2012 ABSTRACTS - Universität Leipzig

Saxon Biotechnology Symposium June 20, 2012

ABSTRACTS 2012

Editors: Andrea A. Robitzki Mario Mörl Tilo Pompe Michael Schroeder Jochen Guck Konstantinos Anastassiadis

I MPRINT Editors

Prof. Dr. Andrea A. Robitzki Director Center for Biotechnology and Biomedicine, Universität Leipzig



Prof. Dr. Mario Mörl



Professorship for Biochemistry and Molecular Biology Institute of Biochemistry, Universität Leipzig



Prof. Dr. Tilo Pompe



Professorship for Biophysical Chemistry



Institute of Biochemistry, Universität Leipzig



Scientific Director



Biotechnology Center, Technische Universität Dresden

Prof. Dr. Michael Schroeder



Prof. Dr. Jochen Guck



Professorship for Cellular Machines Biotechnology Center, Technische Universität Dresden



Dr. Konstantinos Anastassiadis



Research Group Leader, Genetic Engineering of Stem Cells Biotechnology Center, Technische Universität Dresden

Compilation

Dr. Svenne Eichler



Chief Executive Officer Center for Biotechnology and Biomedicine, Universität Leipzig



Head of Administration Biotechnology Center, Technische Universität Dresden

Katharina Plesse



Iris Kochinka



Center for Biotechnology and Biomedicine, Universität Leipzig



Center for Biotechnology and Biomedicine, Universität Leipzig

Print

Merkur Druck- und Kopierzentrum GmbH

Editorial Deadline

May 2, 2012

ISBN

978-3-00-038523-0

Layout

Antje Ferrier

C O NTE NT





Foreword17

1.

Cells as Active Sensors19

1.1

Real-time monitoring of cellular inflammatory and degenerative processes on microarrays Andrea A. Robitzki20

1.2

Towards functional microbial sensor cell arrays Shimshon Belkin, Tal Elad, Sahar Melamed, Sharon Yagur-Kroll, Yosi Shacham-Diamand21

1.3

Recombinant yeast cells as a tool for biosensor applications Gerhard Rödel22

1.4

Arsolux: cell-based detection system for arsenic contamination Hauke Harms, Konrad Siegfried, Antonis Chatzinotas,   Sonja Hahn-Tomer23

1.5

Living lasers – towards laser-based biosensors  Malte C. Gather24

2.

Live Cell Analysis27

2.1

Analysing infection of macrophages by Salmonella  Pietro Cicuta28

2.2 Chromobodies ® and the Fluorescent 2-Hybrid (F2H ) assay: New technologies for real time analysis of cellular components in living cells  Kourosh Zolghadr29 2.3

Automatic tracking and quantification of dynamic cellular characteristics  Ingo Röder30

2.4

Genetic tools to study the brain: from transgenic mice to metabolic imaging  Johannes Hirrlinger31

3

3.

Molecular Medicine and Protein Design33

3.1

Post-translational modified peptides to develop efficient IVD to follow up disease activity of autoimmune diseases: a challenge for theragnostics  Anna Maria Papini34

3.2

Technologies for the development of tailored enzymes and strains  Thomas Greiner-Stöffele35

5.5

Polyunsaturated fatty acids impact the intracellular trafficking of Phospholipase D1 differently  Shereen Basiouni, Herbert Fuhrmann, Julia Schumann48

5.6

Identification of novel viral protease inhibitors via dynamic fragment ligation  Daniel Becker, Jörg Rademann, Christoph Arkona49

5.7

Influence of opioid receptor activation and inhibition on synaptic transmission under hypoxia  Marcus Bloßfeld, Andreas Merkenschlager, Karen Nieber50

3.3

Designer recombinases reverse HIV infection  Frank Buchholz36

3.4

Genome surgery using designer nucleases: ZFN s or TALEN s?  Claudio Mussolino37

5.8

Cytotoxic effects of curcumin on human retinal pigment epithelial cells  Rui Chen, Margrit Hollborn, Andreas Bringmann, Leon Kohen, Peter Wiedemann51

4.

Evening Lecture39

5.9

4.1

Phosphonate chemistry in medicine: from bone actives to antiviral agents  Charles E. McKenna40

A tripeptide switch region to induce agonism and inverse agonism at the ghrelin receptor  Sylvia Els, Enrico Schild, Pia S. Petersen, Constance Chollet, Birgitte Holst, Thue W. Schwartz, Annette G. Beck-Sickinger52

5.10

5.

Molecular Medicine43

5.1

PUFA promote the phagocytosis but not the killing of the persistent pathogens R. equi and P. aeruginosa  Stephanie Adolph, Herbert Fuhrmann, Julia Schumann44

A murine model of Cryptococcus neoformans-related immune reconstitution inflammatory syndrome (IRIS)  Maria Eschke, Andreas Grahnert, Daniel Piehler, Tina Richter, Gabriele Köhler, Werner Stenzel, Uwe Müller, Gottfried Alber53

5.11

A new approach for screening of colorectal cancer circulating tumor cell (CTC) using EpCAM -pluri­Beads  Nadezhda Frolova, Christoph Mohr, Anne Rasser, Ulrich Sack, Jan-Michael Heinrich54

5.12

biocrea: Creating novel drugs for debilitating CNS diseases  Thorsten Hage, Martin Gunthorpe55

5.13

Analysis of HeLa rho0 cell lines after transfer of wild type mitochondria  Sandra Heller, Susanna Schubert, Mario Krehan, Ingo Schäfer, Jessica Zenker, Peter Seibel56

5.14

Switching Y-receptor selectivity of short NPY analogs: acylation versus carbaboranylation  Sven Hofmann, René Frank, Evamarie Hey-Hawkins,  Annette G. Beck-Sickinger57

5.15

Cells for the analysis of diabetes: The understanding of disease development and progression  Matthias Jung, Diana Oelschlägel, Bernadette Harwardt,  Insa S. Schroeder58

5.2

Polymer-based nanoparticles as delivery platform for small

RNA molecules (siRNA s, miRNA s) in vivo: development of novel

therapeutic strategies based on gene silencing  Achim Aigner, Sabrina Höbel, Ulrike Weirauch, Anja Frömberg, Alexander Ewe45 5.3

5.4

4

Simplet (smp) is required for caudal fin regeneration and for canonical Wnt signaling by regulating nuclear localization of ß-catenin  Christopher Antos, Beate Küchler, Caghan Kizil, Günes Özhan-Kizil, Jia-Jiun Yan, Andrew C. Oates, Enrico Moro, Francesco Argenton, Michael Brand, Gilbert Weidinger46 CD160130: a selective inhibitor of the tumor-type hERG1 channels,

active in human leukemias  Annarosa Arcangeli, Luca Gasparoli, Massimo D’Amico, Andrea Becchetti, Olivia Crociani, Marika Masselli, Serena Pillozzi, Kenneth Mugridge, Wolfgang Tiedke47

5

5.16

Titanocene dichloride derivatives with improved cytotoxic activity  Goran N. Kaluđerović, Santiago Gómez-Ruiz, Beatriz Gallego, Željko Žižak, Evamarie Hey-Hawkins59

5.27

How fatty acids modulate macrophage-mediated immune defense – a mechanistic trial  Axel Schöniger, Herbert Fuhrmann, Julia Schumann 70

5.17

Generation of ρo-cells without nuclear genome interference  Mario Krehan, Ingo Schäfer, Susanna Schubert, Sandra Heller, Jessica Zenker, Astrid Schön, Peter Seibel60

5.28

Membrane fatty acid patterns and macrophage activity  Julia Schumann, Axel Schöniger, Stephanie Adolph, Herbert Fuhrmann71

5.29

5.18

Cisplatin – COX inhibitor conjugates  Wilma Neumann, Evamarie Hey-Hawkins61

The role of BIM-EL and BCL2-α on the efficacy of erlotinib and gefitinib in lung cancer  Jacinta Simasi, Andreas Schubert, Adrian Gillissen, Karen Nieber72

5.19

Synthesis and evaluation of PET probes for imaging of the PDE10A in brain: An inter­disciplinary project  Karen Nieber, Fritzi Siegert, Detlef Briel, Ghadir Barbar Askar, Norbert Sträter, Michael Zahn, Matthias Scheunemann, Peter Brust62

5.30

Validation of ncRNA s relevant to Alzheimer´s disease  Igor Turković, Uwe Ueberham, Thomas Arendt73

5.31

Glial but not neuronal osmotic cell volume regulation in the inner nuclear layer of the rat retina  Stefanie Vogler, Antje Grosche, Thomas Pannicke, Peter Wiedemann, Andreas Reichenbach, Andreas Bringmann74

5.32

Photoactive phosphotyrosine mimetics  Stefan Wagner, Jörg Rademann, André Horatscheck, Jutta Ortwein, Boo Geun Kim, Michael Lisurek, Samuel Beligny, Anja Schütz75

5.33

Development of a MDCKII cell in vitro model for the assessment of BCRP mediated drug transport in the lactating mammary gland of dairy animals  Louise Waßermann, Stefan Lindner, Sandra Halwachs,  Kerstin U. Honscha, Walther Honscha76

5.34

Investigating protein stability of a FOXP2 disease variant  Sven Weyer, Wolfgang Enard77

5.35

Structural studies of DnaK in complex with small proline-rich antimicrobial peptides  Michael Zahn, Daniel Knappe, Nicole Berthold, Ralf Hoffmann, Norbert Sträter78

6.

Bioanalytics81

6.1

Identification and characterization of dityrosine cross-linked peptides using mass spectrometry  Andrea Annibal, Ravi Chand Bollineni, Maria Fedorova, Ralf Hoffmann82

6.2

A qualitative and quantitative study of carbonyl tagging reagents for redox proteomics  Ravi Chand Bollineni, Maria Fedorova, Ralf Hoffmann83

5.20

Synthesis of a multifunctional peptide by two distinct click-reactions  Mareen Pagel, Rayk Hassert, Manfred Wießler,  Annette G. Beck-Sickinger63

5.21

Novel kinase-inhibitors and their application for the individualised therapy of the malignant melanoma  Sarah Pönick, Heinz-Georg Jahnke, Jan Maschke, Michael Kendler, Jan C. Simon, Andrea A. Robitzki64

® chamomile component affects contraction 5.22 Myrrhinil-Intest  and morphology of rat ileum / jejunum ex vivo preparations in a concentration dependent manner  Mikolaj Raszek, Karen Nieber, Karl-Heinz Goos65

5.23

Genetic analysis of antibody genes from influenza NP-specific B-cells  Sven Reiche, Yamen Dwai, Christian Jassoy66

5.24

Saffron extract acts neuroprotective against hypoxia on neuronal cells in vitro  Franziska Reitmajer, Marcus Bloßfeld, Jörg Flemmig, Maria Schönberg, Andreas Hensel, Jürgen Arnhold, Karen Nieber67

5.25

Th17 cells can compensate for Th1 deficiency in pulmonary cryptococcosis  Tina Richter, Daniel Piehler, Andreas Grahnert, Maria Eschke, Uwe Müller, Katarzyna Warszawska, Gabriele Köhler, Robert Sabat, Gottfried Alber68

5.26

6

Expression of recombinant rotavirus proteins in mammalian cell lines  Antje Rückner, Thomas W. Vahlenkamp69

7

6.3

A peptide-protein assay for identification of antimicrobial peptides by fluorescence quenching  Kristin Dobslaff, Thomas Kreisig, Nicole Berthold, Ralf Hoffmann, Thole Züchner84

6.14

Oncocin derivative Onc72 is highly active against Escherichia coli in a systemic septicaemia infection mouse model  Daniel Knappe, Uwe Müller, Michael Zahn, Stefanie Fritsche, Norbert Sträter, Gottfried Alber, Ralf Hoffmann95

6.4

Expression of P2X receptor ectodomains  Christoph Döhler, Matthias Zebisch, Norbert Sträter85

6.15

6.5

Development of new MS-based analytical techniques to study lipid- and protein-bound carbonyls  Maria Fedorova, Ivana Milic, Ravi Chand Bollineni, Ralf Hoffmann86

Quantitative profiling of arachidonic acid metabolites by hybrid triple quadrupole / linear ion trap mass spectrometry  Linda Kortz, Juliane Dorow, Joachim Thiery, Uta Ceglarek96

6.16

6.6

Qualitative and quantitative characterization of protein glycation patterns in human plasma  Andrej Frolov, Maria Fedorova, Matthias Blüher, Ralf Hoffmann87

Development of an ITC based enzyme assay for nucleoside triphosphate diphosphohydrolases (NTPD ases)  Michel Krauß, Matthias Zebisch, Norbert Sträter97

6.17

A novel affinity-optimized homogeneous immunoassay  Thomas Kreisig, Ralf Hoffmann, Thole Züchner98

6.7

In vitro formation, detection, and identification of lysine-derived advanced glycation endproducts  Uta Greifenhagen, Rico Schmidt, Ralf Hoffmann, Andrej Frolov88

6.18

6.8

Profiling of free oxysterols in plasma by fast liquid chromatographytandem mass spectrometry and preanalytical aspects  Christin Helmschrodt, Susen Becker, Joachim Thiery, Uta Ceglarek89

Potential of using chip calorimetry as a quality control tool for encapsulated cell products  Johannes Lerchner, Eva-Maria Brandtner, Florian Mertens, Walter H. Günzburg99

6.19

6.9

A-YES ® and A-YAS ® test systems – innovative biosensors for the

Analysis of oxidation products of 1-palmitoyl-­2-linoleylphosphatidylcholine and their reaction products with cysteine, histidine and lysine residues  Ivana Milic, Ralf Hoffmann, Maria Fedorova100

6.20

Immobilization of green alga Chlorella vulgaris within alginate / silica hybrid materials for cell-based detection systems  Angela Pannier, Ulrich Soltmann, Bettina Soltmann,  Mechthild Schmitt-Jansen, Rolf Altenburger101

6.21

Live cell based fluorescent adenosine triphosphate (ATP) biosensors in microfluidic free-flow-electrophoresis chips  Stefan Jezierski, Anke Klein, Stefan Nagl, Michael Schaefer, Detlev Belder92

Screening for inhibitors of human ecto-5’-nucleotidase  Jan Pippel, Karen Yates, Matthias Zebisch, Christa E. Müller, Norbert Sträter102

6.22

Improvement of succinic acid production by yeast Yarrowia lipolytica through optimization of α-ketoglutaric acid production  Svetlana V. Kamzolova, Maria N. Chiglintseva, Julia N. Lunina, Igor G. Morgunov93

Improvement of the signal to noise ratio of a highly sensitive protease assay by different additives  Agneta Prasse, Thomas Zauner, Renate Berger-Hoffmann, Katrin Müller, Ralf Hoffmann, Thole Züchner103

6.23

Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria  Konrad Siegfried, Carola Endes, Abul Fateh Md. Khaled Bhuiyan, Anke Kuppardt, Jürgen Mattusch, Jan Roelof van der Meer, Antonis Chatzinotas, Hauke Harms104

6.10

6.11

6.12

6.13

8

measurement of estrogenic and androgenic activity in water  Karina Hettwer, Steffen Uhlig, Martin Jähne, Sven Krügener, Kirsten Simon90 Qualitative study of combinations of herbal components of STW 5 in LPS -stimulated CaCo-2 cells  Stefanie Hoser, Victoria Winkelmann, Heba Abdel-Aziz, Olaf Kelber, Dieter Weiser, Karen Nieber91

Quantitative proteomics reveals novel functions of osteoclastassociated receptor in STAT signaling and cell adhesion in human endothelial cells  Stefanie Kliemt, Claudia Göttsch, Kathrin Sinningen,  Martin von Bergen-Tomm, Lorenz C. Hofbauer, Stefan Kalkhof94

9

7.

Bioinformatics107

7.1

AP2D – A novel program for high specific primer design 

7.2

7.3

7.4

7.5

Janine Brettschneider, Bianca Liebscher, Gabriel Kind, Dirk Labudde108 Drug repositioning through incomplete bi-cliques in an integrated drug-target-disease Network Simone Daminelli, V. Joachim Haupt, Matthias Reimann, Michael Schroeder109 Combined proteome and metabonome analysis of a time- and concentration resolved exposure scenario with the carcinogenic contaminant Benzo[a]pyrene  Franziska Dautel, Stefan Kalkhof, Salvatore Loguercio,  Sven Baumann, Saskia Trump, Wolfgang Otto, Susanne Rudzok, Irina Lehmann, Andreas Beyer, Martin von Bergen-Tomm110 DARIO: A ncRNA detection and analysis tool for next-generation sequencing experiments  Mario Fasold, David Langenberger, Hans Binder, Peter F. Stadler, Steve Hoffmann111

7.8

7.9

10

Parameterization of an avascular tumor model from data  Nick Jagiella, Benedikt Müller, Margareta Müller,  Irene Vignon-Clementel, Dirk Drasdo117

7.11

Approach for the identification of human pathogen fungi based on quality-verified DNA sequences  Bianca Liebscher, Katja Köpke, Dirk Labudde118

7.12

The spider silk on molecular level  Tony Petzold, Franka Eichler, Dirk Labudde119

7.13

Google goes cancer: Improving outcome prediction for cancer patients by network-based ranking of marker genes  Janine Roy, Christof Winter, Zerrin Isik, Michael Schroeder120

7.14

Self-organizing maps: Portraying the OMEs with individual resolution  Henry Wirth, Lydia Hopp, Hans Binder121

8.

Tissue and Cell Engineering123

8.1

A novel microfluidic cell culture device for in vitro reconstruction of liver sinusoids  Jan Böttger, Julia Schütte, Karin Benz, Christian Freudigmann, Britta Hagmeyer, Simon Werner, Peter Röhnert, Holger Becker, Christoph Höppner, Martin Stelzle, Rolf Gebhardt124

8.2

Detection of transplanted mesenchymal stem cells (MSC) by PCR  Claire Fabian, Melanie Hartman, Arnd Hinze, Alexandra Stolzing125

8.3

Designing 3D biomimetic microenvironments for in vitro studies of cell-cell interaction  Katja Franke, Liv Kalbitzer, Michael Ansorge, Tilo Pompe126

8.4

Human iPS cells differentiated into microglia as a therapy for Alzheimer´s disease  Yahaira Naaldijk, Alexandra Stolzing127

8.5

Development of a bioreactor for pattern-dependent stimulation of 3D chondrocyte constructs  Oliver Petters, Nico Wüstneck, Frank Peinemann, Ronny Schulz128

8.6

Modelling circadian rhythm in colonic crypts  Jens Przybilla, Markus Löffler, Gabriela Aust, Jörg Galle129

8.7

Aging signature of human induced pluripotent stem cells  Leili Rohani, Antje Arnold, Alexandra Stolzing130

Structure topology prediction of discriminative protein sequence motifs in domains of unknown function  Steffen Grunert, Florian Heinke, Dirk Labudde112

7.6 eGOR – Predicting the total potential energy of a proteins native state by sequence  Florian Heinke, Steffen Grunert, Dirk Labudde113 7.7

7.10

An integrative multi-scale approach to liver modeling in 3D  Stefan Höhme, Lars Ole Schwen, Lorenza A. D´Alessandro, Adrian Friebel, Johannes Neitsch, Andreas Raue, Seong-Hwan Rho, Felix Gremse, Iris von Recklinghausen, Seddik Hammad, Ahmed Ghallab, Patricio Godoy, Raymond Reif, Iryna Ilkavets, Steven Dooley, Fabian Kiessling, Jens Timmer, Tobias Preusser, Jan G. Hengstler, Ursula Klingmüller, Dirk Drasdo114 Regeneration after partial hepatectomy: from cell to organ scale  Stefan Höhme, Marc Brulport, Alexander Bauer, Essam Bedawy, Wiebke Schormann, Matthias Hermes, Verena Puppe,  Rolf Gebhardt, Sebastian Zellmer, Michael Schwarz,  Ernesto Bockamp, Tobias Timmel, Jan G. Hengstler, Dirk Drasdo115 Direct and inverse modeling to test parameter inference from perfusion imaging  Nick Jagiella, Irene Vignon-Clementel, Hendrik Laue,  Fabian Kiessling, Dirk Drasdo116

11

8.8

8.9

UTX knock out phenotype in mice and Neural Stem Cells 

Kamola Saydaminova, Katrin Neumann, Francis Stewart, Konstantinos Anastassiadis131 Is it possible to transform bone marrow stem-cells into venous endothelial cells?  Franziska Schlegel, Stefan Dhein, Ömir Akhavuz,  Friedrich-Wilhelm Mohr, Pascal Maria Dohmen132

9.

Genome and Protein Engineering141

9.1

Functional linkage of adenine nucleotide binding sites in the mammalian muscle 6-Phospho­fruc­to­kinase  Antje Brüser, Jürgen Kirchberger, Marco Kloos, Norbert Sträter, Torsten Schöneberg142

9.2

Proline-rich insect antimicrobial peptides display potent antibacterial activity in vivo without being immunomodulatory for innate immune cells in vitro  Stefanie Fritsche, Daniel Knappe, Heiner von Buttlar,  Nicole Berthold, Uwe Müller, Ralf Hoffmann, Gottfried Alber143

8.10

Transcatheter implantation of an injectable tissue engineered heart valve in a mini pig in vivo model  Franziska Schlegel, Aida Salameh, Katja Oelmann, Phillip Kiefer, Stefan Dhein, Friedrich-Wilhelm Mohr, Pascal Maria Dohmen133

9.3

8.11

hiPS cell-derived neuronal networks: a prospective basis for the investigation of neurodegenerative diseases  Diana Seidel, Heinz-Georg Jahnke, Delphine Laustriat,  Marc Pechanski, Simone Haupt, Oliver Brüstle, Andrea A. Robitzki134

Development of a novel system for enantioselective catalysis using artificial metalloenzymes  Maika Genz, David Singer, Joscha Holldorf, Evamarie Hey-Hawkins, Ralf Hoffmann, Norbert Sträter144

9.4

8.12

Effects of insulin and dexamethasone on growth and metabolism of fetal bovine liver cells cultivated in Williams’ Medium E  Katja Stöckel, Martin Köhne, Herbert Fuhrmann135

The zebrafish CreZoo: Generation of novel CreERT2 - driver lines expressing in various tissues  Stefan Hans, Peggy Jungke, Michael Brand145

9.5

8.13

Neuroprotection against iron-induced cell death by perineuronal nets  Anne Suttkus, Susanne Rohn, Carsten Jäger, Thomas Arendt, Markus Morawski136

In silico selection of a theophylline riboswitch  Manja Malchau, Sven Findeiß, Nadine Weissheimer, Peter F. Stadler, Mario Mörl146

9.6

8.14

Introduction of sulfated glycosaminoglycans in growth substrates impairs the TGFβ1 driven differentiation of human dermal fibroblasts to myofibroblasts  Anja van der Smissen, Vera Hintze, Dieter Scharnweber, Stephanie Möller, Matthias Schnabelrauch, Ulf Anderegg137

Development of a prokaryotic expression system for in vivo protein biotinylation of dengue virus proteins for serological diagnostics  Awadalkareem Mohammed Eljamal, Alexandros Hadjilaou, Sven Reiche, Christian Jassoy147

9.7

Role of the H3K4 methyltransferase MLL4 in mouse embryogenesis  Christian Much 148

9.8

Subtoxic concentrations of Benzene and Toluene affect cellular metabolism and induce oxidative stress by Nrf2 pathway in lung epithelial cells (A549)  Kalaimathi Murugesan, Stefanie Kliemt, Sven Baumann, Iljana Mögel, Irina Lehmann, Martin von Bergen-Tomm, Janina M. Tomm149

9.9

Characterization of the interaction between interleukin-8 and glycosaminoglycans  Karoline Nordsieck, Annelie Pichert, Daniel Huster,  Annette G. Beck-Sickinger150

9.10

Microbial induced mineralization for the development of a sustainable self-healing mortar  Karen Stumm, Andreas Hecker, Markus Gläser, Horst-Michael Ludwig151

8.15

Genetic modification of human pluripotent stem cells to correct X-linked chronic granulomatous disease (X-CGD)-associated mutations  Sergii Velychko, Maria Rostovskaya, Sebastian Brenner, Konstantinos Anastassiadis138

8.16

Development of an intra-operative, stem cell-based therapy for the treatment of focal cartilage defects in sheep model  Matthias Zscharnack, Kathrin Godthardt, Oliver Petters, Bastian Marquass, Ronny Schulz139

12

13

9.11

10. 10.1

10.2

Regulated intramembrane proteolysis in the ECF sigma factor mediated envelope stress response in Bacillus subtilis  Thomas Wiegert152

Biophysics155 Resolving length changes of single DNA molecules with angstrom accuracy in real time  Hergen Brutzer, Alexander Huhle, Daniel Klaue, Ralf Seidel156 Cell traction force measurements of malignant and benign breast cell lines  Anya Burkart, Thomas Fuhs, Mareike Zink, Josef A. Käs157

10.3

On the biomechanics of stem cell niche formation in the gut: Modelling growing organoids  Peter Buske, Jens Przybilla, Markus Löffler, Jörg Galle158

10.4

Development of a hES derived cardiomyocyte screening platform for the electrophysiological and impedimetric detection of active pharmaceutical ingredients adverse effects  Stephan Fleischer, Heinz-Georg Jahnke, Daniella Steel, Peter Sartipy, Andrea A. Robitzki159

10.5

Lipidlayer and antibody functionalized LbL-microcarriers as novel drug delivery systems  Martin Göse, Jacqueline Leßig, Daniel Huster, Uta Reibetanz160

10.11 Cellular adhesion – A key mechanism for compartmentalization and tumor spreading?  Steve Pawlizak, Anatol Fritsch, Mareike Zink, Josef A. Käs166 10.12 The trans-dienelactone hydrolase from Cupriavidus necator: a novel metalloenzyme  Christian Roth, Silke Wegener, Michael Schlömann, Norbert Sträter167 10.13 Dynamic force spectroscopy on fluorescence labeled tau-peptides and monoclonal antibodies measured by using Optical Tweezers  Tim Stangner, Carolin Wagner, David Singer, Christof Gutsche, Olaf Ueberschär, Ralf Hoffmann, Friedrich Kremer168 10.14 Novel electroactive polymer-based nanopore membranes for the controlled size selective biomolecule filtration  Marek Staude, Christo Tchernev, Christoph Prönnecke, Sabine Schmidt, Henning Ebert, Heinz-Georg Jahnke, Andrea A. Robitzki169 10.15 Embryotoxicity testing with 3D stem cell structures  Silvia Vinz, Randy Kurz, Andrea A. Robitzki170 10.16 The binding of monoclonal antibodies and tau peptides investigated on a single-molecule level  Carolin Wagner, David Singer, Tim Stangner, Ralf Hoffmann, Friedrich Kremer171 10.17 Mathematical modeling of liver tumor growth  William Weens, Dirk Drasdo, Stefan Höhme, Jan G. Hengstler172

10.6

Probing the mechanosensing of neurons with magnetic tweezers  Tina Händler, Bernd Kohlstrunk, Josef A. Käs161

Index175

10.7

TWO in ONE: The development of a microcontroller-based hybrid

Program183

10.8

Active multi-electrode arrays based on zinc oxide  Fabian Klüpfel, Bernd Ulrich Sebastian Schmidt, Alexander Lajn, Holger von Wenckstern, Josef A. Käs, Marius Grundmann163

10.9

Domain motion of 5´-nucleotidase  Ulrike Krug, Antje Keim, Nathan S. Alexander, Richard A. Stein, Hassane Mchaourab, Jens Meiler, Norbert Sträter164

bioelectronic measurement system  Heinz-Georg Jahnke, Ronny Azendorf, Andrea A. Robitzki162

10.10 Control of cell adhesion via non-covalently bound adhesion ligands  Christina Müller, Andreas Müller, Rayk Hassert,  Annette G. Beck-Sickinger, Tilo Pompe165

14

15

FOREWORD

In 2012 the Center for Biotechnology and Biomedicine (BBZ) at the Universität Leipzig and the Biotechnology Center (BIOTEC) of the Technical University of Dresden are continuing their cooperation to hold the jointly organized Biotechnology Symposium in Saxony. This year it will be hosted in Leipzig. The main topics of the conference being “Cells as Active Sensors”, “Live Cell Analysis” and “Molecular Medicine and Protein Design”. The Biotechnology Symposium 2012 reflects Saxony’s excellent basic, applied and integrative research in the fields of nanobiotechnology, nanomedicine and molecular bioengineering, which form the basis for a successful development of and cooperation with the biotechnology industry. Saxony has evolved into a dynamic biotechnology region, providing a platform for the transfer of knowledge and technology in the fields of molecular medicine, bioanalytics, bioinformatics, tissue and cell engineering, genome and protein engineering and biophysics. The result is the sustainable growth of Saxony as a futureoriented biotech location – closely connected with an outstanding research and innovation potential. Biotechnology is emerging as a cross-section technology for the future, inventions and innovations being the motors for biotechnological progress. We trust this symposium will boost the Saxon biotechnological sector and enhance universities, research centers and companies on their path towards Biosax 2030!

