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73 ARTICLES PUBLISHED IN JoVE

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Biology

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Julian Konig 1, Kathi Zarnack 2, Gregor Rot 3, Tomaz Curk 3, Melis Kayikci 1, Blaz Zupan 3, Daniel J. Turner 4, Nicholas M. Luscombe 2, Jernej Ule 1
1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

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Biology

Preparation of Quality Inositol Pyrophosphates
Omar Loss 1, Cristina Azevedo 1, Zsolt Szijgyarto 1, Daniel Bosch 1, Adolfo Saiardi 1
1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

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Neuroscience

Electrophysiological Measurements and Analysis of Nociception in Human Infants
L. Fabrizi *1, A. Worley *2, D. Patten 1, S. Holdridge 1, L. Cornelissen 1, J. Meek 3, S. Boyd 2, R. Slater 1,4
1Neuroscience, Physiology and Pharmacology, University College London, 2Department of Clinical Neurophysiology, Great Ormond Street Hospital, 3Elizabeth Garrett Anderson Obstetric Hospital, University College Hospital, 4Nuffield Department of Anaesthetics, University of Oxford

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

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Medicine

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
Kate Lawrenson 1, Barbara Grun 2, Simon A. Gayther 1
1Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College London

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

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Medicine

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model
Marianne S. Carlon *1, Jaan Toelen *2, Marina Mori da Cunha 2, Dragana Vidović 1, Anke Van der Perren 3, Steffi Mayer 2, Lourenço Sbragia 2, Johan Nuyts 4, Uwe Himmelreich 5, Zeger Debyser 1, Jan Deprest 2
1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

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Biology

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
Jamie Freeman 1, Janos Kriston-Vizi 1, Brian Seed 2, Robin Ketteler 1
1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

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Neuroscience

Chicken Embryo Spinal Cord Slice Culture Protocol
Kristina C. Tubby *1, Dee Norval *1, Stephen R. Price 1
1Research Department of Cell and Developmental Biology, University College London

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

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Medicine

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction
Ryan Hodges 1,2, Masayuki Endo 1, Andre La Gerche 3, Elisenda Eixarch 4,5, Philip DeKoninck 1, Vessilina Ferferieva 3, Jan D'hooge 3, Euan M. Wallace 2, Jan Deprest 1
1Division Woman and Child, Department Women, University Hospitals Leuven, 2The Ritchie Centre, Monash Institute of Medical Research, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia, 3Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 4Fetal and Perinatal Medicine Research Group, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 5Maternal-Fetal Medicine Department, ICGON, Hospital Clínic, Universitat de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

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Engineering

Setting Limits on Supersymmetry Using Simplified Models
Christian Gütschow 1, Zachary Marshall 2,3
1Department of Physics and Astronomy, University College London, 2CERN, 3Physics Division, Lawrence Berkeley National Laboratories

This paper demonstrates a protocol for recasting experimental simplified model limits into conservative and aggressive limits on an arbitrary new physics model. Publicly available LHC experimental results can be recast in this manner into limits on almost any new physics model with a supersymmetry-like signature.

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Bioengineering

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration
Mattia F. M. Gerli *1, Sara M. Maffioletti *1, Queensta Millet 1, Francesco Saverio Tedesco 1,2
1Department of Cell and Developmental Biology, University College London, 2Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Hospital

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

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Bioengineering

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix
Panagiotis Maghsoudlou *1, Giorgia Totonelli *1, Stavros P Loukogeorgakis 1, Simon Eaton 1, Paolo De Coppi 1
1Surgery Unit, Institute of Child Health and Great Ormond Street Hospital, University College London

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

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Immunology and Infection

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection
Joseph W. Ndieyira 1, Moyu Watari 1, Rachel A. McKendry 1
1London Centre for Nanotechnology and Departments of Medicine, University College London

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

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Biology

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors
Açelya Yilmazer 1, Irene de Lázaro 1, Cyrill Bussy 1, Kostas Kostarelos 2
1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

