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RWTH Aachen University

39 ARTICLES PUBLISHED IN JoVE

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Biology

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography
Matthew C. Johnson *1, Frederik Rudolph *1,2, Tina M. Dreaden *3, Gengxiang Zhao *1, Bridgette A. Barry 3, Ingeborg Schmidt-Krey 1,3
1School of Biology, Georgia Institute of Technology, 2Department of Molecular Pharmacology, RWTH Aachen University, 3School of Chemistry and Biochemistry, Georgia Institute of Technology

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 – 90kDa.

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Biology

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Thomas Lenz 1, Peter Poot 2, Olivia Gräbner 1, Mirko Glinski 1, Elmar Weinhold 2, Mathias Dreger 1, Hubert Köster 1
1Department of Biochemistry / Analytics, caprotec bioanalytics GmbH, 2Institute of Organic Chemistry, RWTH Aachen University

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

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Biology

Detection of Histone Modifications in Plant Leaves
Michal Jaskiewicz 1,2, Christoph Peterhansel 3, Uwe Conrath 2
1Department of Botany, RWTH Aachen University, 2Department of Plant Physiology, RWTH Aachen University, 3Department of Botany, Leibniz University

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

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Medicine

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction
Kazuyuki Nagai 1,2, Shintaro Yagi 2, Shinji Uemoto 2, Rene H. Tolba 1
1Institute for Laboratory Animal Science and Experimental Surgery, RWTH-Aachen University, 2Department of Hepato-Biliary-Pancreatic Surgery and Transplantation, Graduate School of Medicine, Kyoto University

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

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Medicine

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis
Sakine Simsekyilmaz 1, Fabian Schreiber 2, Stefan Weinandy 3, Felix Gremse 4, Tolga Taha Sönmez 5, Elisa A. Liehn 1
1Institute for Molecular Cardiovascular Research, RWTH Aachen University, 2Institute for Textile Technology and Mechanical Engineering, RWTH Aachen University, 3Institute for Applied Medical Engineering, Helmholtz-Institute of RWTH Aachen University, 4Department of Experimental Molecular Imaging, RWTH Aachen University, 5Department of Oral and Maxillofacila Surgery, RWTH Aachen University

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

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Bioengineering

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation
Alexander Gruenberger 1, Christopher Probst 1, Antonia Heyer 1, Wolfgang Wiechert 1, Julia Frunzke 1, Dietrich Kohlheyer 1
1Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Juelich GmbH

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

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Bioengineering

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Johannes Felix Buyel 1, Rainer Fischer 1,2
1Institute for Molecular Biotechnology, RWTH Aachen University, 2Institute for Molecular Biology and Applied Ecology, Fraunhofer Gesellschaft

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

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Environment

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants
Megan Garvey 1, Johannes Klinger *1, Holger Klose *1, Rainer Fischer 2, Ulrich Commandeur 1
1Institute for Molecular Biotechnology, Bio7, RWTH Aachen University, 2Fraunhofer Institute for Molecular Biology and Applied Ecology

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

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Biology

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Gisela Maria Hanz *1, Britta Jung *1, Anna Giesbertz 1, Matyas Juhasz 1, Elmar Weinhold 1
1Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

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Medicine

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice
Adelina Curaj 1, Sakine Simsekyilmaz 1, Mareike Staudt 1, Elisa Liehn 1
1Institute for Molecular Cardiovascular Research, RWTH Aachen University, Germany

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

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Neuroscience

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings
Guanxiao Qi 1, Gabriele Radnikow 1, Dirk Feldmeyer 1,2
1Institute of Neuroscience and Medicine (INM-2), Research Centre Jülich, 2Department of Psychiatry, Psychotherapy and Psychosomatics, Medical Faculty, JARA, RWTH Aachen University

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

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Medicine

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis
Carmen G. Tag 1, Sibille Sauer-Lehnen 1, Sabine Weiskirchen 1, Erawan Borkham-Kamphorst 1, René H. Tolba 2, Frank Tacke 3, Ralf Weiskirchen 1
1Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH Aachen University, 2Institute for Laboratory Animal Science and Experimental Surgery, RWTH Aachen University, 3Department of Medicine III, RWTH Aachen University

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

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Immunology and Infection

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice
Felix Heymann *1, Patricia M. Niemietz *1, Julia Peusquens 1, Can Ergen 1, Marlene Kohlhepp 1, Jana C. Mossanen 1, Carlo Schneider 1, Michael Vogt 2, Rene H. Tolba 3, Christian Trautwein 1, Christian Martin 4, Frank Tacke 1
1Department of Medicine III, RWTH University-Hospital Aachen, 2IZKF Aachen Core Facility "Two-Photon Imaging", RWTH University-Hospital Aachen, 3Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 4Institute for Pharmacology, RWTH University-Hospital Aachen

