University of Manitoba 13 articles published in JoVE Immunology and Infection Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System Dawn White1, Ayush Kumar1 1Department of Microbiology, University of Manitoba We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens. Environment Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration Kadir Yanaç1, Jhannelle Francis2, Jocelyn Zambrano-Alvarado2, Qiuyan Yuan1, Miguel Uyaguari-Díaz2 1Department of Civil Engineering, Price Faculty of Engineering, University of Manitoba, 2Department of Microbiology, Faculty of Science, University of Manitoba Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types. Chemistry A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes Franklin Brian Apea-Bah1,2, Pamela Drawbridge1,2, Trust Beta1,2 1Department of Food and Human Nutritional Sciences, University of Manitoba, 2Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes. Immunology and Infection Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices Ali N. Doucet1, Carmine J. Slipski1, George R. Golding1,2, Michael R. Mulvey1,2, Denice C. Bay1 1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 2National Microbiology Laboratory, Public Health Agency of Canada This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening. Bioengineering Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability Tuyet Mai Thi To1, Vladimir Kubyshkin2, Franz-Josef Schmitt3, Nediljko Budisa1,2, Thomas Friedrich1 1Institute of Chemistry, Technische Universität Berlin, 2Department of Chemistry, University of Manitoba, 3Institute of Physics, Martin-Luther-Universität Halle-Wittenberg To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light. Neuroscience Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo Jessica Meza-Resillas*1, Noushin Ahmadpour*1, Michael Stobart1, Jillian Stobart1 1College of Pharmacy, Rady Faculty of Health Sciences, University of Manitoba This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type. Neuroscience Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum Shahin Shabanipour1,2, Azadeh Dalvand1,2, Xiaodan Jiao1,2, Maryam Rahimi Balaei1,2, Seung H. Chung3, Jiming Kong1, Marc. R. Del Bigio2,4, Hassan Marzban1,2 1Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 2The Children's Hospital Research Institute of Manitoba (CHRIM), Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 3Department of Oral Biology, University of Illinois at Chicago, 4Department of Pathology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons. Biochemistry Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay Javad Alizadeh1,2, Shahla Shojaei1,2,3, Simone da Silva Rosa1, Adel Rezaei Moghadam1,2, Amir A. Zeki4, Mohammad Hashemi5, Marek J. Los6,7,8, Joseph W. Gordon1,9, Saeid Ghavami1,2,10 1Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, 2Biology of Breathing Theme, Children Hospital Research Institute of Manitoba, University of Manitoba, 3Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, 4Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Center for Comparative Respiratory Biology and Medicine, 5Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, 6Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, 7Centre de biophysique moléculaire - UPR 4301, Centre national de la recherche scientifique (CNRS) CS80054, 8LinkoCare Life Sciences AB, 9College of Nursing and Children's Hospital Research Institute of Manitoba, Rady Faculty of Health Sciences, University of Manitoba, 10Health Policy Research Center, Institute of Health, Shiraz University of Medical Sciences Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases. 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. Immunology and Infection An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood Ke Yang*1,2,3, Jiandong Wu*3,4, Ling Zhu1, Yong Liu1, Michael Zhang5, Francis Lin3,4,6,7 1Institute of Applied Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 2University of Science and Technology of China, 3Department of Physics and Astronomy, University of Manitoba, 4Department of Biosystems Engineering, University of Manitoba, 5Seven Oaks General Hospital, 6Department of Immunology, University of Manitoba, 7Department of Biological Sciences, University of Manitoba This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip. Neuroscience Low-Density Primary Hippocampal Neuron Culture Reiko T. Roppongi*1,2, Kevin P. Champagne-Jorgensen*1,2, Tabrez J. Siddiqui1,2 1Department of Physiology and Pathophysiology, University of Manitoba, 2Kleysen Institute for Advanced Medicine, Health Sciences Centre This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays. Biology Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands Rushin Shojaii1, Stephanie Bacopulos2,3, Wenyi Yang2,3, Tigran Karavardanyan4, Demetri Spyropoulos5, Afshin Raouf6, Anne Martel1,4, Arun Seth2,3 1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. Medicine Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples Jennifer A. Juno1, Genevieve Boily-Larouche1, Julie Lajoie1, Keith R. Fowke1,2 1Department of Medical Microbiology, University of Manitoba, 2Department of Community Health Sciences, University of Manitoba The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.
