Johns Hopkins University View Institution's Website 91 articles published in JoVE Neuroscience Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays Ruixing Liang1,2, Griffin Mess2,3, Joshua Punnoose2,3, Kelley M. Kempski Leadingham2,3, Constantin Smit2,3, Nitish Thakor1,4, Christa W. Habela3, Betty Tyler3, Yousef Salimpour3, Amir Manbachi1,2,3,4,5,6 1Electrical and Computer Engineering, Johns Hopkins University, 2HEPIUS Innovation Laboratory, Johns Hopkins University School of Medicine, 3Neurosurgery, Johns Hopkins University School of Medicine, 4Biomedical Engineering, Johns Hopkins University School of Medicine, 5Mechanical Engineering, Johns Hopkins University, 6Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine Here, we present a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of focused ultrasound on human-induced pluripotent stem cell (HiPSC) neurons. Bioengineering Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model Meghana Bhimreddy1, Denis Routkevitch1,2,3, Andrew M. Hersh1, Ali Mohammadabadi1,3, Arjun K. Menta1, Kelly Jiang1, Carly Weber-Levine1, A. Daniel Davidar1, Joshua Punnoose1,2,3, Kelley M. Kempski Leadingham1,3, Joshua C. Doloff2, Betty Tyler1, Nicholas Theodore1,3, Amir Manbachi1,2,3,4,5,6 1Department of Neurosurgery, Johns Hopkins University School of Medicine, 2Department of Biomedical Engineering, Johns Hopkins University, 3HEPIUS Innovation Laboratory, Johns Hopkins University School of Medicine, 4Department of Electrical Engineering and Computer Science, Johns Hopkins University, 5Department of Mechanical Engineering, Johns Hopkins University, 6Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization. Cancer Research Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice Rachel Vistein1, Briana Winer2, Stephanie Myers1,3, Juliane Liberto4, Shun Ishiyama1,5,6, Xin Guo1, Harumi Saeki7, Tian-Li Wang4,8, Ie-Ming Shih2,4,8, Kathleen Gabrielson1,4,8 1Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, 2Department of Obstetrics and Gynecology, Johns Hopkins University School of Medicine, 3Texas Tech University School of Veterinary Medicine, 4Department of Pathology, Johns Hopkins University School of Medicine, 5Department of Surgery, Johns Hopkins University School of Medicine, 6Department of Coloproctological Surgery, Juntendo University School of Medicine, 7Department of Human Pathology, Juntendo University School of Medicine, 8Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes. Bioengineering Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System Griffin Mess1,2, Taylor Anderson1,2, Shivani Kapoor3, Rasika Thombre1,2, Ruixing Liang4, Emre Derin1, Kelley M. Kempski-Leadingham1, Santosh K. Yadav5, Betty Tyler1, Amir Manbachi1,2,4,6,7 1Department of Neurosurgery, Johns Hopkins University School of Medicine, 2Department of Biomedical Engineering, Johns Hopkins University, 3Krieger School of Arts and Sciences, Johns Hopkins University, 4Department of Electrical Engineering and Computer Science, Johns Hopkins University, 5Department of Radiology, Cancer Imaging Division, Johns Hopkins University School of Medicine, 6Department of Mechanical Engineering, Johns Hopkins University, 7Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine Here, we describe a protocol that details how to perform sonodynamic therapy in an in vivo mouse glioblastoma model using magnetic resonance-guided focused ultrasound. Biology Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome Ren Dodge1, William B. Ludington1,2 1Department of Embryology, Carnegie Institution for Science, 2Department of Biology, Johns Hopkins University This method quantifies microbial abundance using a 96-well plate format to plate colony forming units (CFUs) and is applied to the Drosophila microbiome in whole fly homogenate samples. CFUs are counted with an automated image analysis software provided here. Environment Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India Karthikeyan D. Rajamani1, Sankar Sambandam1, Krishnendu Mukhopadhyay1, Naveen Puttaswamy1, Gurusamy Thangavel1, Durairaj Natesan1, Rengaraj Ramasamy1, Saritha Sendhil1, Amudha Natarajan1, Vigneswari Aravindalochan1, Ajay Pillarisetti2, Michael Johnson3, Joshua Rosenthal*4, Kyle Steenland5, Ricardo Piedhrahita3, Jennifer Peel6, Maggie L. Clark6, Dana Boyd Barr5, Sarah Rajkumar6, Bonnie Young6, Shirin Jabbarzadeh7, Ghislaine Rosa8, Miles Kirby9, Lindsay J. Underhill10, Anaite Diaz-Artiga11, Amy Lovvorn5, William Checkley12, Thomas Clasen5, Kalpana Balakrishnan1 1Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), 