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Tsinghua University

59 ARTICLES PUBLISHED IN JoVE

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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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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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Bioengineering

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
Bibhudatta Mishra 1, Mostafa Ghannad-Rezaie 2, Jiaxing Li 1, Xin Wang  1, Yan Hao 1, Bing Ye 3,4, Nikos Chronis 2,5, Catherine A. Collins 1
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

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Biology

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)
Lin Zhang 1, Katleen Vranckx 2, Koen Janssens 2, Todd R. Sandrin 1
1School of Mathematical and Natural Sciences, Arizona State University, 2Applied Maths NV

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

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Chemistry

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles
Bryan Wei 1, Michelle K. Vhudzijena 2, Joanna Robaszewski 2, Peng Yin 2,3
1Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 2Wyss Institute for Biologically Inspired Engineering, Harvard University, 3Department of Systems Biology, Harvard Medical School

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

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Biology

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity
Yong Zhang 1, Xueyan Pang 1, Zhenxing Liu 1
1Department of Biology, University of Nevada Reno

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

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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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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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JoVE Journal

Synthesis of High Purity Nonsymmetric Dialkylphosphinic Acid Extractants
Junlian Wang 1, Meiying Xie 1, Xinyu Liu 1, Shengming Xu 2
1School of Civil and Resource Engineering, University of Science and Technology Beijing, 2Institute of Nuclear and New Energy Technology, Tsinghua University

A protocol for the synthesis of high purity nonsymmetric dialkylphosphinic acid extractants is presented, taking (2,3-dimethylbutyl)(2,4,4'-trimethylpentyl)phosphinic acid as an example.

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Chemistry

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture
Yongsan Li 1, Yaling Zhang 2, Yen Wei 1, Lei Tao 1
1The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, 2Institute of Chemical Materials, China Academy of Engineering Physics

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel’s mechanical strength and its application in 3D cell culture are presented.

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Bioengineering

Visual Detection of Multiple Nucleic Acids in a Capillary Array
Jianwei Chen *1,2,3, Ning Shao *1,2,3, Jiaying Hu *4, Rong Li 4, Yuanshou Zhu 1,2,3, Dabing Zhang 4,5, Shujuan Guo 1,2,3, Junhou Hui 6, Peng Liu 6, Litao Yang 4, Sheng-Ce Tao 1,2,3
1Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 2State Key Laboratory of Oncogenes and Related Genes, 3School of Biomedical Engineering, Shanghai Jiao Tong University, 4Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 5Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, 6Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

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Medicine

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction
Lifei Yang *1,2, Jianwen Lu *1,2,3, Yue Wang 1,2,3, Mei Zhang 1,2,3, Yuan Shi 4, Shasha Wei 1,2, Peng Liu 1,2,3, Zheng Wu 3, Yi Lv 1,2,3, Rongqian Wu 1,2
1Shaanxi Provincial Center for Regenerative Medicine and Surgical Engineering, First Affiliated Hospital, Xi'an Jiaotong University, 2Institute of Advanced Surgical Technology and Engineering, First Affiliated Hospital, Xi'an Jiaotong University, 3Department of Hepatobiliary Surgery, First Affiliated Hospital, Xi'an Jiaotong University, 4Organ Transplantation Center, Tianjin First Center Hospital

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

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Bioengineering

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions
Zheng-Ze Pan 1,2, Hirotomo Nishihara 3, Wei Lv 1, Cong Wang 1,2, Yi Luo 1,2, Liubing Dong 1,2, Houfu Song 1,4, Wenjie Zhang 2, Feiyu Kang 1,2,4, Takashi Kyotani 3, Quan-Hong Yang 1,4,5
1Engineering Laboratory for Functionalized Carbon Materials and Shenzhen Key Laboratory for Graphene-based Materials, Graduate School at Shenzhen, Tsinghua University, 2School of Materials Science and Engineering, Tsinghua University, 3Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 4Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, 5School of Chemical Engineering and Technology, Tianjin University

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

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Engineering

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices
Yan Wu 1, Xiang Qian 1, Shengli Mi 1, Min Zhang 1, Shuqing Sun 1, Xiaohao Wang 1,2
1Graduate School at Shenzhen, Tsinghua University, 2The State Key Laboratory of Precision Measurement Technology and Instruments, Tsinghua University