Prof. Dr. Andrea A. Robitzki Prof. Dr. Michael Schroeder Director Director Center for Biotechnology and Biomedicine Biotechnology Center





PRESENTATIONS

1.

Cells as Active Sensors

CELLS AS ACTIVE SENSORS

1.1

Real-time monitoring of cellular inflammatory and degenerative processes on microarrays Andrea A. Robitzki

Injury and inflammatory processes in e. g. the brain have many fold reasons and causes based on molecular changes. The demands on reporting these molecular and physiological alterations in real time as well as online might become actually stronger. Therefore, a broad range of microarrays were developed and fabricated consisting of various microelectrode designs and configurations realizing an optoelectronic recording on viable complex brain slices and / or 3D organotypic neuronal cultures. Tauopathology and beta-amyloid plaque formation might cause a kind of neurodegeneration reflecting Alzheimer’s disease. Thus a better understanding of the role of the conformation of the Tau protein could strengthen the molecular events of a Tau dependent pathology. Herewith, novel 3D human neuroblastoma SH-SY5Y models and rat brain slice cultures expressing e. g. four fold tau gene mutants which might be responsible for a fast and emphasized degeneration of nerve fibers or neuritis. Using the appropriate multielectrode-microarrays concerning different formats, 96-­ well-and 384-well-microelectrode arrays for monolayers and slices or microcavity arrays consisting of transparent substrates and indium tin oxide-electrodes for neurospheres are perfect monitoring tools. The measurement method of choose is impedance spectroscopy indicating the frequency dependent extracellular electrical resistance in an electric field of altered current. Viable complex neuronal models expressing Tau mutants and / or reflecting hyperphosphorylated Tau protein omitting any microtubule binding were investigated on molecular as well as on morphological level.1–3 1 Jahnke HG, Rothermel A, Sternberger I, Mack TG, Kurz RG, et al. (2009) An impedimetric microelectrode-based array sensor for label-free detection of tau hyperphosphorylation in human cells. Lab Chip 9: 1422 – 1428. 2 Krinke D, Jahnke HG, Mack TG, Hirche A, Striggow F, et al. (2010) A novel organotypic tauopathy model on a new microcavity chip for bioelectronic label-free and real time monitoring. Biosens Bioelectron 26: 162 – 168. 3 Jahnke HG, Braesigk A, Mack TG, Ponick S, Striggow F, et al. (2012) Impedance spectroscopy based measurement system for quantitative and label-free real-time monitoring of tauopathy in hippocampal slice cultures. Biosens Bioelectron 32: 250 – 258.

1.2

Towards functional microbial sensor cell arrays Shimshon Belkin, Tal Elad, Sahar Melamed, Sharon Yagur-Kroll, Yosi Shacham-Diamand

Interest in the implementation of whole cell biosensors based on genetically engineered bacteria has been constantly increasing over the last two decades. Such devices sensitively report either on the biological effects of the sample and / or on the presence of pre-determined classes of chemicals. By using live cells it is possible to detect and quantify the very complex series of reactions that can exist only in an intact, functioning organism. Only a sensor of this type can report on the “wellbeing” of a system, on the toxicity of a sample, the genotoxicity of a chemical or the bioavailability of a pollutant. No molecular recognition element or chemical analysis can provide this type of information. Biosensors of the type described here offer unique advantages in the toxicity assessment field. Compared to current toxicity testing methodologies, mostly based on the use of live animals, they present an ethically acceptable in vitro toxicology option that is both cost effective as well as amenable to high throughput testing, and may find uses in environmental, pharmaceutical, security and industrial applications. The small size requirements, rapid responses and sensing versatility of bacterial sensors allow their application not only as single-strain reporters but also as building blocks for live cell arrays, in a manner analogous to DNA- or protein-based biochips. Different research directions taken in order to turn genetically engineered bacterial reporter strains into usable arrays by immobilizing them onto a solid state platform coupled to a signal transduction apparatus will be described. Examples include flow-through bioluminescent biochips for water toxicity monitoring, electrochemical detection of genotoxicity, live cell printing, algorithms for deciphering array signal patterns and molecular enhancements of sensor performance.

Prof. Dr. Andrea A. Robitzki

20

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology

Prof. Dr. Shimshon Belkin

[email protected] www.uni-leipzig.de/~dmpt

[email protected] www.bio.huji.ac.il/staff_in.asp?staff_id=62

Hebrew University Jerusalem Institute of Life Sciences

21

CELLS AS ACTIVE SENSORS

1.3

Recombinant yeast cells as a tool for biosensor applications Gerhard Rödel

Arsolux: cell-based detection system for arsenic contamination Hauke Harms, Konrad Siegfried, Antonis Chatzinotas, Sonja Hahn-Tomer

The yeast Saccharomyces (S.) cerevisiae provides a promising tool as a biological transducer in biosensor applications, e. g. for monitoring fermentation processes or detecting biological relevant amounts of (toxic) agents and pollutants. Yeast cells combine the ease of genetic manipulation, the GRAS status, rapid growth on a broad range of substrates, a long-term stability and excellent immobilization properties with the ability of sensing a wide spectrum of intra- and extracellular substances. Genetically engineered yeast cells can act as sensor cells by expressing marker proteins in response to the presence or absence of a specific analyte. Recently we generated cells monitoring the presence of nutrients such as glucose, nitrogen, phosphor, androgenic substances or toxic pollutants like arsis. In the case of fluorescent marker proteins, sensor cells can ideally be combined with optical biosensor devices. Haploid yeast cells are of either the a or α matingtype. Each cell type secretes a specific pheromone (the a- and α-factor, respectively), which is recognized by specific receptors on the surface of cells with the opposite mating type. Pheromone binding to these receptors leads to the activation of a signal cascade and subsequently to the activation of specific genes. We are exploiting this pheromone-based system for signal amplification and controlled cell-cell communication in sensory applications.

22

1.4

In spite of the substantial efforts of many research groups around the world to develop wholecell living biosensors, so called bioreporter bacteria for the quick analysis of environmental chemicals and their adverse biological effects, virtually none of the technically successful developments has made it into broad-scale application. Indeed, very convincing reasons are needed, if one wants to replace established methods of chemical analysis by products consisting of genetically modified organisms. Such a good reason is the large-scale natural arsenic contamination of groundwater in SouthEast Asian countries, with Bangladesh being a hot-spot where about 70 million people are at risk of taking up more arsenic than recommendable and millions are already severely poisoned. Due to lacking financial means and functional infrastructures, lab-based spectro­ scopic methods of arsenic analysis are no option for the regular testing of an estimated 10 million groundwater tube wells scattered around the country. Here, I introduce a practical bioreporter-based test kit, developed in our lab, and report on our efforts to implement it in a country that urgently needs relief from a major public health tragedy.

Prof. Dr. Gerhard Rödel

Prof. Dr. Hauke Harms

Technische Universität Dresden Institute of Genetics

Helmholtz-Zentrum für Umweltforschung – UFZ Department of Environmental Microbiology

[email protected] www.tu-dresden.de/genetik

[email protected] www.ufz.de/index.php?en=13566

23

CELLS AS ACTIVE SENSORS

1.5

Living lasers – towards laser-based biosensors Malte C. Gather

The invention of the laser was one of the most important breakthroughs in optics in the 20  th century: Lasers have enabled radically new technologies in medicine, communications, material processing, and many other fields. The 21 st century has often been proclaimed as the century of the life sciences and lasers have indeed been used extensively to elucidate biological mechanisms, often at sub-cellular length scales. However, the generation of laser light remained within the realm of inanimate materials and bionic principles have not been applied in any extensive manner to design and realize new lasers. Here, we present a fundamentally new optical device, a biological laser. The optical gain material of this laser is a highly fluorescent material that nature provides us with: the green fluorescent protein (GFP). Lasing is not only observed in recombinant GFP solutions, but also from living mammalian 1 and bacteria 2 cells that are genetically programmed to produce GFP. Besides the fundamental significance of being able to generate laser light in a biological system, we expect that these bio-lasers will become enabling tools for imaging and tracking of biological processes. In the single-cell based laser, intracellular diffraction and scattering impose a unique spatial pattern onto the laser output which is believed to carry information about the cell. This effect might enable a novel modality of high-throughput cytometry providing information complimentary to that obtained in conventional fluorescence activated cell sorting. 1 M . C . Gather, S. H . Yun, Nat. Photonics 5, 406 (2011) 2 M . C . Gather, S. H . Yun, Opt. Lett. 36, 3299 (2011)

Jun.-Prof. Dr. Malte C. Gather Technische Universität Dresden Institute for Applied Photophysics [email protected] www.iapp.de/~mgather

24

PRESENTATIONS

2.

Live Cell Analysis

LIVE CELL ANALYSIS

2.1

Analysing infection of macrophages by Salmonella Pietro Cicuta

2.2 Chromobodies ® and the Fluorescent 2-Hybrid (F2H) assay: New technologies for real time analysis of cellular components in living cells Kourosh Zolghadr

Macrophages are key cells in the frontline immune defence against pathogens. In vivo they encounter a range of debris and pathogens which they phagocytose to destroy or or render inert. An example of where the macrophage-pathogen interaction is particularly important is that between macrophages and bacteria such as Salmonella. The infection process is a very complex system, which in vivo involves a hierarchy of behaviour from single cell response to inflammation in tissues. Even at the cellular level, our understanding of this process is incomplete, due to the challenges of disentangling the properties of two biological species: the outcome of a bacterium / macrophage contact can in principle depend on the epigenetic and cell cycle state of either cell. This makes the interpretation of population-average cell culture experiments particularly hard. We have approached this problem by developing imaging assays in which we track the contacts and fate of macrophages at the single cell level. We have explored the role in infection of both bacteria and macrophage cell structures. On the bacterial side, we studied infection dynamics of various genetic mutants with differing flagellar or motility properties, and lacking a virulence protein that is required for infection. On the macrophage side we are exploring the role of membrane receptors sensitive to LPS which is present on the Salmonella surface.

The understanding of cellular processes in response to external stimuli is one of the major challenges in early drug discovery. To visualize dynamic cellular processes in real time we have developed a new format of intracellular functional antibodies (Chromobodies ®). In combination with High-Throughput Microscopy and automated image analysis we have demonstrated that Chromobodies can be used to target and trace antigens for High-Content Analysis (HCA) in living cells. Our proof-of-principle studies include cell cycle monitoring and real time apoptosis studies. We thereby expand the quality and quantity of information that can be gathered in High-Content Analysis. In addition we developed a new assay to directly visualize protein interactions in living cells. By tethering a fluorescent bait at a defined cellular structure we generate a positional protein-protein interaction biosensor and assay for co-localization of fluorescent prey proteins in living cells. We use this Fluorescent 2-Hybrid (F2H) assay to study changes of complex formation during cell cycle progression and differentiation. Automated image acquisition and analyses allow us to perform high throughput screens for molecules and drugs perturbing specific protein interactions in the cellular environment and in real time. Rothbauer et al., Nature Methods (2006) Zolghadr et al., Mol. Cell. Proteomics (2008) Kirchhofer et al., Nature Structural Molecular Biology (2010) Schmidthals et al., Anal. Bioanal. Chem. (2010)

Dr. Pietro Cicuta

28

University of Cambridge Department of Physics Cavendish Laboratory

Dr. Kourosh Zolghadr

pc245@ cam.ac.uk www.bss.phy.cam.ac.uk/~pc245

[email protected] www.chromotek.com

ChromoTek GmbH

29

LIVE CELL ANALYSIS

2.3

Automatic tracking and quantification of dynamic cellular characteristics Ingo Röder

2.4

Genetic tools to study the brain: from transgenic mice to metabolic imaging Johannes Hirrlinger

The continuous analysis of individual cell fates within a population of cells is still a major experimental challenge. However, new monitoring techniques, such as high-resolution timelapse video microscopy, facilitate the tracking and the quantitative analysis of single cells and their progeny. The obtained information, e. g. on cellular development, divisional his­ tory, and differentiation of individual cells, can be comprised into a pedigree-like structure, denoted as cellular genealogy. To go beyond a pure descriptive analysis of this type of data and to statistically extract reliable information about effecting variables and control mecha­ nisms under­lying cell fate decisions, it is necessary to analyse large numbers of cellular geneal­ ogies. This requires the development and application of automatic cell tracking algo­rithms. In the talk I will present computational methods that allow for the automatic segmen­tation (recognition) and tracking of individual cells and, based hereon, the reconstruction of cellular genealogies from time-lapse video data. In particular, the talk will report about a study that applied the tracking algorithms to human haematopoietic stem and progenitor cells (HSPCs) in bioengineered culture conditions over several days as well as on the tracking of cellular movement in early zebrafish embryos.

The brain is one of the most fascinating but also one of the most complex organs of mammals. Besides neurons, a large population of glial cells crucially contributes to brain function. Transgenic mice with cell type specific expression of fluorescent proteins allow imaging of these different types of cells, their intimate morphological interaction as well as the dynamics of this interaction in brain slices as well as in vivo in the healthy and injured central nervous system. Furthermore, advanced transgenic fate-mapping tools like the splitCre system allow for a highly specific dissection of cell populations based on expression domains defined by the overlapping activity of two promoters. An important aspect of the functional interaction of brain cells is energy metabolism. While classical biochemical approaches to quantify the metabolic state of cells rely on the lysis of many cells or whole tissues, cellular resolution can be achieved using fluorescent imaging approaches. These include the analysis of cellular NAD (P) H -fluorescence as well as recently developed genetically encoded sensors for molecules like glucose, ATP, NADH or glutamate. These tools will contribute to a deeper understanding of how signalling and metabolism in the brain is controlled by different types of brain cells in the context of brain physiology and pathophysiology.

Dr. Johannes Hirrlinger

Prof. Dr. Ingo Röder Technische Universität Dresden Faculty of Medicine Carl Gustav Carus Institute for Medical Informatics and Biometry [email protected] www.tu-dresden.de/die_tu_dresden/fakultaeten/ medizinische_fakultaet/inst/imb

30

Max-Planck-Institut für Experimentelle Medizin Göttingen Department of Neurogenetics [email protected] www.em.mpg.de Universität Leipzig Carl Ludwig Institute for Physiology [email protected] www.cliphys.uniklinikum-leipzig.de/

31

PRESENTATIONS

3.

Molecular Medicine and Protein Design

MOLECUL AR MEDICINE AND PROTEIN DESIGN

3.1

Post-translational modified peptides to develop efficient IVD to follow up disease activity of autoimmune diseases: a challenge for theragnostics Anna Maria Papini

Autoimmune diseases affecting an increasing number of individuals throughout the world, represent a large and diverse group of disorders categorized by tissue injury or pathology. Thus, reliable diagnostic / prognostic tools are necessary not only for an early diagnosis but also for monitoring disease activity. Growing evidences indicate that post-translational modi­­ fi­cations (i. e., acetylation, lipidation, citrullination, glycosylation), either native or aberrant, may play a fundamental role for specific autoantibody recognition in autoimmune diseases. We have recently developed structure based designed N-glucosylated peptides, characterized by β-turn structures,1,2 as Antigenic Probes accurately measuring in sera of a statistically significant population of neuroinflammatory diseases, i. e. Multiple Sclerosis and Rett Syn­ drome, high affinity autoantibodies as biomarkers of disease activity.3-5 This was possible because of an innovative “chemical reverse approach” 5 allowing optimization of the glycopeptide sequence able to detect the most specific and high affinity autoantibody titre in sera.6-9 Moreover, these peptide scaffolds modified with different aberrant PTM s, each one specific for antibody-mediated forms of different immune-mediated diseases (i. e. coeliac disease, primary biliary cyrrhosis, rheumatoid arthritis, etc.) were able to recognise specific biomarkers.10 The collection of the Synthetic Antigenic Probes is proposed to be used in multiple diagnostic / prognostic immunoassays with a “theragnostic” aim.11

3.2

Technologies for the development of tailored enzymes and strains Thomas Greiner-Stöffele

c-LE cta is a white biotech company which provides new, customized enzymes and strains to industrial processes. The company uses a proprietary technology platform to identify new enzymes and strains in biodiversity, to evolve their properties by focused engineering strategies and to develop efficient processes for enzyme and small molecule production. Despite the availability of commercial enzymes used in the manufacturing of fine and specialty chemicals, there is still a huge demand for customized enzymes with improved performance and the technology to provide them in sufficient quantities. The same applies for microbial strains, why their optimization for the efficient production of proteins and of biochemical building blocks has received a considerable attention over the last years, especially with the emergence of ‘all’ omics techniques. c-LE cta’s technology platform enables the fast access to high performance enzymes and strains by using the proprietary Cluster-Screening. Furthermore, c-LE cta is specialized on the rapid scale-up of production processes into commercial scale. In the presentation we will show exemplary successful conducted engineering programs on enzymes in which significant improvements connected to corresponding cost reductions in the target application process were achieved and will report on a screening program in which we could identify > 50 alcohol dehydrogenases within several weeks.

1 (a) Mazzucco, S. et al. Bioorg. Med. Chem. Lett. 9 (1999) 167; (b) Carotenuto, A. et al. J. Med. Chem. 44 (2001) 2378; Lolli, F. et al. Neurology 65 (2005) 781. 2 (a) Lolli, F. et al. P. N. A. S.  U. S. A. 102 (2005) 10273; (b) Carotenuto, A. et al. J. Med. Chem. 49 (2006) 5072. 3 (a) Papini, A. M. Nat. Med. 11 (2005) 13; (b) Papini, A. M.; Rovero, P.; Chelli, M.; Lolli, F. PCT WO 03/000733. 4 Lolli, F. et al. J. Neuroimmunology 167 (2005) 131. 5 Alcaro, M. C. et al. Chemistry Today 25 (2007) 14. 6 Carotenuto, A. et al. J. Med. Chem. 51 (2008) 5304. 7 F. Gori, et al. Journal of Neuroimmunology (2011) 233, 216. 8 A. M. Papini. J. Pept. Sci. (2009), 15, 621. 9 A. M. Papini Patent application 2012. 10 F. Nuti, et al. Biopolymers (Peptide Science) (2010), 94, 791. 11 A. M. Papini et al. (a) EP 2284188 A1 - US 2011028409 A1; (b) EP2402368 A1 - WO2012001103 A1; (c) EP2050761 B1.

Prof. Dr. Anna Maria Papini Università degli Studi di Firenze Polo Scientifico e Tecnologico, Department of Chemistry [email protected] www.peptlab.eu

34

Dr. Thomas Greiner-Stöffele c-LEcta GmbH Leipzig [email protected] www.c-lecta.com

35

MOLECUL AR MEDICINE AND PROTEIN DESIGN

3.3

Designer recombinases reverse HIV infection Frank Buchholz

3.4

Genome surgery using designer nucleases: ZFNs or TALENs? Claudio Mussolino

HIV / A IDS has remained an incurable disease despite the successful implementation of highly active antiretroviral therapy (HAART) into clinical practice. This is due to the fact that the HIV provirus stably integrates into the chromosomes of host cells. As a consequence,

patients have to take lifelong medication, causing serious side effects and reducing the life expectancy of affected individuals. We have used directed molecular evolution to generate designer recombinases able to excise HIV-1 proviral DNA from infected cells. I will present the development of a second generation Tre recombinase that targets a highly conserved sequence in the long terminal repeat of most HIV-1 isolates. Furthermore, I will report the successful excision of HIV-1 proviral DNA in vivo. In particular, pronounced anti-retroviral effects were seen in both, humanized Rag2-/- γc-/- mice engrafted either with Tre-transduced primary CD4+ T cells, or with Tre-transduced CD34+ hematopoietic stem and progenitor cells. These results support the use of Tre-recombinases in a clinical eradication approach with the aim to provide a cure for HIV / A IDS.

Designer nucleases represent a valuable tool to induce targeted genome modifications for human gene therapy. Zinc-finger nucleases (ZFNs) have traditionally been used to specifically modify the human genome. More recently, a novel DNA binding motif derived from transcription activator-like effectors (TALE s) of the bacterial genus Xanthomonas has been employed to direct cleavage activity to desired genomic locations. In order to compare these two designer nuclease platforms side-by-side, we engineered TALE -based nucleases (TALENs) and ZFNs that target the three human loci CCR5, IL2RG and AAVS1, and profiled activity and cytotoxicity associated with their use in human cells. For both classes of designer nucleases the allelic gene disruption activity in HEK293T cells ranged from 21 % to 29 % at the three loci analyzed. Notably, however, TALENs outperformed ZFNs with regards to cytotoxicity. While the cell survival rate upon delivery of ZFN pairs dropped to ~ 50 %, it remained at ~ 85 % with the TALENs. For the CCR5 -specific designer nucleases a cell cycle analysis in HeL a FUCCI cells was performed showing no cell cycle aberrations associated with the use of TALENs while an S-G2-M arrest three days post transfection was measured in ZFNs treated cells. Moreover, cells expressing ZFNs showed a slight increase in the number of apoptotic cells. A known off-target site of the CCR5 -specific ZFN pair has been identified in the highly similar CCR2 locus. Among the few CCR5 -specific TALENs tested, we identified two pairs that revealed only minimal cleavage activity at CCR2, suggesting superior cleavage specificity of these nucleases. Taken together these results demonstrate that TALENs can be easily generated to cleave human loci with similar activity as ZFNs but with improved specificity and cytotoxicity profiles. These desirable characteristics mark the great potential of this novel platform for tailored editing of the human genome.

Prof. Dr. Frank Buchholz Technische Universität Dresden Faculty of Medicine University Cancer Center (UCC)

36

www.krebscentrum.de

Dr. Claudio Mussolino

Max-Planck-Institut für Molekulare Zellbiologie und Genetik

Medizinische Hochschule Hannover Institute of Experimental Hematology

[email protected] www.mpi-cbg.de

[email protected] www.mh-hannover.de/experimentalhematology.html

37

PRESENTATIONS

4.

