McMaster University View Institution's Website 27 articles published in JoVE Biochemistry Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice Liza-Anastasia DiCecco*1,2, Samantha Berry*1, G. M. Jonaid*1,3, Maria J. Solares1,4, Liam Kaylor1,4, Jennifer L. Gray5, Carol Bator6, William J. Dearnaley1, Michael Spilman7, Madeline J. Dressel-Dukes8, Kathryn Grandfield2, Sarah M. McDonald Esstman9, Deborah F. Kelly1,5,6 1Department of Biomedical Engineering, Pennsylvania State University, 2Department of Materials Science and Engineering, McMaster University, 3Bioinformatics and Genomics Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 4Molecular, Cellular, and Integrative Biosciences Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 5Materials Research Institute, Pennsylvania State University, 6Huck Institutes of the Life Sciences, Pennsylvania State University, 7Applications team, Direct Electron, 8Application Scientist, Protochips, Inc., 9Department of Biology, Wake Forest University Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy. Biology Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure Himeshi Samarasinghe1, Gregory Korfanty1, Jianping Xu1 1Department of Biology, McMaster University This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments. Immunology and Infection Antibiotic Dereplication Using the Antibiotic Resistance Platform Haley L. Zubyk1, Georgina Cox2, Gerard D. Wright1 1Department of Biochemistry and Biomedical Sciences, M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 2Department of Molecular and Cellular Biology, University of Guelph We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient. Chemistry High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry Meera Shanmuganathan1, Sabrina Macklai1, Clara Barrenas Cárdenas1, Zachary Kroezen1, Marcus Kim2, William Zizek3, Howard Lee3, Philip Britz-McKibbin1 1Department of Chemistry and Chemical Biology, McMaster University, 2Agilent Technologies Inc., 3Seroclinix Corporation Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry. Biology Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1 Lucas Le Nagard1, Viviana Morillo-López2, Cecile Fradin1, Dennis A. Bazylinski2 1Department of Physics & Astronomy, McMaster University, 2School of Life Sciences, University of Nevada Las Vegas We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium. Engineering A 3D-printed Chamber for Organic Optoelectronic Device Degradation Testing Emma Mogus1, Benjamin Torres-Kulik1, Christopher Gustin2, Ayse Turak1 1Department of Engineering Physics, McMaster University, 2 Here, we present a protocol for the design, manufacture, and use of a simple, versatile 3D-printed and controlled atmospheric chamber for the optical and electrical characterization of air-sensitive organic optoelectronic devices. Bioengineering Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning Daryl Sivakumaran1, Emilia Bakaic1, Scott B. Campbell1, Fei Xu1, Eva Mueller1, Todd Hoare1 1Department of Chemical Engineering, McMaster University Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers. Genetics Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease Allison A. Dilliott1,2, Sali M.K. Farhan3, Mahdi Ghani4, Christine Sato4, Eric Liang5, Ming Zhang4, Adam D. McIntyre1, Henian Cao1, Lemuel Racacho6,7, John F. Robinson1, Michael J. Strong1,8, Mario Masellis9,10, Dennis E. Bulman6,7, Ekaterina Rogaeva4, Anthony Lang10,11, Carmela Tartaglia4,10, Elizabeth Finger12,13, Lorne Zinman9, John Turnbull14, Morris Freedman10,15, Rick Swartz9, Sandra E. Black9,16, Robert A. Hegele1,2 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, 5 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. Chemistry Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry Holly A. Bilton*1, Zainab Ahmad*1, Nancy Janzen1, Shannon Czorny1, John F. Valliant1 1Department of Chemistry and Chemical Biology, McMaster University Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry. Biology Colorimetric Detection of Bacteria Using Litmus Test Kha Tram1, Sepehr Manochehry1, Qian Feng2, Dingran Chang1, Bruno J. Salena3, Yingfu Li1 1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University, 3Department of Medicine, McMaster University We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads. Behavior Combining Multiple Data Acquisition Systems to Study Corticospinal Output and Multi-segment Biomechanics Michael J. Asmussen1, Aaron Z. Bailey1, Peter J. Keir1, Jim Potvin1, Tim Bergel2, Aimee J. Nelson1 1Department of Kinesiology, McMaster University, 22Els Espots The use of transcranial magnetic stimulation (TMS) to study human motor control requires the integration of data acquisition systems to control TMS delivery and simultaneously record human behavior. The present manuscript provides a detailed methodology for integrating data acquisition systems for the purpose of investigating human movement via TMS. Behavior Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression Caitlin L. Carew1, Erica L. Tatham2, Andrea M. Milne1, Glenda M. MacQueen3, Geoffrey B.C. Hall4 1Department of Psychiatry and Behavioural Neurosciences, McMaster