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University of Melbourne

51 ARTICLES PUBLISHED IN JoVE

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Biology

Quantification of γH2AX Foci in Response to Ionising Radiation
Li-Jeen Mah 1,2, Raja S. Vasireddy 1,2,3, Michelle M. Tang 1,3, George T. Georgiadis 1, Assam El-Osta 2,3, Tom C. Karagiannis 1,2
1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct

Quantification of DNA double-strand streaks using γH2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of γH2AX foci after exposure of cells to radiation.

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Biology

Quantitation of γH2AX Foci in Tissue Samples
Michelle M. Tang 1,2, Li-Jeen Mah 1,3, Raja S. Vasireddy 1,2,3, George T. Georgiadis 1, Assam El-Osta 2,3, Simon G. Royce 3,4,5, Tom C. Karagiannis 1,3
1Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenetics in Human Health and Disease, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, The University of Melbourne, 4Department of Allergy and Immunology, Murdoch Children's Research Institute, Royal Children's Hospital, 5Department of Pediatrics, The University of Melbourne

Quantitation of DNA double-strand breaks on the basis of γH2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of γH2AX foci in tissue samples.

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Biology

Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation
Raja S. Vasireddy 1,2,3, Michelle M. Tang 1,2, Li-Jeen Mah 2,3, George T. Georgiadis 2, Assam El-Osta 1,3, Tom C. Karagiannis 2,3
1Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, University of Melbourne

Microscopic analysis of γH2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced γH2AX formation within the nucleus.

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Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
Kirsty R. Short 1, Dimitri A. Diavatopoulos 2, Patrick C. Reading 1, Lorena E. Brown 1, Kelly L. Rogers 3, Richard A. Strugnell 1, Odilia L.C. Wijburg 1
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

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Biology

Clonogenic Assay: Adherent Cells
Haloom Rafehi 1,2, Christian Orlowski 1,2,3, George T. Georgiadis 1, Katherine Ververis 1,4, Assam El-Osta 2,3, Tom C. Karagiannis 1,2
1Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Department of Pathology, The University of Melbourne, 3Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 4Department of Anatomy and Cellular Biology, The University of Melbourne

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

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Bioengineering

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
Wah Chin Boon 1, Karolina Petkovic-Duran 2, Yonggang Zhu 2, Richard Manasseh 3, Malcolm K. Horne 1, Tim D. Aumann 1
1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

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Biology

Extraction of Tissue Antigens for Functional Assays
Andra Necula 1, Rochna Chand 1, Batool Albatat 1, Stuart I. Mannering 1,2
1Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 2Department of Medicine, University of Melbourne

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

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Biology

Glycan Profiling of Plant Cell Wall Polymers using Microarrays
Isabel E. Moller 1,2, Filomena A. Pettolino 3, Charlie Hart 1, Edwin R. Lampugnani 1, William G.T. Willats 4, Antony Bacic 1,2
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

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Neuroscience

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster
Christof Rickert 1, Thomas Kunz 1, Kerri-Lee Harris 2, Paul Whitington 2, Gerhard Technau 1
1Institute of Genetics, University of Mainz, 2Department of Anatomy and Neuroscience, University of Melbourne

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

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Medicine

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis
David A. X. Nayagam 1,2,3, Ceara McGowan 1, Joel Villalobos 1, Richard A. Williams 2,3, Cesar Salinas-LaRosa 2,3, Penny McKelvie 2,3, Irene Lo 2,3, Meri Basa 2,3, Justin Tan 1, Chris E. Williams 1,3,4
1Bionics Institute, 2Department of Anatomical Pathology, St Vincent's Hospital Melbourne, 3Department of Pathology, University of Melbourne, 4Medical Bionics Department, University of Melbourne

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

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Medicine

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice
Hao Chen 1, Jessica N. Perez 1, Eleni Constantopoulos 1, Laurel McKee 1, Jessica Regan 1, Patricia B. Hoyer 1, Heddwen L. Brooks 1, John Konhilas 1
1Department of Physiology, University of Arizona

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

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Immunology and Infection

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins
N. Bishara Marzook *1, Dean J. Procter *1, Helena Lynn 1, Yui Yamamoto 2, Jacquelyn Horsington 3, Timothy P. Newsome 1
1School of Molecular Bioscience, University of Sydney, 2Department of Medicine, Center for Vascular Research, 3Asia Pacific Centre for Animal Health, Faculty of Veterinary Science, University of Melbourne

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here.