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

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JoVE Journal

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure
Jiang Li 1, Jia Man 2, Zhongnan Li 2, Haosheng Chen 2
1School of Mechanical Engineering, University of Science and Technology Beijing, 2State Key Laboratory of Tribology, Tsinghua University

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

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Bioengineering

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices
Gordon D. Hoople *1, Andrew Richards *2, Yan Wu 2, Albert P. Pisano 3, Kun Zhang 2
1Department of Engineering, University of San Diego, 2Department of Bioengineering, University of California, San Diego, 3Department of Mechanical and Aerospace Engineering, University of California, San Diego

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

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Medicine

Photoselective Vaporesection of the Prostate via an End-firing Lithium Triborate Crystal Laser
Xin Wang  1, Yao-Guang Zhang 1, Sheng-Cai Zhu 1, Ben Wan 1, Ming Liu 1, Jian-Ye Wang 1
1Department of Urology, Beijing Hospital, National Center of Gerontology

Here we present a protocol to perform Photoselective Vaporesection of the Prostate (PVRP) for benign prostatic hyperplasia (BPH) treatment.

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Immunology and Infection

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model
Shouming Zhang 1,2,3, Xueping Gao 1,2,3, Genhui Xiao 1,2,3,4, Chengping Lu 1,2,3, Huochun Yao 1,2,3, Hongjie Fan 1,2,3,5, Zongfu Wu 1,2,3
1College of Veterinary Medicine, Nanjing Agricultural University, 2Key Lab of Animal Bacteriology, Ministry of Agriculture, 3OIE Reference Lab for Swine Streptococcosis, 4School of Medicine, Tsinghua University, 5Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

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Behavior

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism
Peng Zhou *1, Weiyi Ma *2, Likan Zhan *3, Huimin Ma *4
1Department of Foreign Languages and Literatures, Tsinghua University, 2School of Human Environmental Sciences, University of Arkansas, 3Institute for Speech Pathology and the Brain Science, Beijing Language and Culture University, 4Department of Electronic Engineering, Tsinghua University

We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism.

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Cancer Research

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics
Xin Wang  *1, Jiamin Zhou *2, Chen Qi *1, Gelin Wang 1,3
1School of Pharmaceutical Sciences, Tsinghua University, 2Department of Radiology, University of California, San Francisco, 3Beijing Advanced Innovation Center for Structural Biology

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

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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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Chemistry

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation
Shoichi Tokunaga *1, Mengyan Zeng *1,2, Yoshimitsu Itoh 1, Fumito Araoka 3, Takuzo Aida 3
1Department of Chemistry and Biotechnology, The University of Tokyo, 2Department of Chemistry, Tsinghua University, 3RIKEN Center for Emergent Matter Science

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

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Behavior

Dissociation of the Confounding Influences of Expectancy and Integrative Difficulty Residing in Anomalous Sentences in Event-related Potential Studies
Yunlong Huang 1, Minghu Jiang 1, Qian Guo 2, Yuling Wang 1, Fan-Pei Gloria Yang 3
1Center for Psychology and Cognitive Science, School of Humanities, Tsinghua University, 2Department of Foreign Languages and Literatures, School of Humanities, Tsinghua University, 3Center for Cognition and Mind Sciences, Lab for Language and Cognitive Neurology, National TsingHua University

We present a protocol to dissociate the intertwining factors of integrative difficulty and unexpectedness in semantically anomalous sentences by applying multiple repetitions to enhance participant's expectancy for anomalous sentences. The dissociation helps to investigate the major contributor of elicited event-related potentials (ERP) effects such as N400 in language studies.

