University of Liverpool View Institution's Website 19 articles published in JoVE Biology Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics Revathy Krishnamurthi1, Enrique González-Tortuero2, Grace Plahe2, Ian B. Goodhead2, Joanne L. Fothergill1, Chloë E. James2, Heather E. Allison1 1Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences (IVES), University of Liverpool, 2School of Science, Engineering, and Environment, University of Salford This protocol enables the impact of prophages on their hosts to be revealed. Bacterial cultures are synchronized using conditions that best support the lysogenic state, limiting spontaneous induction. RT-qPCR unequivocally distinguishes prophage-restricted genes and those uncoupled from phage control from those that are expressed during the lytic replication cycle. Engineering Microembossing: A Convenient Process for Fabricating Microchannels on Nanocellulose Paper-Based Microfluidics Wenwen Yuan1,2,3, Hang Yuan1, Sixuan Duan1,2, Ruiqi Yong1, Jia Zhu1,2,4, Eng Gee Lim1,2, Ivona Mitrovic2, Pengfei Song1,2 1School of Advanced Technology, Xi’an Jiaotong - Liverpool University, 2Department of Electrical Engineering and Electronics, University of Liverpool, 3 This protocol describes a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanofibrillated cellulose paper, achieving a minimum width of 200 µm. Environment Dissolved Solute Sampling Across an Oxic-Anoxic Soil-Water Interface Using Microdialysis Profilers Sha Zhang1,2, Zhaofeng Yuan2, Yujia Cai2, Hao Liu2, Ziyan Liu2, Zheng Chen2 1Department of Geography and Planning, University of Liverpool, 2Department of Health and Environmental Sciences, Xi’an Jiaotong-Liverpool University A microdialysis profiler is described to sample dissolved porewater solutes across an oxic-anoxic soil-water interface in situ with minimal disturbance. This device is designed to capture rapid changes in concentration-depth profiles induced by disturbances at the soil-water interface and beyond. Biology Determining Temperature Preference of Mosquitoes and Other Ectotherms Amirah Haziqah-Rashid1, Kinga Stobierska1, Lorna Glenn1, Soeren Metelmann1,2, Ken Sherlock3, Ewa Chrostek1, Marcus S. C. Blagrove1 1Department of Evolution, Ecology, and Behavior, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, 2COVID-19 Outbreak Surveillance Team, UK Health Security Agency, 3Department of Livestock and One Health, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors. Chemistry Synthesis of Metal Nanoparticles Supported on Carbon Nanotube with Doped Co and N Atoms and its Catalytic Applications in Hydrogen Production Pui-Ching Poon*1, Ka-Man Lee*1, Yuanhao Wang2, William Wai Yan Lam1, Peter Shu-Wai Leung1, Xiao-Ying Lu1, Weiqun Li3, B. Layla Mehdi3, Yang Lu4, Chi-Wing Tsang1, Eugene Yin Cheung Wong2 1Faculty of Science and Technology, Technological and Higher Education Institute of Hong Kong (THEi), 2Department of Supply Chain and Information Management, The Hang Seng University of Hong Kong, 3Department of Mechanical, Materials and Aerospace Engineering, University of Liverpool, 4Laboratory of Building Environment and New Energy Resources, School of Civil Engineering, Faculty of Infrastructure Engineering, Dalian University of Technology Here, we present a protocol to synthesize Co nanoparticles supported on carbon nanotubes with Co- and N- dopants for hydrogen productions. Biochemistry An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter Florence B Kineavy1, Alex A Davies1, Molly R Mitchell1, Daniel Lay1, Matt J Dominguez2, Elliott J Stollar1 1School of Life Sciences, University of Liverpool, 2Health Sciences Center, Texas Tech University A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation. Immunology and Infection Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis Denise A. Marston1, Daisy L. Jennings1, Nikki C. MacLaren1, Daniel Dorey-Robinson1, Anthony R. Fooks1,2, Ashley C. Banyard1, Lorraine M. McElhinney1,2 1Wildlife Zoonoses & Vector-Borne Diseases Research Group, Animal and Plant Health Agency, 2Institute of Infection and Global Health, University of Liverpool This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results. Chemistry Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers David A. Gregory1, Piyush Kumar1, Ana Jimenez-Franco1, Yi Zhang1, Yu Zhang 2, Stephen J. Ebbens1, Xiubo Zhao1 1Department of Chemical and Biological Engineering, University of Sheffield, 2School of Engineering, University of Liverpool This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications. Medicine Transdermal Measurement of Glomerular Filtration Rate in Mice Lauren Scarfe1,2, Daniel Schock-Kusch3, Lorenzo Ressel4, Jochen Friedemann3, Yury Shulhevich3, Patricia Murray2, Bettina Wilm2, Mark de Caestecker1 1Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, 2Department of Cellular and Molecular Physiology, University of Liverpool, 3MediBeacon GmbH, 4Department of Veterinary Pathology and Public Health, Institute of Veterinary