (smFISH). Quantification of smFISH provides the means to analyze neuron-to-neuron variability in the activation of the HSR and enables us to study the transcriptional induction and localization of HSP70 mRNA in primary neurons. This information might be critical to find the druggable steps for developing effective therapies to treat age-related NDs.

The role of Claudin-3 and glycoproteins in neural tube closure

Elizabeth-Ann Legere^1,2, Aimée K. Ryan^1,2
¹McGill University
²Research Institute, McGill University Health Center
Corresponding Author: Elizabeth-Ann Legere, email elizabeth-ann.legere@mail.mcgill.ca

Abstract

The neural tube is the embryonic precursor to the central nervous system, which is formed following elevation and fusion of the lateral edges (neural folds) of the neural plate. Our lab is investigating the role of the claudin (cldn) family of tight junction proteins in neural tube development. In chick embryos, depletion of Cldn3 causes neural tube defects at the final stage of neural tube development, neural fold fusion. The mechanism of chick neural fold fusion is still not fully understood. In scanning electron microscope (SEM) images, we identified a fibrous mesh between the closing neural folds of wild-type chick embryos which was absent in the Cldn3-depleted embryos. Research from the 1970s showed that the mesh is a glycoprotein cell-surface matrix, involved in temporary adhesion between the neural folds as they fuse. We hypothesize that Cldn3 is required for the formation of a glycoprotein mesh which is critical for neural fold fusion. Using fluorescently-tagged carbohydrate-binding lectin proteins, I am examining the pattern of expression of glycan chains along the neural tube. I will then determine whether this pattern changes in the Cldn3-depleted embryo and how the glycoprotein chains are involved in neural tube development. My preliminary results show that subsets of glycan chains are strongly expressed at the closing neural folds and that interrupting these glycan chains through lectin binding causes neural tube defects. This research is working towards a better understanding of the mechanisms in chick neural tube closure, and the role that Cldn3 is playing in this process.

Loss of Planar Cell Polarity Gene Fuzzy Leads to the Embryonic Kidney Defects

Rhythm Sharma^1,2, Elena Torban^1,2
¹Research Institute of McGill University Health Centre (RI-MUHC), Glen site
²Department of Medicine, McGill University, Montreal
Corresponding Author: Rhythm Sharma, email rhythm.sharma@mail.mcgill.ca

Abstract

Congenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of the end-stage renal disorder (ESRD) in children, including cystic kidney diseases caused by mutant genes that affect the primary cilia on renal tubular cells. We discovered hypoplastic cystic kidneys in the mice mutant for the Planar Cell Polarity (PCP) effector gene, Fuzzy. Fuzzy is known to participate in ciliogenesis by controlling the trafficking of certain molecules needed for the assembly of the primary cilium. However, how Fuzzy regulates ciliogenesis and causes cysts in the kidney is not well understood.
Our preliminary observations show that Fuzzy might act via p190a to control ciliogenesis. P190A is a GTPase activating protein which acts as an inhibitor of RhoA GTPase and regulates actin polymerization at the base of the cilium required for proper cilia elongation. Loss of p190a gene causes shorter cilia, renal hypoplasia and glomerulocystic kidney which phenotypically similar to the E16.5 Fuzzy-/- kidneys. We showed by CoIP that Fuzzy interacts with p190A and that ciliogenesis is rescued in unciliated Fuzzy-/- MEFs by inhibitors of Rho Kinase, a downstream RhoA GAP effector. By crossing p190A and Fuzzy mutant mice, we are currently analysing whether there is a genetic interaction between these genes. With this approach we will establish the mechanism whereby Fuzzy controls ciliogenesis and will understand the unknown role of Fuzzy in kidney development and diseases.

Study of decay mechanism of HSP70 mRNA in Saccharomyces Cerevisiae

RuoChen Xiao¹, Maria Vera Ugalde¹
¹Biochemistry, CRBS
Corresponding Author: Alan RuoChen Xiao, email ruochen.xiao@mail.mcgill.ca

Abstract

Environmental stressors can disrupt cellular protein homeostasis. In response to stress, cells activate a survival mechanism known as the heat shock response (HSR). The HSR mitigates the negative effects through the induction of heat shock proteins (HSPs), especially that of HSP70. Once the stress ceases, the expression of inducible HSP70 stops. Consequences of HSP70 mis-regulation are tumor formation in mammalian cells and abnormal development in drosophila.
My project aims to uncover the mechanism that regulates HSP70. During cell recovery, cells downregulate the synthesis of HSP70 though general CAP-dependent translation restarts. We propose that the efficient degradation of HSP70 mRNA as a mechanism to limit its expression. My hypothesis is that the decay of HSP70 mRNA is linked to the resume of protein synthesis through a mRNA surveillance mechanism known as no-go Decay (NGD). Studies showed NGD occurs when stalled ribosomes collided during translation elongation. Ribosome stalling can be caused by rare codons (e.g. CAA) and codons encoding basic residues. We studied the sequence of the gene ssa4 encoding inducible HSP70 in Saccharomyces cerevisiae, and found it is rich in pro-stalling codons. We suggest the restart of CAP-dependent translation during recovery allows more ribosomes to participate in translation elongation which increases the possibility of ribosome stalling and collision on the transcript. We created yeast strains with depletion of NGD factors to screen for their function during HSP70 mRNA decay using single molecular fluorescent microscopy and northern blot. The findings provide insights on the regulation of HSP70 expression.

Uncovering the Structural Determinants for Functional Selectivity in the Angiotensin II Type 1 Receptor

Yubo (Frank) Cao¹, Aaron Cho², and Stephane A. Laporte^1,2,3
¹Department of Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal, Quebec
²Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, Quebec
³Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec
Corresponding Author: Yubo (Frank) Cao, email yubo.cao@mail.mcgill.ca

Abstract

Hypertension and its associated cardiovascular risks can be attributed to the hormone angiotensin II (AngII), which regulates blood volume and vascular resistance through the AngII type 1 receptor (AT1R). As a G protein-coupled receptor (GPCR), AT1R signals by coupling to G proteins (Gαq/11, Gα12/13, Gαi1/2/3) and β-arrestins (β-arr1 and 2). While most drugs that target AT1R act by blocking the activation of the receptor and attenuating all downstream signaling pathways indiscriminately, recent studies highlight that certain AT1R-mediated pathways may in fact promote beneficial cardio-protective effects. Thus, recent efforts have focused on developing compounds that activate specific subsets of AT1R signaling pathways through a phenomenon known as biased signaling or functional selectivity. However, development of efficacious biased compounds have been challenging, since the structural determinants for activating specific AT1R signaling pathways remain unknown. Here, we begin to uncover the structural determinants for such selective signal transduction by generating an alanine scan library of 359 amino acids in AT1R and characterizing the signaling of these alanine mutants using an array of bioluminescence resonance energy transfer (BRET)-biosensors. By analyzing three major G protein signaling pathways and β-arrestin recruitment, we identify potential orthosteric binding sites and key residues in AT1R that elicit unique signal transduction behaviors. Furthermore, by stratifying the different mutants according to their signaling profiles, we establish networks of residues that are critical for distinct signaling pathways. Altogether, our findings provide a better mechanistic understanding of AT1R activation and offer new insights towards the development of efficacious biased compounds for better-targeted therapeutics.

The role of the meiCT gene HORMAD1 and meiomitosis in genomic instability and carcinogenesis

Jennifer Gantchev¹, Amelia Martinez Villarreal¹, Ivan V. Litvinov^1,2
¹McGill University Health Centre, Montreal, QC;
²Division of Dermatology, Department of Medicine, McGill University Health Centre, Montreal, QC
Corresponding Author: Jennifer Gantchev, email jennifer.theoret@mail.mcgill.ca

Abstract

Cancer meiomitosis is defined as the synchronized activation of both mitotic and meiotic machineries in neoplastic cells to confer a selective advantage related to increased genomic instability. MeiCT (meiosis-specific cancer/testis) genes that specialize in the reductional division of germ cells in meiosis I are ectopically expressed in several cancers. HORMAD1, a meiosis specific protein that functions to ensure that a sufficient number of DSBs are formed for proper meiotic progression and to maintain genome integrity throughout the processes of homologous recombination and crossover formation. Studies show that HORMAD1 is significantly upregulated in several cancers and is regarded as a potentially important oncogene that plays a role in sustaining increased genomic instability. With the use of shRNA mediated knockdown of HORMAD1 in cancer cells, we used current chemotherapies to evaluate the effects of HORMAD1 on genomic instability and survival in cancer. Our results demonstrate that HORMAD1 expression in breast and head and neck squamous cell carcinoma cancer cell lines influences genomic instability by modulating the amount of double strand breaks, the number of chromatin bridges, the formation of micronuclei as well as cell survival and proliferation in response to etoposide and the poly (ADP ribose) polymerase (PARP) inhibitors Olaparib and Nirapaib. We conclude that HORMAD1 helps cancer cells acquire treatment resistance and subsequent survival by harnessing a functional and balanced level of genomic instability.

