Experimental Medicine Biomedical Graduate Conference (AMBGC) | March 21st, 2023
Experimental Medicine Graduate Students' Society (EMGSS)
Published online: March 21st, 2023Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins
2Nencki Institute of Experimental Biology, Poland,
3University of Texas, USA
Corresponding Author: Huy Bui, email huy.bui@mcgill.ca
Abstract
Cilia are hair-like organelles responsible for cellular motility and sensory functions. The cytoskeletal core of the cilium consists of nine hollow doublet microtubules arranged in a ring around two central singlets. It was recently discovered through cryogenic electron microscopy that there exist many different microtubule inner proteins (MIPs) inside each doublet microtubule. These MIPs form a meshwork of luminal proteins weaving into the tubulin lattice. Knockout of MIPs such as FAP52 and FAP45 destabilizes the doublet microtubule and causes a reduced swimming phenotype observed in both Tetrahymena thermophila and Chlamydomonas reinhardtii. The interaction of MIPs with the tubulin lattice suggests that some MIPs are essential for assembly and stability of the doublet microtubule. However, the individual functions of MIPs are largely unknown. I identified and modeled MIPs that are potentially responsible for the assembly and stability of the Tetrahymenadoublet microtubule in the outer junction region. Gene knockout of the conserved outer junction protein CFAP77 reduces swimming and ciliary beating in Tetrahymena. Cryogenic electron tomography of cilia from the CFAP77 knockout strain reveals damage to doublet microtubules compared to wild type. These results suggest that CFAP77 has an important role in outer junction stability and ciliary beating.
Characterization of Von Economo Neurons in Depression and Schizophrenia
2McGill University, Montreal, QC, Canada
Corresponding Author: Canonne Candice, email candice.canonne@mail.mcgill.ca
Abstract
Von Economo neurons (VENs) are a type of neurons found in a few regions of the human brain including the anterior cingulate cortex (ACC) and frontal insula (FI). While ACC and FI are part of the salience network that regulates the switching between the executive and default mode network, studies increasingly implicate VENs in schizophrenia (SCZ) and to a lesser extend major depressive disorder (MDD), two illnesses presenting alterations of the default mode network activation. Recently, a transcriptomic analyze of VENs enabled the identification of differentially expressed genes in VENs vs pyramidal neurons, in particular a higher expression of genes associated with SCZ and MDD. However, the involvement of VENs in these disorders remains to be largely characterized. In my project, I started by assessing if VENs density is illness-dependent in FI and ACC, and tested candidate markers for VENs. Well-characterized post-mortem brain samples from the ACC and FI for each group were provided by the Douglas-Bell Canada Brain Bank. A quantification of Nissl-stained VEN densities using QuPath was performed, as well as a DAB-stained VENs quantification with 2 candidate markers, VAT1L and NMB. Differences in the distribution of VENs within the gyrus for each group of study were found, as well as a heterogeneity of VENs positive or not for VAT1L and NMB. These results could be explained by the potential existence of VENs subtypes, a possibility that I also aim to investigate.
The effects of e-cigarette use on cardiopulmonary health
2Lady Davis Institute for Medical Research, Montreal, QC, Canada
3Research Institute of McGill University Health Center
Corresponding Author: Dr. Koren Mann & Dr. Carolyn Baglole, email vincenza.caruana@mail.mcgill.ca
Abstract
E-cigarettes are designed to simulate the act of cigarette smoking without the injurious health impacts of tobacco. Although e-cigarettes were intended to be a cessation tool for smokers, their sleek design and flavorings made them popular with teens and young adults in the US and Canada. Yet, the health effects of e-cigarette use in non-smokers remains unclear. Several studies, including our previous work have correlated e-cigarette aerosols to increased inflammation and pathological conditions such as myocardial infarctions and organ fibrosis. Therefore, this project investigates the impact of a chronic, high-level e-cigarette exposure on multiple pulmonary and cardiovascular outcomes, including fibrosis and atherosclerosis. To model atherosclerosis, 5-week-old male and female C57BL/6J mice were injected with AAV-PCSK9 and fed a high fat diet. One week later, the mice were exposed to room air or the e-cigarette aerosol twice daily for 16 weeks. The e-cigarette brands STLTH and Vuse were used as they are among the most popular e-cigarette brands used today. After the exposure period, metal deposition, inflammatory and fibrotic markers in the lungs will be assessed. The extent of the atherosclerotic plaque and its constituents will be evaluated by en face oil red O staining, picrosirius red, α-smooth muscle actin, and MOMA-2 staining. These results have the potential to make significant impacts in understanding the long-term health consequences of vaping.