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Immunology and Infection

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy
Jiahuan Chen 1, Ying Xu 2, Yan Shi 2, Tie Xia 2
1State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 2Institute for Immunology, School of Medicine, Tsinghua University

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

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Immunology and Infection

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
Xue Bai *1,2, Lin Zhang *3, Jin Hu 2, Xiuxiu Zhao 2, Jinheng Pan 2, Haiteng Deng 3, Shan Feng 1,2
1Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 2Mass Spectrometry Core Facility, School of Life Sciences, Westlake University, 3MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

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Education

Optimized Sealing Process and Real-Time Monitoring of Glass-to-Metal Seal Structures
Zhichun Fan 1, Kangjia Hu 2, Zhiyong Huang 1, Yong Zhang 2, He Yan 1
1Institute of Nuclear and New Energy Technology, Key Laboratory of Advanced Reactor Engineering and Safety of Ministry of Education, Collaborative Innovation Center for Advanced Nuclear Energy Technology, Tsinghua University, 2Institute of Nuclear and New Energy Technology, Beijing Key Laboratory of Fine Ceramics, Tsinghua University

Key procedures to optimize the sealing process and achieve real-time monitoring of the metal-to-glass seal (MTGS) structure are described in detail. The embedded fiber Bragg grating (FBG) sensor is designed to achieve online monitoring of temperature and high-level residual stress in the MTGS with simultaneous environmental pressure monitoring.

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Neuroscience

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats
Peng Liu *1, Xiu-Chun Song *2, Xiao-Shuai Yang 1, Qi-Long Cao 1, Yong-Yong Tang 1, Xiao-Dun Liu 3, Min Yang 1, Wen-Qiang An 1, Bai-Xiang Dong 1, Xiu-Yun Song 1
1Beijing Yinfeng Dingcheng Biological Engineering Technology Ltd., 2Hulunbuir People's Hospital, 3Shandong Qilu Stem Cell Engineering Co. Ltd.

The purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

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Neuroscience

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System
Yi Chen *1, Curt Mazur *2, Yi Luo *1, Linhong Sun 1, Mingdi Zhang 1, Alex McCampbell 1, Giulio Srubek Tomassy 1
1Biogen, Inc., 2Ionis Pharmaceuticals

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

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Biology

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection
Xin Liu 1, Xue Bai 1, Mingqi Li 1, Huimin Li 1, Yong Zhang 1,2, Baofeng Yang 1,3
1Department of Pharmacology, The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, College of Pharmacy, Harbin Medical University, 2Institute of Metabolic Disease, Heilongjiang Academy of Medical Science, 3Department of Pharmacology and Therapeutics, Melbourne School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences University of Melbourne

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

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Immunology and Infection

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy
Liangfei Niu 1, Cong Wang 1, Kaile Zhang 1,2, Miaomiao Kang 1,2, Rui Liang 1, Xiaonan Zhang 1, Bo Yan 1
1Shanghai Public Health Clinical Center, Fudan University, 2School of Life Sciences, Bengbu Medical College

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

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Genetics

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification
Xianju Bi 1,2, Xuechun Zhang 1, Xiaohua Shen 1
1Tsinghua-Peking Center for Life Sciences, School of Medicine and School of Life Sciences, Tsinghua University, 2Department of Molecular Biology, University of Texas Southwestern Medical Center

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

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Genetics

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)
Yaolei Mi 1,2, Zhihui Zhu 3, Guangtao Qian 3, Yu Li 4, Xiangxiao Meng 1, Jianping Xue 3, Qingfu Chen 5, Wei Sun 1, Yuhua Shi 1,2
1Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, 2Artemisinin Research Center, China Academy of Chinese Medical Sciences, 3College of Life Science, Huaibei Normal University, 4Economic Crop Research Institute Sichuan Academy of Agriculture Sciences, 5Research Center of Buckwheat Industry Technology, Guizhou Normal University

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

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Genetics

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila
Lin Zhang 1, Kenny Dao 1, Angela Kang 1, Andre C. Loyola 1, Robin Shang 1, Jinghong Li 1, Willis X. Li 1
1Department of Medicine, University of California San Diego

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

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Biology

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes
Xinyi Liu 1,2, Si Wu 1,2, Wenqian Li 1,2, Meihong Zhang 1,2, Yan Wu 1,2, Ning Zhou 3, Pa Wu 1,2
1Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation, College of Life Science, Hunan Normal University, 2State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Provincial Key Laboratory for Microbial Molecular Biology, College of Life Science, Hunan Normal University, 3Department of Infectious Diseases, the Second Xiangya Hospital, Central South University

