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31 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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring
Michael Boska 1, Yutong Liu 1, Mariano Uberti 1, Balarininvasa R. Sajja 1, Shantanu Balkundi 2, JoEllyn McMillan 2, Howard E. Gendelman 2
1Department of Radiology, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

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Biology

A Protocol for Computer-Based Protein Structure and Function Prediction
Ambrish Roy 1,2, Dong Xu 1, Jonathan Poisson 1, Yang Zhang 1,2
1Center for Computational Medicine and Bioinformatics, University of Michigan , 2Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

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Environment

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses
Hui Jiang 1, Hugues Barbier 2, Thomas Brutnell 1
1Donald Danforth Plant Science Center, 2Boyce Thompson Institute

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

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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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Biology

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Yohei Niikura 1, Katsumi Kitagawa 1
1Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

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Chemistry

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering
Youngkyu Han 1, Suk-kyun Ahn 2,3, Zhe Zhang 1,4, Gregory S. Smith 1, Changwoo Do 1
1Biology and Soft Matter Division, Neutron Science Directorate, Oak Ridge National Laboratory, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Polymer Science and Engineering, Pusan National University, 4Jülich Center for Neutron Science, Forschungszentrum Jülich

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

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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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Fabrication of 109° Periodic Domain Walls with a Bottom Electrode in BiFeO3 Thin Films
Deyang Chen *1, Chao Chen *1, Peilian Li 1, Zhen Fan 1, Xingsen Gao 1, Jun-Ming Liu 1,2
1Institute for Advanced Materials, South China Academy of Advanced Optoelectronics, South China Normal University, 2Laboratory of Solid State Microstructures and Innovation Center of Advanced Microstructures, Nanjing University

109° periodic domain walls can successfully be fabricated by introducing a dielectric La-BiFeO3 layer between a bottom electrode and a ferroelectric BiFeO3 layer, enabling the study of the switching behavior of a 109° domain structure and the investigation of the electric field control of exchange bias in a ferromagnet/BiFeO3 system.

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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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Biology

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents
Feng Pan *2, Jie Zhang *3, Yuyan Liu 1, Liangsheng Lu 1, Xuefeng Qiu 4, Kangtai Lv 5, Qipeng Zhang 1
1State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, Nanjing University, 2Department of Andrology, The Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, 3School of Pharmaceutical Sciences, Nanjing Tech University, 4Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, 5Department of Ultrasound, Nanjing Qixia District Maternity and Child Health Care Hospital

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

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Biology

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice
Daniel Villarreal 1, Geetali Pradhan 2, Chia-Shan Wu 1,2, Clinton D Allred 1, Shaodong Guo 1, Yuxiang Sun 1,2
1Department of Nutrition and Food Science, Texas A&M University, 2Children's Nutrition Research Center, Baylor College of Medicine

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse.

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JoVE Core

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection
Yang Zhang *1, Lifeng Shen *2, Ping'er Wang 3, Weixing Xi 4, Zhongming Yu 1, Xiaowen Huang 1, Xuping Wang 1, Dan Shou 1
1Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, 2Department of Orthopaedic Surgery, Tongde Hospital of Zhejiang Province, 3Institute of Orthopaedics and Traumatology, First Affiliated Hospital of Zhejiang Chinese Medical University, 4Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

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Immunology and Infection

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals
Jingshu Chen *2, Lei Zhong *1, Jing Wu 1, Sui Ke 2, Benjamin Morpurgo 3, Andrei Golovko 3, Nengtai Ouyang 4, Yuxiang Sun 2, Shaodong Guo 2, Yanan Tian 1,2
1Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, Hunan Agriculture University, 2Texas A&M University, 3Texas A&M Institute for Genomic Medicine, 4Sun Yat-Sen Memorial Hospital

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

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Engineering

A Soft Tooling Process Chain for Injection Molding of a 3D Component with Micro Pillars
Yang Zhang 1, David Bue Pedersen 1, Michael Mischkot 1, Matteo Calaon 1, Federico Baruffi 1, Guido Tosello 1
1Department of Mechanical Engineering, Technical University of Denmark

A protocol for fabricating injection molding inserts for complex geometry with micro features on surfaces employing Additive Manufacturing (AM) is presented.

