University of Alberta View Institution's Website 67 articles published in JoVE Biochemistry ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography Sarah Wazir1, Danny Farhat1, Mahalashmi Srinivasan1,2, Jyh-Yeuan Lee1 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. Neuroscience Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning Aislinn D. Maguire1, Jason R. Plemel1,2,3, Bradley J. Kerr1,4,5 1Neuroscience and Mental Health Institute, University of Alberta, 2Department of Medicine, Division of Neurology, University of Alberta, 3Department of Medical Microbiology and Immunology, University of Alberta, 4Department of Pharmacology, University of Alberta, 5Department of Anesthesiology and Pain Medicine, University of Alberta This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation. Medicine A Piglet Perinatal Asphyxia Model to Study Cardiac Injury and Hemodynamics after Cardiac Arrest, Resuscitation, and the Return of Spontaneous Circulation Eydis Oddsdottir Stenersen1,2, Annette Olsen2, Maria Melheim3, Rønnaug Solberg3,4, Ingrid Dannevig5, Georg Schmölzer6,7, Po-Yin Cheung6,7, Britt Nakstad8,9, Ola Didrik Saugstad10,11, Arild Rønnestad1,2, Anne Lee Solevåg2 1Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 2Department of Neonatal Intensive Care, Division of Pediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, 3Department of Pediatric Research, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Rikshospitalet, 4Department of Pediatrics, Vestfold Hospital Trust, 5Department of Anesthesiology, Oslo University Hospital Rikshospitalet, 6Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, 7Centre for the Studies of Asphyxia and Resuscitation, Neonatal Research Unit, Royal Alexandra Hospital, 8Division of Paediatric and Adolescent Medicine, Institute of Clinical Medicine, University of Oslo, 9Department of Paediatrics and Adolescent Health, University of Botswana, 10Department of Pediatric Research, Oslo University Hospital, University of Oslo, 11Department of Pediatrics, Robert H Lurie Medical Research Center, Northwestern University Chicago This piglet model involves surgical instrumentation, asphyxiation until the cardiac arrest, resuscitation, and post-resuscitation observation. The model allows for multiple sampling per animal, and by using continuous invasive arterial blood pressure, ECG, and non-invasive cardiac output monitoring, it provides knowledge about hemodynamics and cardiac pathophysiology in perinatal asphyxia and neonatal cardiopulmonary resuscitation. Medicine Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction Elizabeth Hillier1,2, Jason Covone1, Matthias G. Friedrich1,3 1Faculty of Medicine and Health Sciences, McGill University, 2Faulty of Medicine and Dentistry, University of Alberta, 3Departments of Medicine and Diagnostic Radiology, McGill University The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function. Medicine Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism Keir A. Forgie1,2, Nicholas Fialka3, Max Buchko1,2, Sayed Himmat4, Sanaz Hatami4, Xiao Qi4, Xiuhua Wang4, Katie-Marie Buswell5, Ryan Edgar5, Darren H. Freed1,2,6,7, Jayan Nagendran1,2,6,7 1Division of Cardiac Surgery, Department of Surgery, Faculty of Medicine, University of Alberta, 2Mazankowski Alberta Heart Institute, 3Faculty of Medicine and Dentistry, University of Alberta, 4Department of Surgery, Faculty of Medicine, University of Alberta, 5Ray Rajotte Surgical-Medical Research Institute (SMRI), University of Alberta, 6Alberta Transplant Institute, 7Canadian Donation and Transplantation Research Program This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting. Medicine Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP Keir A. Forgie1,2, Nicholas Fialka3, Mubashir Khan4, Max Buchko1,2, Sanaz Hatami4, Sayed Himmat4, Xiao Qi4, Xiuhua Wang4, Katie-Marie Buswell5, Ryan Edgar5, Daina Domahidi5, Darren H. Freed1,2,6,7, Jayan Nagendran1,2,6,7 1Division of Cardiac Surgery, Department of Surgery, Faculty of Medicine, University of Alberta, 2Mazankowski Alberta Heart Institute, 3Faculty of Medicine and Dentistry, University of Alberta, 4Department of Surgery, Faculty of Medicine, University of Alberta, 5Ray Rajotte Surgical-Medical Research Institute (SMRI), University of Alberta, 6Alberta Transplant Institute, 7Canadian Donation and Transplantation Research Program This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting. Immunology and Infection Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells Shammy Raj1, Lei Lu2, Larry D. Unsworth1 1Department of