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University of Pittsburgh School of Medicine

44 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction
Xin Meng 1, Gongpu Zhao 1, Peijun Zhang 1
1Department of Structural Biology, University of Pittsburgh School of Medicine

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

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Biology

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
David Svilar 1,2, Conchita Vens 3, Robert W. Sobol 1,2,4
1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

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Immunology and Infection

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy
Peter A. Keyel 1, Michelle E. Heid 1, Simon C. Watkins 2, Russell D. Salter 1
1Department of Immunology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

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Engineering

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation
Jonathan A. Kopechek 1, Peng Zhang 1, Mark T. Burgess 1, Tyrone M. Porter 1
1Department of Mechanical Engineering, Boston University

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

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Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
Sangmi Jun 1, Gongpu Zhao 1, Jiying Ning 1, Gregory A. Gibson 2, Simon C. Watkins 2, Peijun Zhang 1
1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

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Biology

High Throughput Microinjections of Sea Urchin Zygotes
Nadezda A. Stepicheva 1, Jia L. Song 1
1Department of Biological Sciences, University of Delaware

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

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JoVE Journal

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Kristine M. Cihil 1,2, Agnieszka Swiatecka-Urban 1,2
1Department of Nephrology, Children's Hospital of Pittsburgh of UPMC, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

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Medicine

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography
Ravirajsinh N. Jadeja 1, Vikrant Rachakonda 2, Zsolt Bagi 3, Sandeep Khurana 1
1Section of Gastroenterology and Hepatology, Georgia Regents University, 2Division of Gastroenterology and Hepatology, University of Pittsburgh School of Medicine, 3Vascular Biology Center, Georgia Regents University

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

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Neuroscience

Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking
Megan L. Brady 1, Charles E. Moon 1, Tija C. Jacob 1
1Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine

Here we demonstrate the use of fluorescent Alexa dye coupled to α-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds α-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

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Medicine

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
F. Aura Kullmann 1, Stephanie L. Daugherty 2, William C. de Groat 2, Lori A. Birder 1,2
1Department of Medicine, Renal division, University of Pittsburgh School of Medicine, 2Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology, and pathology.

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Developmental Biology

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow
Christopher C. Rymer 1,2, Sunder Sims-Lucas 1,2
1Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, 2Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

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Medicine

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye
Larry Kagemann 1,2, Bo Wang 2, Gadi Wollstein 1, Hiroshi Ishikawa 1,2, Brandon Mentley 1, Ian Sigal 1,2,3, Richard A Bilonick 1,4, Joel S Schuman 1,2,3
1Department of Ophthalmology, UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, 3The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 4Deptartment of Biostatistics, Graduate School of Public Health, University of Pittsburgh

Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

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Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation
Arun Sharma *1, Guang Li *1, Kuppusamy Rajarajan *1, Ryoko Hamaguchi 1, Paul W. Burridge 1, Sean M. Wu 1,2
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Institute of Stem Cell Biology and Regenerative Medicine, Cardiovascular Medicine Division, Department of Medicine, Child Health Research Institute, Stanford University School of Medicine

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

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Medicine

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side
Stefanie Dinner 1, Julia Borkowski 1, Carolin Stump-Guthier 1, Hiroshi Ishikawa 2, Tobias Tenenbaum 1, Horst Schroten 1, Christian Schwerk 1
1Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University, 2Department of NDU Life Sciences, Nippon Dental University

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

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Developmental Biology

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment
Destiny L. Baars 1, Kendra A. Takle *1,2, Jonathon Heier *1,3, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

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Biochemistry

Measuring Protein Binding to F-actin by Co-sedimentation
Jonathon A. Heier 1, Daniel J. Dickinson 2, Adam V. Kwiatkowski 1
1Department of Cell Biology, University of Pittsburgh School of Medicine, 2Department of Biology, University of North Carolina at Chapel Hill

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

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Immunology and Infection

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins
Jill A. Dembowski 1, Neal A. Deluca 1
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

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Immunology and Infection

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates
Alexander G. White *1, Pauline Maiello *1, M. Teresa Coleman 1, Jaime A. Tomko 1, L. James Frye 1, Charles A. Scanga 1, Philana Ling Lin 2, JoAnne L. Flynn 1
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 2Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

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Medicine

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation
Wyatt E. Lanik 1, Lily Xu 2, Cliff J. Luke 1, Elise Z. Hu 2, Pranjal Agrawal 2, Victoria S. Liu 2, Rajesh Kumar 1, Alexa M. Bolock 1, Congrong Ma 3, Misty Good 1
1Division of Newborn Medicine, Department of Pediatrics, Washington University School of Medicine, 2Washington University, 3Division of Newborn Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

