Chinese Academy of Sciences

52 articles published in JoVE

• Biology

  
  Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis Chun Wang*¹, Jie Lv*¹, Wei Zhuang^1,2, Ling Xie¹, Guangyu Liu¹, Kaidireya Saimaier¹, Sanxing Han¹, Changjie Shi¹, Qiuhong Hua¹, Ru Zhang¹, Guangfeng Shi³, Changsheng Du^{1 1}Putuo People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, ²National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, ³Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Fudan University The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

• 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 1}Key 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.

• 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 Ge³, Kenji Ogino^{1,2 1}Graduate 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.

• Engineering

  
  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 1}State 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.

• Biochemistry

  
  Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees Yingying Song¹, Ruiping Li^1,2, Lili Li¹, Fang Ouyang³, Xingyuan Men^{1 1}Institute of Plant Protection, Shandong Academy of Agricultural Sciences, ²College of Plant Protection, Shandong Agricultural University, ³State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

• Biology

  
  Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection Jing Zhao^1,2,3, Jie Yang^1,2,3, Lili Zhang^1,2, Rongxiang Fang^1,2, Yan Huo^{1,2 1}State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, ²National Plant Gene Research Center, ³University of the Chinese Academy of Sciences The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

• Environment

  
  Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell Liyi Wang^1,2, Nana Xue^1,2, Wenfeng Li², Rehemanjiang Wufuer¹, Daoyong Zhang^{1,3 1}Xinjiang 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.

• Developmental Biology

  
  Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish Wenyi Wang¹, Tao Kang¹, Lin Bai¹, Wei Hu², Yayoi Obata³, Jianzhen Li^{1 1}College of Life Sciences, Northwest Normal University, ²State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, ³Department of Bioscience, Tokyo University of Agriculture Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

• 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 Liu², Yingze Zhao², Yan Chai⁴, Jianxin Lyu¹, Jianxun Qi⁴, William J. Liu^{1,2 1}School 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.

• 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 Lin¹, Fei Li¹, Zelin Zhang¹, Xilong Yu^{1,2 1}Institute 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.

• Biochemistry

  
  Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis Chuan-Chih Hsu^1,3, Chia-Feng Tsai², W. Andy Tao^3,4, Pengcheng Wang^{5 1}Department of Plant Biology, Carnegie Institute for Science, ²Graduate School of Pharmaceutical Sciences, Kyoto University, ³Department of Biochemistry, Purdue University, ⁴Department of Chemistry, Purdue University, ⁵Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

• Engineering

  
  Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes Xiang Zhang*^1,2, Zhaolong Chen*³, Hongliang Chang^1,2, Jianchang Yan^1,2, Shenyuan Yang^2,4, Junxi Wang^1,2, Peng Gao⁵, Tongbo Wei^{1,2 1}State Key Laboratory of Solid-State Lighting, Institute of Semiconductors, Chinese Academy of Sciences, ²Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Science, ³Center for Nanochemistry (CNC), Beijing National Laboratory for Molecular Science, College of Chemistry and Molecular Engineering, Peking University, ⁴State Key Laboratory of Superlattices and Microstructures, Institute of Semiconductors, Chinese Academy of Sciences, ⁵Electron Microscopy Laboratory, School of Physics, Peking University A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

• Genetics

  
  Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease Rafael J. L. Morcillo¹, Achen Zhao^1,2, María I. Tamayo-Navarrete³, José M. García-Garrido³, Alberto P. Macho^{1 1}Shanghai 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.

• Engineering

  
  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 Wu¹, Huan Jiang¹, Pu Zhang¹, Liang Hu^{1 1}National 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.

• Developmental Biology

  
  Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue Yiding Qi*¹, Xiang Miao*², Lian Xu³, Mengxia Fu³, Shi Peng⁴, Kailei Shi⁵, Jun Li⁴, Maoqing Ye⁵, Ruogu Li^{1 1}Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiao Tong University, ²Shanghai Institute of Nutrition and Health, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, ³Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, ⁴Department of Cardiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, ⁵Department of Cardiology, Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital Affiliated to Fudan University We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre^+/-;Rosa26^RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

• Neuroscience

  
  Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation Wanting Chen¹, Rui Chen¹, Qinghua He^{1,2,3,4,5 1}Faculty of Psychology, Southwest University, ²Key Laboratory of Cognition and Personality, Ministry of Education, Southwest University, ³Southwest University Branch, Collaborative Innovation Center of Assessment toward Basic Education Quality, Beijing Normal University, ⁴Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, ⁵Chongqing Collaborative Innovation Center for Brain Science Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

• Developmental Biology

  
  An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons Wai Wa Ray Chan*¹, Wen Li*^1,2, Dik Long Dennis Chau*¹, Kwok-Fai Lau^{1 1}School of Life Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, ²Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

• Chemistry

  
  Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method Zhihui Tu^1,2, Jian Yin³, Liangxu Xie^{4 1}State 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.