Chemical and Materials Engineering, Donadeo Innovation Centre for Engineering, University of Alberta, 2School of Life Science and Engineering, Southwest Jiaotong University Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors. Medicine A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers Jerry T. Dang1, Valentin Mocanu1, Breanna Fang2, Michael Laffin1, Shahzeer Karmali1, Karen Madsen3, Daniel W. Birch1 1Department of Surgery, University of Alberta, 2Faculty of Medicine and Dentistry, University of Alberta, 3Department of Medicine, University of Alberta Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates. Bioengineering Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture Leshern Karamchand1, Ashley Wagner1, Syed Benazir Alam1, Marianna Kulka1,2 1Nanotechnology Research Center, National Research Council Canada, 2Department of Medical Microbiology and Immunology, University of Alberta This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (FcεRI). Medicine Focal Laser Ablation of Prostate Cancer: An Office Procedure Wayne G. Brisbane1, Shyam Natarajan1, Alan Priester1, Ely R. Felker1, Adam Kinnaird2, Leonard S. Marks1 1University of California Los Angeles, 2University of Alberta This article presents and describes an outpatient treatment for prostate cancer using focal laser ablation. Laser catheter placement is guided by MRI-ultrasound fusion imaging in a fashion similar to prostate needle biopsy. Treatment is monitored in real-time with a thermal probe, placed adjacent to the laser fiber. Cancer Research Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model Konstantin Stoletov1, Lian Willetts2, Perrin H. Beatty1, John D. Lewis1 1Department of Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Department of Cell Biology & Anatomy, Cumming School of Medicine, University of Calgary This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing. Engineering Cryogenic Liquid Jets for High Repetition Rate Discovery Science Chandra B. Curry*1,2, Christopher Schoenwaelder*1,3, Sebastian Goede4, Jongjin B. Kim1, Martin Rehwald5,6, Franziska Treffert1,7, Karl Zeil5, Siegfried H. Glenzer1, Maxence Gauthier1 1SLAC National Accelerator Laboratory, 2University of Alberta, 3Friedrich-Alexander-Universität Erlangen-Nürnberg, 4European XFEL, 5Helmholtz-Zentrum Dresden-Rossendorf, 6Technische Universität Dresden, 7Technische Universität Darmstadt This protocol presents the operation and principles of micron-scale cylindrical and planar cryogenic liquid jets. Until now, this system has been used as a high repetition rate target in laser-plasma experiments. Anticipated cross-disciplinary applications range from laboratory astrophysics to material science, and eventually next-generation particle accelerators. Behavior Fully Automated Leg Tracking in Freely Moving Insects using Feature Learning Leg Segmentation and Tracking (FLLIT) Animesh Banerjee1, Shuang Wu2, Li Cheng3, Sherry Shiying Aw1 1Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 2Bioinformatics Institute, Agency for Science, Technology and Research, 3Department of Electrical and Computer Engineering, University of Alberta We describe detailed protocols for using FLLIT, a fully automated machine learning method for leg claw movement tracking in freely moving Drosophila melanogaster and other insects. These protocols can be used to quantitatively measure subtle walking gait movements in wild type flies, mutant flies and fly models of neurodegeneration. Chemistry 18F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor (SiFA) for Positron Emission Tomography David Connolly*1, Justin J. Bailey*1, Harun Ilhan2,3, Peter Bartenstein2,3, Carmen Wängler4, Björn Wängler5, Melinda Wuest1, Frank Wuest1, Ralf Schirrmacher1 1Department of Oncology, University of Alberta, 2Department of Nuclear Medicine, University Hospital of Munich, 3ENETS Centre of Excellence, Interdisciplinary Center of Neuroendocrine Tumors of the GastroEnteroPancreatic System, LMU Munich, 4Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, 5Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University The synthesis of fluorine-18 (18F) labeled radiopharmaceuticals for positron emission tomography typically requires months of experience. When incorporated into a radiotracer, the silicon-fluoride acceptor (SiFA) motif enables a simple 18F-labeling protocol that is independent of costly equipment and preparatory training, while reducing precursor quantity needed and utilizing milder reaction conditions. Medicine Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture Sadman