Prediction of late gadolinium enhancement (LGE) using non-contrast CMR biomarkers
Corresponding Author: Katerina Eyre, email katerina.eyre@muhc.mcgill.ca
Abstract
Late gadolinium enhancement (LGE) cardiovascular magnetic resonance imaging (CMR) is limited by administration of a contrast agent. The purpose of this study was to identify and rank non-contrast biomarkers to train a machine learning (ML) algorithm that predicts LGE in American Heart Association (AHA) segments. Patients referred for a CMR exam with ischemic cardiomyopathy (ICMP) were recruited. Short- and long-axis cine images, T1 and T2 maps and LGE images were acquired. All data were analyzed using certified software (cvi42, Circle Cardiovascular Imaging Inc., Calgary, AB, Canada). The CMR and individual biomarkers were inputted into the Cardiom AI system (Area19 Medical Inc.) to train an ML algorithm to classify AHA segments as having positive or negative LGE. A Shapley analysis was used to identify the salient biomarkers which led to LGE prediction. 78 participants were enrolled in this study (mean age = 61y., 66 male). The ML algorithm was able to correctly classify LGE AHA segments with an area-under-the-curve (AUC) of 0.84 (Fig.1A). Circumferential strain was found to be the biomarker which contributed the most to LGE prediction, with decreasing strain values implying a greater likelihood of positive LGE (Fig.2A). This study demonstrates a strong potential of non-contrast CMR biomarkers to predict irreversible myocardial tissue damage on a segmental level. This suggests that a multi-parametric, contrast-free CMR approach has the diagnostic potential to replace LGE.
The Effect of Obstructive Sleep Apnea on Cognitive Functioning among Parkinson’s Disease Individuals: Evidence from the Canadian Longitudinal Study on Aging (CLSA).
2Translational Research in Respiratory Diseases, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada,
3Department of Medicine and Department of Epidemiology, Biostatistics & Occupational Health, McGill University Health Centre, Montreal, Quebec, Canada,
4Montreal Neurological Hospital, McGill University Centre, Montreal, Quebec, Canada,
5Research Center, CIUSSS Nord-de-l'Ile-de-Montreal, Montreal, Quebec, Canada,
6Department of Neurology and Neurosurgery, McGill University, Montreal General Hospital, Montreal, QC,
7Respiratory Division and Sleep Laboratory, McGill University Health Centre, Montreal, Quebec, Canada
Corresponding Author: Teresa Gomes, email teresa.gomes@mail.mcgill.ca
Abstract
Background: Obstructive sleep apnea (OSA) is associated with cognitive decline in the general older population and with lower cognition in PD patients in clinical cohorts. We aimed to evaluate associations between high risk for OSA and cognition in individuals with Parkinson’s disease (PD) from a population cohort. Methodology: Cross-sectional data from the Canadian Longitudinal Study of Aging (CLSA) included identifying participants with PD at baseline or at 3-year follow-up using a validated algorithm and cognitive test battery, allowing for domain-specific analyses. High risk of OSA was determined using the STOP-B28 (Snoring, Tiredness, Observed apneas, high blood Pressure and Body Mass Index [BMI]≥28) >2. Linear regression was performed to assess relationships between STOP-B28 dichotomized or continuous scores and cognitive measures, adjusted for potential cofounders. Results: We identified 150 individuals with PD. Overall, 76 had a high risk of OSA (mean age 70.9 (9.0) years, 69.7% male) and 73 did not (mean age 69.1 (8.9) years, 63.0% male). There was no significant association between STOP-B28 >2 and cognitive measures. Higher STOP-B28 scores were associated with longer time to answer on the STROOP-word in adjusted analyses (p=0.03). Conclusion: No association emerged between STOP-B28 >2 and cognitive measures in individuals with PD in the CLSA. Higher STOP-B28 scores were associated with poorer results on an executive function test.
CTCF copy number loss delays breast cancer initiation and suppresses metastasis
2Lady Davis Research Institute, Jewish General Hospital, Montréal, QC, Canada
3Department of Medicine, McGill University, Montréal, QC, Canada
Corresponding Author: Michael Witcher, email michael.witcher@mcgill.ca
Abstract
CCCTC-binding factor (CTCF) is a putative tumor suppressor and CTCF+/- mice develop lymphomas. While CTCF undergoes copy number loss (CNL) in over 50% of breast cancers, the role of CTCF CNL in breast tumorigenesis is not understood. CTCF is involved in hierarchical chromatin organization. It anchors static loops called topologically-associated domains (TAD) and smaller, dynamic loops within TADs called subTADs. These chromatin loops insulate interactions between local regulatory elements. CTCF thus prevents aberrant promoter-enhancer contacts and coordinates many transcriptional networks. Here, for the first time, we investigate the role of CTCF CNL in breast cancer initiation and metastasis in vivo. We developed a new transgenic CTCF+/- breast cancer mouse model by crossing mice carrying floxed CTCF alleles with mice carrying a MMTV-NEU-IRES-Cre (NIC) transgene. These NIC/CTCF+/- mice express an activated HER2/Neu oncogene and loss of a single CTCF allele in the mammary gland. We monitored these mice for tumor initiation and volume, and lung metastasis. Surprisingly, NIC/CTCF+/- displayed a delayed tumor onset and a reduced number of metastases, but once tumors were initiated, tumor volumes were consistent with CTCF+/+/Neu mice. Consistent with these data, CTCF-/-/Neu mice failed to grow tumors. Overall, our mouse model indicates a role for CTCF in promoting tumor initiation, which contradicts the current paradigm of CTCF function that is based mainly on in vitro studies.
Extracellular vesicles: A window into the etiology of Major Depressive Disorder
2McGill Group for Suicide Studies, Douglas Mental Health University Institute, Montreal, Quebec, Canada.
3Department of Psychiatry, McGill University, Montreal, Quebec, Canada.
