The University of Queensland 28 articles published in JoVE Medicine Modified Posterior Vertebral Column Resection for Patients with Thoracolumbar Kyphotic Deformity Xiaoming Tian*1, Yachong Huo*1, Felicity Y. Han2, Dalong Yang1, Wenyuan Ding1, Sidong Yang1 1Department of Spinal Surgery, The Third Hospital of Hebei Medical University, 2Australian Institute for Bioengineering and Nanotechnology, The University of Queensland We describe a modified technique for resecting the posterior vertebral column unilaterally based on a modified trephine for patients with thoracolumbar kyphotic deformity. Cancer Research Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids Gency Gunasingh1, Alexander P. Browning2, Nikolas K. Haass1 1The University of Queensland Diamantina Institute, The University of Queensland, 2School of Mathematical Sciences, Queensland University of Technology Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing. Cancer Research Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells Malcolm Lim1, Nicholas Fletcher2, Amy McCart Reed1, Melanie Flint3, Kristofer Thurecht2, Jodi Saunus*1,4, Sunil R. Lakhani*1,5 1UQ Centre for Clinical Research, The University of Queensland, 2Centre for Advanced Imaging, Australian Institute for Bioengineering and Nanotechnology and ARC Training Centre for Innovation in Biomedical Imaging Technology, The University of Queensland, 3School of Biomedical Sciences, The University of Queensland, 4Mater Research and Mater Research Institute, The University of Queensland, 5 Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors. Neuroscience Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro Mohammed R. Shaker*1, Zoe L. Hunter*1, Ernst J. Wolvetang1 1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro. Biochemistry The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination Ziyi Wang1,2, Qinghua Liu3,4, Liang Wang3,5, Robert G. Gilbert1,2,6, Mitchell A. Sullivan7 1School of Chemistry and Molecular Biosciences, The University of Queensland, 2Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, 3Jiangsu Provincial Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, 4Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, 5Department of Bioinformatics, School of Medical Informatics and Engineering, Xuzhou Medical University, 6Joint International Research Laboratory of Agriculture and Agri-Product Safety, College of Agriculture, Yangzhou University, 7Glycation and Diabetes Group, Mater Research Institute-The University of Queensland, Translational Research Institute An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial. Cancer Research Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma Giulia Pericoli*1, Roberta Ferretti*1, Andrew S. Moore2,3, Maria Vinci1 1Department of Onco-hematology, Gene and Cell Therapy, Bambino Gesù Children's Hospital, IRCCS, 2 This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration. Biology Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation Anthony L. Hessel1,4, Daniel Hahn1,2, Markus de Marées3 1Faculty of Sport Science, Human Movement Science, Ruhr University Bochum, 2School of Human Movement and Nutrition Sciences, The University of Queensland, 3Faculty of Sport Science, Sports Medicine and Sports Nutrition, Ruhr University Bochum, 4Institute of Physiology II, University of Muenster This technical report describes a variation of the modified Bergström technique for the biopsy of the musculus tibialis anterior that limits fiber damage. Neuroscience Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound Gerhard Leinenga1, Liviu-Gabriel Bodea1, Wee Kiat Koh1, Rebecca M. Nisbet1, Jürgen Götz1 1Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology. Medicine Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology Michael A. Pearen*1, Hong Kiat Lim*1, Francis D. Gratte2,3, Manuel A. Fernandez-Rojo1,4,5, Sujeevi K. Nawaratna6, Geoffrey N. Gobert7, John K. Olynyk8,9, Janina E. E. Tirnitz-Parker2,10, Grant A. Ramm1,4 1Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, 2School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, 3School of Veterinary and Life Sciences, Murdoch University, 4School of Medicine, The University of Queensland, 5Madrid Institute for Advanced Studies (IMDEA) in Food, CEI UAM+CSIC, 6School of Medicine, Griffith University, 7 This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology. Immunology and Infection Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes M. Rhia