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University of Pennsylvania Perelman School of Medicine

18 ARTICLES PUBLISHED IN JoVE

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Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans
Marni J. Falk 1,2, Meera Rao *1, Julian Ostrovsky *1, Evgueni Daikhin 1, Ilana Nissim 1, Marc Yudkoff 1,2
1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

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Immunology and Infection

Quantitative Measurement of the Immune Response and Sleep in Drosophila
Tzu-Hsing Kuo 1, Arun Handa 1, Julie A. Williams 1
1Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.

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Medicine

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers
Ilia Fishbein 1, Scott P. Forbes 1, Richard F. Adamo 1, Michael Chorny 1, Robert J. Levy 1, Ivan S. Alferiev 1
1Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

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Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits
Joshua B. Slee 1,2, Ivan S. Alferiev 1,2, Robert J. Levy 1,2, Stanley J. Stachelek 1,2
1Division of Cardiology, Department of Pediatrics, The Children's Hospital of Philadelphia, 2University of Pennsylvania Perelman School of Medicine

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

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Biology

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis
Simone Sidoli 1, Natarajan V. Bhanu 1, Kelly R. Karch 1, Xiaoshi Wang 1, Benjamin A. Garcia 1
1Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

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Biology

Assays for the Degradation of Misfolded Proteins in Cells
Lili Guo 1,2, Wil Prall 1, Xiaolu Yang 1
1Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine, 2Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

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Genetics

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity
Sara Lynn N. Farwell 1, Joshua B. Slee 2, Yaqiu Li 1, Linda J. Lowe-Krentz 1
1Department of Biological Sciences, Lehigh University, 2Department of Natural Science, DeSales University

A construct encoding TMEM184A with a GFP tag at the carboxy-terminus designed for eukaryotic expression, was employed in assays designed to confirm the identification of TMEM184A as a heparin receptor in vascular cells.

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Biochemistry

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Robert Warneford-Thomson 1,4, Chongsheng He 1,2, Simone Sidoli 1,3, Benjamin A. Garcia 1,3, Roberto Bonasio 1,2
1Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 3Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, 4Graduate Group in Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

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Genetics

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome
Vid Leko 1,2, Smitha Sripathy 1, Robin L. Adrianse 1, Taylor Loe 1, Angela Park 1, Uyen Lao 1, Eric J. Foss 1, Marisa S. Bartolomei 3, Antonio Bedalov 1,4
1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 3Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 4Departments of Medicine and Biochemistry, University of Washington

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

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Neuroscience

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
Joseph R. Patterson 1, Nicole K. Polinski 2, Megan F. Duffy 1,3, Christopher J. Kemp 1, Kelvin C. Luk 4, Laura A. Volpicelli-Daley 5, Nicholas M. Kanaan 1, Caryl E. Sortwell 1,3,6
1Department of Translational Science and Molecular Medicine, Michigan State University, 2The Michael J. Fox Foundation for Parkinson's Research, 3Neuroscience Program, Michigan State University, 4Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, 5Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, 6Mercy Health Hauenstein Neuroscience Medical Center

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

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Medicine

Cooling or Warming the Esophagus to Reduce Esophageal Injury During Left Atrial Ablation in the Treatment of Atrial Fibrillation
Jason Zagrodzky 1, Mark M. Gallagher 2, Lisa W. M. Leung 2, Tiffany Sharkoski 3, Pasquale Santangeli 4, Cory Tschabrunn 4, Jose M. Guerra 5, Bieito Campos 5, John MacGregor 6, Jamal Hayat 2, Brad Clark 7, Alex Mazur 8, Marcel Feher 9, Martin Arnold 9, Mark Metzl 10, Jose Nazari 10, Erik Kulstad 11
1St. David's South Austin Medical Center, 2St George's University Hospitals NHS Foundation Trust, St. George's, University of London, 3Hospital of the University of Pennsylvania, Perelman Center for Advanced Medicine, 4University of Pennsylvania Perelman School of Medicine, 5Hospital de la Santa Creu I Sant Pau, Universitat Autònoma de Barcelona, CIBERCV, 6PeaceHealth Medical Group, St. Joseph Medical Center, 7St. Vincent Hospital, 8University of Iowa, 9University Hospital Erlangen, 10NorthShore University Health System, 11Department of Emergency Medicine, University of Texas, Southwestern Medical Center

The goal of this protocol is to describe the use of esophageal temperature modulation to counteract esophageal thermal injury from left atrial ablation for the treatment of atrial fibrillation.

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Neuroscience

Cerebrovascular Reactivity Measurement with Functional Near Infrared Spectroscopy
Franck Amyot 1, Cora Davis 1, Mike Sangobowale 4, Carol Moore 2, Erika Silverman 4, Amir Gandjbakhche 3, Ramon Diaz-Arrastia 4, Kimbra Kenney 1,2
1National Intrepid Center of Excellence, Walter Reed National Military Medical Center, 2Uniformed Services University of the Health Sciences, 3The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 4University of Pennsylvania Perelman School of Medicine

Presented here is a protocol for imaging and measurement of cerebrovascular reactivity in humans with functional Near Infrared Spectroscopy (fNIRS). fNIRS is a novel imaging modality that captures the concentration changes of hemoglobin species in the brain’s outermost cortex under specific stimuli.

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Immunology and Infection

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach
Yasmine Issah 1, Amruta Naik 1, Soon Yew Tang 2,3, Kaitlyn Forrest 1, Katherine N. Theken 2,3, Shaon Sengupta 1,3,4
1The Children's Hospital of Philadelphia, 2Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 3Institute of Translational Medicine and Therapeutics (ITMAT), University of Pennsylvania, 4Department of Pediatrics, University of Pennsylvania Perelman School of Medicine

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

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Neuroscience

A Rapid Food-Preference Assay in Drosophila
John O. Mack 1, Yali V. Zhang 1,2
1Monell Chemical Senses Center, 2Department of Physiology, The Diabetes Research Center, University of Pennsylvania Perelman School of Medicine

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

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Behavior

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease
Manuela Lavorato *1, Neal D. Mathew *1, Nina Shah 1, Eiko Nakamaru-Ogiso 1,2, Marni J. Falk 1,2
1Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania Perelman School of Medicine

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

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Biology

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue
Jazmine-Saskya N. Joseph-Chowdhury 1, Stephanie Stransky 1, Sarah Graff 1, Ronald Cutler 1, Dejauwne Young 1, Julie S. Kim 1, Carlos Madrid-Aliste 1, Jennifer T. Aguilan 1, Edward Nieves 1, Yan Sun 1, Edwin J. Yoo 1, Simone Sidoli 1
1Department of Biochemistry, Albert Einstein College of Medicine

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

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Biology

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium
Kole H. Buckley 1, Keely A. Beyries 1, Sandra Ryeom 2, Sam S. Yoon 2, Bryson W. Katona 1
1Division of Gastroenterology and Hepatology, University of Pennsylvania Perelman School of Medicine, 2Department of Surgery, Columbia University Irving Medical Center

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

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Developmental Biology

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells
Rong Qiu 1, Chayanne Petit 1, Christopher S. Thom 1,2
1Division of Neonatology, Children’s Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania Perelman School of Medicine

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

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