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

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Bioengineering

Hybrid µCT-FMT imaging and image analysis
Felix Gremse *1, Dennis Doleschel *1, Sara Zafarnia 1, Anne Babler 2, Willi Jahnen-Dechent 2, Twan Lammers 1,3, Wiltrud Lederle 1, Fabian Kiessling 1
1Experimental Molecular Imaging, RWTH Aachen University, 2Institute for Biomedical Engineering - Biointerface Laboratory, RWTH Aachen University, 3Utrecht Institute for Pharmaceutical Sciences, Utrecht University

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (µCT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

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Medicine

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice
Chichi Xie 1, Weiwei Wei 1, Andrea Schenk 3, Lars Ole Schwen 3, Sara Zafarnia 2, Michael Schwier 3, Felix Gremse 2, Isabel Jank 1, Olaf Dirsch 4, Uta Dahmen 1
1Department of General, Visceral and Vascular Surgery, Jena University Hospital, 2Experimental Molecular Imaging, RWTH Aachen University, 3Fraunhofer Institute for Medical Image Computing MEVIS, 4Institute of Pathology, Klinikum Chemnitz gGmbH

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using µCT and histological serial sections.

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Biology

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts
Johannes F. Buyel 1,2
1Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

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Biology

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts
Johannes F. Buyel 1,2, Jürgen Hubbuch 3, Rainer Fischer 1,2
1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University, 3Department of Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT)

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 °C.

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Chemistry

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels
Otto L. J. Virtanen 1, Ashvini Purohit 1, Monia Brugnoni 1, Dominik Wöll 1, Walter Richtering 1
1Institute of Physical Chemistry, RWTH Aachen University

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

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Neuroscience

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ
Tobias Ackels 1,2, Daniela R. Drose 1, Marc Spehr 1
1Department of Chemosensation, Institute for Biology II, RWTH Aachen University, 2Mill Hill Laboratory, The Francis Crick Institute

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

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Medicine

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model
Adelina Curaj 1, Wu Zhoujun 1, Mareike Staudt 1, Elisa A. Liehn 1,2
1Institute for Molecular Cardiovascular Research, RWTH Aachen University, 2Human Genetic Laboratory, University of Medicine and Pharmacy

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

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Biochemistry

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties
Johannes F. Buyel 1,2, Hannah M. Gruchow 1, Martin Wehner 3
1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University, 3Fraunhofer Institute for Laser Technology ILT, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

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Medicine

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video
Zuzanna Rowinska 1,2, Simone Gorressen 3, Marc W. Merx 4, Thomas A. Koeppel 5, Alma Zernecke 6, Elisa A. Liehn 2
1Department of Vascular Surgery and Interdisciplinary Vein Center, St. Josef-Hospital, Ruhr- University Bochum, 2Institute of Molecular Cardiovascular Research, University Hospital, RWTH Aachen University Department of Medicine, 3Institute of Pharmacology and Clinical Pharmacology, Düsseldorf University Hospital, 4Department of Cardiology, Vascular Medicine and Intensive Care Medicine, Hospital Robert Koch Gehrden, 5Division of Vascular Surgery, Asklepios Klinik St. Georg, 6Institute of Experimental Biomedicine, Würzburg University

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

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Bioengineering

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology
Johannes Hemmerich *1,2, Lars Freier *1,2, Wolfgang Wiechert 1,2,3, Eric von Lieres 1,2, Marco Oldiges 1,2,4
1IBG-1: Biotechnology, Forschungszentrum Jülich, 2Research Center Jülich, Bioeconomy Science Center (BioSC), 3Computational Systems Biotechnology (AVT.CSB), RWTH Aachen University, 4Institute for Biotechnology, RWTH Aachen University

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

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Immunology and Infection

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices
Vanessa Neuhaus *1, Olga Danov *1, Sebastian Konzok 1, Helena Obernolte 1, Susann Dehmel 1, Peter Braubach 2, Danny Jonigk 2, Hans-Gerd Fieguth 3, Patrick Zardo 4, Gregor Warnecke 4, Christian Martin 5, Armin Braun 1,6, Katherina Sewald 1
1Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of REBIRTH Cluster of Excellence, 2Institute for Pathology, Hannover Medical School, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), 3Division of Thoracic and Vascular Surgery, Klinikum Region Hannover (KRH), 4Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), 5Institute of Pharmacology and Toxicology, RWTH Aachen University, 6Institute for Immunology, Hannover Medical School

