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Perelman School of Medicine at the University of Pennsylvania

9 ARTICLES PUBLISHED IN JoVE

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Biology

Purification of Hsp104, a Protein Disaggregase
Elizabeth A. Sweeny 1, Morgan E. DeSantis 1, James Shorter 1
1Department of Biochemistry and Biophysics, University of Pennsylvania

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

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Neuroscience

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury
Colin J. Smith 1,2, Brian N. Johnson 1, Jaclynn A. Elkind 1, Jill M. See 1, Guoxiang Xiong 1, Akiva S. Cohen 1,3
1Division of Neurology, Children's Hospital of Philadelphia, 2Neuroscience Graduate Group, Perelman School of Medicine at the University of Pennsylvania, 3Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

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Biology

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
Meredith E. Jackrel 1, Amber Tariq 1, Keolamau Yee 1, Rachel Weitzman 1, James Shorter 1
1Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

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Medicine

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy
Xuehai Chen 1, Rong Yu 2, Ziqiang Xu 3, Yan Zhang  3, Chengyang Liu 4, Bicheng Chen *1, Hao Jin *3
1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, 2Reproductive Center, The First Affiliated Hospital of Wenzhou Medical University, 3Department of Transplantation, The First Affiliated Hospital of Wenzhou Medical University, 4Department of Surgery, Perelman School of Medicine at the University of Pennsylvania

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

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Medicine

Taste Exam: A Brief and Validated Test
Jennifer E. Douglas 1,2, Corrine J. Mansfield 2, Charles J. Arayata 2, Beverly J. Cowart 2, Lauren R. Colquitt 2, Ivy W. Maina 1,2, Mariel T. Blasetti 3, Noam A. Cohen 3, Danielle R. Reed 2
1Perelman School of Medicine at the University of Pennsylvania, 2Monell Chemical Senses Center, 3Department of Otorhinolaryngology-Head and Neck Surgery, Division of Rhinology, Hospital of the University of Pennsylvania

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject's reported sensation and taste receptor genotype.

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Cancer Research

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy
Saba Ghassemi 1, Michael C. Milone 1
1Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

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Neuroscience

Maintenance of a Lateral Fluid Percussion Injury Device
Anthony M. Farrugia 2, Samuelle A. S. Delcy 2,3, Brian N. Johnson 1, Akiva S. Cohen 1,2
1Division of Neurology, Children’s Hospital of Philadelphia, 2Department of Anesthesiology, Perelman School of Medicine at the University of Pennsylvania, 3Pharmacology Graduate Group, Perelman School of Medicine at the University of Pennsylvania

Proper care and maintenance are essential for a lateral fluid percussion injury (LFPI) device to function reliably. Here, we demonstrate how to properly clean, fill, and assemble an LFPI device, and ensure that it is adequately maintained for optimal results.

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Medicine

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor
Rene Machietto 1, Nicholas Giacobbe 1, Jessica Perazzelli 2, Ted J. Hofmann 2, Allison Barz Leahy 2,3, Stephan A. Grupp 1,2,3, Yongping Wang 1,3,4, Stephan Kadauke 1,3,4
1Cell and Gene Therapy Laboratory, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, 2Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, 3Perelman School of Medicine at the University of Pennsylvania, 4Division of Transfusion Medicine, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

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Cancer Research

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines
Kyra Harvey 1,2,3, Katherine Labella 1,2,3, Angela Liou 1,2,3, Stephanie Brosius 1,2,3, Thomas De Raedt 1,2,3
1Division of Oncology, Children’s Hospital of Philadelphia, 2Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, 3School of Medicine, University of Pennsylvania

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

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