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Huazhong University of Science and Technology

49 ARTICLES PUBLISHED IN JoVE

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Biology

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF
Wen Xiong 1, Yan Gao 1, Xun Cheng 1, Charles Martin 1, Dongmei Wu 1, Shuyuan Yao 1, Min-Ju Kim 2, Yang Liu 1
1Research and Development, Stemgent, 2Product Marketing, Stemgent

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

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Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans
Samuel H. Chung *1,2, Lin Sun *1,2, Christopher V. Gabel 1,2
1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

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Bioengineering

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars
Mahtab Hassanpourfard 1, Xiaohui Sun 2, Amin Valiei 1, Partha Mukherjee 3, Thomas Thundat 1, Yang Liu 2, Aloke Kumar 4
1Department of Chemical and Material Engineering, University of Alberta, 2Department of Civil and Environmental Engineering, University of Alberta, 3Department of Mechanical Engineering, Texas A&M University, 4Department of Mechanical Engineering, University of Alberta

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

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Bioengineering

Porous Silicon Microparticles for Delivery of siRNA Therapeutics
Jianliang Shen *1,2, Xiaoyan Wu *1,3, Yeonju Lee 1, Joy Wolfram 1,4, Zhizhou Yang 1, Zong-Wan Mao 2, Mauro Ferrari 1,5, Haifa Shen 1,6
1Department of Nanomedicine, Houston Methodist Research Institute, 2MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, 3Pediatrics Department of Union Hospital, Huazhong University of Science and Technology, 4CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology of China, 5Department of Medicine, Weill Cornell Medical College, 6Department of Cell and Developmental Biology, Weill Cornell Medical College

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

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Medicine

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury
Klaus Zweckberger 1, Yang Liu 1, Jian Wang 1, Nicole Forgione 1, Michael G. Fehlings 1,2,3
1Department of Genetics and Development, Toronto Western Research Institute and Spinal Program, University Health Network, Krembil Neuroscience Centre, 2Department of Surgery, University of Toronto, 3Institute of Medical Sciences, University of Toronto

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

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Medicine

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect
Bo Zhang 1,2, Jonathan P. Davis 1, Mark T. Ziolo 1
1Department of Physiology & Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, 2Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

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Bioengineering

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii
Swayamdipta Bhaduri 1, Nandini Debnath 1, Sushanta Mitra 2, Yang Liu 3, Aloke Kumar 1
1Department of Mechanical Engineering, University of Alberta, 2Department of Mechanical Engineering, York University, 3Department of Civil and Environmental Engineering, University of Alberta

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

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Bioengineering

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning
Daryl Sivakumaran 1, Emilia Bakaic 1, Scott B. Campbell 1, Fei Xu 1, Eva Mueller 1, Todd Hoare 1
1Department of Chemical Engineering, McMaster University

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

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Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing
Kristin A. Knouse 1,2,3, Jie Wu 4, Austin Hendricks 5
1Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical Institute, 3Division of Health Sciences and Technology, Harvard Medical School, 4The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 5BioMicro Center, Department of Biology, Massachusetts Institute of Technology

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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Genetics

Protocols for C-Brick DNA Standard Assembly Using Cpf1
Shi-Yuan Li 1, Guo-Ping Zhao 1, Jin Wang 1
1Institute 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.

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Medicine

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System
Wusong Zou 1, Yanqun Yang 1, Ye Gu 1, Pengfei Zhu 1, Mingjing Zhang 1, Zhong Cheng 1, Xiaoyan Liu 1, Yijun Yu 1, Xiaohong Peng 1
1Heart Center at Puai Hospital, Wuhan Puai Hospital, Jianghan University

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

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Developmental Biology

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging
Nan Zhang 1,2, Liakot A Khan 1, Edward Membreno 1, Gholamali Jafari 1, Siyang Yan 1, Hongjie Zhang 1,3, Verena Gobel 1
1Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital, Harvard Medical School, 2College of Life Sciences, Jilin University, 3Faculty of Health Sciences, University of Macau

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

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Developmental Biology

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging
Nan Zhang 1,2, Edward Membreno 1, Susan Raj 1, Hongjie Zhang 1,3, Liakot A Khan 1, Verena Gobel 1
1Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital for Children, Harvard Medical School, 2College of Life Sciences, Jilin University, 3Faculty of Health Sciences, University of Macau

