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Duke University School of Medicine

18 ARTICLES PUBLISHED IN JoVE

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Neuroscience

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia
Stephan J. Guyenet 1, Stephanie A. Furrer 2, Vincent M. Damian 1, Travis D. Baughan 2, Albert R. La Spada *3, Gwenn A. Garden *2
1Department of Biochemistry, University of Washington, 2Department of Neurology, University of Washington, 3Division of Genetics, Departments of Pediatrics and Cellular and Molecular Medicine, and the Institute for Genomic Medicine, University of California, San Diego - Rady Children’s Hospital

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

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Immunology and Infection

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum
Gregory LaMonte *1,2, Katelyn A. Walzer *1,2, Joshua Lacsina 3, Christopher Nicchitta 3, Jen-Tsan Chi 1,2
1Department of Molecular Genetics and Microbiology, Duke University School of Medicine, 2Center for Genomic and Computational Biology, Duke University School of Medicine, 3Department of Cell Biology, Duke University School of Medicine

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

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Genetics

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
Sriram Vijayraghavan 1, Boris Kantor 1
1Duke Viral Vector Core, Department of Neurobiology, Duke University School of Medicine

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

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Cancer Research

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation
Jayanth S. Shankara Narayanan 1, Partha Ray 1, Ibtehaj Naqvi 2, Rebekah White 1
1Moores Cancer Center, University California San Diego, 2Duke University School of Medicine

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

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Genetics

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation
Edward G Jones 1, Andrew P Landstrom 2
1Department of Pediatrics, Baylor College of Medicine, 2Department of Pediatrics, Division of Cardiology, Duke University School of Medicine

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant "hotspots" within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

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Genetics

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models
Lidia Tagliafierro 1,2, Ekaterina Ilich 3, Malik Moncalvo 3, Jeffrey Gu 1, Ahila Sriskanda 1,2, Carole Grenier 4, Susan K. Murphy 4, Ornit Chiba-Falek 1,2, Boris Kantor 3
1Department of Neurology, Duke University Medical Center, 2Center for Genomic and Computational Biology, Duke University Medical Center, 3Viral Vector Core, Duke University Medical Center, 4Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Duke University Medical Center

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

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Behavior

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease
Virginia Wertman 1,2, Anastasia Gromova 1,2,3, Albert R. La Spada 1,2,4,5, Constanza J. Cortes 1,2
1Department of Neurology, Duke University School of Medicine, 2Duke Center for Neurodegeneration & Neurotherapeutics, Duke University School of Medicine, 3Biomedical Sciences Graduate Program, University of California San Diego, 4Department of Neurobiology, Duke University School of Medicine, 5Department of Cell Biology, Duke University School of Medicine

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

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Bioengineering

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions
Morgan Burt 1, Rohan Bhattachaya 1, Arinze E. Okafor 1, Samira Musah 1,2
1Department of Biomedical Engineering, Duke University, 2Department of Medicine, Division of Nephrology, Duke University School of Medicine

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (>90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

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Biology

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures
Wendi Guo 1,2, Colleen Wu 1,2,3
1Department of Orthopedic Surgery, Duke University School of Medicine, 2Department of Pharmacology and Cancer Biology, Duke University School of Medicine, 3Department of Cell Biology, Duke University School of Medicine

A routine culture of bone marrow stromal cells (BMSCs) leads to the isolation of heterogeneous cell populations, with many cells being of hematopoietic origins. Here, we describe a method that utilizes low oxygen tension to greatly reduce hematopoietic contaminants in murine BMSC cultures.

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Bioengineering

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells
Yasmin Roye 1, Samira Musah 1,2,3,4
1Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 2Department of Medicine, Division of Nephrology, Duke University School of Medicine, 3Department of Cell Biology, Duke University, 4Center for Biomolecular and Tissue Engineering, Duke University

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

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Medicine

Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis
Rebecca G. Theophanous 1, Vinca W. Chow 2, David L. Convissar 3, Stephen C. Haskins 4,5, Robert A. Jones 6, Hari K. P. Kalagara 7, Yuriy S. Bronshteyn 8
1Department of Emergency Medicine, Duke University School of Medicine, 2Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, 3Department of Anesthesiology, Massachusetts General Hospital, 4Department of Anesthesiology, Critical Care and Pain Management, Hospital for Special Surgery, 5Department of Anesthesiology, Weill-Cornell Medical College, 6Department of Emergency Medicine, MetroHealth Medical Center/Case Western Reserve University, 7Department of Anesthesiology, Mayo Clinic, 8Department of Anesthesiology, Duke University School of Medicine

Traditionally, lower extremity deep venous thrombosis (DVT) is diagnosed by radiology-performed venous duplex ultrasound. Providers appropriately trained in focused point-of-care ultrasound (POCUS) can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. We describe the scanning technique for focused POCUS DVT lower extremity examination.