Sakib1, Yang Yu3,4, Anna Voigt2, Mark Ungrin2,3,4, Ina Dobrinski1,2 1Biochemistry and Molecular Biology, University of Calgary, 2Comparative Biology and Experimental Medicine, University of Calgary, 3Biomedical Engineering Graduate Program, University of Calgary, 4Alberta Diabetes Institute, University of Alberta Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system. Neuroscience Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain Adrianna Giuffre1,2, Lauran Cole1,2,3, Hsing-Ching Kuo4,5, Helen L. Carlson2,4,5, Jeff Grab6, Adam Kirton2,3,4,5,7, Ephrem Zewdie3,4,5 1Department of Neurosciences, University of Calgary, 2Hotchkiss Brain Institute, University of Calgary, 3Cumming School of Medicine, University of Calgary, 4Department of Pediatrics, University of Calgary, 5Alberta Children's Hospital Research Institute, University of Calgary, 6Faculty of Medicine and Dentistry, University of Alberta, 7Department of Clinical Neurosciences, University of Calgary We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children. Editorial JoVE Methods Collection Highlights: Protein-Protein Interactions Alastair Barr1, Michael Overduin2 1School of Life Sciences, University of Westminster, 2Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta Medicine Brachial Artery Catheterization in Swine Richard R.E. Uwiera1, Amirali Toossi2,3, Dirk G. Everaert3,4, Trina C. Uwiera5, Vivian K. Mushahwar2,3,6 1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Neuroscience and Mental Health Institute, University of Alberta, 3Sensory Motor Adaptive Rehabilitation Technology (SMART) Network, University of Alberta, 4Division of Physical Medicine and Rehabilitation, University of Alberta, 5Division of Pediatric Surgery, Department of Surgery, University of Alberta, 6Division of Physical Medicine and Rehabilitation, University of Alberta The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements. Developmental Biology Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis Kevin H. Yoon1, Sonya A. Widen1,2, Melissa M. Wilson1, Jennifer C. Hocking2,3,4,5, Andrew J. Waskiewicz1,2,6 1Department of Biological Sciences, University of Alberta, 2Women & Children's Health Research Institute, University of Alberta, 3Division of Anatomy, Department of Surgery, University of Alberta, 4Department of Cell Biology, University of Alberta, 5Department of Medical Genetics, University of Alberta, 6Neuroscience and Mental Health Research Institute, University of Alberta Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus. Behavior A Vibrotactile Feedback Device for Seated Balance Assessment and Training Andrew D. Williams1, Albert H. Vette1,2,3 1Department of Biomedical Engineering, University of Alberta, 2Department of Mechanical Engineering, University of Alberta, 3Glenrose Rehabilitation Hospital, Alberta Health Services A sitting platform has been developed and assembled that passively destabilizes sitting posture in humans. During the user's stabilizing task, an inertial measurement unit records the device's motion, and vibrating elements deliver performance-based feedback to the seat. The portable, versatile device may be used in rehabilitation, assessment, and training paradigms. Medicine Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism Sanaz Hatami1, Christopher W. White1, Martin Ondrus1, Xiao Qi1, Max Buchko1, Sayed Himmat1, Lily Lin2, Katie Cameron3, David Nobes4, Hyun-Joong Chung5, Jayan Nagendran1,6, Darren H. Freed1,3,6,7 1Department of Surgery, Faculty of Medicine, University of Alberta, 2Department of Pediatrics, Faculty of Medicine, University of Alberta, 3Department of Biomedical Engineering, Faculty of Medicine, University of Alberta, 4Department of Mechanical Engineering, Faculty of Engineering, University of Alberta, 5Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, 6Canadian National Transplant Research Program, 7Department of Physiology, Faculty of Medicine, University of Alberta Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP. Biochemistry Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering Tyler Mrozowich1, Steffane McLennan2, Michael Overduin2,4, Trushar R. Patel1,3,4 1Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 2Department of Biochemistry, University of Alberta, 3Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, 4DiscoveryLab, Faculty of Medicine & Dentistry, University of Alberta Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution. Biochemistry Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST) Graeden Winkelaar1, Catharine Trieber1, Jitendra Kumar1, Michael