Evening Lecture

EVENING LECTURE

4.1

Phosphonate chemistry in medicine: from bone actives to antiviral agents Charles E. McKenna

Phosphonate analogues of biological phosphates are valuable therapeutic agents for a wide range of diseases. However, they also exhibit certain limitations in this role that make them compelling targets for enhancements by suitable chemical strategies. In one example, bisphosphonates (BPs) constitute an important class of anti-resorptive drugs, useful against osteoporosis and increasingly important in treating cancer. At the same time, they can be exploited to design nucleotide analogues with systematically variable stereoelectronic properties and biochemical specificities, resulting in unique and subtle probes of enzymatic structure and function. As bone active drugs, their skeletal distribution, cellular uptake and mechanisms of action remain to be fully elucidated, prompting creation of imaging probes capable of mimicking some or all of their pharmacological properties. Recently, we introduced a “magic linker” synthesis of novel fluorescent conjugates formed from heterocyclic BP drugs such as risedronate or zoledronate. Labeling of BPs and derivatives having different bone affinities by dyes with distinguishable emission spectra generates a fluorescent probe “toolkit”, allowing simultaneous detection of the individual BPs in bone, bone tissues, and cells. Application of these sensors in a number of biological systems in vivo and in vitro is providing exciting new insights into the localization, function and unrealized potential of BP drugs. In a second example, acyclic nucleoside phosphonates (ANPs) are effective antiviral and anti-cancer drugs, but their low permeability significantly limits their utility. A new prodrug approach will be described, that when applied to the clinical ANP cidofovir results in enhanced oral bioavailability, and in some cases, dramatically increased antiviral potency.

Prof. Dr. Charles E. McKenna University of Southern California Department of Chemistry [email protected] http://chem.usc.edu

40

POSTERS

5.

Molecular Medicine

MOLECUL AR MEDICINE

5.1

PUFA promote the phagocytosis but not the

killing of the persistent pathogens R. equi and P. aeruginosa Stephanie Adolph, Herbert Fuhrmann, Julia Schumann

Phagocytosis is a major effector mechanism of macrophages. At this, the bactericidal prop­ erties of the immune cells are of particular relevance with respect to the pathogens R. equi or P. aeruginoae, which are known to survive or even to replicate inside macrophages. Phagocytosis is a membrane-mediated process. Thus, we assumed that the enrichment of macro­phage plasma membrane with polyunsaturated fatty acids (PUFA) may support the engulfment and the killing of microorganisms by the immune cells. In the present study the macrophage cell line RAW 264.7 was used as a model system. Both the phagocytosis rate and the bactericidal capacity of PUFA-supplemented macrophages against viable pathogens of the virulent R. equi strain ATTC 33710, the non-virulent R. equi strain ATTC 6939 as well as the virulent P. aeruginosa strain ATTC 10145 was investigated. The number of engulfed and surviving microorganisms were determined in time series by means of the plate counting test and the MTT-assay. For all pathogens tested the data gained by both assays consistently show that supplementation of macrophages with PUFA promotes the phagocytic capacity of the cells. However, the bactericidal properties of the RAW 264.7 were not affected by the PUFA enrichment. No significant differences in the survival and replication rates of the internalized microorganisms could be observed. Hence, our clinical-related study provides evidence that PUFA supplementation improves the phagocytic activity of macrophages in case of infection but is not sufficient to promote the killing of the persistent pathogens.

Stephanie Adolph

44

5.2

Polymer-based nanoparticles as delivery platform for small RNA molecules (siRNA s, miRNA s) in vivo: development of novel therapeutic strategies based on gene silencing Achim Aigner, Sabrina Höbel, Ulrike Weirauch, Anja Frömberg, Alexander Ewe

In molecular medicine, a promising approach is based on the therapeutic exploration of RNA interference (RNA i) for the knockdown of pathologically overexpressed genes. Likewise, many miRNA s have been shown to be downregulated under pathological conditions, offer­ ing possibilities for miRNA replacement therapy. However, the application of small RNA s requires the development of delivery strategies and the identification of suitable target genes. We have established the non-covalent complexation of therapeutic RNA s with polyethylenimines (PEI) as in vivo delivery platform. PEI s are synthetic polymers with protonable amino groups and a high cationic charge density. This allows the formation of nanoscale interpolyelectrolyte complexes (‘nanoplexes’) with nucleic acids, mediating their full protection against nucleolytic degradation and delivery to target tissues as well as their cellular uptake and intracellular release. Critical for in vivo applications are the PEI s or PEI derivatives being used. The therapeutic efficacy of PEI / siRNA-mediated RNA i has been demonstrated in various mouse tumor xenograft models. Among others, this includes VEGF in prostate or pancreatic xenografts, the HER-2 receptor in ovarian carcinoma as well as survivin or the growth factor pleiotrophin (PTN) in glioblastoma. Likewise, the in vivo application of miRNA-145 or miR-33a results in efficient miRNA delivery and in antitumor effects. In conclusion, the systemic delivery of siRNA or miRNA represents an attractive approach in molecular medicine, and PEI-based nanoplexes are a promising delivery platform for therapeutic RNA molecules.

Prof. Dr. Achim Aigner

Universität Leipzig Faculty of Veterinary Medicine Institute of Physiological Chemistry

Universität Leipzig Faculty of Medicine Rudolf Boehm Institute of Pharmacology and Toxicology Department of Clinical Pharmacology

[email protected] www.uni-leipzig.de/~vpci

[email protected] www.uni-leipzig.de/~pharma

45

MOLECUL AR MEDICINE

5.3

Simplet (smp) is required for caudal fin regeneration and for canonical Wnt signaling by regulating nuclear localization of ß-catenin Christopher Antos, Beate Küchler, Caghan Kizil, Günes Özhan-Kizil, Jia-Jiun Yan, Andrew C. Oates, Enrico Moro, Francesco Argenton, Michael Brand, Gilbert Weidinger

Tissue formation and regeneration requires coordination of progenitor cell proliferation and patterning. We previously showed that the gene simplet (smp) is required for both cell proliferation and patterning in regenerating zebrafish fins; however, the mechanisms through which smp regulates these phenomena are unknown. Using zebrafish embryos, we now show that smp is required for regulating Wnt / ß-catenin signal transduction. Morpholino knockdown of smp results in reduced activation of Wnt-dependent reporters in transgenic fish and of endogenous Wnt-target genes in vivo, and this effect is associated with the loss of nuclear localization of ß-catenin. Knockdown also prevented the induction of the Wntdependent reporters by overexpression of Wnt and a stabilized version of ß-catenin. Coexpression experiments showed that smp enhanced canonical Wnt signaling. Expression of Smp-GFP in cultured cells and embryos showed localization both in the nucleus and in the cytoplasm, and the nuclear localization of Smp-GFP coinsided with the nuclear localization of endogenous ß-catenin. Immunohistochemistry experiments also indicate that Smp is localized in the nucleus with endogenous ß-catenin in regenerating fins. Smp contains a single nuclear localization signal (NLS) and mutation of this signal prevented the nuclear localization of Smp and of endogenous ß-catenin. Furthermore, loss of the NLS in the Smp protein reduced Wnt signaling and phenocopied the loss-of-function wnt and smp phenotypes. Lastly, co-immunoprecipitation experiments revealed an interaction between Smp and ß-catenin. Together, these results show that smp is required for Wnt signaling by regulating ß-catenin nuclear localization and indicate that smp is involved in regulating Wnt signaling in developing and regenerating tissues.

46

5.4

CD160130 : a selective inhibitor of the tumor-type hERG1 channels, active in human leukemias

Annarosa Arcangeli, Luca Gasparoli, Massimo D’Amico, Andrea Becchetti, Olivia Crociani, Marika Masselli, Serena Pillozzi, Kenneth Mugridge, Wolfgang Tiedke hERG1 channels, besides sustaining the cardiac Ikr , are aberrantly expressed in human cancers, where they regulate several aspects of neoplastic cell behavior. Consequently, hERG1 is considered a potential target for antineoplastic therapy. hERG1 is also considered an anti-target due to the negative cardiac side-effects which follow its blocking within the heart. To circumvent this problem, we identified a strategy based on the biophysical and structural differences between the “cardiac” and the “tumor” hERG1, noticeably by the preferential expression of the hERG1B isoform in leukemias. We investigated the pharmacological effects of CD160130, a phosphodiesterase-4 inhibitor which also demonstrates hERG1 channel binding affinity, and antineoplastic activity on primary B-cell chronic lymphocytic leukemia cells. We evaluated the killing ability of CD160130 on four human leukemic cell lines; CD160130 displayed a good killing ability and a ten-fold greater inhibition on currents of the hERG1B isoforms compared to hERG1A with an IC50 value similar to the ED 50 obtained for leukemia cell killing. When tested on FLG 29.1 cells (which exclusively expresses the hERG1B isoform), CD160130 blocked hERG1 currents with an IC50 value almost identical to that determined in hERG1B transfected cells and similar to the ED 50 for leukemia cell killing thus confirming the selectivity of CD160130 for the hERG1B tumor-type isoform. No killing effect was exerted by CD140793, a close analogue of CD160130 which possesses no hERG1 inhibitory activity. These findings point towards CD160130 as a potential first-in-class selective tumor-type hERG1 channel inhibitor for the treatment of leukemias.

Dr. Christopher Antos

Prof. Dr. Annarosa Arcangeli

Technische Universität Dresden Center for Regenerative Therapies Dresden (CRTD)

Università degli Studi di Firenze Department of Experimental Pathology and Oncology

[email protected] www.crt-dresden.de

[email protected] www.patgen.unifi.it

47

MOLECUL AR MEDICINE

5.5

Polyunsaturated fatty acids impact the intracellular trafficking of Phospholipase D1 differently Shereen Basiouni, Herbert Fuhrmann, Julia Schumann

Phospholipase D (PLD), a receptor-regulated signalling enzyme, is involved in numerous cellular processes including exocytosis and phagocytosis. In mast cells, the two isoforms PLD1 and PLD2 appear to be essential for the stimulated secretion of granules. Using the canine mastocytoma cell line C2 , a model for atopic dermatitis in dogs, the aim of the present work was to investigate the modulatory effect of polyunsaturated fatty acids (PUFA) on intracellular PLD trafficking. Supplementation of the cell culture medium with α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) resulted in an enrichment of mast cell membranes with the fatty acid added, at this modulating the lipid composition of both raft and non-raft membrane domains. To investigate the effect of the membrane remodeling on PLD trafficking, the C2 cells were transfected with EGP plasmids encoding PLD1 or PLD2 , stimulated with mastoparan and imaged using confocal microscopy. Before stimulation PLD1 is situated within intracellular vesicular structures; PLD2 is located at the plasma membrane. After stimulation, PLD1 migrates to the plasma membrane, whereas PLD2 maintains at its peripheral location. Enrich­ ment of the mast cells with PUFA affected the stimulation-induced trafficking of PLD1. All PUFA examined, except AA , prevented the peripheral migration thus retaining PLD1 intracellular. For PLD2 no PUFA effects could be observed. In conclusion, membrane remodeling due to PUFA supplementation affects intracellular PLD1 trafficking which in turn impact mast cell function.

5.6

Identification of novel viral protease inhibitors via dynamic fragment ligation Daniel Becker, Jörg Rademann, Christoph Arkona

RNA-viruses like West-Nile- or Coxsackie-Virus infect millions of humans annually, thus

causing a variety of diseases. Therefore the development of novel antiviral agents is essential for tackling the increasing number of infections. Recently, our group has introduced dynamic ligation screening for the detection of reversibly ligated fragments in a biochemical assay through their competition with a fluorogenic enzyme substrate.1 As a member of the SILVER consortium, we now utilize this concept to identify novel inhibitors for viral proteases. The SILVER project is dedicated to set up a state-of-theart inhibitor lead design program that will contribute to the control of diseases caused by RNA viruses. In detail, we aim to identify inhibitors for the non-structural protein NS3 (WNV) and the 3C protease (CV) via the concept of dynamic fragment ligation. We have synthesized a number of small molecule “warheads” carrying a functional group essential for irreversible inhibition of the viral protease, and an aldehyde group capable of ligating amine-fragments to enhance the inhibitory effect through steric and electronic interactions. Inhibition is determined in a fluorescence assay, utilizing a peptide substrate functionalized with AMC.2 The viral protease is incubated with peptide substrate, amine fragment and the low-affinity warhead. Reduced enzyme activity indicates the cooperative action of an inhibition-enhancing amine-fragment. This method enables the identification of specific protease inhibitors in a high-throughput screen. 1 Rademann et al. Angew. Chem. 2009,121,6464 2 Rademann et al. Angew. Chem. 2008,120,3319

Shereen Basiouni

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Daniel Becker

Universität Leipzig Faculty of Veterinary Medicine Institute of Physiological Chemistry

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy Medicinal Chemistry

[email protected] www.uni-leipzig.de/~vpci

[email protected] www.uni-leipzig.de/agrademann

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5.7

Influence of opioid receptor activation and inhibition on synaptic transmission under hypoxia Marcus Bloßfeld, Andreas Merkenschlager, Karen Nieber

Activation of delta opioid receptors by [D-Ala2, D-Leu5] enkephalin (DADLE) has been reported to have neuroprotective effects on rat embryonic cortical neurons under hypoxia. A neurotoxic impact has also been shown for micromolar concentrations.1 There are limited information about effects of naloxon during hypoxia. The aim of the present study was to examine the influence of DADLE and naloxone on the amplitude of electrically-evoked postsynaptic potentials (PSPs) under normoxic (95 % O2 , 5 % CO2 ) and hypoxic (1 % O2 , 5 % CO2 , 94 % N2 ) conditions. The experiments were done using intracellular recordings on cortical pyramidal cells in brain slices of adult rats. DADLE (1 µM) decreased the amplitude of PSPs. Naloxone (100 µM) inhibited synaptic transmission in neurons of rats younger than 10 weeks whereas in neurons of older rats no influence could be found. DADLE (1 µM) and naloxone (100 µM) did not modulate membrane potential and input resistance. Exposure of the slices to hypoxia resulted in a decreased amplitude of PSPs. DADLE , superfused 5 min before hypoxia, did not alter the hypoxia-decreased PSPs. Contrary, naloxone increased the reduced amplitude of PSPs. This effect was age-independent. Our results indicate that during hypoxia naloxon (100 µM) but not DADLE (1 µM) has a neuroprotective effect. 1 Sun K, Su D and Wang X. 2009. Delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) reduced oxygen-glucose deprivation caused neuronal injury through the MAPK pathway.

Marcus Bloßfeld Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy Pharmacology for Natural Sciences [email protected] www.uni-leipzig.de/~pharm

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5.8

Cytotoxic effects of curcumin on human retinal pigment epithelial cells Rui Chen, Margrit Hollborn, Andreas Bringmann, Leon Kohen, Peter Wiedemann

The bioflavonoid curcumin from turmeric is an ingredient of curry powder. Due to its antiinflammatory, antioxidant and anti-carcinogenic effects, curcumin may be a promising agent against cancer and age-dependent cellular diseases. However, it is not known whether curcumin has effects on retinal cells. We investigated the effects of curcumin on cultured human retinal pigment epithelial (RPE) cells. RPE cell proliferation was slightly increased by curcumin at 10 µM and significantly reduced above 50 µM. This decrease could not be altered by treatment with PDGF, a strong stimulator of RPE cell proliferation. Curcumin decreased dose- and time-dependently the viability of RPE cells via induction of early necrosis (at concentrations above 10 µM) and delayed apoptosis (above 1 µM). Curcumin reduced the secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF or chemical hypoxia, respectively. Curcumin increased the activation of p38 MAPK and decreased the phosphorylation of Akt protein. Activation of p38 MAPK was blocked by the selective inhibitor SB203580. The mRNA expression of different growth factors and MMP9 was altered by curcumin. Curcumin (at 50 µM) stimulated the chemotaxis of RPE cells. The results show that curcumin has various different effects on RPE cells in vitro including induction of necrosis and apoptosis at relatively low doses. The toxic effects on RPE cells suggest that eyes should be monitored carefully in clinical studies.

Rui Chen Universität Leipzig Faculty of Medicine Department of Ophthalmology [email protected] www.augenklinik.uniklinikum-leipzig.de

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5.9

A tripeptide switch region to induce agonism and inverse agonism at the ghrelin receptor Sylvia Els, Enrico Schild, Pia S. Petersen, Constance Chollet, Birgitte Holst, Thue W. Schwartz, Annette G. Beck-Sickinger

The ghrelin receptor is a GPCR possessing an unique constitutive activity. Signaling of the receptor is controlled by its endogenous ligand ghrelin and considerably contributes to the regulation of appetite, food intake and energy homeostasis. Thus, reducing the basal activity can be an approach to decrease body weight and to develop an anti-obesity drug. Inverse agonists are able to reduce basal signaling of a receptor. Holst et al. introduced variants of substance P acting as inverse agonists, e. g. KwF wLL -NH 2 . 1  We describe the design of highly potent ligands for the ghrelin receptor. Small changes in the switch region -wFw- of the hexapeptide KwF wLL -NH 2 could easily swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. In activity assays, introduction of β-(3-benzothienyl)-D-alanine, 3,3-diphenyl-D-alanine and 1-naphthyl-D-alanine at position 2 resulted in highly potent and efficient inverse agonists, the substitution of D-Trp of position 4 with 1-naphthyl-D-alanine and 2-naphthyl-D-alanine induces agonism. Thereby, modification of only few atoms can decide between agonism and inverse agonism. Only minor changes in the peptide sequence can have a major impact on biological actions. Furthermore, the peptide with highest potency and affinity, K-(D -1-Nal)F wLL -NH 2, was tested in acute food intake studies, revealing an almost 5-fold decrease of acute food intake compared to vehicles. 1 Holst et al., J. Biol. Chem., 282, 15799 (2007)

Sylvia Els

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5.10 A murine model of Cryptococcus neoformansrelated immune reconstitution inflammatory syndrome (IRIS) Maria Eschke, Andreas Grahnert, Daniel Piehler, Tina Richter, Gabriele Köhler, Werner Stenzel, Uwe Müller, Gottfried Alber Cryptococcus neoformans is an opportunistic fungal pathogen that can cause fatal meningitis in immunocompromised hosts. Especially in sub-Saharan countries cryptococcosis is a major health problem with more than half a million death cases of C. neoformans-infected AIDS patients every year. The clinical management of the HIV pandemic is considerably complicated by the fact that following antiretroviral -therapy a significant proportion of patients with cryptococcal co-infection paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS). The underlying mechanisms are as of yet poorly understood. We have established a mouse model of cryptococcal IRIS based on lymphocyte-deficient mice (i. e. RAG -1 - /- mice) which are immune-reconstituted by adoptive transfer of wild-type splenocytes six weeks after low-dose infection with C. neoformans strain 1841. Following immune reconstitution mice develop a severe wasting disease that manifests as rapid weight loss, mortality and multi-organ inflammation and thus is pathologically similar to clinical IRIS. To characterize the cell type(s) responsible for IRIS induction, adoptive transfer of purified splenic CD4+ T helper cells was performed. Rapid wasting upon T helper cell reconstitution revealed a central role of these cells in IRIS pathogenesis and was associated with an increased production of pro-inflammatory cytokines such as IFN -gamma and interleukin-6. Further analysis of our murine cryptococcal IRIS model will yield a first cellular and molecular understanding of disease development which is necessary in order to improve HIV treatment strategies.

Maria Eschke

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Immunology Molecular Pathogenesis

[email protected] www.biochemie.uni-leipzig.de

[email protected] www.uni-leipzig.de/~blessing

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5.11 A new approach for screening of colorectal cancer circulating tumor cell (CTC) using EpCAM -pluri­Beads Nadezhda Frolova, Christoph Mohr, Anne Rasser, Ulrich Sack, Jan-Michael Heinrich

The presence of adequate methodological tools is the most important prerequisite for successful achievement of actual goals. Developing of methods for circulating tumor cells (CTC) screening is the key problem in cancer research. Typically, appearance of CTCs in blood indicates a poor prognosis in cancer development. Recently, several innovative ap­ proaches to detect rare cells in blood were developed. Some of them are based on physical or biological properties of epithelial cells. For example, epithelial cell adhesion molecule (EpCAM) antigen (an oncogenic signaling molecule for cancer cells) is being used as a target for immunotherapy treatment of human carcinomas. Here we are presenting a new approach of colon carcinoma circulating tumor cells (CTC) screening using pluriBeads carrying a tumor-associated EpCAM antibody. This method is based on a non-magnetic cell separation technology. It does not require any sample pretreatment. EpCAM -pluriBeads can be added directly to a whole blood sample. The method is also suitable for single cell isolation from different biological fluids. Moreover, its sensitivity can be additionally increased via raising of the sample volume. The bound EpCAM -positive colon carcinoma cells can be easily detached from pluriBeads and then involved in further molecular-genetic experiments aiming detection of their mutation status. In case of colon carcinoma, KRAS mutation status is predictive of response to anticancer therapy. Thus, the new method can be judged as a fast and effective tool for early cancer diagnostics.

Dr. Nadezhda Frolova

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5.12 biocrea: Creating novel drugs for debilitating CNS diseases Thorsten Hage, Martin Gunthorpe

biocrea is a leading pharmaceutical company that creates first-in-class drug candidates for the treatment of debilitating disorders of the central nervous system (CNS). We are starting from the therapeutic concept for which we are synthesizing our small molecules on the basis of our proprietary compound libraries and expert molecular modeling before we apply a rigorous lead optimization process. biocrea licenses or sells these candidate drugs to larger pharmaceutical companies. GlaxoSmithKline, Wyeth, Pfizer and Boehringer-Ingelheim were our partners over the last years. biocrea is currently focused on Huntington’s disease and other neurological and psychiatric diseases and is implementing a ‘patient-to-screen’ phenotypic drug discovery platform. biocrea collaborates worldwide with excellent academic groups to expand and complement its knowledge about disease mechanisms, new targets and new technologies. Our scientists participate in and contribute to international key meetings in neuroscience and drug discov­ ery. We are looking always for new partnerships – please talk to us.

Dr. Thorsten Hage

pluriSelect GmbH

biocrea GmbH Disease Area CNS

[email protected] www.pluriselect.com

[email protected] www.biocrea.com

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5.13 Analysis of HeLa rho0 cell lines after transfer of wild type mitochondria Sandra Heller, Susanna Schubert, Mario Krehan, Ingo Schäfer, Jessica Zenker, Peter Seibel

Background: The mitochondrion harbours important biochemical processes and acts as keyplayer in the programmed cell death. Cells without mitochondrial DNA (ρ0 cells) lack a functional respiratory chain and require in addition metabolic supplementation with pyruvate and uridine for cell viability. One possible method to generate cells without mitochondrial DNA (mtDNA) is to cultivate cells on growth medium that includes chemicals like ethidiumbromide or ditercalinium that interfere with DNA replication. A new method takes advantage of a restrictionendonuclease directed to the mitochondrial matrix. This allowed us to generate ρ0 cells without the toxicological side effects caused by the chemical method. Methods: A HeLa cell line devoid of mitochondrial DNA (mtDNA) after cultivation with ethidiumbromide and transfected with targeted restrictionendonuclease, respectively was fused with cytoplasts of a HeLa wild type cell line and analysed by Clark electrode and FACS analysis. Results: Fusion of HeLa ρ0 cells and cytoplasts of HeLa wild type cells was monitored by confocal microscopy with differently stained mitochondria. Fused cells were selected by cultivation in media without uridine. Analysis of cell growth revealed no significant differences for HeLa fused cells in comparison to wild type cells. However cell growth of fused cell lines transfected with targeted restrictionendonuclease is slightly decreased in media without uridine. Conclusions: Further experiments should be carried out like determination of enzyme activity or ATP level to improve characterisation of both ρ0 cell lines fused with wild type mitochondria.

Sandra Heller

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5.14 Switching Y-receptor selectivity of short NPY analogs: acylation versus carbaboranylation Sven Hofmann, René Frank, Evamarie Hey-Hawkins, Annette G. Beck-Sickinger

Neuropeptide Y (NPY) is known to be the naturally potent agonist of the neuropeptide Y receptor 1, 2 and 5 (Y1R , Y2R, Y5R) whereas the pancreatic polypeptide (PP) mainly stimulates the neuropeptide Y 4 receptor (Y4R). Apart from the Y receptor body distribution and their physiological functions, especially the Y1R and the Y4R were shown to be promising pharmaceutical targets. The Y1R is particularly over-expressed on primary and disseminated mamma-carcinoma cells 1 and can be utilized as peptide shuttle to selectively address breast cancer tissue for both diagnosis and therapy.2 The Y4R plays a crucial role in food intake and is a key player in obesity signaling. Despite their distinct physiological and pathophysiological functions, both receptors show a similar ligand-binding mode which exacerbates the development of highly selective NPY analogs. Due to increased synthesis costs of full-length NPY analogs, short NPY analogs are of great interest. Based on the previously developed first Y1R selective NPY ago-nist [Pro30, Nle31, Bpa32, Leu34]-NPY (28 – 36) 3 we now discovered a key to selectively switch between Y1R- and the Y4R-activity. Herein, the single incorporation of either an acyl chain or an ortho-carbaboranyl-Nε-modified L-lysine (K-Nε(Cpa)) 4 in a crucial position could be shown to selectively modulate the activation potency. By performing receptor activation studies with stably transfected Y receptor-positive COS-7 cells, we could confirm the desired selectivity switch between Y1R and Y4R. EC50 values in range of the [Pro30, Nle31, Bpa32, Leu34]-NPY (28 – 36) lead structure could be determined. 1 Reubi, J. C., Gugger, M., Waser, B., Schaer, J. C. (2001) Y-1-mediated effect of neuropeptide Y in cancer: Breast carcinomas as targets. Cancer Res. 61: 4636 – 4641. 2 Khan, I. U., Zwanziger, D., Böhme, I., Javed, M., Naseer, H., Hyder, S. W., Beck-Sickinger, A. G. (2010) Breast cancer diagnosis by neuropeptide Y analogues – first clinical approach. Angew. Chem. Int. Ed. 49: 1155 – 1158. 3 Zwanziger, D., Böhme, I., Lindner, D., Beck-sickinger, A. G. (2009) The First Selective Agonist of the Neuropeptide Y1Receptor with Reduzed Size. J. Pept. Sci. 15: 856 – 866. 4 Ahrens, V. M., Frank, R., Stadlbauer, S., Beck-Sickinger, A. G., Hey-Hawkins, E. (2011) Incorporation of ortho-CarbaboranylNε-Modified L-Lysine into Neuropeptide Y Receptor Y1- and Y2-Selective Analogues. J. Med. Chem. 54: 2368 – 2377.