• 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 Sun⁴, Zhongwei Fan^{1,2,3 1}The 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.

• Chemistry

  
  Separation of Uranium and Thorium for ²³⁰Th-U Dating of Submarine Hydrothermal Sulfides Lisheng Wang^1,2,3, Xuefeng Wang^1,2, Jun Ye⁴, Zhibang Ma^1,2, Weifang Yang⁵, Jule Xiao^{1,2 1}Key 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.

• Environment

  
  A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans Shan Gao*¹, Weiyang Chen*², Nan Zhang¹, Chi Xu³, Haiming Jing^1,4, Wenjing Zhang^1,4, Gaochao Han^1,4, Matthew Flavel⁵, Markandeya Jois⁵, Yingxin Zeng¹, Jing-Dong J. Han³, Bo Xian³, Guojun Li^{1,4 1}Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control/Beijing Center of Preventive Medicine Research, China, ²College of Computer Science and Technology, Qilu University of Technology(Shandong Academy of Sciences), China, ³Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China, ⁴Beijing Key Laboratory of Environmental Toxicology, School of Public Health, Capital Medical University, China, ⁵School of Life Sciences, La Trobe University, Australia A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

• Immunology and Infection

  
  Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster Shuo Yang*^1,2,3, Yaya Zhao*^1,2, Junjing Yu⁴, Zhiqin Fan², Si-tang Gong¹, Hong Tang^1,2, Lei Pan^{1,2,3 1} This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

• 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 1}CAS 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.

• 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 Zhang³, Xuejing Lu^1,2, You Wan^4,5,6, Li Hu^{1,2,7 1}CAS 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.

• Engineering

  
  Quantitative Analysis by Thermogravimetry-Mass Spectrum Analysis for Reactions with Evolved Gases Rongbin Li¹, Qian Huang², Kai Wei², Hongde Xia^{2 1}School of Metallurgical and Ecological Engineering, University of Science and Technology Beijing, ²Institute of Engineering Thermophysics, Chinese Academy of Sciences Precise determination of the evolved gases' flow rate is key to study the details of reactions. We provide a novel quantitative analysis method of equivalent characteristic spectrum analysis for thermogravimetry-mass spectrum analysis by establishing the calibration system of the characteristic spectrum and relative sensitivity, for obtaining the flow rate.

• Immunology and Infection

  
  Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice Yongli Zhang*^1,2, Hangjie Zhang*², Wenqiang Ma*³, Kefang Liu^1,2, Min Zhao⁴, Yingze Zhao², Xuancheng Lu⁵, Fuping Zhang⁶, Xiangdong Li³, George F. Gao^1,2,4,6, William J. Liu^{1,2 1}School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, ²NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, ³State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, ⁴Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, ⁵Laboratory Animal Center, Chinese Center for Disease Control and Prevention, ⁶CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1^-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

• Developmental Biology

  
  A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes Jiayin Yang*^1,2,6, Lai-Yung Wong*², Xiao-Yu Tian*³, Rui Wei^1,2, Wing-Hon Lai², Ka-Wing Au², Zhiwei Luo^4,5, Carl Ward^4,5, Wai-In Ho², David P. Ibañez^4,5, Hao Liu^4,5, Xichen Bao^4,5, Baoming Qin^4,5, Yu Huang³, Miguel A. Esteban^4,5,7, Hung-Fat Tse^{1,2,6,7 1}Department of Medicine, University of Hong Kong-Shenzhen Hospital, ²The Cardiology Division, Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, ³School of Biomedical Sciences, Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, ⁴Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health and Guangzhou Medical University, ⁵Laboratory of RNA, Chromatin, and Human Disease, CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, ⁶Research Centre of Heart, Brain, Hormone, and Healthy Ageing, Li Ka Shing Faculty of Medicine, University of Hong Kong, ⁷Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, University of Hong Kong and Guangzhou Institutes of Biomedicine and Health Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