Corresponding Author: Gustavo Turecki, email gustavo.turecki@mcgill.ca
Abstract
Major Depressive Disorder (MDD) is a leading cause of disability worldwide. MicroRNA’s (miRNA) are disrupted in MDD and can be packaged into extracellular vesicles (EVs), along with other bioactive molecules, such as proteins. We hypothesize that EV cargo from the anterior cingulate cortex, a brain region highly implicated in MDD, will have a disease specific profile that could mediate disease development. EVs were isolated from post-mortem human brain tissue via size exclusion chromatography. The quality was assessed by western blots, transmission electron microscopy (TEM), and microfluidic resistive pulse sensing. MiRNA and Protein profiling and differential analysis of EV cargo was then performed. Preliminary differential analyses revealed sex-specific dysregulation of miRNAs, as well as in proteomic profiles of EVs in MDD. MiR-92a-3p was found to be downregulated in females, while miR-129-5p was downregulated in males. Interestingly, both miRNAs are involved in processes related to neurodevelopment and neurotransmitter signaling. Future directions point towards identifying mechanisms implicated in this dysregulation. This will be the first study to profile brain-derived EV miRNA and protein in the context of depression. This could provide novel mechanistic insights into the pathophysiology of MDD, which could serve as a starting point for the development of targeted therapeutic strategies as well as prevention measures.
Non-invasive Localization of the Epileptogenic Zone of Clinically Complex Epilepsy Patients Using [11C]ABP688 PET
2Positron Emission Tomography Unit, McConnell Brain Imaging Center, Montreal Neurological Institute, McGill University, Montreal, QC, Canada,
3Translational Neuroimaging Laboratory, McGill Center for Studies in Aging, Douglas Mental Health University Institute, McGill University, Montreal, QC, Canada
Corresponding Author: Eliane Kobayashi, email eliane.kobayashi@mcgill.ca
Abstract
Background: Temporal lobe epilepsy (TLE) is a common type of focal epilepsy in adults. Treatment options in TLE patients with a known focus location includes surgical intervention. A subset of these patients with bilateral and/or neocortical foci are considered clinically complex and require intracranial electroencephalography recordings to locate the epileptogenic zone (EZ). The [11C]ABP688 radioligand binds allosterically to mGluR5, allowing for quantification of mGluR5 availability, a non-invasive measure to determine surgical candidacy. We tested whether [11C]ABP688 PET could provide additional information for patients in whom localization of the EZ is more challenging. Methods: Ten patients who had undergone [11C]ABP688 PET and magnetic resonance imaging (MRI) were retrospectively selected. [11C]ABP688 binding potential (BPND) maps were generated and qualitatively analyzed for abnormalities within the mesial and neocortical temporal structures. Results: [11C]ABP688 binding reduction concordant with the clinical hypothesis was observed in presumed seizure onset areas in our cohort. All 10 patients exhibited variable degrees of reduction in [11C]ABP688 BPND in mesial temporal structures as well as the neocortex. Conclusion: This proof-of-concept study demonstrates the value of mGluR5 imaging, especially for use in determining surgical candidacy in more intricate TLE cases and is one of the first to observe [11C]ABP688 BPND reductions in both mesial and neocortical TLE.
Deciphering the role of epigenetic combination therapies in ovarian cancer
Corresponding Author: Marios Langke, email marios.lange@mail.mcgill.ca
Abstract
High grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer, accompanied by low responsiveness to the current standard of care and high rates of mortality. It is clear that epigenetic processes play a central role in tumor progression and as such, there is much interest in drugs targeting the epigenome as anti-cancer therapeutics. Our lab is quite interested in the potential of BET inhibitors (BETi), that act by repressing the transcription of oncogenic networks to treat ovarian cancer. Towards this goal, we carried out a CRISPR screen with BETi to look for potential synthetic lethalities. We found the transcriptional co-activators CDK7 and CREBBP (CBP) among our top hits as potential synthetic lethalities. We hypothesize that BET proteins (primarily BRD4) may co-operate with CDK7 and CBP for the regulation of key oncogenes. As a first step we tested BETi in combination with inhibitors of CDK7 or CBP against syngeneic models of murine ovarian cancer. We found that BETi+CBPi achieve a profound synergy in vitro across all cell lines tested. We are currently exploring the efficacy of this combination in vivo. We also also exploring the mechanism whereby these agents achieve synergy through RNA-seq and ChIP-seq. These experiments will provide us with novel insights regarding the efficacy of CREBBPi+BETi combination treatment, thus opening the possibilities for their use in the clinic.
A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine
2Cell and Systems Biology, University of Toronto, Toronto, ON, Canada
3Biological Sciences, Brock University, St. Catharines, ON, Canada
4Food Science and Agricultural Chemistry, McGill University, Montreal, QC, Canada
Corresponding Author: Mehran Dastmalchi, email mehran.dastmalchi@mcgill.ca
Abstract
Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, and a lesion-mimic phenotype. Its lesions are the site of high of akuammicine concentrations, where LC/qTOF-MS profiling revealed enrichment of 25 other MIAs, displaying higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Finally, the mutant can be a source for akuammicine, a stereochemically complex molecule that is a promising medicinal target, as a weak opioid agonist with a preference for the non-addictive κ-opioid receptor. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.