Overduin1 1Department of Biochemistry, University of Alberta Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments. Immunology and Infection Antigenic Liposomes for Generation of Disease-specific Antibodies Kyle J. Bednar*1, Lakeya Hardy*2,3, Johanna Smeekens*3,4, Dharmendra Raghuwanshi5, Shiteng Duan6, Mike D. Kulis3,4, Matthew S. Macauley5,7 1Janssen R&D, 2Department of Microbiology and Immunology, University of North Carolina, 3UNC Food Allergy Initiative, University of North Carolina, 4Department of Pediatrics, University of North Carolina, 5Department of Chemistry, University of Alberta, 6Department of Molecular Medicine, Scripps Research Institute, 7Department of Medical Microbiology and Immunology, University of Alberta Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models. Environment Determination of the Settling Rate of Clay/Cyanobacterial Floccules Tiffany Playter1, Kurt Konhauser1, George W. Owttrim2, Denise S. Whitford2, Tyler Warchola1, Cheryl Hodgson1,3, Aleksandra M. Mloszewska4, Bruce Sutherland1, J.-P. Zonneveld1, S. George Pemberton1, Murray K. Gingras1 1Department of Earth and Atmospheric Sciences, University of Alberta, 2Department of Biological Sciences, University of Alberta, 3Department of Earth Sciences, Simon Fraser University, 4Earth Sciences Department, University of Toronto The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules. Medicine Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice. Shereen M. Hamza1, John E. Hall2 1Department of Pediatrics, University of Alberta, 2Department of Physiology & Biophysics, University of Mississippi Medical Center, 3Mississippi Center for Obesity Research Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined. Engineering All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics Mohammad Rashidi1,2, Wyatt Vine1, Jacob A.J. Burgess3,4,5, Marco Taucer1,2,6, Roshan Achal1, Jason L. Pitters2, Sebastian Loth3,4, Robert A. Wolkow1,2 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. Bioengineering Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation Kyle M. Koss1,2, Matthew A. Churchward1,2, Andrea F. Jeffery1,3, Vivian K. Mushahwar1,2,4, Anastasia L. Elias3, Kathryn G. Todd1,2,5 1Department of Psychiatry, University of Alberta, 2Alberta Innovates-Health Solutions Interdisciplinary Team in Smart Neural Prostheses (Project SMART), University of Alberta, 3Department of Chemical and Materials Engineering, University of Alberta, 4Division of Physical Medicine and Rehabilitation, University of Alberta, 5Centre for Neuroscience, University of Alberta Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging. Bioengineering A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact Henry Y. Yu1, Brooklynn M. Knowles1, Christopher R. Dennison1 1Department of Mechanical Engineering, University of Alberta Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact. Behavior Neurodevelopmental Reflex Testing in Neonatal Rat Pups Antoinette T. Nguyen1, Edward A. Armstrong1, Jerome Y. Yager1 1Department of Pediatrics, University of Alberta Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here. Neuroscience Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils Sara Breid1, Maria E. Bernis1, Julius B. Tachu1, Maria C. Garza2, Holger Wille2, Gültekin Tamgüney1 1German Center for Neurodegenerative Diseases (DZNE), 2Centre for Prions and Protein Folding Diseases & Department of Biochemistry, University of Alberta Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system. Engineering In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions Cedric Laborde-Boutet1, William C. McCaffrey1 1Department of Chemical and Materials Engineering, University of Alberta This article describes a setup and method for the in situ visualization of oil samples under a variety of temperature and pressure conditions that aim to emulate refining and upgrading processes. It is primarily used for studying isotropic and anisotropic media involved in the fouling behavior of petroleum feeds. Cancer Research Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers Richard Wang1, Harris Wang1, Zhixiang Wang1 1Department of Medical Genetics, Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. Medicine Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise Vincent Tedjasaputra1,2, Sean van Diepen3, Sophie É Collins1,4, Wade M. Michaelchuk1,2, Michael K. Stickland1,5 1Division of Pulmonary Medicine, University of Alberta, 2Faculty of Physical Education and Recreation, University of Alberta, 3Divisions of Critical Care and Cardiology, University of Alberta, 