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

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Medicine

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo
Tim R. Glowka 1, Pascal Paschenda 2, Michael Czaplik 3, Jörg C. Kalff 1, René H. Tolba 2
1Department of Surgery, University of Bonn, 2Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 3Department of Anesthesiology, RWTH Aachen University

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

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Developmental Biology

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues
Bartosz Ditkowski 1, Tiago R Veloso 1, Martyna Bezulska-Ditkowska 1,2, Andreas Lubig 3, Stefan Jockenhoevel 3, Petra Mela 3, Ramadan Jashari 4, Marc Gewillig 1, Bart Meyns 5, Marc F Hoylaerts 2, Ruth Heying 1
1Cardiovascular Developmental Biology, Department of Cardiovascular Sciences, KU Leuven, 2Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, 3Department of Biohybrid & Medical Textiles, AME - Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, 4European Homograft Bank, Saint Jean Clinique, 5Division of Clinical Cardiac Surgery, Department of Cardiovascular Sciences, KU Leuven

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

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Neuroscience

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy
Vanessa Reindl 1,2, Kerstin Konrad 1,2, Christian Gerloff 2,3, Jana A. Kruppa 1,2,4, Laura Bell 1, Wolfgang Scharke 1
1Child Neuropsychology Section, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Medical Faculty, RWTH Aachen University, 2JARA-Brain Institute II, Molecular Neuroscience and Neuroimaging, RWTH Aachen & Research Centre Juelich, 3Lehrstuhl II für Mathematik, RWTH Aachen University, 4Translational Brain Research in Psychiatry and Neurology, Department of Child and Adolescent Psychiatry, Psychosomatics, and Psychotherapy, University Hospital Aachen

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

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Biochemistry

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study
Matthias Knödler 1,3, Clemens Rühl 2, Patrick Opdensteinen 1,3, Johannes F. Buyel 1,3
1Institute for Molecular Biotechnology, RWTH Aachen University, 2Sanofi Deutschland GmbH, 3Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

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Neuroscience

Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy
Paul V. Ritschl *1,2,3, Lena Hofhansel *2,4, Bernhard Flörchinger 5, Rupert Oberhuber 2, Robert Öllinger 1, Johann Pratschke 1, Katja Kotsch 6
1Department of Surgery Campus Charité Mitte/Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 2Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, 3Charité Clinician Scientist Program, Berlin Institute of Health, 4Department of Psychiatry, Psychotherapy and Psychosomatics, RWTH Aachen University, 5Department of Cardiothoracic Surgery, University Medical Center Regensburg, 6Department of General, Visceral and Vascular Surgery, Charité - Universitätsmedizin Berlin

We present a murine model of brain death induction in order to evaluate the influence of its pathophysiological effects on organs as well as on consecutive grafts in the context of solid organ transplantation.

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Medicine

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access
Anne Cornelissen *1, Roberta Florescu *1, Nicole Schaaps 1, Mamdouh Afify 1, Sakine Simsekyilmaz 1, Elisa Liehn *1,2,3, Felix Vogt *1
1Division of Cardiology, Angiology, and Critical Care, University Hospital RWTH Aachen, 2Department for Operative Intensive Medicine, University Hospital RWTH Aachen, 3Institute for Pathology "Victor Babes"

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

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Chemistry

Fractionation of Lignocellulosic Biomass using the OrganoCat Process
Leonie Schoofs 1, Dennis Weidener 1, Ulrich Schurr 1, Holger Klose 1, Philipp M. Grande 1
1Institut für Bio- und Geowissenschaften, Pflanzenwissenschaften (IBG-2), Forschungszentrum Jülich

OrganoCat is a method for the pretreatment and fractionation of lignocellulose under mild conditions into lignin, fermentable sugars, and cellulose pulp. In a biogenic, biphasic solvent system of water and 2-methyltetrahydrofuran with 2,5-furancarboxylic acid as catalyst, the OrganoCat products are separated in situ for straightforward product recovery.

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Biology

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells
Boheng Liu 1,2, Nadine Mundt 3,4, Melissa Miller 1, David E. Clapham 5, Yuriy Kirichok 6, Polina V. Lishko 1
1Department of Molecular and Cell Biology, University of California, Berkeley, 2Department of Neurobiology, Peking University, 3Institute for Biology II / Chemosensation Lab, RWTH Aachen University, 4Research Training Group 2416 MultiSenses-MultiScales, RWTH Aachen University, 5Howard Hughes Medical Institute, Janelia Research Campus, 6Department of Physiology, University of California, San Francisco

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

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Medicine

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS)
Martin Russ *1, Emilia Boerger *1, Philip von Platen 2, Roland C. E. Francis 1, Mahdi Taher 1, Willehad Boemke 1, Burkhard Lachmann 1, Steffen Leonhardt 2, Philipp A. Pickerodt 1
1Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt Universität zu Berlin and Berlin Institute of Health Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, 2Chair for Medical Information Technology, RWTH Aachen University

A combination of surfactant washout using 0.9% saline (35 mL/kg body weight, 37 °C) and high tidal volume ventilation with low PEEP to cause moderate ventilator induced lung injury (VILI) results in experimental acute respiratory distress syndrome (ARDS). This method provides a model of lung injury with low/limited recruitability to study the effect of various ventilation strategies for extended periods.