Science, University of Liverpool Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor. Immunology and Infection Quantification of Efferocytosis by Single-cell Fluorescence Microscopy Kyle Taruc1, Charles Yin1, Daniel G. Wootton2,3, Bryan Heit1 1Department of Microbiology and Immunology and the Center for Human Immunology, University of Western Ontario, 2Institute of Infection and Global Health, University of Liverpool, 3Department of Respiratory Research, Aintree University Hospital NHS Foundation Trust Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways. Bioengineering Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies I-Ning Lee*1,2, Joseph Hosford*1, Shuai Wang3, John A. Hunt4, Judith M. Curran2, William P. Heath3, Lu Shin Wong1 1Manchester Institute of Biotechnology & School of Chemistry, University of Manchester, 2School of Engineering, University of Liverpool, 3School of Electrical and Electronic Engineering, University of Manchester, 4School of Science and Technology, Nottingham Trent University Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies. Medicine Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development Sarah Arrowsmith*1, Peter Keov2, Markus Muttenthaler3,4, Christian W. Gruber*2,5 1Harris-Wellbeing Preterm Birth Research Centre, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, 2School of Biomedical Sciences, The University of Queensland, 3Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, 4Institute for Molecular Bioscience, University of Queensland, 5Center for Physiology and Pharmacology, Medical University of Vienna This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads. Developmental Biology Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans Ana Soriano-Arroquia1, Peter D. Clegg1, Andrew P. Molloy1,2, Katarzyna Goljanek-Whysall1 1Institute of Ageing and Chronic Disease, University of Liverpool, 2Aintree University Hospital This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies. Biology Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition Josephine C. Moran1, Emma L. Crank1, Hanaa A. Ghabban1, Malcolm J. Horsburgh1 1Institute of Integrative Biology, University of Liverpool The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition. Bioengineering Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture Michael P. Storm1, Ian Sorrell2, Rebecca Shipley3, Sophie Regan2, Kim A. Luetchford1, Jean Sathish2, Steven Webb4, Marianne J. Ellis1 1Department of Chemical Engineering and Centre for Regenerative Medicine, University of Bath, 2MRC Centre for Drug Safety Science and Institute of Translational Medicine, University of Liverpool, 3Mechanical Engineering, University College London, 4Department of Applied Mathematics, Liverpool John Moores University The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture. Neuroscience Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila Rudi L. Verspoor1, Chloe Heys1, Thomas A. R. Price1 1Institute of Integrative Biology, University of Liverpool This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia. Medicine Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield Andrea M. Collins1,2, Jamie Rylance3, Daniel G. Wootton4, Angela D. Wright3,5, Adam K. A. Wright1,3, Duncan G. Fullerton3,6, Stephen B. Gordon3 1Biomedical Research Centre in Microbial Diseases, National Institute for Health Research, 2Respiratory Infection Group, Royal Liverpool and Broadgreen University Hospital Trust, 3Respiratory Infection Group, Liverpool School of Tropical Medicine, 4Institute of Infection and Global Health, University of Liverpool, 5Comprehensive Local Research Network, Royal Liverpool and Broadgreen University Hospital Trust, 6Department of Respiratory Research, University Hospital Aintree We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects. Medicine Experimental Human Pneumococcal Carriage Jenna F. Gritzfeld1, Angie D. Wright1,2,3, Andrea M. Collins1,2,4, Shaun H. Pennington1, Adam K.A. Wright5, Aras Kadioglu6, Daniela M. Ferreira1, Stephen B. Gordon1 1Respiratory Infection Group, Liverpool School of Tropical Medicine, 2Royal Liverpool and Broadgreen, University Hospital Trust, 3Comprehensive Local Research Network, 4NIHR Biomedical Research Centre in Microbial Diseases, Royal Liverpool and Broadgreen University Hospitals NHS Trust, 5Institute of Lung Health, Respiratory Biomedical Unit, University Hospitals of Leicester NHS Trust & University of Leicester, 6Department of Clinical Infection Microbiology & Immunology, Institute of Infection & Global Health, University of Liverpool Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol. Immunology and Infection Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung Sebastian Kirchner1, Joanne L Fothergill2, Elli A. Wright1, Chloe E. James1, Eilidh Mowat1, Craig Winstanley1 1Institute of Infection and Global Health, University of Liverpool, 2NIHR Biomedical Research Centre in Microbial Disease, University of Liverpool Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.