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Chinese Academy of Agricultural Sciences

27 ARTICLES PUBLISHED IN JoVE

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Bioengineering

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

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

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Biology

Crystallization of Membrane Proteins in Lipidic Mesophases
Wei Liu 1, Vadim Cherezov 1
1Molecular Biology, The Scripps Research Institute

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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Medicine

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD
Min Jia 1, Fei Meng 2, Stanley E. Smerin 1, Guoqiang Xing 1, Lei Zhang 1, David M. Su 2, David Benedek 1, Robert Ursano 1, Yan A. Su 2, He Li 1
1Department of Psychiatry, Center for the Study of Traumatic Stress, Uniformed Services University of the Health Sciences, Bethesda, Maryland, 2Department of Gene and Protein Biomarkers, GenProMarkers, Inc.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

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Immunology and Infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model
Dimitrios N. Vatakis 1,2,3, Gregory C. Bristol 1,2, Sohn G. Kim 1,2, Bernard Levin 1,2, Wei Liu 4, Caius G. Radu 4, Scott G. Kitchen 1,2,3, Jerome A. Zack 1,2,5
1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

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Biology

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response
Ritesh Kumar *1, Shu Wei Wu *1, Arya Bagus Boedi Iswanto 1, Dhinesh Kumar 1, Xiao Han 2, Jae-Yean Kim 1
1Division of Applied Life Science (BK21 plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, 2Biotechnology Research Institute, Chinese Academy of Agricultural Sciences

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

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Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Andrii Ishchenko 1,2, Vadim Cherezov 1,2, Wei Liu 3
1The Bridge Institute, University of Southern California, 2Department of Chemistry, University of Southern California, 3School of Molecular Sciences, Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

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Engineering

Cooling an Optically Trapped Ultracold Fermi Gas by Periodical Driving
Jiaming Li 1, Leonardo F. de Melo 1, Le Luo 1
1Department of Physics, Indiana University–Purdue University Indianapolis (IUPUI)

We present a parametric driving method to cool an ultracold Fermi gas in a crossed-beam optical dipole trap. This method selectively removes high-energy atoms from the trap by periodically modulating the trap depth with frequencies that are resonant with the anharmonic components of the trapping potential.

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JoVE Journal

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm
Xianming Yang 1, Pengjun Xu 2, Robert I. Graham 3, He Yuan 1, Kongming Wu 1
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 3Crop and Environment Sciences, Harper Adams University

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

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Bioengineering

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening
Yaqian Li *1,2, Xiaojun Yan *1, Wei Liu *1, Lyu Zhou 1,3, Zhifeng You 1, Yanan Du 1,2
1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3School of Life Sciences, Tsinghua University

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

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Medicine

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo
Mingming Liu 1, Xiaoyan Zhang 1, Bingwei Li 1, Bing Wang 1, Qingbin Wu 1, Fei Shang 1, Ailing Li 1, Hongwei Li 1, Ruijuan Xiu 1
1Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet β cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

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Chemistry

Extraction of Ramie Fiber in Alkali Hydrogen Peroxide System Supported by Controlled-release Alkali Source
Chaoran Meng 1, Zhaoling Li 1, Chaoyun Wang 2, Chongwen Yu 1,2, Xuerong Bi 1, Siyi Wang 1
1College of Textiles, Donghua University, 2Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences

Presented here is a protocol for extraction of ramie fiber in alkali hydrogen peroxide system supported by controlled-release alkali source.

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Environment

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens
Cai Wang 1, Huifang Wang 1, Tao Ma 1,2, Qiang Xiao 3, Panrong Cao 4, Xuan Chen 5, Hongpeng Xiong 1, Wenquan Qin 1, Zhaohui Sun 1, Xiujun Wen 1
1Guangdong Key Laboratory for Innovation Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, 2College of Agriculture, South China Agricultural University, 3Tea Research Institute, Chinese Academy of Agricultural Sciences, 4College of Horticulture, South China Agricultural University, 5College of Coast and Environment, Louisiana State University

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

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Environment

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions
Zhongyi Wang *1, Jiaming Li *2, Lu’an Qian *3, Linna Liu 1, Jun Qian 1, Bing Lu 1, Zhendong Guo 1
1Academy of Military Medical Sciences, 2Chinese Academy of Agricultural Sciences, 3The Experimental High School Attached to Beijing Normal University