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Biology

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis
Shinichiro Yokota 1,2, Yoshihiro Ono 2, Toshimasa Nakao 2, Peng Zhang 3, George K. Michalopoulos 4,5, Zahida Khan 3,4,5,6
1Department of Surgery, Jichi Medical University, 2Department of Surgery, University of Pittsburgh School of Medicine, 3Department of Pediatrics, University of Pittsburgh School of Medicine, 4Department of Pathology, University of Pittsburgh School of Medicine, 5Pittsburgh Liver Research Center, University of Pittsburgh, 6McGowan Institute for Regenerative Medicine, University of Pittsburgh

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

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Biology

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium
Peng Shang 1, Nadezda A. Stepicheva 1, Stacey Hose 1, J. Samuel Zigler, Jr. 2, Debasish Sinha 1,2
1Department of Ophthalmology, University of Pittsburgh School of Medicine, 2Wilmer Eye Institute, The Johns Hopkins University School of Medicine

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

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Biology

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima
Jee-Hyun Um *1,2, Young Yeon Kim *1,2, Toren Finkel 3, Jeanho Yun 1,2
1Peripheral Neuropathy Research Center, College of Medicine, Dong-A University, 2Department of Biochemistry, College of Medicine, Dong-A University, 3Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

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Medicine

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice
Sidney Mahan 1, Mingjun Liu 1, Richard A. Baylis 2,3, Delphine Gomez 1,4
1Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, 2Robert M. Berne Cardiovascular Research Center, University of Virginia, 3Department of Biochemistry and Molecular Genetics, University of Virginia, 4Division of Cardiology, University of Pittsburgh School of Medicine

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

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Education

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency
Marianne Theile 1, Linus Wiora 1, Dominik Russ 1, Jonas Reuter 1, Hiroshi Ishikawa 2, Christian Schwerk 3, Horst Schroten 3, Stefan Mogk 1
1Interfaculty Institute of Biochemistry, University of Tübingen, 2Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 3Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

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Developmental Biology

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells
Wei Feng 1, Andrew Przysinda 1, Guang Li 1
1Department of Developmental Biology, University of Pittsburgh School of Medicine

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

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Genetics

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens
Jinxia Shi 1, Yijuan Jia 1, Di Fang 1, Shidan He 1, Peng Zhang 1,2, Yushuang Guo 3, Yongli Qiao 1
1Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, 2College of Agriculture, Yangtze University, 3Laboratory of Molecular Genetics, China National Tobacco Corporation, Guizhou Institute of Tobacco Science

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

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Biology

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide
Kyle S. Feldman 1, Maliha Zahid 1
1Department of Developmental Biology, University of Pittsburgh School of Medicine

We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.

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Biology

Measuring RAN Peptide Toxicity in C. elegans
Paige Rudich 1, Carley Snoznik 2, Noah Puleo 2, Todd Lamitina 1,2
1Graduate Program in Cell Biology and Molecular Physiology, University of Pittsburgh, 2Department of Pediatrics, University of Pittsburgh School of Medicine

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

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Immunology and Infection

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells
Tiao Li 1, Chen Long 1, Kristen V. Fanning 1, Chunbin Zou 1
1Acute Lung Injury Center of Excellence, Pulmonary, Allergy, Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

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Biology

The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells
Nilay Mitash 1,2, Joshua E. Donovan 1,3, Agnieszka Swiatecka-Urban 1,3
1Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, 2Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, 200 Lothrop Street, University of Pittsburgh, Pittsburgh, PA, 15261, 3Department of Pediatric Nephrology, University of Virginia, Charlottesville, VA, 22908

Here, we describe the Ago2-miRNA-co-IP assay designed to quantify an active pool of specific miRNAs induced by TGF-β1 in human bronchial epithelial CFBE41o- cells. This assay provides functional information on the recruitment of miRNA to the RNA induced silencing complex, quantified by qRT-PCR using specific miRNA primers and TaqMan hydrolysis probes.

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Biology

In vivo Evaluation of Mucociliary Clearance in Mice
Kyle S. Feldman 1, Maliha Zahid 1
1Department of Developmental Biology, University of Pittsburgh School of Medicine

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

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JoVE Core

A Modeling and Simulation Method for Preliminary Design of an Electro-Variable Displacement Pump
Xu Han 1, Peng Zhang 2, Tatiana Minav 3, Yongling Fu 1, Jian Fu 1
1School of Mechanical Engineering and Automation, Beihang University, 2Beijing Institute of Precision Mechatronics and Controls, 3Innovative Hydraulics and Automation, Tampere University

A simulation model specifically supporting the preliminary design of an electro-variable displacement pump (EVDP) is developed and partially verified by experiments. The control performance, life, reliability, etc., can all be evaluated using the proposed model, which covers the main performance requirements under the EVDP preliminary design task.