2Division of Environmental Health Sciences, University of California, Berkeley, 3Berkeley Air Monitoring Group, 4Division of International Epidemiology and Population Studies, National Institutes of Health, 5Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 6Department of Environmental and Radiological Health Sciences, Colorado State University, 7Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, 8Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 9Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, 10Cardiovascular Division, Washington University School of Medicine, Washington University, 11Centro de Estudios en Salud, Universidad del Valle de Guatemala, 12Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes. Editorial Novel Methodological Perspectives In The Study Of Mosquito Biology Yuemei Dong1, Eric P. Caragata2 1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University, 2Department of Entomology & Nematology, Florida Medical Entomology Laboratory, Institute of Food and Agricultural Sciences, University of Florida Neuroscience A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging Rebecca Kerestes1, Shuo Han2, Srinivas Balachander3, Carlos Hernandez-Castillo4, Jerry L. Prince5,6, Jörn Diedrichsen7, Ian H. Harding1,8 1Department of Neuroscience, Central Clinical School, Monash University, 2Department of Biomedical Engineering, The Johns Hopkins University, 3Department of Psychiatry, National Institute of Mental Health & Neuro Sciences (NIMHANS), 4Faculty of Computer Science, Dalhousie University, 5Department of Electrical and Computer Engineering, The Johns Hopkins University, 6Department of Computer Science, The Johns Hopkins University, 7Brain and Mind Institute, Department for Statistical and Actuarial Sciences, Department for Computer Science, Western University, 8Monash Biomedical Imaging, Monash University A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification. Neuroscience Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons Arens Taga*1, Christa W. Habela*1, Alexandra Johns1, Shiyu Liu1, Mollie O'Brien1, Nicholas J. Maragakis1 1Department of Neurology, Johns Hopkins University We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording. Cancer Research A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate Shun Ishiyama1,2,3,4, Casey Kissel5, Xin Guo1, Alexis Howard6, Harumi Saeki7, Tomoaki Ito8, Polina Sysa-Shah9, Hajime Orita10, Kazuhiro Sakamoto4, Kathleen Gabrielson1,2 1Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 3Department of Surgery, Johns Hopkins University School of Medicine, 4Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 5Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, 6Tuskegee College of Veterinary Medicine, 7Department of Human Pathology, Juntendo University Faculty of Medicine, 8Department of Surgery, Juntendo University Shizuoka Hospital, Juntendo University School of Medicine, 9Department of Urology, The Johns Hopkins School of Medicine, 10Department of Gastroenterology and Minimally Invasive Surgery, Juntendo University Faculty of Medicine Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described. Biology Super-Resolution Live Cell Imaging of Subcellular Structures Rajesh Ranjan1, Xin Chen1 1Department of Biology, The Johns Hopkins University Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue. Biology Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding Abhai K Tripathi1, Godfree Mlambo1, Sachie Kanatani1, Photini Sinnis1, George Dimopoulos1 1Johns Hopkins Malaria Research Institute, W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum. Immunology and Infection Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms Dominic Thomas1, Ting Yu Wu1, Merylin Cottagiri1, Maeva Nyandjo1, Dolores B. Njoku1,2 1Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, 2Department of Pediatrics and Pathology, Johns Hopkins University We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques. Biology Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing Mi-Kyung Shin1, Lenise J. Kim1, Candela Caballero-Eraso2, Vsevolod Y. Polotsky1 1Department of Medicine, Johns Hopkins University, 2Instituto de Biomedicina de Sevilla (IBiS), Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Hospital Universitario Virgen del Rocío/Universidad de Sevilla Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed 'loss of function' experiments measuring the effect of leptin on HVR after CB denervation and 'gain of function' experiments measuring HVR after overexpression of the