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University of Chinese Academy of Sciences

54 ARTICLES PUBLISHED IN JoVE

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Biology

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
Yue Zhang *1, Luv Kashyap *2, Annabel A. Ferguson 2, Alfred L. Fisher 2
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

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Medicine

Technical Aspects of the Mouse Aortocaval Fistula
Kota Yamamoto 1,2, Xin Li 1,3, Chang Shu 3, Tetsuro Miyata 2, Alan Dardik 1,4
1The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, 2Department of Vascular Surgery, The University of Tokyo, 3Department of Vascular Surgery, Central South University, 4VA Connecticut Healthcare Systems

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

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Bioengineering

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
Yunfeng Chen *1, Baoyu Liu *2, Lining Ju *3, Jinsung Hong *1, Qinghua Ji 4,5, Wei Chen 6, Cheng Zhu 1
1Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 2Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 3Charles Perkins Centre, The University of Sydney, 4Institute of Biophysics, Laboratory of RNA Biology, Chinese Academy of Sciences, 5University of Chinese Academy of Sciences, 6School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

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Medicine

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail
Chun Yu Wong 1,2,3, Margreet R. de Vries 2,3, Yang Wang 4, Joost R. van der Vorst 3, Alexander L. Vahrmeijer 3, Anton-Jan van Zonneveld 1,2, Jaap F. Hamming 3, Prabir Roy-Chaudhury 4, Ton J. Rabelink 1,2, Paul H. A. Quax 2,3, Joris I. Rotmans 1,2
1Department of Nephrology, Leiden University Medical Center, 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 3Department of Surgery, Leiden University Medical Center, 4Division of Nephrology, University of Cincinnati

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

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Neuroscience

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals
Feng Liu 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

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JoVE Core

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization
Feng Liu 1, Sunzida Ferdous 2, Xianjian Wan 1, Chenhui Zhu 3, Eric Schaible 3, Alexander Hexemer 3, Cheng Wang 3, Thomas P. Russell 1,2
1Materials Sciences Division, Lawrence Berkeley National Laboratory, 2Department of Polymer Science and Engineering, University of Massachusetts, Amherst, 3Advanced Light Source, Lawrence Berkeley National Laboratory

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

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Genetics

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Huan-Huan Wei *1, Yuanlong Liu *1, Yang Wang 2, Qianyun Lu 1, Xuerong Yang 1, Jiefu Li 1, Zefeng Wang 1
1Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences (SIBS), 2Institute of Cancer Stem Cell, Second Affiliated Hospital, Cancer Center, Dalian Medical University

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

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Medicine

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava
Hualong Bai 1,2,3,4,5, Xin Li 6, Takuya Hashimoto 1,2,5, Haidi Hu 1,2,5, Trenton R. Foster 1,2,5, Jesse J. Hanisch 1,2,5, Jeans M. Santana 1,2,5, Alan Dardik 1,2,5
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, First Affiliated Hospital of Zhengzhou University, 4Basic Medical College of Zhengzhou University, 5VA Connecticut Healthcare Systems, West Haven, CT, 6Department of Vascular Surgery, Xiangya Second Hospital of Central South University, Changsha, China

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

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Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro
Zhaofeng Jia 1,2, Yujie Liang 3, Bin Ma 4,5, Xiao Xu 2,6, Jianyi Xiong 2, Li Duan 2, Daping Wang 1,2
1Guangzhou Medical University, 2Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopeadic Engineering, Department of Orthopedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 3Department of Chemistry, The Chinese University of Hong Kong, 4School of Biomedical Engineering, Shanghai Jiao Tong University, 5Renji Hospital Clinical Stem Cell Research Center, Shanghai Jiao Tong University School of Medicine, 6Shantou University Medical College

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

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Environment

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor
Yue Zhang 1,2, Pengfei Liu 1, Zhaoheng Gong 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

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Environment

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber
Yue Zhang 1,2, Zhaoheng Gong 1, Suzane de Sa 1, Adam P. Bateman 1, Yingjun Liu 1, Yongjie Li 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

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Developmental Biology

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis
Bao Li Zhang 1,2,3, Da Yuan Gao 1,2,3, Xiao Xu Zhang 1,3, Shuo Shi 4, Winnie Shum 1
1School of Life Science and Technology, ShanghaiTech University, 2Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences, 4Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