Sven Hofmann

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Molecular Cell Therapy

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry Biochemistry and Bioorganic Chemistry

[email protected] www.uni-leipzig.de/~mct

[email protected] www.biochemie.uni-leipzig.de/agbs

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5.15 Cells for the analysis of diabetes: The understanding of disease development and progression Matthias Jung, Diana Oelschlägel, Bernadette Harwardt, Insa S. Schroeder Pluripotent human embryonic stem (ES) cells can be used to generate a variety of cell types of the human body including insulin producing cells. Therefore, the development in vivo is mimicked to obtain beta-like cells. Because disease-specific induced pluripotent stem (iPS) cells share almost all properties of ES cells but also contain the patients genetic background, they are an ideal tool to reproduce diabetes mellitus in vitro to explore the pathogenesis for the improvement of anti-diabetic therapies. We used human pluripotent stem cell (ES & iPS) for the differentiation of pancreatic progenitors in 2D- and 3D-models in co-culture with other cell types and analysed the mRNA and protein expression of pancreatic markers. Reprogramming was induced in MSC s and fibroblasts using episomal vectors, microRNA s, and hypoxia. Pluripotency was characterised by mRNA and protein levels of pluripotency markers, alkaline phosphatase staining, and expression of pluripotency-associated microRNA s. Pancreatic progenitors expressed markers such as Pdx1, Nkx6.1, and Mist1. The use of 3D-matrices could enhance pancreatic markers by the addition of endothelial cells. Reprogrammed MSCs and fibroblasts expressed Oct4 and Nanog mRNA and protein. Func­ tionality of microRNA s was shown by the repression of predicted target genes (Wdr61 and others, about 50 – 80 %) and the induction of precursor microRNA s. In summary, we provide potential new strategies for the analysis of the normal and altered development of beta-like cells for the analysis of diabetes.

5.16 Titanocene dichloride derivatives with improved cytotoxic activity Goran N. Kaluđerović, Santiago Gómez-Ruiz, Beatriz Gallego, Željko Žižak, Evamarie Hey-Hawkins

Since the pioneering work of Köpf and Köpf-Maier and the phase I clinical trials carried out for titanocene dichloride in 1993, metallocene and non-metallocene titanium (IV) complexes have been studied intensively due to their anticancer properties.1 One of the most active fields in titanium (IV) anticancer chemistry is focused on the design of new compounds with different substituents which may increase their cytotoxicity in comparison with that of titanocene dichloride.2 It has been observed that titanocene complexes having polar substituents or electron-withdrawing groups in their structure present higher in vitro anticancer activity.1 Three different titanium (IV) carboxylate complexes have been synthesised and structurally characterised: 3 (1) [Ti(η5 -C5 H 5)2(O2CCH 2SMes)2] (2) [Ti(η5 -C5 H4Me)2(O2CCH 2SMes)2] (3) [Ti(η5 -C5 H 5)(η5 -C5 H4SiMe3)(O2CCH 2S-Mes)2] Mes = 2,4,6-Me3C 6 H 2

The cytotoxic activity of 1 – 3 was tested against human tumour cell lines (adenocarcinoma HeLa, myelogenous leukemia K562, malignant melanoma Fem-x) and normal immuno­ competent cells (peripheral blood mononuclear cells PBMC), observing that from the studied complexes 3 exhibited highest cytotoxic activity (against K562 IC50 = 87.9 ± 3.6 µM). An increment on the cytotoxic activity was observed through the substitution of the chloride by carboxylate ligands (e. g., titanocene dichloride → 1). 1 K. Strohfeldt, M. Tacke, Chem. Soc. Rev. 2008, 37, 1174. 2 G. N. Kaluđerović, R. Paschke. Curr. Med. Chem. 2011, 18, 4738. 3 S. Gómez-Ruiz, B. Gallego, Ž. Žižak, E. Hey-Hawkins, Z. D. Juranić, G. N. Kaluđerović, Polyhedron 2010, 29, 354.

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Matthias Jung

Dr. Goran N. Kaluđerović

Martin-Luther-Universität Halle-Wittenberg Faculty of Medicine Institute of Anatomy and Cell Biology

Universität Leipzig Faculty of Chemistry and Mineralogy Institute of Inorganic Chemistry

[email protected] www.medizin.uni-halle.de/index.php?id=1125#c956

[email protected] www.uni-leipzig.de/chemie/hh

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5.17 Generation of ρo-cells without nuclear genome interference Mario Krehan, Ingo Schäfer, Susanna Schubert, Sandra Heller, Jessica Zenker, Astrid Schön, Peter Seibel

Mitochondria are the primary energy-generating system in most eukaryotic cells and participate in intermediary metabolism, calcium signaling and apoptosis. Mitochondrial DNA (16569 bp in Homo sapiens) contains 37 genes that are important for normal mitochondrial function. Mutations in this genome can affect the whole organism and cause severe neuromuscular deseases to early death. Our strategy for therapy is to destroy and replace defective mitochondrial DNA . The first step of this project will be the generation of ρ0 cells. These are cells without mtDNA . Object of the project will be optimisation for a faster and gentle production of ρ0 cells compared to the chemical method. This requires generation of megamitochondria, microinjection of a purified recombinant mtDNA-specific Endoribo-nuclease into mitochondria and selection of ρ0 clone. The selection of transformed clones will be facilitated by marking cells with a nontoxic dye during injection. After having a ρ0 cell line the next objective will be the delivery of a new or modified genome. For this, several methods have been developed in our group. Alternatively, ρ0 cells can be used to study several regulatory processes such as ageing and apoptosis.

5.18 Cisplatin – COX inhibitor conjugates Wilma Neumann, Evamarie Hey-Hawkins

Cisplatin is one of the most widely used drugs in the treatment of cancer. However, conventional platinum-based chemotherapy is often associated with strong side effects, and the efficacy of anti-tumour drugs is strongly limited by intrinsic and treatment-induced resistance of tumour cells. An enzyme which is overexpressed in several tumour tissues and is involved in tumour onset and progression is COX-2 , an isoform of cyclooxygenase (COX).1 Clinical studies revealed that targeting the COX-2 pathway is a promising strategy for the prevention and treatment of tumours. Furthermore, promising results were obtained with combinatorial treatments with chemotherapeutic agents, such as cisplatin, and COX inhibitors.2 These observed synergistic effects could be improved by combining both molecules in one compound. A covalent bond between the chemotherapeutic agent and the COX inhibitor should ensure a concerted transport of the drugs through the body and uptake into tumour cells. Furthermore, binding of the coupled COX inhibitor at COX-2 could prevent an efflux of the platinum complex. Besides an increased accumulation of the drugs in the tumour cells, coupling of cisplatin with COX inhibitors could further enhance the efficacy of the chemotherapeutic agent by enabling a simultaneous action of both drugs in COX-2 overexpressing tumour cells. 1 N. Ghosh, R. Chaki, V. Mandal, S. C. Mandal, Pharmacol. Rep. 2010, 62, 233 – 244. 2 M. Ogino, S. Minoura, Int. J. Clin. Oncol. 2001, 6, 84 – 89.

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Mario Krehan

Wilma Neumann

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Molecular Cell Therapy

Universität Leipzig Faculty of Chemistry and Mineralogy Institute of Inorganic Chemistry

[email protected] www.uni-leipzig.de/~mct

[email protected] www.uni-leipzig.de/chemie/hh

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5.19 Synthesis and evaluation of PET probes for imaging of the PDE10A in brain: An inter­disciplinary project Karen Nieber, Fritzi Siegert, Detlef Briel, Ghadir Barbar Askar, Norbert Sträter, Michael Zahn, Matthias Scheunemann, Peter Brust Phosphodiesterases (PDE s) are a family of enzymes subdivided into 11 distinct families according to structural and functional properties. These enzymes metabolically inactivate widely occurring intracellular second messengers. Of all known PDE families, PDE10A has the most restricted distribution with high mRNA expression only in the brain and testes. PDE10A mRNA expression and protein are highly expressed in medium spiny neurons of the striatum. Noninvasive imaging of PDE10A using Positron Emission Tomography (PET) would allow for studying the distribution of this enzyme in neuronal and psychiatric disorders. Therefore we might still identify derivatives with sufficient potency and selec­ tivity for PDE10A , resulting in a PET tracer showing a reasonable pharmacodynamic, pharmacokinetic and toxicological profile. We decided to initiate a chemical exploration around a lead compound. The project includes four subprojects. The aim of the subproject 1 (Prof. Briel, Prof. Sträter) was to synthesize analogues that can be easily radiolabeled with 18F. The subproject 2 (Prof. Nieber) was aimed to evaluate in vitro and in vivo toxicity of selected analogues. The subproject 3 (Prof. Brust) was responsible for radiolabeling a potential candidate with 18F and to measure the in vivo uptake and binding in brain of the most promising derivatives. The in vitro PDE10A potency and selectivity was determined in co-operation with biocrea GmbH. The data of this project are essential to assess the suitability of a potential candidate as PET agent for imaging PDE10A in vivo.

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5.20 Synthesis of a multifunctional peptide by two distinct click-reactions Mareen Pagel, Rayk Hassert, Manfred Wießler, Annette G. Beck-Sickinger

Since the design and synthesis of new and complex biomolecules is steadily increasing, the investigation of selective methods to introduce multifunctionality is required. Click chemistry gained thereby remarkably importance. The Diels-Alder reaction with inverse electron demand (DAR inv) and the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) fulfil the criteria of a click-reaction. We could combine these orthogonal bioconjugation techniques to synthesize a multifunctional biomolecule. The CuAAC was thereby used to modify a SiO2-binding peptide with a fluorescent dye to facilitate the subsequent detection of the immobilized peptide on the inorganic surface. Furthermore, the peptide was conjugated to a cyclic integrin ligand, by DAR inv to gain a bioactive molecule. Finally, in vitro studies with osteoblasts showed improved cell attachment and spreading on the SiO2 surface promoted by the previously performed peptide coating. Hereby we developed a unique strategy to build up a multifunctional molecule in a selective, easy and efficient way.

Prof. Dr. Karen Nieber

Mareen Pagel

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy Department of Pharmacology for Natural Sciences

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry Biochemistry and Bioorganic Chemistry

[email protected] www.uni-leipzig.de/~pharm

[email protected] www.biochemie.uni-leipzig.de/agbs

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5.21 Novel kinase-inhibitors and their application for the individualised therapy of the malignant melanoma Sarah Pönick, Heinz-Georg Jahnke, Jan Maschke, Michael Kendler, Jan C. Simon, Andrea A. Robitzki The malignant melanoma causes the majority of skin cancer related deaths. Due to the fact that each tumour has its own characteristics concerning genetic properties and sensitivity to active pharmaceutical ingredients (API), there is a strong demand for personalised and targeted therapies. In this context we developed a screening system that allows direct testing of oncogene directed therapeutics on primary melanoma cell lines. Using impedance spectroscopy as a non-invasive, label-free detection technique in combi­ nation with our self-developed 2D microelectrode array we are able to monitor API efficacy for more than four days. Additionally, we established protocols for detection of b-raf and c-kit mutations for analysis of tumour heterogeneity and patient dependent target validation of novel APIs like the kinase inhibitors PLX4720 and Imatinib. We could correlate oncogene directed mutations like b-raf V600E within melanoma meta­ stasis derived cell lines with specific sensitivity to the mutation specific kinase inhibitor PLX4720. Furthermore, we could show that our self-generated melanoma cell lines carry no mutation in c-kit and thereby demonstrate no sensitivity to the applied Imatinib. These alterations in proliferation and apoptosis induced by the tested API s could be quantified by impedance spectroscopy. The impedimetric screening on melanoma metastasis derived cell lines with or without specific oncogene mutations revealed the target dependent response to novel kinase-inhibitors. Based on these results, our direct biosensoric chemosensitivity testing system is a promising tool for the patient specific tumour treatment.

Sarah Pönick Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology [email protected] www.uni-leipzig.de/~dmpt

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® chamomile component affects 5.22 Myrrhinil-Intest  contraction and morphology of rat ileum / jejunum ex vivo preparations in a concentration dependent manner

Mikolaj Raszek, Karen Nieber, Karl-Heinz Goos Myrrhinil-Intest® is a prescribed phytotherapy for the treatment of inflammatory intestinal diseases. However, how its functional effects are exerted has not been fully elucidated. One of its active ingredients, lyophilised extract of chamomile flowers, was tested on the acute inflammation of rat ileum / jejunum preparations, experimentally induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS, 10 mM, 30 min). At low concentrations tested (8.75 – 70 µg / mL) chamomile exhibited a basal tone enhancement of both the untreated controls and TNBS -treated preparations. At higher concentrations this trend subsided at first with the inflamed segments only (140 µg / mL), with subsequent inhibition of basal tone for both untreated and TNBS -treated preparations (210 – 280 µg / mL). Histological analysis revealed that the concentration-dependent effect correlated with a protective function of chamomile from TNBS -induced morphological damage (100 mM, 30 min). Thus whilst pre-treatment with 70 µg / mL exhibited protective function, higher levels of chamomile (280 µg / mL) did not. 10 mM TNBS also appeared to have 1.6-fold reduction effect on acetylcholine (ACh)-induced contraction (EC50 of 0.9 µM vs. 0.58 µM for untreated controls). This could be reversed with chamomile pretreatment, however, this observation was not statistically significant. The importance of a chamomile treatment concentration-dependence is further underscored by the fact that TNBS -inflamed segments also exhibit increased sensitivity to chamomile inhibition of ACh-induced contractions (IC50 of 106 µg / mL vs. 175 µg / mL for untreated controls), particularly in the higher concentration range.

Dr. Mikolaj Raszek Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy [email protected] www.uni-leipzig.de/~pharm

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5.23 Genetic analysis of antibody genes from influenza NP-specific B-cells Sven Reiche, Yamen Dwai, Christian Jassoy

Background: Little is known about the diversity of the human antibody (ab) repertoire against single proteins. Such data about the diversity of the humoral immune response will give important insights into the induced variety of the antigen specific ab repertoire especially for the development and improvement of vaccines. Methods: We isolated influenza nucleoprotein (NP)-specific memory B-cells from the blood using immunomagnetic sorting. The isolated NP specific B-cell population was highly pure (> 95 %, determined by ELISpot assay). Gene fragments comprising the variable regions of antibody heavy and light chains from a single cell were amplified, sequenced and cloned into a modified eukaryotic expression vector carrying an ab expression cassette. Recombinant antibodies were produced in 293 T cells co-transfected with a combination of vectors carrying paired light and heavy chains. The ab function was examined by ELISA and WB. Result: We obtained > 100 sequences from NP-specific B-cells. Alignments revealed that the obtained sequences were highly diverse and represented segments from almost all of the existing different V, D, and J gene families of human antibody chains. From these sequences we cloned and expressed 16 genetically different NP-specific monoclonal antibodies (mabs). Conclusions: The established method enables the generation of recombinant mabs and the determination of the human antigen-specific diversity of memory B-cells. The method should be adaptable to other antigens. The obtained recombinant human antibodies can be produced under defined conditions making them suitable as potential therapeutics.

Dr. Sven Reiche

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5.24 Saffron extract acts neuroprotective against hypoxia on neuronal cells in vitro Franziska Reitmajer, Marcus Bloßfeld, Jörg Flemmig, Maria Schönberg, Andreas Hensel, Jürgen Arnhold, Karen Nieber Ischaemic stroke represents the third most cause of death in industrialized nations and is considered to be the most prominent reason for permanent disabilities in adulthood. The only treatment option for ischaemic stroke is systemic thrombolysis. Due to the narrow time frame and the limited treatmental success, novel therapeutical strategies are necessary. Saffron obtained from the dried stigmata of Crocus sativus L. (Iridaceae) seems to be a possible therapeutical approach. Saffron showed neuroprotective properties in brain slice preparations and in clinical trials. The aim of the present study was to examine the influence of a hydro-ethanolic saffron extract (CSE) on rat neuroblastoma cells (B104) under normoxic and hypoxic conditions (OGD, oxygen-glucose-deprivation). Additionally, the effect of CSE on glutamate-mediated excitotoxicity was investigated. Cells were stained with JC-1 dye and cell vitality was determined using flow cytometry. CSE (500 µg / ml) decreased the cell vitality of B104 cells under normoxic conditions by 5 % compared to control. The incubation with glutamate (200 mM) reduced the cell vitality by 80 %. CSE (500 µg / ml) further enhanced this effect by 10 %. OGD reduced time-dependently the cell vitality, which was significant after 8 hours. CSE (500 µg / ml) increased the vitality under OGD. Our results indicate cytoprotective effects for CSE (500 µg / ml) on rat neuronal cells under hypoxic conditions and confirm further results on pyramidal cells in rat brain slices.

Franziska Reitmajer

Universität Leipzig Faculty of Veterinary Medicine Institute of Virology

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy Pharmacology for Natural Sciences

[email protected] www.uni-leipzig.de/~virology

[email protected] www.uni-leipzig.de/~pharm

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5.25 Th17 cells can compensate for Th1 deficiency in pulmonary cryptococcosis Tina Richter, Daniel Piehler, Andreas Grahnert, Maria Eschke, Uwe Müller, Katarzyna Warszawska, Gabriele Köhler, Robert Sabat, Gottfried Alber Cryptococcus neoformans is one of the most important opportunistic fungal pathogens during HIV infection. Each year, approximately one million cases of cryptococcal meningitis result in more than 600,000 deaths. In the murine model protective immunity against C. neoformans is dependent on T helper (Th) 1 cells, whereas Th2 cells are detrimental. There is some evidence for Th17 cells contributing to protection. IL-17A was shown to be beneficial for cryptococcal clearance. However, the function of IL-22 is largely unknown. To monitor Th17 induction over time, we intranasally infected wild-type C57BL / 6 mice, re-stimulated lung leukocytes and analyzed cryptococcal-specific production of IL-22 and IL-17A. We found peak values of IL-22 and IL-17A already at 7 days post infection (dpi). Until 70 dpi both Th17 cytokines gradually decreased. It is known that both, Th1 and Th2 cells suppress Th17 responses. To analyze the in vivo function of Th17 cytokine production double knockout mice with a reduced Th1 (IL-12p35- /-) and a reduced Th2 (IL-4Rα - /-) response were created and infected with C. neoformans. IL-12p35 / IL-4Rα - /- mice showed significantly increased serum concentrations of IL-22 compared to single knockout mice. Higher IL-22 production indicates the induction of an elevated Th17 response in the absence of Th1 and Th2 cells. Interestingly, IL-12p35 / IL4Rα - /- mice were found to be almost as resistant as IL-4Rα - /- mice, whereas IL-12p35- /were highly susceptible (75 vs. 100 vs. 8 % survival at 112 dpi). The data suggest that Th17 cells may compensate for Th1 deficiency in pulmonary cryptococcosis.

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5.26 Expression of recombinant rotavirus proteins in mammalian cell lines Antje Rückner, Thomas W. Vahlenkamp

Rotaviruses of the family reoviridae are one of the leading causes of gastroenteritis in children under the age of three. High mortality rates, especially in developing countries, are due to rapid dehydration caused by acute watery diarrhea. Rotaviruses are potentially zoonotic, infecting among other animals, calves and piglets. All members of reoviridae possess a common segmented genome of double stranded RNA , which allows for the occurrence of reassortments between rotavirus strains. Analysis of the dynamics and the frequency of these events are of particular importance for the creation of effective vaccines. Until now, it has been exceptionally difficult to prepare an in vitro study of reassortment due to the lack of a helper virus-free reverse genetics system. Here we present a reverse genetics system intended for the expression of recombinant rotaviruses proteins in various mammalian cell lines. To this end, rotavirus genes were cloned under control of the T7-promoter ensuring a directed transcription. The functionality of the resulting plasmids as well as the expression of the recombinant proteins were demonstrated via immunofluorescence employing polyclonal antisera. The structural protein VP6 has displayed varying distributions within cells: the wild-type VP6 tends to accumulate more within the cytoplasm while the recombinant protein seems to coalesce with cellular structures. To which extent these are specific and if synthesis of completely recombinant virus particles may be influenced by the variable localization of the individual proteins will be the focus of future studies.

Tina Richter

Antje Rückner

Universität Leipzig Faculty of Veterinary Medicine Institute of Immunology

Universität Leipzig Faculty of Veterinary Medicine Institute of Virology

[email protected] www.vmf.uni-leipzig.de/ik/wimmunologie

[email protected] www.uni-leipzig.de/~virology

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5.27 How fatty acids modulate macrophage-mediated immune defense – a mechanistic trial Axel Schöniger, Herbert Fuhrmann, Julia Schumann

We recently demonstrated that polyunsaturated fatty acids (PUFA) exert anti-inflammatory and immune suppressive actions on macrophage immune defense. The underlying mecha­ nisms, however, are largely unknown. Therefore, the objective of the present study is to elucidate the mechanisms of action which underlie the immune modulatory effects of the fatty acids. Fluorescence microscopic examinations show that protein kinase C (PKC) is not affected by PUFA enrichment of macrophages. There was no influence of PUFA supple­ mentation on the dynamic PKC trafficking in response to stimulation. In contrast, the tolllike receptor (TLR) signaling as well as the MAP kinase pathway emerged to be affected by the enrichment of the macrophages with PUFA . The modulation of the TLR and MAP kinase pathways seems to be partially mediated by a down-regulation of TLR4 gene expression. Furthermore, the disruption of lipid rafts due to PUFA supplementation is likely to perturbate the interaction of TLR4 with its adapter protein CD14. Hence, our data support the idea that the immune suppressive actions of PUFA are mediated on the membrane level.

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5.28 Membrane fatty acid patterns and macrophage activity Julia Schumann, Axel Schöniger, Stephanie Adolph, Herbert Fuhrmann

Objective: Nutritional fatty acids impact membrane lipid composition of body cells, including immune cells. This provides a link between dietary fatty acid uptake, inflammation and immunity. In the present study we reveal the significance of macrophage membrane fatty acid patterns on gene expression and cytokine synthesis. Items highlighted include signal transduction processes, macrophage activation as well as macrophage defense mechanisms. Procedure: The murine macrophage cell line RAW264.7 was used. Cells were supplemented with PUFA in a concentration of 15 µmol / l for 72 h and stimulated in the last 24 h of incubation with LPS, PMA or viable bacteria of the species R. equi or P. aeruginosa. Genes analyzed include the genes for the surface molecules Fc receptor, MHCII and CD86, the adapter proteins MyD88 and RICK as well as the antimicrobial peptide lysozyme. Cytokines analyzed include the pro-inflammatory mediators IL-1β, IL-6 and TNF-α as well as the antiinflammatory mediator IL-10. Results: We identified unsaturated fatty acids to act immune-suppressive on activated macrophages at this down-regulating the synthesis of (i) the pro-inflammatory cytokines IL‑1β, IL-6 and TNF-α, (ii) the co-stimulatory molecule CD86 as well as (iii) the anti­ microbial polypeptide lysozyme. Of particular importance, the anti-inflammatory effects of the PUFA could be seen in case of infection of RAW264.7 with viable R. equi and P. aeruginosa as well. Conclusion: Our data endorse a directed supply of fatty acids to immunocompromised individuals as a supportive therapy of chronic infections caused by persistent pathogens.

Axel Schöniger

Dr. Julia Schumann

Universität Leipzig Faculty of Veterinary Medicine Institute of Physiological Chemistry

Universität Leipzig Faculty of Veterinary Medicine Institute of Physiological Chemistry

[email protected] www.uni-leipzig.de/~vpci

[email protected] www.uni-leipzig.de/~vpci

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5.29 The role of BIM-EL and BCL2-α on the efficacy of erlotinib and gefitinib in lung cancer Jacinta Simasi, Andreas Schubert, Adrian Gillissen, Karen Nieber

Introduction: The use of erlotinib and gefitinib in the treatment of lung cancer faces challenges which include varied response to the drugs by patients and resistance which lead to low efficacy of the drugs. In this study, factors that influence secondary resistance were investigated. Methodology: Viability, apoptosis and gene expression were analyzed after exposing the cells to the drugs, using MTT test, caspase 3 / 7 activity assay and Q-PCR methods respectively. Results: HCC827 cell line developed resistance to erlotinib and gefitinib growth inhibition of the drugs on the resistant cells lines reduced. After developing resistance the activity of caspase 3 and 7 increased significantly in the parent cell line but not in the resistant cell lines. BIM-EL was upregulated in the parent cell line but downregulated in the resistant cell whereas BCL2 -α did not show any changes in expression levels in the parent cell line, but upregulation was observed in the resistant cell lines. After developing resistance BIM-EL expression increased although the differences were not significant. After drug application the expression decreased. BCL2 -α expression increased after developing resistance and after drug application the expression increased further. Conclusion: Secondary resistant to the drugs in lung cancer was evident. The drugs inhibit growth of cancer cells and induce apoptosis. BIM-EL expression variations shows that BIM-EL mediates the activation of the intrinsic apoptotic pathway, BCL2 -α counter reacts by inhibiting the activity of BIM-EL . This counter-effect of BCL2 -α is one of the causes of secondary resistance to the drugs.