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

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Behavior

Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research
Ao Zhu 1, Annebella Tsz Ho Choi 1, Ko-Hsuan Ma 1, Shi Cao 2, Han Yao 1, Jian Wu 3, Jibo He 1
1Psychology Department, School of Social Sciences, Tsinghua University, 2Department of Systems Design Engineering, University of Waterloo, 3Haier Innovation Design Center, Haier Company

This protocol describes a driving simulation platform and a tactile vibrating toolkit for the investigation of driving-related research. An exemplar experiment exploring the effectiveness of tactile warnings is also presented. 

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Biochemistry

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy
Feng Ge 1, Jianfeng Xue 1, Yan He 1
1Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

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Behavior

An Assessment Method and Toolkit to Evaluate Keyboard Design on Smartphones
Yincheng Wang 1, Ke Wang 1, Yuqi Huang 1, Di Wu 2, Jian Wu 3, Jibo He 1
1Department of Psychology, School of Social Sciences, Tsinghua University, 2Department of Computer Science, Beijing Normal University, 3Haier Innovation Design Center, Haier Company

The presented protocol integrates various evaluation methods and demonstrates a method to evaluate the keyboard design on smartphones. Pairs matched by English characters are proposed as the input material, and the transition time between two keys is used as the dependent variable.

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Bioengineering

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting
Tiankun Liu *1,2, Yongchun Shao *1,2, Zitong Wang 1,2, Yuqiu Chen 1,2, Yuan Pang 1,2, Ding Weng 1,2, Wei Sun 1,2,3
1Biomanufacturing Center, Dept. of Mechanical Engineering, Tsinghua University, 2Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, 3Department of Mechanical Engineering, Drexel University

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 µm can be obtained and collected for further culturing.

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Behavior

Interaction between Phonological and Semantic Processes in Visual Word Recognition using Electrophysiology
Yuling Wang 1, Minghu Jiang 1, Xinyi Xu 1, Yunlong Huang 2
1Center for Psychology and Cognitive Science, Lab of Computational Linguistics, Tsinghua University, 2Advanced Innovation Center for Future Education, Beijing Normal University

We present a protocol to explore the relative activation sequence of phonology and semantics in visual word recognition. The results show that consistent with interactive accounts, semantic and phonological representations may be processed interactively, and higher-level linguistic representations may affect early processing.

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Behavior

Individualized rTMS Treatment for Depression using an fMRI-Based Targeting Method
Xin Luo 1, Yiru Hu 1, Runhua Wang 1, Min Zhang 1, Xiaomei Zhong 1, Bin Zhang 1
1Affiliated Brain Hospital of Guangzhou Medical University

The present protocol describes the application of repetitive transcranial magnetic stimulation (rTMS), where a subregion of the dorsolateral prefrontal cortex (DLPFC) with the strongest functional anticorrelation with the subgenual anterior cingulate cortex (sgACC) was located as the stimulation target under the assistance of a fMRI-based neuronavigation system.

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Bioengineering

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)
Xingjin Jian *1,2, Xiaojie Guo *1,2,3, Jia Wang 4, Zheng Lin Tan 1,2,6, Xin-hui Xing 1,2,5, Liyan Wang 3, Chong Zhang 1,2,5
1Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, 2Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University, 3Luoyang TMAXTREE Biotechnology Co., Ltd., 4Biochemical Engineering Research Group, School of Chemical Engineering and Technology, Xi’an Jiaotong University, 5Center for Synthetic & Systems Biology, Tsinghua University, 6School of Life Science and Technology, Tokyo Institute of Technology

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

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Biochemistry

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature
Linta M. Biju *1, Cong Wang *1, Weijia Kang 1, Irin P. Tom 1, Indika Kumarapperuma 1, Xiaojing Yang 1,2, Zhong Ren 1,3
1Department of Chemistry, University of Illinois at Chicago, 2Department of Ophthalmology and Vision Sciences, University of Illinois at Chicago, 3Renz Research, Inc.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

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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

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
Yanan Zhu *1, Fajin Li *2,3, Xuerui Yang 2,3, Zhengtao Xiao 1
1School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, 2MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, 3Joint Graduate Program of Peking-Tsinghua-National Institute of Biological Science

Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome level.

