Potato Virus X-Based microRNA Silencing (VbMS) In Potato. J Vis Exp. 2020 May 11;(159): Authors: Zhao J, Rios CG, Song J Abstract Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we...
Cryogenic Liquid Jets for High Repetition Rate Discovery Science. J Vis Exp. 2020 May 09;(159): Authors: Curry CB, Schoenwaelder C, Goede S, Kim JB, Rehwald M, Treffert F, Zeil K, Glenzer SH, Gauthier M Abstract This protocol presents a detailed procedure for the operation of continuous, micron-sized cryogenic cylindrical and planar liquid jets. When operated as described here, the jet exhibits high laminarity and stability for centimeters. Successful...
In Vitro ELISA Test to Evaluate Rabies Vaccine Potency. J Vis Exp. 2020 May 11;(159): Authors: Jallet C, Tordo N Abstract The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives...
Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells. J Vis Exp. 2020 May 07;(159): Authors: Takizawa H, Sato M, Aoki Y Abstract Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry...
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage. J Vis Exp. 2020 May 06;(159): Authors: Matozel EK, Dale N, Price AC Abstract Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target...
Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes. J Vis Exp. 2020 May 05;(159): Authors: Liu Y, Liang Y, Wang M, Wang C, Wei H, Naruse K, Takahashi K Abstract Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human...
Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells. J Vis Exp. 2020 May 11;(159): Authors: Li T, Long C, Fanning KV, Zou C Abstract Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in...
Detection of Vascular Pathways of Oral Mucosa Influencing Soft- and Hard Tissue Surgeries by Latex Milk Injection. J Vis Exp. 2020 May 07;(159): Authors: Shahbazi A, Pilsl U, Molnár B, Feigl G Abstract In this methodological study, the purpose was to visualize the macroscopic vascular pathway of the oral mucosa. Corpses were injected and fixed with Thiel's solution for embalming to keep the natural color, fresh texture, and elasticity of the tissues....
In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish. J Vis Exp. 2020 May 08;(159): Authors: Qin X, Laroche FFJ, Peerzade SAMA, Lam A, Sokolov I, Feng H Abstract Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application....
Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration. J Vis Exp. 2020 May 09;(159): Authors: Frank D, Melamed I, Gruenbaum BF, Grinshpun J, Kuts R, Shvartsur R, Azab AN, Assadi MH, Vinokur M, Boyko M Abstract Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves...
Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series. J Vis Exp. 2020 May 10;(159): Authors: Partridge JD, Harshey RM Abstract Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement....
Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration. J Vis Exp. 2020 May 05;(159): Authors: Ali SA, Stebbins AW, Hanks JE, Kupfer RA, Hogikyan ND, Feldman EL, Brenner MJ Abstract This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal...
In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems. J Vis Exp. 2020 May 05;(159): Authors: Clerc EE, Raina JB, Lambert BS, Seymour J, Stocker R Abstract Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous...
In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation. J Vis Exp. 2020 May 06;(159): Authors: Kalebic N, Langen B, Helppi J, Kawasaki H, Huttner WB Abstract Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in...
Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM). J Vis Exp. 2020 May 08;(159): Authors: Beattie R, Streicher C, Amberg N, Cheung G, Contreras X, Hansen AH, Hippenmeyer S Abstract Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage...
Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy. J Vis Exp. 2020 May 08;(159): Authors: Makowski Ł, Trojanowski D, Zakrzewska-Czerwińska J Abstract Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus...
Optimizing Brillouin Optical Time-Domain Analyzers Based on Gain Spectrum Engineering. J Vis Exp. 2020 May 11;(159): Authors: Feng C, Schneider T Abstract Demonstrated is a unique method for sensing performance enhancement in Brillouin optical time-domain analyzers (BOTDA). A Brillouin gain spectrum (BGS) is superimposed with two symmetric Brillouin loss spectra (BLS). This leads to a complex engineered spectrum shape that is more resistant to the...
Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings. J Vis Exp. 2020 May 07;(159): Authors: Kraus L, Monni L, Schneider UC, Onken J, Spindler P, Holtkamp M, Fidzinski P Abstract Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are...
Magnetic Adjustment of Afterload in Engineered Heart Tissues. J Vis Exp. 2020 May 05;(159): Authors: Becker B, Rodriguez ML, Werner TR, Stenzig J, Eschenhagen T, Hirt MN Abstract Afterload is known to drive the development of both physiological and pathological cardiac states. As such, studying the outcomes of altered afterload states could yield important insights into the mechanisms controlling these critical processes. However, an experimental...
Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System. J Vis Exp. 2020 May 05;(159): Authors: Altman T, Maimon R, Ionescu A, Pery TG, Perlson E Abstract Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for...
Introducing Point Mutations into Human Pluripotent Stem Cells using Seamless Genome Editing. J Vis Exp. 2020 May 10;(159): Authors: Wang Y, Smith AJH, Hay DC Abstract Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4α)...
Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using χCRAC. J Vis Exp. 2020 May 09;(159): Authors: McKellar SW, Ivanova I, van Nues RW, Cordiner RA, Worboys W, Langford A, Jensen TH, Granneman S Abstract The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production,...
Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment. J Vis Exp. 2020 May 06;(159): Authors: Barosova H, Drasler B, Petri-Fink A, Rothen-Rutishauser B Abstract A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs]...
An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats. J Vis Exp. 2020 May 08;(159): Authors: Weigle CA, Lieke T, Vondran FWR, Timrott K, Klempnauer J, Beetz O Abstract Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic...
Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer. J Vis Exp. 2020 May 07;(159): Authors: van de Locht M, de Winter JM, Rassier DE, Helmes MHB, Ottenheijm CAC Abstract Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile...
Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ. J Vis Exp. 2020 May 08;(159): Authors: Espada J, Carrasco E, Calvo-Sánchez MI, Fernández-Martos S, Montoya JJ Abstract Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in...
Measuring Crop Motility and Food Passaging in Drosophila. J Vis Exp. 2020 May 09;(159): Authors: Cai J, Xi J, Wei Y Abstract Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The...
Induction of Leptomeningeal Cells Modification Via Intracisternal Injection. J Vis Exp. 2020 May 07;(159): Authors: Zamboni M, Santopolo G, Frisén J Abstract The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the...
Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory. J Vis Exp. 2020 May 11;(159): Authors: Nyhus E, Engel WA, Pitfield TD, Vakkur IMW Abstract Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol...
Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion. J Vis Exp. 2020 May 10;(159): Authors: Zhou L, Yau A, Zhang W, Chen Y Abstract A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 µm in length with inner hydrophobic...
Enucleation of the Prostate for the Treatment of Benign Prostatic Hyperplasia Using a 980 nm Diode Laser. J Vis Exp. 2020 May 05;(159): Authors: Zhang Y, Fu C, Wang J, Wu P, Chen X, Hou H, Song X, Cheng W, Wang J, Liu M Abstract In the aging male population, the occurrence of lower urinary tract symptoms (LUTS) caused by benign prostatic hyperplasia (BPH) is a common problem. Here, we introduce a new technique called 980 nm diode laser enucleation...
Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells. J Vis Exp. 2020 May 09;(159): Authors: Xiong J, Li Y, Tan X, Fu L Abstract Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods...
Simulator Training for Endovascular Neurosurgery. J Vis Exp. 2020 May 06;(159): Authors: Elsawaf Y, Rennert RC, Steinberg JA, Santiago-Dieppa DR, Olson SE, Khalessi AA, Pannell JS Abstract Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk environments. In the field of endovascular neurosurgery, the demand for consequence- and risk-free learning environments...
Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System. J Vis Exp. 2020 May 08;(159): Authors: Schalla MA, Kühne SG, Friedrich T, Hanel V, Kobelt P, Goebel-Stengel M, Rose M, Stengel A Abstract The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to...
Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System.
Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System. J Vis Exp. 2020 May 10;(159): Authors: Meseguer-Ripolles J, Wang Y, Sorteberg A, Sharma A, Ding NL, Lucendo-Villarin B, Kramer P, Segeritz CP, Hay DC Abstract Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability....
In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation. J Vis Exp. 2020 May 11;(159): Authors: Bączyk M, Krutki P Abstract Intracellular recording of spinal motoneurons in vivo provides a "gold standard" for determining the cells' electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording...
Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates. J Vis Exp. 2020 May 08;(159): Authors: Pillon MC, Stanley RE Abstract Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies...
Induction of Complete Transection-Type Spinal Cord Injury in Mice. J Vis Exp. 2020 May 06;(159): Authors: Reshamwala R, Eindorf T, Shah M, Smyth G, Shelper T, St John J, Ekberg J Abstract Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from...
Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques. J Vis Exp. 2020 May 07;(159): Authors: Faber CL, Matsen ME, Meek TH, Krull JE, Morton GJ Abstract Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include...
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Medicine by Alexandros G. Sfakianakis,Anapafseos 5 Agios Nikolaos 72100 Crete Greece,00302841026182,00306932607174,alsfakia@gmail.com,