L. Stone1, Wanida Phetsang1, Matthew A. Cooper1, Mark A. T. Blaskovich1 1Centre for Superbug Solutions, Institute for Molecular Biosciences, The University of Queensland Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy. Bioengineering Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes Jennifer Walshe1, Najla Al Khaled Abdulsalam2, Shuko Suzuki1, Traian V. Chirila1,3,4,5,6,7,8, Damien G. Harkin1,3,4 1Queensland Eye Institute, 2King Faisal University, 3School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 4Institute of Health and Biomedical Innovation, Queensland University of Technology, 5Science and Engineering Faculty, Queensland University of Technology, 6Faculty of Medicine, University of Queensland, 7Australian Institute for Bioengineering and Nanotechnology, University of Queensland, 8Faculty of Science, University of Western Australia This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented. Developmental Biology Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells Hannah C. Leeson1,2, Tailoi Chan-Ling3,4, Michael D. Lovelace3,5,6, Jeremy D. Brownlie7, Michael W. Weible II*1,4,7, Ben J. Gu*8 1Griffith Institute for Drug Discovery, Griffith University, 2Australian Institute for Bioengineering and Nanotechnology, University of Queensland, 3Discipline of Anatomy and Histology, School of Medical Science, University of Sydney, 4Bosch Institute, University of Sydney, 5 Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads. Medicine Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis Benson U.W. Lei1,2, Miko Yamada1, Van L.T. Hoang3, Paul J. Belt4, Mark H. Moore5, Lynlee L. Lin2, Ross Flewell-Smith1, Nhung Dang1,2, Shoko Tomihara2, Tarl W. Prow1,2 1Future Industries Institute, University of South Australia, 2Faculty of Medicine, University of Queensland, 3Faculty of Health, Queensland University of Technology, 4Department of Plastic and Reconstructive Surgery, Princess Alexandra Hospital, 5Australian Craniofacial Unit, Women's and Children's Hospital In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner. Medicine An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay Lien Anh Ha Do1,2, Reuben Tse1, Jordan Nathanielsz1, Jeremy Anderson1, Darren Suryawijaya Ong1, Keith Chappell3, Kim Mulholland1,2,4, Paul V. Licciardi1,2 1 This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types. Medicine Cardiac Magnetic Resonance Imaging at 7 Tesla Daniel Stäb1,2, Aiman Al Najjar1, Kieran O'Brien1,3, Wendy Strugnell4, Jonathan Richer3, Jan Rieger5, Thoralf Niendorf5, Markus Barth1 1The Centre for Advanced Imaging, The University of Queensland, Brisbane, Australia, 2Department of Diagnostic and Interventional Radiology, University Clinic Würzburg, Würzburg, Germany, 3Siemens Healthcare Pty Ltd, Brisbane, Australia, 4Richard Slaughter Centre of Excellence in CVMRI, The Prince Charles Hospital, Brisbane, Australia, 5MRI.Tools GmbH, Berlin, Germany The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept. Environment A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid Babasaheb M. Matsagar1, Shahriar A. Hossain2,3, Tofazzal Islam4, Yusuke Yamauchi2,3,5,6, Kevin C.-W. Wu1 1Department of Chemical Engineering, National Taiwan University, 2International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 3Australian Institute for Innovative Materials (AIIM), University of Wollongong, 4Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, 5School of Chemical Engineering & Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, 6Department of Plant and Environmental New Resources, Kyung Hee University We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Brønsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl). Environment Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects Andrew Allan Walker1, Max Rosenthal1, Eivind E. A. Undheim2, Glenn F. King1 1Institute for Molecular Bioscience, The University of Queensland, 2Centre for Advance Imaging, The University of Queensland Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection. 