Investigation of the sexual dimorphic tumor suppressor role of DDX3X in melanoma
2McGill Genome Centre, Department of Human Genetics McGill University, Montreal, Quebec, Canada,
3McGill University Health Centre, Lady Davis Institute, McGill University, Montreal, Quebec, Canada
Corresponding Author: Marine Lingrand, email marine.lingrand@mail.mcgill.ca
Abstract
Patient sex remains a poorly understood prognostic factor in melanoma. Men with melanoma have at all stages poorer prognosis than females. Recently, our group identified new significantly mutated genes by performing a mutational analysis of 1,014 melanoma exomes from five studies (Alkallas et al., 2020). We found that loss-of-function (LoF) mutations in the X-linked-DEAD-box RNA helicase, DDX3X, are solely found in male patients. We provided evidence that DDX3X can escape X-inactivation, which would protect females from complete DDX3X loss in the case of a single mutational event in melanoma. To date, the reported functions of DDX3X include RNA regulation of translation and mediators of important cancer signalling pathways. However, the role of DDX3X in melanoma remains unclear. We hypothesize that DDX3X is a sexual dimorphic tumor suppressor gene, which may explain in part, some of the observed differences in incidence and outcome between female and male melanoma patients. To examine this, we generated stable DDX3X knock-outs (KOs) and inducible DDX3X knockdowns in male and female melanoma and melanocyte lines. We observed DDX3X loss leads to increase proliferation, migration, and invasion only in male melanoma and melanocyte lines supporting its sexual dimorphic tumor suppressor role in melanoma.
What is a gamete-specific gene doing in Cutaneous T-Cell Lymphoma development?
2Cancer Research Program, Research Institute McGill University Health Centre, Montreal, QC, Canada
3Division of Dermatology, Faculty of Medicine and Health Sciences, McGill University, Montreal, QC, Canada
Corresponding Author: Amelia Martinez Villarreal, email amelia.martinezvillarreal@mail.mcgill.ca
Abstract
Cutaneous T-Cell Lymphomas (CTCL) is a group of rare lymphomas characterized by the accumulation of T cells in the skin. Transcriptomic analyses of patient samples identified the ectopic expression of gamete genes. We aim to understand the role of Gamete Specific Factor 1 (GTSF1) in development of CTCL. In gametes, GTSF1 controls retrotransposon expression during development. We performed shRNA-mediated knockdown of GTSF1 in three CTCL cells lines. Then, we performed RNA-Seq followed by differential gene expression and pathway enrichment analysis. We analyzed the effects of knockdown with qRT-PCR, immunoblotting, luciferase retrotransposition assay, ELISA, flow cytometry and mass-spectrometry. Surprisingly, our experiments suggest that GTSF1 is not silencing retrotransposons in CTCL. However, seems GTSF1 regulates the Th phenotype of CTCL cells. Specifically, pathway enrichment analysis shows an enrichment of pathways such as response to cytokine and Interferon gamma signaling. Detailed analysis shows an increase in production of IFNγ and TNF. Interestingly, as the disease progresses there is a decrease in Th1 cytokine production (IFNγ and TNF) and an enhanced Th2 cytokine production (IL-4 and IL-5). This shift from a Th1 to a Th2 response causes severe immunodeficiency. Our data suggest GTSF1 is regulating, in part, the Th2 phenotype shift associated with a worse prognosis. Because of its gamete-restricted expression, GTSF1 presents an ideal candidate for targeted therapy.
Targeting Shp2 sensitizes Class 3 BRAF mutant cancers to BRAF and MEK inhibitors
2Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, QC, Canada,
3Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada,
4Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada
5Department of Medicine, University of Toronto, Toronto, ON, Canada
Corresponding Author: nan, email nan
Abstract
BRAF, a constituent of the MAPK signaling pathway, is one of the most frequently mutated oncogenes. Class 3 BRAF mutations hyperactivate the MAPK pathway by binding to and activating WT BRAF and CRAF in the presence of active RAS. While BRAF+MEK inhibitors (e.g., binimetinib and encorafenib (B+E)) improve survival in patients with Class 1 BRAF mutant cancers, there are no standard therapies for Class 3 BRAF mutant cancers. Therefore, we sought to identify effective treatments for Class 3 BRAF mutant cancers. We analyzed functional genomics data and found several components of the Shp2 signaling complex (PTPN11, GAB1, GRB2) as being significantly more essential for the growth of Class 3 vs. Class 1 BRAF mutant cells. Shp2, a protein tyrosine phosphatase, functions upstream of RAS to activate the MAPK pathway. We evaluated the efficacy of Shp2 inhibitors (SHP099, TNO155 (TNO)) across BRAF mutant cell lines and observed significantly lower IC50 values in Class 3 (n=7) vs. Class 1 (n=3) BRAF mutant cells. Compared to B+E or TNO, B+E+TNO more effectively inhibited the growth of Class 3 BRAF mutant cells in vitro. Lastly, we demonstrated that B+E+TNO induced significantly more tumor growth inhibition than B+E in several Class 3 BRAF mutant PDXs. Together, these data highlight Shp2 as a potential therapeutic target for Class 3 BRAF mutant cancers. Future research will explore molecular mechanisms of sensitivity and resistance to Shp2i in Class 3 mutant tumors in vitro and in vivo.
Temporal and spatial probing into cancer-associated fibroblast heterogeneity using multiplex imaging
2Segal Cancer Centre, Lady Davis Institute and Jewish General Hospital, Montréal, QC, Canada.
3Montreal Rossy Cancer Network, Montréal, QC, Canada.