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Bioengineering

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock
Dong Wei 1,2, Jinjie Gu 1, Zhongxi Zhang 1, Chenhong Wang 1, Dexin Wang 1, Chul Ho Kim 3, Biao Jiang 1, Jiping Shi 1,4, Jian Hao 1
1Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Biorefinery Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 4School of Life Science and Technology, ShanghaiTech University

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

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Education

The Modular Design and Production of an Intelligent Robot Based on a Closed-Loop Control Strategy
Libo Zhang 1, Junjie Zhu 2, Hao Ren 2, Dongdong Liu 3, Dan Meng 4, Yanjun Wu 1, Tiejian Luo 5
1Institute of Software, Chinese Academy of Sciences, 2Hunan University, 3Fuyang Normal University, 4Anhui Normal University, 5University of Chinese Academy of Sciences

We present a protocol on modular design and production of intelligent robots to help scientific and technical workers design intelligent robots with special production tasks based on personal needs and individualized design.

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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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Developmental Biology

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis
Junhua Lv 1,2, Feng Liu 1,2
1State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

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Genetics

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Xuan Yao 1,2, Xing Wang 1,2, Junlai Liu 2,3,4, Linyu Shi 1, Pengyu Huang 3, Hui Yang 1
1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 2College of Life Sciences, University of Chinese Academy of Sciences, 3School of Life Science and Technology, Shanghai Tech University, 4Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

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Behavior

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila
Lifen Jiang *1,2, Yinpeng Zhan *2,3, Yan Zhu 2,3
1School of Life Science, University of Science and Technology of China, 2State key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

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Immunology and Infection

Generation of Monoclonal Antibodies Against Natural Products
Yue Zhang 1, Peng Cao 2, Fang Lu 3, Xin Yan 4, Bingqian Jiang 3, Jinjun Cheng 3, Huihua Qu 5
1School of Life Science, Beijing University of Chinese Medicine, 2Third Affiliated Hospital, Beijing University of Chinese Medicine, 3School of Basic Medical Sciences, Beijing University of Chinese Medicine, 4School of Chinese Materia Medica, Beijing University of Chinese Medicine, 5Center of Scientific Experiment, Beijing University of Chinese Medicine

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

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Biology

A Method for Islet Transplantation to the Omentum in Mouse
Ying Lu *1, Shangyou Zou *1, Suzanne Bertera *2, Rita Bottino 2, David K.C. Cooper 3, Zhengzhao Liu 1, Yi Huang 1, Chengjun Wang 1, Chungu Hong 1, Tian He 1, Hancheng Zhang 1, Qin Huo 4, Hongxing Fu 5, Zhiming Cai 1, Lisha Mou 1
1Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen University Health Science Center, 2Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, 3Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, 4College of Life Science and Oceanography, Shenzhen University, 5School of Pharmcy, Wenzhou Medical College

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

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Biology

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model
Zhaofeng Jia 1,2,3, Yujie Liang 4,5, Xingfu Li 2,3, Xiao Xu 1,2,3, Jianyi Xiong 2,3, Daping Wang 1,2,3, Li Duan 2,3
1Postgraduate institution, Guangzhou Medical University, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, 3Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopaedic Engineering, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), 4Department of Chemistry, Chinese University of Hong Kong, 5Shenzhen Kangning Hospital, Shenzhen Mental Health Center

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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Engineering

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy
Tianzhuo Zhao 1,2,3, Xin Li 1,2, Qixiu Zhong 1,2, Hong Xiao 1,3, Shuzhen Nie 1,3, Fuqiang Lian 1,3, Sining Sun 4, Zhongwei Fan 1,2,3
1The Academy of Opto-electronics, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3National Engineering Research Center for DPSSL, 4Beijing GK Laser Technology Co., Ltd.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

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Biology

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level
Huijun Xue *1,2, Zhengyan Zhan *1, Kaining Zhang 1,2, Yu Vincent Fu 1,2
1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 2Savaid Medical School, University of Chinese Academy of Sciences

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified λ DNA substrate and a Quantum-dot labeled protein.