• Bioengineering

  
  Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods Baozhen Zhang¹, Zhilong Chen^1,2, Jinyu Han^1,3, Mengxia Li¹, Nihar R. Nayak⁴, Xiujun Fan^{1 1}Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, ²College of Veterinary Medicine, Hunan Agricultural University, ³Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, College of Chemistry and Material Sciences, Heilongjiang University, ⁴Department of Obstetrics and Gynecology, Wayne State University School of Medicine We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

• Environment

  
  Evolution of Staircase Structures in Diffusive Convection Shuang-Xi Guo¹, Sheng-Qi Zhou¹, Xian-Rong Cen¹, Yuan-Zheng Lu^{1 1}State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences Diffusive convection (DC) widely occurs in natural processes and engineering applications, characterized by a series of staircases with homogeneous convecting layers and stratified interfaces. An experimental procedure is described to simulate the evolution process of the DC staircase structure, including the generation, development and disappearance, in a rectangular tank.

• 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 1}State 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.

• Engineering

  
  Generation and Coherent Control of Pulsed Quantum Frequency Combs Benjamin MacLellan*¹, Piotr Roztocki*¹, Michael Kues^1,2, Christian Reimer¹, Luis Romero Cortés¹, Yanbing Zhang¹, Stefania Sciara^1,3, Benjamin Wetzel^1,4, Alfonso Cino³, Sai T. Chu⁵, Brent E. Little⁶, David J. Moss⁷, Lucia Caspani⁸, José Azaña¹, Roberto Morandotti^{1,9,10 1}Institut National de la Recherche Scientifique - Centre Énergie, Matériaux et Télécommunications (INRS-EMT), ²School of Engineering, University of Glasgow, ³Department of Energy, Information Engineering and Mathematical Models, University of Palermo, ⁴School of Mathematical and Physical Sciences, University of Sussex, ⁵Department of Physics and Material Science, City University of Hong Kong, ⁶State Key Laboratory of Transient Optics and Photonics, Xi'an Institute of Optics and Precision Mechanics, Chinese Academy of Science, ⁷Centre for Micro Photonics, Swinburne University of Technology, ⁸Institute of Photonics, Department of Physics, University of Strathclyde, ⁹Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, ¹⁰National Research University of Information Technologies, Mechanics and Optics A protocol is presented for the practical generation and coherent manipulation of high-dimensional frequency-bin entangled photon states using integrated micro-cavities and standard telecommunications components, respectively.

• Biochemistry

  
  An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C Cuiping Li¹, Weiyi Lai¹, Hailin Wang^{1 1}State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

• 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 1}School 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.

• Immunology and Infection

  
  An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection Chongfeng Xu¹, Junhong Ai², Qingxun Zhang¹, Ting Li³, Xiaorong Wu¹, Zhengde Xie², Ziyuan Duan^{1 1}Genetic Resources Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, ²MOE Key Laboratory of Major Diseases in Children, National Key Discipline of Pediatrics, National Clinical Research Center for Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, ³State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

• Neuroscience

  
  An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice Nastasia K-H Lim^1,2, Visse Moestrup^3,4, Xiao Zhang¹, Wen-An Wang⁵, Arne Møller^3,4,6, Fu-De Huang^{1,4 1}Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Chinese Academy of Science, ²Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences, Chinese Academy of Science, ³Center of Functionally Integrative Neuroscience, Department of Clinical Medicine, Aarhus University, ⁴Sino-Danish Center for Education and Research (SDC), ⁵Department of Neurology, Xinhua Hospital Chongming Branch Affiliated to Shanghai Jiao Tong University School of Medicine, ⁶Department of Nuclear Medicine and PET-centre, Aarhus University Hospital This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

• 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 Shi¹, Pengyu Huang³, Hui Yang^{1 1}Institute 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.

• Developmental Biology

  
  Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis Junhua Lv^1,2, Feng Liu^{1,2 1}State 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.

• Engineering

  
  The Modular Design and Production of an Intelligent Robot Based on a Closed-Loop Control Strategy Libo Zhang¹, Junjie Zhu², Hao Ren², Dongdong Liu³, Dan Meng⁴, Yanjun Wu¹, Tiejian Luo^{5 1}Institute 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.

