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20 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
Ronald W. Jensen 1, Jason Rivest 1, Wei Li 1, Varalakshmi Vissa 1
1Department of Microbiology, Immunology and Pathology, Colorado State University

Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies
Wei Li 1, Shu Zhu 1, Yusong Zhang 1, Jianhua Li 1, Andrew E. Sama 1, Ping Wang 1, Haichao Wang 1
1The Feinstein Institute for Medical Research, North Shore – LIJ Health System

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

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Immunology and Infection

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy
Peter A. Keyel 1, Michelle E. Heid 1, Simon C. Watkins 2, Russell D. Salter 1
1Department of Immunology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

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Biology

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
Graham Bailes 1, Margaret Lind 1, Andrew Ely 1, Marianne Powell 2, Jennifer Moore-Kucera 3, Carol Miles 2, Debra Inglis 2, Marion Brodhagen 1
1Biology Department, Western Washington University, 2Washington State University Northwestern Research and Extension Center, 3Department of Plant and Soil Science, Texas Tech University

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

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JoVE Core

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots
Jing Zhao 1, Wei Li 1,2, Xiaoqian Mao 1, Mengfan Li 1
1School of Electrical Engineering and Automation, Tianjin University, 2Department of Computer & Electrical Engineering and Computer Science, California State University

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

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Medicine

Ferric Chloride-induced Murine Thrombosis Models
Wei Li 1,2, Marvin Nieman 3, Anirban Sen Gupta 4
1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 3Department of Pharmacology, Case Western Reserve University, 4Department of Biomedical Engineering, Case Western Reserve University

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

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Education

Reaction Kinetics and Combustion Dynamics of I4O9 and Aluminum Mixtures
Dylan K. Smith 1, Michelle L. Pantoya 1, Jeffrey S. Parkey 2, Mehmet Kesmez 2
1Department of Mechanical Engineering, Texas Tech University, 2Lynntech Inc.

A protocol for measuring flame speeds of a reactive mixture composed of tetraiodine nonoxide (I4O9) and aluminum (Al) is presented. A method for resolving reaction kinetics using differential scanning calorimetry (DSC) is also presented. It was found that I4O9 is 150% more reactive than other iodine(V) oxides.

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JoVE Journal

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development
Vishnu V. Krishnamurthy *1, Aurora J. Turgeon *2, John S. Khamo 1, Payel Mondal 1, Savanna R. Sharum 1, Wenyan Mei 2, Jing Yang 2, Kai Zhang 1,3,4
1Department of Biochemistry, University of Illinois at Urbana-Champaign, 2Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, 3Neuroscience Program, University of Illinois at Urbana-Champaign, 4Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

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Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Wei Li 1,2, Chen Zhang 1, Xiulian Sun 3
1Department of Neurology, Qilu Hospital of Shandong University, 2Department of Neurology, Qingdao Municipal Hospital, 3Brain Research Institute, Qilu Hospital of Shandong University

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

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Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis
Zemfira N. Karamysheva 2, Elena B. Tikhonova 1, Petar N. Grozdanov 1, James C. Huffman 2,3, Kristen R. Baca 1,3, Alexander Karamyshev 1, R. Brian Denison 1, Clinton C. MacDonald 1, Kai Zhang 2, Andrey L. Karamyshev 1
1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 2Department of Biological Sciences, Texas Tech University, 3CISER (Center for the Integration of STEM Education & Research), Texas Tech University

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

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Engineering

Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy
Kai Zhang 1,2, Nima Davoudzadeh 1,2, Guillaume Ducourthial 1,2, Bryan Q. Spring 1,2,3
1Translational Biophotonics Cluster, Northeastern University, 2Department of Physics, Northeastern University, 3Department of Bioengineering, Northeastern University

A method is presented to build a custom low-cost, mode-locked femtosecond fiber laser for potential applications in multiphoton microscopy, endoscopy, and photomedicine. This laser is built using commercially available parts and basic splicing techniques.

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Chemistry

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium
Kiyoharu J. Miyagishima 1, Congxiao Zhang *2, Volha V. Malechka *3, Kapil Bharti 2, Wei Li 1
1Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, 2Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health, 3Human Visual Function Core, National Eye Institute, National Institutes of Health

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

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Engineering

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices
Celine R. Garcia *1, Zhenya Ding *1, Hilario C. Garza 1, Wei Li 1
1Department of Chemical Engineering, Texas Tech University

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of <10 µm were achieved.

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Engineering

Calibration Procedures for Orthogonal Superposition Rheology
Ran Tao 1,2, Aaron M. Forster 1
1Material Measurement Laboratory, National Institute of Standards and Technology, 2Department of Chemical Engineering, Texas Tech University

We present a detailed calibration protocol for a commercial orthogonal superposition rheology technique using Newtonian fluids including end-effect correction factor determination methods and recommendations for best practices to reduce experimental error.

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Environment

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method
Lavanya Dampanaboina 1, Ning Yuan 2, Venugopal Mendu 2
1Department of Plant and Soil Science, Texas Tech University, 2Fiber and Biopolymer Research Institute (FBRI), Department of Plant and Soil Science, Texas Tech University

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

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Biochemistry

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)
Lavanya Dampanaboina 1, Ning Yuan 2, Venugopal Mendu 2
1Department of Plant and Soil Science, Texas Tech University, 2Fiber and Biopolymer Research Institute (FBRI), Department of Plant and Soil Science, Texas Tech University

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

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Biochemistry

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter
Florence B Kineavy 1, Alex A Davies 1, Molly R Mitchell 1, Daniel Lay 1, Matt J Dominguez 2, Elliott J Stollar 1
1School of Life Sciences, University of Liverpool, 2Health Sciences Center, Texas Tech University

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

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Engineering

Solution Blow Spinning of Polymeric Nano-Composite Fibers for Personal Protective Equipment
Zois Tsinas 1,2, Ran Tao 1,3, Amanda L. Forster 1
1Material Measurement Laboratory, National Institute of Standards and Technology, 2Theiss Research, 3Department of Chemical Engineering, Texas Tech University

The primary goal of this study is to describe a protocol to prepare polymeric fiber mats with consistent morphology via solution blow spinning (SBS). We aim to use SBS to develop novel, tunable, flexible polymeric fiber nanocomposites for various applications, including protective materials, by incorporating nanoparticles in a polymer-elastomer matrix.

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Biology

Primary Culture of Porcine Retinal Pigment Epithelial Cells
Feng Wen *1,2,3, Yanzi Wang *1,2,3, Danxue He 1,2,3, Chunyan Liao 1,2,3, Weijie Ouyang 1,2,3, Zuguo Liu 1,2,3, Wei Li 1,2,3, Yi Liao 1,2,3
1Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, School of Medicine, Xiamen University, 2Department of Ophthalmology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, 3Xiamen University Affiliated Xiamen Eye Center, School of Medicine, Xiamen University

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

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Immunology and Infection

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major
Chaitanya S. Haram 1, Samrat Moitra 1, Rilee Keane 1, Elana Breslav 1, Kai Zhang 1, Peter A. Keyel 1
1Department of Biological Sciences, Texas Tech University

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

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