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Developmental Biology

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity
Mengrou Liu *1, Chunsen Zhu *1, Shun Bai *2, Xin Li 1, Kaiqiang Fu 1, Lan Ye 1, Ke Zheng 1
1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 2Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

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Behavior

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats
Lupeng Yue 1,2, Fengrui Zhang 3, Xuejing Lu 1,2, You Wan 4,5,6, Li Hu 1,2,7
1CAS Key Laboratory of Mental Health, Institute of Psychology, 2Department of Psychology, University of Chinese Academy of Sciences, 3Research Center of Brain Cognitive Neuroscience, Liaoning Normal University, 4Neuroscience Research Institute, Peking University, 5Department of Neurobiology, School of Basic Medical Sciences, Peking University, 6Key Laboratory for Neuroscience, Ministry of Education/National Health and Family Planning Commission, Peking University, 7Department of Pain Management, State Key Clinical Specialty in Pain Medicine, Second Affiliated Hospital of Guangzhou Medical University

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

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Education

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides
Lisheng Wang 1,2,3, Xuefeng Wang 1,2, Jun Ye 4, Zhibang Ma 1,2, Weifang Yang 5, Jule Xiao 1,2
1Key Laboratory of Cenozoic Geology and Environment, Institute of Geology and Geophysics, Chinese Academy of Sciences, 2Institutions of Earth Science, Chinese Academy of Sciences, 3University of Chinese Academy of Sciences, 4First Institute of Oceanography, State Oceanic Administration, 5Key Laboratory of Submarine Geosciences, Second Institute of Oceanography, State Oceanic Administration

The protocol describes a method to purify and separate the U and Th nuclide in submarine hydrothermal sulfide sample with Fe co-precipitation and extraction chromatography for 230Th-U disequilibrium dating.

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Behavior

A Conflict Model of Reward-seeking Behavior in Male Rats
Shaofei Jiang 1,2, Yue Zhang 1,2, Xigeng Zheng 1,2, Haoshuang Luo 1,2, Zhengkui Liu 1,2, Yunjing Bai 1,2
1CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, 2Department of Psychology, University of Chinese Academy of Sciences

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

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Immunology and Infection

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue
Xin Li 1, Zhengmei Mao 2, Li Yang 1, Kai Sun 1
1Center for Metabolic and Degenerative Diseases, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 2Microscopy Core, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

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JoVE Journal

Thermocapillary Convection Space Experiment on the SJ-10 Recoverable Satellite
Li Duan *1,2, Yongli Yin *3, Jia Wang *1, Qi Kang 1,2, Di Wu 1, Huan Jiang 1, Pu Zhang 1, Liang Hu 1
1National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, 2School of Engineering Sciences, University of Chinese Academy of Sciences, 3China Astronaut Research and Training Center

A protocol for the space payload design, the space experiment on thermocapillary convection, and analyses of experimental data and images are presented in this paper.

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Chemistry

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method
Zhihui Tu 1,2, Jian Yin 3, Liangxu Xie 4
1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3XtalPi Inc. (Shenzhen Jingtai Technology Co., Ltd.), 4Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology

A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

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Genetics

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease
Rafael J. L. Morcillo 1, Achen Zhao 1,2, María I. Tamayo-Navarrete 3, José M. García-Garrido 3, Alberto P. Macho 1
1Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín (CSIC)

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

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Immunology and Infection

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice
Meng Wang *1, Yuanyuan Huang *1, Wangpeng Gu 2,3, Haikun Wang 1
1CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 2School of Life Science, University of Science and Technology of China, 3State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

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JoVE Journal

Rapid Repetition Rate Fluctuation Measurement of Soliton Crystals in a Microresonator
Peng Xie *1,2, Xinyu Wang *1,2, Weiqiang Wang 1,2, Wenfu Zhang 1,2, Zhizhou Lu 1,2, Yang Wang 1,2, Wei Zhao 1,2
1State Key Laboratory of Transient Optics and Photonics, Xi'an Institute of Optics and Precision Mechanics (XIOPM), Chinese Academy of Sciences (CAS), 2University of Chinese Academy of Sciences

Here, we present a protocol to generate soliton crystals in a butterfly-packaged micro-ring resonator using a thermal tuned method. Further, the repetition rate fluctuations of a soliton crystal with a single vacancy are measured using a delayed self-heterodyne method.