leptin receptor in CB. Behavior Evaluating Skilled Prehension in Mice Using an Auto-Trainer Robert Hubbard1, Jason Dunthorn2, Richard J. O’Brien3, Dan Tasch2, Uri Tasch2, Steven R. Zeiler1 1Department of Neurology, Johns Hopkins University, 2Step Analysis, LLC, 3Department of Neurology, Duke University Method to assess the impact of training on motor skills is a useful tool. Unfortunately, most behavioral assessments can be labor intensive and/or expensive.We describe here a robotic method of assessing prehension (reach-to-grasp) skill in mice. Bioengineering Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings Viviana Zlochiver*1, Stacie L. Kroboth*1, Christopher R. Beal1, Jonathan A. Cook1, Rosy Joshi-Mukherjee1,2,3 1Aurora Research Institute, Advocate Aurora Health Care, 2Department of Biomedical Engineering, College of Engineering and Applied Science, University of Wisconsin-Milwaukee, 3Department of Medicine-Cardiovascular, School of Medicine, Johns Hopkins University This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days. Bioengineering 3D Analysis of Multi-cellular Responses to Chemoattractant Gradients Tae-Yun Kang1, David Ellison2, Sung Hoon Lee1, Andrew J. Ewald2,3, Andre Levchenko1 1Department of Biomedical Engineering and Yale Systems Biology Institute, Yale University, 2Department of Biomedical Engineering, Johns Hopkins University, 3Center for Cell Dynamics and Department of Cell Biology, Johns Hopkins University We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs. Neuroscience Localization of the Locus Coeruleus in the Mouse Brain Katharina Schmidt1, Bilal Bari2, Martina Ralle3, Clorissa Washington-Hughes1, Abigael Muchenditsi1, Evan Maxey4, Svetlana Lutsenko1 1Department of Physiology, Johns Hopkins University, School of Medicine, 2Department of Neuroscience, Johns Hopkins University, 3Department of Molecular and Medical Genetics, OHSU, 4X-ray science division, Advanced Photon Source, Argonne National Laboratory The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus. Engineering Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles John W. Hickey1,2,3,4, Jonathan P. Schneck2,4 1Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, 2Institute for Cell Engineering, School of Medicine, Johns Hopkins University, 3Institute for Nanobiotechnology, Johns Hopkins University, 4Department of Pathology, School of Medicine, Johns Hopkins University Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy. Medicine Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine Robert A. Kaiser1,2, Shennen A. Mao1, Jaime Glorioso3, Bruce Amiot1, Clara T. Nicolas1, Kari L. Allen1, Zeji Du1, Caitlin J VanLith1, Raymond D. Hickey1, Scott L. Nyberg1, Joseph B. Lillegard1,2,4 1Department of Surgery, Mayo Clinic, 2 This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications. Biology In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge Oliver A. Kent1, Charles Steenbergen2, Samarjit Das2 1Princess Margaret Cancer Centre, University of Toronto, 2Department of Pathology, Department of Cardiology, Johns Hopkins University Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition. Medicine Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI—Application in Premanifest Huntington's Disease Dan Wu1, Andreia V. Faria1, Laurent Younes2,3,4, Christopher A. Ross5, Susumu Mori1,6, Michael I. Miller2,3,7 1The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 2Center for Imaging Science, Johns Hopkins University, 3Institute for Computational Medicine, Johns Hopkins University, 4Department of Applied Mathematics and Statistics, Johns Hopkins University, 5Division of Neurobiology, Departments of Psychiatry, Neurology, Neuroscience and Pharmacology, and Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, 6F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, 7Department of Biomedical Engineering, Johns Hopkins University This paper describes a statistical model for volumetric MRI data analysis, which identifies the "change-point" when brain atrophy begins in premanifest Huntington's disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images. Developmental Biology Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis Erica L. McGrath1,2, Junling Gao1, Ping Wu1,3 1Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 2Department of Neurology, Johns Hopkins University, 3Beijing Institute for Brain Disorders, Capital Medical University This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection. Environment Nanopore DNA Sequencing for Metagenomic Soil