University of Sao Paulo

25 articles published in JoVE

• 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 Ferreira¹, Andre V. Rodrigues², Tatiana Teixeira Torres^{1 1}Department 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.

• Medicine

  
  Robotic Myotomy and Partial Fundoplication for Achalasia Rubens Antonio Aissar Sallum¹, Felipe Alexandre Fernandes¹, Leonardo Torres Branco¹, Luna Serena Arguelho Pereira¹, Camila Maria Arruda Vilanova de Câmara¹, Ítalo Beltrão Pereira Simões¹, José Donizeti de Meira Junior¹, Melissa Mello Mazepa¹, Leonardo Ervolino Corbi¹, Rodrigo Nicida Garcia¹, Flávio Roberto Takeda^{1 1}Gastroenterology 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.

• Biology

  
  Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions Bianca Raissa Nogueira¹, Amanda Aparecida de Oliveira¹, Janiele Pereira da Silva², Odair Correa Bueno^{1 1}Institute of Biosciences, Department of General and Applied Biology, Laboratory of Leaf-cutting Ants, São Paulo State University, ²Institute of Psychology, Laboratory Walter Hugo Cunha, São Paulo University Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

• 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 Correia¹, Felipe R. Teixeira^{1,2 1}Department 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.

• 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 Pinhel¹, Jean-Baptiste Cazier^2,3, Lígia Moriguchi Watanabe¹, Albert Nobre Menezes⁴, Carlos Roberto Bueno⁵, Carolina Ferreira Nicoletti¹, Bruno Affonso Parenti de Oliveira¹, Isabelle Mello Schineider⁶, Isabella Harumi Yonehara Noma⁷, Igor Caetano Dias Alcarás⁸, Fernando Barbosa⁹, Carla Barbosa Nonino^{6 1}Department 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.

• Immunology and Infection

  
  High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions André Nicolau Aquime Gonçalves^1,2, Vanessa Escolano Maso³, Ícaro Maia Santos de Castro^2,3, Amanda Pereira Vasconcelos³, Rodrigo Luiz Tomio Ogava^2,3, Helder I Nakaya^{2,3,4 1}Laboratory 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.

• Biology

  
  Double-Staining Method to Detect Pectin in Plant-Fungus Interaction João Paulo Rodrigues Marques¹, Laura Galvan Nuevo^{2 1}Faculty 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.

• 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 Quinet², Carlos F. M. Menck^{1 1}Department 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.

• Immunology and Infection

  
  High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication Rafaela S. Fernandes¹, Gabriela D. Noske¹, Victor O. Gawriljuk¹, Ketllyn I. Z. de Oliveira¹, Andre S. Godoy¹, Nathalya C. M. R. Mesquita¹, Glaucius Oliva^{1 1}São Carlos Institute of Physics, University of Sao Paulo In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

• Neuroscience

  
  The 6-hydroxydopamine Rat Model of Parkinson's Disease Rayanne Poletti Guimarães¹, Danilo Leandro Ribeiro¹, Keila Bariotto dos Santos¹, Lívea Dornela Godoy¹, Mirella Rosine Corrêa¹, Fernando Eduardo Padovan-Neto^{1 1}Department 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.

• 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 Guimarães¹, Fernando Eduardo Padovan-Neto^{1 1}Department 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.

• Medicine

  
  Low-Cost Single-Port (LoCoSP) Device for a Transcervical Approach in Minimally Invasive Transhiatal Esophagectomy Flavio Roberto Takeda¹, Rubens Antonio Aissar Sallum¹, Felipe Alexandre Fernandes¹, Ivan Cecconello^{1 1}Gastroenterology 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.

• Immunology and Infection

  
  Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions Camila Gachet-Castro*¹, Lays Adrianne Mendonça Trajano-Silva*¹, Munira Muhammad Abdel Baqui^{1 1}Laboratory of Cell and Molecular Biology of Trypanosomatids, Department of Cellular and Molecular Biology and Pathogenic Bioagents, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

• 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 Barros¹, Izabella Karina Xavier de Oliveira¹, Meire Ioshie Hiyane¹, Niels Olsen Saraiva Camâra^{1,2 1}Department 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.

• Biology

  
  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 Cavalheiro², Marlise I. Klein^{1 1}Department 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.