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

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Biochemistry

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

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

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Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay
Yanxin Lu 1,2, Yang Liu 1, Chunzhang Yang 1
1Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, 2Basic Medical Science Department, Zunyi Medical College-Zhuhai Campus

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

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Neuroscience

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury
Chen Chen 1,2, Yi Ping Zhang 3, Yan Sun 1,4, Wenhui Xiong 1, Lisa B. E. Shields 3, Christopher B. Shields 3,5, Xiaoming Jin 1, Xiao-Ming Xu 1
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, and Department of Neurological Surgery, Indiana University School of Medicine, 2Program in Medical Neuroscience, Stark Neurosciences Research Institute, Indiana University School of Medicine, 3Norton Neuroscience Institute, Norton Healthcare, 4Department of Human Anatomy & Histoembryology, School of Basic Medical Sciences, Fudan University, 5Department of Neurological Surgery, University of Louisville School of Medicine

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

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Biology

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents
Feng Pan *2, Jie Zhang *3, Yuyan Liu 1, Liangsheng Lu 1, Xuefeng Qiu 4, Kangtai Lv 5, Qipeng Zhang 1
1State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, Nanjing University, 2Department of Andrology, The Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, 3School of Pharmaceutical Sciences, Nanjing Tech University, 4Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, 5Department of Ultrasound, Nanjing Qixia District Maternity and Child Health Care Hospital

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

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Chemistry

Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations
Ren Wei Toh 1, Jie Sheng Li 1, Jie Wu 1
1Department of Chemistry, National University of Singapore

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

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Medicine

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats
Xiao-Jing Lin 1,2,3, Shaonan Wen 2,6, Ling-Xiao Deng 3, Heqiao Dai 3, Xiaolong Du 3, Chen Chen 3,4, Melissa J. Walker 3,4, Ting-Bao Zhao 1,5, Xiao-Ming Xu 3
1Department of Spinal Cord Injury and Repair, Trauma and Orthopedics Institute of Chinese PLA, 960th Hospital, Joint Logistics Support Force of PLA, 2Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Academy of Military Sciences, 3Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman Campbell Brain and Spine, Indiana University School of Medicine, 4Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, 5Department of Orthopedics, the Third Affiliated Hospital, Shandong University, 6Beijing Computing Center, Beijing Academy of Science and Technology

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

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Bioengineering

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart
Yichen Ding 1, Zachary Bailey 2, Victoria Messerschmidt 2, Jun Nie 3, Richard Bryant 2, Sandra Rugonyi 4, Peng Fei 3, Juhyun Lee 1,2, Tzung K. Hsiai 1
1Department of Bioengineering, University of California Los Angeles, 2Department of Bioengineering, University of Texas at Arlington, 3School of Optical and Electronic Information, Huazhong University of Science and Technology, 4Department of Biomedical Engineering, OSHU

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

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Engineering

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro
Zilan Xiong *1, Shasha Zhao *2, Xu Yan 3
1State Key Laboratory of Advanced Electromagnetic Engineering and Technology, Huazhong University of Science and Technology, 2College of Life Science and Health, Wuhan University of Science and Technology, 3Department of Pathophysiology, Beijing Neurosurgical Institute/Beijing Tiantan Hospital, Capital Medical University

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

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Neuroscience

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5
Shuiqing Chi 1, Mijing Fang 1, Kang Li 1, Li Yang 2, Shao-tao Tang 1
1Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center

This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung's disease has preferable sensitivity and specificity rates.