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Environment

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry
Colin Ruprecht 1, Takayuki Tohge 1, Alisdair Fernie 1, Cara L. Mortimer 2, Amanda Kozlo 2, Paul D. Fraser 2, Norma Funke 1, Igor Cesarino 3,4, Ruben Vanholme 3,4, Wout Boerjan 3,4, Kris Morreel 3,4, Ingo Burgert 5,6, Notburga Gierlinger 5,6, Vincent Bulone 7, Vera Schneider 8, Andrea Stockero 8, Juan Navarro-Aviñó 9, Frank Pudel 10, Bart Tambuyser 11, James Hygate 12, Jon Bumstead 13, Louis Notley 13, Staffan Persson 1,14
1Max Planck Institute for Molecular Plant Physiology, 2School of Biological Sciences, Plant Molecular Science, Centre for Systems and Synthetic Biology, Royal Holloway, University of London, 3Department of Plant Systems Biology, VIB, 4Department of Plant Biotechnology and Bioinformatics, UGhent, 5Institute for Building Materials, ETH Zurich, 6Applied Wood Materials, EMPA, 7Division of Glycoscience, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), 8European Research and Project Office GmbH, 9ABBA Gaia S.L., 10Pflanzenöltechnologie, 11Capax Environmental Services, 12Green Fuels, 13Neutral Consulting Ltd, 14Plant Cell Biology Research Centre, School of Botany, University of Melbourne

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

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Immunology and Infection

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays
Danika L. Hill 1,2, Emily M. Eriksson 1,2, Louis Schofield 1,2
1Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, 2Department of Medical Biology, University of Melbourne

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

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Neuroscience

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice
Doris Tomas 1, Augustinus H. Prijanto 1, Emma L. Burrows 1, Anthony J. Hannan 1, Malcolm K. Horne 1, Tim D. Aumann 1
1Florey Institute of Neuroscience and Mental Health, The University of Melbourne

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

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Medicine

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks
David A.X. Nayagam 1,2, Irfan Durmo 3, Ceara McGowan 1, Richard A. Williams 2,4, Robert K. Shepherd 1,5,
1Bionics Institute, 2Department of Pathology, The University of Melbourne, 3Cochlear Limited, 4Department of Anatomical Pathology, St Vincent's Hospital Melbourne, 5Medical Bionics Department, The University of Melbourne

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

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Medicine

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice
Matthias Ernst 1,2,3, Adele Preaudet 1, Tracy Putoczki 1,2
1The Walter and Eliza Hall Institute for Medical Research, 2The Department of Medical Biology, University of Melbourne, 3Olivia Newton-John Cancer Research Institute

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

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Neuroscience

Generation of Local CA1 γ Oscillations by Tetanic Stimulation
Robert J. Hatch 1, Christopher A. Reid 1, Steven Petrou 1
1The Florey Institute of Neuroscience and Mental Health, University of Melbourne

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 γ oscillations.

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Developmental Biology

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes
Sandy S.C. Hung 1, Alice Pébay 1, Raymond C.B. Wong 1
1Centre for Eye Research Australia & Department of Ophthalmology, University of Melbourne