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Immunology and Infection

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes
Andreas Kühbacher 1,2,3, Edith Gouin 1,2,3, Jason Mercer 4, Mario Emmenlauer 5, Christoph Dehio 5, Pascale Cossart 1,2,3, Javier Pizarro-Cerdá 1,2,3
1Unité des Interactions Bactéries Cellules, Pasteur Institute, 2INSERM U604, 3Institut National de la Recherche Agronomique (INRA), USC2020, 4Institute of Biochemistry, ETH Zürich, 5Focal Area Infection Biology, Biozentrum, University of Basel

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

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Neuroscience

Preparation of Neuronal Co-cultures with Single Cell Precision
Ngoc-Duy Dinh 1, Ya-Yu Chiang 1,2, Heike Hardelauf 1, Sarah Waide 1, Dirk Janasek 1, Jonathan West 1,3
1Leibniz-Institut für Analytische Wissenschaften, ISAS, 2Department of Biochemical Engineering, University College London, 3Institute for Life Sciences, University of Southampton

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

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Neuroscience

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration
Miguel Tillo 1, Quenten Schwarz 1,2, Christiana Ruhrberg 1
1UCL Institute of Ophthalmology, University College London, 2Department of Human Immunology, Centre for Cancer Biology, South Australia

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

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Bioengineering

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder
Koon-Yang Lee 1,2, Siti Rosminah Shamsuddin 3, Marta Fortea-Verdejo 1, Alexander Bismarck 1,3
1Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, University of Vienna, 2Department of Chemical Engineering, University College London, 3Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

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Behavior

Cortical Source Analysis of High-Density EEG Recordings in Children
Joe Bathelt 1, Helen O'Reilly 2, Michelle de Haan 1
1Cognitive Neuroscience & Neuropsychiatry Section, UCL Institute of Child Health, 2Academic Division of Neonatology, Institute for Women's Health, University College London

In recent years, there has been increasing interest in estimating the cortical sources of scalp measured electrical activity for cognitive neuroscience experiments. This article describes how high density EEG is acquired and how recordings are processed for cortical source estimation in children from the age of 2 years at the London Baby Lab.

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Behavior

Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course
Magdalena W. Sliwinska 1, Sylvia Vitello 1, Joseph T. Devlin 1
1Cognitive, Perceptual & Brain Sciences, University College London

Transcranial magnetic stimulation (TMS) is a technique for non-invasively disrupting neural information processing and measuring its effect on behavior. When TMS interferes with a task, it indicates that the stimulated brain region is necessary for normal task performance, allowing one to systematically relate brain regions to cognitive functions.

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Immunology and Infection

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat
Fatma Dalgakiran 1, Luci A. Witcomb 1, Alex J. McCarthy 1, George M. H. Birchenough 2, Peter W. Taylor 1
1School of Pharmacy, University College London, 2Mucin Biology Group, University of Gothenburg

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

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JoVE Journal

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin
Thomas Ramezani 1, Derek W. Laux 1, Isabel R. Bravo 1, Masazumi Tada 2, Yi Feng 1
1MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, 2Department of Cell & Developmental Biology, University College London

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

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Neuroscience

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Laura E. Brown 1, Celine Fuchs 1, Martin W. Nicholson 1, F. Anne Stephenson 1, Alex M. Thomson 1, Jasmina N. Jovanovic 1
1UCL School of Pharmacy, University College London

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

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Biology

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay
Sonia Christou-Savina 1, Philip L. Beales 1, Daniel P. S. Osborn 2
1Genetics and Genomic Medicine, Institute of Child Health, University College London, 2Molecular Cell Science Research Centre, St. George's University of London

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

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Neuroscience

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons
Rahul Mohan *1, Andrew P. Tosolini *1, Renée Morris 1
1Translational Neuroscience Facility, School of Medical Sciences, University of New South Wales

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

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Neuroscience

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester
Joseph Jebelli *1, Thomas Piers *2, Jennifer Pocock 3
1Department of Neurology, University of Washington, 2Therapeutic Innovation Group, School of Life and Medical Sciences, University College London, 3Department of Neuroinflammation, University College London

