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Charles University

14 ARTICLES PUBLISHED IN JoVE

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Medicine

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine
Pavel Hála 1,2, Mikuláš Mlček 1, Petr Ošťádal 1,2, David Janák 1,3, Michaela Popková 1, Tomáš Bouček 1, Stanislav Lacko 1, Jaroslav Kudlička 1, Petr Neužil 1,2, Otomar Kittnar 1
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.

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Developmental Biology

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells
Polina Zjablovskaja *1,2, Petr Danek *1, Miroslava Kardosova 1, Meritxell Alberich-Jorda 1,2
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.

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Biochemistry

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling
Marie Stiborova 1
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.

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Medicine

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations
Petr Stastny 1, Michal Lehnert 2, James J. Tufano 1
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.

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Biology

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages
Jarmila Kralova 1,2, Daniela Glatzova 1,2,3, Simon Borna 1,2, Tomas Brdicka 1
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.

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Neuroscience

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection
Adriana Roithová 1,2, David Staněk 1
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.

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Immunology and Infection

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing
Jarmila Princová 1, Martin Schätz 1, Ondřej Ťupa 1, Martin Převorovský 1
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.

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Bioengineering

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications
Goutam Pramanik 1, Alena Keprova 1, Jan Valenta 2, Vaclav Bocan 3, Klaudia Kvaková 1,4, Lenka Libusova 3, Petr Cigler 1
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.

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Biology

Simple Detection of Primary Cilia by Immunofluorescence
Alzbeta Filipova 1, Daniel Diaz Garcia 2, Josef Dvorak 3, Stanislav Filip 4, Marcela Jelicova 1, Zuzana Sinkorova 1
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.

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Bioengineering

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver
Marek Novák 1, Jozef Rosina 1, Robert Gürlich 2, Ivana Cibulková 3, Jan Hajer 3
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.

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Biology

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells
Moustafa Elkalaf 1,2, Karolína Vaněčková 1,2, Pavla Staňková 1, Zuzana Červinková 1, 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.

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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 Studenovska 4, David Rais 4, Štěpán Popelka 4, Lucie Tichotová 5,6, Yaroslav Nemesh 5,6, Anastasiia Kolesnikova 5,6, Ruslan Nyshchuk 5,6, Anna Brymová 5,6, Zdeňka Ellederová 5, Jana Čížková 5, Jana Juhásová 5, Štefan Juhás 5, Pavla Jendelová 7, Richárd Nagymihály 8, Igor Kozak 9, Slaven Erceg 10, Susanne Binder 11, Brigitte Müller 1, Knut Stieger 1, Jan Motlik 5, 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.

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Medicine

Translational Rabbit Model of Chronic Cardiac Pacing
Vinisha Somaya 1, Michaela Popkova 1, David Janak 2,3, Ilona Princova 2,4, Mikulas Mlcek 2, Jan Petru 1, Petr Neuzil 1, Otomar Kittnar 2, Pavel Hala 1
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.

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Cancer Research

Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software
Esraa Ali 1, Lenka Červenková 2,3, Richard Pálek 2,4, Filip Ambrozkiewicz 1, Sergii Pavlov 1, Wenjing Ye 1, Petr Hošek 2, Ondrej Daum 5,6, Václav Liška 2,4, Kari Hemminki 1,7, Andriy Trailin 1
1Laboratory of Translational Cancer Genomics, Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 2Laboratory of Cancer Treatment and Tissue Regeneration, Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 3Department of Pathology, Third Faculty of Medicine, Charles University, 4Department of Surgery and Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 5Sikl's Institute of Pathology, Faculty of Medicine and Teaching Hospital in Pilsen, Charles University, 6Bioptická Laboratoř s.r.o., 7Department of Cancer Epidemiology, German Cancer Research Center

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

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