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

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Neuroscience

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations
Valerio Conti *1, Aurelie Carabalona *2,3,15, Emilie Pallesi-Pocachard 2,3,4, Richard J. Leventer 5,6,7, Fabienne Schaller 2,3,8, Elena Parrini 1, Agathe A. Deparis 2,3, Françoise Watrin 2,3, Emmanuelle Buhler 2,3,8, Francesca Novara 9, Stefano Lise 10, Alistair T. Pagnamenta 10, Usha Kini 11, Jenny C. Taylor 10, Orsetta Zuffardi 9,12, Alfonso Represa 2,3, David Antony Keays 13, Renzo Guerrini 1,14, Antonio Falace 2,3, Carlos Cardoso 2,3
1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5Royal Children's Hospital, 6Murdoch Children's Research Institute, 7University of Melbourne, 8Plateforme postgenomique INMED, 9University of Pavia, 10Wellcome Trust Centre for Human Genetics, 11Oxford Radcliffe NHS Trust, 12IRCCS Casimiro Mondino Foundation, 13Research Institute of Molecular Pathology, 14IRCCS Stella Maris, 15Columbia University

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

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JoVE Core

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques
Sunil Ratnayake 1, Kristina Ford 1, Antony Bacic 1
1ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

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Bioengineering

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber
Weiqing Zhan *1, Diego Marre *1, Geraldine M. Mitchell 1,2,3, Wayne A. Morrison 1,2,3, Shiang Y. Lim 1,2
1O'Brien Institute Department, St Vincent's Institute of Medical Research, 2Department of Surgery, University of Melbourne, 3Faculty of Health Sciences, Australia Catholic University

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

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Neuroscience

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats
Christine T. Nguyen 1, Tina I. Tsai 1, Zheng He 1, Algis J. Vingrys 1, Pei Y. Lee 1, Bang V. Bui 1
1Department of Optometry and Vision Sciences, University of Melbourne

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

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Behavior

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats
Jason Charng 1, Zheng He 1, Bang Bui 1, Algis Vingrys 1, Magnus Ivarsson 1, Rebecca Fish 2, Rachel Gurrell 2, Christine Nguyen 1
1Department of Optometry and Vision Sciences, University of Melbourne, 2Pfizer Neusentis

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

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Immunology and Infection

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing
Patrice Newton 1, Eleanor A. Latomanski 1, Hayley J. Newton 1
1Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

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JoVE Journal

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood
Clovis S. Palmer 1,2,3, Joshua J. Anzinger 4, Tiffany R. Butterfield 4, Joseph M. McCune 5, Suzanne M. Crowe 1,2,6
1Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, 2Department of Infectious Diseases, Monash University, 3Department of Microbiology and Immunology, University of Melbourne, 4Department of Microbiology, The University of the West Indies, 5Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, 6Department of Medicine, Monash University

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

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Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences
Stefanie Dudczig 1,2, Peter D. Currie 2, Lucia Poggi 3, Patricia R. Jusuf 1,2
1School of Biosciences, The University of Melbourne, 2Australian Regenerative Medicine Institute (ARMI), Monash University, 3The David J Apple Center for Vision Research, Department of Ophthalmology, Heidelberg University

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

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Medicine

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection
Hao Chen 1, Robert Klein 1, Stacy Arnold 1, Yiying Wang 2, Setsuko Chambers 3,4, Wenxin Zheng 5,6
1Department of Pathology, University of Arizona College of Medicine, 2Department of Obstetrics and Gynecology, Henan Provincial People's Hospital, 3Department of Obstetrics and Gynecology, University of Arizona College of Medicine, 4University of Arizona Cancer Center, 5Department of Pathology, University of Texas Southwestern Medical Center, 6Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

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Chemistry

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene
Rebecca A. Ryan 1, Sophie Williams 1, Andrew V. Martin 1, Ruben A. Dilanian 1, Connie Darmanin 2, Corey T. Putkunz 1, David Wood 3, Victor A. Streltsov 4, Michael W.M. Jones 5, Naylyn Gaffney 6, Felix Hofmann 7, Garth J. Williams 8, Sebastien Boutet 9, Marc Messerschmidt 10, M. Marvin Seibert 11, Evan K. Curwood 11, Eugeniu Balaur 2, Andrew G. Peele 5, Keith A. Nugent 2, Harry M. Quiney 1, Brian Abbey 2
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.