Jacinta Simasi

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5.30 Validation of ncRNA s relevant to Alzheimer´s disease Igor Turković, Uwe Ueberham, Thomas Arendt

Alzheimer´s disease (AD) is a progressive neurodegenerative disorder among elderly people. The neuropathological characteristics of AD are deposition of Aß-plaques, formation of neurofibrillary tangles (containing hyperphosphorylated microtubuleassociated Tau protein) which are associated with a loss of differentiation control, activation of cell cycle proteins and neuronal apoptosis. Although the molecular mechanisms are unknown, neoplastic transformation in postmitotic cells contributes to neurodegenerative cell death in AD. Here we demonstrate that a specific miRNA which has a crucial role in promoting neoplastic transformation and predominate tumor suppressor functions in tumor development, is elevated in AD. Additionally, this miRNA can regulate the expression of the amyloid precursor protein (APP) and BACE1 thus promoting the pro-amyloidogenic pathway. In parallel, it inhibits neuronal differentiation and structural neuronal plasticity. Using microarray analysis we identified further miRNA s being potentially involved in the regulation of neuronal differentiation. The present study supports the concept of a molecular regulatory level shared between neoplastic transformation of diving cells and degenerative cell death of neurons and provides a new potential target for disease modifying strategies in AD. This study was supported by the BMBF (01EW0907 ) in the frame of ERA -Net Neuron, the Project BBZ07: 14494 (University Leipzig) and the AFI -Project 984 000-150.

Igor Turković

Fraunhofer-Institut für Zelltherapie und Immunologie (IZI) Vascular Biology Group

Universität Leipzig Faculty of Medicine Paul Flechsig Institute for Brain Research

[email protected] www.izi.fraunhofer.de

[email protected] www.uni-leipzig.de/~pfi

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5.31 Glial but not neuronal osmotic cell volume regulation in the inner nuclear layer of the rat retina Stefanie Vogler, Antje Grosche, Thomas Pannicke, Peter Wiedemann, Andreas Reichenbach, Andreas Bringmann Intense neuronal activity in the retina is associated with a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., 1999, Vis. Neurosci. 16: 1157 – 1167). Here, we compared the osmotic and receptor-mediated regulation of the volume of Müller and bipolar cell somata in acutely isolated slices of the rat retina. Superfusion of retinal slices with a hypoosmotic extracellular solution induced swelling of bipolar cell somata but not of Müller cell somata. Exposure of the slices to a hypoosmotic solution containing barium ions induced swelling of both Müller cell and bipolar cell somata. Müller and bipolar cell somata also displayed hypoosmotic swelling in slices of 3 days-postischemic retinas. The hypoosmotic swelling of Müller cell somata was blocked by administration of VEGF, glutamate, ATP, and adenosine, respectively. In contrast, these receptor agonists did not block the hypoosmotic swelling of bipolar cell somata. It is suggested that Müller cells but not bipolar cells possess endogenous cell volume-regulatory mechanisms which prevent cellular swelling under hypoosmotic conditions.

5.32 Photoactive phosphotyrosine mimetics Stefan Wagner, Jörg Rademann, André Horatscheck, Jutta Ortwein, Boo Geun Kim, Michael Lisurek, Samuel Beligny, Anja Schütz

Protein tyrosine phosphorylation and dephosphorylation constitutes one of the most important switches of cellular activity, whereby failure in the regulation of this process leads to a number of diseases like cancer and diabetes. Since the molecular interactions that lead to activity of phosphotyrosine recognition domains are regulated in a complex manner there is a strong demand for chemical probes that can be used for the identification and quantification of active phosphotyrosine recognition domains in cells. In this work we identified benzoylphosphonate as the first photoactive phosphotyrosine bioisoster.1 While benzoylphosphonate itself shows Ki values in mM-range, the affinity towards phosphotyrosine recognizing proteins could be increased upon irradiation up to 400 fold leading to inhibition constants in a low µM-range. First we synthesized a fluorescein-labeled benzoylphosphonate to investigate the degree of covalent protein modification in a time- and concentration-dependant manner. After the successful fluorescence-labeling of the proteintyrosinphosphatase PTP1B we synthesized a non-natural amino acid building block 4 phosphono-carbonyl phenylalanine (pcPhe). The incorporation of the benzoylphosphonate into peptide sequences led to high affinity PAL probes for STAT5b with binding constants between 0.90 µM and 11 µM. The affinity of these photoactive peptides was investigated before and after irradiation and the selectivity and kinetics of the covalent crosslinking reaction was studied in protein mixtures. 1 Rademann et al. Angew. Chem. 2012, in revision

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Stefanie Vogler

Stefan Wagner

Universität Leipzig Faculty of Medicine Paul Flechsig Institute for Brain Research Department of Neurophysiology

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy Medicinal Chemistry

[email protected] www.uni-leipzig.de/~pfi

[email protected] www.uni-leipzig.de/agrademann

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5.33 Development of a MDCKII cell in vitro model for the assessment of BCRP mediated drug transport in the lactating mammary gland of dairy animals Louise Waßermann, Stefan Lindner, Sandra Halwachs, Kerstin U. Honscha, Walther Honscha The presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. The ATP-binding cassette (ABC) transport protein Breast Cancer Resistance Protein (BCRP; ABCG2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. BCRP is known to play a major role in the active secretion of a variety of xenobiotics into human milk. So far there is little information about the transport activity and substrate specificity of dairy BCRP. Therefore we aimed to establish a MDCKII cell in vitro model expressing BCRP of dairy animals. BCRP mRNA was isolated from bovine, caprine and ovine mammary gland. Fulllength clones were generated using RACE (rapid amplification of cDNA ends) PCR . The final full-length bovine, ovine and caprine ABCG2 cDNA-clone sequences were submitted to the NCBI genebank (EU570105, GQ141082 and GQ241418). Stable transfection of BCRP in MDCKII cells was performed and the subcellular localization of BCRP at the apical plasma membrane was identified by confocal laser scanning microscopy. BCRP-mediated transport of the substrate Hoechst 33342 was measured and the selectivity was determined by the BCRP inhibitor Ko143. Inhibition studies using Hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine BCRP. To further characterize BCRP carrier activity, bidirectional transport studies were performed with Transwell filter inserts. Our results may enlarge the understanding of drug transport into milk.

Louise Waßermann Universität Leipzig Faculty of Veterinary Medicine Institute of Pharmacology, Pharmacy and Toxicology [email protected] www.uni-leipzig.de/~vetppt

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5.34 Investigating protein stability of a FOXP2 disease variant Sven Weyer, Wolfgang Enard

FOXP2 is a transcription factor that is expressed in the brain and is linked to speech and language. A specific point mutation, referred to as the “KE mutation”, results in an arginine-

to-histidine substitution and causes developmental verbal dyspraxia. Affected persons can’t manage the fast orofacial movements to produce proper speech and have severe language impairments. In this study, a FOXP2 disease variant was produced by introducing the KE mutation into full length human FOXP2 cDNA . Both were expressed as GST fusion proteins in E. coli BL21, purified on a glutathione-coupled Sepharose column and cleaved from the GST tag by PreScission Protease. The purified proteins were then compared respective to their stability with the Thermoflour assay, set up on a Stratagene qPCR machine and using SYPRO Orange as fluorescent protein-binding dye.

Sven Weyer Max-Planck-Institut für evolutionäre Anthropologie Department of Evolutionary Genetics [email protected] www.eva.mpg.de

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5.35 Structural studies of DnaK in complex with small proline-rich antimicrobial peptides Michael Zahn, Daniel Knappe, Nicole Berthold, Ralf Hoffmann, Norbert Sträter

Bacterial infections are a major cause of death worldwide. Due to increasing resistance against the commercially available antibiotics over the past few decades, novel antimicrobial drug classes with new mode of actions are required for future treatments. Small proline rich antimicrobial peptides (PR-AMPs) from mammals and insects were identified to target the E. coli Hsp70 chaperone DnaK after cell penetration. Binding of the peptides to DnaK compromises the activity of the chaperone and thus the viability of the bacterial cells, in particular under conditions of stress. The non-lytic cell penetration of PR-AMPs to Gram-negative bacteria makes them a promising drug candidate against human infections. Therefore, structural information about the interactions between peptide inhibitors and DnaK are necessary for a better understanding of the mode of actions. After recombinant expression of the substrate binding domain in E. coli and subsequent purification by IMAC and gelfiltration, we crystallized the domain with several PR-AMPs. Elucidation of the binding mode of the peptides and characterization of the substrate specificity of DnaK will allow a structure-guided development of peptide inhibitors as antimicrobial agents targeting DnaK.

Michael Zahn Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers [email protected] www.uni-leipzig.de/~straeter

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POSTERS

6.

Bioanalytics

BIOANALYTICS

6.1

Identification and characterization of dityrosine cross-linked peptides using mass spectrometry Andrea Annibal, Ravi Chand Bollineni, Maria Fedorova, Ralf Hoffmann

Oxidation of tyrosine residues by reactive oxygen species (ROS) represents a valid biomarker for protein oxidative damage in vivo. Tyrosine radicals can cross-link two tyrosine residues forming dityrosine, for example by UV and γ-irradiation, hydroxyl radicals, or by activation of different cellular peroxidases. Although dityrosine can be detected by its fluorescence (λ ex / em = 325 / 410 nm), mapping of the modification sites within proteins has not been accomplished yet. Whereas mass spectrometry (MS) is routinely used to map protein post-translational modification (PTM) sites, the direct analysis of dityrosine residues is challenged by cross-linked peptides that do not follow the linear protein sequence. Four different peptide sequences containing tyrosine residues were oxidized in vitro using the horseradish peroxidase (HRP) / H2O2 system. Reaction products were monitored by MALDI-TOF-MS and fluorescence spectroscopy. The fragmentation characteristics of the dityrosine containing peptides by CID and ETD were studied on an ESI-LTQ -Orbitrap-MS. Incubation of model peptides with HRP / H2O2 yielded several oxidations products of tyrosine residues. However, only a low ratio of HRP and H2O2 favored the formation of dityrosine. When mixing two Tyr-containing peptides both homo- and heterodimers were obtained. Interestingly, each peptide followed a different kinetic of cross-link formation, as monitored by MALDI-TOF-MS and fluorescence spectroscopy. Tandem mass spectra acquired in CID - and ETD -mode allowed us to localize the positions and provided even general rules for the analysis of this oxidative PTM in complex biological samples.

6.2

A qualitative and quantitative study of carbonyl tagging reagents for redox proteomics Ravi Chand Bollineni, Maria Fedorova, Ralf Hoffmann

The term “carbonylation” refers to the post-translational modifications yielding reactive aldehydes, ketones or lactams. Proteome wide analyses of carbonylated proteins usually rely on different chemical tagging strategies to facilitate their enrichment, identification and quantification by mass spectrometry (MS). A detailed study, however, on the efficiency, specificity and MS behavior of different chemical tagging reagents is missing. Here we studied these questions using model peptides containing aldehyde, ketone and lactam functional groups and three widely used reagents with different carbonyl-specific groups (hydrazine, hydrazide, and hydroxyl amine). The reactivity of these tagging reagents was judged by MALDI-MS and quantitative labeling efficiency was studied by RP-HPLC. Furthermore, the MS / MS characteristics were studied on an LTQ -Orbitrap-MS using different fragmentation techniques such as CID, and ETD. Based on our results, hydrazine functionalized reagent reacted with all different carbonyl groups, whereas biotin conjugated hydrazide and hydroxyl amine derivatives reacted only with aldehydes and ketones. Quantitative derivatization of all different carbonyl groups with a single tagging reagent was not possible. Considering the fragmentation behavior ETD fragmentation technique would be of better choice for peptides labeled with biotin conjugated tagging reagents, as cleavage of the tag and as well as the modification site were less pronounced. For hydrazine tagged peptides during CID or HCD fragmentation no cleavage at the modifications site was observed.

This publication is supported by the SMWK (Saxon Ministry of Research and the Fine Arts), the BMBF (Federal Ministry of Education and Research), and LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERFD ) and by means of the Free State of Saxony within the framework of the excellence initiative.

Dr. Andrea Annibal Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics [email protected] www.uni-leipzig.de/~bioanaly

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Ravi Chand Bollineni Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry [email protected] www.uni-leipzig.de/~bioanaly

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BIOANALYTICS

6.3

A peptide-protein assay for identification of antimicrobial peptides by fluorescence quenching Kristin Dobslaff, Thomas Kreisig, Nicole Berthold, Ralf Hoffmann, Thole Züchner

Peptide-protein interactions play a major role in a variety of biological processes. Therefore, the analysis of such interactions in high-throughput assays is of high importance in the field of medical research. Here we present a novel homogenous quenching assay for the determination of specific interactions between peptides and proteins using the model system of proline-rich antimicrobial peptides (AMP) and their biological target protein DnaK. In detail, the AMPs were modified with fluorescein, whereas the protein was labeled with the non-fluorescent Black Hole Quencher 10 (BHQ10). In the case of a specific peptideprotein interaction, a dynamic dipole-dipole based quenching process occurs between the excited state of the fluorophor and the ground state of the quencher. In comparison to the conventional polarized-light based fluorescence anisotropy method for analysis of peptideprotein interactions, the novel quenching assay shows a good correlation regarding Kd -­value determination and a higher specificity. In addition, the application of the quenching principle offers the possibility of establishing a fast prescreening method leading to a dramatic reduc­ tion of the sample amount of 98 % compared to the established fluorescence polarization assay and making the optimized quenching assay highly suitable for high-throughput analysis of large peptide libraries.

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6.4

Expression of P2X receptor ectodomains Christoph Döhler, Matthias Zebisch, Norbert Sträter

P2X receptors are trimeric cation-selective ion channels regulated by extracellular ATP.

Their involvement in a lot of physiological diseases including chronic pain makes them an attractive drug target. Protein crystallography in combination with structure-based drug design is a helpful tool to discover new subtype-specific and highly affine agonists and antagonists. In 2009, the 3.1 Å structure of the zebrafish P2X4 was determined. Despite high functional insight, the binding site of the natural agonist ATP remains speculative. High resolution complex structures of ideally all seven human P2X receptor subtypes are required for investigations of subtype-specific binding modes and the design of novel lead molecules. Since the expression, purification and crystallization of full-length P2X receptors is very difficult, we are testing strategies to express the ectodomains (ECD s) of the receptors. Human P2X ECD s could not be expressed in functional, i. e. folded, form in E. coli in our hands. We are therefore currently testing eukaryotic expression systems for the preparation of functional P2X ectodomains. To enhance the folding properties, the P2X ECD s of the trimeric receptor could be cloned as a concatameric construct. This is possible due to the close distance between the N- and C-terminus of different monomers. The fusion of three P2X ECD s into a concatameric protein leads to decrease of intermolecular distances and to higher chance of protein folding. Specific heteromeric P2X receptors are attractive drug target as well and may be prepared by using this strategy.

Kristin Dobslaff

Christoph Döhler

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers

[email protected] www.uni-leipzig.de/~bioanaly

[email protected] www.uni-leipzig.de/~straeter

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BIOANALYTICS

6.5

Development of new MS -based analytical techniques to study lipid- and protein-bound carbonyls Maria Fedorova, Ivana Milic, Ravi Chand Bollineni, Ralf Hoffmann

Oxidative stress, an imbalance between the generation of reactive oxygen species (ROS) and their elimination by antioxidants, has been linked to the pathophysiology of many human disorders. One of the most hazardous oxidative modifications are reactive carbonyls (aldehydes / ketones), which are considered a major hallmark of ROS -related diseases. Here we present novel mass spectrometry (MS)-based strategies to assess the changes in proteins and lipids leading to “carbonyl stress”. In the study of protein carbonylation, derivatization with dinitrophenylhydrazine (DNPH) was favorable, as the reagent excess provided a selective matrix to specifically analyze DNP-modified peptides by MALDI-MS. Therefore, the derivatized peptides could be enriched (MALDI MS -based enrichment) and identified by RPC-ESI-MS. This approach was tested on the proteome of HeLa cells treated with Cu(II)-ions and allowed to identify hundreds of carbonylated proteins representing different functional groups. In the study of carbonylated lipid peroxidation products (oxoLPP), specific derivatization allowed a shotgun approach using ESI-LTQ -OrbitrapMS. The superior ionization efficiencies and the identification of specific reporter ions allowed us to identify 120 oxoLPP in the mixture of 4 in vitro oxidized phospholipids. Biological relevance of detected oxoLPP was proved by identification of the corresponding adducts on ApoB-100 protein of LDL complex isolated from the blood of healthy donors. Presented novel proteomics and lipidomics strategies allow a robust and sensitive analysis of carbonylated species in qualitative and quantitative terms.

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6.6

Qualitative and quantitative characterization of protein glycation patterns in human plasma Andrej Frolov, Maria Fedorova, Matthias Blüher, Ralf Hoffmann

Glycation (non-enzymatic glycosylation) is a reversible reaction of protein amino groups with reducing sugars. D-Glucose, the most abundant monosaccharide in blood, is readily involved in this process yielding relatively stable Amadori compounds. The latter undergo further reactions producing diverse advanced glycation end-products (AGE s), known to be markers of diabetes. Though multiple glycation sites in plasma proteins were described, their distribution in healthy and diabetic individuals is still not characterized. Here we present a new approach to identify and relatively quantify Amadori peptides in diabetic patients and healthy individuals. Plasma proteins obtained from diabetic and healthy individuals were digested with trypsin and glycated peptides were subjected to RP-HPLC-ESI-MS / MS analysis after enrichment on m-aminophenylboronic acid-agarose. Amadori peptides were identified by their MS / MS fragmentation patterns while quantification was performed by MS -scans. First, the plasma of five diabetic patients was analyzed. Altogether, 18 Amadori peptides were identified and relatively quantified. Eleven lysines were glycated at similar quantities in all samples, with Lys549 H-KAmadori QTALVELVK-OH being the most abundant. The study was further extended to the groups of diabetic and healthy individuals, each consisting of five persons. The diabetic samples resembled the previously identified pattern of HSA glycation sites, but revealed also those in some other abundant proteins. The glycation level varied for the modification sites and was lower for healthy individuals.

Dr. Maria Fedorova

Dr. Andrej Frolov

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

[email protected] www.uni-leipzig.de/~bioanaly

[email protected] www.uni-leipzig.de/~bioanaly

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BIOANALYTICS

6.7

In vitro formation, detection, and identification of lysine-derived advanced glycation endproducts Uta Greifenhagen, Rico Schmidt, Ralf Hoffmann, Andrej Frolov

Glycation (non-enzymatic glycosylation) refers to a chemical reaction of reducing sugars with amino groups of peptides and proteins. Resulting sugar amines (Amadori and Heyns compounds) undergo slow degradation, oxidation and cross-linking reactions yielding advanced glycation endproducts (AGE s). Intermediates of this degradation are reactive dicarbonyls (e. g. glyoxal and methylglyoxal) formed in processes of monosaccharide autoxidation and lipid peroxidation. Though the resulting AGE s are known markers of diabetes, Alzheimer’s disease and ageing1, reliable sequencing strategies for AGE -peptides are not available. Therefore, approaches to identify carboxymethyllysine (CML) and carboxyethyllysine (CEL) residues were established and validated on the protein level. Following synthesis2 and purification, the fragmentation patterns of CML and CEL -peptides were analyzed by ESI -QqTOF- and -LTQ -Orbitrap-MS / MS. The side-chain modifications proved to be stable under CID conditions. Thereby the corresponding sites could be unambiguously identified by characteristic mass increments. The same sequencing strategy was applied to human serum albumin (HSA) glycated in vitro which was analyzed by RP-HPLC-ESI-LTQ Orbitrap-MS in an information-dependent acquisition (IDA) experiment with subsequent database search for carboxymethyl- and carboxyethyl-modified peptides. The results were proven by manual interpretation of the spectra. Thereby, multiple carboxymethylation and -ethylation sites were identified. 1 2

6.8

Profiling of free oxysterols in plasma by fast liquid chromatography-tandem mass spectrometry and preanalytical aspects Christin Helmschrodt, Susen Becker, Joachim Thiery, Uta Ceglarek

A number of pathophysiological effects in atherogenesis and inflammation are attributed to oxysterols which can be formed enzymatically and non-enzymatically from cholesterol. The analysis of oxysterols is hampered by their low physiological concentrations and the susceptibility to in-vitro autoxidation of cholesterol. The aim of this study was the development of a simple, and reliable high-throughput method for quantification of free oxysterols in human plasma and to standardize preanalytical conditions for oxysterol analysis. Chromatographic separation of 9 oxysterols was performed by a monolithic column RP-18e Chromolith (Merck, Germany) and was coupled to an API 4000 ® (AB SCIEX) mass spectro­ meter. A protein precipitation step was used as sample preparation procedure. Simultaneous analysis of free cholesterol and the free 9 oxysterols was enabled within 7 min. The limit of detection ranged from 0.1 ng / mL (7-ketocholesterol) to 2 ng / mL (4-β-hydroxycholesterol). The linear range varied from 0.5 – 5 ng / mL (LLQ ) to 2000 ng / mL (ULQ ). In freshly prepared plasma samples, free oxysterols were stable for one hour when stored at 4 °C prior to further sample clean up. This validated fast liquid chromatography combined with tandem mass spectrometry method, including a short and gentle sample preparation, is suitable for a rapid and sensitive profiling of free oxysterols and cholesterol in plasma. The addition of butyl hydroxy toluol (BHT) did not increase the stability of these analytes in plasma samples during storage at 4 °C for one hour. Up to three freeze-thaw cycles do not affect analyte levels.

P. Ulrich, A. Cerami; Rec. Progr. Horm. Res. 2001, 56, 1 P. Gruber, T. Hofmann; J. Pept. Res. 2005, 66, 111

Uta Greifenhagen

88

Christin Helmschrodt

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

Universität Leipzig Faculty of Medicine Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics LIFE – Leipzig Research Center for Civilization Diseases

[email protected] www.uni-leipzig.de/~bioanaly

[email protected] www.uni-leipzig-life.de

89

BIOANALYTICS

6.9

A-YES ® and A-YAS ® test systems – innovative

biosensors for the measurement of estrogenic and androgenic activity in water Karina Hettwer, Steffen Uhlig, Martin Jähne, Sven Krügener, Kirsten Simon

The detection of estrogens and androgens in drinking and surface water is an important task due to their potential to alter the endocrine system of humans and animals. In cooperation with the IPK (Gatersleben, Germany) we developed and validated innovative yeast based assays for the detection of cumulated estrogenic or androgenic activity in mineral, curative and environmental water. The screening assays are based on the non-conventional salt tolerant yeast Arxula adeninivorans. The human estrogen receptor α (hERα) or alternatively the androgen receptor (hAR) and a phytase as reporter enzyme were integrated in the genome of A. adeninivorans. In the presence of estrogenic or androgenic compounds, receptor-ligand-complexes induce the expression of the secretory reporter enzyme. The estrogen assays are available for different types of samples. A-YES ®_aqua 1.0 and A-YES ®_aqua 2.0 were designed for the detection of estrogenic activity in mineral water. The assays differ in the sensitivity of the method, whereby the A-YES ®_aqua 2.0 is more sensitive. A-YES ®_matrix 1.1 was designed for the detection of estrogenic activity in environmental water samples such as surface-, ground- or brackish water as well as curative water. The limit of detection was determined to be 2.7 ng / L 17-ß-Estradiol (E2) and the quantification interval ranges from 3.7 till 52.1 ng / L E2 . The detection of the cumulative androgenic activity in mineral water can be performed using the A-YAS ®_aqua, with a limit of detection of 31.3 ng / L 5-α-Dihydrotestosterone (DHT) and a quantification range from 44.2 till 325.8 ng / L DHT.

6.10 Qualitative study of combinations of herbal components of STW 5 in LPS -stimulated CaCo-2 cells Stefanie Hoser, Victoria Winkelmann, Heba Abdel-Aziz, Olaf Kelber, Dieter Weiser, Karen Nieber Inflammatory processes are often involved in gastrointestinal disorders. Lipopolysaccharide (LPS) is a common in-vitro model for studying inflammation due to its induction of cytokine release via NFκB pathway. The aim of this study was to investigate the contribution of the herbal components of STW 5 (Iberogast ®) to its anti-inflammatory effect. Concentration response relationships were determined and, based on the isobologram method, combinations of two extracts were examined. LDH assay was used as a cytotoxicity test in CaCo-2 cells after stimulation with LPS (10 ng / ml) for 2 hours. STW 5 (500.5 µg / ml) and STW 5 -II (511.1 µg / ml, lacking angelica, milk thistle and celandine) inhibited LPS -induced LDH release by up to 74 % in a concentration dependent manner. Lemon balm leaves (59.9 µg / ml, equivalent concentration to STW 5) showed a similar effect with a maximum inhibition of 75 %. Angelica roots (84.7 µg / ml), milk thistle fruits (15.2 µg / ml), peppermint leaves (38.9 µg / ml), chamomile flowers (116.3 µg / ml) and Iberis amara (29.1 µg / ml) caused a decrease of LPS -ind. LDH release of 25 – 37 %. The combination of peppermint leaves with either Iberis amara or with milk thistle fruits had a synergistic effect of 28 – 32 % and 13 – 16 %, respectively. The combination of chamomile flowers and Iberis amara an additive effect, whereas chamomile flowers combined with angelica roots acted antagonistically. Our results indicate that the individual herbal components contribute differently to the antiinflammatory effect of STW 5. The extracts contained in STW 5 can produce synergistic effects as well as additive or antagonistic interactions depending on their combination.

