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21 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Sittiporn Pattaradilokrat 1, Jian Li 1,2, Xin-zhuan Su 1
1National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2School of Life Science, Xiamen University

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

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Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
Ronald W. Jensen 1, Jason Rivest 1, Wei Li 1, Varalakshmi Vissa 1
1Department of Microbiology, Immunology and Pathology, Colorado State University

Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.

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Bioengineering

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma
Jian Li 1, Alexey Shashurin 1, Madhusudhan Kundrapu 1, Michael Keidar 1
1Department of Mechanical and Aerospace Engineering, The George Washington University

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies
Wei Li 1, Shu Zhu 1, Yusong Zhang 1, Jianhua Li 1, Andrew E. Sama 1, Ping Wang 1, Haichao Wang 1
1The Feinstein Institute for Medical Research, North Shore – LIJ Health System

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

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Engineering

Characterization of Full Set Material Constants and Their Temperature Dependence for Piezoelectric Materials Using Resonant Ultrasound Spectroscopy
Liguo Tang 1, Wenwu Cao 2
1Key Laboratory of Underwater Acoustic Communication and Marine Information Technology, Xiamen University, 2Department of Mathematics and Material Research Institute, The Pennsylvania State University

This protocol describes the procedure of measuring the temperature dependence of the full set material constants of piezoelectric materials using resonant ultrasound spectroscopy (RUS).

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JoVE Core

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots
Jing Zhao 1, Wei Li 1,2, Xiaoqian Mao 1, Mengfan Li 1
1School of Electrical Engineering and Automation, Tianjin University, 2Department of Computer & Electrical Engineering and Computer Science, California State University

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

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Medicine

Ferric Chloride-induced Murine Thrombosis Models
Wei Li 1,2, Marvin Nieman 3, Anirban Sen Gupta 4
1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 3Department of Pharmacology, Case Western Reserve University, 4Department of Biomedical Engineering, Case Western Reserve University

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

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Bioengineering

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
Jian Li 1, Holger Jahr 2,3, Wei Zheng 4, Pei-Gen Ren 1
1Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2Department of Orthopedic Surgery, Maastricht UMC+, 3Department of Orthopaedic Surgery, University Hospital RWTH, 4Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

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Medicine

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients
Elena K. Schneider 1, Felisa Reyes-Ortega 1, Jian Li 1,2, Tony Velkov 1
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 2Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

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Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Wei Li 1,2, Chen Zhang 1, Xiulian Sun 3
1Department of Neurology, Qilu Hospital of Shandong University, 2Department of Neurology, Qingdao Municipal Hospital, 3Brain Research Institute, Qilu Hospital of Shandong University

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

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Biochemistry

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers
Huijuan You *1, Shimin Le *2,3, Hu Chen 2,4, Linyan Qin 1, Jie Yan 2,4,5
1School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 2Mechanobiology Institute, National University of Singapore, 3Department of Physics, National University of Singapore, 4Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics, Xiamen University, 5Center for Bioimaging Sciences, National University of Singapore

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

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JoVE Journal

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction
Dan Zhou *1,2, Lei Tan *1, Jian Li *3, Tanbin Liu 1, Yi Hu 1, Yalan Li 1, Sachiyo Kawamoto 4, Chengyu Liu 5, Shiyin Guo 3, Aibing Wang 1
1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), 2Department of Pathology, Georgetown University Medical School, 3College of Food Science and Technology, Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC), National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core, National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH)

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

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JoVE Core

Home-Based Prescribed Pulmonary Exercise in Patients with Stable Chronic Obstructive Pulmonary Disease
Xiaodan Liu 1,2, Peijun Li 3, Jian Li 3, Lu Xiao 1, Ning Li 3, Yufan Lu 3, Zhengrong Wang 3, Jianqing Su 3, Zhenwei Wang 4, Chunlei Shan 1,2, Weibing Wu 3
1School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, 2Institute of Rehabilitation Medicine, Shanghai Academy of Traditional Chinese Medicine, 3Department of Sports Medicine, Shanghai University of Sport, 4Department of Respiratory Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine

Presented here is a protocol to investigate the effects of home-based prescribed pulmonary exercise in stable chronic obstructive pulmonary disease (COPD) patients, which is modified based on traditional Chinese exercises according to dyspnea and limited exercise capacity observed in COPD patients.