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Medicine

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets
Benedikt Freystetter 1, Maximilian Grab 1,2, Linda Grefen 1, Lara Bischof 1, Lorenz Isert 3, Petra Mela 2, Deon Bezuidenhout 4, Christian Hagl 1,5, Nikolaus Thierfelder 1
1Department of Cardiac Surgery, Ludwig Maximilians University Munich, 2Chair of Medical Materials and Implants, Technical University Munich, 3Faculty for Chemistry and Pharmacy, Ludwig Maximilians University Munich, 4Cardiovascular Research Unit, University of Cape Town, 5DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance

The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup.

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Bioengineering

Interlinked Macroporous 3D Scaffolds from Microgel Rods
Dirk Rommel 1,2, Sitara Vedaraman 1,2, Matthias Mork 1,2, Laura De Laporte 1,2,3
1DWI - Leibniz Institute for Interactive Materials, 2Institute for Technical and Macromolecular Chemistry, RWTH Aachen University, 3Institute of Applied Medical Engineering, Department of Advanced Materials for Biomedicine, RWTH Aachen University

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

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Biochemistry

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro
Armand M. G. Jaminon *1, Nikolas Rapp *1, Asim C. Akbulut 1, Robert Dzhanaev 2, Chris P. Reutelingsperger 1, Willi Jahnen-Dechent 2, Leon J. Schurgers 1,3
1Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 2Helmholtz Institute for Biomedical Engineering, Biointerface Group, RWTH Aachen University, 3Institute of Experimental Medicine and Systems Biology, RWTH Aachen University

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

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Chemistry

Screening of Coatings for an All-Solid-State Battery Using In Situ Transmission Electron Microscopy
Shibarata Basak 1,2, Junbeom Park 1, Janghyun Jo 2, Osmane Camara 1, Amir H. Tavabi 2, Hermann Tempel 1, Hans Kungl 1, Chandramohan George 3, Rafal E. Dunin-Borkowski 2, Joachim Mayer 2,4, Rüdiger-A. Eichel 1,5
1Institute of Energy and Climate Research - Fundamental Electrochemistry (IEK-9), Forschungszentrum Jülich GmbH, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, 3Dyson School of Design Engineering, Imperial College London, 4Central Facility for Electron Microscopy (GFE), RWTH Aachen University, 5Institute of Physical Chemistry, RWTH Aachen University

Utilizing the volume change of Si nanoparticles during (de)lithiation, the present protocol describes a screening method of potential coatings for all-solid-state batteries using in situ transmission electron microscopy.

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Bioengineering

Multimodal Cross-Device and Marker-Free Co-Registration of Preclinical Imaging Modalities
Mirko Thamm 1,2, Justin J. Jeffery 3, Yapei Zhang 4, Bryan R. Smith 4, Stephen Marchant 5, Fabian Kiessling 1,6, Felix Gremse 1,2
1Institute for Experimental Molecular Imaging, RWTH Aachen University, 2Gremse-IT GmbH, 3Carbone Cancer Center, University of Wisconsin, 4Department of Biomedical Engineering and Institute for Quantitative Health Science and Engineering, Michigan State University, 5MediLumine Inc., 6Fraunhofer Institute for Digital Medicine MEVIS

The combination of multiple imaging modalities is often necessary to gain a comprehensive understanding of pathophysiology. This approach utilizes phantoms to generate a differential transformation between the coordinate systems of two modalities, which is then applied for co-registration. This method eliminates the need for fiducials in production scans.

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Neuroscience

Home-Based EEG Hyperscanning for Infant-Caregiver Social Interactions
Vaidehi Ramanarayanan *1, Qian Chern Oon *1, Amritha Varshini Devarajan 1, Stanimira Georgieva 1,2, Vanessa Reindl 1,3
1Department of Psychology, School of Social Sciences, Nanyang Technological University, 2Department of Pediatrics, University of Cambridge, 3Child Neuropsychology Section, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Medical Faculty, RWTH Aachen University

This protocol describes how synchronized electroencephalography, electrocardiography, and behavioral recordings were captured from infant-caregiver dyads in a home setting.

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