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Brown University

27 ARTICLES PUBLISHED IN JoVE

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Biology

Gene-gun Transfection of Hippocampal Neurons
Powrnima Joshi 1, Anna Dunaevsky 1
1Dept of Neuroscience, Brown University

Gene-gun Transfection of Hippocampal Neurons

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Biology

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
Katherine H. Watkins 1, Allan Stewart 1, William G. Fairbrother 1,2
1Department of Molecular and Cellular Biology, Brown University, 2Center for Computational Molecular Biology, Brown University

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

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Biology

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo
Ashly Brown 1, Stephen Brown 2, Debra Ellisor 2, Nellwyn Hagan 1, Elizabeth Normand 1, Mark Zervas 2
1Department of Neuroscience, Division of Biology and Medicine, Brown University, 2Department of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

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Biology

A Protocol for the Production of KLRG1 Tetramer
Stephanie C. Terrizzi 1, Cindy Banh 1, Laurent Brossay 1
1Department of Molecular Microbiology and Immunology, Brown University

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

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Biology

Visualizing RNA Localization in Xenopus Oocytes
James A. Gagnon 1, Kimberly L. Mowry 1
1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

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Behavior

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale
Barry M. Lester 1,2, Lynne Andreozzi-Fontaine 1,2, Edward Tronick 3, Rosemarie Bigsby 1,2
1Center for the Study of Children at Risk, Alpert Medical School, Brown University, 2Women & Infants Hospital of Rhode Island, 3University of Massachusetts, Boston

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

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Behavior

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze
Tara K. Jacobson *1, Jonathan W. Ho *1, Brendon W. Kent 1, Fang-Chi Yang 1, Rebecca D. Burwell 1,2
1Department of Cognitive, Linguistic & Psychological Sciences, Brown University, 2Department of Neuroscience, Brown University

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

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Medicine

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest
Brenda Cordeiro 1, Richard Clements 1,2
1Cardiothoracic Surgery Research Center, Brown University, 2Rhode Island Hospital and Alpert Medical School, Brown University

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

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Bioengineering

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix
David A. Stout 1, Jennet Toyjanova 2, Christian Franck 3
1Department of Mechanical and Aerospace Engineering, California State University, Long Beach, 2Ximedica, 3School of Engineering, Brown University

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

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Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila
Eugenia Villa-Cuesta 1, David M. Rand 2
1Biology Department, Adelphi University, 2Department of Ecology and Evolutionary Biology, Brown University

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

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Immunology and Infection

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials
M. Heath Farris 1, Kara A. Ford 2, Richard C. Doyle 3
1Department of Advanced Technology, The MITRE Corporation, 2Center for Biomedical Engineering, Brown University, 3The MITRE Corporation

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

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Immunology and Infection

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells
Collins Cheruiyot 1, Zemplen Pataki 1, Robert Williams 1, Bharat Ramratnam 2,3, Ming Li 3
1Brown University, 2COBRE Center for Cancer Research, Lifespan Laboratories, Rhode Island and Miriam Hospitals, 3Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School, Brown University

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

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Environment

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx
Paul K. Wojtal 1,2, David J. Miller 2,3, Mary O'Connor 1, Sydney C. Clark 2,3, Meredith G. Hastings 2,3
1Department of Chemistry, Brown University, 2Institute at Brown for Environment and Society, Brown University, 3Department of Earth, Environmental, and Planetary Sciences, Brown University

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

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JoVE Core

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules
Michael A. Monn 1, Jarod Ferreira 1, Jianzhe Yang 1, Haneesh Kesari 1
1School of Engineering, Brown University

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 µN up to 10 N and can therefore accommodate a variety of fiber sizes.

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Education

The Diffusion of Passive Tracers in Laminar Shear Flow
Manuchehr Aminian 1,2, Francesca Bernardi 1, Roberto Camassa 1, Daniel M. Harris 1,3, Richard M. McLaughlin 1
1Department of Mathematics, University of North Carolina at Chapel Hill, 2Department of Mathematics, Colorado State University, 3School of Engineering, Brown University

A protocol for the study of the diffusion of passive tracers in laminar pressure-driven flow is presented. The procedure is applicable to various capillary pipe geometries.