Endocrinology and Energy Homeostasis

Aspartyl/asparaginyl ß-hydroxylase (ASPH) and humbug are two novel gamma-carboxylated proteins expressed in pancreatic ß-cells

Kevin Guo^1,2, Julie Lacombe², Denis Faubert², Mathieu Ferron^1,2
¹Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC;
²Institut de recherches cliniques de Montréal, Montreal, QC
Corresponding Author: Kevin Guo, email kevin.guo@ircm.qc.ca

Abstract

Background: Vitamin K (VK) is essential for the conversion of glutamic acid (Glu) into gamma-carboxyglutamic acid (Gla) residues. Recent findings suggest a beneficial role of VK in preventing diabetes, but the mechanism at play is unknown. We have observed that mice lacking Ggcx in the pancreas have reduced insulin secretion and a decreased β-cell mass. Since none of the known Gla proteins are expressed in β-cell, the goal of this study was to identify new Gla proteins expressed in β-cells.
Methods and results: Gla proteins were immunoprecipitated (IP) and characterized using a proteomic approach, leading to the identification of two novel carboxylated proteins encoded by the same gene: aspartyl/asparaginyl β-hydroxylase (ASPH) and humbug. ASPH hydroxylates aspartate and asparagine in Ca+2 binding EGF-like domains, while humbug regulates intracellular Ca2+ fluxes. Quantitative PCR and RNA sequencing on mouse islets confirmed that both ASPH and humbug are highly expressed in islets. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of ASPH and humbug revealed clusters of Gla residues in the N- and C-terminal regions of a shared Glu rich domain. Neither ASPH nor humbug were gamma-carboxylated in the presence of warfarin, an inhibitor of gamma-carboxylation, or following deletion of their Glu rich domain.
Conclusion: We have identified two novel Gla proteins expressed in islets. Our current work is aimed at determining if gamma-carboxylation directly regulates the function of ASPH and humbug. We are also generating mice lacking either of these two proteins to assess their role in β-cell function in vivo.

Epidemiology, Bioethics, and Clinical Research

Pathogenic Short Tandem DNA Repeats in Premutation Carriers: Medical Actionability and Inclusion in AMCG Secondary Findings Reports

Joshua Moise-Silverman¹
¹McGill University, Division of Experimental Medicine, Biomedical Ethics Unit
Corresponding Author: Joshua Moise-Silverman, email joshua.moise-silverman@mail.mcgill.ca

Abstract

The definition of genetic actionability, identifying and preventing diseases related to genetic aberrations, has important ramifications as it informs which anomalous genetic sequencing findings are to be reported to a patient. Currently, this definition is inadequate as it does not allow for genetic anticipation, nor does it consider issues of procreative beneficence. The exclusion of monogenetic disorders, caused by short tandem repeats (STRs), from the reportable secondary findings list created by the American College of Genetics and Genomics (AMCG) highlights the insufficiency of the current definition of medical actionability.
Examination of cases where STRs are found, but the patient is phenotypically normal highlights an opportunity for report of secondary findings. Review of cases with premutation carriers, where the number of STR repeats can increase in subsequent generations increasing the likelihood of disease in offspring, highlights how interaction with the proband can prevent disease in offspring. Additionally, some STR related diseases increase a premutation carriers’ risk for developing secondary conditions. Woman who carry STR premutations responsible for Fragile-X syndrome often suffer from primary ovarian insufficiency. This is a treatable, thus actionable condition. Patients made aware of their premutation status can pursue fertility preservation options and utilize fetal genetic tests.
Medical actionability is a shifting concept, increasing the scope of actionability to include treatment of premutation caused illness and the prevention of disease in offspring can improve outcomes. This redefinition allows premutation carriers to seek treatment and make medically relevant decisions for themselves and potential offspring.

Prevalence and associated factors of non-alcoholic fatty liver disease in South Asian women with polycystic ovary syndrome: A cross-sectional study using Transient Elastography

Mohamed Shengir¹, Srinivasan Krishnamurthy², Peter Ghali³, Marc Deschenes³, Philip Wong³, Tianyan Chen³, Giada Sebastiani³
¹Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada.
²Department of Obstetrics and Gynaecology, McGill University Health Centre, Montreal, Quebec, Canada.
³Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada.
Corresponding Author: Mohamed Shengir, email mohamed.shengir@mail.mcgill.ca

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease globally and the leading indication for liver transplantation in women. Polycystic ovary syndrome (PCOS) seems to be a risk factor for NAFLD due to shared metabolic abnormalities. Data on NAFLD in PCOS women from South Asian descents are scarce.
Objectives: Determine the prevalence of NAFLD and significant liver fibrosis (SLF), as well as cofactors of NAFLD, among premenopausal South Asian women with PCOS.
Methods: South Asian women diagnosed with PCOS according to Rotterdam criteria were consecutively recruited. Transient elastography (TE) with controlled attenuation parameter (CAP) was performed to identify NAFLD. Other causes of hepatic steatosis were excluded. Clinical, serological, and biochemical evaluation were performed at enrollment. NAFLD was defined as CAP ³ 248 dB/m and SLF as TE measurement ³ 8.0 kPa. Cofactors were determined through multivariate logistic regression analysis.
Results: 101 PCOS patients (mean age 36.3 years) were included. Prevalence of NAFLD, severe NAFLD, and SLF were 61.4%, 36.6% and 6.9%, respectively. After adjusting for PCOS duration, insulin resistance and free androgen index (FAI), cofactors independently associated with NAFLD were higher waist circumference (adjusted odds ratio [aOR] 1.08, 95% CI 1.01-1.17), higher triglycerides (aOR 7.15, 95% CI 1.52-33.55) and ALT (aOR 2.52, 95% CI 1.04-6.11). NAFLD+ had higher lifetime CVD risk than no NAFLD counterpart (P-value 0.004).
Conclusions: Despite the young age, NAFLD is a frequent comorbidity in South Asian women with PCOS. Non-invasive screening strategies could help early diagnosis, cardiovascular risk stratification and initiation of interventions.

Geographic Variations in Bone Mineral Density and Prevalent Fractures in Canada

Nazila Hassanabadi¹, Claudie Berger², Alexandra Papaioannou³, Angela M Cheung⁴, Elham Rahme^1,2, William D Leslie⁵, David Goltzman^1,2, Suzanne N Morin^1,2
¹Department of Medicine, McGill University
²Research Institute of the McGill University Health Centre
³Department of Medicine, McMaster University
⁴Department of Medicine, University of Toronto
⁵Department of Medicine, University of Manitoba
Corresponding Author: Nazila Hassanabadi, email nazila.hassanabadi@mail.mcgill.ca

Abstract

Purpose: The prevalence of osteoporosis and fractures differs across countries and regions. We aimed to describe sex-specific total hip bone mineral density(Hip-BMD) and prevalent major osteoporotic fractures(MOF) patterns across 7 Canadian provinces.
Methods: We used baseline data of 27,244 Canadians (13,828 women,13,416 men) aged 50 years+ participating in the Canadian Longitudinal Study of Aging (2012-15). Linear and logistic regression models were used looking for associations of the province of residence and Hip-BMD and prevalent MOF(self-reported) stratified by sex and adjusted for important covariates.
Results: The mean(SD) age of participants was 64.4(9.4) years in women and 64.8(9.4) in men. Mean BMI(kg/m2) was lowest (27.4[5.1]) in British-Columbia(BC) and highest (29.0[6.1]) in Manitoba(MB). In BC, both women and men had the lowest mean Hip-BMD and prevalence of MOF of all provinces. The mean FRAX® score was 10%(7) in women and 6%(3) in men. Alberta(AB)(12.3%) and Quebec(QC)(12.0%) had the highest proportion of participants reporting falls, while MB(8.6%) had the fewest. Adjusted linear regressions demonstrated significant differences in Hip-BMD across provinces; women and men from BC and AB, and women from MB and NS had lower BMD than ON. Adjusted Odds Ratios(OR[95%CI]) for prevalent MOF were significantly lower in women (0.56[0.40-0.79]) and men (0.35[0.21-0.58]) from BC and men from NS (0.52[0.28-0.98]) compared to ON. Results were similar when restricting the analyses to participants of White ancestry only.
Conclusion: Small geographical variations in Hip-BMD and prevalent MOF between provinces persisted after adjusting for important covariates. Unmeasured individual, social and environmental factors could possibly explain these observations.

