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20 ARTICLES PUBLISHED IN JoVE

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Medicine

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)
Yana Yunusova 1,2, Jordan R. Green 3, Jun Wang 3, Gary Pattee 4, Lorne Zinman 2,5
1Department of Speech-Language Pathology, University of Toronto, 2ALS/ MN Clinic, Sunnybrook Health Science Centre, 3Department of Special Education and Communication Disorders, University of Nebraska-Lincoln, 4Department of Neurology, Munroe-Meyer Institute, University of Nebraska Medical Center, 5Department of Neurology, University of Toronto

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

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Bioengineering

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3
Jialie Luo 1, Yingmin Zhu 1, Michael X. Zhu 1, Hongzhen Hu 1
1Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

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Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Shenglan Li *1,2, Haipeng Xue *1,2, Bo Long 1,2,5, Li Sun 1,2,6, Tai Truong 1,2,4,7, Ying Liu 1,2,3
1Department of Neurosurgery, The University of Texas Health Science Center at Houston, 2Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 3The Senator Lloyd & B. A. Bentsen Center for Stroke Research, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 4Summer Research Program, Office of Educational Programs, The University of Texas Health Science Center at Houston, 5Department of Anesthesiology, Shengjing Hospital, China Medical University, 6Department of Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 7Biology Department, University of West Georgia

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

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Bioengineering

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography
Dustin R. Morado 1, Bo Hu 1, Jun Liu 1
1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

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Chemistry

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray
Lisa S. Ramirez 1,2, Jun Wang 1,2
1Department of Chemistry, University at Albany, State University of New York, 2Multiplex Biotechnology Laboratory, Cancer Research Center

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

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Neuroscience

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains
Antonio J. Tito 1, Shebna Cheema 2, Mian Jiang 2, Sheng Zhang 1,3
1Center for Metabolic and Degenerative Diseases, The Brown Foundation Institute of Molecular Medicine, Programs in Human and Molecular Genetics and Neuroscience, The Graduate School of Biomedical Sciences at Houston, The University of Texas Health Science Center at Houston, 2Department of Natural Sciences, University of Houston - Downtown, 3Department of Neurobiology and Anatomy, McGovern Medical School, The University of Texas Health Science Center at Houston

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

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Medicine

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening
Hui Jiang *1, Yicun Wang *1, Zhantao Deng *1,2,3, Jiewen Jin 2,3, Jia Meng 1, Shuo Chen 1, Jun Wang 1, Yang Qiu 1, Ting Guo 1, Jianning Zhao 1
1Department of Orthopedics, Jinling Hospital, School of Medicine, Nanjing University, 2Center for Translational Medicine, Nanjing University Medical School, 3Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

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Developmental Biology

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Qinggang Dai *1, Yujiao Han *2, Furong Xie *1, Xuhui Ma 3, Zhan Xu 2, Xiao Liu 1, Weiguo Zou 2, Jun Wang 1
1Department of Pediatric Dentistry, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, National Clinical Research Center of Stomatology, 2State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, 3Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

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Chemistry

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control
Xinyi Ding 1,2,3, Yuxiang Sun 1,2,3, Yanming Chen 1,2,3, Wanchuan Ding 1,2,3, Steven Emory 1,2,3,4, Tianhao Li 1,2,3, Zixing Xu 1,2,3, Ning Han 1,2,3, Jun Wang 1,2,3, Gang Ruan 1,2,3
1Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, 2Institute of Materials Engineering, College of Engineering and Applied Sciences, Nanjing University, 3Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing University, 4Department of Chemistry, Western Washington University

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

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Cancer Research

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells
Ruoji Zhou 1,2, An Xu 1, Jian Tu 1,3, Mo Liu 1, Julian A Gingold 4, Ruiying Zhao 1, Dung-Fang Lee 1,2,5,6
1Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, 2Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center UTHealth, 3Department of Musculoskeletal Oncology, The First Affiliated Hospital of Sun Yat-sen University, 4Women's Health Institute, Cleveland Clinic Foundation, 5Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, 6Center for Precision Health, School of Biomedical Informatics and School of Public Health, The University of Texas Health Science Center at Houston

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

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Genetics

Culturing and Manipulation of O9-1 Neural Crest Cells
Bao H. Nguyen 1, Mamoru Ishii 2, Robert E. Maxson 2, Jun Wang 1
1Molecular Physiology and Biophysics, Baylor College of Medicine, 2Department of Biochemistry and Molecular Biology, University of Southern California

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

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Biology

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis
Jeffrey R. Millman 1,2, Teresa Doggett 3, Christina Thebeau 3, Sheng Zhang 3, Clay F. Semenkovich 1, Rithwick Rajagopal 3
1Division of Metabolism, Endocrinology and Lipid Research, Department of Medicine, Washington University School of Medicine, 2Department of Biomedical Engineering, Washington University in Saint Louis, 3Department of Ophthalmology and Visual Science, Washington University School of Medicine

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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Behavior

Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants
Jiazhi Chen *1,2, Xinyu Chen *3, Siyuan Lv 2, Yuzhen Zhang 1, Hao Long 1, Kaijun Yang 1, Songtao Qi 1, Wangming Zhang 2, Jun Wang 1
1Department of Neurosurgery, Nanfang Hospital, Southern Medical University, 2Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, 3Faculty of Brain Sciences, University of College London

Here, we present a protocol to demonstrate 3D printing in the construction of deep brain stimulation implants.