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Immunology and Infection

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing
Lin Sun 1, Naoko Kono 2, Hiroyuki Toh 3, Hanbing Xue 1, Kaori Sano 4,5, Tadaki Suzuki 4, Akira Ainai 4, Yasuko Orba 6, Junya Yamagishi 7,8, Hideki Hasegawa 4,5, Yoshimasa Takahashi 9, Shigeyuki Itamura 2, Kazuo Ohnishi 9,10
1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Center for Influenza Virus Research, National Institute of Infectious Diseases, 3School of Science and Technology, Kwansei Gakuin University, 4Department of Pathology, National Institute of Infectious Diseases, 5Division of Infectious Diseases Pathology, Department of Global Infectious Diseases, Tohoku University Graduate School of Medicine, 6Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, 7Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, 8Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, 9Department of Immunology, National Institute of Infectious Diseases, 10Faculty of Life and Environmental Sciences, University of Tsukuba

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

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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 Zhang 1, Chi Xu 3, Haiming Jing 1,4, Wenjing Zhang 1,4, Gaochao Han 1,4, Matthew Flavel 5, Markandeya Jois 5, Yingxin Zeng 1, Jing-Dong J. Han 3, Bo Xian 3, Guojun Li 1,4
1Beijing 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.

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Behavior

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier
Xiaoyan Liu *1, Huanhuan Li *1, Jie Wu 1, Yijun Yu 1, Mingjing Zhang 1, Wusong Zou 1, Ye Gu 1
1Heart Center, Wuhan Fourth Hospital, Puai Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

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Cancer Research

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research
Zhuo Li *1, Xiao Yang *1, Yilu Zhang 1, Xiaolong Yang 1, Xinxin Cui 1, Yanjuan Zhang 1, Wenfeng Gong 1, Huichen Bai 1, Ning Liu 1, Zhiyu Tang 1, Mingming Guo 1, Kang Li 1, Tong Zhang 1, Lai Wang 1, Xiaomin Song 1
1BeiGene (Beijing) Co., Ltd.

We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

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Medicine

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice
Fuliang Luo 1, Xin Wang 1, Xiaokang Luo 1, Bo Li 1, Desheng Zhu 2, Hansong Sun 3, Yue Tang 1
1Beijing Key Laboratory of Pre-clinical Research and Evaluation for Cardiovascular Implant Materials, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Laboratory Animal Center, Peking University, 3Center of Cardiac Surgery for Adults, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

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Medicine

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Di Liu *1, Xizhu Xu *2, Yuejin Li 2, Jie Zhang 1, Xiaoyu Zhang 1, Qihuan Li 1, Haifeng Hou 2, Dong Li 2, Wei Wang 1,2,3, Youxin Wang 1
1Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, 2School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, 3School of Medical and Health Sciences, Edith Cowan University

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

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Behavior

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training
Haifeng Zhang 1,2, Jinlei Nie 3, Zhaowei Kong 4, Xiangui Zhu 1, Yang Liu 1, Qingde Shi 3
1Physical Education College, Hebei Normal University, 2Hebei Provincial Key Lab of Measurement and Evaluation in Human Movement and Bio-Information, 3School of Health Sciences and Sports, Macao Polytechnic Institute, 4Faculty of Education, University of Macau

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

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JoVE Core

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice
Jie Zhang 1,2, Fang Fang 1,3, Liang Li 1, Haoliang Huang 1, Hannah C. Webber 1, Yang Sun 1,4, Vinit B. Mahajan 1,4, Yang Hu 1
1Department of Ophthalmology, Stanford University School of Medicine, 2Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 3Department of Ophthalmology, Second Xiangya Hospital of Central South University, 4Department of Ophthalmology, Veterans Affairs Palo Alto Health Care

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

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Engineering

In Situ Measurement of Vacuum Window Birefringence using 25Mg+ Fluorescence
W. H. Yuan 1, H. L. Liu 1, W. Z. Wei 1, Z. Y. Ma 1, P. Hao 1, Z. Deng 1, K. Deng 1, J. Zhang 1, Z. H. Lu 1
1MOE Key Laboratory of Fundamental Physical Quantities Measurement, Hubei Key Laboratory of Gravitation and Quantum Physics, PGMF and School of Physics, Huazhong University of Science and Technology

Presented here is a method to measure the birefringence of vacuum windows by maximizing the fluorescence counts emitted by Doppler cooled 25Mg+ ions in an ion trap. The birefringence of vacuum windows will change the polarization states of the laser, which can be compensated by changing the azimuthal angles of external wave plates.