4Faculty of Rehabilitation Medicine, University of Alberta, 5G.F. MacDonald Centre for Lung Health To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses. Biology A Web Tool for Generating High Quality Machine-readable Biological Pathways Miguel Ramirez-Gaona1, Ana Marcu1, Allison Pon1, Jason Grant1, Anthony Wu1, David S. Wishart1,2 1Department of Computer Science, University of Alberta, 2Department of Biological Sciences, University of Alberta PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms. Developmental Biology Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform Reza Salehi*1, Stephen C.M. Tsoi*1, Marcos G. Colazo2, Divakar J. Ambrose1,2, Claude Robert3, Michael K. Dyck1 1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Livestock Research Branch, Alberta Agriculture and Forestry, 3Laboratory of Functional Genomics of Early Embryonic Development, Université Laval Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA. Environment Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils Sylvie A. Quideau1, Anne C.S. McIntosh2, Charlotte E. Norris1, Emily Lloret3, Mathew J.B. Swallow4, Kirsten Hannam5 1Department of Renewable Resources, University of Alberta, 2Department of Science, Augustana Faculty, University of Alberta, 3Laboratoire Génie Civil et géo-Environnement, Université de Lille, 4Department of Earth and Environmental Sciences, Mount Royal University, 5Forest Ecology & Production, Great Lakes Forestry Centre, Natural Resources Canada Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography. Bioengineering Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii Swayamdipta Bhaduri1, Nandini Debnath1, Sushanta Mitra2, Yang Liu3, Aloke Kumar1 1Department of Mechanical Engineering, University of Alberta, 2Department of Mechanical Engineering, York University, 3Department of Civil and Environmental Engineering, University of Alberta Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge. Chemistry An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions Mohameedyaseen Syedbasha1, Janina Linnik1,2,3, Deanna Santer4, Daire O'Shea5, Khaled Barakat4,6, Michael Joyce4, Nina Khanna7, D. Lorne Tyrrell4, Michael Houghton4, Adrian Egli1,8 1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, and Swiss Institute of Bioinformatics, 3Swiss Institute of Bioinformatics, 4Li Ka Shing Institute for Virology, University of Alberta, 5Regional Infectious Diseases Unit, University of Edinburgh, 6Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 7Infection Biology, Department of Biomedicine, University of Basel, 8Clinical Microbiology, University Hospital Basel The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions. Medicine Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs Richard R. Uwiera1, Rabban Mangat1, Sandra Kelly1, Trina C. Uwiera2, Spencer D. Proctor1 1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Department of Surgery, University of Alberta We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph. Developmental Biology Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR Milena S. Blanes*1, Stephen C.M. Tsoi*1, Michael K. Dyck1 1Department of Agricultural, Food and Nutritional Science, University of Alberta This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification. Biology Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI) Hilmar Strickfaden1, Zhi Zhong Xu1, Michael J. Hendzel1 1Department of Oncology, Faculty of Dentistry and Medicine, University of Alberta Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example. Bioengineering Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration Sébastien Meghezi1, Dawit G. Seifu1,2, Nina Bono1,3, Larry Unsworth2,4,5, Kibret Mequanint6, Diego Mantovani1,2 1Laboratory for Biomaterials and Bioengineering, Department Min-Met-Materials Eng & CHU de Québec Research Center, Canada Research Chair I for the Innovation in Surgery, Laval University, 2NSERC CREATE Program for Regenerative Medicine (NCPRM), Laval University, 3Department Electronics, Information and Bioengineering, Politecnico di Milano, 4Department of Chemical and Materials Engineering, University of Alberta, 5National Institute for Nanotechnology, National Research Council (Canada), 6Department of Chemical and Biochemical Engineering, University of Western Ontario In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described. Neuroscience Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders Surya T. Madabattula1, Joel C. Strautman1, Andrew M. Bysice1, Julia A. O’Sullivan1, Alaura Androschuk1, Cory Rosenfelt1, Kacy Doucet1, Guy Rouleau2, Francois