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Immunology and Infection

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System
David Chetrit 1, Donghyun Park 1,2, Bo Hu 3, Jun Liu 1,2, Craig R. Roy 1
1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

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JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
Jing Liu 1,2, Dalibor Kurepa 3, Francesco Feletti 4,5, Almudena Alonso-Ojembarrena 6, Jovan Lovrenski 7, Roberto Copetti 8, Erich Sorantin 9, Javier Rodriguez-Fanjul 10, Karishma Katti 3, Andrea Aliverti 4, Huayan Zhang 11,12, Misun Hwang 13, Tsu F. Yeh 14, Cai-Bao Hu 15, Xing Feng 16, Ru-Xin Qiu 1,2, Jing-Han Chi 17, Li-Li Shang 18, Guo-Rong Lyu 19, Shao-Zheng He 20, Yan-Fen Chai 21, Zhan-Jun Qiu 22, Hai-Ying Cao 2,23, Yue-Qiao Gao 1,2, Xiao-Ling Ren 1,2, Guo Guo 1,24, Li Zhang 1,2, Ying Liu 1,2, Wei Fu 1,2, Zu-Lin Lu 1,2, Hong-Lei Li 1,2
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, 5Dipartimento di Diagnostica per Immagini, Ausl della Romagna, S. Maria delle Croci Hospital, 6Neonatal Intensive Care Unit, Puerta del Mar University Hospital, 7Faculty of Medicine, University of Novi Sad, Serbia, Institute for Children and Adolescents Health Care of Vojvodina, 8Emergency Department, University Hospital of Cattinara, 9Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 10Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 11Center for Newborn Care, Guangzhou Women and Children's Medical Center, 12Division of Neonatology, Children's Hospital of Philadelphia, 13Section of Neonatal Imaging, Department of Radiology, Children's Hospital of Philadelphia, 14Maternal Child Health Research institute, Taipei Medical University and China Medical University, 15Intensive Care Unit, Zhejiang Hospital, 16Department of Neonatology, Children's Hospital of Soochow University, 17Department of Neonatology and NICU, Bayi Children's Hospital Affiliated to the Seventh Medical Center of Chinese PLA General Hospital, 18Intensive Care Unit, Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Second Affiliated Hospital of Fujian Medical University, 21Department of Emergency Medicine, Tianjin Medical University General Hospital, 22Department of Emergency and Critical Care Medicine, Affiliated Hospital of Traditional Chinese Medicine, 23Department of Ultrasound, GE Healthcare, 24The Neonatal Intensive Care Unit, Fifth Medical Center of Chinese PLA General Hospital

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

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Bioengineering

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures
Sriram Kumar 1, Ying Liu 1, Rebecca E. Taylor 1,2,3
1Department of Mechanical Engineering, Carnegie Mellon University, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Electrical and Computer Engineering, Carnegie Mellon University

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

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Developmental Biology

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells
Tram P. Le *1, Xiaolei Zhao *1, Shannon Erhardt 1,2, Jianhua Gu 3, Huie Wang 3, Tina O. Findley 1, Jun Wang 1,2
1Department of Pediatrics, McGovern Medical School, The University of Texas Health Science Center at Houston, 2The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, 3SEM AFM Core in the Houston Methodist Hospital Research Institute

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

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Genetics

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform
Chengming Xu *1, Riqing Wei *2, Hui Lin 1, Leiyu Deng 1, Li Wang 3, Deyang Li 4, Honghui Den 5, Wensong Qin 1, Ping Wen 1, Ying Liu 1, Yingsong Wu 2, Qiang Ma 2, Jinliang Duan 1
1Centre for Women, Children, and Reproduction, Guangxi Key Laboratory of Metabolic Diseases Research, 2Department of Biopharmaceutics, School of Laboratory Medicine and Biotechnology, Southern Medical University, 3Department of Obstetrics and Gynecology, Chinese PLA General Hospital, 4Clinical Laboratory, Northern Theater Air Force Hospital, 5Guangzhou Darui Reproduction Technology Co., Ltd.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

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Developmental Biology

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte
Laura Adams *1, Ying Liu *1, Barbara Durrant 2, Elena Ruggeri 2, Carly Young 2, Rebecca Krisher 3, Irina Polejaeva 1
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University, 2Reproductive Sciences, Beckman Center for Conservation Research, San Diego Zoo Wildlife Alliance, 3Genus PLC

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P < 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

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