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. Neuroscience In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal Adekunle T. Bademosi1, Elsa Lauwers2, Rumelo Amor3, Patrik Verstreken2, Bruno van Swinderen3, Frédéric A. Meunier1 1Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, 2VIB Centre for Brain and Disease Research, KU Leuven Department of Neurosciences, Leuven Institute for Neurodegenerative Disease (LIND), 3Queensland Brain Institute, The University of Queensland Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva. Biology The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury Jully Gogoi-Tiwari1, Julia Köhn-Gaone1, Corey Giles2, Dirk Schmidt-Arras3, Francis D. Gratte1,4, Caryn L. Elsegood1, Geoffrey W. McCaughan5,6,7, Grant A. Ramm8,9, John K. Olynyk10,11, Janina E.E. Tirnitz-Parker1,12 1School of Biomedical Sciences & Curtin Health Innovation Research Institute, Curtin University, 2School of Public Health & Curtin Health Innovation Research Institute, Curtin University, 3Institute of Biochemistry, Christian-Albrechts-University, 4School of Veterinary and Life Sciences, Murdoch University, 5Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, 6Royal Prince Alfred Hospital, 7A.W. Morrow Gastroenterology and Liver Centre, 8QIMR Berghofer Medical Research Institute, 9Faculty of Medicine and Biomedical Sciences, The University of Queensland, 10Fiona Stanley and Fremantle Hospitals, 11School of Medical and Health Sciences, Edith Cowan University, 12School of Medicine and Pharmacology, University of Western Australia Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses. Biochemistry Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells Thomas P. Karbanowicz1, Ala Lew-Tabor1,2, Manuel Rodriguez Valle1 1Queensland Alliance for Agriculture & Food Innovation, The University of Queensland, 2Centre for Comparative Genomics, Murdoch University A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications. Medicine Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury Kai-Zheong Lim*1, Christopher D. Daly*1,2,3, Peter Ghosh4, Graham Jenkin3, David Oehme5, Justin Cooper-White6,7, Taryn Naidoo6, Tony Goldschlager1,8 1Department of Surgery, Monash University, 2Department of Neurosurgery, Monash University, 3The Ritchie Centre, Hudson Institute of Medical Research, 4Proteobioactives, Pty Ltd, 5 Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration. Bioengineering Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example Emma L. C. J. Blundell1, Robert Vogel2,3, Mark Platt1 1Department of Chemistry, School of Science, Loughborough University, 2Izon Science Limited, 3School of Mathematics and Physics, The University of Queensland Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles. Developmental Biology Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue Rebecca A. Pelekanos1, Varda S. Sardesai1, Kathryn Futrega2, William B. Lott2, Michael Kuhn2, Michael R. Doran2,3 1UQ Centre for Clinical Research, The University of Queensland, 2Translational Research Institute, Queensland University of Technology, 3Translational Research Institute, Mater Medical Research - University of Queensland Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues. Developmental Biology The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish Christian Lawrence1, Jason Best1, Jason Cockington2, Eric C. Henry3, Shane Hurley1, Althea James1, Christopher Lapointe1, Kara Maloney1, Erik Sanders4 1 Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae. Medicine Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids Kimberley A. Beaumont1,2, Andrea Anfosso1,2, Farzana Ahmed3, Wolfgang Weninger*1,4,5, Nikolas K. Haass*1,3,5 1The Centenary Institute, 2Sydney Medical School, University of Sydney, 3The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, 4Department of Dermatology, Royal Prince Alfred Hospital, 5Discipline of Dermatology, University of Sydney We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids. Medicine Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets Oanh H. Do1, Jiun T. Low1, Peter Thorn1 1School of Biomedical Sciences, The University of Queensland We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy. Behavior Transcranial Direct Current Stimulation and Simultaneous Functional Magnetic Resonance Imaging Marcus Meinzer1,2, Robert Lindenberg2, Robert Darkow2, Lena Ulm2, David Copland1, Agnes Flöel2 1Centre for Clinical Research, University of Queensland, 2Department of Neurology, NeuroCure Clinical Research Centre, and Centre for Stroke Research Berlin, Charité Universitätsmedizin Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique. It has successfully been used in basic research and clinical settings to modulate brain function in humans. This article describes the implementation of tDCS and simultaneous functional magnetic resonance imaging (fMRI), to investigate the neural basis of tDCS effects.