Corresponding Author: Paige McCallum, email paige.mccallum@mail.mcgill.ca
Abstract
Cancer-associated fibroblasts (CAFs), abundantly found in the tumor microenvironment, are highly versatile plastic cells that play a crucial role in enhancing tumor phenotypes. We have previously characterized the CAF compartment of the TME in a murine breast cancer model using transcriptomic and proteomic methods. We aim to better understand the functional role of CAF subsets by characterizing their spatial locations in a 4T1 mouse breast cancer model using 7-color multiplex immunofluorescence staining (IF) on fresh frozen tissues at distinct stages of tumor development. This will allow us to visualize CAF subset location vis-à-vis other key components of the TME, affording insights into CAF-TME interplay. In parallel, to increase the translational relevance of our findings, we intend to comprehensively characterize the TME of aggressive human breast cancer using a 50-plex Co-Detection by indEXing (CODEX) panel, with a focus on CAFs. Integrating insights between murine and human breast cancer will allow us to better understand breast cancer pathology and the functions CAFs play in facilitating it. Integrating digital pathology and machine learning, our results show that the CAF subsets identified by single-cell RNAseq and flow cytometry can be visualized and quantified in situ. This study provided an in-depth characterization of CAFs based on molecular features and their interactions within the TME in breast cancer.
Development, characterization and antimicrobial activity of α-tocoferol and cinnamaldehyde nanocapsules
2Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa 58051-900, PB, Brazil
3Department of Otolaryngology and Head and Neck Surgery and Lady Davis Institutes for Medical Research of the Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada
Corresponding Author: Isis Muniz, email isisamuniz@gmail.com
Abstract
Background: Mucositis can be quite severe increasing the risk of infections and the treatment involves managing the symptoms and prevent complications. This study aimed to develop and characterize new formulations of nanocapsules combining α-tocopherol (α-toc) and cinnamaldehyde (CNM) coated with chitosan in order to enhance antimicrobial effects in oral cavity. Methodology: The characterization of the nanocapsules was carried out through the particle size, polydispersion index, zeta potential, and infrared spectroscopy by Fourier transform (FTIR). Furthermore, studies involving sol-gel phases, stability, rheological tests, and antimicrobial activity were done. Results: Particles with size ranging from 135.7 to 1997nm and zeta potential with values between -3.76 to +11.1 and FTIR confirmed the encapsulation of assets in dispersions. The sol-gel phase transition occurred at temperatures between 20-30°C and remained in the form of a solution when stored at room temperature for 15.9 to 47.27s. It was observed great stability regarding pH, homogeneity, odor, and coloration. In the rheological analyses, the dispersions of nanocapsules had a pseudoplastic behavior. The CNM nanocapsules (MIC 3.12µg/mL) showed an increased antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida albicans compared with free CNM (MIC 39µg/mL). Conclusion: Nanocapsules have superior characteristics and antimicrobial effects for future clinical application in oral cavity.
Adaptation and translation, and Validation of the Patient Reported Experience Measure for Children and Young People (PREM-CYP) at The Montreal Children’s Hospital
2Department of Pediatric Surgery, Harvey E. Beardmore Division of Pediatric Surgery, McGill University Health Center, Montreal, QC, Canada,
3Great Ormond Street Hospital for Children (GOSH), London, United Kingdom
Corresponding Author: Zanib Nafees, email zanib.nafees@mail.mcgill.ca
Abstract
INTRODUCTION: Patient Reported Experience Measures (PREMs) objectively evaluate children and youth’s perception of their care. However, use of PREMs is limited and generalizing any local data to larger populations remains to be accomplished. PREMs are useful as they reflect on the quality of care, are feasible, and are time effective. The utility of PREMs is seen in public reporting, institutional benchmarking, and health care plans. PREMs data can improve patients’ experiences in waiting times, communication, and team coordination. METHODS: This study will be a mixed-method research design that uses qualitative methods in Phases 1 and 2, adapting the existing PREM-CYP instrument to the MCH and translating it to French. Quantitative methods for Phase 3 will be used to cross-validate the MCH English and French versions. Research Participants: Around 12-15 patients in the age groups (8-11, 12-13, 14-16 years) at the pediatric surgery clinics will take part in each session; think-aloud testing, cognitive testing, and bilingual validation). RESULTS: We anticipate that by examining the meaning of the healthcare experiences of the children and young people (CYP), we will be able to generalize the results to other CYP in different settings. CONCLUSIONS: Our dissemination goals are: 1) to ensure that the voices of CYP are heard regarding their patient reported experiences (PRE), 2) to increase the acceptability of generalized data over a larger population by translating PREM-CYP_MTL from English to French, and 3) to ensure instrument compatibility with the CYP population of Montreal by having a bilingually validated PREM-CYP_MTL.
Deciphering the Role of Faciogenital Dysplasia 1 (FGD1) in Melanoma Progression
2Department of Clinical Laboratory Medicine, McGill University Health Center, Montreal, Quebec, Canada.