Analysis P.J. Cummings1, J. Olszewicz1, K.M. Obom1 1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated. Bioengineering Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy Lijuan He*1,2, Alexandra Sneider*1, Weitong Chen1, Michelle Karl1, Vishnu Prasath3, Pei-Hsun Wu1,2, Gunnar Mattson3, Denis Wirtz1,2,4 1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 2Johns Hopkins Physical Sciences - Oncology Center, Johns Hopkins University, 3Department of Biomedical Engineering, Johns Hopkins University, 4Departments of Oncology and Pathology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis. Neuroscience Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System Bruna Soares Landeira1, Jéssica Alves de Medeiros Araújo1, Timm Schroeder2, Ulrich Müller3, Marcos R. Costa1 1Brain Institute, Federal University of Rio Grande do Norte, 2Department of Biosystems Science and Engineering, ETH Zurich, 3The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia. Bioengineering Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting Chin Siang Ong1,2, Takuma Fukunishi1, Andrew Nashed1, Adriana Blazeski3, Huaitao Zhang1, Samantha Hardy1, Deborah DiSilvestre2, Luca Vricella1, John Conte1, Leslie Tung3, Gordon Tomaselli2, Narutoshi Hibino1 1Division of Cardiac Surgery, Johns Hopkins Hospital, 2Division of Cardiology, Johns Hopkins Hospital, 3Department of Biomedical Engineering, Johns Hopkins University This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease. Biology High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake Jessica P. Otis1, Steven A. Farber1,2 1Department of Embryology, Carnegie Institution for Science, 2Department of Biology, Johns Hopkins University Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques. Biology Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast Derek C. Prosser1, Kristie Wrasman1, Thaddeus K. Woodard1, Allyson F. O’Donnell2, Beverly Wendland1 1Department of Biology, The Johns Hopkins University, 2Department of Biological Sciences, Duquesne University Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry. Neuroscience Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue F. Javier Rubio*1, Xuan Li*1, Qing-Rong Liu1, Raffaello Cimbro2, Bruce T. Hope1 1Behavioral Neuroscience Research Branch, Intramural Research Program, NIDA, NIH, DHHS, 2Division of Rheumatology, School of Medicine, Johns Hopkins University Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects. Medicine Murine Prostate Micro-dissection and Surgical Castration Kenneth C. Valkenburg1, Sarah R. Amend1, Kenneth J. Pienta1 1Department of Urology, Johns Hopkins University This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols. Immunology and Infection Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation Sarah R. Amend1, Kenneth C. Valkenburg1, Kenneth J. Pienta1 1Department of Urology, Johns Hopkins University Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space. Bioengineering Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications Xiomara Calderón-Colón1, Giorgio Raimondi2, Jason J. Benkoski1, Julia B. Patrone3 1Research and Exploratory Development Department, The Johns Hopkins Applied Physics Laboratory, 2Department of Plastic and Reconstructive Surgery, Johns Hopkins School of Medicine, 3Asymmetric Operations Sector, The Johns Hopkins Applied Physics Laboratory In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. Medicine Instillation and Fixation Methods Useful in Mouse Lung Cancer Research Nathachit Limjunyawong1, Jason Mock2, Wayne Mitzner1 1Bloomberg School of Public Health, Environmental Health Sciences, Johns Hopkins University, 2Department of Medicine, Pulmonary Diseases and Critical Care Medicine, University of North Carolina School of Medicine The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements. Medicine Use of Electromagnetic Navigational Transthoracic Needle Aspiration (E-TTNA) for Sampling of Lung Nodules Sixto Arias1, Hans Lee1, Roy Semaan1, Bernice Frimpong1, Ricardo Ortiz1, David Feller-Kopman1, Karen Oakjones-Burgess1, Lonny Yarmus1 1Section of Interventional Pulmonology, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University We describe the novel use of electromagnetic navigational guided transthoracic needle aspiration for the pathologic assessment of human lung nodules. Neuroscience Minimally-invasive Technique for Injection into Rat Optic Nerve Kateryna Raykova1, Melina V. Jones1, Hwa Huang1, Paul F. Hoffman1, Michael Levy1 1Department of Neurology, Johns Hopkins University Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid. Medicine Measurement of the Pressure-volume Curve in Mouse Lungs Nathachit Limjunyawong1, Jonathan Fallica1, Maureen R. Horton1, Wayne Mitzner1 1Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung’s structural changes with developing pathology. Neuroscience Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders Soumya Narayan1, Charlee McLean2, Akira Sawa1, Sandra Y. Lin3, Narayan Rai2, MariaMananita S. Hipolito2, Nicola Cascella4, John J.I. Nurnberger, Jr.5, Koko Ishizuka1, Evaristus A. Nwulia2 1Department of Psychiatry, Johns Hopkins University, 2Department of Psychiatry and Behavioral Sciences, Howard University, 3Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, 4Department of Psychiatry, Sheppard Pratt Hospital, 5Department of Psychiatry, Indiana University In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD. Medicine Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound Maggie M. Kuo1, Viachaslau Barodka2, Theodore P. Abraham3, Jochen Steppan2, Artin A. Shoukas1, Mark Butlin4, Alberto Avolio4, Dan E. Berkowitz1,2, Lakshmi Santhanam1,2 1Department of Biomedical Engineering, Johns Hopkins University, 2Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, 3Department of Medicine (Cardiology), Johns Hopkins University, 4The Australian School of Advanced Medicine, Macquarie University We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured. Bioengineering Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System Kristina M. Obom1, Patrick J. Cummings1, Janelle A. Ciafardoni1, Yasunori Hashimura2, Daniel Giroux2 1Center for Biotechnology Education, Johns Hopkins University, 2PBS Biotech, Inc. Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells. Behavior Performing Behavioral Tasks in Subjects with Intracranial Electrodes Matthew A. Johnson1, Susan Thompson1, Jorge Gonzalez-Martinez2, Hyun-Joo Park1, Juan Bulacio2, Imad Najm2, Kevin Kahn4, Matthew Kerr4, Sridevi V. Sarma4, John T. Gale1,3 1Department of Neurosciences, Cleveland Clinic Foundation, 2Epilepsy Center, Cleveland Clinic Foundation, 3Department of Neurosciences and Center for Neurological Restoration, Cleveland Clinic Foundation, 4Department of Biomedical Engineering, Johns Hopkins University Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population. Immunology and Infection Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells Christian Schütz1, Martin Fleck2,3, Jonathan P. Schneck1, Mathias Oelke1 1Department of Pathology, Institute of Cell Engineering, Johns Hopkins University, 2Department of Internal Medicine I, University of Regensburg, 3Department of Rheumatology, Asklepios Medical Center Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system. Medicine Fast and Accurate Exhaled Breath Ammonia Measurement Steven F. Solga1, Matthew L. Mudalel1, Lisa A. Spacek2, Terence H. Risby3 1 Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors. Bioengineering Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor Scherrine A. Tria1, Marc Ramuz1, Leslie H. Jimison2, Adel Hama1, Roisin M. Owens1 1Department of Bioelectronics, Ecole Nationale Superieure des Mines, 2Research and Exploratory Development Division, Applied Physics Laboratory, Johns Hopkins University The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured. Bioengineering Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides Yang Li1, Catherine A. Foss2, Martin G. Pomper2,3, S. Michael Yu1,3 1Department of Bioengineering, University of Utah, 2Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 3Institute for NanoBiotechnology, Johns Hopkins University This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands. Engineering Fabrication and Visualization of Capillary Bridges in Slit Pore Geometry David J. Broesch1, Joelle Frechette1 1Chemical and Biomolecular Engineering Department, Johns Hopkins University A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. The creation of capillary bridges relies on the formation of pillars to provide a directional physical and chemical heterogeneity to pin the fluid. Capillary bridges are formed and manipulated using microstages and visualized using a CCD camera. Medicine Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers Nancy A. Hodgson1, Douglas A. Granger2,3,4 1Center for Innovative Care in Aging, Johns Hopkins University School of Nursing, 2Institute for Interdisciplinary Salivary Bioscience Research, Arizona State University, 3Johns Hopkins University School of Nursing, 4Johns Hopkins University Bloomberg School of Public