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

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Behavior

Using Fiberless, Wearable fNIRS to Monitor Brain Activity in Real-world Cognitive Tasks
Paola Pinti 1,2, Clarisse Aichelburg 3, Frida Lind 3, Sarah Power 1, Elizabeth Swingler 3, Arcangelo Merla 2, Antonia Hamilton 3, Sam Gilbert 3, Paul Burgess 3, Ilias Tachtsidis 1
1Department of Medical Physics and Biomedical Engineering, Malet Place Engineering Building, University College London, 2Infrared Imaging Lab, Institute for Advanced Biomedical Technology (ITAB), Department of Neuroscience, Imaging and Clinical Sciences, University of Chieti-Pescara, 3Institute of Cognitive Neuroscience, Alexandra House, University College London

Monitoring brain activity outside the lab without physical constraints presents methodological challenges. A fiberless, wearable functional Near Infrared Spectroscopy (fNIRS) system was used to measure brain activity during an ecological prospective memory task. It was demonstrated that this system could be used to monitor brain activity during non-lab based experiments.

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Bioengineering

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture
Michael P. Storm 1, Ian Sorrell 2, Rebecca Shipley 3, Sophie Regan 2, Kim A. Luetchford 1, Jean Sathish 2, Steven Webb 4, Marianne J. Ellis 1
1Department of Chemical Engineering and Centre for Regenerative Medicine, University of Bath, 2MRC Centre for Drug Safety Science and Institute of Translational Medicine, University of Liverpool, 3Mechanical Engineering, University College London, 4Department of Applied Mathematics, Liverpool John Moores University

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

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Medicine

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery
Mark Nowell 1, Roman Rodionov 1, Gergely Zombori 2, Rachel Sparks 2, Michele Rizzi 1, Sebastien Ourselin 2, Anna Miserocchi 3, Andrew McEvoy 3, John Duncan 1
1Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, 2Center of Medical Imaging and Computing, UCL, 3Department of Neurosurgery, National Hospital for Neurology and Neurosurgery

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

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Neuroscience

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices
Daniel Bosch 1, Douglas Asede 2, Ingrid Ehrlich 1
1Hertie Institute for Clinical Brain Research and Werner Reichardt Centre for Integrative Neuroscience, University of Tuebingen, 2Max Planck Florida Institute for Neuroscience

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

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Neuroscience

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala
Sabine Schönherr 1, Anna Seewald 1, Yu Kasugai 1, Daniel Bosch 2, Ingrid Ehrlich 2, Francesco Ferraguti 1
1Department of Pharmacology, Medical University of Innsbruck, 2Hertie Institute for Clinical Brain Research and Centre for Integrative Neuroscience, University of Tübingen

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

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Medicine

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation
James N. Fullerton 1, Elisabetta Segre 1, Roel P.H. De Maeyer 1, Alexander A.N. Maini 1, Derek W. Gilroy 1
1Centre for Clinical Pharmacology, Division of Medicine, University College London

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

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Neuroscience

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections
Sarah Jolly *1, Alexander Fudge *1, Nigel Pringle 1, William D. Richardson 1, Huiliang Li 1
1Wolfson Institute for Biomedical Research, University College London

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

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Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart
Sindhu Ramesh *1, Anamika Singh *1, Dasan M. Cibi 1, Derek J. Hausenloy 1,2,3, Manvendra K. Singh 1,2
1Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 2National Heart Research Institute Singapore, National Heart Centre Singapore, 3The Hatter Cardiovascular Insititute, University College London

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

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Engineering

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging
Colin R. Crick *1, Sacha Noimark *2,3, William J. Peveler *3, Joseph C. Bear 3, Aleksandar P. Ivanov 1, Joshua B. Edel 1, Ivan P. Parkin 3
1Department of Chemistry, Imperial College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Chemistry, University College London

