Indiana University School of Medicine

20 ARTICLES PUBLISHED IN JoVE

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Chemistry

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
Elijah N. McCool *1, Rachele Lubeckyj *1, Xiaojing Shen 1, Qiang Kou 2, Xiaowen Liu 2,3, Liangliang Sun 1
1Department of Chemistry, Michigan State University, 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 3Center for Computational Biology and Bioinformatics, Indiana University School of Medicine

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

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Biochemistry

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation
Ying Fan *1, Francesca Tonelli *1, Shalini Padmanabhan 2, Marco A.S. Baptista 2, Lindsey Riley 2, Danielle Smith 3, Connie Marras 4, Andrew Howden 5, Dario R. Alessi 1, Esther Sammler 1,6
1MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2The Michael J. Fox Foundation for Parkinson's Research, 3Department of Medical and Molecular Genetics, Indiana University School of Medicine, 4Morton and Gloria Shulman Movement Disorders Centre and the Edmond J. Safra Program in Parkinson's Disease, Toronto Western Hospital, University of Toronto, 5Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, 6Division of Molecular and Clinical Medicine, School of Medicine, Ninewells Hospital and Medical School, University of Dundee

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

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JoVE Core

Setup and Execution Of the Blindfolded Code Training Exercise
Patrick G. Hughes 1, Kate E. Hughes 2, Rami A. Ahmed 3
1Florida Atlantic University Schmidt College of Medicine, 2Department of Emergency Medicine, University of Arizona College of Medicine, 3Department of Emergency Medicine, Indiana University School of Medicine

The blindfolded code training exercise, which blindfolds the team leader in a code resuscitation simulation, is an advanced teaching technique to improve closed-loop communication, organizational skills, and critical thinking. 

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Biology

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis
Kaley L. Liang *1, Julia O. Bursova *1, Frank Lam 1, Xingjuan Chen 1, Alexander G. Obukhov 1
1The Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

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Biology

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue
Daria Barwinska 1, Michael J. Ferkowicz 1, Ying-Hua Cheng 1, Seth Winfree 1,2, Kenneth W. Dunn 1, Katherine J. Kelly 1, Timothy A. Sutton 1, Brad H. Rovin 3, Samir V. Parikh 3, Carrie L. Phillips 4, Pierre C. Dagher 1, Tarek M. El-Achkar 1, Michael T. Eadon 1,
1Department of Medicine, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 3Division of Nephrology, Department of Medicine, Ohio State University Wexner Medical Center, 4Division of Pathology, Indiana University School of Medicine

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

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Medicine

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization
Ziqian Zeng 1,2, Tanmaye Nallan Chakravarthula 1,2, Nathan J. Alves 1,2
1Emergency Medicine Department, Indiana University School of Medicine, 2Weldon School of Biomedical Engineering, Purdue University

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

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JoVE Core

Setup and Execution of the Rapid Cycle Deliberate Practice Death Notification Curriculum
Patrick G. Hughes 1, Kate E. Hughes 2, Mary J. Hughes 3,4, Lindsay Weaver 4, Lauren E. Falvo 4, Anna M. Bona 4, Dylan Cooper 4, Cherri Hobgood 4, Rami A. Ahmed 4
1Florida Atlantic University Schmidt College of Medicine, 2Department of Emergency Medicine, University of Arizona College of Medicine, 3Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, 4Indiana University School of Medicine

The goal is to demonstrate how to apply the rapid cycle deliberate practice debriefing technique to the GRIEV_ING death notification curriculum.

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Behavior

The Participant-Reported Implementation Update and Score (PRIUS): A Novel Method for Capturing Implementation-Related Data Over Time
Edward J. Miech 1,2,3,4,5, Nicholas A. Rattray 1,2,3,6, Dawn M. Bravata 1,2,3,5, Jennifer Myers 1,3, Teresa M. Damush 1,2,3,5
1VA Precision Monitoring (PRIS-M) QUERI, Richard L. Roudebush VA Medical Center, 2William M. Tierney Center for Health Services Research, Regenstrief Institute, 3VA Health Services Research & Development Center for Health Information and Communication (CHIC), Richard L. Roudebush VA Medical Center, 4Department of Emergency Medicine, Indiana University School of Medicine, 5Department of Internal Medicine and Geriatrics, Indiana University School of Medicine, 6Department of Anthropology, Indiana University-Purdue University Indianapolis

This protocol describes a novel method for collecting and analyzing data related to ongoing implementation called the Participant-Reported Implementation Update and Score (PRIUS). The PRIUS method allows for the efficient and systematic capture of data over time and from multiple viewpoints in healthcare settings.

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Medicine

A Murine Tail Lymphedema Model
Aladdin H. Hassanein *1, Mithun Sinha *1, Colby R. Neumann 1, Ganesh Mohan 1, Imran Khan 1, Chandan K. Sen 1
1Department of Surgery, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine

Lymphedema is extremity swelling caused by lymphatic dysfunction. We describe a chronic murine tail model of lymphedema and the novel use of tissue nanotransfection technology (TNT) for genetic cargo delivery to the tail.

