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University of Ottawa

45 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology
Sophie Imbeault *1, Nicolas Valenzuela *2, Stephen Fai 2, Steffany A.L. Bennett 1
1Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism, Carleton University

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

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Immunology and Infection

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
Caroline Gravel *1, Changgui Li *2, Junzhi Wang 2, Anwar M Hashem 1,3,4, Bozena Jaentschke 1, Gary Van Domselaar 5, Runtao He 5, Xuguang Li 1,3
1Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health canada, 2National Institute for the Control of Pharmaceutical and Biological Products, The State Food and Drug Administration, Beijing, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Microbiology Department, Faculty of Medicine, King Abdulaziz University, 5National Microbiology Laboratory, Public Health Agency of Canada

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

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Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells
Carmen G. Palii 1, Roya Pasha 1, Marjorie Brand 1,2
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

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Medicine

Ex Vivo Infection of Live Tissue with Oncolytic Viruses
Jean-Simon Diallo 1, Dominic Roy 1, Hesham Abdelbary 1, Naomi De Silva 1, John C. Bell 1
1Center for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI)

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

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Biology

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification
Ashleigh C. McLean 1,2,3, Nicolas Valenzuela 3,4, Stephen Fai 3,4, Steffany A.L. Bennett 1,3
1Department of Biochemistry, Microbiology and Immunology, Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, 2Department of Cellular and Molecular Medicine, University of Ottawa , 3CIHR Program in Neurodegenerative Lipidomics, University of Ottawa , 4Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

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JoVE Core

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows
Katie L. Pitts 1, Marianne Fenech 1,2
1Department of Chemical and Biological Engineering, University of Ottawa , 2Department of Mechanical Engineering, University of Ottawa

Micro-particle image velocimetry (μPIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

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Engineering

Fabrication of VB2/Air Cells for Electrochemical Testing
Jessica Stuart 1, Ruben Lopez 2, Jason Lau 1, Xuguang Li 2, Mahesh Waje 2, Matthew Mullings 2, Christopher Rhodes 2, Stuart Licht 1
1Department of Chemistry, The George Washington University, 2Lynntech

A protocol is presented to study multi-electron metal/air battery systems by using previous technology developed for the zinc/air cell. Electrochemical testing is then performed on fabricated batteries to evaluate performance.

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Immunology and Infection

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)
Thien-Fah Mah 1
1Department of Biochemistry, Microbiology and Immunology, University of Ottawa

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

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Neuroscience

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish
James J. Jun 1,2,3, André Longtin 1,2,3, Leonard Maler 2,3
1Department of Physics, University of Ottawa, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Centre for Neural Dynamics, University of Ottawa

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal's electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

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Engineering

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores
Eric Beamish 1, Harold Kwok 1, Vincent Tabard-Cossa 1, Michel Godin 1,2
1Department of Physics, University of Ottawa, 2Ottawa-Carleton Institute of Biomedical Engineering, University of Ottawa

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

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Medicine

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies
Lee-Hwa Tai 1, Christiano Tanese de Souza 1, Shalini Sahi 1, Jiqing Zhang 1,2,4, Almohanad A Alkayyal 1,3,5, Abhirami Anu Ananth 1,3, Rebecca A.C. Auer 1,6
1Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 4Department of Neurosurgery, The Second Hospital of Shandong University, 5Department of Medical Laboratory Technology, University of Tabuk, 6Department of Surgery, Ottawa General Hospital

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

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Bioengineering

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology
Dominique Tremblay 1, Charles M. Cuerrier 1,2, Lukasz Andrzejewski 1, Edward R. O'Brien 3, Andrew E. Pelling 1,4,5
1Centre for Interdisciplinary NanoPhysics, Department of Physics, University of Ottawa, 2University of Ottawa Heart Institue, University of Ottawa, 3Libin Cardiovascular Institute of Alberta, University of Calgary, 4Department of Biology, University of Ottawa, 5Institute for Science, Society and Policy, University of Ottawa

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

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Biology

High-throughput Titration of Luciferase-expressing Recombinant Viruses
Vanessa Garcia 1,2, Ramya Krishnan 1,2, Colin Davis 1,2, Cory Batenchuk 1,2, Fabrice Le Boeuf 1,3, Hesham Abdelbary 1,3, Jean-Simon Diallo 1,2
1Center for Innovative Cancer Research, Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 3Faculty of Medicine, University of Ottawa

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

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Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models
Devon S. Svoboda 1, Alysen Clark 1, David S. Park 1, Ruth S. Slack 1
1Department of Cellular and Molecular Medicine, University of Ottawa

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

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Neuroscience

Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees
Levente L. Orbán 1, Catherine M.S. Plowright 1
1School of Psychology, University of Ottawa

This video describes Radio-Frequency Identification (RFID) and motion-sensitive video recording methods to monitor choice behavior by bumblebees.