Corresponding Author: Guy Namir, email guy.namir@mail.mcgill.ca
Abstract
Cutaneous melanoma is one of the most metastatic cancers. In Canada in 2021, there were 8,700 melanoma new cases and 1,240 deaths. It was shown that sex is an independent predictor of survival. Accumulating data suggests that melanoma might be facilitated by X-linked genes, such as FGD1, FGD1, a Guanine nucleotide Exchange Factor, activates the GTPase Cdc42 that targets a few cellular signaling pathways. Due to FGD1’s involvement in cell cycle, morphology, motility, and extracellular matrix degradation, its amplification is related to several cancers’ progression. However, the role of FGD1 in melanoma development remains unknown. We analyzed FGD1 mRNA expression from hundreds of melanoma samples, using the TCGA and GSEA databases. ATAC-seq analysis shows that the FGD1 locus is more accessible in melanoma tumors compared to other cancers and potentially translated to high protein levels. Moreover, we show a correlation between high FGD1 levels to poor survival rates. Furthermore, we show that high and low expression of FGD1 contributes to proliferative and invasive transcriptional states respectively. We develop several FGD1 KO and overexpression melanoma cell lines, to show the effect and mechanism of FGD1 on proliferation, cell cycle, migration, and invasion. We will validate the results using in vivo models and patients samples. Discovering the role of FGD1 in melanoma progression will assist us to identify new biomarkers and innovative treatments to treat melanoma.
D-mannose supplementation decreases atherosclerotic lesions in ApoE-/- mice through regulation of gut microbiota composition and inflammation
Corresponding Author: Stephanie Lehoux, email stephanie.lehoux@mcgill.ca
Abstract
D-mannose, a C−2 epimer of glucose, alters gut microbiota and exhibits anti-inflammatory properties. Given these qualities, we hypothesized that mannose would alleviate the pro-atherogenic effects of high fat diet (HFD). ApoE-/- mice were fed a HFD supplemented with 0, 5, or 20% mannose. No differences in body weight, lipid levels, or glucose tolerance were observed among the groups. Atherosclerotic plaque size of mice treated with 5 or 20% mannose was half of that of 0% mannose mice. Furthermore, plaques of 20% mannose mice contained increased a-smooth muscle actin compared with 0%, suggesting increased stability. Blood Ly6CHI pro-inflammatory monocytes and neutrophils were reduced in 5 or 20% mannose-treated mice vs 0%. Importantly, mannose abated changes in the intestinal microbiome brought about by HFD. The Firmicutes/Bacteroidetes ratio rose significantly in mice with HFD+0% mannose (6.1±1.5) compared with chow+0% mannose (2.0±0.3) but was no longer elevated in HFD+20% mannose (2.5±0.1). Concurrently, HFD induced a 2-fold increase in plasma LPS levels and higher proportions of intestinal F4/80+ macrophages expressing the LPS receptor TLR4 compared with chow. 20% mannose prevented these effects. Our results show that mannose supplementation reduces atherosclerosis and improves plaque stability in ApoE-/- mice fed a HFD. These protective effects may occur through the regulation of gut microbiota, reducing LPS-induced intestinal inflammation, and abating monocytosis and neutrophilia.
Role of the human telomerase insertion in fingers domain in telomerase activation at telomeres
2Jewish General Hospital, Lady Davis Institute, Montreal, QC, Canada,
3Université de Montréal, Département de Biochimie et Médecine Moléculaire, Montreal, QC, Canada.
Corresponding Author: Chantal Autexier, email chantal.autexier@mcgill.ca
Abstract
Background The proper regulation of telomeres and telomerase is essential in healthy cells. Telomerase is a ribonucleoprotein (RNP) that elongates telomeres, since otherwise the telomeres shorten after every round of replication which leads to cell senescence. For telomerase to be active, the holoenzyme is minimally composed of two subunits: human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR). After assembly and localization to the telomeres, hTERT acts as the core catalytic subunit and uses hTR as a template to add new TTAGGG DNA repeats onto the telomeres. We are interested in how residues within the insertion in fingers domain (IFD), a region of hTERT, regulate retention and activity at the telomeres since this region is known to regulate telomerase activity in vitro and localization to the telomeres. Methodology To test this hypothesis, we are assessing the activity of mutant hTERT compared to the wild-type hTERT at the telomeres by measuring the incorporation of mutant telomere repeats using a mutant hTR RNA template. We are also using a single-molecule tracking assay to quantify if IFD mutants are defective in recruitment and/or retention at the telomeres. Conclusion By elucidating the role of the IFD, this study aims to provide insight to the overall regulation of telomerase.
Dissect the role of an amphipathic alpha-helix inside the Med19 IDR in Cdk8 function and understanding the role of Cdk8 in transcription
2Division of Experimental Medicine at McGill University, Montreal, QC, Canada
Corresponding Author: Francois Robert, email francois.robert@ircm.qc.ca
Abstract
Mediator is a transcriptional co-regulator found throughout eukaryotes. Although subunit composition varies between organisms (yeast have 25 subunits and up to 30 subunits in higher mammals), the structure of Mediator is well-conserved. Organized into 4 modules (essential Head and Middle modules with the regulatory Tail and Kinase modules (CKM)), Mediator is generally required for RNA Polymerase II (RNAPII) transcription. CKM consists of 4 subunits including the enzyme Cdk8. Work from our lab using truncation mutants and Cdk8-growth assays has identified a portion of Middle module subunit Med19 that regulates Cdk8 function. Co-immunoprecipitations show Med19 is not required for the interaction between Mediator and CKM. We narrowed down the Med19 determinant for Cdk8 function to a small region, predicted to form a Molecular Recognition Motif (MoRF) and expected to fold into an amphipathic alpha helix. Changing the hydrophobic residues of this helix to alanines led to loss of Cdk8 function. We hypothesize the MoRF interacts with a factor that is key for CKM function. My first objective is to identify the factor(s) that interact with the MoRF using crosslinking mass spectrometry and dissect how it/they regulate(s) Cdk8. We also aim to understand the impact of this mutant and subsequent of Cdk8 function on Mediator interactions with RNAPII and the impact on transcription.
