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21 ARTICLES PUBLISHED IN JoVE

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Medicine

Vascular Occlusion Training for Inclusion Body Myositis: A Novel Therapeutic Approach
Bruno Gualano 1,2, Carlos Ugrinowitsch 1, Manoel Neves Jr. 2, Fernanda R. Lima 2, Ana Lúcia S. Pinto 2, Gilberto Laurentino 1, Valmor A.A. Tricoli 1, Antonio H. Lancha Jr. 1, Hamilton Roschel 1,2
1School of Physical Education and Sport, University of São Paulo, 2Division of Rheumatology, School of Medicine, University of São Paulo

The present article presents the details pertaining to the application of resistance training associated to vascular occlusion in IBM patients.

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Biology

Determining the Contribution of the Energy Systems During Exercise
Guilherme G. Artioli 1, Rômulo C. Bertuzzi 2, Hamilton Roschel 1,3, Sandro H. Mendes 1, Antonio H. Lancha Jr. 1, Emerson Franchini 4
1Laboratory of Applied Nutrition, School of Physical Education and Sport, University of Sao Paulo, 2Aerobic Performance Research Group, School of Physical Education and Sport, University of Sao Paulo, 3Laboratory of Neuromuscular Adaptations to Strength Training, School of Physical Education and Sport, University of Sao Paulo, 4Martial Arts and Combat Sports Research Group, School of Physical Education and Sport, University of Sao Paulo

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

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Medicine

Oral Biofilm Formation on Different Materials for Dental Implants
Thalisson S. O. Silva 1, Alice R. Freitas 1, Marília L. L. Pinheiro 1, Cássio do Nascimento 1, Evandro Watanabe 2, Rubens F. Albuquerque 1
1Department of Dental Materials and Prosthodontics, School of Dentistry of Ribeirão Preto, University of São Paulo, 2Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

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Developmental Biology

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells
Amanda B.T. Hill 1,2, Jonathan E.B.T. Hill 1, Fabiana F. Bressan 3, Maria Angélica Miglino 4, Joaquim M. Garcia 1
1The School of Agricultural and Veterinary Studies (FCAV), Jaboticabal, São Paulo State University (UNESP), 2CRRF, Université de Montréal, 3FZEA, University of São Paulo, 4FMVZ, University of São Paulo

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

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Neuroscience

Short-Term Free-Floating Slice Cultures from the Adult Human Brain
Artur Fernandes 1,2, Niele Dias Mendes 1,3, Glaucia Maria Almeida 1, Giovanna Orlovski Nogueira 1, Carla de Moraes Machado 4, Jose de Anchieta de Castro Horta-Junior 4, João Alberto Assirati Junior 5, Norberto Garcia-Cairasco 2, Luciano Neder 3, Adriano Sebollela 1
1Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, 2Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, 3Department of Pathology and Forensic Medicine, Ribeirão Preto Medical School, University of São Paulo, 4Department of Anatomy, Institute of Biosciences, São Paulo State University, 5Clinical Hospital at the Ribeirão Preto Medical School, University of São Paulo

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

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Medicine

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish
Camila Morales Fénero *1, Barbara Nunes Padovani *1, Mariana Abrantes do Amaral *1,2, Guilherme José Bottura de Barros 1, Izabella Karina Xavier de Oliveira 1, Meire Ioshie Hiyane 1, Niels Olsen Saraiva Camâra 1,2
1Department of Immunology, University of São Paulo, 2Department of Medicine, Nephrology Division, Federal University of São Paulo

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

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Integrated One-Pot Process to Fabricate and Impregnate Starch Aerogels in Supercritical Carbon Dioxide
Maria E. Villegas *1,2, Alessandra L. Oliveira *2, Reinaldo C. Bazito *1, Pedro Vidinha *1
1Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, 2Faculty of Zootechnics and Food Engineering, University of São Paulo

This protocol describes the development of a one-pot strategy for the fabrication and impregnation of starch aerogels. Some modifications were made within the traditional fabrication process, which allowed the integration of the three critical steps (gelatinization, retrogradation, and drying) of aerogel fabrication into a single step.