The Effect of Overdose Education and Naloxone Distribution Distribution: An Umbrella Review of Systematic Reviews

Amir Razaghizad MSc¹, Sarah B. Windle MPH¹, Kristian B. Filion PhD^1,2, Genevieve Gore MLIS³, Irina Kudrina MDCM^4,5, Elena Paraskevopoulos MD⁴, Jonathan Kimmelman PhD⁶, Marc O. Martel PhD⁷, Mark J. Eisenberg MD^1,2,8
¹Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada
²Departments of Medicine and of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, QC, Canada
³Schulich Library of Physical Sciences, Life Sciences, and Engineering, McGill University, Montreal, QC, Canada
⁴Department of Family Medicine, Faculty of Medicine, McGill University, Montreal, QC, Canada
⁵Department of Anesthesia, Faculty of Medicine, McGill University, Montreal, QC, Canada
⁶Studies of Translation, Ethics and Medicine Biomedical Ethics Unit, McGill University, Montreal, QC, Canada
⁷Faculty of Dentistry and Department of Anesthesia, McGill University, Montreal, QC, Canada
⁸Division of Cardiology, Jewish General Hospital, McGill University, Montreal, QC, Canada
Corresponding Author: Amir Razaghizad, email amirzadd@gmail.com

Abstract

Background: Opioids contribute to more than 50,000 deaths annually in North America. While the expansion of naloxone-based interventions for overdose rescue, including overdose education and naloxone distribution (OEND) programs, have been recommended in response to the opioid crisis, their effectiveness remains unclear.
Methods: We conducted an umbrella review of systematic reviews to summarize the effect of naloxone-based interventions for overdose rescue, and to identify areas for OEND program optimization. We systematically searched PubMed, Embase, PsycINFO, Epistemonikos, the Cochrane Database of Systematic Reviews, and hand-searched the bibliographies of relevant articles. Eligible reviews reported systematic search strategies and inclusion criteria, reported primary study assessments, and evaluated the use of naloxone for overdose rescue in adults.
Results: Six systematic reviews containing 87 unique studies were included. We found that OEND programs: produce long-term knowledge improvement regarding opioid overdose; improve participants’ attitudes towards naloxone; provide sufficient training for participants to safely and effectively manage overdoses; and effectively reduce opioid-related mortality. High-concentration intranasal naloxone appeared to have similar efficacy to intramuscular naloxone at the same dose (2 mg/ml), while lower-concentration intranasal naloxone was less effective. Evidence was limited for other naloxone formulations, as well as the need for hospital care after overdose reversal. The majority of available evidence pertained to self-identified persons who use heroin, with limited data available for users of prescription opioids.
Conclusion: Evidence from systematic reviews suggest that the expansion of OEND programs in high-risk populations is effective for reducing opioid-related mortality; however, additional high-quality research is required to optimize program delivery.

A Randomized Controlled Trial Testing the Effectiveness of an Insulin-Dose Learning Algorithm in Adults with Type 1 Diabetes

Alessandra Kobayati^1,2, Anas El Fathi³, Michael Tsoukas^1,2, Ahmad Haidar^1,2,3
¹Department of Medicine
²Research Institute of the McGill University Health Centre
³Department of Biomedical Engineering
Corresponding Author: Alessandra Kobayati, email alessandra.kobayati@hotmail.com

Abstract

Background: Type 1 diabetes is an autoimmune condition that results in little to no insulin production from the pancreas. Intensive insulin therapy is essential for life, and the prevailing delivery method is via multiple daily injections. Despite advances in diabetes technology, most individuals still struggle to achieve optimal glucose control. Thus, our lab has developed a learning algorithm that is integrated with our novel mobile application to recommend weekly personalized insulin dose adjustments based on the previous week’s meal, insulin and glucose data. Methods: We are conducting a three-month, randomized, controlled, two-way, parallel trial in 84 adults, aged 18 years or older with type 1 diabetes who have suboptimal control (HbA1C ≥ 7.5%) using daily insulin injections. The purpose of the study is to examine the effectiveness of this decision support system in free-living conditions with the aim of improving overall glucose control and treatment satisfaction. Participants are currently being recruited at the Research Institute of the McGill University Health Centre, where they are randomized in a 1:1 ratio to either intervention arm. 13 participants (mean baseline HbA1c: 9.0%) have been enrolled in the study; 6 were allocated to the algorithm group, and 7 were allocated to the control group. The primary outcome is the change in HbA1c at 12 weeks from baseline. The data will be analyzed on a modified intention-to-treat basis, wherein a minimum of 10 completed weeks is required to be included. A p-value of less than 0.05 will be deemed significant to detect superiority.

Genetics and Gene Expression

Defining the mechanisms of RNA targeting to cancer cell derived extracellular vesicles

Juan-Carlos A. Padilla^1,2, Louis-Philip Benoit Bouvrette^1,3, Jonathan Boulais¹, Eunjeong Kwon¹, Eric Lécuyer^1,2,3
¹Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC H2W 1R7, Canada
²Division of Experimental Medicine, McGill University, Montréal, QC H4A 3J1, Canada
³Département de Biochimie, Université de Montréal, Montréal, QC H3T 1J4, Canada
Corresponding Author: Juan-Carlos A. Padilla, email Juan-Carlos.Padilla@ircm.qc.ca

Abstract

Background & Aims: Most cells release extracellular vesicles (EVs) that can mediate the transfer of diverse molecular species to other cells in their environment. However, the molecular mechanisms driving the selective packaging of RNAs into EVs remain largely unknown. The subcellular targeting of RNAs is mediated by cis-regulatory motifs (CRMs) residing within the RNA molecule, which are recognized by trans-acting RNA binding proteins (RBPs). We hypothesize that RNA incorporation into EVs is achieved via conserved transport pathways involving specific CRMs and RBPs. To address this hypothesis, we aim to: 1) define conserved CRMs required for RNA localization to EVs, and 2) identify RBPs required for selective RNA incorporation within EVs. Methods & Results: We have used RNA-seq to identify large collections of EV-enriched RNAs (exRNAs). To functionally characterize CRMs involved in EV targeting, we are conducting in silico analysis to identify sequence motifs overrepresented in our datasets followed by validation with RT-qPCR. These studies will identify functional CRMs that match consensus binding motifs of specific RBPs. To investigate the function of suspected RBPs in exRNA-targeting, we will conduct loss-of-function studies of select EV-enriched RBPs identified via mass spectrometry profiling. We will then profile the recruitment of the selected exRNAs. Conclusion: Understanding the pathways by which specific RNAs are targeted to EVs is an emerging area of cancer research. This project will lay the foundation for the development of novel approaches to profile and manipulate the RNA composition of EVs, thus offering great promise for innovations in diagnostics and therapeutics.