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

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Behavior

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease
Dennis Chan 1, Laura Marie Gallaher 2, Kuven Moodley 2, Ludovico Minati 3, Neil Burgess 4, Tom Hartley 5
1Department of Clinical Neurosciences, University of Cambridge, 2Clinical Imaging Sciences Centre, Brighton and Sussex Medical School, 3U.O. Direzione Scientifica, Fondazione IRCCS Istituto Neurologico Carlo Besta, 4Institute of Cognitive Neuroscience, University College London, 5Department of Psychology, University of York

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

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Medicine

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status
Wendy E. Heywood *1, Anna Baud *1, Emily Bliss 1, Ernestas Sirka 1, Jonathan M. Schott 2, Henrik Zetterberg 3, Daniela Galimberti 4, Neil J. Sebire 5, Kevin Mills 1
1Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College London

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

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Developmental Biology

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System
Enrica Bertin 1, Martina Piccoli 1, Chiara Franzin 1, Andras Nagy 2, Maria Mileikovsky 2, Paolo De Coppi 3, Michela Pozzobon 1
1Stem Cell and Regenerative Medicine Laboratory, Fondazione Istituto di Ricerca Pediatrica Citta della Speranza, 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 3Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Programme, UCL Institute of Child Health and Great Ormond Street Hospital

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

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Developmental Biology

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors
Thomas Duncan 1, Aileen Lowe 1, Marshall A. Dalton 2, Michael Valenzuela 1
1Regenerative Neuroscience Group, University of Sydney, 2Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

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Immunology and Infection

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator
Juliet R. Foote 1, Adam P. Levine 1, Philippe Behe 1, Michael R. Duchen 2, Anthony W. Segal 1
1Centre for Molecular Medicine, Division of Medicine, University College London, 2Cell and Developmental Biology, University College London

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

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Biology

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy
Matthew McKenzie 1,2, Sze C. Lim 1, Michael R. Duchen 3
1Centre for Genetic Diseases, Hudson Institute of Medical Research, 2The Department of Molecular and Translational Sciences, Monash University, 3Department of Physiology, University College London

Mitochondria can utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using fluorescent dyes and confocal microscopy.

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Bioengineering

Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration
Daniil I. Nikitichev *1, Dzhoshkun I. Shakir *1, François Chadebecq 1, Marcel Tella 1, Jan Deprest 1,2, Danail Stoyanov 3, Sébastien Ourselin 1, Tom Vercauteren 1
1Translational Imaging Group, CMIC, University College London, 2Department of Obstetrics and Gynecology, University Hospitals Leuven, 3Surgical Robot Vision Group, CMIC, University College London

This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

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Engineering

Electrospinning of Photocatalytic Electrodes for Dye-sensitized Solar Cells
Nicolò Canever 1, Fraser Hughson 1, Thomas J. Macdonald 2, Thomas Nann 1
1The MacDiarmid Institute for Advanced Materials & Nanotechnology, School of, Victoria University of Wellington, 2Department of Chemistry, University College London

The overall goal of this project was to use electrospinning to fabricate a photoanode with improved performance for dye-sensitized solar cells.

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Developmental Biology

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells
Yu Wang 1, Sharmin Alhaque 1, Kate Cameron 1, Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Hassan Rashidi 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

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Biology

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper
Robert D. M. Gray 1,2, Jason Mercer 1, Ricardo Henriques 1,3
1MRC Laboratory for Molecular Cell Biology, University College London, 2Centre for Mathematics and Physics in Life Sciences and Experimental Biology (CoMPLEX), University College London, 3Department of Cell and Developmental Biology, University College London

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

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Bioengineering

The Bionic Clicker Mark I & II
Elliott G. Magee 1, S. Ourselin 2, Daniil Nikitichev 2, T. Vercauteren 2, Anne Vanhoestenberghe 1
1Aspire Create, University College London, 2Translational Imaging Group, CMIC, University College London

A device was created to demonstrate electromyography-based control to a lay audience. After the success of the initial device, a second device was made with greater flexibility in functionality for demonstration and research purposes. This protocol describes the process of building and calibrating both devices.