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Biology

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology
Qiang Yan *1, Yingying He *2,3, Yang Yue 2,3, Luyao Jie 1, Tingmei Wen 2,3, Yumiao Zhao 2,3, Min Zhang 2, Tingting Zhang 2
1State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, School of Life Sciences, Henan University, 2Research Institute of Applied Biology, Shanxi University, 3School of Life Science, Shanxi University

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

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Medicine

Microscopic Replantation of Penile Glans Amputation Due to Circumcision
Da-Chun Jin *1, Bo Zhou *1, Jun Li 2, Cong-Cong Bao 1, Yong Luo 1, Yong Zhang 1, Peng Wang 1, Gang Bi 1, Yan-Feng Li 1
1Department of Urology, Daping Hospital, Army Medical University, 2Department of Urology, Chongqing 13th People's Hospital

The present protocol describes the emergency management of microscopic replantation of penile glans amputation due to circumcision.

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Medicine

The Miniature Pig: A Large Animal Model for Cochlear Implant Research
Xiaojun Ji *1,2,3,4, Yi Luo *1,2,3,4, Weiwei Guo 1,2,3,4, Fei Ji 1,2,3,4, Shuolong Yuan 1,2,3,4, Liangwei Xu 1,2,3,4, Wei Chen *1,2,3,4
1College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, 2National Clinical Research Center for Otolaryngologic Diseases, 3Key Lab of Hearing Science, Ministry of Education, 4Beijing Key Lab of Hearing Impairment for Prevention and Treatment

Miniature pigs (mini-pigs) are an ideal large animal model for research into cochlear implants. Cochlear implantation surgery in mini-pigs can be utilized to provide initial evidence of the safety and potential performance of novel electrode arrays and surgical approaches in a living system similar to human beings.

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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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Chemistry

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center
Chunli Song *1, Zhanfeng Hou *1,2, Zijun Jiao *1, Zhaodi Liu 3, Chenshan Lian 1,2, Min Zhang 1, Wei Liang 3, Feng Yin 1,2, Zigang Li 1,2
1Pingshan Translational Medicine Center, Shenzhen Bay Laboratory, 2Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 3Department of Radiation Oncology, the First Affiliated Hospital, Anhui Medical University

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

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Medicine

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica
Fuyu Jin 1, Yaqian Li 1, Tian Li 1, Xinyu Yang 1, Wenchen Cai 1, Shifeng Li 1, Xuemin Gao 1, Fang Yang 1, Hong Xu 1, Heliang Liu 1
1Hebei Key Laboratory for Organ Fibrosis Research, School of Public Health, North China University of Science and Technology

This study describes a technique to establish a silicosis rat model with the inhalation of silica through the whole body in an inhalation chamber. The rats with silicosis could closely mimic the pathological process of human silicosis in an easy, cost-effective manner with good repeatability.

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Medicine

Development of a Surgical Technique for Subretinal Implants in Rats
Juan Carlos Martinez Camarillo 1,2, Yuntao Hu 1,5, Biju B. Thomas 1,2, Danhong Zhu 3, David R. Hinton 2,3, Debbie Mitra 1,2, Jose Miguel Mora Correa 2, Deepthi S. Rajendran Nair 1,2, Jane Lebkowski 4, Mark S. Humayun 1,2
1USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, 2USC Dr. Allen and Charlotte Ginsburg Institute for Biomedical Therapeutics, University of Southern California, 3Department of Pathology, Keck School of Medicine of the University of Southern California, 4Regenerative Patch Technologies LLC, 5Eye center, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

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Engineering

A Micro-Displacement Measurement Based on the Shadow Technique
Yong Yang 1, Meirong Zhao 1, Yinguo Huang 1, Yu Tian 2, Yelong Zheng 1
1State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University, 2State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University

High-precision micro-displacement measurement is significant in the field of aerospace engineering, ultra-precision machining, and micro-assembly. The present protocol describes measuring micro displacements based on the shadow technique.