The genetic and molecular analyses of RAD51C and RAD51D in ovarian cancer cases from a founder population identifies two new functionally relevant missense variants

Wejdan M. Alenezi^1,2,3, Larissa Milano^4,5, Caitlin Fierheller^1,2, Corinne Serruya², Timothée Revil^1,6, Kathleen K. Oros⁷, Javad Nadaf^1,6, Suzanna L. Arcand⁶, Liliane Meunier⁸, Dan Spiegelman^1,9, Simon Gravel^1,6, Anne-Marie Mes-Masson^8,9, Diane Provencher^8,10, William Foulkes^1,2,7,11,12, Zaki El Haffaf^8,13, Guy Rouleau^1,9, Luigi Bouchard^14,15,16, Celia M.T. Greenwood^1,7,17,18, Jean-Yves Masson^4,5, Jiannis Ragoussis^1,6 and Patricia N. Tonin^1,2,12
¹Department of Human Genetics, McGill University, Montréal, Québec, Canada
²Cancer Research Program, The Research Institute of McGill University Health Centre, Montréal, Québec, Canada
³Department of Medical Laboratory Technology, Taibah University, Medina, Saudi Arabia
⁴Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec City, Québec, Canada
⁵Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion, Oncology Axis, Québec City, Québec, Canada
⁶McGill Genome Centre, McGill University, Montréal, Québec, Canada
⁷Lady Davis Institute for Medical Research of the Jewish General Hospital, Montréal, Québec, Canada
⁸Centre de recherche du Centre hospitalier de l'Université de Montréal and Institut du cancer de Montréal, Montreal, Quebec
⁹Montreal Neurological Institute, McGill University, Montréal, Québec, Canada; Département de Médecine, Université de Montréal, Montréal, Québec, Canada
¹⁰Division de obstétrique et d’gynécologie, Université́ de Montréal, Montréal, Québec, Canada
¹¹Department of Medical Genetics, McGill University Health Centre, Montréal, Québec, Canada
¹²Department of Medicine, McGill University, Montréal, Québec, Canada
¹³Service de Médecine Génique, Centre Hospitalier de l’Université́ de Montréal, Montréal, Québec, Canada
¹⁴Département de biochimie, Université de Sherbrooke, Sherbrooke, Canada
¹⁵Département de biologie médicale, CIUSSS Saguenay-Lac-Saint-Jean Hôpital Universitaire de Chicoutimi, Saguenay, Canada
¹⁶Centre de Recherche du CHUS, Sherbrooke, Canada
¹⁷Gerald Bronfman Department of Oncology, McGill University, Montréal, Québec, Canada
¹⁸Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montréal, Québec, Canada
Corresponding Author: Wejdan Alenezi, email wagdan.alenizy@mail.mcgill.ca

Abstract

Objective: To investigate rare potentially pathogenic variants in BRIP1, RAD51C and RAD51D, proposed ovarian cancer (OC) predisposing genes, in French Canadians (FC) of Quebec with OC.
Methods: We performed whole exome sequencing (WES) of germline DNA from BRCA1 and BRCA2 mutation-negative OC patients from: 17 FC families with at least two OC cases and 53 FC early-onset cases (diagnosed before age of 50 years). Candidate variants were selected based on minor allele frequency <0.01 and predicted as potentially pathogenic different in silico tools. Due to founder effect in FCs resulting in recurrence of risk alleles, candidate variants were screened in 811 sporadic FC OC cases enriched for high grade serous OC subtype and 1025 FC cancer-free controls. Selected variants with unknown biological function were assayed in cellulo for biological impact.
Results: Five potentially pathogenic variants in RAD51C (n=2) or RAD51D (n=3), but not in BRIP1, were carried by three familial (17.6%) and six early-onset (11.3%) OC cases. In carriers, loss of the wild-type allele was observed in their tumours. The carrier frequency of RAD51D or RAD51C variants were significantly higher in OC cases than in cancer-free controls (p<0.001). Molecular assays of two missense variants with unknown biological function showed that an exonic RAD51C variant affected splicing resulting in skipping exon 4, and a RAD51D variant isoform affected the protein stability and sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitor.
Conclusion: Our strategy of applying WES and bioinformatics analyses along with in cellulo assays to familial and early-onset OC cases from a founder population further support the role of RAD51C and RAD51D as ovarian cancer predisposing genes.

Phosphorylation of eIF2alpha safeguards the navie state of pluripotency

Mehdi Amiri¹, Stephen J Kiniry², Tayebeh Basiri¹, Qiyun Deng¹, Pavel V Baranov², Soroush Tahmasebi³, Nahum Sonenberg¹
¹McGill University, Biochemistry, Montreal, Canada
²University College Cork, Biochemistry and Cell Biology, Cork, Ireland
³The University of Illinois, College of Medicine, Pharmacology, Chicago, IL
Corresponding Author: Mehdi Amiri, email mehdi.amiri@mail.mcgill.ca

Abstract

Embryonic stem cells (ESCs) maintain a low translation rate and subtle changes in the expression of stem cell factors can greatly influence ESCs' self-renewal and differentiation. One of the well-characterized translation control mechanisms is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2). Phosphorylation of eIF2α at serine 51 inhibits global translation, however, promotes the translation of some mRNAs containing upstream open reading frame (uORFs) at their 5’UTRs. The importance of this phosphorylation event in regulating ESC is not well defined. To establish the role of eIF2α phosphorylation in ESCs, we have derived mouse ESCs from eIF2α+/+ (WT) and eIF2αS51A/S51A mice. When cultured in 2i (GSK3 and MEK inhibitors )+LIF medium eIF2αS51A/S51A ESCs have increased global translation compared to WT ESCs but the expression of the key pluripotency factors remained the same. Withdrawal of 2i, two factors that protect an undifferentiated state, significantly reduced the level of Nanog in eIF2αS51A/S51A ESCs compared to WT ESCs. We employed genome-wide translatome analysis to identify a subset of mRNAs that are translationally regulated by phosphorylation of eIF2α. In addition to known p-eIF2α resistant mRNAs (e.g. ATF4 and ATF5), we identified a large number of p-eIF2α resistant mRNAs that play critical roles in self-renewal and differentiation of ESCs. We also uncovered novel p-eIF2α sensitive genes that control fundamental cellular processes such as chromosome organization. In addition, our data suggest that the translation of different components of large protein complexes are synchronized through phosphorylation of eIF2α.

DNA methylation is essential for cell viability of human trophoblast stem cells

Deepak Saini¹, Dr. William A. Pastor^1,2
¹Department of Biochemistry
²Goodman Cancer Research Center
Corresponding Author: Deepak Saini, email deepak.saini@mail.mcgill.ca

Abstract

DNA methylation, an epigenetic mechanism mediated by DNA methyltransferases (DNMTs), is required for proper mammalian embryonic development. De novo methylation occurs in post-implantation blastocysts, generating unique methylation profiles in both the inner cell mass (ICM) which forms embryonic tissue, and the trophoblast which contributes to the placenta. Murine trophoblast stem cells (mTSCs) lacking DNA methylation survive normally and contribute to placenta, indicating that DNA methylation is not essential for the generation of the mouse placenta. To determine the role of DNA methylation in human placental cells, we used nucleofection-based delivery of CRISPR/Cas9 ribonucleoprotein particles to target DNMT1, an enzyme responsible for DNA methylation maintenance, in bulk populations of human trophoblast stem cells (hTSCs). We show that this system is highly efficient in generating DNMT1 knockouts (DNMT1 KO) in the majority of the heterogenous bulk population. Despite this, we have been unable to isolate single-cell clonal lines of DNMT1 KO hTSCs. By continuously culturing the heterogeneous DNMT1 KO hTSC bulk population and taking weekly genotyping timepoints, we have observed a gradual decrease in the prevalence of the DNMT1 KO allele, 2.5 weeks post-nucleofection. These preliminary results suggest hTSCs show reduced cell viability when DNMT1 is ablated; unlike mTSCs, which can survive without the DNMT genes. In future work of this study, we hope to identify both the methylation profile and changes in gene expression networks of DNMT1 KO bulk populations using Bisulfite sequencing and RNA-seq. These experiments will help us identify potential DNA methylation markers responsible for this phenotype.