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JoVE Core

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries
Sabrina M. Ribeiro *1, Érick D. O. Fratucelli *1, Júlia M. Fernandes *2, Paula C. P. Bueno 2,3, Alberto José Cavalheiro 2, Marlise I. Klein 1
1Department of Dental Materials and Prosthodontics, School of Dentistry, São Paulo State University (UNESP), 2Department of Organic Chemistry, Institute of Chemistry, São Paulo State University (UNESP), 3Faculty of Pharmaceutical Sciences of Ribeiraão Preto (FCFRP-USP), Department of Physics and Chemistry, University of São Paulo

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

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Developmental Biology

Differentiation of Porcine Induced Pluripotent Stem Cells (piPSCs) into Neural Progenitor Cells (NPCs)
Lucas Simões Machado 1, Kaiana Recchia 1,2, Naira Caroline Godoy Pieri 2, Ramon Cesar Botigelli 2,3, Raquel Vasconcelos Guimarães de Castro 2, Jéssica Brunhara Cruz 1, Laís Vicari de Figueiredo Pessôa 2, Fabiana Fernandes Bressan 1,2
1Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, 2Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, 3Department of Pharmacology, Institute of Biosciences, São Paulo State University

This protocol describes a method for chemical differentiation and culture of neural progenitor cells derived from porcine induced pluripotent stem cells (piPSCs).

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Immunology and Infection

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions
André Nicolau Aquime Gonçalves 1,2, Vanessa Escolano Maso 3, Ícaro Maia Santos de Castro 2,3, Amanda Pereira Vasconcelos 3, Rodrigo Luiz Tomio Ogava 2,3, Helder I Nakaya 2,3,4
1Laboratory of Pathology of Infectious Diseases, Department of Pathology, Medical School, University of São Paulo, 2Scientific Platform Pasteur USP, 3Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, 4Hospital Israelita Albert Einstein

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

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Medicine

Low-Cost Single-Port (LoCoSP) Device for a Transcervical Approach in Minimally Invasive Transhiatal Esophagectomy
Flavio Roberto Takeda 1, Rubens Antonio Aissar Sallum 1, Felipe Alexandre Fernandes 1, Ivan Cecconello 1
1Gastroenterology Department, University of São Paulo

Here, we describe a step-by-step description of transhiatal esophagectomy and the development of a low-cost single-port device for a transcervical approach in a minimally invasive transhiatal esophagectomy.

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Genetics

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies
Natália Yumi Noronha *1, Guilherme da Silva Rodrigues *1, Marcela Augusta de Souza Pinhel 1, Jean-Baptiste Cazier 2,3, Lígia Moriguchi Watanabe 1, Albert Nobre Menezes 4, Carlos Roberto Bueno 5, Carolina Ferreira Nicoletti 1, Bruno Affonso Parenti de Oliveira 1, Isabelle Mello Schineider 6, Isabella Harumi Yonehara Noma 7, Igor Caetano Dias Alcarás 8, Fernando Barbosa 9, Carla Barbosa Nonino 6
1Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, 2Centre for Computational Biology, University of Birmingham, 3Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, 4Cancer Genetics and Evolution Laboratory, Cancer Research UK, Institute of Genetics & Molecular Medicine, The University of Edinburgh, 5Ribeirão Preto School of Physical Education and Sport, University of São Paulo, 6Health Sciences Department, Ribeirao Preto Medical School, University of Sao Paulo, 7Faculty of Pharmaceutical Sciences, University of São Paulo, 8Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, 9Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo

The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

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Neuroscience

The 6-hydroxydopamine Rat Model of Parkinson's Disease
Rayanne Poletti Guimarães 1, Danilo Leandro Ribeiro 1, Keila Bariotto dos Santos 1, Lívea Dornela Godoy 1, Mirella Rosine Corrêa 1, Fernando Eduardo Padovan-Neto 1
1Department of Psychology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

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Biology

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease
Keila Bariotto-dos-Santos *1, Danilo Leandro Ribeiro *1, Rayanne Poletti Guimarães 1, Fernando Eduardo Padovan-Neto 1
1Department of Psychology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

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Biology

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction
João Paulo Rodrigues Marques 1, Laura Galvan Nuevo 2
1Faculty of Animal Science and Food Engineering, Department of Basic Sciences, University of São Paulo, 2Laboratory of Nuclear Instrumentation, Center of Nuclear Energy in Agriculture, University of São Paulo

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

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Genetics

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
Davi J. Martins *1, Stephanie Tirman *2, Annabel Quinet 2, Carlos F. M. Menck 1
1Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, 2Division of Oncology, Department of Internal Medicine, Washington University in St. Louis

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

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Biochemistry

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
Patrícia M. S. dos Passos *1, Valentine Spagnol *2, Camila R.S.T.B de Correia 1, Felipe R. Teixeira 1,2
1Department of Genetic and Evolution, Federal University of São Carlos, 2Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

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Medicine

Robotic Myotomy and Partial Fundoplication for Achalasia
Rubens Antonio Aissar Sallum 1, Felipe Alexandre Fernandes 1, Leonardo Torres Branco 1, Luna Serena Arguelho Pereira 1, Camila Maria Arruda Vilanova de Câmara 1, Ítalo Beltrão Pereira Simões 1, José Donizeti de Meira Junior 1, Melissa Mello Mazepa 1, Leonardo Ervolino Corbi 1, Rodrigo Nicida Garcia 1, Flávio Roberto Takeda 1
1Gastroenterology Department, University of São Paulo

Surgical myotomy with a partial fundoplication may be used in selected patients as a definitive treatment for achalasia. This article provides a step-by-step description of a robotic myotomy and partial fundoplication in a 32-year-old patient with megaesophagus.

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JoVE Core

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells
Rafael Badain 1, Daniel S. C. Damineli 1,2, Maria Teresa Portes 3, José Feijó 4, Stefano Buratti 5, Giorgia Tortora 6, Hugo Neves de Oliveira 1, Roberto M. Cesar Jr 1
1Institute of Mathematics and Statistics, University of São Paulo, 2Center for Mathematics, Computing and Cognition, Federal University of ABC, 3Department of Botany, Institute of Biosciences, University of São Paulo, 4Cell Biology and Molecular Genetics Department, University of Maryland, 5Department of Biosciences, University of Milan, 6Department of Physics, Politecnico di Milano

Current methods for analyzing the intracellular dynamics of polarized single cells are often manual and lack standardization. This manuscript introduces a novel image analysis pipeline for automating midline extraction of single polarized cells and quantifying spatiotemporal behavior from time lapses in a user-friendly online interface.

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Medicine

Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography
Yousif Al-Khoury 1,2, Stephanie Finzel 3, Camille Figueiredo 4, Andrew J. Burghardt 5, Kathryn S. Stok 6, Lai-Shan Tam 7, Isaac Cheng 7, Justin J. Tse 1, Sarah L. Manske 1
1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong

Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis.

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Behavior

Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences
Vanessa A. S. Cunha *1, Sophie Tandonnet *1, Diniz Lima Ferreira 1, Andre V. Rodrigues 2, Tatiana Teixeira Torres 1
1Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, 2Department of Zoology, Institute of Biosciences, University of São Paulo

Herein, two protocols for assessing food source and oviposition preferences in larvae and females of blowflies are detailed. These comprise four choices with two interacting factors: substrate type and temperature. The assays enable the determination of the food source preference of the larvae and the oviposition site preference for the females.

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