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Biology

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium
Lyndsay L. Leach 1, G. Burch Fisher 2, Jeffrey M. Gross 1,3
1Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, 2Earth Research Institute, University of California, Santa Barbara, 3Department of Developmental Biology, University of Pittsburgh School of Medicine

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

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Biology

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging
Yuewen Sheng 1, Kyle Morris 1, Julika Radecke *1, Peijun Zhang *1,2,3
1Electron Bio-Imaging Centre, Diamond Light Source Ltd, Harwell Science & Innovation Campus, 2Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, 3Chinese Academy of Medical Sciences Oxford Institute, University of Oxford

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 Å resolution.

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Bioengineering

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs
Meagan J. Makarcyzk 1,2, Zhong Alan Li 1,3, Ilhan Yu 1, Haruyo Yagi 1, Xiurui Zhang 1, Lauren Yocum 1, Eileen Li 1, Madalyn R. Fritch 1, Qi Gao 4, Bruce A. Bunnell 5, Stuart B. Goodman 4,6, Rocky S. Tuan 1,8, Peter G. Alexander 1,7, Hang Lin 1,2,7
1Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 2Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, 3Department of Neurobiology, University of Pittsburgh School of Medicine, 4Department of Orthopaedic Surgery, Stanford University, 5Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, 6Department of Bioengineering, Stanford University, 7McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 8The Chinese University of Hong Kong

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

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Medicine

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents
Stephanie L. Daugherty 1, Keara M. Healy 1, Jonathan M. Beckel 1
1Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

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Immunology and Infection

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major
Chaitanya S. Haram 1, Samrat Moitra 1, Rilee Keane 1, Elana Breslav 1, Kai Zhang 1, Peter A. Keyel 1
1Department of Biological Sciences, Texas Tech University

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

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Medicine

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells
Marcelo D. Carattino 1,2, Wily G. Ruiz 1, Gerard Apodaca 1,2
1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

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Biology

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction
Wily G. Ruiz 1, Dennis R. Clayton 1, Marianela G. Dalghi 1, Nicolas Montalbetti 1, Marcelo D. Carattino 1, Gerard Apodaca 1
1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine

Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products.

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Biology

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids
Erin Crawford *1, Heather L. Mentrup *2,3, Elizabeth A. Novak 2,3, Kevin P. Mollen 2,3
1Division of Pediatric Gastroenterology, Hepatology and Nutrition, UPMC Children’s Hospital of Pittsburgh, 2Division of Pediatric Surgery, UPMC Children’s Hospital of Pittsburgh, 3Department of Surgery, University of Pittsburgh School of Medicine

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

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Biology

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid
Alejandro Pizzoni 1, Nyla Naim 1,2, Xuefeng Zhang 1, Daniel L Altschuler 1
1Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, 2Addgene

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

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Neuroscience

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion
Jeremy Y. Gedeon 1,2,3, Jorge Baruch Pineda-Farias 2,3, Michael S. Gold 2,3
1Center for Neuroscience at the University of Pittsburgh, 2Department of Neurobiology, University of Pittsburgh School of Medicine, 3Pittsburgh Center for Pain Research, University of Pittsburgh

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

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Bioengineering

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform
Ting Zhang 1, Vinitha Dhamotharan 1, Yu Xiao 1,2, Sydne Ballengee 1,3, Jill Pollon 1,4, George K. Gittes 1
1Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, 2North Allegheny High School, 3Wright State University Boonshoft School of Medicine, 4College of Engineering, University of Michigan

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

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Developmental Biology

Methods to Enable Spatial Transcriptomics of Bone Tissues
Luigi Mancinelli 1,2, Karen Elizabeth Schoedel 3,4, Kurt Richard Weiss 5,6,7, Giuseppe Intini 1,2,8,9,10
1Department of Periodontics and Preventive Dentistry, University of Pittsburgh School of Dental Medicine, 2Center for Craniofacial Regeneration, University of Pittsburgh School of Dental Medicine, 3UPMC Presbyterian, 4UPMC Shadyside, 5Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, 6UPMC Hillman Cancer Center, 7Departments of Anatomic Pathology and General Surgical Oncology, University of Pittsburgh, 8Department of Medicine, University of Pittsburgh School of Medicine, 9University of Pittsburgh UPMC Hillman Cancer Center, 10McGowan Institute for Regenerative Medicine, University of Pittsburgh

Here, we describe a method that allows for the decalcification of freshly obtained bone tissues and the preservation of high-quality RNA. A method is also illustrated for sectioning Formalin Fixed Paraffin Embedded (FFPE) samples of non-demineralized bones to obtain good quality results if fresh tissues are not available or cannot be collected.

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