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University of Arkansas for Medical Sciences

23 ARTICLES PUBLISHED IN JoVE

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Biology

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity
Lauren P. Blair 1, Nathan L. Avaritt 1, Alan J. Tackett 1
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

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Medicine

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain
Karl A. Sillay 1,2, S. Gray McClatchy 3, Brandon A. Shepherd 1, Garrett T. Venable 1, Tyler S. Fuehrer 4
1University of Tennessee Health Science Center, 2Semmes-Murphey Clinic, 3University of Arkansas for Medical Sciences, 4Restorative Neurosciences Foundation

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

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Neuroscience

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons
Francisco J. Urbano 1, Brennon R. Luster 2, Stasia D'Onofrio 2, Susan Mahaffey 2, Edgar Garcia-Rill 2
1IFIBYNE-CONICET, University of Buenos Aires, 2Center for Translational Neuroscience, University of Arkansas for Medical Sciences

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

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Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System
Isabelle R. Miousse 1, Igor Koturbash 1, Marie-Cécile Chalbot 2, Martin Hauer-Jensen 3, Ilias Kavouras 2, Rupak Pathak 3
1Department of Occupational and Environmental Health, University of Arkansas for Medical Sciences, 2Department of Environmental Health Sciences, University of Alabama at Birmingham, 3Division of Radiation Health, University of Arkansas for Medical Sciences

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

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Genetics

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice
Rupak Pathak 1, Igor Koturbash 2, Martin Hauer-Jensen 1,3
1Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, 2Department of Environmental Health, Fay W. Boozman School of Public Health, University of Arkansas for Medical Sciences, 3Surgical Service, Central Arkansas Veterans Healthcare System

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

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Genetics

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
Muhammad Khairul Ramlee 1, Jing Wang 1, Alice M. S. Cheung 1, Shang Li 1
1Cancer & Stem Cell Biology Programme, Duke-NUS Medical School

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

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Chemistry

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro
Rafaela G. Feresin 1, Shirin Pourafshar 2,3, Jingwen Huang 2, Yitong Zhao 2, Bahram H. Arjmandi 2,3, Gloria Salazar 2,3
1Department of Dietetics and Nutrition, University of Arkansas for Medical Sciences, 2Department of Nutrition, Food and Exercise Sciences, Florida State University, 3Center for Advancing Exercise and Nutrition Research on Aging (CAENRA), Florida State University

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

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Neuroscience

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method
Thomas R. Groves 1,2,3, Jing Wang 1,2, Marjan Boerma 1,2, Antiño R. Allen 1,2,3
1Division of Radiation Health, University of Arkansas for Medical Sciences, 2Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 3Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

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Biology

Clock Scan Protocol for Image Analysis: ImageJ Plugins
Maxim Dobretsov 1, Georg Petkau 2, Abdallah Hayar 3, Eugen Petkau 1
1Department of Anesthesiology, University of Arkansas for Medical Sciences, 2Department of Lymphocyte Development, Max Plank Institute for Infection Biology, 3Department of Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences

This paper describes two novel ImageJ plugins for 'Clock Scan' image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

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Cancer Research

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation
Magdalena Delgado *1, Anisha Kothari *1, Walter N. Hittelman 2, Timothy C. Chambers 1
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 2Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

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Neuroscience

Intracranial Pharmacotherapy and Pain Assays in Rodents
Erik Martinez 1, Haocheng Zhou 1, Jing Wang 1,2
1Department of Anesthesiology, Perioperative Care and Pain Medicine, New York University School of Medicine, 2Department of Neuroscience and Physiology, New York University School of Medicine

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

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Biology

Quantification of Proliferative and Dead Cells in Enteroids
Hua-Shan Li *1, Shao-Fang Xu *1, Jian-Ying Sheng *1, Zhi-Hui Jiang 1, Jing Wang 1, Ning Ding 1, Tao Wang 1, Matthew A. Odenwald 2, Jerrold R. Turner 2,3, Wei-Qi He 1, Hong Xu 1, Juan-Min Zha 1
1Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genomic Resource Center, Medical College of Soochow University, Department of Oncology, The First Affiliated Hospital of Soochow University, 2Department of Pathology, University of Chicago, 3Department of Pathology, Brigham and Women's Hospital–Harvard Medical School