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Chemistry

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis
Miguela Martin 1, David Perez-Guaita 1, Dean W. Andrew 3, Jack S. Richards 3,4, Bayden R. Wood 1, Philip Heraud 1,2
1Centre for Biospectroscopy, Monash University, 2Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, 3Centre for Biomedical Research, Burnet Institute, 4Department of Medicine, University of Melbourne

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

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Bioengineering

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology
Vijay Rajagopal 1,2,3, Gregory Bass 2,3, Shouryadipta Ghosh 1,2,3, Hilary Hunt 2,4, Cameron Walker 5, Eric Hanssen 6, Edmund Crampin 2,3,4,7,8, Christian Soeller 9
1Cell Structure and Mechanobiology Group, University of Melbourne, 2Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, 3Department of Biomedical Engineering, University of Melbourne, 4School of Mathematics and Statistics, Faculty of Science, University of Melbourne, 5Department of Engineering Science, University of Auckland, 6Advanced Microscopy Facility, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 7ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of Melbourne, 8School of Medicine, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, 9Living Systems Institute, University of Exeter

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

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Biology

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy
Adam C. Parslow 1,2, Andrew H.A. Clayton 3, Peter Lock 4, Andrew M. Scott 1,2,5,6,7
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.

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JoVE Core

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires
Madhur Upadhyay 1, Raja Shah 2, Sachin Agarwal 3, Meenakshi Vishwanath 4, Po-Jung Chen 5, Takafumi Asaki 6, Donald Peterson 7
1Division of Orthodontics, University of Connecticut Health, 2Private Practice, 3Department of Orthodontics, University of Melbourne, 4Department of Orthodontics, University of Nebraska Medical Center, 5Department of Craniofacial Sciences, University of Connecticut Health, 6Biomedical Engineering, University of Hartford, 7Department of Mechanical Engineering, College of Engineering and Engineering Technology, Northern Illinois University

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

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Genetics

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Sarah E. Ashley 1,2, Braydon A. Meyer 2,3, Justine A. Ellis 2,3,4, David J. Martino 2,3,5
1Molecular Genetics of Chronic Inflammation and Allergic Disease, Max-Delbrück Center for Molecular Medicine, 2Murdoch Childrens Research Institute, 3Department of Paediatrics, University of Melbourne, 4Centre for Social and Early Emotional Development, Faculty of Health, Deakin University, 5Department of Paediatrics, University of Western Australia

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

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Cancer Research

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System
Hao Chen *1, Thomas M.B. Ware *1, Josephine Iaria 1, Hong-Jian Zhu 1
1Department of Surgery (RMH), University of Melbourne

Here, we present a protocol for live cell imaging of TGF-β/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

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Medicine

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice
Zheng He 1, Da Zhao 1, Anna K. van Koeverden 1, Christine T Nguyen 1, Jeremiah K. H. Lim 1, Vickie H. Y. Wong 1, Algis J. Vingrys 1, Bang V. Bui 1
1Department of Optometry and Vision Sciences, University of Melbourne

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

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Immunology and Infection

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing
Georges Khoury 1, Damian F.J. Purcell 1
1Department of Microbiology and Immunology, University of Melbourne

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

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Medicine

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay
Lien Anh Ha Do 1,2, Reuben Tse 1, Jordan Nathanielsz 1, Jeremy Anderson 1, Darren Suryawijaya Ong 1, Keith Chappell 3, Kim Mulholland 1,2,4, Paul V. Licciardi 1,2
1Murdoch Children's Research Institute, 2Department of Paediatrics, University of Melbourne, 3School of Chemistry and Molecular Bioscience, University of Queensland, 4London School of Hygiene and Tropical Medicine

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.

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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. Leeson 1,2, Tailoi Chan-Ling 3,4, Michael D. Lovelace 3,5,6, Jeremy D. Brownlie 7, 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, 5Applied Neurosciences Program, Peter Duncan Neurosciences Research Unit, St. Vincent's Centre for Applied Medical Research, 6School of Medical Sciences, The University of New South Wales (UNSW) Medicine, Sydney, New South Wales, 7School of Environment and Science, Griffith University, Brisbane, Queensland, 8Florey Institute of Neuroscience and Mental Health, University of Melbourne

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.