Stefanie Hoser Karina Hettwer

90

QuoData GmbH Research and Development

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Pharmacy Pharmacology for Natural Sciences

[email protected] www.quodata.de

[email protected] www.uni-leipzig.de/~pharm

91

BIOANALYTICS

6.11 Live cell based fluorescent adenosine triphosphate (ATP) biosensors in microfluidic free-flowelectrophoresis chips Stefan Jezierski, Anke Klein, Stefan Nagl, Michael Schaefer, Detlev Belder The miniaturisation towards chip based micro free-flow electrophoresis (µFFE) leads to a reduced need of sample amount, buffers, and separation time compared to classic FFE . Due to the microscale dimensions, the demand on the sensitivity of the used detection techniques is very high. As powerful optical detection method, fluorescence detection is often used. Therefore the analyte has to be fluorescent or to be fluorescently labelled. Using microchipintegrated optical biosensors, labelling procedures of the sample can be circumvented and the analytes can be separated in their native form. Using cells as probes can be advantageous in identifying biological active compounds in the sample. In this work we present the implementation of adherently bonded cells inside a µFFE chip for sensing purposes. Human embryonic kidney cell lines, loaded with different Ca2+ sensitive dyes, were used to detect adenosine triphosphate (ATP) without previous labelling. ATP binding to cell surface and triggers internal Ca2+ -release, causing a change of the dyes fluorescence intensity. Electrophoretic deflection of ATP in a low ionic strength solution was performed. It was possible to identify the position of the analyte stream. For the first time adherently bonded cells were implemented in a microfluidic environment for sensing purposes in combination with an electrophoretic separation technique. This approach enables the label-free detection of biologically relevant molecules in samples and provides the possibility to separate them from other parts of the matrix by µFFE chips.

6.12 Improvement of succinic acid production by yeast Yarrowia lipolytica through optimization of α-ketoglutaric acid production Svetlana V. Kamzolova, Maria N. Chiglintseva, Julia N. Lunina, Igor G. Morgunov Succinic acid (SA) is a common metabolite formed by microorganisms, plants, and animals. SA and its derivatives are widely used in industries producing biodegradable plastics, foods, pharmaceutical products, and cosmetics. SA is applied in medicine as an antistress, antihypoxic, and immunity-improving agent. SA is produced petrochemically from butane through maleic anhydride. However, much attention has been focused on the microbiological production of SA . Recently, co-authors of the present study developed the process of SA production, which included the synthesis of alpha-ketoglutaric acid (KGA) from ethanol by Yarrowia lipolytica and subsequent oxidation of the acid by hydrogen peroxide to SA .1 The maximum concentration of SA and its yield were found to be 63.4 g / L and 58 % of the ethanol consumed, respectively. The aim of the present work was to improve of SA production through the optimization of KGA production by Y. lipolytica. We studied the effect of ethanol, iron and zinc, the level of aeration and pH medium on KGA synthesis as well as the cell requirement for a thiamine and nitrogen by Y. lipolytica VKM Y-2412 . We developed a high-effective process of SA production leads to the accumulation of 139 g / L SA with the yield of 70 %. This work has been supported by the Russian Foundation for Basic Research (grant No. 12-08-01157 ). 1 Kamzolova SV, Yusupova AI, Vinokurova NG, Fedotcheva NI, Kondrashova MN, Finogenova TV, Morgunov IG (2009) Chemically assisted microbial production of succinic acid by the yeast Yarrowia lipolytica grown on ethanol. Appl Microbiol Biotechnol 83:1027-1034.

92

Stefan Jezierski

Dr. Svetlana V. Kamzolova

Universität Leipzig Faculty of Chemistry and Mineralogy Institute of Analytical Chemistry

Russian Academy of Sciences G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms

[email protected] www.uni-leipzig.de/~belder

[email protected] www.ras.ru/en/index.aspx

93

BIOANALYTICS

6.13 Quantitative proteomics reveals novel functions of osteoclast-associated receptor in STAT signaling and cell adhesion in human endothelial cells

6.14 Oncocin derivative Onc72 is highly active against Escherichia coli in a systemic septicaemia infection mouse model

Stefanie Kliemt, Claudia Göttsch, Kathrin Sinningen, Martin von Bergen-Tomm, Lorenz C. Hofbauer, Stefan Kalkhof

Daniel Knappe, Uwe Müller, Michael Zahn, Stefanie Fritsche, Norbert Sträter, Gottfried Alber, Ralf Hoffmann

Previous studies indicate a novel role for the osteoclast-associated receptor (OSCAR) in oxidative stress-mediated atherogenesis. However, the functional role of OSCAR in endothelial cells is unknown. Here we characterized OSCAR signaling in human endothelial cells using a proteomic approach. OSCAR was either overexpressed or silenced and the functional effects were assessed by an in-depth proteomic study using stable isotope labeling with amino acids in cell culture (SILAC). The analysis of the subcellular protein fractions from the membrane, the cytosol, and the nucleus of human endothelial cells yielded 4,975 unique proteins, of which OSCAR overexpression regulated 145 proteins, while OSCAR silencing regulated 110 proteins. These proteins were mainly involved in cellular proliferation, inflammatory response and cell-tocell signaling. Interestingly, signal transducer and activator of transcription 1 (STAT 1) and 3 (STAT 3) were reciprocally regulated by OSCAR modulation. Thus, STAT 1 and several interferon-induced proteins showed a clear inverse correlation to OSCAR expression, which was confirmed by Western Blot analysis. In contrast, OSCAR overexpression activated STAT 3. Furthermore, OSCAR overexpression increased integrin-signaling and up-regulated proteins involved in cell adhesion, which correlated with an increased adhesion of monocytes to the endothelium after OSCAR overexpression. In conclusion endothelial cell-derived OSCAR was found to be involved in the STAT signaling pathway and to affect monocyte adhesion. This indicates a novel role of OSCAR in the vascular-immune cross-talk.

The rapid emergence of drug-resistant bacterial strains demands the development of new antimicrobial compounds acting by novel mechanisms. Oncocin is a new antimicrobial proline-rich designer peptide which we developed to treat Gram-negative infections by Enterobacteriaceae (e. g. Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae) and non-fermenting species (e. g. Acinetobacter baumannii and Pseudomonas aeruginosa). These pathogens represent a severe threat due to the rapid spread of multi- and pan-resistant strains. Here, we describe the in vivo efficacy and the target inhibition mode of novel oncocin derivatives with (i) superior antimicrobial activities, (ii) high serum stabilities, (iii) no eukaryotic cell toxicity, and (iv) fast bactericidal activity. NMRI mice treated with i. p. bolus injections with single doses of 20 mg or 40 mg Onc72 per kg did not result in any abnormal animal behavior. No mouse became moribund or died within the studied time period. Histopathological investigations revealed no toxic effects. Moreover, oncocin was not cytotoxic against human cell lines and murine bone marrowderived dendritic cells. The same mouse strain was used to establish a lethal peritoneal sepsis model. When infected with E. coli (ATCC 25922; 106 cfu in 2.5 % mucin) none of the untreated animals survived the next 24 h, whereas all animals treated three times with doses of 5 mg / kg Onc72 or more survived the observation period of five days. No bacteria were detected in the blood of treated animals after day 5. The effective dose (ED 50) was around 2 mg / kg indicating a safety margin larger than 20.

Dr. Daniel Knappe Stefanie Kliemt

94

Helmholtz-Zentrum für Umweltforschung – UFZ Department of Proteomics

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

[email protected] www.ufz.de/index.php?de = 21816

[email protected] www.uni-leipzig.de/~bioanaly

95

BIOANALYTICS

6.15 Quantitative profiling of arachidonic acid metabolites by hybrid triple quadrupole / linear ion trap mass spectrometry Linda Kortz, Juliane Dorow, Joachim Thiery, Uta Ceglarek

Objective: Eicosanoids are enzymatic products of arachidonic acid (AA) bound in cell mem­branes to phospholipids. They play important functional roles in inflammation, cellular proliferation, and intracellular signalling. The simultaneous analysis of a multitude of eicosanoids, in contrast to the isolated view of one single pathway, will help to understand molecular mechanisms underlying the complex AA metabolic system. The quantitative analysis of AA metabolites is of interest for different sample types e. g. supernatants of cell culture experiments or human body fluids. Method: Plasma or urine samples were processed by protein precipitation (plasma samples) and off-line solid phase extraction (both plasma and urine specimen). Fast chromatographic separation was achieved on a Kinetex C-18 core-shell column with a Shimadzu Prominence UFLC system. MS analysis was performed on a 5500 QTrap ® instrument applying electro­ spray ionization in negative mode. Results: About 50 multiple reaction monitoring (MRM) transitions for the quantitation of eicosanoids are included in the presented method. Additionally, product ion spectra can be generated by changing to ion trap mode during an analytical run, thus allowing verification of each lipid through the characteristic fragment pattern. Conclusion: Our analytical method using the hybrid QTrap ® technology allows the simul­ taneous acquisition of both quantitative data and structural information of AA metabolites in human biological fluids.

Linda Kortz Universität Leipzig Faculty of Medicine Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics LIFE – Leipzig Research Center for Civilization Diseases [email protected] www.uni-leipzig-life.de

96

6.16 Development of an ITC based enzyme assay for nucleoside triphosphate diphosphohydrolases (NTPD ases) Michel Krauß, Matthias Zebisch, Norbert Sträter

For the regulation of the extracellular levels of nucleotides membrane-bound ectonucleotidases such as nucleoside triphosphate diphosphohydrolases play an important physiological role. CD39 or NTPD ase1, the prototype member of the eukaryotic NTPD ase family is the dominant NTPD ase of the vasculature. Based on the hydrolysis of proinflammatory ATP and platelet-activating ADP to AMP the platelet-aggregation is blocked and blood flow is supported. Thus, an understanding of the structure and dynamics of CD39 is vitally important. Here, we present the crystal structure of a variant of soluble NTPD ase1 lacking a putative membrane interaction loop (MIL). This loop was identified between the two lobes of the catalytic domain. Measurements of ATPase and ADPase activities where performed using a newly established kinetic assay via isothermal titration calorimetry (ITC). In addition, a comparison of the domain orientations of the four independent proteins in the crystal asymmetric unit provides first direct experimental evidence for a domain motion of NTPD ases. An interdomain rotation angle of up to 7.4 ° affects the active site cleft between the two lobes of the protein. Comparison with a previously solved bacterial NTPD ase structure indicates that the domains may undergo relative rotational movements of more than 20 °. Our data support the idea that the influence of transmembrane helix dynamics on activity is achieved by coupling to a domain motion.

Michel Krauß Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers [email protected] www.uni-leipzig.de/~straeter

97

BIOANALYTICS

6.17 A novel affinity-optimized homogeneous immunoassay Thomas Kreisig, Ralf Hoffmann, Thole Züchner

For the detection of antigens, a variety of different heterogeneous immunoassay techniques can be used. These assays are sensitive and provide linear ranges spanning three orders of magnitude. Besides these advantages, the drawback remains that multiple incubation and washing steps are required and hence heterogeneous immunoassays are very timeconsuming with several hours of assay time. A direct and fast readout of the immunoreaction is possible using homogeneous immunoassays without separation steps. Here we present a homogeneous competitive immunoassay for the determination of phosphorylated tau peptides. A phosphorylation-specific antibody and the corresponding peptide probe are labeled with two dyes: One linked to the antibody as acceptor and a donor fluorophore coupled to a specifically designed peptide probe. The intensity of the fluorescence resonance energy transfer between the labeled antibody and the donor peptide probe depends on the analyte concentration. The design of the assay allows a quantitative analysis of the antigen within 90 seconds.

6.18 Potential of using chip calorimetry as a quality control tool for encapsulated cell products Johannes Lerchner, Eva-Maria Brandtner, Florian Mertens, Walter H. Günzburg

Pharmaceutical products based on encapsulated cells belong to a new class of therapeutics called “advanced therapy medicinal products” (Salmons et al., 2007). Encapsulated cell products can be used to treat a wide spectrum of diseases with diverse cause. The encapsulation technology can also be used to produce missing enzymes and growth factors, or to augment their production. The immune system can also be bolstered by the production of cytokines from encapsulated cells. Another attractive feature of the encapsulation technology is that physiological regulations of the released therapeutic agent can be achieved in response to a metabolic stimulus. In order to prove the potential of using calorimetry as a quality control tool for a GMP production of encapsulated cell products, we measured the metabolic heat rate of encapsulated cells using a chip calorimeter. Because of their real-time capabilities, if at all, only miniaturized calorimeters seem to suit the challenges of a fully automated production line. In our experiments, “human embryonic kidney” (HEK) cells encapsulated in biologically inert cellulose sulphate polymers were used. Capsules with a diameter of 0.8 mm each containing about 5,000 cells were taken from a cultured batch. To conduct the heat measure­ ments, about 14 capsules each were placed inside the measurement chamber of a prototype chip calorimeter developed at the TU Bergakademie Freiberg. Salmons, B., Hauser, O., Guenzburg, W. H. et al., 2007. GMP production of an encapsulated cell therapy product: Issues and considerations. BioProcessing Journal 4:36-43.

Thomas Kreisig Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute for Bioanalytical Chemistry Ultrasensitive Protein Detection Unit [email protected] www.uni-leipzig.de/uspdu

98

Dr. Johannes Lerchner Technische Universität Bergakademie Freiberg Faculty of Chemistry and Physics Institute of Physical Chemistry [email protected] http://tu-freiberg.de/fakult2/phych

99

BIOANALYTICS

6.19 Analysis of oxidation products of 1-palmitoyl-­ 2-linoleyl-phosphatidylcholine and their reaction products with cysteine, histidine and lysine residues Ivana Milic, Ralf Hoffmann, Maria Fedorova

Carbonyl-containing lipid peroxidation products (LPP), formed during oxidative stress, can readily react with the nucleophilic amino acids, changing the structures and functional activities of proteins. However, exact molecular species responsible for protein modifications are poorly investigated. Here we studied formation of LPP caused by overnight incubation of linoleic acid and 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine (PLPC) with or without H2O2 in the presence or absence of CuCl, using shotgun MS -approach. The PLPC derived carbonyls were derivatized with dinitrophenylhydrazine (DNPH) and extracted following standard protocol. Underivatized and derivatized LPP were analyzed using ESI-LTQ -Orbitrap-MS. Furthermore three synthetic peptides containing Cys, His or Lys residues were incubated with linoleic acid and PLPC under optimized oxidative conditions, and analyzed by nanoRPC-ESI -Orbitrap-MS. Using shotgun lipidomics strategy we identified 55 different LPP, 26 containing reactive carbonyl groups. The strongest oxidation conditions yielded 51 LPP, whereas air oxidation produced 12 LPP. Independent of the oxidation conditions, around half of all LPP were short-chain (oxidative cleavage) and the others long-chain (oxygen addition). Using LC -MS approach we have detected and identified 10 LPP-derived peptide modifica­tions at lysine, 9 at cysteine and 3 on histidine residue. Three high molecular weight (PC -bound) carbonyls were detected on Lys-containing peptide. Three LPP-derived mass shifts (86, 88 and 102 u) which have not previously reported, were obtained at Cys residues.

6.20 Immobilization of green alga Chlorella vulgaris within alginate / silica hybrid materials for cell-based detection systems Angela Pannier, Ulrich Soltmann, Bettina Soltmann, Mechthild Schmitt-Jansen, Rolf Altenburger Thin layers and arrays of individual spots of alginic acid with embedded microalgae have been deposited onto glass slides. The arrays have been patterned by using a non-contact microdosage system (Nanoplotter 2.0, G eSIM , Germany) which even allows printing solutions of high viscosity such as alginic acid. The alginic acid was subsequently cross-linked using amino-functionalized silica sols to obtain alginate hydrogels which are reinforced by silica. The alginate / silica hybrid materials were more stable in salt-containing solutions compared to alginate gels cross-linked by traditional usage of Ca2+ -ions. The applicability of that approach to design cell-based detection systems has been investigated by immobilizing green microalga Chlorella vulgaris as sensing microorganisms within the newly developed alginate / silica hybrid matrices. Toxicity testings have been carried out by applying heavy metals (e. g., Cu) and herbicides (e. g., atrazine). The pulse amplitude modulation (PAM)method has been used to evaluate the impact on chlorophyll a fluorescence as the presence of these substances affects the photosynthetic activity of green algae and other photosynthetic organisms.

This publication is supported by LIFE – Leipzig Research Center for Civilization Diseases, Universität Leipzig. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERFD ) and by means of the Free State of Saxony within the framework of the excellence initiative.

Ivana Milic

100

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

Angela Pannier

[email protected] www.uni-leipzig.de/~bioanaly

[email protected] www.gmbu.de

GMBU e.V.

Functional Coatings

101

BIOANALYTICS

6.21 Screening for inhibitors of human ecto-5’nucleotidase Jan Pippel, Karen Yates, Matthias Zebisch, Christa E. Müller, Norbert Sträter

The eukaryotic ecto-5’-nucleotidase (e5NT) is an extracellular enzyme which catalyzes the hydrolysis of AMP to adenosine and phosphate.[1] Since the effects of adenosine are often opposite to those of ATP, e5NT might be an appealing new drug target with various fields of application. For the production of recombinant e5NT an E. coli expression system was established including refolding procedures. Different variants were expressed and tested for their suitability for crystallization. Structures with resolutions of up to 1.8 Å were determined at Bessy PX beamlines. The molecular characterization revealed that e5NT is composed of two domains. Via a large (~ 100 °) rotation of the N-terminal domain, the enzyme is able to switch between an open and closed conformation. Based on these results we hope to support rational inhibitor design by determining complex structures. Therefore, we identified several potential e5NT inhibitors via high throughput inhibitor screening which are now used in co-crystallization and soaking experiments. Also inhibitors that mimic the natural substrate are of interest as such complex structures could provide further insights into the substrate binding mode and control of substrate specificity. In contrast to the bacterial 5‘-nucleotidases, e5NT is specific for AMP and does not hydrolyze ADP or ATP.

6.22 Improvement of the signal to noise ratio of a highly sensitive protease assay by different additives Agneta Prasse, Thomas Zauner, Renate Berger-Hoffmann, Katrin Müller, Ralf Hoffmann, Thole Züchner Proteases are well known tools in analytical sciences and established as reagents in a variety of assays. At the same time, proteases play an essential role in a numerous of diseases, so their sensitive detection is of high biotechnological and biomedical interest. We recently published a protease assay based on fluorescence resonance energy transfer which allowed detection down to 10 amol of enteropeptidase in E. coli lysate [Zauner et al. 2011]. This sensitivity is based on an enzyme cascade for signal amplification. The second enzyme of the cascade, trypsinogen, is activated by enteropeptidase and the resulting trypsin cleaves a designed FRET-peptide and therefore causes an increase of donor fluorescence. This increase corresponds to the enteropeptidase activity. Although the sensitivity and selectivity of the described assay were already high, further steps were taken to improve the assay parameters. In detail, different additives were tested to reduce the signal to noise ratio of the assay as well as to reduce the background signals caused by trypsinogen autoactivation. This resulted in a significant improvement of the signal to noise ratio for the detection of enteropeptidase in biological samples. Zauner T, Berger-Hoffmann R, Müller K, Hoffmann R, Zuchner T., Analytical Chemistry, 2011 (83), 7356-7363

1 Sträter, N. (2006) Ecto-5‘-nucleotidase: structure function relationships. Purinergic Signalling. 2: 343-350. We gratefully thank the “Deutsche Forschungsgesellschaft” (DFG) for funding.

102

Jan Pippel

Agneta Prasse

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Bioanalytics

[email protected] www.uni-leipzig.de/~straeter

[email protected] www.uni-leipzig.de/~bioanaly

103

BIOANALYTICS

6.23 Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria Konrad Siegfried, Carola Endes, Abul Fateh Md. Khaled Bhuiyan, Anke Kuppardt, Jürgen Mattusch, Jan Roelof van der Meer, Antonis Chatzinotas, Hauke Harms A test kit based on living, lyophilized bacterial bioreporters emitting bioluminescence as a response to arsenite and arsenate was applied during two field campaigns in six villages across Bangladesh. Bioreporter field measurements of arsenic in groundwater from tube wells were in satisfying agreement with results of spectroscopic analyses of the same samples conducted in the lab. The practicability of the bioreporter test in terms of logistics and material requirements, suitability for high sample throughput and waste disposal was much better than that of two commercial chemical test kits that were included as references. The campaigns furthermore demonstrated large local heterogeneity of arsenic in groundwater, underscoring the use of well switching as an effective remedy to avoid high arsenic exposure.

Dr. Konrad Siegfried Helmholtz-Zentrum für Umweltforschung – UFZ Department of Environmental Microbiology ARSO lux [email protected] www.arsolux.ufz.de

104

POSTERS

7.

Bioinformatics

BIOINFORMATICS

7.1

AP2 D – A novel program for high specific primer

design

Janine Brettschneider, Bianca Liebscher, Gabriel Kind, Dirk Labudde

PCR is widely used in life sciences and now, in the age of “Omics”, it is an important

part in experimental procedures. A critical factor of the efficiency and accuracy is linked with the design of primers. Only 70 – 75 % of the designed PCR reactions work without subsequent optimizations. A major error source occurs due to unspecific DNA hybridization. Therefore we developed a special primer program, AP ²D, which designs primer with high quality. Involved features are: with a BLAST request the Genbank is searched for possible false hybridization. The request is carried out with the previously designed best primer pairs and the results are automatically analyzed. The user receives the results reporting whether potential hybridizations with the forward or the reverse primer exist. The validity of positive targets will be checked as well. Furthermore AP ²D is developed for large studies. The user can save all settings and results (e. g. designed primer, specificity result), thus it is user friendly to share information with other AP ²D users. Our software is free, is regularly improved and is going to be available at www.bioforscher.de soon.

108

7.2

Drug repositioning through incomplete bi-cliques in an integrated drug-target-disease Network Simone Daminelli, V. Joachim Haupt, Matthias Reimann, Michael Schroeder

Recently, there has been much interest in gene-disease networks and polypharmacology as a basis for drug repositioning. Here, we integrate data from structural and chemical databases to create a drug-target-disease network for 147 promiscuous drugs, their 553 protein targets, and 44 disease indications. Visualizing and analyzing such complex networks is still an open problem. We approach it by mining the network for network motifs of bi-cliques. In our case, a bi-clique is a subnetwork in which every drug is linked to every target and disease. Since the data is incomplete, we identify incomplete bi-cliques, whose completion introduces novel, predicted links from drugs to targets and diseases. We demonstrate the power of this approach by repositioning cardiovascular drugs to parasitic diseases, by predicting the cancer-related kinase PIK3CG as novel target of resveratrol, and by identifying for five drugs a shared binding site in four serine proteases and novel links to cancer, cardiovascular, and parasitic diseases.

Janine Brettschneider

Simone Daminelli

Hochschule Mittweida Faculty of Mathematics, Sciences and Computer Science

Technische Universität Dresden Biotechnology Center (BIOTEC)

[email protected] www.bioforscher.de

[email protected] www.biotec.tu-dresden.de

109

BIOINFORMATICS

7.3

Combined proteome and metabonome analysis of a time- and concentration resolved exposure scenario with the carcinogenic contaminant Benzo[a]pyrene Franziska Dautel, Stefan Kalkhof, Salvatore Loguercio, Sven Baumann, Saskia Trump, Wolfgang Otto, Susanne Rudzok, Irina Lehmann, Andreas Beyer, Martin von Bergen-Tomm

Within the Helmholtz-Alliance on Systems biology, the network “Contaminant molecules” aims for a comprehensive mechanistic understanding of the cellular effects of the carcino­ genic contaminant benzo[a]pyrene (B[a]P). Reproducible quantitative protein expression data were generated with the gel-free approach SILAC (stable isotope labeling by amino acids in cell culture) and pulsed-SILAC using murine Hepa1c1c7 cells and a toxic (5 μM) and a subacute concentration (50 nM) of B[a]P over a period of 2 to 24 h for later model development predicting the effects of structurally related chemicals on cellular systems. In addition, 163 cellular metabolites were monitored in response to B[a]P. Various pathway analysis including Ingenuity and STEM (Short Time-series Expression Miner) were per­ formed. Expression profiles of 1,133 and 1,114 proteins were analyzed of which 576 pro­ teins were found to be regulated in response to B[a]P, 190 for the higher and 150 for the lower concentration respectively and 236 for both concentrations. Several metabolites such as amino acids, acylcarnitins, and glycerophospholipids showed severe B[a]P-induced alterations. Known cellular responses including oxidative stress or cell cycle perturbations were confirmed and further investigated on a large timescale. The combined results provide mechanistic explanations on how B[a]P may change metabolism which in turn modulates the cells’ fate. This study emphasizes the benefit of a combined proteomic and metabonomic analysis for the elucidation of various cellular and toxicological outcomes in time- and concentration exposure scenarios.

