Charles University 15 articles published in JoVE Medicine Translational Rabbit Model of Chronic Cardiac Pacing Vinisha Somaya1, Michaela Popkova1, David Janak2,3, Ilona Princova2,4, Mikulas Mlcek2, Jan Petru1, Petr Neuzil1, Otomar Kittnar2, Pavel Hala1 1Department of Cardiology, Na Homolce Hospital, 2Department of Physiology, First Faculty of Medicine, Charles University, 3Department of Cardiovascular Surgery, Second Faculty of Medicine, Charles University, 4Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University We present a minimally invasive leporine model of long-term cardiac pacing that can be utilized for artificial pacing and heart failure development in preclinical studies. Bioengineering Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care Lyubomyr Lytvynchuk*1,2, Zbynek Stranak*3, Hana Studenovska4, David Rais4, Štěpán Popelka4, Lucie Tichotová5,6, Yaroslav Nemesh5,6, Anastasiia Kolesnikova5,6, Ruslan Nyshchuk5,6, Anna Brymová5,6, Zdeňka Ellederová5, Jana Čížková5, Jana Juhásová5, Štefan Juhás5, Pavla Jendelová7, Richárd Nagymihály8, Igor Kozak9, Slaven Erceg10, Susanne Binder11, Brigitte Müller1, Knut Stieger1, Jan Motlik5, Goran Petrovski*8, Taras Ardan*5 1Eye Clinic, Department of Ophthalmology, University Hospital Giessen and Marburg GmbH, 2Karl Landsteiner Institute for Retinal Research and Imaging, 3Department of Ophthalmology, University Hospital Kralovske Vinohrady and Third Faculty of Medicine, Charles University in Prague, 4Institute of Macromolecular Chemistry, Czech Academy of Sciences, 5Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 6Department of Cell Biology, Faculty of Science, Charles University, 7Institute of Experimental Medicine, Czech Academy of Sciences, 8Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 9Moorfields Eye Hospitals UAE, 10Stem Cell Therapies in Neurodegenerative Diseases Lab, Research Center “Principe Felipe”, 11Eye Center Donaustadt, Department of Ophthalmology, Sigmund Freud University The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs. Bioengineering Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver Marek Novák1, Jozef Rosina1, Robert Gürlich2, Ivana Cibulková3, Jan Hajer3 1Department of Medical Biophysics and Medical Informatics, Third Faculty of Medicine, Charles University, 2Department of General Surgery, Third Faculty of Medicine, Charles University, University Hospital Královské Vinohrady, 3Department of Internal Medicine, Third Faculty of Medicine, Charles University, University Hospital Královské Vinohrady The manuscript presents a miniature implantable pH sensor with ASK modulated wireless output together with a fully passive receiver circuit based on zero-bias Schottky diodes. This solution can be used as a basis in the development of in vivo calibrated electrostimulation therapy devices and for ambulatory pH monitoring. Biology Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells Moustafa Elkalaf1,2, Karolína Vaněčková1,2, Pavla Staňková1, Zuzana Červinková1, Jan Polák*2,3, Otto Kučera*1 1Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University, 2Department of Pathophysiology, Third Faculty of Medicine, Charles University, 3Department of Internal Medicine, University Hospital Kralovske Vinohrady In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines. Biology Simple Detection of Primary Cilia by Immunofluorescence Alzbeta Filipova1, Daniel Diaz Garcia2, Josef Dvorak3, Stanislav Filip4, Marcela Jelicova1, Zuzana Sinkorova1 1Department of Radiobiology, Faculty of Military Health Sciences in Hradec Kralove, University of Defence, 2Department of Clinical Biochemistry and Diagnostics, University Hospital, 3Department of Oncology, Thomayer Hospital, Charles University, 4Department of Oncology and Radiotherapy, Faculty of Medicine, Charles University Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope. Bioengineering Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications Goutam Pramanik1, Alena Keprova1, Jan Valenta2, Vaclav Bocan3, Klaudia Kvaková1,4, Lenka Libusova3, Petr Cigler1 1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 2Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University, 3Department of Cell Biology, Faculty of Science, Charles University, 4First Faculty of Medicine, Charles University A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described. Neuroscience Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection Adriana Roithová1,2, David Staněk1 1Laboratory of RNA Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, 2Department of Genetics and Microbiology, Faculty of Science, Charles University Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell. Immunology and Infection Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing Jarmila Princová1, Martin Schätz1, Ondřej Ťupa1, Martin Převorovský1 1Faculty of Science, Charles University Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells. Biology Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages Jarmila Kralova1,2, Daniela Glatzova1,2,3, Simon Borna1,2, Tomas Brdicka1 1Laboratory of Leukocyte Signalling, Institute of Molecular Genetics of the ASCR, 2Faculty of Science, Charles University, 3Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry ASCR In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro. Biology Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins Stanislav Vosolsobě1, Kateřina Schwarzerová1, Jan Petrášek1 1Department of Experimental Plant Biology, Faculty of Science, Charles University Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker. Medicine Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations Petr Stastny1, Michal Lehnert2, James J. Tufano1 1Faculty of Physical Education and Sport, Charles University, 2Faculty of Physical Culture, Palacky University Olomouc The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds. Biochemistry Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling Marie Stiborova1 1Department of Biochemistry, Charles University Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification. Developmental Biology Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells Polina Zjablovskaja*1,2, Petr Danek*1, Miroslava Kardosova1, Meritxell Alberich-Jorda1,2 1Department of Hemato-Oncology, Institute of Molecular Genetics of the ASCR, 2Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation. Medicine Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine Pavel Hála1,2, Mikuláš Mlček1, Petr Ošťádal1,2, David Janák1,3, Michaela Popková1, Tomáš Bouček1, Stanislav Lacko1, Jaroslav Kudlička1, Petr Neužil1,2, Otomar Kittnar1 1Department of Physiology, First Faculty of Medicine, Charles University, 2Department of Cardiology, Na Homolce Hospital, 3Department of Cardiovascular Surgery, Second Faculty of Medicine, Charles University Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment. Medicine Transvaginal Mesh Insertion in the Ovine Model Iva Urbankova1,2, Geertje Callewaert1,3, Nikhil Sindhwani1, Alice Turri1, Lucie Hympanova1,2, Andrew Feola1, Jan Deprest1,3 1Centrum for Surgical Technologies, Department of Development and Regeneration, Clinical Specialties Research Groups, Faculty of Medicine, KU Leuven, 2Institute for the Care of Mother and Child and Third Faculty of Medicine, Charles University, Prague, 3Pelvic Floor Unit, University Hospitals KU Leuven This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.