Bolduc1 1Department of Pediatrics, University of Alberta, 2Montreal Neurological Institute and Hospital, McGill University We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. Engineering Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates Robert F. Peters1,2, Luis Gutierrez-Rivera1,2, Steven K. Dew1,2, Maria Stepanova1,2 1Department of Electrical and Computer Engineering, University of Alberta, 2National Institute for Nanotechnology, National Research Council of Canada We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated. Environment A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry Anna R. Bramucci1, Leen Labeeuw1, Teaghan J. Mayers1, Julie A. Saby1, Rebecca J. Case1 1Department of Biological Sciences, University of Alberta The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures. Medicine Murine Ileocolic Bowel Resection with Primary Anastomosis Troy Perry1, Anna Borowiec1, Bryan Dicken1, Richard Fedorak2, Karen Madsen2 1Department of Surgery, University of Alberta, 2Department of Medicine, University of Alberta Ileocolic resection is commonly performed in several human diseases; however, little is known regarding the impact of intestinal resection on surgical illnesses. This article provides instruction on executing the procedure in mice with high success, providing a means to study the effects of ileocolic resection in models of disease. Bioengineering Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars Mahtab Hassanpourfard1, Xiaohui Sun2, Amin Valiei1, Partha Mukherjee3, Thomas Thundat1, Yang Liu2, Aloke Kumar4 1Department of Chemical and Material Engineering, University of Alberta, 2Department of Civil and Environmental Engineering, University of Alberta, 3Department of Mechanical Engineering, Texas A&M University, 4Department of Mechanical Engineering, University of Alberta Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. Chemistry A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence Zachary J. Storms1, Elliot Cameron1, Hector de la Hoz Siegler1,2, William C. McCaffrey1 1Department of Chemical and Materials Engineering, University of Alberta, 2Department of Chemical and Petroleum Engineering, University of Calgary A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance. Medicine A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies Mukti R. Parikh1, Andrew R. Belch2,3, Linda M Pilarski2,3, Julia Kirshner1 1Department of Biological Sciences, Purdue University, 2Department of Oncology, University of Alberta, 3Cross Cancer Institute In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue. Biology Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae Miguel F. Gonzales1, Teresa Brooks2, Stefan U. Pukatzki2, Daniele Provenzano1,3 1Department of Biological Sciences, University of Texas Brownsville, 2Department of Medical Microbiology and Immunology, University of Alberta, 3Department of Biomedical Sciences, University of Texas Brownsville Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories. Neuroscience Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells Birbickram Roy1, Declan William Ali1 1Department of Biological Sciences, University of Alberta We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo. Engineering Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates Ryan Starko-Bowes1, Sandipan Pramanik1 1Electrical and Computer Engineering, University of Alberta We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics. Immunology and Infection A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry Erick García-García1, Eileen Uribe-Querol2, Carlos Rosales3 1Department of Biological Sciences, University of Alberta, 2División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, 3Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei Engineering Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing Shalon McFarlane1, C.P.K. Manchee1, Joshua W. Silverstone1, Jonathan Veinot1, Al Meldrum1 1Department of Physics, University of Alberta Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium. Immunology and Infection Examination of Thymic Positive and Negative Selection by Flow Cytometry Qian Hu*1, Stephanie A. Nicol*1, Alexander Y.W. Suen*1, Troy A. Baldwin1 1Department of Medical Microbiology and Immunology, University of Alberta We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background. Medicine Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis Xiaoqin Hua1,2, Tobias Deuse1,2, Evangelos D. Michelakis3, Alois Haromy3, Phil S. Tsao4, Lars Maegdefessel4, Reinhold G. Erben5, Claudia Bergow5, Boris B. Behnisch6, Hermann