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Developmental Biology

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem
Yang Wang 1, Arun Sampathkumar 1
1Max Planck Institute of Molecular Plant Physiology

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

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Determining the Influence of Soil Microbial Biomass Size on Soil Organic Matter Priming and Plant Residue Decomposition
Lu-Jun Li 1,2, Xia Zhu-Barker 3, Rongzhong Ye 4, William R. Horwath 3
1National Field Observation and Research Station of Hailun Agroecosystems, Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Department of Land, Air and Water Resources, University of California, Davis, 4Department of Plant & Environmental Sciences, Pee Dee Research & Education Center, Clemson University

This protocol describes a method to determine the influence of ryegrass residue addition on soil organic matter mineralization (i.e., priming effect) as well as explore the changes in soil microbial biomass size induced by soil organic matter priming, which involves artificially changing the size of microbial biomass.

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Immunology and Infection

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous β2-Microglobulin
Di Zhang *1,2, Kefang Liu *3,4, Dan Lu *2,5, Pengyan Wang 1,2, Qingxu Zhang 1,2, Peipei Liu 2, Yingze Zhao 2, Yan Chai 4, Jianxin Lyu 1, Jianxun Qi 4, William J. Liu 1,2
1School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 2NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 3Faculty of Health Sciences, University of Macau, 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 5Savaid Medical School, University of Chinese Academy of Sciences

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential β2-microglobulin (β2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous β2m were investigated.

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Engineering

Improving the Combustion Performance of a Hybrid Rocket Engine using a Novel Fuel Grain with a Nested Helical Structure
Zezhong Wang 1,2, Xin Lin 1, Fei Li 1, Zelin Zhang 1, Xilong Yu 1,2
1Institute of Mechanics, Chinese Academy of Sciences, 2School of Engineering Science, University of Chinese Academy of Sciences

A technique utilizing a solid fuel grain with a novel nested helical structure to improve the combustion performance of a hybrid rocket engine is presented.

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Environment

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell
Liyi Wang 1,2, Nana Xue 1,2, Wenfeng Li 2, Rehemanjiang Wufuer 1, Daoyong Zhang 1,3
1Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Key Laboratory of Microbial Technology for Industrial Pollution Control of Zhejiang Province, College of Environment, Zhejiang University of Technology

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

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Bioengineering

Microfluidic Model to Mimic Initial Event of Neovascularization
Ping Zhao 1, Xing Zhang 1, Xiao Liu 1, Li Wang 4, Haoran Su 1, Liyi Wang 1, Dongrui Zhang 1, Xiaoyan Deng 3, Yubo Fan 1,2
1Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Chinese Education Ministry, School of Biological Science and Medical Engineering, Beihang University, 2School of Engineering Medicine, Beihang University, 3Artificial Intelligence Key Laboratory of Sichuan Province, School of Automation and Information Engineering, Sichuan University of Science and Engineering, 4Beijing Research Center of Urban System Engineering

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

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Biology

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture
Li Duan 1,2, Xingfu Li 1,2, Xiao Xu 1,2, Limei Xu 1,2, Daping Wang 1,2, Kan Ouyang 1,2, Yujie Liang 1,3
1Department of Orthopedics, The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, Shenzhen Second People's Hospital, 3Department of Child and Adolescent Psychiatry, Shenzhen Kangning Hospital, Shenzhen Mental Health Center, Shenzhen Key Laboratory for Psychological Healthcare & Shenzhen Institute of Mental Health

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

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Biology

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Raphael Nyaruaba 1,2,3, Xiaohong Li 1, Caroline Mwaliko 2,3,4, Changchang Li 1, Matilu Mwau 5, Nelson Odiwour 1,2,3, Elishiba Muturi 1,2,3, Caroline Muema 1,2,3, Junhua Li 1, Junping Yu 1, Hongping Wei 1
1Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, 2International College, University of Chinese Academy of Sciences, 3Sino-Africa Joint Research Center, 4CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 5Center for Infectious and Parasitic Diseases Control Research, Kenya Medical Research Institute

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

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Cancer Research

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice
Lisha Wang 1, Zhiming Wang 1, Junyi Guo 2, Huayu Lin 1, Shuqiong Wen 2, Qiao Liu 1, Yiding Li 3, Qing Wu 1, Leiqiong Gao 1, Xiangyu Chen 1, Luoyingzi Xie 1, Qin Tian 1, Jianfang Tang 1, Zhirong Li 1, Li Hu 1, Juan Wang 4, Lifan Xu 1, Qizhao Huang 5, Lilin Ye 1
1Institute of Immunology, Third Military Medical University, 2Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Stomatological Hospital, Sun Yat-Sen University, 3Shigatse Branch, Xinqiao Hospital, Third Military Medical University, 4Department of Emergency Medicine, Southwest Hospital, Third Military Medical University, 5Cancer Center, The General Hospital of Western Theater Command

