University of Sheffield View Institution's Website 19 articles published in JoVE Immunology and Infection A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics Hannah C. Regan1,2, Annette F. Taylor1,2, Esther Karunakaran1,2 1Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, 2Department of Chemical and Biological Engineering, University of Sheffield The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials. Biochemistry Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox Mahmoud A. S. Abdelhamid*1, Alice V. Rhind-Tutt*1, Benjamin Ambrose1, Timothy D. Craggs1 1Sheffield Institute for Nucleic Acids, Department of Chemistry, University of Sheffield This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis. Immunology and Infection Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis Katarzyna Okurowska1,2, Sanhita Roy3, Praveen Thokala4, Lynda Partridge1,5, Prashant Garg3, Sheila MacNeil1,6, Peter N. Monk1,7, Esther Karunakaran1,2 1Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, 2Department of Chemical and Biological Engineering, University of Sheffield, 3Hyderabad Eye Research Foundation, L V Prasad Eye Institute, 4Health Economics and Decision Science, School of Health and Related Research, University of Sheffield, 5Department of Molecular Biology and Biotechnology, University of Sheffield, 6Department of Materials Science and Engineering, University of Sheffield, 7Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Engineering Scalable Quantum Integrated Circuits on Superconducting Two-Dimensional Electron Gas Platform Kaveh Delfanazari1,2, Pengcheng Ma2, Reuben Puddy2, Teng Yi2, Moda Cao2, Yilmaz Gul3, Carly L. Richardson4, Ian Farrer2,5, David Ritchie2, Hannah J. Joyce1, Michael J. Kelly1,2, Charles G. Smith2 1Centre for Advanced Photonics and Electronics, Engineering Department, University of Cambridge, 2Department of Physics, Cavendish Laboratory, University of Cambridge, 3Department of Electronic and Electrical Engineering, University College London, 4Department of Materials Science and Metallurgy, University of Cambridge, 5Department of Electronic and Electrical Engineering, University of Sheffield Quantum integrated circuits (QICs) consisting of array of planar and ballistic Josephson junctions (JJs) based on In0.75Ga0.25As two-dimensional electron gas (2DEG) is demonstrated. Two different methods for fabrication of the two-dimensional (2D) JJs and QICs are discussed followed by the demonstration of quantum transport measurements in sub-Kelvin temperatures. 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. Chemistry Preparation of Functional Silica Using a Bioinspired Method Joseph R.H. Manning1, Eleni Routoula1, Siddharth V. Patwardhan1 1Department of Chemical and Biological Engineering, University of Sheffield Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine 'additive', which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives. Bioengineering Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications Caroline J. Wilcock1, Piergiorgio Gentile1, Paul V. Hatton1, Cheryl A. Miller1 1Bioengineering and Healthcare Technologies, School of Clinical Dentistry, University of Sheffield This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry. Neuroscience In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments Sara Nullens1, Annemie Deiteren2, Wen Jiang3, Christopher Keating4, Hannah Ceuleers1, Sven Francque5, David Grundy3, Joris G. De Man1, Benedicte Y. De Winter1 1Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, 2Visceral Pain Group, Discipline of Medicine, University of Adelaide, 3Department of Biomedical Sciences, University of Sheffield, 4Department of Pharmacy, Pharmacology and Postgraduate Medicine, University of Hertfordshire, 5Department of Gastroenterology and Hepatology, Antwerp University Hospital Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity. Engineering Blast Quantification Using Hopkinson Pressure Bars Samuel D. Clarke1, Stephen D. Fay1,2, Samuel E. Rigby1, Andrew Tyas1,2, James A. Warren1,2, Jonathan J. Reay2, Benjamin J. Fuller1, Matthew T. A. Gant3, Ian D. Elgy3 1Department of Civil & Structural Engineering, University of Sheffield, 2Blastech Ltd., 3Blast and IED Protection, Physical Sciences Department, Defence Science and Technology Laboratory (Dstl) This protocol details the use of Hopkinson pressure bars to measure reflected blast loading from near-field explosive events. It is capable of interpolating a pressure-time