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Developmental Biology

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode
Jiaheng Xie 1, Patricia R. Jusuf 1, Patrick T. Goodbourn 2, Bang V. Bui 3
1School of Biosciences, University of Melbourne, 2Melbourne School of Psychological Sciences, University of Melbourne, 3Department of Optometry and Vision Sciences, University of Melbourne

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

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Immunology and Infection

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester
Anthony R. Di Carluccio 1, Eleonora Tresoldi 1, Michelle So 1, Stuart I. Mannering 1,2
1Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 2Department of Medicine, University of Melbourne, St. Vincent's Hospital

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

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JoVE Core

Quantifying the Relative Thickness of Conductive Ferromagnetic Materials Using Detector Coil-Based Pulsed Eddy Current Sensors
Nalika Ulapane 1, Karthick Thiyagarajan 2, David Hunt 2, Jaime Valls Miro 2
1Melbourne School of Engineering, University of Melbourne, 2Center for Autonomous Systems, University of Technology Sydney

Here, we present a protocol to quantify the relative thickness (i.e., thickness as a percentage with respect to a reference) of conductive ferromagnetic materials using detector coil-based pulsed eddy current sensors, while overcoming the calibration requirement.

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Bioengineering

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior
Francesco Carrara 1, Douglas R. Brumley 2, Andrew M. Hein 3, Yutaka Yawata 4,5, M. Mehdi Salek 1, Kang Soo Lee 1, Elzbieta Sliwerska 1, Simon A. Levin 6, Roman Stocker 1
1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, 2School of Mathematics and Statistics, University of Melbourne, 3Institute of Marine Sciences, University of California, Santa Cruz, 4Faculty of Life and Environmental Sciences, University of Tsukuba, 5Microbiology Research Center for Sustainability, University of Tsukuba, 6Department of Ecology and Evolutionary Biology, Princeton University

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

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JoVE Core

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats
Martin M. Bertrand 1,2, Janet R. Keast 1
1Department of Anatomy and Neuroscience, University of Melbourne, 2Department of Visceral Surgery, CHU de Nîmes

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

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Medicine

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery
Julia A. Dubowitz 1,2,3, Fabian Jost-Brinkmann 1,4,5, Alexandra I. Ziegler 1, Ryan D. Gillis 1, Bernhard Riedel 1,2,3,6, Erica K. Sloan 1,2
1Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, 2Department of Anaesthesia, Division of Cancer Surgery, Peter MacCallum Cancer Centre, 3Department of Critical Care, Melbourne Medical School, University of Melbourne, 4Medical Department, Division of Hepatology and Gastroenterology, Charité - Universitätsmedizin Berlin, 5Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 6Sir Peter MacCallum Department of Oncology, University of Melbourne

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

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Biology

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay
Sebastian Bass-Stringer 1,2, Colleen J. Thomas 2,3, Clive N. May 3, Paul Gregorevic 4, Kate L. Weeks 1,5,6, Julie R. McMullen 1,2,5,6,7
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.

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Developmental Biology

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model
Meaghan J. Griffiths *1,2, Jordan N. Higgins *1, Fiona L. Cousins 3, Lauren R. Alesi 1, Amy L. Winship *1, Karla J. Hutt *1
1Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, 2Gynaecology Research Centre, Royal Women’s Hospital and Department of Obstetrics and Gynaecology, University of Melbourne, 3The Ritchie Centre, Hudson Institute for Medical Research and Department of Obstetrics and Gynaecology, Monash University

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

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Medicine

Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography
Yousif Al-Khoury 1,2, Stephanie Finzel 3, Camille Figueiredo 4, Andrew J. Burghardt 5, Kathryn S. Stok 6, Lai-Shan Tam 7, Isaac Cheng 7, Justin J. Tse 1, Sarah L. Manske 1
1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong

Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis.