• Neuroscience

  
  Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay Yuetian Zhang*¹, Yi Pan*¹, Hiroaki Matsunami^2,3, Hanyi Zhuang^{1,4 1}Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao Tong University School of Medicine, ²Department of Molecular Genetics and Microbiology, Duke University Medical Center, ³Department of Neurobiology, Duke Institute for Brain Sciences, Duke University Medical Center, ⁴Institute of Health Science, Chinese Academy of Science/Shanghai Jiao Tong University School of Medicine Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

• Behavior

  
  An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration Wenze Cao*^1,2,3, Li Song*^1,2,3, Jingjing Cheng^1,2,3, Na Yi^1,2,3, Luyi Cai^1,2,3, Fu-de Huang^4,5, Margaret Ho^{1,2,3 1}Research Center for Translational Medicine, Tongji University School of Medicine, ²Key Laboratory of Arrhythmias of the Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, ³Department of Anatomy and Neurobiology, Tongji University School of Medicine, ⁴Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Chinese Academy of Sciences, ⁵Sino-Danish College, University of Chinese Academy of Sciences, Chinese Academy of Sciences This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

• Developmental Biology

  
  A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells Qing Cissy Yu¹, Wenqian Song¹, Dengwen Lai¹, Yi Arial Zeng^{1 1}State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

• 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 Shi⁴, Winnie Shum^{1 1}School 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.

• Developmental Biology

  
  Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells Yueying Li², Yong Hai¹, Jiayu Chen³, Tie Liu^{1 1}Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University, ²Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, ³Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

• Bioengineering

  
  Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock Dong Wei^1,2, Jinjie Gu¹, Zhongxi Zhang¹, Chenhong Wang¹, Dexin Wang¹, Chul Ho Kim³, Biao Jiang¹, Jiping Shi^1,4, Jian Hao^{1 1}Lab 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.

• Immunology and Infection

  
  An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood Ke Yang*^1,2,3, Jiandong Wu*^3,4, Ling Zhu¹, Yong Liu¹, Michael Zhang⁵, Francis Lin^{3,4,6,7 1}Institute of Applied Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, ²University of Science and Technology of China, ³Department of Physics and Astronomy, University of Manitoba, ⁴Department of Biosystems Engineering, University of Manitoba, ⁵Seven Oaks General Hospital, ⁶Department of Immunology, University of Manitoba, ⁷Department of Biological Sciences, University of Manitoba This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

• Genetics

  
  Protocols for C-Brick DNA Standard Assembly Using Cpf1 Shi-Yuan Li¹, Guo-Ping Zhao¹, Jin Wang^{1 1}Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences, Chinese Academy of Sciences CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

• Medicine

  
  Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis Aoyuan Cui¹, Zhimin Hu¹, Yamei Han¹, Yi Yang², Yu Li^{1 1}CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Chinese Academy of Sciences, ²Suzhou Niumag Analytical Instrument Corporation Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

• Immunology and Infection

  
  A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines Xi Liu^1,2, Chongfeng Xu², Ziyuan Duan^{2 1}Wuhan Institute of Virology, Chinese Academy of Sciences, ²Genetic Resources Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

• 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 Chen⁶, Cheng Zhu^{1 1}Woodruff 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.

• Biology

  
  Measuring Spatial and Temporal Ca²⁺ Signals in Arabidopsis Plants Xiaohong Zhu¹, Aaron Taylor², Shenyu Zhang¹, Dayong Zhang^1,3, Ying Feng^1,4, Gaimei Liang^1,5, Jian-Kang Zhu^{1,6 1}Department of Horticulture and Landscape Architecture, Purdue University, ²Bindley Bioscience Center, Purdue University, ³Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, ⁴College of Environmental & Resource Science, Zhejiang University, ⁵Dryland Agriculture Research Centre, Shanxi Academy of Agricultural Sciences, ⁶Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences Ca²⁺ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca²⁺ signals in Arabidopsis cells and tissues using the genetically encoded Ca²⁺ indicators Aequorin or Case12.

• Biology

  
  Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC) Zach Hensel¹, Xiaona Fang^1,2,3, Jie Xiao^{1 1}Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, ²Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, ³Department of Physics, Jilin University We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI ¹.

• Biology

  
  Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment Zachery Oestreicher¹, Steven K. Lower^1,2, Wei Lin³, Brian H. Lower^{2 1}School of Earth Sciences, The Ohio State University, ²School of Environment & Natural Resources, The Ohio State University, ³Institute of Geology and Geophysics, Chinese Academy of Sciences We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.