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Genetics

Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size (LEfSe) in Microbiome Data
Fang Chang *1, Shishi He *2, Chenyuan Dang 3
1Marine Resources Research Centre, Tianjin Research Institute for Water Transport Engineering, M.O.T., 2Key Laboratory of Regional Energy Systems Optimization, Ministry of Education, College of Environmental Science and Engineering, North China Electric Power University, 3School of Environmental Science and Engineering, Huazhong University of Science and Technology

LEfSe (LDA Effect Size) is a tool for high-dimensional biomarker mining to identify genomic features (such as genes, pathways, and taxonomies) that significantly characterize two or more groups in microbiome data.

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Neuroscience

Free-floating Immunostaining of Mouse Brains
Longlong Tu 1, Nan Zhang 1,2,3, Kristine M Conde 1, Jonathan C Bean 1, Chunmei Wang 1, Yong Xu 1,4
1USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 2Department of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 3Hubei Provincial Clinical Research Center for Diabetes and Metabolic Disorder, 4Department of Molecular and Cellular Biology, Baylor College of Medicine

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

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Medicine

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma
Yalan Yu *1, Hui Shen *1, Li Liu 1, Ping Luo 1, Sanyun Wu 1, Jing He 1,2, Xiqin Tong 1, Yufeng Shang 1, Liang Shao 1, Fuling Zhou 1
1Department of Hematology, Zhongnan Hospital of Wuhan University, 2Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

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Medicine

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice
Bingjie Lv *1, Jin Zhou *1, Shaolin He *1, Yuqi Zheng 1, Wenlin Yang 1, Shangwei Liu 1, Chengxing Liu 1, Boyuan Wang 1, Dazhu Li 1, Jibin Lin 1
1Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

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Developmental Biology

In Ovo Intravascular Injection in Chicken Embryos
Kai Jin 1,2, Jing Zhou 1,2, Gaoyuan Wu 1,2, Ziyi Lian 1,2, Zongyi Zhao 1,2, Shujian Zhou 1,2, Chen Chen 1,2, Hongyan Sun 1,2, Yingjie Niu 1,2, Qishenng Zuo 1,2, Yani Zhang 1,2, Jiuzhou Song 3, Guohong Chen 1,2, Bichun Li 1,2,4
1Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, 2Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, 3Animal & Avian Sciences, University of Maryland, College Park, 4College of Biotechnology, Jiangsu University of Science and Technology

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

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Neuroscience

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination
Hang Zhang 1, Sheng Yang 1, Man Chen 1, Dai-Shi Tian 1, Chuan Qin 1
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

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Biology

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping
Chao Song 1,2, Arianna Valeri 1, Fanxin Long 1
1Translational Research Program of Pediatric Orthopedics, Department of Surgery, The Children's Hospital of Philadelphia, 2Department of Orthopedic Surgery, Tongji Hospital, Huazhong University of Science and Technology

This protocol describes an easy-to-use method to examine substrate oxidation by tracking 14COproduction in vitro.

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Medicine

Clinical Application of 24 G Cannula Needle and 3-0 Polypropylene Suture in Vas Deferens Exploration
Jin Wang *1, Wenjia Li *2, Yao He 1, Yuanbin Xia 1, Lin Sun 1, Youpeng Zhang 1, Zhaohui Zhu 1
1Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

This protocol presents the clinical application of a 24 G cannula and 3-0 polypropylene suture as a simple and effective method for the exploration of the vas deferens.

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Neuroscience

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice
Xiao-Wei Pang 1, Man Chen 1, Yun-Hui Chu 1, Yue Tang 1, Chuan Qin 1, Dai-Shi Tian 1
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.

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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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Cancer Research

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice
Qibin Fu 1, Yan Zhang 1, Tuchen Huang 1, Yang Liu 1
1Sino-French Institute of Nuclear Engineering and Technology, Sun Yat-sen University

Here a protocol is presented to build a fast and non-destructive system for measuring cell or nucleus compressibility based on acoustofluidic microdevice. Changes in mechanical properties of tumor cells after epithelial-mesenchymal transition or ionizing radiation were investigated, demonstrating the application prospect of this method in scientific research and clinical practice.

