La Trobe University View Institution's Website 18 articles published in JoVE Biochemistry Fiber Type Identification of Human Skeletal Muscle Heidy K. Latchman*1, Stefan G. Wette*1, Dion J. Ellul1, Robyn M. Murphy1, Noni T. Frankenberg1 1Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, School of Agriculture, Biomedicine and Environment, La Trobe University This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting. Medicine Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans Lachlan Van Schaik1,3, Christine Kettle1, Rod A. Green1, Helen R. Irving1, Joseph A. Rathner1,2 1La Trobe Institute for Molecular Science, Department of Rural Clinical Sciences, La Trobe University, 2School of Biomedical Sciences, Department of Physiology, The University of Melbourne, 3Melbourne Medical School, Department of Rural Health, The University of Melbourne Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans. Behavior Usability Evaluation of Augmented Reality: A Neuro-Information-Systems Study Jun Wu*1, Di Zhang*1, Tao Liu2,3,4, Helen Hong Yang5, Yi Wang1, Huili Yao1, Shinan Zhao1 1Economics and Management School, Jiangsu University of Science and Technology, 2School of Health, Fujian Medical University, 3School of Management, Shanghai University, 4School of Management, Zhejiang University, 5Business School, La Trobe University This study presents an experimental paradigm for a usability test combining subjective and objective evaluations. The objective evaluation adopted Neuro-Information-Systems (NeuroIS) methods, and the subjective evaluation adopted a usability questionnaire and a NASA-Task Load Index (NASA-TLX) scale. Environment Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night Kylie A. Robert1, Alicia M. Dimovski1, Joel A. Robert2, Stephen R. Griffiths1 1Department of Ecology, Environment and Evolution, La Trobe University, 2Independent researcher To study the impacts of artificial light at night (ALAN) on nocturnal flying insects, sampling needs to be confined to nighttime. The protocol describes a low-cost automated flight intercept trap that allows researchers to sample at user-defined periods with increased replication. Biology A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay Sebastian Bass-Stringer1,2, Colleen J. Thomas2,3, Clive N. May3, Paul Gregorevic4, Kate L. Weeks1,5,6, Julie R. McMullen1,2,5,6,7 1Baker Heart and Diabetes Institute, 2Department of Physiology, Anatomy and Microbiology, La Trobe University, 3Florey Institute of Neuroscience and Mental Health, University of Melbourne, 4Department of Physiology, Centre for Muscle Research (CMR), The University of Melbourne, 5Department of Diabetes, Central Clinical School, Monash University, 6Baker Department of Cardiometabolic Health, The University of Melbourne, 7Department of Physiology and Department of Medicine Alfred Hospital, Monash University A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6. Bioengineering Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron Peter Berntsen1, Rama Sharma1, Michael Kusel2, Brian Abbey1, Connie Darmanin1 1Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia, 3086, 2Kusel Design, Niddrie, Melbourne, Australia, 3042 The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron. Biology Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants Lim Chee Liew1,2, Yan Wang1,2, Marta Peirats-Llobet1, Oliver Berkowitz1,2,3, James Whelan1,2,3, Mathew G. Lewsey1,3 1Department of Animal, Plant and Soil Science, AgriBio Building, La Trobe University, 2Australian Research Council Centre of Excellence in Plant Energy Biology, La Trobe University, 3Australian Research Council Research Hub for Medicinal Agriculture, AgriBio Building, La Trobe University Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes. Environment A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans Shan Gao*1, Weiyang Chen*2, Nan Zhang1, Chi Xu3, Haiming Jing1,4, Wenjing Zhang1,4, Gaochao Han1,4, Matthew Flavel5, Markandeya Jois5, Yingxin Zeng1, Jing-Dong J. Han3, Bo Xian3, Guojun Li1,4 1Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control/Beijing Center of Preventive Medicine Research, China, 2College of Computer Science and Technology, Qilu University of Technology(Shandong Academy of Sciences), China, 3Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China, 4Beijing Key Laboratory of Environmental Toxicology, School of Public Health, Capital Medical University, China, 5School of Life Sciences, La Trobe University, Australia A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes. Medicine Non-invasive Assessment of Dorsiflexor Muscle Function in Mice Frederico Gerlinger-Romero1, Alex B. Addinsall2, Richard M. Lovering3, Victoria C. Foletta4, Chris van der Poel5, Paul A. Della-Gatta4, Aaron P. Russell4 1School of Exercise and Nutrition Sciences, Deakin University, 2Centre for Molecular and Medical Research, School of Medicine, Deakin University, 3Department of Orthopaedics, School of Medicine, University of Maryland, 4Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, 5Department of Physiology, Anatomy and Microbiology, La Trobe University Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse. Biochemistry Detection and Isolation of Apoptotic Bodies to High Purity Thanh Kha Phan1, Ivan KH Poon1, Georgia K Atkin-Smith1 1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity. Biology Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy Adam C. Parslow1,2, Andrew H.A. Clayton3, Peter Lock4, Andrew M. Scott1,2,5,6,7 1Tumour Targeting Laboratory, Olivia Newton-John Cancer Research Institute, 2School of Cancer Medicine, La Trobe University, 3Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, 4LIMS Bioimaging Facility, La Trobe Institute for Molecular Science, La Trobe University, 5Department of Medical Oncology, Olivia Newton-John Cancer and Wellnes Centre, Austin Health, 6Department of Medicine, University of Melbourne, 7Department of Molecular Imaging and Therapy, Austin Health Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface. Bioengineering Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen Noordiana Nordin1, Nor Azah Yusof2,3, Son Radu1, Roozbeh Hushiarian4 1Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 2Laboratory of Functional Device, Institute of Advanced Technology, Universiti Putra Malaysia, 3Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 4La Trobe Institute for Molecular Science, La Trobe University A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented. Biochemistry Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana Wenhui Lyu1, Jennifer Selinski1, Lu Li1, David A. Day2, Monika W. Murcha3, James Whelan1, Yan Wang1 1ARC Centre of Excellence in Plant Energy Biology, Department of Animal, Plant and Soil Science, School of Life Science, La Trobe University, 2School of Biological Sciences, Flinders University, 3ARC Centre of Excellence in Plant Energy Biology, University of Western Australia As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction. Chemistry Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene Rebecca A. Ryan1, Sophie Williams1, Andrew V. Martin1, Ruben A. Dilanian1, Connie Darmanin2, Corey T. Putkunz1, David Wood3, Victor A. Streltsov4, Michael W.M. Jones5, Naylyn Gaffney6, Felix Hofmann7, Garth J. Williams8, Sebastien Boutet9, Marc Messerschmidt10, M. Marvin Seibert11, Evan K. Curwood11, Eugeniu Balaur2, Andrew G. Peele5, Keith A. Nugent2, Harry M. Quiney1, Brian Abbey2 1ARC Centre of Excellence in Advanced Molecular Imaging, School of Physics, University of Melbourne, 2Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, 3Department of Physics, Imperial College London, 4Florey Institute of Neuroscience and Mental Health, 5Science and Engineering Faculty, Queensland University of Technology, 6Swinburne University of Technology, 7Department of Engineering Science, University of Oxford, 8Brookhaven National Laboratory, 9Linac Coherent Light Source, SLAC National Accelerator Laboratory, 10BioXFEL Science and Technology Center, 11Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, 12Australian Synchrotron We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1. Environment Functionalization and Dispersion of Carbon Nanomaterials Using an Environmentally Friendly Ultrasonicated Ozonolysis Process Eudora S. Y. Yeo1, Gary I. Mathys2, Narelle Brack3, Erik T. Thostenson4,5,6, Andrew N. Rider1 1Aerospace Division, Defence Science and Technology Group, 2Maritime Division, Defence Science and Technology Group, 3Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, 4Department of Mechanical Engineering, University of Delaware, 5Department of Materials Science and Engineering, University of Delaware, 6Center for Composite Materials, University of Delaware Here, a novel method for the functionalization and stable dispersion of carbon nanomaterials in aqueous environments is described. Ozone is injected directly into an aqueous dispersion of carbon nanomaterial that is continuously recirculated through a high-powered ultrasonic cell. Genetics The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development Antanas Spokevicius1, Lynette Taylor1, Emma Melder1, Kim Van Beveren1, Josquin Tibbits2, Nicky Creux3,4, Gerd Bossinger1 1School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne, 2Victorian AgriBiosciences Centre, La Trobe University R&D Park, 3College of Biological Sciences, Department of Plant Biology, University of California, Davis, 4Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species. Neuroscience A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes Alexander R. Harris1,3, Simeon J. Morgan1,3, Gordon G. Wallace3, Antonio G. Paolini1,3,4 1School of Psychological Science, La Trobe University, 2Intelligent Polymer Research Institute, University of Wollongong, 3ARC Centre of Excellence for Electromaterials Science, 4Health Innovations Research Institute, College of Science, Engineering, and Health, RMIT University Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes. Neuroscience Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach Simeon J. Morgan1, Antonio G. Paolini1 1School of Psychological Science, La Trobe University The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.