Reichenspurner1,2, Robert C. Robbins7, Sonja Schrepfer1,2,7 1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine, 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model. Neuroscience Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition Gloria Wong1,2, Sanda Dolcos1,3, Ekaterina Denkova1, Rajendra Morey4,5,6, Lihong Wang4,5, Gregory McCarthy6,7, Florin Dolcos1,2,3,8,9 1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University, 5Department of Psychiatry and Behavioral Sciences, Duke University, 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants. Medicine A Swine Model of Neonatal Asphyxia Po-Yin Cheung1, Richdeep S. Gill2, David L. Bigam2 1Departments of Pediatrics, Pharmacology and Surgery, University of Alberta, 2Department of Surgery, University of Alberta Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations. Medicine Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas Eric B. Jelin1, Samuel C. Schecter1, Kelly D. Gonzales1, Shinjiro Hirose1, Hanmin Lee1, Geoffrey A. Machin2, Larry Rand3, Vickie A. Feldstein4 1Division of Pediatric and Fetal Surgery, Department of Surgery, University of California, San Francisco, 2Department of Pathology, University of Alberta, 3Department of Obstretics and Gynecology, University of California, San Francisco, 4Department of Radiology, University of California, San Francisco Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins’ circulations. Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome. Neuroscience Brain Imaging Investigation of the Neural Correlates of Emotion Regulation Sanda Dolcos1, Keen Sung2, Ekaterina Denkova3, Roger A. Dixon4,5, Florin Dolcos1,6,7 1Department of Psychology, University of Illinois, Urbana-Champaign, 2Department of Computing Science, University of Alberta, Edmonton, 3Department of Psychiatry, University of Alberta, Edmonton, 4Department of Psychology, University of Alberta, Edmonton, 5Centre for Neuroscience, University of Alberta, Edmonton, 6Neuroscience Program, University of Illinois, Urbana-Champaign, 7Beckman Institute, University of Illinois, Urbana-Champaign We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients. Neuroscience Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection Ekaterina Denkova1, Trisha Chakrabarty1, Sanda Dolcos1,2, Florin Dolcos1,2,3,4 1Department of Psychiatry, University of Alberta, Edmonton, 2Psychology Department, University of Illinois, Urbana-Champaign, 3Neuroscience Program, University of Illinois, Urbana-Champaign, 4Beckman Institute for Advanced Science & Technology, University of Illinois, Urbana-Champaign We present a protocol that allows investigation of the neural correlates of recollecting emotional autobiographical memories, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants. Neuroscience Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions Keen Sung1, Sanda Dolcos2, Sophie Flor-Henry3, Crystal Zhou3, Claudia Gasior4, Jennifer Argo5, Florin Dolcos2,6,7 1Department of Computing Science, University of Alberta, 2Department of Psychology, University of Illinois, 3Centre for Neuroscience, University of Alberta, 4Department of Psychology, University of Alberta, 5Department of Marketing, Business Economics, and Law, University of Alberta, 6Neuroscience Program, University of Illinois at Urbana-Champaign, 7Beckman Institute, University of Illinois at Urbana-Champaign This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions. Biology Swimming Performance Assessment in Fishes Keith B. Tierney1 1Department of Biological Sciences, University of Alberta The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour. Neuroscience Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion Andrea Shafer1, Alexandru Iordan2, Roberto Cabeza3, Florin Dolcos1,4 1Centre for Neuroscience, University of Alberta, 2Neuroscience Program, University of Illinois, Urbana-Champaign, 3Center for Cognitive Neuroscience, Duke University, 4Psychology Department, Neuroscience Program, & Beckman Institute, University of Illinois, Urbana-Champaign We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations. Biology Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death Aja M. Rieger1, Kimberly L. Nelson1, Jeffrey D. Konowalchuk1, Daniel R. Barreda1,2 1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models. Biology Catheterization of Intestinal Loops in Ruminants Richard R. E. Uwiera1, John P. Kastelic2, G. Douglas Inglis2 1Department of Agricultural, Food and Nutritional Science, University of Alberta, 2Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.