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Neuroscience

Implantation Surgery for Abdominal Vagus Nerve Stimulation and Recording Studies in Awake Rats
Tomoko Hyakumura 1,2, James B. Fallon 1,2,3, Sophie C. Payne 1,2
1Bionics Institute, 2Medical Bionics Department, University of Melbourne, 3Department of Otolaryngology, University of Melbourne

The present protocol describes the surgical technique for implanting an electrode array onto the abdominal vagus nerve in rats, along with methods for chronic electrophysiology testing and stimulation using the implanted device.

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Medicine

Regenerative Peripheral Nerve Interface: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain
Emily Pettersen 1,2,3,4, Paolo Sassu 5, Francesca Alice Pedrini 1,5, Hannes Granberg 1,2, Carina Reinholdt 2,6, Juan Manuel Breyer 7, Aidan Roche 8, Andrew Hart 9,10, Adil Ladak 11, Hollie A. Power 11, Michael Leung 12, Michael Lo 12, Ian Valerio 13, Kyle R. Eberlin 13, Jason Ko 14, Gregory A. Dumanian 14, Theodore A. Kung 15, Paul Cederna 15, Max Ortiz-Catalan 1,4,16,17
1Center for Bionics and Pain Research, 2Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, 3Department of Electrical Engineering, Chalmers University of Technology, 4Bionics Institute, 5IV Clinica Ortoplastica, IRCCS Istituto Ortopedico Rizzoli, 6Department of Hand Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, 7Department of Orthopedic Surgery, Hand Unit, Worker Hospital, 8College of Medicine and Veterinary Medicine, The Queen's Medical Research Institute, The University of Edinburgh, 9Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, 10College of Medicine, Veterinary & Life Sciences, The University of Glasgow, 11Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 12Plastics and Reconstructive Surgery, Dandenong Hospital, Monash Health, 13Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital & Harvard Medical School, 14Division of Plastic Surgery, Department of Surgery, Northwestern Feinberg School of Medicine, 15Section of Plastic Surgery, Department of Surgery, Michigan Medicine, 16Medical Bionics Department, University of Melbourne, 17Prometei Pain Rehabilitation Center

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular transposition.

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Medicine

Targeted Muscle Reinnervation: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain
Emily Pettersen 1,2,3,4, Paolo Sassu 5, Francesca Alice Pedrini 1,5, Hannes Granberg 1,2, Carina Reinholdt 2,6, Juan Manuel Breyer 7, Aidan Roche 8, Andrew Hart 9,10, Adil Ladak 11, Hollie A. Power 11, Michael Leung 12, Michael Lo 12, Ian Valerio 13, Kyle R. Eberlin 13, Theodore A. Kung 14, Paul Cederna 14, Jason M. Souza 15,16, Oskar Aszmann 17, Jason Ko 18, Gregory A. Dumanian 18, Max Ortiz-Catalan 1,4,19,20
1Center for Bionics and Pain Research, 2Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, 3Department of Electrical Engineering, Chalmers University of Technology, 4Bionics Institute, 5IV Clinica Ortoplastica, IRCCS Istituto Ortopedico Rizzoli, 6Department of Hand Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, 7Department of Orthopedic, Hand Unit, Worker Hospital, 8College of Medicine and Veterinary Medicine, The Queen's Medical Research Institute, The University of Edinburgh, 9Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, 10College of Medicine, Veterinary & Life Sciences, The University of Glasgow, 11Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 12Plastics and Reconstructive Surgery, Dandenong Hospital, Monash Health, 13Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital &, Harvard Medical School, 14Section of Plastic Surgery, Department of Surgery, Michigan Medicine, 15Department of Surgery, Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, 16Department of Plastic and Reconstructive Surgery, Ohio State University, 17Clinical Laboratory for Bionic Extremity Reconstruction, Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University Vienna, 18Division of Plastic Surgery, Department of Surgery, Northwestern Feinberg School of Medicine, 19Medical Bionics Department, University of Melbourne, 20Prometei Pain Rehabilitation Center

The protocol outlines the surgical procedure for the treatment of postamputation pain using Targeted Muscle Reinnervation (TMR). TMR will be compared with two other surgical techniques, specifically Regenerative Peripheral Nerve Interface (RPNI) and neuroma excision, followed by immediate burying within muscle under the context of an international, randomized controlled trial.

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