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Medicine

Transvaginal Mesh Insertion in the Ovine Model
Iva Urbankova 1,2, Geertje Callewaert 1,3, Nikhil Sindhwani 1, Alice Turri 1, Lucie Hympanova 1,2, Andrew Feola 1, Jan Deprest 1,3
1Centrum for Surgical Technologies, Department of Development and Regeneration, Clinical Specialties Research Groups, Faculty of Medicine, KU Leuven, 2Institute for the Care of Mother and Child and Third Faculty of Medicine, Charles University, Prague, 3Pelvic Floor Unit, University Hospitals KU Leuven

This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.

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Neuroscience

A Prediction Error-driven Retrieval Procedure for Destabilizing and Rewriting Maladaptive Reward Memories in Hazardous Drinkers
Ravi K. Das 1, Grace Gale 1, Vanessa Hennessy 1, Sunjeev K. Kamboj 1
1Clinical Psychopharmacology Unit, University College London

This manuscript describes a memory retrieval procedure for destabilizing robust reward memories and rewriting them with counterconditioning prior to their reconsolidation.

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Neuroscience

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices
Faye McLeod *1, Aude Marzo *1, Marina Podpolny 1, Soledad Galli 1, Patricia Salinas 1
1Department of Cell and Developmental Biology, University College London

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

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Neuroscience

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis
Mathew Tata 1, Christiana Ruhrberg 1
1UCL Institute of Ophthalmology

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

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Engineering

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver
Wenfeng Xia 1,2, Simeon J. West 3, Malcolm C. Finlay 2,4, Rosalind Pratt 5,6, Sunish Mathews 1,2, Jean-Martial Mari 7, Sebastien Ourselin 1,2,6, Anna L. David 1,5,8,9, Adrien E. Desjardins 1,2
1Wellcome/EPSRC Centre for Interventional and Surgical Sciences, University College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Anaesthesia, University College Hospital, 4St Bartholomew's Hospital and Queen Mary University of London, 5Institute for Women's Health, University College London, 6Centre for Medical Imaging Computing, University College London, 7GePaSud, University of French Polynesia, 8Department of Development and Regeneration, KU Leuven (Katholieke Universiteit), 9NIHR University College London Hospitals Biomedical Research Centre

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

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Biology

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay
James T. Brash 1, Christiana Ruhrberg 1, Alessandro Fantin 1
1UCL Institute of Ophthalmology, University College London

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

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Immunology and Infection

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo
Line L. Holm 1,2,3, Milica Vukmanovic-Stejic 4, Thomas Blauenfeldt 1, Thomas Benfield 2,3, Peter Andersen 1, Arne N. Akbar 4, Morten Ruhwald 1
1Department of Infectious Disease Immunology, Center for Vaccine Research, Statens Serum Institut, 2Department of Infectious Diseases, Hvidovre Hospital, 3Institute of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 4Division of Infection and Immunity, University College London

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

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JoVE Core

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction
Cecilia S.N. Cheung 1,2, Donald J. Weidner 1, Li Li 1, Philip G. Meredith 3, Haiyan Chen 1, Matthew Whitaker 1, Xianyin Chen 4
1Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook University

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

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Bioengineering

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects
Prasad Sawadkar 1,3, Jason Wong 2, Vivek Mudera 1
1Division of Surgery and Interventional Science, University College London, 2Blond McIndoe Laboratories, Division of Cell Matrix and Regenerative Medicine, MAHSC, University of Manchester, 3Department of Surgical Research, Northwick Park Institute for Medical Research, Northwick Park Hospital

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

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JoVE Core

Scalable Quantum Integrated Circuits on Superconducting Two-Dimensional Electron Gas Platform
Kaveh Delfanazari 1,2, Pengcheng Ma 2, Reuben Puddy 2, Teng Yi 2, Moda Cao 2, Yilmaz Gul 3, Carly L. Richardson 4, Ian Farrer 2,5, David Ritchie 2, Hannah J. Joyce 1, Michael J. Kelly 1,2, Charles G. Smith 2
1Centre for Advanced Photonics and Electronics, Engineering Department, University of Cambridge, 2Department of Physics, Cavendish Laboratory, University of Cambridge, 3Department of Electronic and Electrical Engineering, University College London, 4Department of Materials Science and Metallurgy, University of Cambridge, 5Department of Electronic and Electrical Engineering, University of Sheffield

Quantum integrated circuits (QICs) consisting of array of planar and ballistic Josephson junctions (JJs) based on In0.75Ga0.25As two-dimensional electron gas (2DEG) is demonstrated. Two different methods for fabrication of the two-dimensional (2D) JJs and QICs are discussed followed by the demonstration of quantum transport measurements in sub-Kelvin temperatures.