history at any point on a reflective boundary and as such can be used to fully characterize the spatial and temporal variations in loading produced. Engineering Preparation and 3D Tracking of Catalytic Swimming Devices Andrew Campbell1, Richard Archer1, Stephen Ebbens1 1Department of Chemical and Biological Engineering, University of Sheffield A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented. Neuroscience Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo Mikko Juusola1,2, An Dau2, Lei Zheng1, Diana Rien1,2 1National Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, 2Department of Biomedical Science, The University of Sheffield Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes. Developmental Biology Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients Simon W. Fellgett1, Simon A. Ramsbottom2, Richard J. Maguire3, Stephen Cross4, Peter O'Toole5, Mary E. Pownall5 1Department of Biomedical Science, The Bateson Centre, University of Sheffield, 2Institute of Genetic Medicine, Newcastle University, 3Department of Cardiovascular Science, The Bateson Centre, University of Sheffield, 4School of Biochemistry, University of Bristol, 5Biology Department, University of York The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients. Biology Comparing the Affinity of GTPase-binding Proteins using Competition Assays Rosalind C. Williamson1, Mark D. Bass2 1School of Biochemistry, University of Bristol, 2Department of Biomedical Science, University of Sheffield This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate. Bioengineering Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model Nicola H. Green1, Bernard M. Corfe2, Jonathan P. Bury3, Sheila MacNeil1 1Department of Materials Science and Engineering, University of Sheffield, 2Department of Oncology and Insigneo Institute for in silico Medicine, University of Sheffield, 3Department of Histopathology, Sheffield Teaching Hospitals NHS Foundation Trust This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus. Engineering Casting Protocols for the Production of Open Cell Aluminum Foams by the Replication Technique and the Effect on Porosity Erardo M. Elizondo Luna1, Farzad Barari2, Robert Woolley2, Russell Goodall1 1Department of Materials Science and Engineering, The University of Sheffield, 2Department of Mechanical Engineering, The University of Sheffield Replication is one of the processing techniques used for the production of porous metal sponges. In this paper one implementation of the method for the production of open celled porous aluminum is shown in detail. Immunology and Infection Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI Sari Sabban1, Hongtu Ye2, Birgit Helm2 1Biological Department, King Abdulaziz University, 2The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen. Bioengineering Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration Ílida Ortega1, Farshid Sefat1, Pallavi Deshpande1, Thomas Paterson1, Charanya Ramachandran3, Anthony J. Ryan2, Sheila MacNeil1, Frederik Claeyssens1 1Department of Materials Science and Engineering, University of Sheffield, 2Department of Chemistry, University of Sheffield, 3L. V. Prasad Eye Institute We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures. Engineering Using Neutron Spin Echo Resolved Grazing Incidence Scattering to Investigate Organic Solar Cell Materials Andrew J. Parnell1, Adam Hobson2, Robert M. Dalgliesh3, Richard A. L. Jones1, Alan D. F. Dunbar2 1Department of Physics and Astronomy, University of Sheffield, 2Department of Chemical and Biological Engineering, The University of Sheffield, 3ISIS Pulsed Neutron and Muon Source Science and Technology Facilities Council, Rutherford Appleton Laboratory Progress has been made in utilizing spin echo resolved grazing incidence scattering (SERGIS) as a neutron scattering technique to probe the length-scales in irregular samples. Crystallites of [6,6]-phenyl-C61-butyric acid methyl ester have been probed using the SERGIS technique and the results confirmed by optical and atomic force microscopy. Bioengineering Postproduction Processing of Electrospun Fibres for Tissue Engineering Frazer J. Bye1, Linge Wang2, Anthony J. Bullock1, Keith A. Blackwood1, Anthony J. Ryan3, Sheila MacNeil1 1Materials Science and Engineering, University of Sheffield, 2Department of Biomedical Science, University of Sheffield, 3Department of Chemistry, University of Sheffield Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.