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Biology

A Modified Technique for Transverse Aortic Constriction in Mice
Kudusi Abuduwufuer *1,2, James Jiqi Wang *1,2, Huihui Li *1,2, Chen Chen 1,2
1Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Hubei Key Laboratory of Genetics and Molecular Mechanism of Cardiological Disorders, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

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Engineering

A Teleoperated Robotic System-Assisted Percutaneous Transiliac-Transsacral Screw Fixation Technique
Binbin Liu 1, Fei Xu 1, Yang Liu 1, Tianqi Wang 2, Yajing Cao 2, Zehang Zheng 1, Hanqing Xu 1, Chuang Huang 1, Zhengqiang Luo 1
1Department of Orthopedics, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 2Wuhan United Imaging Healthcare Surgical Technology Co. Ltd.

Teleoperated robotic system-assisted percutaneous transiliac-transsacral screw fixation is a feasible technique. Screw channels can be implemented with high accuracy owing to the excellent freedom of movement and stability of the robotic arms.

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Cancer Research

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression
Lulu Zhang *1, Siqi Zhang *2, Wenyu Wu 1, Xingkai Wang 2, Jieting Shen 2, Dongyuan Wang 4, Kuan Hu *2,3, Ming-Rong Zhang *3, Feng Wang *1, Rui Wang *2
1Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, 2State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Department of Advanced Nuclear Medicine Sciences, Institute for Quantum Medical Science, National Institutes for Quantum Science and Technology, 4Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

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Biology

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia
Yangpeng Li *1,2, Jun Yang *1, Rui Zhang 1, Tangting Chen 1, Shiyu Zhang 1, Yuqing Zheng 1, Qiang Wen 3, Tao Li 1, Xiaoqiu Tan 1,2, Ming Lei 1,4, Xianhong Ou 1
1Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, 2Department of Cardiology, the Affiliated Hospital of Southwest Medical University, 3Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 4Department of Pharmacology, University of Oxford

This protocol introduces dual-dye optical mapping of mouse hearts obtained from wild-type and knock-in animals affected by catecholaminergic polymorphic ventricular tachycardia, including electrophysiological measurements of transmembrane voltage and intracellular Ca2+ transients with high temporal and spatial resolution.

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Medicine

Isolation and Identification of Limbal Niche Cells
Guanyu Su 1, Wei Wang 1, Lingjuan Xu 1, Rong Liu 1, Yongyao Tan 1, Bihui Jin 1, Chunxiu You 1, Xiao Zhou 1, Yihong Xiong 2, Huatao Xie 3, Guigang Li 1
1Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Institute of Medicine Nursing, Hubei University of Medicine, 3Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

Here, we present a protocol to isolate and identify the human limbal niche cells.

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Engineering

A Flexible Wearable Supernumerary Robotic Limb for Chronic Stroke Patients
Hongge Ru 1, WeiJian Gao 2, Weixuan Ou 2, Xingyue Yang 1, Andong Li 1, Zhongzheng Fu 1, Jun Huo 1, Bo Yang 1, Yanzhao Zhang 3, Xiling Xiao 3, Zhaohui Yang 3, Jian Huang 1
1Key Laboratory for Image Processing and Intelligent Control of Education Ministry of China, School of Artificial Intelligence and Automation, Huazhong University of Science and Technology, 2School of Mechanical Science and Engineering, Huazhong University of Science and Technology, 3Wuhan Union Hospital, Huazhong University of Science and Technology Tongji Medical College First Clinical College Union Hospital

This protocol introduces a flexible wearable supernumerary robotic limb tailored to assist in finger rehabilitation for stroke patients. The design incorporates a bending sensor to facilitate seamless human-robot interaction. Validation through experiments involving both healthy volunteers and stroke patients underscores the efficacy and dependability of the proposed study.

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Medicine

Roller Microneedle Combined with Tranexamic Acid Solution in Treating Melasma
Wenhao Zheng *1, Sutao Liu *1, Liming Tian 2,3, Dan Ke 1
1Department of Dermatology, Chongqing Hospital of Traditional Chinese Medicine, 2Department of Dermatology, Hospital of Traditional Chinese and Western Medicine of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, 3Hubei Provincial Key Laboratory of Skin Infection and Immunity, Wuhan No.1 Hospital

This article presents a protocol to conduct the treatment of melasma by using roller microneedles in combination with tranexamic acid solution and evaluate the efficacy.

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