Ectopic PRAME expression impairs anti-cancer retinoid activity in keratinocyte carcinomas
Corresponding Author: Brandon Ramchatesingh, email brandon.ramchatesingh@mail.mcgill.ca
Abstract
Background: As potent regulators of epidermal turnover, retinoids have been contemplated as treatments for keratinocyte carcinomas. Preferentially Expressed Antigen in Melanoma (PRAME), a germline-restricted retinoid signaling repressor, is ectopically expressed in keratinocyte carcinomas. The functions and therapeutic relevance of PRAME in these cancers has not been studied. Methods: To investigate the impact of PRAME on malignant progression and therapy response, cell lines representative of human basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and immortalized keratinocytes were subjected to PRAME overexpression and knockdown. Cells were treated with all trans retinoic acid (ATRA) or tazarotene. Assays for cell proliferation, death and differentiation were conducted. Results: PRAME overexpression conferred resistance to the anti-growth activity of retinoids in SCC and keratinocyte cell lines. PRAME knockdown potentiated the anti-growth activity of retinoids in BCC and SCC cell lines. Additionally, retinoid-induced changes in cytokeratin profile were abrogated by PRAME overexpression, and conversely, potentiated by PRAME knockdown. EPZ 6438, an epigenetic therapy, conferred sensitivity to ATRA in a PRAME-positive cSCC cell line. Conclusions: We propose that PRAME confers resistance to retinoid therapies. PRAME may serve as a useful prognostic biomarker or target to sensitize cancer cells to retinoid therapeutics.
Associations of maternal gestational diabetes with offspring diabetes across two pregnancies: preliminary work
2Research Institute McGill University Health Center, Montreal, QC, Canada
3Montreal Children’s Hospital, McGill University Health Centre, Montreal, QC, Canada
Corresponding Author: Laura Rendon, email laura.rendon@mail.mcgill.ca
Abstract
Over 25% of children with type 1 diabetes (T1DM) in Quebec are diagnosed at the time of emergency presentation with diabetic ketoacidosis (DKA) (CJD, 2022). Knowledge of T1DM indicators may promote earlier detection. Our group identified a link between gestational diabetes (GDM) and pediatric diabetes (CMAJ, 2019). Whether this association comes from household/genetic factors or the intrauterine environment, is unknown. To gain insight into this issue, we are examining the associations of GDM during a sibling’s pregnancy with T1DM in the subject. We hypothesize that if household/genetic factors are responsible, having an exposed sibling could be a risk for T1DM in the subject, even in the absence of GDM during the index pregnancy. Using provincial health insurance administrative databases, we identified 485222 families with two consecutive deliveries from 1990 to 2012. Follow-up was until April 1st, 2019. We have calculated descriptive statistics and incidence rates (IR), for different exposure categories. A total of 2230 subjects developed diabetes. Diabetes IR was higher in all exposure categories compared to the control (2.71). Interestingly, IR for diabetes were similar for those exposed to GDM (3.83) and those whose sibling was exposed to GDM (3.63). This suggests that household/genetic factors may be more important than in utero glycemia in terms of contributions to diabetes risk. We are constructing Cox models to examine this further, with adjustments for covariates.
Harnessing the onset of metastasis in uveal melanoma: CD36 as a promising target.
2Lady Davis Institute for Medical Research,
3Jewish General Hospital Clinical Research Unit,
4Jewish General Hospital BioBanks
Corresponding Author: Shin, Heejin Hayley, email heejin.shin@mail.mcgill.ca
Abstract
Uveal melanoma is the most common intra-ocular malignant cancer type in adults. 50% of patients experience metastasis to the liver quite early and from hereon, approximately 80% of patients die within the first year. To make matters worse, there are no standardized testing platforms nor an effective therapy. Hence, we’ve developed a multi-coloured FACS panel composed of multiple classical cell state markers in order to characterize more than 10 uveal melanoma cell lines derived from patient primary tumors or metastasized tumors. Careful phenotyping of all cell lines with the panel gave us a broad spectrum of expression levels for each marker amongst the different cell lines. This observed heterogeneity has been the proposed mechanism underlying the ability of cancer cells to metastasize. Particularly, CD36 was highly expressed in cell lines as well as moderate expression in some of the others. CD36 has been studied in other cancer types such as cervical cancer and glioblastoma multiforme, to increase metastatic potential as well as having a negative correlation with patient survival. Hence, we depleted CD36 in our uveal melanoma cell line and saw that this affected the tumor cells’ ability to migrate and reduced their ability to invade observed through a Boyden-chamber invasion assay. Currently, we have taken this study in vivo to study the effect of CD36 depletion to study metastatic potential, tumor microenvironment, and phenotypic characterization of tumors.
SARS-CoV-2 variant natural evolution in K18-ACE2 mice increases virulence and produces treatment resistance-linked variant alleles
2McGill University Genome Centre, Montreal, QC, Canada.
3Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada.
4Axe maladies infectieuses et immunitaires, Centre de Recherche du Centre Hospitalier Universitaire de Québec- Université Laval, Canada.
5Centre de Recherche ARThrite-Arthrite, Recherche et Traitements, Université Laval, Québec, QC, Canada.