Investigating the genetic landscape of French-Canadian Hereditary Breast Cancer cases that harbour pathogenic BRCA2-mutations

Neil Recio^1,2, Timothée Revil^1,3, Jiannis Ragoussis^1,3, Patricia Tonin^1,3,4
¹Department of Human Genetics, McGill University, Montréal, Quebec
²Cancer Research Program, The Research Institute of McGill University Health Centre, Montréal, Quebec
³McGill University Genome Centre, Montréal, Quebec; 4. Department of Medicine, McGill University, Montréal, Quebec
Corresponding Author: Neil Recio, email neil.recio@mail.mcgill.ca

Abstract

Specific pathogenic mutations in BRCA1, BRCA2, and PALB2 have been shown to recur in the French-Canadian (FC) hereditary breast cancer (HBC) population of Quebec due to common ancestors. Interestingly, rare reports of HBC cases harbouring co-occurring pathogenic mutations in two BC predisposing genes (i.e., BRCA1-BRCA2, and BRCA2-PALB2) have been reported, though the influence on disease progression is unknown. We performed whole exome sequencing and bioinformatic analyses on germline DNA from index cases from 57 FC HBC families (>2 BC cases) known to carry a FC recurrent pathogenic mutation in BRCA1/BRCA2. We filtered for rare (MAF<1%), protein-coding alleles and selected for variants that were predicted to be pathogenic and conserved by in silico tools, prioritizing variants that occurred in DNA repair pathway genes (n=276). We identified 8 rare, potentially pathogenic alleles among ATM, BARD1, CHEK2, ERCC6, MUTYH, MLH1, and NSMCE2 in 10 cases (17.5%), of which 9 were BRCA2-mutation carriers. A targeted mutational screening for candidate variants in additional FC-HBC cases that are BRCA-mutation negative (N=12) or unselected for BRCA-mutation status (N=93) and FC women with no personal history of cancer (N=52) identified only one additional carrier for the CHEK2 p.Q83Xfs variant. For BRCA2-mutation carriers, an analysis to determine which molecular pathways are commonly enriched with mutations identified focal adhesion in 92.5% of cases. We show that FC HBC cases harbouring a pathogenic BRCA2 mutation also carry rare, potentially pathogenic mutations in other DNA repair genes. Their role in modifying risk or disease progression in BRCA2 carriers warrants further investigation.

A non-canonical role for the EDC4 decapping factor in regulating MARF1-mediated mRNA decay

William R. Brothers¹, Steven Hébert¹, Claudia L. Kleinman^1,2, Marc R. Fabian^1,3,4
¹Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
²Department of Human Genetics, McGill University, Montreal, Quebec, Canada
³Department of Biochemistry, McGill University, Montreal, Quebec, Canada
⁴Department of Oncology, McGill University, Montreal, Quebec, Canada
Corresponding Author: Will Brothers, email william.brothers@mail.mcgill.ca

Abstract

The enhancer of mRNA decapping 4 (EDC4) is a core component of processing (P)-bodies that binds the DCP2 decapping enzyme and stimulates mRNA decay. EDC4 also interacts with mammalian MARF1, a recently identified endoribonuclease that promotes oogenesis and contains a number of RNA binding domains, including two RRMs and multiple LOTUS domains. How EDC4 regulates MARF1 action and the identity of MARF1 target mRNAs is not known. Our transcriptome-wide analysis identifies bona fide MARF1 target mRNAs and indicates that MARF1 predominantly binds their 3’ UTRs via its LOTUS domains to promote their decay. We also show that a MARF1 RRM plays an essential role in enhancing its endonuclease activity. Importantly, we establish that EDC4 binding to MARF1 impairs MARF1-mediated repression by preventing its LOTUS domains from binding to target mRNAs. Thus, EDC4 not only serves as an enhancer of mRNA turnover that binds DCP2, but also as a repressor that binds MARF1 to prevent the decay of MARF1 target mRNAs.

Microbiology and Immunology

Tungsten Alters DNA Damage Repair Mechanisms

Rowa Bakadlag^1,2, Hsiang Chou^1,2, Steven Findlay^1,2, John Heath^1,2, Alexander Orthwein^1,2,3, Koren K. Mann^1,2,3
¹Lady Davis Institute for Medical Research
²Division of Experimental Medicine
³Gerald Bronfman Department of Oncology; McGill University, Quebec, Canada
Corresponding Author: Rowa Bakadlag, email rowa.bakadlag@mail.mcgill.ca

Abstract

Tungsten was linked to several pediatric preB lymphocytic leukemia clusters but was not designated as the causative agent. Tungsten increases DNA damage and gH2AX levels in developing B cells in vitro and in vivo. B cell development depends on DNA damage and its subsequent repair by non-homologous end joining (NHEJ) to facilitate immunoglobulin recombination. Thus, we hypothesize that tungsten impedes DNA damage repair through the NHEJ pathway. RNA sequencing of murine preB cells exposed to tungsten in vivo showed decreased expression of several members of the NHEJ pathway. In addition, tungsten enhances neocarzinostatin-induced DNA-damage as assessed by increased gH2AX through flow cytometry, and may reduce repair. We further showed that tungsten abrogates NHEJ repair and demonstrated that tungsten decreases NHEJ-dependent class switch recombination to IgA. Together, our data suggest that tungsten inhibits NHEJ resulting in increased DNA damage in B cells.

HIV-1 sequesters a specific antiviral microRNA to overcome a block in viral transcription

Owen R. S. Dunkley^1,2, Sergio P. Alpuche-Lazcano^1,2, Anne Gatignol^1,2,3
¹Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, QC, Canada
²Lady Davis Institute for Biomedical Research, Montreal, QC, Canada
³Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada
Corresponding Author: Owen Dunkley, email owen.dunkley@mail.mcgill.ca

Abstract

Background: HIV-1 interferes with numerous host processes including the RNA interference (RNAi) pathway. We have previously identified an interaction between the HIV-1 Gag protein and Dicer, a key RNAse for RNAi function. Here, we explore the downstream effects of the formation of the Gag-Dicer complex through a microRNA preferentially bound to the complex that targets the transcriptional Super Elongation Complex (SEC).
Methods: Predicted microRNA targets were confirmed using microRNA mimics and antimiRNAs in combination with reverse-transcription quantitative polymerase chain reaction (qRT-PCR), gene reporter assays (GRAs) with fragments of a predicted target mRNA and Western Blot assays (WB). The effect of the microRNA mimic on HIV-1 expression with or without Gag and the indirect effects of Gag on cellular mRNA and HIV-1 gene expression were tested with qRT-PCR and WB.
Results:b A microRNA that preferentially binds to the Gag-Dicer complex was predicted to target several host mRNAs, including AF4/FMR2 Family Member 4 (AFF4) which encodes a scaffold member of the SEC. Knockdown of AFF4 by microRNA mimics was confirmed, as was the downstream effect of this microRNA on HIV-1 expression. qRT-PCRs and WB of cells expressing Flag, Flag-Gag, the HIV-1 genome or HIV-1 with Gag inactivated showed a reduction of the microRNA’s effects on AFF4 and downstream HIV-1 expression in cells expressing Gag.
Conclusions: We show that by interacting with Dicer, HIV-1 Gag modifies the Dicer-binding microRNA landscape, in turn inhibiting the effects of a specific microRNA on AFF4. This sequestration indicates a novel pathway by which HIV-1 regulates transcriptional elongation.

Identification of a new strain of Mucispirillum schaedleri in a mouse model of chronic granulomatous disease

Aléhandra Desjardins¹, Robert J. Palmer², Yu Han³, Chantal Massé¹, Sean Conlan⁴, Steven M. Holland⁵, Julie A. Segre⁴, Emilia L. Falcone¹
¹Montreal Clinical Research Institute (IRCM), Immunity and Viral Infections, Montréal, Canada
²National Institutes of Health (NIH), National Institute of Dental and Craniofacial Research, Bethesda, MD, USA
³Food and Drug Administration (FDA), Center for Devices and Radiological Health, Silver Spring, MD, USA
⁴National Institutes of Health (NIH), National Human Genome Research Institute (NHGRI), Bethesda, MD, USA
⁵National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA
Corresponding Author: Aléhandra Desjardins, email alehandra.desjardins@ircm.qc.ca

Abstract

Background: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any one of the five subunits forming the NADPH oxidase complex 2 (NOX2; gp91phox, p47phox, p22phox, p67phox, p40phox), which leads to decreased phagocyte-derived reactive oxygen species (ROS) formation. As a result, patients with CGD have severe infections and a 50% rate of inflammatory bowel disease (IBD). The microbiome appears to play an important role in CGD-associated IBD. Our evaluation of the intestinal microbiome of gp91phox-/- mice that are resistant to chemical and infectious colitis models showed that these mice were colonized with Mucispirillum schaedleri, an intestinal commensal reported to have both protective and pathogenic properties depending on the host environment.
Methods: We performed shotgun metagenomic sequencing on cecal contents from gp91phox-/- mice and whole genome sequencing of an M. schaedleri isolate from the same mice. We computationally identified unique genes in the M. schaedleri strain isolated from gp91phox-/- mice and confirmed by PCR that they are present in the gp91phox-/- -derived strain but not the type strain. We compared the metabolic profiles of gp91phox-/- -derived and type strains using a Biolog panel, measured ROS metabolism using an Amplex Red assay, and evaluated colitis susceptibility in gp91phox-/- mice decolonized of M. schaedleri from birth.
Results: We identified 321 unique genes in the metagenome of the gp91phox-/- -derived M. schaedleri strain and confirmed the presence of a subset of these genes by PCR. Metabolic profiling of the gp91phox-/- -derived M. schaedleri strain demonstrated increased activity in the glyoxylate and dicarboxylate (p=0.0001), pyruvate (p=0.0004) and methane (p=0.006) metabolic pathways, as well as decreased H2O2 metabolism compared to the type strain. We also observed that gp91phox-/- mice decolonized of M. schaedleri from birth have increased susceptibility to chemical colitis.
Conclusion: We have identified a new strain of the intestinal commensal M. schaedleri that may have adapted to the NOX2-defective host, and as such, has acquired new functional properties that may confer protection in the context of CGD colitis.