• Biochemistry

  
  Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes Felipe A. Carvalho¹, Moises R. A. Barros¹, Thierry P. F. Girotto¹, M. Griselda Perona¹, Carla C. Oliveira^{1 1}Department of Biochemistry, Institute of Chemistry, University of Sao Paulo Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

• Immunology and Infection

  
  In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice Juliana Ide Aoki¹, Ahyun Hong¹, Ricardo Andrade Zampieri¹, Lucile Maria Floeter-Winter¹, Maria Fernanda Laranjeira-Silva^{1 1}Department of Physiology, Institute of Bioscience, University of Sao Paulo Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

• Neuroscience

  
  Short-Term Free-Floating Slice Cultures from the Adult Human Brain Artur Fernandes^1,2, Niele Dias Mendes^1,3, Glaucia Maria Almeida¹, Giovanna Orlovski Nogueira¹, Carla de Moraes Machado⁴, Jose de Anchieta de Castro Horta-Junior⁴, João Alberto Assirati Junior⁵, Norberto Garcia-Cairasco², Luciano Neder³, Adriano Sebollela^{1 1}Department 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.

• Immunology and Infection

  
  Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter Tiago Franco de Oliveira*^1,2, Antonio Anax Falcão de Oliveira*¹, Miriam Lemos³, Mariana Veras³, Paulo Hilário Nascimento Saldiva^3,4, Marisa Helena Gennari de Medeiros⁵, Paolo Di Mascio⁵, Ana Paula de Melo Loureiro^{1 1}Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, ²Departamento de Farmacociências, Universidade Federal de Ciências da Saúde de Porto Alegre, ³Laboratório de Poluição Atmosfêrica Experimental - LIM05, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, ⁴Instituto de Estudos Avançados, Universidade de São Paulo, ⁵Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 1,N⁶-etheno-2'-deoxyadenosine (1,N⁶-dAdo) and 1,N²-etheno-2'-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

• Neuroscience

  
  Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain Faddi G. Saleh Velez^1,2, Camila B. Pinto^1,3, Emma S. Bailin⁴, Marionna Münger¹, Andrew Ellison⁵, Beatriz T. Costa¹, David Crandell⁶, Nadia Bolognini^7,8, Lotfi B. Merabet⁴, Felipe Fregni^{1 1}Laboratory of Neuromodulation & Center for Clinical Research Learning, Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, ²University of Chicago Medical Center, Department of Neurology, University of Chicago, ³Department of Neuroscience and Behavior, Psychology Institute, University of Sao Paulo, ⁴The Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, ⁵Center for Biomedical Imaging, Department of Anatomy and Neurobiology, Boston University School of Medicine, ⁶Spaulding Rehabilitation Hospital, Harvard Medical School, ⁷Department of Psychology & Milan Center for Neuroscience, University of Milano-Bicocca, ⁸Neuropsychological Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto Auxologico Italiano We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

• Developmental Biology

  
  Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells Amanda B.T. Hill^1,2, Jonathan E.B.T. Hill¹, Fabiana F. Bressan³, Maria Angélica Miglino⁴, Joaquim M. Garcia^{1 1}The 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.

• Medicine

  
  Oral Biofilm Formation on Different Materials for Dental Implants Thalisson S. O. Silva¹, Alice R. Freitas¹, Marília L. L. Pinheiro¹, Cássio do Nascimento¹, Evandro Watanabe², Rubens F. Albuquerque^{1 1}Department 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.

• Neuroscience

  
  RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord Felipe M. Vieceli¹, C.Y. Irene Yan^{1 1}Department of Cell and Developmental Biology, Universidade de São Paulo This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

• Bioengineering

  
  Flying Insect Detection and Classification with Inexpensive Sensors Yanping Chen¹, Adena Why², Gustavo Batista³, Agenor Mafra-Neto⁴, Eamonn Keogh^{1 1}Department of Computer Science and Engineering, University of California, Riverside, ²Department of Entomology, University of California, Riverside, ³Institute of Mathematics and Computer Sciences, University of São Paulo - USP, ⁴ISCA Technologies We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

• Environment

  
  Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil Danilo O. Carvalho^1,2, Derric Nimmo¹, Neil Naish¹, Andrew R. McKemey¹, Pam Gray¹, André B. B. Wilke³, Mauro T. Marrelli³, Jair F. Virginio⁴, Luke Alphey^1,5, Margareth L. Capurro^{2,6 1}Oxitec Ltd, ²Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, ³Departamento de Epidemiologia, Universidade de São Paulo, ⁴Moscamed Brasil, ⁵Deptartment of Zoology, University of Oxford, ⁶Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM) To achieve population suppression of Aedes aegypti using the RIDL^® (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.