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Biology

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
Mame Daro Faye 1, Tyson E Graber 2, Martin Holcik 3
1Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Montreal Neurological Institute, 3Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Pediatrics, University of Ottawa

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

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Developmental Biology

A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish
Velislava Tzaneva 1, Steve F. Perry 1
1Department of Biology, University of Ottawa

This manuscript describes a protocol to track the re-distribution of branchial ionocytes and their innervation using a time differential staining technique coupled with full bilateral gill denervation.

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Bioengineering

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation
Rym Mehri 1, Catherine Mavriplis 1, Marianne Fenech 1
1Department of Mechanical Engineering, University of Ottawa

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

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Behavior

Evaluation of a Smartphone-based Human Activity Recognition System in a Daily Living Environment
Edward D. Lemaire 1, Marco D. Tundo 2, Natalie Baddour 2
1The Ottawa Hospital Research Institute, University of Ottawa, 2Department of Mechanical Engineering, University of Ottawa

A standardized evaluation method was developed for Wearable Mobility Monitoring Systems (WMMS) that includes continuous activities in a realistic daily living environment. Testing with a series of daily living activities can decrease activity recognition sensitivity; therefore, realistic testing circuits are encouraged for valid evaluation of WMMS performance.

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Biology

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs
Jennifer J.P. Collins 1,2, Marius A. Möbius 1,3,4, Bernard Thébaud 1,2,5
1Sinclair Centre for Regenerative Medicine, Ottawa Hospital Research Institute, 2University of Ottawa, 3Department of Neonatology and Pediatric Critical Care Medicine, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, 4DFG Research Center and Cluster of Excellence for Regenerative Therapies (CRTD), Technische Universität, Dresden, 5Children's Hospital of Eastern Ontario Research Institute

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

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Behavior

Substantiating Appropriate Motion Capture Techniques for the Assessment of Nordic Walking Gait and Posture in Older Adults
Christopher M. Dalton 1, Julie Nantel 1
1School of Human Kinetics, Faculty of Health Sciences, University of Ottawa

The aim was to substantiate optimal use of data collection techniques for Nordic walking gait and posture analysis. Three-dimensional motion capture should be used during short duration analysis (i.e. single gait cycle), while accelerometry should be employed for longer duration analysis (i.e. repeated cycles) like a 6 Minute Walk Test.

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Medicine

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior
Matthew M. Piaseczny *1,2, Graciella M. Pio *1,2, Jenny E. Chu 1,2, Ying Xia 1, Kim Nguyen 1,3, David Goodale 1, Alison Allan 1,2,4,5
1London Regional Cancer Program, London Health Sciences Centre, 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, 3Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, 4Department of Oncology, Schulich School of Medicine & Dentistry, Western University, 5Lawson Health Research Institute

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

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Genetics

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
Mohamed I. Hassan *1, Fern R. McSorley *1, Kinya Hotta 2, Christopher N. Boddy 1
1Department of Chemistry and Biomolecular Sciences, University of Ottawa, 2School of Biosciences, The University of Nottingham Malaysia Campus

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

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Neuroscience

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons
Matthew Laaper 1,2, Takrima Haque 1, Ruth S. Slack 3, Arezu Jahani-Asl 1,2,4
1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Integrated Program in Neuroscience, McGill University, 3Department of Cellular and Molecular Medicine, University of Ottawa, 4Department of Oncology, Faculty of Medicine, McGill University

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

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Engineering

All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics
Mohammad Rashidi 1,2, Wyatt Vine 1, Jacob A.J. Burgess 3,4,5, Marco Taucer 1,2,6, Roshan Achal 1, Jason L. Pitters 2, Sebastian Loth 3,4, Robert A. Wolkow 1,2
1Department of Physics, University of Alberta, 2National Institute for Nanotechnology, National Research Council of Canada, Edmonton, 3Max Planck Institute for the Structure and Dynamics of Matter, 4Max Planck Institute for Solid State Research, 5Department of Physics and Astronomy, University of Manitoba, 6Joint Attosecond Science Laboratory, University of Ottawa

We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.