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Biology

Artificial Intelligence Approaches to Assessing Primary Cilia
Ruchi Bansal 1, Staci E. Engle 1, Tisianna K. Kamba 1, Kathryn M. Brewer 1, Wesley R. Lewis 2, Nicolas F. Berbari 1,3,4
1Department of Biology, Indiana University-Purdue University Indianapolis, 2Nikon Instruments Inc., 3Stark Neurosciences Research Institute, Indiana University, 4Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

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Biology

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence
Cassandra Warden *1, Alan J. Simmons *2, Lejla Pasic 3, Ashley Pitzer 4,6, Sean S. Davies 4, Justin H. Layer 5, Raymond L. Mernaugh 3, Annet Kirabo 4,6
1Vanderbilt Eye Institute, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Department of Biochemistry, Vanderbilt University, 4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 5Division of Hematology and Oncology, Indiana University School of Medicine, 6Department of Molecular Physiology and Biophysics, Vanderbilt University

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

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Medicine

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes
Mark C, Wagner 1, Ruben M. Sandoval 1, Silvia B. Campos-Bilderback 1, Bruce A. Molitoris 1
1Department of Medicine, Division of Nephrology, Indiana University School of Medicine

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

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Behavior

The Motivation for Alcohol Reward: Predictors of Progressive-Ratio Intravenous Alcohol Self-Administration in Humans
Bethany L. Stangl 1, Nia D. Byrd 1, Soundarya Soundararajan 1, Martin H. Plawecki 2, Sean O’Connor 2,3, Vijay A. Ramchandani 1
1Human Psychopharmacology Laboratory, Division of Intramural Clinical and Biological Research, NIAAA, 2Dept. of Psychiatry, Indiana University School of Medicine, 3R.L. Roudebush VA Medical Center

This study aims to show that the Progressive-Ratio Computer-assisted Alcohol-Infusion System (CAIS) paradigm is a reliable and sensitive method that can be used to examine the motivating properties associated with alcohol self-administration in humans.

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Medicine

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit
Julian Dilley *1,2,3, Hessam Noori-Dokht *3,4,6, Abhijit Seetharam 1, Margaret Bello 1,3, Aaron Nanavaty 1,3, Roman M. Natoli 2,3, Todd McKinley 1,2,3, Zachary Bault 5, Diane Wagner 2,3,6, Uma Sankar 1,3
1Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, 2Department of Orthopaedic Surgery, Indiana University School of Medicine, 3Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, 4School of Mechanical Engineering, Purdue University, 5Large Animal Resource Center, Indiana University School of Medicine, 6Department of Mechanical and Energy Engineering, Indiana University-Purdue University Indianapolis

The open medial femoral condyle impact model in rabbits is reliable for studying post-traumatic osteoarthritis (PTOA) and novel therapeutic strategies to mitigate PTOA progression. This protocol generates an isolated cartilage defect of the posterior medial femoral condyle in rabbits using a carriage-based drop tower with an impactor head.

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Genetics

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting
Kyungchan Kim 1, Solaema Taleb 1, Jisun So 1, Jamie Wann 1, Hyun Cheol Roh 1
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

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Immunology and Infection

Swine Model of Biofilm Infection and Invisible Wounds
Mohamed El Masry 1, Pramod Bhasme 1, Shomita S. Mathew-Steiner 1, Jessica Smith 1, Thomas Smeenge 1, Sashwati Roy 1, Chandan K. Sen 1
1Indiana Center for Regenerative Medicine & Engineering, Indiana University Health Comprehensive Wound Center, Department of Surgery, Indiana University School of Medicine

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

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Bioengineering

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model
Alexei Christodoulides 1, Abigail R. Hall 1, Anushri Umesh 1,2, Nathan J. Alves 1,2
1Department of Emergency Medicine, Indiana University School of Medicine, 2Weldon School of Biomedical Engineering, Purdue University

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

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Biochemistry

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry
Tripthi Battapadi 1, Madhumita Sridharan 1, Degang Liu 2, John Turchi 3, Lata Balakrishnan 1
1Department of Biology, Indiana University Indianapolis, 2Sartorius Corporation, 3Department of Medicine, Indiana University School of Medicine

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

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Biology

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors
Laura C. Brown 1, Jay Chopra 2, Rachel E. Horness 3, Julia C. van Kessel 4
1Department of Chemistry, Indiana University, 2Indiana University School of Medicine, 3Department of Chemistry, Carleton College, 4Department of Biology, Indiana University

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

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Neuroscience

Optimizing Mouse Urodynamic Techniques for Improved Accuracy
Aytak Khabbaz 1,2, Kristen Lynn Cohen 1,2, Shengqi Zhang 1,2,3, Samhita Chakraborty 1,2, Yihong Zhang 1,2, Lingxiao Deng 1,2
1Department of Neurological Surgery, Indiana University School of Medicine, 2Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery, Goodman and Campbell Brain and Spine, Indiana University School of Medicine, 3Department of Rehabilitation Medicine, Zhongda Hospital Southeast University

This protocol provides a guide for waterproofing the skin with cyanoacrylate to prevent urine absorption by fur and skin. It includes instructions for applying the glue to the skin, implanting a bladder catheter, and electrodes for cystometry and external urethral sphincter electromyography recordings in awake mice.

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