Australian National University View Institution's Website 16 articles published in JoVE Biology Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells Yoshika Janapala*1, Katrina Woodward*1, Jiwon Lee2, Melanie Rug2, Thomas Preiss1,3, Nikolay E. Shirokikh1 1Division of Genome Sciences and Cancer, The John Curtin School of Medical Research, The Australian National University, 2Centre for Advanced Microscopy, The Australian National University, 3Victor Chang Cardiac Research Institute We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry. Biology Identification of Alternative Splicing and Polyadenylation in RNA-seq Data Gunjan Dixit1, Ying Zheng1, Brian Parker2, Jiayu Wen1 1Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, 2Department of Biology, New York University Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions. Biology Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker Jennifer Eom1, Vaughan Feisst1, Louis Ranjard2, Kerry Loomes1, Tanvi Damani1, Victoria Jackson-Patel1,3, Michelle Locke4,5, Hilary Sheppard1, Pritika Narayan1,6, P. Rod Dunbar1,7 1School of Biological Sciences, The University of Auckland, 2Research School of Biology, The Australian National University, 3Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, 4Department of Plastic Surgery, Counties Manukau District Health Board, 5Department of Surgery, Faculty of Medical and Health Sciences, The University of Auckland, 6Biomedical Imaging Research Unit, Faculty of Medical and Health Sciences, The University of Auckland, 7Maurice Wilkins Centre, The University of Auckland Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine. Biology Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine Pietro Ariani*1, Elodie Vandelle*1, Darren Wong2, Alejandro Giorgetti1, Andrea Porceddu3, Salvatore Camiolo3, Annalisa Polverari1 1Dipartimento di Biotecnologie, Università degli Studi di Verona, 2Ecology and Evolution, Research School of Biology, The Australian National University, 3Dipartimento di Agraria, SACEG, Università degli Studi di Sassari This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis Tóxicos in Levadura (ATL) E3 ubiquitin ligases. Environment Techniques for Investigating the Anatomy of the Ant Visual System Fiorella Ramirez-Esquivel1, Willi A. Ribi1, Ajay Narendra2 1Research School of Biology, Australian National University, 2Department of Biological Sciences, Macquarie University This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest. Neuroscience Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain Alexandru S. Colibaba*1, Aicee Dawn B. Calma*1, Alexandra L. Webb1, Krisztina Valter1 1Medical School, College of Medicine, Biology and Environment, Australian National University This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology. Engineering Light Enhanced Hydrofluoric Acid Passivation: A Sensitive Technique for Detecting Bulk Silicon Defects Nicholas E. Grant1 1Research School of Engineering, Australian National University A RT liquid surface passivation technique to investigate the recombination activity of bulk silicon defects is described. For the technique to be successful, three critical steps are required: (i) chemical cleaning and etching of silicon, (ii) immersion of silicon in 15% hydrofluoric acid and (iii) illumination for 1 min. Medicine A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data Tim Spelman1,2, Orla Gray3, Robyn Lucas4, Helmut Butzkueven1,2 1Department of Neurology, Royal Melbourne Hospital, 2Department of Medicine (RMH), The University of Melbourne, 3Department of Neurology, Ulster Hospital, 4National Centre for Epidemiology and Population Health, Australian National University Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude. Immunology and Infection In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry Patrick M. Lelliott1, Brendan J. McMorran1, Simon J. Foote1,2, Gaetan Burgio1 1Australian School of Advanced Medicine, Macquarie University, 2John Curtin School of Medical Research, Australian National University The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria. Medicine Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions Lavinia Fiorani*1, Rita Maccarone*1, Nilisha Fernando2,3, Linda Colecchi1, Silvia Bisti1,2, Krisztina Valter2,3,4 1 This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described. Immunology and Infection The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo Benjamin J. C. Quah1, Danushka K. Wijesundara1, Charani Ranasinghe1, Christopher R. Parish1 1Department of Immunology, John Curtin School of Medical Research, Australian National University The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses >250 parameters simultaneously by flow cytometry. Engineering Construction and Characterization of External Cavity Diode Lasers for Atomic Physics Kyle S. Hardman1, Shayne Bennetts1, John E. Debs1, Carlos C. N. Kuhn1, Gordon D. McDonald1, Nick Robins1 1Department of Quantum Science, The Australian National University This is an instructional paper to guide the construction and diagnostics of external cavity diode lasers (ECDLs), including component selection and optical alignment, as well as the basics of frequency reference spectroscopy and laser linewidth measurements for applications in the field of atomic physics. Biology Identification of Post-translational Modifications of Plant Protein Complexes Sophie J. M. Piquerez1, Alexi L. Balmuth2, Jan Sklenář2, Alexandra M.E. Jones1,2, John P. Rathjen3, Vardis Ntoukakis1 1School of Life Sciences, University of Warwick, 2The Sainsbury Laboratory, Norwich Research Park, 3Research School of Biology, The Australian National University We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners. Behavior Measuring Sensitivity to Viewpoint Change with and without Stereoscopic Cues Jason Bell1,2, Edwin Dickinson2, David R. Badcock2, Frederick A. A. Kingdom3 1Research School of Psychology, Australian National University, 2School of Psychology, University of Western Australia, 3McGill Vision Research, Department of Ophthalmology, McGill University We discuss a novel method forviewpoint-rotation of visual stimuli, and demonstrate using a mirror stereoscopethe three-dimensional percept of rotation-in-depth. The technique can be used to investigate the role of stereoscopic cues in encoding viewpoint-rotated figures. Engineering Gradient Echo Quantum Memory in Warm Atomic Vapor Olivier Pinel1, Mahdi Hosseini1, Ben M. Sparkes1, Jesse L. Everett1, Daniel Higginbottom1, Geoff T. Campbell1, Ping Koy Lam1, Ben C. Buchler1 1ARC Centre for Quantum Computation and Communication Technology, Department of Quantum Science, The Australian National University The gradient echo memory is a protocol for storing optical quantum states of light in atomic ensembles. Quantum memory is a key element of a quantum repeater, which can extend the range of quantum key distribution. We outline the operation of the scheme when implemented in a 3-level atomic ensemble. Immunology and Infection The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation Benjamin J. C. Quah1, Christopher R. Parish1 1Department of Immunology, John Curtin School of Medical Research, Australian National University CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.