Neuroscience

Perceptual brain mechanisms of emotions conveyed by gait patterns of virtual pedestrians

Yu-Tzu Wu^1,2, Dr. Sylvain Baillet³, Dr. Anouk Lamontagne^1,2
¹School of Physical and Occupational Therapy, McGill University ,Montréal, Canada
²Centre de recherche interdisciplinaire en réadaptation du Montréal métropolitain (CRIR), Jewish Rehabilitation Hospital (JRH)—CISSS de Laval, Laval, Canada
³McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montréal, Canada
Corresponding Author: Yu-Tzu Wu, email yu-tzu.wu2@mail.mcgill.ca

Abstract

People can make social judgements and infer emotional states by observing the movement characteristics of a human walker (biological motion (BM)). Neuroimaging studies revealed a neural network specifically associated with the emotion perception from BM. However, how such a network interacts with other brain regions involved in motion interpretation and social interaction remains to be elucidated. Moreover, there is a paucity of research examining neurobiological sex differences in BM perception. Magnetoencephalography (MEG) will be used to measure brain activation while healthy young adults (n=16) passively view and discriminate virtual walkers displaying different emotions through their gait patterns (neutral, angry, happy). Our primary objective is to map brain activation regions and characterize the brain signal changes involved in emotion perception from BM, while focusing on event-related potential (ERPs) and functional connectivity (FC). The secondary objective is to determine potential interactions between sex/gender of observers vs. emotion walker on brain activation. We hypothesize that the emotional vs. neutral walkers evokes larger amplitudes and shorter latencies of early (N170 in occipitotemporal) and late components of ERPs (N300 in pSTS). The FC will reveal a network associated with the perception of BM (e.g. FBA, EBA, pSTS, hMT/V5+) and social/emotion representation (e.g. amygdala). Furthermore, female observers are hypothesized to elicit greater FC compared to males, in response to both male and female walkers. Results will enhance our understanding of the standard brain mechanism in emotion perception from body movement and serve as a basis for comparison to understand deficits in social interactions in patient population (e.g., traumatic brain injury).

OMICS, Bioinformatics and Computational Biology

Individual deconvolution of epigenomic activities (IDEA) for predicting epigenetic signatures in major depressive disorder

Sydney Sue^1,2, Yue Li³, Gustavo Turecki²
¹Human Genetics, McGill University
²Douglas Mental Health University Institute
³Computer Science, McGill University
Corresponding Author: Sydney Sue, email sydney.sue@mail.mcgill.ca

Abstract

Psychiatric disorders are difficult to study due to their heterogeneity. In particular, major depressive disorder (MDD) is highly prevalent and likely develops from the interaction of genetic variants in association with environmental mechanisms. The pathophysiology of MDD is bridged between genetic and environmental influences through the epigenome. These factors are implicated in the etiology of MDD, particularly in individuals who experienced early-life adversity (ELA). Approaches combining multiple epigenetic marks to define risk signatures have been limited. We aim to predict epigenomic signatures, specifically histone marks and chromatin states, specific to ELA encountered by depressed individuals who died by suicide, using in-house data by Lutz et al. (2018-in preparation). Six histone marks were measured, but due to a lack of tissue availability, samples were pooled together for each mark. However, the transcriptome was measured individually. The epigenetic marks were measured in the amygdala - a critical brain region for emotional regulation, implicated in ELA. A novel method to discover epigenomic signatures, the Individual Deconvolution of Epigenomic Activities (IDEA) model, will be used to deconvolve the pooled samples into individual specific histone profiles. IDEA will impute proximally and distally acting histone mark activities, based on RNA-seq data using variational inference and a neural network. This novel approach will contribute to our understanding of how exposure to ELA contributes to psychopathologies such as MDD and will serve to further demonstrate the potential for machine learning to be used as a valuable resource in the study and treatment of psychiatric pathologies.

Oncology

Deciphering metastasis maintenance events in adolescents and young adults’ sarcomas

Masoumeh Aghababazadeh¹, Livia Garzia¹
¹McGill University Health Centre, Montreal, QC
Corresponding Author: Masoumeh Aghababazadeh, email masoumeh.aghababazadeh@mail.mcgill.ca

Abstract

Sarcomas affect approximately 200 children, adolescents and young adults in Canada every year. Bone and soft tissues like fat, muscles or connective tissue are the sarcomas tissues of origin. Among the over 50 types of sarcoma, we focus on a highly malignant form, known as CIC-rearranged sarcomas. It mostly affects adolescents in the second decade of life and it is curable in only 40% of cases. These cancers respond poorly to available therapies and are highly prone to metastasize. Unfortunately, there is no long-term survival chances for metastatic patients. Due to the fact that surgery is not beneficial in the metastatic stage, there are not many available samples for genomic analysis. To overcome this challenge, we aimed to establish a CIC-rearranged sarcoma model that would also be metastatic in experimental animals, with the overarching goal to shed light on the genetic alterations responsible for metastasis maintenance and therapy failure. Using a new genomic engineering tool called Lentihop system, we transformed the cells of origin of CIC-rearranged sarcomas (mesenchymal stromal cells) by combining the CIC fusion with a transposon based somatic mutagenesis system. In this approach, we will generate random mutations and the clones with suitable genes for mesenchymal stromal cells transformation will have a selective advantage in vivo and make the primary tumor in rodent hosts. In a parallel approach, we developed a transposon based functional genomics screenings in a therapy naïve CIC-DUX4 cell line. This cell line is non-metastatic at baseline, allowing us to use it for our forward genetic screening approach. We expect that the engineered cell line will display a more aggressive phenotype due to the increased mutational burden. We are further comparing mutations after the cell line is injected in vivo, and the animals treated with a mouse adapted chemotherapy protocol. This project will allow us to reproduce the metastases evolution process of human sarcomas in vivo. Targeted sequencing of transposon insertion sites and comparison of the recurrently mutated genes between primary tumors and metastatic tumors will reveal driver mutations causing metastasis and chemoresistance.

Genetic Drivers of Medulloblastoma Metastasis

Castillo Orozco Ana Isabel¹, Garzia Livia¹
¹RI-MUHC, Glen Site. Department of Human Genetics, McGill University
Corresponding Author: Ana Isabel Castillo Orozco, email ana.castilloorozco@mail.mcgill.ca

Abstract

Tumours of the CNS represent the most common solid malignancies in childhood. Medulloblastoma (MB), a highly aggressive brain tumour located in the cerebellum, accounts for nearly 20 percent of all pediatric brain tumours. Far from being a single entity, Medulloblastoma characterizes by presenting a high intertumoral heterogeneity comprising at least four molecular subgroups (WNT, SHH, Group 3 and Group 4), and further 12 subtypes. These entities harbour distinct molecular features and clinical outcomes, being Group 3 the MB type with the lowest survival rates. Besides, the presentation of metastatic dissemination (leptomeningeal disease, LMD) is a determining predictor of poor prognosis independently from subgroup affiliation. Interestingly, recent studies have identified that primary and metastatic MB tumours differ significantly from one another, contrary to what was previously assumed. Consequently, targeted therapy addressed to targets identified in primary MBs might be ineffective for patients affected with LMD. Unfortunately, metastases are rarely resected as there are no clinical reasons to do so, and samples from autopsies are similarly uncommon due to ethical and logical issues. To improve our understanding of the molecular events that drive primary tumours to metastasize, this work aims to perform genome-wide molecular profiling of primary vs metastatic cells from already developed Group 3 PDXes models. Using sequencing technologies such as RNAseq and ChIP-seq we aim to profile the transcriptome and epigenome of primary tumours and mets to identify and subsequently validate candidate metastatic drivers. Hopefully, this approach will allow us to find therapeutic targets to treat or prevent metastatic Medulloblastoma.