Health We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility. Neuroscience Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition Daniel A. Lee1,2, Juan Salvatierra2, Esteban Velarde3, John Wong3, Eric C. Ford4, Seth Blackshaw2,5 1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis. Medicine A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research Zuhaib Ibrahim1, Damon S. Cooney1, Jaimie T. Shores1, Justin M. Sacks1, Eric G. Wimmers1, Steven C. Bonawitz1, Chad Gordon1, Dawn Ruben1, Stefan Schneeberger1, W. P. Andrew Lee1, Gerald Brandacher1 1Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA. Bioengineering Operation of a Benchtop Bioreactor Kristina M. Obom1, Andrew Magno2, Patrick J. Cummings1 1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University, 2ATR Biotech Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture’s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system. Biology Pyrosequencing for Microbial Identification and Characterization Patrick J. Cummings1, Ray Ahmed2, Jeffrey A. Durocher2, Adam Jessen2, Tamar Vardi2, Kristina M. Obom1 1Center for Biotechnology Education, Krieger School of Arts and Sciences, Johns Hopkins University, 2Qiagen Sciences, Inc. Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated. Medicine A Simple Method of Mouse Lung Intubation Sandhya Das1, Kelvin MacDonald2, Herng-Yu Sucie Chang1, Wayne Mitzner1 1Department of Environmental Health Sciences, Program in Respiratory Biology and Lung Disease, Johns Hopkins Bloomberg School of Public Health, 2Department of Pediatrics, Oregon Health Sciences University This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea. Biology Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC) Zach Hensel1, Xiaona Fang1,2,3, Jie Xiao1 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 3Department of Physics, Jilin University We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1. Medicine Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling Alexander Michael Guendel*1, Kyle S. Martin*1, Joshua Cutts2, Patricia L. Foley3, Alexander M. Bailey1, Feilim Mac Gabhann4, Trevor R. Cardinal2, Shayn M. Peirce1 1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy. Bioengineering Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry Ron B. Shmueli1,2, Nupura S. Bhise1,2, Jordan J. Green1,2,3,4 1Biomedical Engineering Department, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Institute for Nanobiotechnology, Johns Hopkins University School of Medicine A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials. Medicine Angiogenesis in the Ischemic Rat Lung John Jenkins1, Elizabeth Wagner1 1Johns Hopkins Asthma and Allergy Center, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization. Chemistry Origami Inspired Self-assembly of Patterned and Reconfigurable Particles Shivendra Pandey1, Evin Gultepe1, David H. Gracias1,2 1Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 2Department of Chemistry, The Johns Hopkins University We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale. Biology Super-resolution Imaging of the Bacterial Division Machinery Jackson Buss1, Carla Coltharp1, Jie Xiao1 1Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins. Immunology and Infection A Simple Chelex Protocol for DNA Extraction from Anopheles spp. Mulenga Musapa1, Taida Kumwenda1, Mtawa Mkulama1, Sandra Chishimba1, Douglas E. Norris2, Philip E. Thuma1, Sungano Mharakurwa1 1Malaria Institute at Macha, 2Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR. Bioengineering Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations Fijoy Vadakkumpadan1, Hermenegild Arevalo1, Natalia A. Trayanova1 1Institute for Computational Medicine and the Department of Biomedical Engineering, Johns Hopkins University A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level. Medicine Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue Elaine Bigelow1,2, Katherine M. Bever1,2, Haiying Xu1,2,3, Allison Yager1, Annie Wu1,2,4, Janis Taube3,5, Lieping Chen6, Elizabeth M. Jaffee1,2,5,7,8, Robert A. Anders1,2,5,8, Lei Zheng1,2,4,5,7 1The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Johns Hopkins University School of Medicine, 3Department of Dermatology, The Johns Hopkins University School of Medicine, 4Department of Surgery, Johns Hopkins University School of Medicine, 5The Sol Goldman Pancreatic Cancer Center, The Johns Hopkins University School of Medicine, 6Yale Cancer Center, Yale School of Medicine, 7The Skip Viragh Center for Pancreatic Cancer, The Johns Hopkins University