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Medicine

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis
Ziming Chen *1,2, Zhantao Deng *1, Yuanchen Ma 1, Junxing Liao 1, Qingtian Li 1, Mengyuan Li 1, Hua Liu 1, Ganghong Chen 1, Chaoming Zeng 1, Qiujian Zheng 1
1Department of Orthopedics, Guangdong General Hospital (Guangdong Academy of Medical Sciences), 2Shantou University Medical College

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

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Medicine

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography
Jie Sun 1, Guang Ming Yang 2, Tao Tao 1, Li Sha Wei 1, Yang Pan 2, Min Sheng Zhu 1
1Model Animal Research Center and MOE Key Laboratory of Model Animal and Disease Study, Nanjing University, 2School of Pharmacy, Nanjing University of Chinese Medicine

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

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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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Chemistry

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules
Xin Guo 1, Xue-Mei Wang 1, Shou-Jun Xiao 1
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

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Biology

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow
Lining Wang 1,2, Suyang Zheng 1,2, Yang Guo 1,2, Yalan Pan 1,2, Jie Sun 1,2, Weimin Xu 1,2, Jinlan Lu 3, Weidong Li 3, Yong Ma 1,2,4
1Laboratory of New Techniques of Restoration & Reconstruction, Institute of Traumatology & Orthopedics, Nanjing University of Chinese Medicine, 2TCM Nursing Intervention Laboratory of Chronic Disease Key Laboratory, Nanjing University of Chinese Medicine, 3Jiangsu Key Laboratory of Chinese Medicine Processing, Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, 4Department of Traumatology and Orthopedics, Affiliated Hospital of Nanjing University of Chinese Medicine

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

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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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Biology

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut
Qiuping Zhang 1, Qian Lu 2, Yubin Luo 1
1Department of Rheumatology and Immunology, West China Hospital, Sichuan University, 2Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

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Biochemistry

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
Yibing Deng 1, Bin Zheng 1, Yutong Liu 1, Shengchao Shi 1, Jingyuan Nie 1, Tao Wu 1, Peng Zheng 1
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

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JoVE Core

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)
Yohei Niikura *1, Lei Fang *2, Risa Kitagawa *3, Peizhao Li 1, Yao Xi 1, Ju You 1, Yan Guo 2, Katsumi Kitagawa 3
1MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, 2Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, 3Greehey Children's Cancer Institute, Department of Molecular Medicine, UT Health Science Center San Antonio

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

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Cancer Research

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate
Shun Ishiyama 1,2,3,4, Casey Kissel 5, Xin Guo 1, Alexis Howard 6, Harumi Saeki 7, Tomoaki Ito 8, Polina Sysa-Shah 9, Hajime Orita 10, Kazuhiro Sakamoto 4, Kathleen Gabrielson 1,2
1Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 3Department of Surgery, Johns Hopkins University School of Medicine, 4Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 5Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, 6Tuskegee College of Veterinary Medicine, 7Department of Human Pathology, Juntendo University Faculty of Medicine, 8Department of Surgery, Juntendo University Shizuoka Hospital, Juntendo University School of Medicine, 9Department of Urology, The Johns Hopkins School of Medicine, 10Department of Gastroenterology and Minimally Invasive Surgery, Juntendo University Faculty of Medicine

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

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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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Neuroscience

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)
Nannan Zhang 1, Zhe Zhang 1,2, Ilker Ozden 2, Shinghua Ding 1,2
1Dalton Cardiovascular Research Center, University of Missouri-Columbia, 2Department of Biomedical, Biological and Chemical Engineering, University of Missouri-Columbia

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

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Medicine

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer
Chenhao Hu *1,2,3, Zhe Zhang *1,2,3, Lei Zhang 1,2,3, Ruihan Liu 2,3, Jun Yan 1,2,3, Qi Sun 1,2,3, Guanghui Wang 1, Junjun She 1,2,3
1Department of General Surgery, The First Affiliated Hospital of Xian Jiaotong University, 2Center for Gut Microbiome Research, Med-X Institute, The First Affiliated Hospital of Xian Jiao tong University, 3Department of High Talent, The First Affiliated Hospital of Xian Jiaotong University

The robotic technique described herein aims to detail a stepwise approach to robot-assisted total mesorectal excision and lateral pelvic lymph node dissection for locally advanced (T3/T4) rectal cancer located below the peritoneal reflection.

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Biology

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance
Yutong Liu 1, Mingming Sun 1, Zipeng Jiang 1, Xinfang Wang 2, Bingguang Xiao 3, Aiguo Yang 1, He Meng 1, Lirui Cheng 1
1Key Laboratory of Tobacco Genetic Improvement and Biotechnology, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 2Institute of Rural Development, Zhejiang Academy of Agricultural Sciences, 3Yunnan Academy of Tobacco Agricultural Sciences

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

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Developmental Biology

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research
Yang Zhang *1,2,3, Zhe Zhang *1,2,3, Nannan Kang 1,2,3, Xiaoqiang Sheng 1,2,3
1Center for Reproductive Medicine and Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 2State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 3Center for Molecular Reproductive Medicine, Nanjing University

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

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