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Genetics

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization
Fani Memi 1, Daniela Tirziu 2, Irinna Papangeli 3
1Department of Cell and Developmental Biology, University College London, 2Yale Cardiovascular Research Group, Section of Cardiovascular Medicine, Yale School of Medicine, 3Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Yale School of Medicine

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

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Neuroscience

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons
Georgia Economides *1, Svenja Falk *1, Audrey Mercer 1
1Department of Pharmacology, University College London

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

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Genetics

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria
Peggy Paschke 1, David A. Knecht 2, Thomas D. Williams 1, Peter A. Thomason 3, Robert H. Insall 3, Jonathan R. Chubb 4,5, Robert R. Kay 1, Douwe M. Veltman 1
1MRC Laboratory of Molecular Biology, 2Department of Molecular and Cell Biology, University of Connecticut, 3Cancer Research UK Beatson Institute Glasgow, 4MRC Laboratory for Molecular Cell Biology, University College London, 5Department of Cell and Developmental Biology, University College London

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

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Developmental Biology

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells
Balta Lucendo-Villarin 1, Hassan Rashidi 1,2, Sharmin Alhaque 1,3, Lena Fischer 1,4, Jose Meseguer-Ripolles 1, Yu Wang 1, Cliona O'Farrelly 4, Michael Themis 3, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2UCL Great Ormond Street Institute of Child Health, University College London, 3Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, 4School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

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Medicine

Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking
Susan Heavey 1, Aiman Haider 2, Ashwin Sridhar 3, Hayley Pye 1, Greg Shaw 1, Alex Freeman 2, Hayley Whitaker 1
1Molecular Diagnostics and Therapeutics Group, University College London, 2Research Department of Pathology, University College London, 3Department of Uro-Oncology, UCLH NHS Foundation Trust

This method involves utilization of clinical diagnostic data for prostate cancer patients in order to guide sampling procedures, when biobanking tissue following radical prostatectomy. This overcomes issues with previously published methods around efficiency and availability of fresh tissue for a wider range of downstream applications.

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Behavior

Implementation of a Real-Time Psychosis Risk Detection and Alerting System Based on Electronic Health Records using CogStack
Tao Wang 1, Dominic Oliver 2, Yamiko Msosa 1, Craig Colling 3, Giulia Spada 2, Łukasz Roguski 4, Amos Folarin 1, Robert Stewart 3,5, Angus Roberts 1,3, Richard J. B. Dobson 1,3,4,6, Paolo Fusar-Poli 2,3,7,8
1Department of Biostatistics and Health Informatics, Institute of Psychiatry, Psychology and Neuroscience, King's College London, 2Early Psychosis: Interventions and Clinical-detection (EPIC) lab, Department of Psychosis Studies, Institute of Psychiatry, Psychology & Neuroscience, King's College London, 3National Institute for Health Research, Maudsley Biomedical Research Centre, South London and Maudsley National Health Service (NHS) Foundation Trust, 4Institute of Health Informatics, University College London, 5Department of Psychological Medicine, Institute of Psychiatry, Psychology and Neuroscience, King's College London, 6Health Data Research UK London, University College London, 7OASIS service, South London and Maudsley National Health Service (NHS) Foundation Trust, 8Department of Brain and Behavioral Sciences, University of Pavia

We demonstrate how to deploy a real-time psychosis risk calculation and alerting system based on CogStack, an information retrieval and extraction platform for electronic health records.