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Immunology and Infection

Optimization of the Cuff Technique for Murine Heart Transplantation
Yunhan Ma *1,2, Baiyi Xie *1,2, Helong Dai *3,4,5, Chenxi Wang 6, Shujiao Liu 6, Tianshu Lan 7, Shuangyue Xu 1,2, Guoliang Yan 1,2,6, Zhongquan Qi 1,2,8
1Organ Transplantation Institute, School of Medicine, Xiamen University, 2Fujian Key Laboratory of Organ and Tissue Regeneration, 3Department of Kidney Transplantation, Center of Organ Transplantation, The Second Xiangya Hospital of Central South University, 4Clinical Research Center for Organ Transplantation in Hunan Province, 5Clinical Immunology Center, Central South University, 6School of Medicine, Xiamen University, 7Xiamen Medical College, 8Medical College, Guangxi University

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

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Chemistry

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium
Kiyoharu J. Miyagishima 1, Congxiao Zhang *2, Volha V. Malechka *3, Kapil Bharti 2, Wei Li 1
1Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, 2Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health, 3Human Visual Function Core, National Eye Institute, National Institutes of Health

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

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Medicine

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery
Tao Liu *1,2,3,4,5, Xueling Song *1,2,3,4,5, Xiaoying Zheng *1,2,3,4,5, Liying Yan 1,2,3,4,5, Caihong Ma 1,2,3,5, Rong Li 1,2,3,4,5, Jie Yan 1,2,3,4,5, Jie Qiao 1,2,3,4,5
1Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology, 3Key Laboratory of Assisted Reproduction, Ministry of Education, 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, 5Research Units of Comprehensive Diagnosis and Treatment of Oocyte Maturation Arrest, Chinese Academy of Medical Sciences

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

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Engineering

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices
Celine R. Garcia *1, Zhenya Ding *1, Hilario C. Garza 1, Wei Li 1
1Department of Chemical Engineering, Texas Tech University

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of <10 µm were achieved.

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Bioengineering

Building a Simple and Versatile Illumination System for Optogenetic Experiments
Phillip Kyriakakis 1, Lourdes Fernandez de Cossio 2, Patrick Wade Howard 1, Sivleng Kouv 1, Marianne Catanho 1, Vincent J. Hu 3, Robert Kyriakakis 1, Molly E. Allen 1, Yunhan Ma 4, Marcelo Aguilar-Rivera 1, Todd P. Coleman 1
1Department of Bioengineering, University of California, San Diego, 2University of California, San Diego, 3Department of Mathematical Computational, and Systems biology, University of California, Irvine, 4Department of Biomedical Engineering, Duke University

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

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Neuroscience

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study
Sara Oberrauch 1, Jing Lu 1, Linda Cornthwaite-Duncan 1, Maytham Hussein 1, Jian Li 2, Gauri Rao 3, Tony Velkov 1
1Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 2Department of Microbiology, Biomedicine Discovery Institute, Monash University, 3UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

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Biology

Primary Culture of Porcine Retinal Pigment Epithelial Cells
Feng Wen *1,2,3, Yanzi Wang *1,2,3, Danxue He 1,2,3, Chunyan Liao 1,2,3, Weijie Ouyang 1,2,3, Zuguo Liu 1,2,3, Wei Li 1,2,3, Yi Liao 1,2,3
1Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, School of Medicine, Xiamen University, 2Department of Ophthalmology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, 3Xiamen University Affiliated Xiamen Eye Center, School of Medicine, Xiamen University

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

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Immunology and Infection

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection
Rajnikant Sharma 1, Shekhar Yeshwante 1, Quentin Vallé 1, Maytham Hussein 2, Varsha Thombare 2, Sean Michael McCann 1, Robert Maile 3,4,5, Jian Li 6, Tony Velkov 2, Gauri Rao 1
1UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Microbiology & Immunology, University of North Carolina School of Medicine, 4Department of Surgery, University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

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