Neural activity is tumor suppressive in Shh Medulloblastoma

Claire Chabot¹, Livia Garzia^1,3,4
¹McGill Faculty of Medicine, Department of Human Genetics
²RI-MUHC
³Cancer Research Program
Corresponding Author: Claire Chabot, email Claire.chabot@mail.mcgill.ca

Abstract

Medulloblastoma (MB) is the most common malignant pediatric brain tumor and cause of death in infants, children and adolescents affected by cancer. Patients systematically undergo surgical resection of the brain tumor, craniospinal irradiation, and chemotherapy; a toxic treatment plan for the developing brain. Out of the four MB molecular subgroups, the second most frequently occurring is Shh in approximately 20% of all cases. The cells of origin within this subgroup are cerebellar granule neuron precursors (CGNPs) which actively proliferate in response to Shh during mammalian brain development. Most pediatric cancers originate from altered developmental mechanisms when progenitors cannot complete differentiation and proliferate uncontrollably. An unexplored avenue for MB therapy is to push the tumor cells beyond this differentiation block during the initial stages of transformation. My hypothesis is that neural activity impairs oncogenic transformation of cerebellar neural progenitors cells and is tumor suppressive in Shh MB. I am investigating the effects of neurotransmitters on the proliferation and differentiation of CGNPs in mouse pups. Our lab has also developed mice expressing optogenetic proteins in CGNPs to characterize the effects of membrane depolarization through photostimulation. The role of neural activity will also be observed through optogenic stimulation on human Shh MB cell lines. Their engraftment potential will be studied post-implanatation in the cerebellum of immunocrompromised mice. Unpublished results from our lab indicate that spontaneous neural activity in the developing cerebellum could counterbalance Shh induced proliferation and drive cell-cycle exit and differentiation.

Oncogenic Impact of CTCF Dysfunction

Benjamin Lebeau¹, Maïka Jangal¹, Tiejun Zao¹, Chenxi Zheng¹, Michael Witcher^1,2
¹McGill University and Lady Davis Institute
²Corresponding Author
Corresponding Author: Benjamin Lebeau, email benjamin.lebeau@mail.mcgill.ca

Abstract

Background: CTCF is a protein whose normal function is commonly disrupted in a wide array of cancer, including loss of heterozygosity in 50% of breast cancers. Substantial evidence demonstrates that CTCF acts as a tumor suppressor gene. However, the role of CTCF deletion in cancer has not been clearly elucidated. Our novel investigation will uncover the mechanism of action of CTCF in cancer progression by studying CTCF single allele deletion.
Methods and Results: As a model for CTCF LOH, we used CRISPR/Cas9 to knockdown WT CTCF from a single allele in a non-transformed breast epithelial cell line, MCF10A. CTCF +/- cells showed increased invasiveness and mammosphere growth in vitro. This translated into an ability to initiate tumors at incomplete penetrance and increased metastatic abilities in vivo. RNA-seq indicated that the classical oncogene SNAI1 was strongly upregulated in CTCF+/- cells and that the PI3K pathway might be also activated, which was subsequently validated by Western Blot. ChIP-Seq revealed that CTCF binding was lost around key PI3K genes and from the SNAI1 promoter. Based on this, we tested the sensitivity of CTCF +/- cells to inhibition of the downstream PI3K effector, mTor, using Torin1. As expected, the invasiveness of CTCF+/- cells was highly sensitive to Torin1, Snail knockdown or CTCF addback. At the epigenetic level, CTCF loss leads to aberrant accumulation of H3K4me3 or H3K27ac at key oncogenes: promoting their upregulation. We are currently examining the changes in 3D chromatin structure surrounding PI3K genes and SNAI1 using Hi-C.
Conclusion: Overall, these studies reveal mechanistically how CTCF dysfunction drives oncogenic progression and provides insight into therapeutic avenues targeting CTCF +/-tumors.

INVESTIGATING ROLES OF 4E-BP1 IN BREAST CANCER METASTASIS

Predrag Jovanovic^1,2, Sophie Guenin^1,3, Valerie Sabourin¹ , Stephanie Totten^1,2, Josie Ursini-Siegel^1,2,3,4, Ivan Topisirovic^1,2,3,4
¹Jewish General Hospital, Lady Davis Institute, Montreal, Canada
²McGill University, Experimental Medicine, Montreal, Canada
³McGill University, Biochemistry, Montreal, Canada
⁴McGill University, Oncology, Montreal, Canada
Corresponding Author: Predrag Jovanovic, email predrag.jovanovic@mail.mcgill.ca

Abstract

Metastasis is the cause of ~90% of deaths among breast cancer patients. It is a multi-step process that requires successful orchestration of several key events. A cancer cell must [1] escape the primary tumor site (invasion), [2] enter circulation (intravasation), [3] travel to distant sites in the body (translocation), [4] exit circulation (extravasation), [5] colonize in the new organ, and [6] form a macrometastasis. These steps make metastasis a strenuous process for cancer cells. Translation can rapidly and reversibly perturb the proteome and is thus thought to play a major role in response to stress to which cancer cells are exposed during metastatic dissemination. Eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4EBPs) play a major role in regulation of translation by impeding assembly of the eIF4F complex that recruits mRNA to the ribosome. In mammals, these proteins are represented by a family of three members, 4E-BP1, 2, and 3. Literature has supported the role of eIF4E in promoting progression and metastasis of breast cancer, among others. However, the role of the 4E-BPs in this process is less understood. The polyomavirus Middle T-antigen (PyMT) transgenic mouse model is used for its ability to recapitulate each step of the metastatic cascade, from initial malignant transformation in the mammary tissue to metastatic outgrowth within the lungs. Unexpectedly, we observed that the germline loss of 4E-BP1/2 significantly reduced lung metastases in PyMT mice, without impacting the growth of the primary breast tumors. We next reconstituted 4E-BP1 expression in 4E-BP1/2- deficient breast cancer cells and observed a previously unappreciated duality in the role of 4E-BP1 in breast cancer metastasis. Specifically, restoring 4E-BP1 expression in 4E-BP1/2 null cells promoted spontaneous metastasis of breast cancer cells to the lung from the primary tumor site. In turn, 4E-BP1 re-expression inhibited lung colonization and metastatic outgrowth in a tail vein injection model. This suggests that 4E-BP1 is necessary for the early steps of the metastatic cascade, including invasion, intravasation and/or survival in the blood stream in a cell autonomous manner. Currently, the molecular mechanisms underlying these observations are being investigated.

High-throughput screen of primary human acute myeloid leukemia stem cells identifies novel anti-LSC compounds

Isabella Iasenza^1,2, Safia Safa³, Frederic Barabé^4,5,6, Sonia Cellot⁷, Brian Wilhelm^3,8, Kolja Eppert^2,9
¹Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada
²Research Institute of the McGill University Health Centre, Montreal, QC, Canada
³Laboratory for High Throughput Biology, Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, QC, Canada
⁴Centre de recherche en infectiologie du CHUL, Centre de recherche du CHU du Québec, Quebec City, QC, Canada
⁵CHU de Québec Hôpital Enfant-Jésus, Québec City, QC, Canada
⁶Department of Medicine, Université Laval, Quebec City, QC, Canada
⁷Department of Pediatrics, Division of Hematology, Ste-Justine Hospital, Université de Montréal, QC, Canada
⁸Department of Medicine, Faculty of Medicine, Université de Montréal, Montreal, QC, Canada
⁹Department of Pediatrics, McGill University, Montreal, QC, Canada
Corresponding Author: Isabella Iasenza, email isabella.iasenza@mail.mcgill.ca

Abstract

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and expansion of immature myeloblasts in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies targeting the unique biology of LSCs are needed while sparing hematopoietic stem cells (HSCs). We therefore isolated the LSC containing fraction of a primary human AML sample (>90% purity and 70% recovery), where the CD34+ cells are functionally validated to be enriched for LSCs in long-term xenotransplant assays, (Eppert, K., et al., 2011), and performed a high throughput screen of 11,166 chemical molecules. We also counter screened against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells. From this, 61 hits with >70% inhibition on CD34+ 8227 cells and <30% inhibition on CD34+ CB cells were identified. The compounds include histone deacetylase inhibitors, anti-apoptotic protein inhibitors and protein kinase inhibitors. We also identified 33 hits with >50% expansion on CD34+ 8227 cells but <30% inhibition on CD34+ CB cells, reminiscent of the differentiation driven by glucocorticoids we have previously identified in an in silico anti-LSC screen (Laverdière, I. & Boileau, M., et al., 2018). We will now re-validate these hits in vitro, where the most efficient compounds will be chosen for anti-LSC validation in a panel of primary AMLs.