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Neuroscience

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
Nicolás Stuardo 1,2, Guillermo Moya-Alvarado 1,3, Carolina Ramírez 1,3, Giampietro Schiavo 4, Francisca C. Bronfman 1,3
1Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, 2Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Center for Aging and Regeneration (CARE UC), Pontificia Universidad Católica de Chile, 3Institute of Biomedical Sciences. Faculty of Medicine and Faculty of Life Sciences, Universidad Andrés Bello, 4Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and UK Dementia Research Institute at UCL, University College London Campus

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

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Environment

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica
Aki Masunaga 1, Andrew W. Liu 1, Yongkai Tan 1, Andrew Scott 1, Nicholas M. Luscombe 1,2,3
1Okinawa Institute of Science and Technology Graduate University, 2Francis Crick Institute, 3UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

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Bioengineering

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning
Eleanor C. Mackle *1,2, Jonathan Shapey *1,2,3,4, Efthymios Maneas 1,2, Shakeel R. Saeed 3,5,6, Robert Bradford 3, Sebastien Ourselin 4, Tom Vercauteren 4, Adrien E. Desjardins 1,2
1Wellcome / EPSRC Centre for Interventional and Surgical Sciences, University College London, 2Department of Medical Physics and Biomedical Engineering, University College London, 3Department of Neurosurgery, National Hospital for Neurology and Neurosurgery, 4School of Biomedical Engineering & Imaging Sciences, King's College London, 5The Ear Institute, University College London, 6The Royal National Throat, Nose and Ear Hospital, London

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

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Immunology and Infection

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43
Vanessa S. Terra 1, Charles D. Plumptre 2, Emma C. Wall 3, Jeremy S. Brown 2, Brendan W. Wren 1
1Faculty of Infections and Tropical Diseases, Department of Infection Biology, London School of Hygiene and Tropical Medicine, 2Division of Medicine, University College London, 3Division of Infection and Immunity, University College London UCL

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

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Immunology and Infection

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model
Chantelle E Bowers 1, Virginia L Calder 1, John Greenwood 1, Malihe Eskandarpour 1
1UCL Institute of Ophthalmology, University College London

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

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Biochemistry

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry
Danye Qiu 1, Verena B. Eisenbeis 1, Adolfo Saiardi 2, Henning J. Jessen 1
1Institute of Organic Chemistry, University of Freiburg, 2Medical Research Council, Laboratory for Molecular Cell Biology, University College London

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

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Neuroscience

Expanding the Toolkit for In Vivo Imaging of Axonal Transport
Andrew P. Tosolini 1,2, David Villarroel-Campos 1,2,3, Giampietro Schiavo 1,2,3, James N. Sleigh 1,2,3
1Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, 2UCL Queen Square Motor Neuron Disease Centre, University College London, 3UK Dementia Research Institute, University College London

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

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Biology

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy
Gary Hong Chun Chung 1, Paul Gissen 1,2, Christopher J. Stefan 1, Jemima J. Burden 1
1MRC Laboratory for Molecular Cell Biology, University College London, 2NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

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Developmental Biology

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System
Olivia L. Hagen 1, Yehyun Kim 1, Elaine Kushkowski 1,2, Hannah Rouse 3, Kara L. Cerveny 1
1Department of Biology, Reed College, 2Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago, 3Department of Cell and Developmental Biology, University College London

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

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Neuroscience

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals
Rhys Burrows 1, Chi-Hsuan Ma 1, Yujiao Jennifer Sun 1
1Institute of Ophthalmology, University College London

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

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Biology

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase
Denise R. B. Pilger 1, Christin Luft 1, Robin Ketteler 1,2
1Laboratory for Molecular Cell Biology, University College London, 2Department of Human Medicine, Medical School Berlin

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

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Medicine

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice
Fady Guirguis 1,2, Véronique Bolduc 1, Matthew J. Slarve 3, Haiyan Zhou 4,5, Francesco Muntoni 2,4, Carsten G. Bönnemann 1
1Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 3Department of Molecular Microbiology and Immunology, University of Southern California, 4NIHR Great Ormond Street Hospital Biomedical Research Centre, 5Genetics and Genomic Medicine Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

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