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Cancer Research

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy
Kristina J. Allan 1,2, Douglas J. Mahoney 1,4, Stephen D. Baird 1, Charles A. Lefebvre 1, David F. Stojdl 1,2,3
1Children's Hospital of Eastern Ontario (CHEO) Research Institute, 2Department of Biology, Microbiology and Immunology, University of Ottawa, 3Department of Pediatrics, University of Ottawa, 4Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

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JoVE Journal

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Allison A. Dilliott 1,2, Sali M.K. Farhan 3, Mahdi Ghani 4, Christine Sato 4, Eric Liang 5, Ming Zhang 4, Adam D. McIntyre 1, Henian Cao 1, Lemuel Racacho 6,7, John F. Robinson 1, Michael J. Strong 1,8, Mario Masellis 9,10, Dennis E. Bulman 6,7, Ekaterina Rogaeva 4, Anthony Lang 10,11, Carmela Tartaglia 4,10, Elizabeth Finger 12,13, Lorne Zinman 9, John Turnbull 14, Morris Freedman 10,15, Rick Swartz 9, Sandra E. Black 9,16, Robert A. Hegele 1,2
1Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, 3Analytic and Translational Genetics Unit, Center for Genomic Medicine, Harvard Medical School, Massachusetts General Hospital, Stanley Centre for Psychiatric Research, Broad Institute of MIT and Harvard, 4Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 5School of Medicine, Faculty of Health Sciences, Queen's University, 6Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 7CHEO Research Institute, Faculty of Medicine, University of Ottawa, 8Department of Clinical Neurological Sciences, Western University, 9Division of Neurology, Department of Medicine, Sunnybrook Health Sciences Centre, University of Toronto, 10Division of Neurology, Department of Medicine, University of Toronto, 11Morton and Gloria Shulman Movement Disorders Centre, Toronto Western Hospital, 12Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, 13Parkwood Institute, St. Joseph's Health Care, 14Department of Medicine, Division of Neurology, McMaster University, 15Division of Neurology, Department of Medicine, Baycrest Health Sciences, 16Canadian Partnership for Stroke Recovery Sunnybrook Site, Sunnybrook Health Science Centre, University of Toronto

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

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Genetics

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping
Ceres Kosuta 1,2, Kate Daniel 1, Devon L. Johnstone 1,2, Kevin Mongeon 1,2, Kevin Ban 1,2, Sophie LeBlanc 1, Stuart MacLeod 1, Karim Et-Tahiry 2, Marc Ekker 2, Alex MacKenzie 1, Izabella Pena 1,2
1Children's Hospital of Eastern Ontario Research Institute, 2Department of Biology, University of Ottawa

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

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Medicine

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat
T. Mark Campbell 1,2,3, Katherine Reilly 2,3, Louis Goudreau 4, Odette Laneuville 2,5, Hans Uhthoff 2,3, Guy Trudel 2,3,4
1Elizabeth Bruyère Hospital, 2The Bone and Joint Research Laboratory, University of Ottawa, 3Department of Medicine, University of Ottawa, 4The Ottawa Hospital Rehabilitation Centre, 5Department of Biology, University of Ottawa

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

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Environment

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium
Benjamin R. Stenzler 1, Jessica Gaudet 1, Alexandre J. Poulain 1
1Department of Biology, University of Ottawa

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

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Cancer Research

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor
Taha Azad *1, Kazem Nouri *1, Helena J. Janse van Rensburg 1, Yawei Hao 1, Xiaolong Yang 1
1Department of Pathology and Molecular Medicine, Queen's University

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

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JoVE Journal

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes
Alexanne Cuillerier 1, Yan Burelle 1
1Interdisciplinary School of Health Science, Faculty of Health Sciences and Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

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Education

Fabricating van der Waals Heterostructures with Precise Rotational Alignment
Justin Boddison-Chouinard 1, Ryan Plumadore 1, Adina Luican-Mayer 1
1Department of Physics, University of Ottawa

In this work we describe a technique that is used to create new crystals (van der Waals heterostructures) by stacking ultrathin layered 2D materials with precise control over position and relative orientation.

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Biology

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice
Ri Youn Kim *1,2, Ju Hee Lee *3,4, Yena Oh 1,2, Hoon-Ki Sung 3,4,5, Kyoung-Han Kim 1,2
1University of Ottawa Heart Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Translational Medicine Program, The Hospital for Sick Children, 4Department of Laboratory Medicine and Pathobiology, University of Toronto, 5Banting and Best Diabetes Centre, University of Toronto

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

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Behavior

Examining Bilingual Language Control Using the Stroop Task
Laura Sabourin *1, Santa Vīnerte *1
1Department of Linguistics, University of Ottawa

This bilingual Stroop task uses Congruent, Incongruent, and Neutral stimuli presented in blocks in the first language (L1) only, the second language (L2) only, and a combination of L1 and L2. This task allows for an examination of language processing and cognitive control in both L1 and L2.