University, 2McMaster Integrative Neuroscience Discovery and Study, McMaster University, 3Department of Psychiatry, University of Calgary, 4Department of Psychology, Neuroscience & Behaviour, McMaster University We aim to identify the neural correlates underlying sustained and transient thought suppression, and thought re-emergence in controls, at-risk and depressed individuals. Activation was greatest for controls compared to the at-risk and the depressed group in the dorsolateral prefrontal cortex during thought suppression and anterior cingulate cortex during thought re-emergence. Behavior Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST) Boris Sakic1, Marcella P. A. Cooper2, Sarah E. Taylor2, Milica Stojanovic2, Bosa Zagorac2, Minesh Kapadia3 1Department of Psychiatry and Behavioral Neurosciences, McMaster University, 2Department of Psychology, Neuroscience & Behaviour, McMaster University, 3Neuroscience Program, McMaster University Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis. Medicine Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses Breanne Cuddington1, Meghan Verschoor1, Karen Mossman1 1Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, Institute for Infectious Disease Research, McMaster University Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model. Immunology and Infection Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae Alicja Puchta1, Chris P. Verschoor1, Tanja Thurn1, Dawn M. E. Bowdish1 1Department of Pathology and Molecule Medicine, McMaster University Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ. Medicine Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus Daniel E. Venegas-Pino1, Nicole Banko1, Mohammed I. Khan2, Yuanyuan Shi2, Geoff H. Werstuck1,2 1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Thrombosis and Atherosclerosis Research Institute, McMaster University We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis. Medicine Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice Jane A. E. Batt1, James Ralph Bain2 1Keenan Research Centre of the LiKaShing Knowledge Institute, St Michaels Hospital, 2Department of Surgery, McMaster University The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. Chemistry A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays Blake-Joseph Helka1, John D. Brennan1 1Department of Chemistry and Chemical Biology, McMaster University A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening. Bioengineering Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion Justin Tong1, Pouya Rezai2, Sangeena Salam1, P. Ravi Selvaganapathy2, Bhagwati P. Gupta1 1Department of Biology, McMaster University, 2Department of Mechanical Engineering, McMaster University A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (“electrotaxis”) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health. Biology Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes Yu Seon Chung1, Alba Guarné1 1Department of Biochemistry and Biomedical Sciences, McMaster University Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation. Medicine Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting Chitra Venugopal1, Nicole M. McFarlane1, Sara Nolte1, Branavan Manoranjan1, Sheila K. Singh1 1Stem Cell and Cancer Research Institute, McMaster University The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells. Immunology and Infection Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation Rahul Kushwah1, Jim Hu2 1Stem Cell and Cancer Research Institute, McMaster University, Hamilton, 2Physiology and Experimental Medicine Research Program, Hospital for Sick Children, University of Toronto We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection. Immunology and Infection Detection of Bacteria Using Fluorogenic DNAzymes Sergio D. Aguirre1, M. Monsur Ali1, Pushpinder Kanda1, Yingfu Li1,2 1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli). Biology Investigating Intestinal Inflammation in DSS-induced Model of IBD Janice J. Kim1, Md. Sharif Shajib1, Marcus M. Manocha1, Waliul I. Khan1 1Farncombe Family Digestive Health Research Institute, Department of Pathology and Molecular Medicine, McMaster University Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease. Biology Primary Human Bronchial Epithelial Cells Grown from Explants Asma Yaghi1, Aisha Zaman1, Myrna Dolovich1 1Medicine, Faculty of Health Sciences, McMaster University Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease. Biology A Tactile Automated Passive-Finger Stimulator (TAPS) Daniel Goldreich1,2, Michael Wong2, Ryan M. Peters2, Ingrid M. Kanics1 1Department of Occupational Therapy, Duquesne University, 2Department of Psychology, Neuroscience and Behaviour, McMaster University We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test. Biology Culture of myeloid dendritic cells from bone marrow precursors Jeanette Boudreau1,2, Sandeep Koshy2,3, Derek Cummings2, Yonghong Wan2 1Medical Sciences Program, McMaster University, 2Centre for Gene Therapeutics, McMaster University, 3Department of Chemical Engineering, University of Waterloo This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.