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Bioengineering

Custom Engineered Tissue Culture Molds from Laser-etched Masters
Nicholas J. Kaiser 1, Fabiola Munarin 1, Kareen L.K. Coulombe 1
1Center for Biomedical Engineering,, Brown University

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

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Biology

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans
Joslyn Mills 1, Erin McConnell 1, Joshua A. Leitão 1, Louis R. Lapierre 1
1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

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Behavior

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits
Hayley A. Bounds 1, Devon L. Poeta 1, Petra M. Klinge 3, Rebecca D. Burwell 1,2
1Cognitive, Linguistic & Psychological Sciences, Brown University, 2Department of Neuroscience, Brown University, 3Department of Neurosurgery, Warren Alpert Medical School, Brown University

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson’s Disease.

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Cancer Research

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures
John P. Zepecki 1, Kristin M. Snyder 2, Nikos Tapinos 1,3
1Molecular Neuro-oncology Laboratory, Brown University, Rhode Island Hospital, 2University of Minnesota, College of Veterinary Medicine, 3Department of Neurosurgery, Brown University

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

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Immunology and Infection

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.
Meredith J. Crane 1, William L. Henry Jr 1, Holly L. Tran 1, Jorge E. Albina 2, Amanda M. Jamieson 1
1Department of Molecular Microbiology & Immunology, Brown University, 2Division of Surgical Research, Department of Surgery, Rhode Island Hospital and The Warren Alpert School of Medicine of Brown University

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

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Neuroscience

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions
Benedetta Assetta *1, Changyong Tang *1,2, Jing Bian *3, Ryan O'Rourke 1, Kevin Connolly 1, Thomas Brickler 3, Sundari Chetty 3,4, Yu-Wen Alvin Huang 1,5,6
1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 2Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 5Department of Neurology, Warren Alpert Medical School of Brown University, 6Center for Translational Neuroscience, Robert J. and Nancy D. Carney Institute for Brain Science and Brown Institute for Translational Science, Brown University

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer’s disease.

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Bioengineering

Biplanar Videoradiography to Study the Wrist and Distal Radioulnar Joints
Bardiya Akhbari 1, Amy M Morton 2, Douglas C Moore 2, Joseph J Crisco 1,2
1Center for Biomedical Engineering, Brown University, 2Department of Orthopedics, The Warren Alpert Medical School of Brown University and Rhode Island Hospital

Biplanar videoradiography (BVR) is an advanced imaging technique for understanding the three-dimensional movement of skeletal bones and implants. Combining density-based image volumes and videoradiographs of the distal upper extremity, BVR is used to study the in vivo motion of the wrist and distal radioulnar joint, as well as joint arthroplasties.

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Bioengineering

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions
Christina M. Bailey-Hytholt 1, Veronica LaMastro 2, Anita Shukla 2
1Department of Chemical Engineering, Worcester Polytechnic Institute, 2Center for Biomedical Engineering, School of Engineering, Brown University

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

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Neuroscience

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice
Ragunathan Padmashri 1, Kevin Tyner 1, Anna Dunaevsky 1
1Department of Neurological Sciences, University of Nebraska Medical Center

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

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Chemistry

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition
Samuel Effman *1, Shawn Avidan *1, Zhen Xiao *1, Vicki Colvin 1,2
1Department of Chemistry, Brown University, 2Center for Biomedical Engineering, School of Engineering, Brown University

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

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Biochemistry

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry
Aniruddha Panda 1,2, Rajan Thakur 1,3, Aastha Kumari 1, Padinjat Raghu 1
1National Centre for Biological Sciences-TIFR, GKVK Campus, 2Department of Cell Biology, Yale University, 3Department of Neuroscience, Brown University

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

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Biology

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism
Susan A. Gerbi 1
1Department of Molecular and Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

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