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Biochemistry

Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay
Manel Zeghal 1, Geneviève Laroche 1, Patrick M. Giguère 1,2
1Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Brain and Mind Research Institute, University of Ottawa

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient β-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

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Chemistry

Hyperspectral Imaging as a Tool to Study Optical Anisotropy in Lanthanide-Based Molecular Single Crystals
Emille M. Rodrigues 1, Nelson Rutajoga 1, David Rioux 2, Jacob Yvon-Leroux 2, Eva Hemmer 1
1Department of Chemistry and Biomolecular Sciences, University of Ottawa, 2Photon etc

Here, we present a protocol to obtain luminescent hyperspectral imaging data and to analyze optical anisotropy features of lanthanide-based single crystals using a Hyperspectral Imaging System.

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Biochemistry

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support
Jordan Demone 1, Kiah A. Barton 1,2, Illimar Altosaar 1,2
1Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 2Proteins Easy Corp

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

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Immunology and Infection

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Taylor R. Jamieson 1,2, Joanna Poutou 1,2, Ricardo Marius 1,2, Xiaohong He 1, Reza Rezaei 1,2, Taha Azad 1,2, Carolina S. Ilkow 1,2
1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

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Immunology and Infection

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter
Reza Rezaei 1,2, Abera Surendran 1,2, Ragunath Singaravelu 1,2, Taylor R. Jamieson 1,2, Parisa Taklifi 3, Joanna Poutou 1,2, Taha Azad 1,2, Carolina S. Ilkow 1,2
1Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 3Department of Biotechnology, University of Tehran

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

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Medicine

Transmission Electron Microscopy: A Surgical Pathology Tool for Neuroblastoma
Consolato M. Sergi *1,2,3,4, Janice Patry *1, Harry Coenraad 1, Jeff McClintock 1, Rod Nicholls 1, Hans-Jörg Steiner 5, Gregor Mikuz 5
1Anatomic Pathology Division of EORLA, Children's Hospital of Eastern Ontario, University of Ottawa, 2Department of Laboratory Medicine and Pathology, University of Alberta, 3Department of Pediatrics, University of Alberta, Stollery Children's Hospital, 4Department of Orthopedics, Tianyou Hospital, Wuhan University of Science and Technology, 5Institute of Pathology, Medical University of Innsbruck

Pediatric small round blue cell tumors are an intriguing and challenging collection of neoplasms. Therefore, transmission electron microscopy (TEM) and professional knowledge of pediatric tumors can be extremely valuable in surgical pathology. Here, we present a protocol to perform TEM for diagnosing neuroblastoma, one of the most common solid tumors in childhood.

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Biology

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk
Aizhu Lu 1,2, Jerry Wang 1,2, Ying Xia 1,2, Ruonan Gu 1, Kyoung-Han Kim 1,2, Erin E. Mulvihill 1,3, Darryl R. Davis 1,2, Rob S. Beanlands 1, Wenbin Liang 1,2
1University of Ottawa Heart Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

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Bioengineering

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo
Maxime Leblanc Latour 1, Maryam Tarar 2, Ryan J. Hickey 1, Charles M. Cuerrier 1, Isabelle Catelas 3,4,5, Andrew E. Pelling 1,2,6,7
1Department of Physics, University of Ottawa, 2Department of Biology, University of Ottawa, 3Department of Mechanical Engineering, University of Ottawa, 4Department of Surgery, University of Ottawa, 5Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 6Institute for Science, Society and Policy, University of Ottawa, 7SymbioticA, School of Human Sciences, University of Western Australia

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

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Biochemistry

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography
Sarah Wazir 1, Danny Farhat 1, Mahalashmi Srinivasan 1,2, Jyh-Yeuan Lee 1
1Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 2Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

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Cancer Research

Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-Derived Extracellular Vesicle-Based Cancer Biotherapeutics
Frederic St-Denis-Bissonnette *1,2, Melanie Kirkby *1,2, Lisheng Wang 2,3,4, Jessie R. Lavoie 1,2
1Biologic and Radiopharmaceutical Drugs Directorate, 2Department of Biochemistry, Microbiology and Immunology Institute, University of Ottawa, 3Centre for Infection, Immunity and Inflammation Institute, University of Ottawa, 4Regenerative Medicine Program, Ottawa Hospital Research Institute

Natural killer cell-derived extracellular vesicles (NK-EVs) hold promising potential as cancer biotherapeutics. This methodology-based study presents a scalable closed-loop biomanufacturing workflow designed to continuously produce and isolate large quantities of high-purity NK-EVs. In-process control testing is performed throughout the biomanufacturing workflow, ensuring the EVs meet quality standards for product release.

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