INVESTIGATING THE EPIGENETIC REPROGRAMMING OF ANKRD30A IN PROSTATE CANCER

Janie Larocque¹, Nadia Boufaied², Swneke D. Bailey³, David P. Labbé^1,2
¹Division of Experimental Medicine, Department of Medicine, McGill University
²Division of Urology, Department of Surgery, McGill University and Research Institute of the McGill University Health Centre (RI-MUHC)
³Division of Thoracic Surgery, Department of Surgery, McGill University and RI-MUHC
Corresponding Author: Janie Larocque, email janie.larocque@mail.mcgill.ca

Abstract

Prostate cancer is the second most common cancer in men worldwide, with over 1 million newly diagnosed cases each year. Aggressive prostate cancer, defined by the progression from localized disease to metastasis, is responsible for the majority of prostate-associated mortality. Epigenetic reprogramming is an emerging mechanism of prostate cancer progression and has been associated with advanced prostate cancer, however, very little is known about the epigenetic reprogramming involved in prostate cancer progression to an aggressive and metastatic disease. We aim to expose the epigenetic changes and the differential usage of regulatory elements that permit the rapid progression of prostate cancer to a metastatic disease. We have reanalyzed a ChIP-seq dataset comparing the H3K27ac profile from 45 indolent and 45 aggressive prostate cancer cases and found a regulatory element enriched with aggressive tumours at the ANKRD30A locus. We determined that ANKRD30A, a transcription factor whose expression is normally restricted to the mammary epithelium and the testis, is aberrantly expressed in DU145, a metastatic prostate cancer cell line. Our goal is to characterize the epigenetic and transcriptional reprogramming driven by ANKRD30A and evaluate the capacity of ANKRD30A to instigate or maintain the aggressive properties of metastatic prostate cancer in vitro. We expect that this project will reveal epigenetic reprogramming as a driver of aggressive prostate cancer associated with rapid disease recurrence. Ultimately, this raises the exciting possibilities that targeted epigenetic profiling could be used to identify prostate cancer patients who would benefit from aggressive treatment modalities or even from epigenome-based personalized medicine.

The integrated stress response is tumorigenic and constitutes a therapeutic liability in KRAS-driven lung cancer.

Nour Ghaddar^1,2, Shuo Wang¹, Bethany Woodvine^3,4, Jothilatha Krishnamoorthy¹, Cedric Darini¹, Vincent Van Hoef⁵, Helmut Popper⁵, Ivan Topisirovic^1,6, Ola Larsson⁷, John Le Quesne^3,4, Antonis E Koromilas^1,6
¹Lady Davis Institute for Medical Research, Oncology, Montreal, Canada
²McGill Univeristy, Medicine, Montreal, Canada
³University of Leicester, Leicester Cancer Research Center, Leicester, United Kingdom
⁴University of Cambridge, MRC Toxicology Unit, Leicester, United Kingdom
⁵Medical University of Graz, Diagnostic and Research Institute of Pathology, Graz, Austria
⁶McGill University, Oncology, Montreal, Canada
⁷Karolinska Institute, Oncology-Pathology, Stockholm, Sweden
Corresponding Author: Nour Ghaddar, email nour.ghaddar@mail.mcgill.ca

Abstract

Mutations in KRAS account for ~25% of lung cancer cases and are refractory to therapy. Tumors with KRAS mutations encounter increased levels of genotoxic, proteotoxic and metabolic stress, which disrupt proliferation and homeostasis. Tumor growth requires induction of pathways to promote adaptation to stress. The Integrated Stress Response (ISR) plays a key role in adaptation to stress via phosphorylation of eukaryotic initiation factor 2 (p-eIF2). When ISR is activated, p-eIF2 mediates a translational-transcriptional reprogramming of genes with roles in survival and adaptation.
We demonstrate that ISR is pivotal in the development of lung adenocarcinoma. Specifically, analysis of 928 lung adenocarcinoma patients showed that increased p-eIF2 associates with a significant decrease in overall survival by ~12 months. These findings underscore the prognostic significance of ISR in the development and treatment of human lung adenocarcinoma. We investigated the role of ISR in mouse models of KRAS driven lung cancer by genetic and pharmacological means. We demonstrate the pro-tumorigenic function of ISR via its ability to stimulate phosphorylation of mitogen activated kinase ERK. Mechanistically, we show that increased p-eIF2 translationally represses dual specificity phosphatase DUSP6, which antagonizes ERK phosphorylation.
In addition to discovering an important mechanism of KRAS-driven lung cancer, our work demonstrates that pharmacological ISR inactivation is highly effective for treatment of KRAS lung cancer in mice. That is, ISR disruption by inhibiting the eIF2α kinase PERK or inactivating the translational effects of p-eIF2 by the integrated stress response inhibitor (ISRIB) substantially reduces lung tumor growth in mice. ISR inhibition is equally effective to inhibition of lung tumors by AMG-510, a potent and specific inhibitor of KRAS G12C.
The significance of our data is highlighted by the ability of ISR inhibitors to impair lung tumor growth with different KRAS mutations. Our findings provide a novel rationale for implementing ISR-based regimens in lung adenocarcinoma treatment.

Investigating the role of the MNK1/2-eIF4E in regulating the mammary gland extracellular matrix (ECM)

Samuel Preston^1,2, Christophe Gonçalves², Vincent Richard³, Rene Zehadi^3,4, Christoph Borchers³, Wilson Miller^1,2,4, Sonia del Rincon^1,2,4
¹Division of Experimental Medicine, McGill University, Montreal, Canada
²Department of Oncology, Lady Davis Institute, Jewish General Hospital, Montreal, Canada
³Segal Cancer Centre Proteomics Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Canada
⁴McGill Centre for Translational Research in Cancer (MCTRC), McGill University, Montreal, Canada
Corresponding Author: Samuel Preston, email samuel.preston@mail.mcgill.ca

Abstract

Background: The ECM is a highly dynamic component of the tumor microenvironment (TME) that adapts to reinforce neoplastic progression. The MNK1/2-eIF4E axis exemplifies how translation initiation can act abnormally to promote cancer. eIF4E phosphorylation, uniquely by MNK1/2, induces the translation of a subset of mRNA involved in invasion and metastasis, some of which are known matrisomal proteins. To date, no one has reported on how translational control, or its deregulation, impacts ECM composition and homeostasis.
Methods: We aim to define ECM signatures that are regulated by the MNK1/2-eIF4E axis. Primary ECM samples were isolated from the mammary glands (MGs) of wild-type and phospho-eIF4E-deficient female mice and subjected to LC-MS/MS proteomic analysis. In tandem, soluble ECM preparations were used to treat human and murine breast cancer cells and primary MG fibroblasts in vitro for subsequent immunoblot, qPCR, transwell migration-invasion and functional assays. Tail vein and orthotopic mammary fat pad injection models were also utilised.
Results: Matrisomal profiling highlights several proteins that are differentially expressed according to eIF4E phosphorylation status. This includes collagen-I which is downregulated in phospho-eIF4E-deficient MG ECM. Phospho-eIF4E-deficient MG fibroblasts produce less collagen-I and secrete a less organised 3D matrix as compared to wild-type. Breast cancer cells treated with phospho-eIF4E-deficient MG ECM have suppressed invasive capacity in vitro and in vivo, without altered proliferation rates or EMT progression, as compared to wild-type.
Conclusion: We have shown that ECM from a phospho-eIF4E deficient MG fosters a less invasive TME. Future work aims to expand our findings into human patients.