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Behavior

Evaluating Flight Performance and Eye Movement Patterns Using Virtual Reality Flight Simulator
Luoma Ke 1, Zhejun Zhang 2, Yidan Ma 1, Yi Xiao 3, Shengjun Wu 4, Xiuchao Wang 4, Xufeng Liu 4, Jibo He 1
1Psychology Department, School of Social Sciences, Tsinghua University, 2Faculty of Psychology, Beijing Normal University, 3National Key Laboratory of Human Factors Engineering, the China Astronaut Research and Training Center, 4Department of Military Medical Psychology, Air Force Medical University

A new virtual reality flight simulator was built, which enables efficient and low-cost evaluation of flight performance and eye movement patterns. It also provides a high-potential research tool for ergonomics and other research.

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Biology

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure
Zhaodi Jiang 1,2
1National Institute of Biological Sciences, Beijing, 2Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

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Neuroscience

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation
Qingyu Yao 1, Xuesong Luo 1, Jie Liu 2,3, Luming Li 1,2
1National Engineering Research Center of Neuromodulation, School of Aerospace Engineering, Tsinghua University, 2IDG/McGovern Institute for Brain Research, Tsinghua University, 3School of Life Sciences, Tsinghua University

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

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Bioengineering

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers
Yuyang Chen *1, Huanye Xu *2, Yuanyuan Zhang *2, Leilei Guo 2, Miaomiao Lan 2, Yanping Yang 2, Wei Liu 2, Xiaojun Yan 2, Yanan Du 1
1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Beijing CytoNiche Biotechnology Co. Ltd.

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

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Neuroscience

Biomarker Identification for Gender Specificity of Alzheimer's Disease Based on the Glial Transcriptome Profiles
Hanjie Liu *1, Hui Yang *2, Shuqing Liu 1, Siyu Li 1, Wen Yang 3, Anwar Ayesha 1, Xin Tan 4, Cen Jiang 1, Yi Liu 5, Lushuang Xie 1
1Chengdu University of Traditional Chinese Medicine, 2Chengdu Shuangliu Hospital of Traditional Chinese Medicine, 3Department of Automation, Tsinghua University, 4Affiliated Reproductive & Women-Children Hospital, Chengdu University of Traditional Chinese Medicine, 5Department of Neurology, Dalian Municipal Central Hospital, Central Hospital of Dalian University of Technology

This study analyzed single-nuclei transcriptomes of thirty-three individuals with Alzheimer's disease (AD), revealing sex-specific DEGs in glial cells. Functional enrichment analysis highlighted synaptic, neural, and hormone-related pathways. Key genes, namely NLGN4Y and its regulators, were identified, and potential therapeutic candidates for gender-specific AD were proposed.

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Biology

Enhancing Density Maps by Removing the Majority of Particles in Single Particle Cryogenic Electron Microscopy Final Stacks
Mengjia Cai *1, Jianying Zhu *2, Qi Zhang *3,4,5,6, Yu Xu 7, Zuoqiang Shi 2,8, Chenglong Bao 2,8,9, Mingxu Hu 1,5,6
1Institute of Bio-Architecture and Bio-Interactions (IBABI), Shenzhen Medical Academy of Research and Translation, 2Yau Mathematical Sciences Center, 3Key Laboratory of Protein Science (Tsinghua University), Ministry of Education, 4School of Life Science, Tsinghua University, 5Beijing Advanced Innovation Center for Structural Biology, 6Beijing Frontier Research Center for Biological Structure, 7Department of Mathematical Sciences, Tsinghua University, 8Yanqi Lake Beijing Institute of Mathematical Sciences and Applications, 9State Key Laboratory of Membrane Biology, School of Life Science

An advanced particle selection method for cryo-EM, namely CryoSieve, improves density map resolution by removing a majority of particles in final stacks, as demonstrated through its application on a real-world dataset.

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Neuroscience

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels
Pengtao Xu *1, MengYuan Guo *1, Wanping Lu *1, Yan Jiang 2, Lei Wang 1, Yunqian Li 1, Tao Lu 1, Xiaoling Liu 1
1School of Life Science, Beijing University of Chinese Medicine, 2School of Pharmaceutical Sciences, Tsinghua University

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

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