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

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Immunology and Infection

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity
Xiaoyan Chang *1, Keru Zhou *1, Yanguang Liu 1,2, Lian Duan 3, Lichun Zhang 2, Guoliang Zhang 3, Haikun Wang 4, Guiqin Wang 1
1Nanjing Advanced Academy of Life and Health, 2Institute of Animal Bio-technology, Jilin Academy of Agricultural Sciences, 3National Clinical Research Center for Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, 4CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, University of Chinese Academy of Sciences

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

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Biochemistry

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds
Yue Zhang *1, Peng Cao *2, Fang Lu 1, Jinjun Cheng 3, Huihua Qu 4
1School of Life Science, Beijing University of Chinese Medicine, 2Third Affiliated Hospital, Beijing University of Chinese Medicine, 3National Institute of TCM Constitution and Preventive Medicine, Beijing University of Chinese Medicine, 4Center of Scientific Experiment, Beijing University of Chinese Medicine

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

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Neuroscience

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches
Xiaoyu Wang *1, Wanying Cao *1, Jingtao Shi *1, Xiaoning Zhang 1, Zhengyang Qu 1, Dongsheng Xu 1, Hongye Wan 1, Yangshuai Su 1, Wei He 1, Xianghong Jing 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 µm tissue sections under confocal microscopy.

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Immunology and Infection

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection
Fei Wang *1, Cihan Yang *1,2, Shunlong Wang 1,2, Qun Wu 1,2, Christabel Ochieng 1,2, Zhiming Yuan 1,2, Han Xia 1,2
1Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

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Biology

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes
Doudou Huang *1, Haixia Ma *1, Lu Zhao 1,2, Xiaoyu Wang 1,2, Yi Huang 1,2, Fei Wang 1, Zhiming Yuan 1,2, Han Xia 1,2
1Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

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Neuroscience

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation
Qiwei Fan *1, Xiaotan Zhang *2, Yuan Wang 3, Xiaoyu Wang 1
1Division of Histology & Embryology, Key Laboratory for Regenerative Medicine of the Ministry of Education, College of Medicine, Jinan University, 2Department of Clinical Pathology, First Affiliated Hospital of Jinan University, 3Program in Neuroscience, Department of Biomedical Sciences, College of Medicine, Florida State University

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

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Bioengineering

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization
Fengqiang Cao *1,2,3, Yali Ming *3,4, Weixiang Gao 3,4, Jia Ge 3, Kenji Ogino 1,2
1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 4University of Chinese Academy of Sciences

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

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Developmental Biology

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)
Yixuan Feng 1,2, Tai Wang 1,2,3, Lingtong Liu 1
1Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 2College of Life Science, University of Chinese Academy of Sciences, 3Innovative Academy of Seed Design, Chinese Academy of Sciences

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

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Environment

Collection of Alfalfa Root Exudates to Study the Impact of Di(2-ethylhexyl) Phthalate on Metabolite Production
Wenjie Ren 1,2, Rui Zhao 1, Ying Teng 1, Yongming Luo 1
1Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, 2College of Resources and Environment, University of Chinese Academy of Sciences

The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

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Chemistry

A Scalable Balz-Schiemann Reaction Protocol in a Continuous Flow Reactor
Zhangtao Zhou 1,2, Yang Wang 1, Shuqiang Liu 1, Andrew Phillis 1,2, Qijun Zhang 1, Weiping Ye 1
1Shenzhen Hwagen Pharmaceutical Co., Ltd, 2Guangdong Raffles Pharmaceutical Technology Co., Ltd

A detailed scalable continuous flow protocol is presented to synthesize an aryl fluoride from an aryl amine through the Balz-Schiemann reaction.

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Biology

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods
Qing Liu 1,3, Lili Zhang 1,2, Rongxiang Fang 1,2,3, Yan Huo 1,2
1State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, 2CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, 3University of Chinese Academy of Sciences

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

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Bioengineering

An Integrated Micro-Device System for Coral Growth and Monitoring
Jinglun Zhao 1, Tao Yuan 2, Hui Huang 2, Xiaolin Lu 1
1State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, 2Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, University of Chinese Academy of Sciences

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

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