110

7.4

DARIO: A ncRNA detection and analysis tool

for next-generation sequencing experiments Mario Fasold, David Langenberger, Hans Binder, Peter F. Stadler, Steve Hoffmann

Small non-coding RNA s (ncRNA s) such as microRNA s, snoRNA s and tRNA s are a diverse collection of molecules with several important biological functions. Current methods for high- throughput sequencing for the first time offer the opportunity to investigate the entire ncRNAome in an essentially unbiased way. However, there is a substantial need for methods that allow a convenient analysis of these overwhelmingly large data sets. Here, we present DARIO, a free web service that allows to study short read data from RNA-seq experiments for small RNA s. It provides a wide range of analysis features, including quality control, read normalization, ncRNA quantification and prediction of putative ncRNA candidates. The DARIO web site can be accessed at http://dario.bioinf.uni-leipzig.de .

Franziska Dautel

Mario Fasold

Helmholtz-Zentrum für Umweltforschung – UFZ Department of Proteomik

Universität Leipzig Interdisciplinary Centre for Bioinformatics (IZBI )

[email protected] www.ufz.de/index.php?de=17656

[email protected] www.izbi.uni-leipzig.de

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BIOINFORMATICS

7.5

Structure topology prediction of discriminative protein sequence motifs in domains of unknown function Steffen Grunert, Florian Heinke, Dirk Labudde

Motivation: Membrane proteins play essential roles in cellular processes of the human body. They are involved to add the specificity and functionality of the cells, including photosynthesis, transport of ions and small molecules, signal transduction and light harvesting. By this the analysis of membrane proteins plays a huge role in genomics and proteomics. A further fact is the discovery of motifs in membrane proteins. They support the understanding of the features that are important for the folded protein in the membrane environment. The protein sequence motif analysis is helpful for target mutagens studies, for investigations of diseases and further studies. Materials & Methods: In the focus of our statistical analysis, 50 protein sequence motifs derived from previous studies were identied and analysed in 32 membrane protein families, including proteins with domains of unknown function (DUF-families). The dataset was derived from Pfam database (http://pfam.sanger.ac.uk ). Results: This analysis led to a novel approach which describes the separation of motifs by a residue specific distribution. Based on these distributions we can predict the location inside of the proteins topology for the majority of identified motifs. Conclusion: Depending on possible topological functionality we are able to perform a prediction for most motifs with an F-score up to over 90 % within all derived protein families. Motifs showing rather small F-scores in prediction cannot be generalized to prediction characteristics. Hence, these motifs point to functional relevance and diverse specificity in membrane protein families.

Steffen Grunert Hochschule Mittweida Faculty of Mathematics, Sciences and Computer Science Bioinformatics [email protected] www.bioforscher.de

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7.6 eGOR – Predicting the total potential energy of a proteins native state by sequence Florian Heinke, Steffen Grunert, Dirk Labudde

Motivation: Experimental structure determination is a time- and resource-consuming procedure. In silico-driven techniques, such as comparative modelling or ab initio-folding, are constricted methods as well. A main limitation of ab initio-folding is that observed convergence of the proteins total potential energy (TPE) during simulation does not necessarily correspond to the proteins native state. In this work, we demonstrate a method for predicting the TPE of a given protein in its natively folded stated based on its sequence. Materials & Methods: We derived a coarse-grained energy model which describes the interaction energy of each residue with respect to its spatial adjacent residues in a given protein structure. The sums of all coarse-grained energies of all investigated proteins correspond very well to TPE s derived by all-atom molecular dynamics (MD) analyses (r² = 0.97). We adapted the GORIII secondary structure prediction algor ithm for predicting discretized coarse-grained energies based on a proteins sequence (eGOR). By applying the correlation elucidated above to the sum of discretized coarse-grained energies predicted by eGOR , the TPE of the protein is predicted. Results: We applied our prediction algor ithm to 220 protein structures. Predicted TPE s and TPE s derived from MD correlate very well (r² = 0.95). Conclusion: The algor ithm presented in this work is suitable for predicting the TPE of a protein in its native fold based on its sequence. It can be applied as a convergence criterion in ab initio-folding and is helpful to distinguish local energy minima from the global energy minimum in spatial folding trajectories of a protein of interest.

Florian Heinke Hochschule Mittweida Faculty of Mathematics, Sciences and Computer Science [email protected] www.bioforscher.de

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BIOINFORMATICS

7.7

An integrative multi-scale approach to liver modeling in 3D

7.8

Regeneration after partial hepatectomy: from cell to organ scale

Stefan Höhme, Lars Ole Schwen, Lorenza A. D’Alessandro, Adrian Friebel, Johannes Neitsch, Andreas Raue, Seong-Hwan Rho, Felix Gremse, Iris von Recklinghausen, Seddik Hammad, Ahmed Ghallab, Patricio Godoy, Raymond Reif, Iryna Ilkavets, Steven Dooley, Fabian Kiessling, Jens Timmer, Tobias Preusser, Jan G. Hengstler, Ursula Klingmüller, Dirk Drasdo

Stefan Höhme, Marc Brulport, Alexander Bauer, Essam Bedawy, Wiebke Schormann, Matthias Hermes, Verena Puppe, Rolf Gebhardt, Sebastian Zellmer, Michael Schwarz, Ernesto Bockamp, Tobias Timmel, Jan G. Hengstler, Dirk Drasdo

During the last years, modeling of different physiological and pathological aspects of the liver advanced significantly with the development of increasingly realistic models on molecular, cellular, tissue and whole organ scale. Nevertheless, model driven liver research is still hampered by a lack of techniques that allow the robust integration of these different scales into unifying frameworks. We here present a three-dimensional individual-cell-based spatio-temporal model of the liver that may serve as such a unifying framework. We illustrate how methods from different scientific fields including graph theory, image processing, image analysis and physics are utilized to link the different scales involved. On the organ scale, the blood vessel network is represented as directed graph while Kirchhoff rule and Poiseuille law are used to mimic the flow of blood through the vessels. Cells are modeled as individual agents capable of moving, growing and dividing. The movement of a cell is modeled by an equation of motion incorporating all physical forces on that cell as well as the cells’ micro-motility and the effect of external stimuli on cell migration. Intracellular signaling is mimicked by a system of ordinary equations or alternatively by Boolean decisions. The model is able to integrate the molecular scale by incorporating signaling pathway models into each individual cell, and to represent the lobule and the lobe architecture. Furthermore, it can be easily linked to macroscopic models on whole organ scale. Thereby, our model framework provides a first step to realistic multi-level multi-scale tissue organization simulations in liver, spanning the molecular and whole organ scale, and including cells as individual agents at the intermediate level.

The liver is a vital organ with a wide range of functions. It plays a key role in detoxification of the blood and is essential for most metabolic functions of the body. One of the outstanding features of the liver is its capacity to regenerate a loss of large parts of its mass within days. This rapid regeneration is of utmost importance for patient survival for example after partial hepatectomy (PH x), a process where parts of the liver are surgically removed for example during liver the treatment of liver cancer. In liver, function and architecture are tightly coupled. We address the question of how the functional components hepatocytes and blood vessels interplay in time and space during the regeneration of liver mass after PH x in an iterative experimental vs. modeling approach. We advanced the single-cell (agent) based spatial-temporal model in 3D established in 1 for liver regeneration after drug induced damage towards regeneration after PH x. The model is constructed based on experimental data, in particular confocal laser scans and whole slide scans, that were quantified by a novel image processing and analysis chain. We find that our model permits a quantitative description of the regeneration process of liver mass and architecture in mouse until the liver size is recovered if we assume that only cells not subject to too strong compression are able to enter the cell cycle. The model reproduces the lobule size, cell size and proliferation pattern experiments. Moreover, we demonstrate that our model, re-calibrated with static data from pig liver lobules and lobes, provided an unexpected, valid prediction about the spatial proliferation pattern.

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1 Höhme, et al. (2010). Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration. Proc. Natl. Acad. Sci. (USA ), 107(23), 10371-10376.

Dr. Stefan Höhme

Dr. Stefan Höhme

Universität Leipzig Interdisciplinary Centre for Bioinformatics (IZBI ) Multicellular Systems Biology

Universität Leipzig Interdisciplinary Centre for Bioinformatics (IZBI ) Multicellular Systems Biology

[email protected] www.hoehme.com

[email protected] www.hoehme.com

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BIOINFORMATICS

7.9

Direct and inverse modeling to test parameter inference from perfusion imaging Nick Jagiella, Irene Vignon-Clementel, Hendrik Laue, Fabian Kiessling, Dirk Drasdo

We here present a methodological approach to evaluate the predictive efficiency of parameter inference methods from dynamic contrast-enhanced (DCE) perfusion images produced from virtual vasculatures. DCE perfusion imaging in patients is a diagnostic instrument to estimate the grade of tumor malignancy and to assess the treatment response. By solving the inverse problem on perfusion images one can infer the structural (plasma or interstitial volume fraction) and functional vessel properties (flow rate, permeability surface). The vasculature is represented by a graph of vessel nodes and segments populating an underlying lattice. Vessel remodeling is rule-based and depends on the functional vessel properties as well as the type of surrounding tissue (normal, tumorous or necrotic). The functional properties as well as the CA dynamics are solved by partial differential equations. As a benchmark for the accuracy of inverse problems, we first solve the direct problem of contrast agent perfusion along a network of either permeable or non-permeable vessels. Then by solving the inverse problem for a non-spatial two-compartment model voxel by voxel and direct comparison between recovered and original parameter maps we study its predictive efficiency for different cases. For purely intra-vascular agents, one gets a good recovery of the volume fractions (plasma, interstitial space) used in the direct model while the flow rate is underestimated the farther the region of interest was away from the feeding artery. For permeable blood vessels the drain of contrast agent along the path of transport leads to further underestimation. perme­ able to the contrast agent the drain of contrast agent along the path of transport leads to an underestimation of all vessel properties (flow, membrane diffusion and volume fraction) in voxels far away from feeding arteries.

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7.10 Parameterization of an avascular tumor model from data Nick Jagiella, Benedikt Müller, Margareta Müller, Irene Vignon-Clementel, Dirk Drasdo

Question: We here present how to parameterize a mathematical agent-based model of growing multi-cellular tumor spheroids (MTCS) almost completely from experimental data to two targeted cell types. Method: As a proof of principle we will present the process chain of model construction and parameterization from different data sources for the avascular growth of the EMT6 / Ro and the SK-MES -1 cell line. In this model, cells are represented individually by agents which populate the sites of an unstructured lattice. The spatio-temporal behavior on the cellular scale is considered by stochastic. The molecular scale is described by deterministic partial differential equations. In a first step, the model was built up and validated with EMT6 / Ro mouse mammary carcinoma MCTS data from literature. In a second step the model was adapted to the SKMES -1 cell line in comparison with growth curves and qualitatively by image analysis of spheroid cryosections stained for apoptosis and proliferation. Results: For the EMT6 / Ro cell line the simulation model predicted the growth kinetics to be controlled by a combination of spatial restrains and nutrient limitation. ATP was found to be the critical resource which the cells try to keep constant over a wide range of oxygen and glucose medium concentrations by switching between aerobic and anaerobic metabolism. Only if both, oxygen and glucose are very limiting saturation was observed which the model could explain by cell-cell-adhesion-driven migration. For the SK-MES -1 cell line, beside ATP, lactate was identified to control the size of the necrotic core.

Nick Jagiella

Nick Jagiella

INRIA BANG Project Team

INRIA BANG Project Team

[email protected] www.rocq.inria.fr/bang/DD/chadha.html

[email protected] www.rocq.inria.fr/bang/DD/chadha.html

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7.11 Approach for the identification of human pathogen fungi based on quality-verified DNA sequences Bianca Liebscher, Katja Köpke, Dirk Labudde

It is a matter of common knowledge, that human pathogen fungi induce relevant diseases, i. e. the sick building syndrome or building related illness syndrome. Concurrent allergies, neurological damages or indisposition may arise. As a consequence, there are high medical costs to therapy the patients. In contrast the generally microbiological detection methods for diagnostics are time consuming and inconvenient. Here, subjective identification and cross contamination are quite common. A new innovative approach is to detect human pathogen fungi using analytical methods established in molecular biology. The ribosomal DNA sequences of 100 human pathogen fungi were analyzed to identify the organisms as accurately as possible. Therefore we compared database sequences from NCBI , ARB SILVA and BOLDSystem as well as own sequencing results. Finally we isolated high quality human pathogen fungi sequences which can be used for primer and probe design. Additionally we deduced criteria to recognize high quality sequences from raw sequencing data. This work could form the basis to develop a detection kit for high specific identification from human pathogen fungi in interiors.

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7.12 The spider silk on molecular level Tony Petzold, Franka Eichler, Dirk Labudde

Synthesis and development of new products in the fields of medicine, biotechnology and further more gain popularity and relevance. Today there is a visible trend in imitating natural materials. A novel aim is to produce the multifunctional spider silk on industrial-scale. The knowledge about the molecular structure is essential for understanding the mechanical properties of the silk. Based on experimental data, we used various bioinformatic methods to gain more insight into the characteristic composition and the complex formation mechanisms of the spider silk proteins. The assembly process of the fiber is fundamental for its final architecture. Inside the spider gland the proteins are naturally highly concentrated in a sodium chloride-enriched environment, without aggregation. Resulting to physiochemical changes in the spider duct, the conformation of the spider silk proteins is rearranged. Thus single spider silk proteins aggregate to form a stable and elastic fiber. In this work we show the correlation between alanine-rich regions on sequence level and the architectural rearrangements.

Bianca Liebscher

Tony Petzold

Hochschule Mittweida Faculty of Mathematics, Sciences and Computer Science

Hochschule Mittweida Faculty of Mathematics, Sciences and Computer Science

[email protected] www.bioforscher.de

[email protected] www.bioforscher.de

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BIOINFORMATICS

7.13 Google goes cancer: Improving outcome prediction for cancer patients by network-based ranking of marker genes Janine Roy, Christof Winter, Zerrin Isik, Michael Schroeder

In the last decade, there has been much work on predicting disease progression and other outcome variables from gene expression to personalize treatment options. Despite first diagnostic kits on the market, found marker genes often show limited prediction accuracy, limited reproducibility, and unclear biological relevance. In order to solve these problems, we developed a novel outcome prediction algor ithm -NetRank- to identify marker genes prognostic for outcome using both expression data and network information. Our approach adapts the random surfer model of Google’s PageRank algor ithm to rank genes according to their prognostic relevance. We applied the algor ithm to gene expression profiles obtained from 30 pancreas cancer patients, and identified seven candidate marker genes. Compared to genes found with state of the art methods, NetRank improved the prediction accuracy by 7 %. When experimentally validating the prognostic value of our seven candidate markers on an independent set of 412 pancreatic cancer samples, we achieved an accuracy superior to established clinical prognostic factors. Besides, we systematically evaluated the prognostic power of networks and NetRank for signature identification on 25 published cancer datasets. We could show that NetRank algor ithm performs better than classic feature selection methods. In addition, reproducibility of signatures created by NetRank significantly in­ creases between different datasets of the same cancer type. In future network-based gene expression analysis will lead to a more detailed understanding of cancer-related processes.

7.14 Self-organizing maps: Portraying the OMEs with individual resolution Henry Wirth, Lydia Hopp, Hans Binder

New high-content technologies provide huge amounts of data per measurement, making traditional analysis inefficient. New strategies of filtering, visualization and functional analysis are inevitable. Our approach applies machine learning known as self-organizing maps (SOMs). SOM s enable the parallel sample- and feature-centered view of molecular phenotypes combined with strong visualization and second-level analysis capabilities. The SOM mapping reduces the initialyl high number of features (e. g., up to hundreds of thousands SNPs or expression values) to a few thousands of meta-features, each representing a cluster of co-regulated single features. These meta-features are visualized as intuitive mosaic pictures, which feature characteristic color patterns for the data landscape of each sample. Analysis techniques normally used at the feature-level such as hierarchical clustering or component analyses provide enhanced sample resolution due to improved signal-to-noise ratios if applied to the meta-features. We applied our analysis to diverse data sets comprising multiple OME s such as transcriptome (e. g. human tissue atlas, progression of prostate cancer and glioblast oma, lymphoma classification or stem cell differentiation), genome (SNP-atlas of human genome diversity), proteome (mass-spectra of Prototheca and Drosophila) and methylome (human prostate cancer and murine stem cells). Our SOM software package provides an intuitive and informative global view of the behavior of a few basal modules of correlated features without loss of primary information. These features can be assigned to well-defined biological functions in most cases. Wirth, H., Löffler, M., Bergen, M. v., & Binder, H., 2011. Expression cartography of human tissues using self-organizing maps. BMC Bioinformatics Wirth, H., Bergen, M. v., Murugaiyan, J., Rösler, U., Stokowy, T. & Binder, H., 2011. MALDI-typing of infectious algae of the genus Prototheca using SOM portraits. Journal of Microbiological Methods Binder, H., Fasold, M., Hopp, L., Cakir, V., Bergen, M. v., Wirth, H., 2011. Molecular phenotypic portraits - exploring the ‘OMEs’ with individual resolution. Proceedings of HIBIT conference 2011 Binder, H., Fasold, M., Hopp, L., Cakir, V., Bergen, M. v., Wirth, H., 2011. Portraying high-dimensional OMICs data with individual resolution. Proceedings of CAMDA conference 2011

Janine Roy Technische Universität Dresden Biotechnology Center (BIOTEC) Bioinformatics [email protected] www.biotec.tu-dresden.de

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Henry Wirth Universität Leipzig Interdisciplinary Centre for Bioinformatics (IZBI ) [email protected] http://som.izbi.uni-leipzig.de

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POSTERS

8.

Tissue and Cell Engineering

TISSUE AND CELL ENGINEERING

8.1

A novel microfluidic cell culture device for in vitro reconstruction of liver sinusoids Jan Böttger, Julia Schütte, Karin Benz, Christian Freudigmann, Britta Hagmeyer, Simon Werner, Peter Röhnert, Holger Becker, Christoph Höppner, Martin Stelzle, Rolf Gebhardt

The high drug failure in clinical trials is calling for new technologies and methodologies to increase the predictability of toxicity assays before administration to humans. To this end, we developed the microfluidic HepaChip ® device, harbouring 3D artificial liver sinusoids composed of endothelial cells (EC) and hepatocytes (HC) which maintain their specific functionality. The HepaChip ® mimics the liver-like micro-environment by (I) comprising 24 micro­ structures with approximately the size of liver sinusoids, (II) integrating microelectrodes to assemble the different liver cell types onto the cell culture areas, (III) providing cells with extracellular matrix and (IV) enabling a microfluidic perfusion. The 3D artificial sinusoids were obtained by assembling the HC onto the ridges using a combination of dielectro­ phoretic and hydrodynamic forces. HC assembled rapidly, attached there and spread out within 2 hours. Subsequently, EC were assembled in the same manner forming a second cell layer around the HC. After 24 hours sinusoid-like 3D hepatocyte cords were established. Due to continuous per­ fusion, the artificial hepatocyte cords exhibit a stable phenotype over several days. This is accompanied by a remarkable increase in metabolic performance for phase I enzyme activities, e. g. CYP1A2 and 3A4, and phase II (SULT, UGT) enzyme activities in HepaChip ® compared to ordinary 2D cultures. The resulting phenotypic stability may raise the predictability of biological and metabolic responses to xenobiotics to an unprecedented level and thus may aid in improving drug development and reduces the risk for patients.

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8.2

Detection of transplanted mesenchymal stem cells (MSC) by PCR Claire Fabian, Melanie Hartman, Arnd Hinze, Alexandra Stolzing

The systemic transplantation of mesenchymal stem cells (MSC) showed benefits in animals with neurodegenerative diseases. To control the success of the transplantation itself and to further analyze how the MSC and the host interact with each other it is necessary to identify and track the transplanted cells. One approach is to use a sex-mismatched set-up. By using a male-female combination it is possible to use animals with the same genetic background without adding any additional marker. The Y-chromosome can be traced as well as quantified by the use of PCR in a specific and highly sensitive manner. The primers for the PCR detect the genes for Y-chromosome linked zinc finger protein 1 and 2 (Zfy 1, 2). Both genes are single copy genes so that one diploid MSC would harbor four copies. The use of genomic DNA (gDNA) as PCR template required special adaptations of the PCR set-up in respect to the annealing temperature of the primers so that even small amounts of male DNA could be traced in a female DNA background. The PCR was sensitive down to 50 pg gDNA . This method was applied in a MSC transplantation trial to exploit the therapeutic effect of these cells. MSC s from male donors were transplanted into female mice by injection into the tail vein. After sacrification of the animals, different tissues like bone marrow (BM) and brain were isolated and gDNA was purified by phenol / chloroform extraction. With this PCR it was possible to identify male DNA of donor cells in the female host background in gDNA in BM as well as brain samples 7 days after transplantation. The same approach can be exploited for the use of human cells.

Dr. Jan Böttger

Dr. Claire Fabian

Universität Leipzig Faculty of Medicine Institute of Biochemistry

Fraunhofer-Institut für Zelltherapie und Immunologie (IZI) Cell Therapy Stem Cell Biology

[email protected] www.uni-leipzig.de/~biochem

[email protected] www.izi.fraunhofer.de

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TISSUE AND CELL ENGINEERING

8.3 Designing 3D biomimetic microenvironments for in vitro studies of cell-cell interaction Katja Franke, Liv Kalbitzer, Michael Ansorge, Tilo Pompe

Analysis of cellular dynamics including adhesion forces, cell migration, and cell differentiation often require defined in vitro approaches to reduce the complexity of the in vivo environment. We design a microwell setup with three-dimensional (3D) networks of collagen fibrils of defined mechanics and network topology combined with gradients of soluble signalling molecules. Using different compositions of collagen type I, heterotypic collagens (e. g. collagen type V), and glycosaminoglycans we modulate fibril morphology and network topology. By that we are able to form soft fibrillar collagen networks with a Youngs modulus around 100 Pa. The open topology allows cells to migrate throughout the 3D collagen network. The 3D network characteristic is overlaid by defined gradients of soluble signalling molecules at physiological concentrations in order to mimic cues of neighbouring cells or tissue compartments. The gradients are established by protein-laden biohybrid hydrogel microparticles based on poly(ethylene glycol)-heparin and gelatine-heparin systems with known release characteristics. At the current stage, signalling molecules of interest comprise SDF-1 and Dkk-1 as important cues of the hematopoietic stem cell niche. With these newly designed in vitro matrices we will implement a range of biophysical and biochemical regulators for cell-cell signalling to be used in a microscopic single cell tracking. The defined biomimetic microenvironments will be applied to investigate cellular regulation in inflammation and wound healing as well as inside the haematopoietic stem cell niche.

Dr. Katja Franke Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry Biophysical Chemistry [email protected] www.biochemie.uni-leipzig.de/agpompe/home.php

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8.4

Human iPS cells differentiated into microglia as a therapy for Alzheimer´s disease Yahaira Naaldijk, Alexandra Stolzing

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting millions of people worldwide. There is no cure for AD, but therapies to treat the symptoms or delay the disease are available. Pathological hallmarks of AD are accumulation of aggregated tau in neurofibrillary tangles and amyloid beta (Aβ) in plaques. During AD progression microglia shows activation and is concentrated in areas affected by pathology but does not lead to a decrease of Aβ plaques. It is believed that microglia undergo senescence and lose the capability to digest cell debris. Given that, we propose that microglia derived from viral iPS cells can replace non-functional microglia and reverse or delay the degeneration due to AD. To analyze the ability of viPS cells to differentiate into functional microglia, viPS cells were differentiated by embryoid body (EBs) formation followed by selection and expansion of microglia-like cells. Microglia-like cells were sorted by CD45 / CD11b+ expression and expanded. Microglia-like cells were analyzed based on phenotype and function. Phagocytosis, oxidative burst and CD phenotype were tested. Microglia-like cells derived from viPS cells showed phagocytotic activity and oxidative burst. In addition, microglia-like cells were positive for microglia-specific CD markers. Microglia cells derived from viPS may be an effective tool for the treatment of AD progression. Furthermore, deriving microglia from patient-specific viPS cells to reduce the possibilities of an immune rejection is an important step towards a successful transplantation therapy in Alzheimer’s disease.

Yahaira Naaldijk Universität Leipzig Translational Center for Regenerative Medicine (TRM ) Cell Therapies for Repair and Replacement [email protected] www.trm.uni-leipzig.de

127

TISSUE AND CELL ENGINEERING

8.5

Development of a bioreactor for pattern-dependent stimulation of 3D chondrocyte constructs Oliver Petters, Nico Wüstneck, Frank Peinemann, Ronny Schulz

Cartilage cells, the chondrocytes, are known to be mechano-sensitive under in vivo and in vitro conditions. For the in vitro cultivation of these cells, it is important to mimic the microenvironment with a suitable bioreactor system (BRS). The aim was to develop such a system by including a modular mechanical stimulation unit. Furthermore the BRS provides the possibility to apply individual oxygen tensions and to stimulate the cells with shear stress by perfusion. All experiments were performed with primary porcine chondrocytes from articular cartilage of freshly slaughtered pigs. Full-thickness cartilage sections were enzymatically digested in 2 mg / ml collagenase-A in DMEM -medium for 24 hours by rotating overhead in a 37 °C incubator. The obtained cell suspension was mixed with pre­warmed agarose type VII solu­ tion to a final gel concentration of 3 % (v / v) with a final cell concentration 4.7 x 10 5 cell per gel. Each BRS contained 6 cell-seeded gels (Ø 10 x 3 mm) for the parallel cultivation over 14 days by mechanical stimulation in sinusoidal and trapezoidal loading pattern (5 min on / 235 min off) with a frequency of 1 Hz and 15 % strain. The results showed just slight differences between static cultivation and the application of different loading patterns on proliferation and the production of freshly produced extracellular matrix (e. g. sulfated glycosaminoglycans). Nevertheless to our current knowledge, these novel bioreactor provides a sterile, scalable and autonomous system with the most sensitive and modular mechanical stimulation while the simultaneous application of shear stress by perfusion and variable oxygen tensions.