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

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Immunology and Infection

Merkel Cell Polyomavirus Infection and Detection
Wei Liu *1, Nathan A. Krump *1, Christopher B. Buck 2, Jianxin You 1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Laboratory of Cellular Oncology, National Cancer Institute

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

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Environment

Preference Assessment of White-backed Planthopper Feeding on Rice
Lei Zhang 1, Chao Wu 1, Yutao Xiao 1
1Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

This study presents a simple and feasible method to assess the nonpreference resistance to white-backed planthoppers who are feeding on rice under laboratory conditions. Improvement of the strategies and makeup of the current method of identification of the resistance to white-backed and brown planthoppers are discussed.

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Biochemistry

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

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

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Behavior

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)
Dong Ai 1,2, Bing Wang 2, Zhennan Fan 1, Yuyao Fu 1, Caihong Yu 1, Guirong Wang 2,3
1School of Chemical and Environmental Engineering, China University of Mining and Technology, 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 3Guangdong Laboratory of Lingnan Modern Agriculture, Shenzhen; Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

Presented here is a protocol of Helicoverpa armigera (Hübner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

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Biology

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts
Lu Zhang 1, Wenwen Liu 1, Xifeng Wang 1
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

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Cancer Research

Longitudinal Intravital Imaging Through Clear Silicone Windows
Laura Maiorino *1,2,3, Margaret Shevik *1,4,5, José M. Adrover 1, Xiao Han 1,6, Elias Georgas 7,8, John Erby Wilkinson 9, Harrison Seidner 1, Leonie Foerschner 1, David A. Tuveson 1, Yi-Xian Qin 8, Mikala Egeblad 1
1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

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Medicine

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

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

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Biology

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

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

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Biochemistry

Purification of Endogenous Drosophila Transient Receptor Potential Channels
Jia Liu *1, Yuyang Liu *1, Weidi Chen 1, Yuzhen Ding 1, Xiaoru Lan 1, Wei Liu 1
1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

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Biology

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis
Jinxi Yuan 1, Jie Zhang 1, Yan Zhang 1, WuYun QiQiGe 1, Wei Liu 2, Shanchun Yan 1, Guirong Wang 2
1Key Laboratory of Sustainable Forest Ecosystem Management - Ministry of Education, Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

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Biology

Quantification of Arbuscular Mycorrhizal Fungi Colonization Rate in the Study of Invasive Alien Plants
Qiao Li 1, Jianying Guo 1, Mengxin Zhao 1
1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences

This protocol provides a simple and easy-to-use approach for determining the colonization rate of Arbuscular mycorrhizal fungi (AMF) in the roots of invasive plants.

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Engineering

Computer Vision-Based Biomass Estimation for Invasive Plants
Zhenyu Huang 1,2, Zhiyong Xu 3, Yanzhou Li 1, Bo Liu 2, Conghui Liu 2, Xi Qiao 1,2, Wanqiang Qian 2, Feng Qin 1,2, Pingdong Li 4, Yiqi Huang 1
1College of Mechanical Engineering, Guangxi University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, 3China Lesso Group Holdings Limited, 4Shenzhen Agricultural Technology Promotion Center

We report detailed procedures for an invasive plant biomass estimation method that utilizes data obtained from unmanned aerial vehicle (UAV) remote sensing to assess biomass and capture the spatial distribution of invasive species. This approach proves highly beneficial for conducting hazard assessment and early warning of invasive plants.

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Environment

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition
Xia Shu 1,2, Huatai Li 1, Jing Wang 3, Shan Wang 4, Yunpeng Liu 1, Ruifu Zhang 1,3
1State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, 2State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, 3Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, 4Jiangsu Engineering and Technology Center for Modern Horticulture, Jiangsu Vocational College of Agriculture and Forestry

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

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Biology

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura
Long-xi Zhang 1, Qian-jin Dong 1, Shuai Yang 1, Wu-nan Che 1, Li-sheng Zhang 2, Jin-cheng Zhou 1,2, Hui Dong 1
1College of Plant Protection, Shenyang Agricultural University, 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

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