School of Medicine, 8Department of Pathology, The Johns Hopkins University School of Medicine B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor’s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here. Neuroscience Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons Saijilafu1, Feng-Quan Zhou1,2 1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown. Medicine Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR Sonia Apaza1, Susan Espetia1, Robert H. Gilman1,2, Sonia Montenegro3, Susana Pineda3, Fanny Herhold1, Romeo Pomari1, Margaret Kosek2, Nancy Vu1, Mayuko Saito1,2,4 1Laboratorio de Investigación y Desarrollo (LID), Universidad Peruana Cayetano Heredia, 2Bloomberg School of Public Health, Johns Hopkins University, 3Laboratorio de Diagnostico Molecular, Facultad de Medicina, University of Concepcion,Chile, 4University of California San Diego School of Medicine A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed. Neuroscience Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers Michael A. Muniak1,2, Zachary M. Mayko3, David K. Ryugo2,4, Christine V. Portfors3 1Department of Neuroscience, Johns Hopkins University, 2Garvan Institute of Medical Research, 3School of Biological Sciences, Washington State University, 4Department of Otolaryngology-HNS, Johns Hopkins University Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse. Neuroscience Derivation of Glial Restricted Precursors from E13 mice André W. Phillips1,2, Sina Falahati1,2, Roshi DeSilva1,3, Irina Shats2, Joel Marx1, Edwin Arauz1, Douglas A. Kerr4, Jeffrey D. Rothstein2,5, Michael V. Johnston1,2,6, Ali Fatemi1,2,6 1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland, 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage. Biology Chromatin Immunoprecipitation (ChIP) using Drosophila tissue Vuong Tran1, Qiang Gan1, Xin Chen1 1Department of Biology, Johns Hopkins University Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system. Bioengineering Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells Robert DeRose1, Christopher Pohlmeyer1, Nobuhiro Umeda1,2, Tasuku Ueno1,2, Tetsuo Nagano2, Scot Kuo1,3, Takanari Inoue1 1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level. Neuroscience In vivo Electroporation of Developing Mouse Retina Jimmy de Melo1, Seth Blackshaw1,2,3,4,5 1Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, 2Department of Neurology, Johns Hopkins School of Medicine, 3Department of Ophthalmology, Johns Hopkins School of Medicine, 4Center for High-Throughput Biology, Johns Hopkins School of Medicine, 5Institute for Cell Engineering, Johns Hopkins School of Medicine A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field. Medicine Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging Cila Herman1, Muge Pirtini Cetingul1 1Department of Mechanical Engineering, The Johns Hopkins University We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease. Medicine Manual Muscle Testing: A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients Nancy Ciesla1, Victor Dinglas1, Eddy Fan1,2, Michelle Kho2,3, Jill Kuramoto4, Dale Needham1,2,3 1Outcomes After Critical Illness and Surgery (OACIS) Group, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 2Critical Care Physical Medicine and Rehabilitation Program, Johns Hopkins Hospital, 3Department of Physical Medicine and Rehabilitation, Johns Hopkins University, 4Department of Rehabilitation Services, University of Maryland Medical System Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale. Immunology and Infection HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL Yen-Ling Chiu1,2, Jonathan P. Schneck3,4, Mathias Oelke3,4 1Immunology Graduate Program, Johns Hopkins University, 2Department of Internal Medicine, Far-Eastern Memorial Hospital, 3Department of Pathology, Johns Hopkins University, 4Institute of Cell Engineering, Johns Hopkins University A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy. Neuroscience Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse Lisa Grant*1, Eunyoung Yi*1, Juan D. Goutman2, Elisabeth Glowatzki1 1Department of Otolaryngology-Head and Neck Surgery., The Johns Hopkins School of Medicine, 2Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea. Neuroscience Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity Tara A. LeGates1, Cara M. Altimus1 1Department of Biology, John Hopkins University This article will review methods that can be used to determine circadian function and light responsiveness in mice. Bioengineering Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells Laura E. Dickinson1, Sharon Gerecht1 1Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences Oncology Center and Institute for NanoBioTechnology, Johns Hopkins University A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing. Immunology and Infection Induction of Experimental Autoimmune Hypophysitis in SJL Mice Melissa A. Landek-Salgado1, Shey-Cherng Tzou1, Hiroaki Kimura1, Patrizio Caturegli1 1Department of Pathology, The Johns Hopkins University This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology. Immunology and Infection Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis Shey-Cherng Tzou1, Melissa A. Landek-Salgado1, Hiroaki Kimura1, Patrizio Caturegli1 1Department of Pathology, The Johns Hopkins University Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins. Neuroscience Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants Oscar Gonzalez-Perez1, Hugo Guerrero-Cazares2, Alfredo Quiñones-Hinojosa2 1 Neuroscience Lab/ Fac. Psicologia, University of Colima, 2Department of Neurosurgery, Johns Hopkins University In this article, we show a method to make glass capillary needles with a 50-μm lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain. Medicine Surgical Management of Meatal Stenosis with Meatoplasty Ming-Hsien Wang1 1Department of Urology, Pediatric Urology, Johns Hopkins School of Medicine Meatoplasty, surgical management of meatal stenosis. Neuroscience Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5 Tarran J. Pierfelice1, Nicholas Gaiano1,2 1Institute for Cell Engineering Neuroregeneration and Stem Cell Programs, Johns Hopkins University School of Medicine, 2Departments of Neurology, Neuroscience, and Oncology, Johns Hopkins University School of Medicine In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5. Biology Isolation of Stem Cells from Human Pancreatic Cancer Xenografts Zeshaan Rasheed1, Qiuju Wang1, William Matsui1 1Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies. Neuroscience Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording Katherine D. Cygnar1, Aaron B. Stephan1, Haiqing Zhao1 1Biology, Johns Hopkins University The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis. Biology Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time Yi-Ping Ho1,2, Hunter H. Chen2,3, Kam W. Leong2, Tza-Huei Wang1,3 1Mechanical Engineering, Johns Hopkins University, 2Biomedical Engineering, Duke University, 3Biomedical Engineering, Johns Hopkins University We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics. Biology The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis Mark F Brady1, Jorge Coronel2, Robert H Gilman3, David AJ Moore4 1The Warren Alpert Medical School of Brown University, 2Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, 3Department of International Health, Johns Hopkins Bloomberg School of Public Health, 4Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method. Biology ALS - Motor Neuron Disease: Mechanism and Development of New Therapies Jeffrey D. Rothstein1 1Department of Neurology, Johns Hopkins University Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies. Biology Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination Suchismita Das1, Lindsey Garver1, Jose Ruiz Ramirez1, Zhiyong Xi1, George Dimopoulos1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated. Biology Protocol for Mosquito Rearing (A. gambiae) Suchismita Das1, Lindsey Garver1, George Dimopoulos1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults. Biology Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination Zhiyong Xi1, Suchismita Das1, Lindsey Garver1, George Dimopoulos1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated. Biology Maintaining Wolbachia in Cell-free Medium Courtney Gamston1, Jason Rasgon1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated. Biology Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive Jason Rasgon1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University In this interview, Jason Rasgon explains the concept of genetic drive and the characteristics of an effective gene drive system. The use of the endosymbiotic bacterium, Wolbachia pipientis, as a means to spread genes through mosquito populations, is hypothesized. Biology Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae) Lindsey Garver1, George Dimopoulos1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated. Biology Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes George Dimopoulos1 1Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.