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JoVE Core

Point-of-Care Lung Ultrasound in Adults: Image Acquisition
Rodrigo O. L. Pereira 1, David L. Convissar 2, Sean Montgomery 3, James T. Herbert 1, Christopher R. Reed 3, Hoang J. Tang 3, Yuriy S. Bronshteyn 1,4
1Department of Anesthesiology, Duke University School of Medicine, 2Department of Anesthesiology, Massachusetts General Hospital, 3Department of Surgery, Duke University School of Medicine, Duke University Health System, 4Department of Anesthesiology, Durham VA

Point-of-care ultrasound (POCUS) of the lungs provides quick answers in rapidly changing clinical scenarios. We present an efficient and informative protocol for image acquisition for use in acute care settings.

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Medicine

Image Acquisition Method for the Sonographic Assessment of the Inferior Vena Cava
Maximilian Hoffman 1, David L. Convissar 2, Marie-Louise Meng 1, Sean Montgomery 4, Yuriy S. Bronshteyn 1,3
1Department of Anesthesiology, Duke University School of Medicine, 2Department of Anesthesiology, Massachusetts General Hospital, 3Department of Anesthesiology, Durham VA, 4Department of Surgery, Duke University School of Medicine

Point-of-care ultrasound evaluation of the inferior vena cava (IVC) is commonly utilized to identify, among other things, the volume status. Imaging should be performed systematically to ensure repeatability. This manuscript reviews the methods and pitfalls of sonographic IVC examination.

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Medicine

Optic Nerve Sheath Point of Care Ultrasound: Image Acquisition
Sean P. Montgomery 1, Brad Moore 2, Spencer M. Hampton 1, Gary Macy 3, Weizhe Li 4, Yuriy S. Bronshteyn 5
1Department of Surgery, Duke University School of Medicine, 2Medical Computing Group, Kitware Incorporated, 3Division of Neurocritical Care, Duke University School of Medicine, 4Department of Neurology, Duke University School of Medicine, 5Department of Anesthesiology, Duke University School of Medicine

Point-of-care ultrasound (POCUS) of the optic nerve sheath diameter (ONSD) has been shown to be useful in identifying patients with increased intracranial pressure (ICP). However, the non-standardized technique for this POCUS has hampered its use. We present a standardized image acquisition protocol for use in the acute care setting.

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Medicine

Focused Assessment with Sonography for Trauma (FAST) Exam: Image Acquisition
John D. Ritchie 1, Charles N. Trujillo 2, David L. Convissar 3, William Shihao Lao 4, Sean Montgomery 5, Yuriy S. Bronshteyn 6
1Trauma, Acute, and Critical Care Surgery, Duke University Hospital, 2Mount Sinai Medical Center, 3Department of Anesthesiology, Massachusetts General Hospital, 4Acute Care Surgery, Atrium Health, 5Department of Surgery, Duke University School of Medicine, 6Department of Anesthesiology, Duke University School of Medicine, Duke University Health System, Durham Veterans Health Administration

The Focused Assessment with Sonography for Trauma (FAST) exam is a diagnostic point-of-care ultrasound examination used to screen for the presence of free fluid in the pericardium and peritoneum. Indications, techniques, and pitfalls of the procedure are discussed in this article.

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Neuroscience

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke
Luca Menozzi *1, Ángela del Águila *2, Tri Vu 1, Chenshuo Ma 1, Wei Yang 2, Junjie Yao 1
1Department of Biomedical Engineering, Duke University, 2Multidisciplinary Brain Protection Program, Department of Anesthesiology, Duke University School of Medicine

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

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Medicine

Point-of-Care Kidney and Genitourinary Ultrasound in Adults: Image Acquisition
Michael Turk 1, Benjamin Catanese 1, Brice Lefler 2, Matthew A. Sparks 1, Yuriy S. Bronshteyn 3, Amber Bowman 2
1Division of Nephrology, Duke University School of Medicine, 2Division of Hospital Medicine, Durham VA Health Care System, Division of General Medicine, Duke University School of Medicine, 3Department of Anesthesiology, Duke University School of Medicine

Point-of-care ultrasound (POCUS) of the renal and genitourinary (renal-GU) system can help to screen for certain causes of kidney dysfunction. However, despite its clinical utility, renal-GU POCUS remains underutilized due to a lack of training among clinicians. To address this gap, this article describes renal-GU image acquisition and interpretation.

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Medicine

Point of Care Transcranial Color-Coded Duplex Ultrasound of the Middle Cerebral Artery
Fawaz Philip Tarzi 1, Sebina Bulic 1, Stacey Ho 1, Pravesh Saini 1, Andrei Alexandrov 2, Yuriy S. Bronshteyn 3
1Department of Neurology, University of Southern California, Keck school of Medicine, 2Department of Neurology, University of Arizona, College of Medicine, 3Department of Anesthesiology, Duke University School of Medicine

Transcranial ultrasound is an essential tool for monitoring patients with various neurological conditions. Although it is commonly used in a protocolized fashion in consultative studies, the brain has been overlooked in many protocols utilizing point-of-care ultrasound (PoCUS). This study proposes a PoCUS image acquisition protocol.

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