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

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Biochemistry

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein
Alev Tascioglu Aliyev 1,2, Francesca LoBianco 1, Kimberly J. Krager 1, Nukhet Aykin-Burns 1
1Division of Radiation Health, Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 2Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

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Cancer Research

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from Sézary Syndrome Patients for Transcriptomic Profiling
Syed J. Mehdi 1, Andrea M. Moerman-Herzog 1, Henry K. Wong 1
1Department of Dermatology, University of Arkansas for Medical Sciences

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with Sézary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

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Neuroscience

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue
Madison Trujillo 1,2,3, Taylor McElroy 1,2,3, Taurean Brown 1,2,3, Pilar Simmons 1,2, Fabio Ntagwabira 1,2,3, Antiño R. Allen 1,2,3
1Division of Radiation Health, University of Arkansas for Medical Sciences, 2Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 3Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

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Medicine

Bridging the Technology Divide in the COVID-19 Era: Using Virtual Outreach to Expose Middle and High School Students to Imaging Technology
Kevin D. Phelan 1, Mohsin Syed 1, Noor Akhter 1, Tiffany W. Huitt 1, Gregory R. Snead 2, Billy R. Thomas 3, Karen L. Yanowitz 4
1Division of Clinical Anatomy, Department of Neurobiology and Developmental Sciences, College of Medicine, University of Arkansas for Medical Sciences, 2Department of Emergency Medicine, College of Medicine, University of Arkansas for Medical Sciences, 3Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, 4Department of Psychology and Counseling, Arkansas State University

This article presents an overview of how synchronous web-based virtual outreach can be used to expose 6th-12th grade students to advanced imaging technologies such as ultrasound, computerized tomography, and electroencephalography. The paper discusses the methods and equipment needed to livestream integrated educational sessions for effective student engagement in STEM.

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Medicine

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis
Smita Joshi *1, Alexis N. Smith 1, Kanakanagavalli Shravani Prakhya 1, Hammodah R. Alfar 1, Joshua Lykins 1, Ming Zhang 1, Irina Pokrovskaya 2, Maria Aronova 3, Richard D. Leapman 3, Brian Storrie 2, Sidney W. Whiteheart *1
1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, 3Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

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Cutting-Edge Technologies Driving Quantitative Mass Spectrometry
Nathan L. Avaritt 1, Stephanie D. Byrum 1
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences

Cutting-Edge Technologies Driving Quantitative Mass Spectrometry

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Neuroscience

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior
Nicole G. Pastorino 1, Saori Tomatsu 1, Amy Lin 1, Jackson Doerr 1, Zachary Waterman 1, Krisztina Sershen 1, Pulak Ray 2, Analiz Rodriguez 3, Deni S. Galileo 1
1Department of Biological Sciences, University of Delaware, 2Helen F. Graham Cancer Center and Research Institute, Christiana Care, 3Department of Neurosurgery, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

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Biology

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells
Qin He 1, Huiliang Zhang 2, Xingyun Qi 1
1Department of Biology, Rutgers University, 2Pharmacology and Toxicology Department, University of Arkansas for Medical Sciences

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

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Medicine

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis
Kelly Ball 1, Irina Pokrovskaya 1, Brian Storrie 1
1Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

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Immunology and Infection

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels
Soumiya Pal 1, Ashim K. Bagchi 1, Amanda J. Stolarz 1
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

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Rapid Formation and Testing of Self-expanding NiTi Frames with a Small Form Factor Suitable for Minimally Invasive Implants
Seyedhamidreza Alaie 1, Jesus Eduardo Mata De la O 2, Subhi J. Al'Aref 3
1Department of Mechanical and Aerospace Engineering, New Mexico State University, 2Department of Mechanical and Aerospace Engineering, New Mexico State University, 3Department of Internal Medicine - Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences

This work illustrates a low-cost fabrication technique for shape-setting nitinol wires/frames with a small form factor using sacrificial fixtures. The technique is demonstrated for the fabrication of self-expanding frames designed for minimally invasive implants with complex shapes.

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