6Division of Rheumatology, Department of Medicine, CHU de Québec-Université Laval, Québec, QC, Canada.
7Canadian Centre for Computational Genomics, McGill University, Montreal, QC, Canada.
8Department of Human Genetics, McGill University, Montreal, QC, Canada.
9Advanced Genomic Technologies Laboratory, McGill Genome Centre, McGill University, Montreal, QC, Canada.
10Département de microbiologie-infectiologie et d'immunologie, Université Laval, QC, Canada.
Corresponding Author: Louis Flamand, email louis.flamand@crchudequebec.ulaval.ca
Abstract
The continuing COVID-19 pandemic is partially due to viral evolution reducing vaccine and treatment efficacy. Others have observed serial infections in mice with reference SARS-CoV-2 lineage IME-BJ05 produce adapted strains with significantly greater infectivity and mortality. We investigated the impact of long-term B.1.351 (Beta) and B.1.617.2 (Delta) lineage viral evolution in K18-ACE2 mice, which express human ACE2 receptor. We infected mice with these unmodified variants in a BSL-3 laboratory and studied viral evolution across 20 passages without selective pressure, sequencing virus at key points. Our IRB determined the research was not gain-of-function. We observed variant alleles with documented contributions towards evading vaccine-induced immunity arise de-novo, including Omicron-characteristic mutation S371F. Passage 20 (P20) viruses were more virulent, and P20 Delta was significantly more resistant to antibody neutralization. These developments occurred rapidly, emulating pandemic progression in non-immune mammals. Our model could be rapidly adapted to include selective pressures to study the evolution of viruses resistant to antivirals or vaccine-induced immunity.
Chemotherapy-induced peripheral neuropathy (CIPN) in cancer survivors: are duloxetine, medical cannabis and exercise safe and effective interventions?
2Division of Palliative Care, Jewish General Hospital, Montreal, Canada
3Division of Supportive and Palliative Care, McGill University Health Centre, Montreal, Canada
4Department of Anesthesia, McGill University, Montreal, Canada
5Research Institute-McGill University Health Centre, McGill University, Montreal, Canada
Corresponding Author: Maria Luisa Vigano, email marialuisa.vigano@mail.mcgill.ca
Abstract
In an observational study, Duloxetine (DLX) (60 gm/day) was provided to cancer survivors diagnosed with CIPN as the treatment standard (phase 1). If DLX failed to improve pain scores by 30% after 4 weeks, CBD or exercise (EX) was prescribed for two months (phase 2). Effectiveness of DLX, CBD or EX was determined by the painDETECT questionnaire, the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity scale, and the Godin-Shephard leisure-time physical activity questionnaire. Tolerability of the three treatments were assessed through weekly follow-ups. Seven participants have completed phase 1. After 4 weeks of DLX, 1) three patients (43%) had a better pain score whereas two patients (29%) experienced worse or unchanged pain, 2) quality of life improved, remained unchanged or deteriorated for three (50%), two (33%) or one (17%) patient, respectively and 3) five patients (71%) were more active. Reported side effects included: nausea (43%), drowsiness (14%), low energy (14%), constipation (14%), sweating (14%), abdominal pain (14%) and abnormal behavior (14%). Five patients (71%) experienced severe side effects and had to stop DLX at 30mg/day. No patients exhibited a ≥30% improvement in painDETECT scores and all moved into phase 2 (CBD or EX). These preliminary results suggest that DLX is partially effective but poorly tolerated in cancer survivors suffering from CIPN.
Regulation of muscle autophagy by BCAS3
2Venetian Institute of Molecular Medicine and Department of Biomedical Science, University of Padova, Padova, Italy
3Département de Sciences de l’activité physique, Faculté des Sciences, UQAM, Québec, Canada
Corresponding Author: Sabah NA Hussain, email sabah.hussain@muhc.mcgill.ca
Abstract
Background: Autophagy is critical for muscle mass, function, and integrity, but the molecular mechanisms regulating autophagy are complex and only partly understood. We recently identified that a regulator of autophagy and skeletal muscle integrity in vivo, termed MYTHO, interacts with breast carcinoma amplified sequence 3 (BCAS3), a highly conserved cytoskeletal protein. The roles that BCAS3 plays in skeletal muscle, however, are unknown. Here, we evaluated the impact of BCAS3 knockdown on skeletal muscle integrity. Methods: BCAS3 was knockdown for 6 weeks in the Tibialis Anterior muscles using intramuscular injections of Adeno-Associated Viruses (AAV). BCAS3-KD’s impact on muscle fiber size and on the proportion of centrally located myonuclei were assessed using immunolabeling approaches on muscle cross-sections. RT-PCR and immunoblots were used to assess markers of autophagy. Results: BCAS3 knockdown was confirmed by RT-qPCR. 6 weeks of BCAS3-KD resulted in significant myofiber atrophy, but no change in the proportion of centrally located nuclei. BCAS3-KD also led to the accumulation of the autophagic marker p62 without altering p62 mRNA levels. No difference was found in LC3B-I and LC3B-II protein expression. Conclusion: Our data show that BCAS3 knockdown is sufficient to trigger myofiber atrophy. This atrophic impact occurs without signs of muscle fiber degeneration / regeneration. Our data also suggest that BCAS3 plays a role in the regulation of skeletal muscle autophagy.
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