Oliver Petters Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Cell Techniques and Applied Stem Cell Biology [email protected] www.uni-leipzig.de/~bader

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8.6

Modelling circadian rhythm in colonic crypts Jens Przybilla, Markus Löffler, Gabriela Aust, Jörg Galle

The intestinal epithelium is an useful system to study self-renewal tissues. In this tissue the stem cells are confined to a well-defined niche at the bottom of invaginations called crypts. The progeny of these stem cells specify into different functional lineages and regenerate the entire tissue within a few days. We here present a modelling framework that allows to model multiple-crypt systems repre­ senting a first step towards a whole-tissue model. We implemented a Cellular Potts Model on a curved surface representing multiple crypts and applied the regulatory mechanisms and organisation concepts of our off-lattice model. This enables us to cover the self-organisation of cell production and loss in the tissue, which is assumed as fixed in the former model. We provide simulation results applying this model to circadian rhythms of intestinal turnover and compare the results to experimental data.

Dr. Jens Przybilla Universität Leipzig Interdisciplinary Centre for Bioinformatics (IZBI ) [email protected] www.izbi.uni-leipzig.de

129

TISSUE AND CELL ENGINEERING

8.7

Aging signature of human induced pluripotent stem cells Leili Rohani, Antje Arnold, Alexandra Stolzing

The reprogramming of differentiated cells to pluripotency promises a hope that induced pluripotent stem cells can be used to generate clinically useful cell types for autologous therapies aimed at repairing deficits arising from injury, illness, and aging. All established iPS cell lines can differentiate into different cell types of three germ layers, but it seems that the epigenetic memory of donor cells has an influence on the differentiation potential of iPS cells. Although different studies suggest a similar differentiation potential between hiPSCs and human embryonic stem cells (hESC), it is unclear whether they can be ex­ panded into homogeneous cell populations without early senescence, suitable for use in drug discovery and clinical translation. Furthermore, there is controversial data about the presence of premature senescence of cells derived from iPS cells. Premature senescence would represent a major drawback for the clinical application of cells derived from iPS. Therefore, it is important to check iPS cells and cells derived from these cells for signs of aging. In this study we first focus on generation of transgene-free mRNA-iPS cells which are big steps towards clinical applications. The next issue is the analysis of senescence and aging signature of iPS cells and iPS -derived cells with different technical method including telomere length analysis, telomerase activity analysis, and DNA repair capacity followed by mitochondria analysis.

8.8

UTX knock out phenotype in mice and Neural

Stem Cells

Kamola Saydaminova, Katrin Neumann, Francis Stewart, Konstantinos Anastassiadis

Methylation of histones has been regarded as stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Histone 3 Lysine 27 trimethylation (H3K27 / me3) is associated with repressed chromatin, this epigenetic state is known to be involved in regulation of pluripotency in embryonic stem cells. Histone 3 Lysine 27 demethylase (Ubiquitously transcribed tetratricopeptide repeat X) UTX located on the X chromosome and its male counterpart UTY, are transcribed during early embryonic development, activating target genes, by removal of methyl mark. Utx null mice have cardiac malfunction phenotype, as well as failure in neural tube closure at E 8.5 1. Whole mount in situ hybridization of mutant embryos, will show expression pattern of UTX target genes such as Wnt 1 and Otx 2. In order to elucidate functions of UTX and UTY demethylases in Neural stem cells (NSC), we used cells derived from transgenic UTX FDC / +, FDC / FDC, FDC / Y mice, generated using Cre / Lox system. Global H3K27 trimethylation will be assessed by Western blot analysis. NSC will be tested for their capacity to self renew and differentiate into Astrocytes and Neurons by immunostaining and FACS analysis for CD44. 1 Lee, S., Lee, J. W. & Lee, S.-K. UTX , a histone H3-lysine 27 demethylase, acts as a critical switch to activate the cardiac developmental program. Developmental cell 22, 25 – 37 (2012).

Leili Rohani Fraunhofer-Institut für Zelltherapie und Immunologie (IZI ) Cell Therapy Stem Cell Biology [email protected] www.izi.fraunhofer.de

130

Kamola Saydaminova Technische Universität Dresden Biotechnology Center (BIOTEC) [email protected] www.biotec.tu-dresden.de

131

TISSUE AND CELL ENGINEERING

8.9

Is it possible to transform bone marrow stem-cells into venous endothelial cells? Franziska Schlegel, Stefan Dhein, Ömir Akhavuz, Friedrich-Wilhelm Mohr, Pascal Maria Dohmen

Objective: This study was performed to investigate the influence of endothelial growth factors to transfer bone marrow stem-cells (BMSC) into venous endothelial cells (EC). Methods: BMSCs were harvested from six minipigs by pelvic bone marrow aspiration. Bone marrow-derived mononuclear cells were isolated by sucrose gradient centrifugation. Endothelial growth factor (16 µg / ml) was added to the DMEM to transform BMSCs into EC s. BMSC transformation and characterisation was examined by immunofluorescence staining for CD31 and von Willebrandt factor (vWF) staining as well as by the expression of endothelial nitric oxide synthase (eNOS). Further NO release was examined by spectrophotometric investigation. To investigate whether endothelial like BMSCs could form angiogenetic tubes in a 3D culture, angiogenesis assay with matrigel was performed. Results: BMSCs exhibited a typical cobblestone-like EC phenotype. Immunofluorescence staining showed that differentiated BMSCs were comparable with mature venous endothelial cells. Comparing eNOS expression of BMSCs and venous ECs (vECs) showed no significant differences between the two cell lines (eNOS positiv BMSCs: 68.4 ± 26.2 %, eNOS positiv vECs: 73.8 ± 33.3 %, p = 0.3). Transformed BMSCs were also positive for the EC marker vWF. Spectrophotometric examination showed comparable NO release for transformed BMSC s and vECs. Angiogenesis assay with matrigel indicated the tube formation of trans­ formed BMSCs in vitro. Conclusion: This study showed that BMSCs could be differentiated into typical phenotype vECs. Further studies are needed to evaluate BMSC transformation in arterial phenotype EC s.

132

8.10 Transcatheter implantation of an injectable tissue engineered heart valve in a mini pig in vivo model Franziska Schlegel, Aida Salameh, Katja Oelmann, Phillip Kiefer, Stefan Dhein, Friedrich-Wilhelm Mohr, Pascal Maria Dohmen

Objective: Today glutaraldehyd fixed pulmonary valves are implanted in clinical trails, however limited by absence of regeneration, remodelling and growth potential. This study was performed to evaluate deliver-related tissue distortion of tissue engineered (TE) pulmo­ nary heart valves during implantation. Methods: The injectable TE heart valves were mounted on a self-expanding nitinol stent (n = 7) and delivered into the pulmonary position of seven pigs (26 – 31 kg) by performing a stenotomy or limited lateral thoracotomy. Prior to implantation, the injectable TE heart valve was crimped by using an applicator. The positioning of the implant was guided by fluoroscopy. Hemodynamic measurements were performed by epicardial echocardiography, angiography and invasive pressure measurements. Finally, the injectable TE heart valves were explanted and examined histologically. Results: Orthotropic delivery of the implanted TE heart valves (Ø 19 mm) was successfully performed in all experiments, except for one due to valve migration because of size dis­crep­ ancy. Angiographically, all other valves (n = 6) showed normal valve function, supported by epicardial echochardiography in which no increase in flow velocity was measured, neither valve regurgitation. Invasive pressure measurements showed a mean pressure gradient of 5 mmHg. Histological evaluation demonstrated no integrity changes of extracellular matrix and absence of collagen and elastin distortion. Conclusion: The implantation of an injectable TE heart valve seems to be possible without tissue formation and distortion due to the delivery system.

Franziska Schlegel

Franziska Schlegel

Universität Leipzig Heart Center Leipzig GmbH Research and Teaching / Surgery

Universität Leipzig Heart Center Leipzig GmbH Research and Teaching / Surgery

[email protected] www.rhoen-klinikum-ag.com/rka/cms/hzl_2/deu

[email protected] www.rhoen-klinikum-ag.com/rka/cms/hzl_2/deu

133

TISSUE AND CELL ENGINEERING

8.11 hiPS cell-derived neuronal networks: a prospective basis for the investigation of neurodegenerative diseases Diana Seidel, Heinz-Georg Jahnke, Delphine Laustriat, Marc Pechanski, Simone Haupt, Oliver Brüstle, Andrea A. Robitzki In recent years, the high potential of human pluripotent stem cells for the research and treatment of a diversity of diseases was discovered. Especially human induced pluripotent stem (hiPS) cells show great promise, since they combine the ability for generation of in vivo-like cells without the ethical constraints of human embryonic stem cells. This enables the establishment of meaningful cell models that overcome the restrictions of animal models but concurrently allow the investigation of key molecular alterations together with high-content / high-throughput screening of active drugs in various pathologies such as neurodegeneration. The generation of an optimal in vitro environment was shown to be crucial for the onset of neuropathologic alterations. This can properly be achieved by hiPS cells differentiated into a matured active neuronal network. In this context, we first optimised the culture conditions of different hiPS cell lines (IMR90-4, IMR90c01, 4603c27, iLB-C1-30m-r12 , iLB-C-31f-r1) switching from a feeder-dependent to a feeder- and xeno-independent system that allows a simple, fast and contamination-free handling and is sufficient for stem cell maintenance over 30 passages. Based on this, neuronal differentiation was implemented comparing two protocols. The success of both, the feeder-free cultivation and the neuronal differentiation of hiPS cells, was proven by immunocytochemical staining of stem and neuronal cell markers. Consequently, these findings provide the baseline for an efficient induction of neuro­ degenerative pathologies in hiPS cells e. g. via the expression of tau mutants or amyloid β oligomers.

Diana Seidel Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology [email protected] www.uni-leipzig.de/~dmpt

134

8.12 Effects of insulin and dexamethasone on growth and metabolism of fetal bovine liver cells cultivated in Williams’ Medium E Katja Stöckel, Martin Köhne, Herbert Fuhrmann

In vitro cultivation of fetal bovine hepatocytes is of interest for studies on liver metabolism and some important metabolic diseases of ruminants. In order to allow these studies, cells must be cultured while maintaining their liver-specific functions and characteristics. This study investigated the effect of supplementation of Williams’ Medium E (WME), with and without a combination of 0,2 U / ml insulin (ins) and 100 nM dexamethasone (dex), at two different concentrations of fetal calf serum (FCS) on growth and metabolism of fetal bovine liver cells. The following supplementation scheme was used: 5 % FCS (A); 10 % FCS (B); 5 % FCS + ins / dex (C); and 10 % FCS + ins / dex (D). Overall cultivation time was four weeks. Cells were counted at week 2, 3 and 4 of the experiment. Urea, lactate and beta-hydro­ xybutyrate were analysed from medium samples. It was possible to cultivate cells in 10 % FCS + ins / dex for two weeks. Supplementation of WME with 10 % FCS promotes cell growth, giving the overall impression of a high lactate and urea production. However, this is achieved by elevated cell numbers rather than a high production rate of these substances per cell. Although WME with 5 %  FCS + ins / dex hampers cell growth, it enhanced the production of urea and lactate per cell. Beta-hydroxybutyrate was below the detection limit in all samples. For short-term experiments on liver metabolism, when low cell counts do not impede the experiment, cultivation in WME supplemented with 5 % FCS, insulin and dexamethasone is preferable.

Katja Stöckel Universität Leipzig Faculty of Veterinary Medicine Institute of Physiological Chemistry [email protected] www.uni-leipzig.de/~vpci

135

TISSUE AND CELL ENGINEERING

8.13 Neuroprotection against iron-induced cell death by perineuronal nets Anne Suttkus, Susanne Rohn, Carsten Jäger, Thomas Arendt, Markus Morawski

Perineuronal nets (PNs) are a specialised form of extracellular matrix, surrounding different types of neurons and mainly consisting of chondroitin sulphate proteoglycans connected to hyaluronan, stabilised by link protein and cross-linked via tenascin-R. Due to their poly­ anionic character, caused by the highly charged chondroitin sulphate glycosaminoglycan and hyaluronan components, PNs might be involved in local ion homeostasis. They are able to scavenge and bind redox-active ions and thus reduce the local oxidative potential. We investigated whether net-enwrapped neurons are less vulnerable against iron-induced oxi­ dative processes. Oxidative stress is a key factor in the development and progression of neuro­ degenerative diseases like Alzheimer’s and Parkinson’s disease. Iron is believed to contribute to oxidative stress in Alzheimer brains by catalysing the generation of free radicals. For examining potential neuroprotective effects of PNs, mice were microinjected with 0.2 µl of a 20 mM solution of FeCl3 into the barrel field while the control group received an equal volume of 0.9 % NaCl. Brains were analysed after time intervals of 24 h and 72 h. Neuronal degeneration was visualised using Fluoro-Jade B staining. The presence of PNs was assessed by Wisteria floribunda agglutinin histochemistry or aggrecan immunocytochemistry. The analysis showed a significant lower degeneration rate of net-ensheathed neurons in comparison to neurons without PNs. The results suggest a neuroprotective mechanism associated with the presence of PNs against iron-induced cell death.

Anne Suttkus

136

8.14 Introduction of sulfated glycosaminoglycans in growth substrates impairs the TGFβ1 driven differentiation of human dermal fibroblasts to myofibroblasts Anja van der Smissen, Vera Hintze, Dieter Scharnweber, Stephanie Möller, Matthias Schnabelrauch, Ulf Anderegg During cutaneous wound healing dermal fibroblasts (dFb) have to differentiate into myofibroblasts (MFb) to gain a contractile, extracellular matrix (ECM) synthesizing pheno­ type. This differentiation is stimulated by transforming growth factor beta 1 (TGFβ1) and results in the expression of the MFb differentiation markers alpha smooth muscle actin (α SMA), ED-A FN - a splice variant of fibronectin, and collagen I (coll). A promising approach for biomaterial design is the application of combined native ECM components. Here, we investigated artificial ECM (aECM) consisting of coll and the glycosaminoglycans (GAG s) hyaluronan or chondroitin sulfate. Additionally, GAG s were chemically modified by the introduction of sulfate groups to obtain high-sulfated GAG derivatives. Sulfate groups in the aECM are expected to bind and concentrate growth factors thus improving their bioactivity. Here, we analyze the effect of aECM on the differentiation of human dFb to MFb in the presence of soluble or aECM -adsorbed recombinant, active, human TGFβ1 within 72 h. Myofibroblast differentiation was evaluated by the expression of α SMA , ED-A FN and coll on mRNA and protein level. The amount of adsorbed TGFβ1 was evaluated by measuring non-adsorbed TGFβ1 with an ELISA . Although adsorbing the highest amount of TGFβ1 aECM s containing high-sulfated GAG s hindered the differentiation of MFb. Therefore, addition of sulfated GAG to aECM could modulate the outcome of the wound and hypertrophic scar formation.

Anja van der Smissen

Universität Leipzig Faculty of Medicine Paul Flechsig Institute for Brain Research

Universität Leipzig Faculty of Medicine Department for Dermatology, Venereology and Allergology

[email protected] www.uni-leipzig.de/~pfi

[email protected] www.hautklinik.uniklinikum-leipzig.de

137

TISSUE AND CELL ENGINEERING

8.15 Genetic modification of human pluripotent stem cells to correct X-linked chronic granulomatous disease (X-CGD)-associated mutations Sergii Velychko, Maria Rostovskaya, Sebastian Brenner, Konstantinos Anastassiadis Development of efficient methods for gene correction of human pluripotent stem cells will make a major step forward for their application in personalized cell-replacement therapy. X-linked chronic granulomatous disease (X-CGD) is a severe inherited disorder, caused by mutations in gp91-phox gene (cytochrome b beta chain, CYBB), which result in a reduced activity of NADPH oxidase in phagocytic cells of the immune system and thus immunodeficiency. The monogenicity of the disease and the fact that only cells of hemato­ poietic origin are affected, makes it a very attractive target for ex vivo gene therapy to generate autologous transplantable corrected cells. In this study, we developed strategies for correction of CYBB deficiency in X-CGD patient specific induced pluripotent stem (iPS) cells by gene targeting and transgenic methods. For gene targeting two different approaches were considered: 1) using conventional targeting construct with 2.6 kb and 1.7 kb homology arms, or 2) BAC targeting method using con­ structs with up to 140 kb homology arm from one side and 7.5 kb from another. To facilitate targeting, TALENs (Transcription Activator-Like Effector Nucleases) will be employed to introduce sequence-specific double strand breaks. Alternatively, for compensation of CYBB deficiency, BAC transgenesis will be applied using the BAC transposition method, which was recently developed in our group. All constructs were created using BAC recombineering technology. TALENs were assembled using Golden Gate method. The described above strategies are currently under validation in human embryonic stem cells as a model.

Sergii Velychko

138

8.16 Development of an intra-operative, stem cell-based therapy for the treatment of focal cartilage defects in sheep model Matthias Zscharnack, Kathrin Godthardt, Oliver Petters, Bastian Marquass, Ronny Schulz An intra-operative, stem cell-based therapy for repair of cartilage defects would have immense benefits. By avoiding an in vitro cultivation and enabling an immediate enrichment of suitable stem cells, the patient would be spared and the manufacturing cost compared to an autologous chondrocyte transplantation would be significantly reduced. In addition, the risk of cellular changes during in vitro culture could be avoided. The idea of this project is, to isolate and enrich mesenchymal CD271+ / CD45- stem cells (CD271-MSCs) from bone marrow aspirate within a short time period, then to transfer the cells immediately to a clinically used collagen matrix and to fill the cartilage defect with the cell-matrix mixture. First experiments included, whether the sorted cells are able to proliferate in the 3D colla­gen gel without previous monolayer expansion phase and to perform chondrogenic differentiation. Therefore, CD271-MSCs from sheep were sorted and enriched with MACS and FACS , seeded in collagen-I gel and stimulated with TGF-beta 3. The sorting of CD271‑MSCs revealed a concentration of 79 ± 21 cells per ml in bone marrow aspirate. Furthermore, these cells showed an increased proliferation rate compared to MSCs from plastic adherence. So, the enhanced proliferation rate could compensate the low initial cell number. In addition, CD271-MSCs proliferated and differentiated in the collagen- I matrix without previous 2D -expansion. So, it could be demonstrated in vitro that it is possible to use isolated CD271-MSC s to generate a cartilage replacement tissue with sufficient cell density without prior monolayer expansion.

Dr. Matthias Zscharnack

Technische Universität Dresden Biotechnology Center (BIOTEC)

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Cell Techniques and Applied Stem Cell Biology

[email protected] www.biotec.tu-dresden.de

[email protected] www.uni-leipzig.de/~bader

139

POSTERS

9.

Genome and Protein Engineering

GENOME AND PROTEIN ENGINEERING

9.1

Functional linkage of adenine nucleotide binding sites in the mammalian muscle 6-Phospho­fruc­to­ kinase Antje Brüser, Jürgen Kirchberger, Marco Kloos, Norbert Sträter, Torsten Schöneberg

6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes catalysing one of the rate-limiting steps of the glycolysis, the phosphorylation of fructose 6-phosphate at position 1. The Pfk activity is modulated by a number of allosteric regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several new adenine nucleotide binding sites. Here, we experimentally addressed the functional relevance of two adenine nucleotide binding sites by site-directed mutagenesis and enzyme kinetic studies. Subsequent characterisation of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation by nucleotides interacting with the other binding site. Such reciprocal linkage of functionally opposite allosteric binding sites is in very good agreement with the Monod-Wyman-Changeux model and was found in crystal structures of prokaryotic Pfk. The crystal structure of eukaryotic Pfk clearly showed that the allosteric nucleotide binding sites did not evolve from the prokaryotic ancestors. Therefore, reciprocal linkage of opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk giving an impressive example of convergent evolution at the molecular level.

Antje Brüser

142

9.2

Proline-rich insect antimicrobial peptides display potent antibacterial activity in vivo without being immunomodulatory for innate immune cells in vitro Stefanie Fritsche, Daniel Knappe, Heiner von Buttlar, Nicole Berthold, Uwe Müller, Ralf Hoffmann, Gottfried Alber

Antimicrobial peptides (AMPs) are conserved innate immune effector molecules which contribute to host defense against various pathogens by direct and indirect antimicrobial activity. Several AMPs were described to be immunomodulatory, which may contribute to effective elimination of the pathogen and suppression of immunopathological effects. We have synthesized optimized proline-rich insect AMPs (PrAMPs) such as the oncocin and apidaecin 1b derivatives Onc72 and Api88. Both, Onc72 and Api88 were able to protect mice in a lethal E. coli infection model. This strong effect may result from direct antimicrobial activity and / or activation of innate immunity. Therefore, we investigated the immunomodulatory capacity of the P rAMPs on key cells of innate immunity such as dendritic cells and macrophages. To this end, we stimulated murine bone marrow-derived dendritic cells (BMDC) and macrophages (BMDM) as well as total splenocytes and peritoneal exudate cells with P rAMPs and compared them to cathelicidin-related antimicrobial peptide (CRAMP). None of the peptides was able to activate dendritic cells, macrophages, spleno­ cytes or peritoneal exudate cells. While the tested P rAMPs did not modulate the LPS -induced immune response, CRAMP showed reduction of the LPS -mediated immune response as published. Moreover, we found that none of the peptides is chemotactic for BMDC. Taken together, strong antimicrobial activity and absent immunomodulatory effects on key innate immune cells will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections.

Stefanie Fritsche

Universität Leipzig Faculty of Medicine Institute of Biochemistry

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Immunology Molecular Pathogenesis

[email protected] www.uni-leipzig.de/~biochem

[email protected] www.uni-leipzig.de/~blessing

143

GENOME AND PROTEIN ENGINEERING

9.3

Development of a novel system for enantioselective catalysis using artificial metalloenzymes Maika Genz, David Singer, Joscha Holldorf, Evamarie Hey-Hawkins, Ralf Hoffmann, Norbert Sträter

Enzyme catalysis is the most efficient strategy for the preparation of enantiopure products by now. Unfortunately, many reactions important for industrial applications are missing from nature’s toolbox.1 Most of those reactions (e. g. hydrogenation, hydroformylation) are catalysed by 4d / 5d transition-metal ions. Incorporation of transition metals into a protein host via a ligand leads to a novel class of hybrid catalysts, so called artificial metalloenzymes.2 The resulting hybrid protein comprises the best parts of both worlds. However, creation of synthetic activity from scratch within an existing protein scaffold still remains a challenging task.3 1 Jing et al., Chem. Eur. J. 2009, 15, 1370-1376 2 Wilson & Whitesides, J. Amer. Chem. Soc. 1978, 4, 306-307 3 Creus & Ward, Org. Biomol. Chem. 2007, 5, 1835-1844

9.4

The zebrafish CreZoo: Generation of novel CreERT2 - driver lines expressing in various tissues Stefan Hans, Peggy Jungke, Michael Brand

Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of the mammalian genome. Recently, we showed that Cre is also highly efficient in the developing and adult zebrafish and temporal control of recombination can be achieved by using the ligand-inducible C reERT2 (Hans et al. 2009, Hans et al. 2011, Knopf et al. 2011, Kroehne et al. 2011). In order to utilize this potential, we aimed to expand the pool of conditional Cre driver lines and performed a gene trap screen to isolate lines expressing Cre in different domains of the developing and adult zebrafish. To this aim we used a vector containing a splice acceptor and a mCherry-tagged variant of C reERT2 (consisting of a single open reading frame coding for mCherry and C reERT2 separated by the viral T2A peptide sequence) followed by a polyadenylation signal (Hans et al., 2011). A variety of mCherry expression patterns were observed indicating that the mCherry-T2aC reERT2 cassette was inserted into various loci in the genome and is expressed under the control of various endogenous promoters. In order to provide the zebrafish community with this helpful set of C reERT2 insertion lines we developed an easy-to-handle database (crezoo.crt-dresden.de) which enables the researcher to search the list of all transgenic lines or single entries by insertions or expression patterns. In most cases the C reERT2 expression profile using in situ hybridization at 24 hpf and 48 hpf is shown, but also additional information (e. g. mCherry or C reERT2 expression at adult stages, transactivation of a Cre-dependent reporter line) is presented.

Maika Genz

144

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers

Dr. Stefan Hans

[email protected] www.uni-leipzig.de/~straeter

[email protected] www.crt-dresden.de

Technische Universität Dresden Center for Regenerative Therapies Dresden (CRTD)

145

GENOME AND PROTEIN ENGINEERING

9.5

In silico selection of a theophylline riboswitch Manja Malchau, Sven Findeiß, Nadine Weissheimer, Peter F. Stadler, Mario Mörl

Riboswitches are RNA-modules able to regulate genes without the need for a protein and are often found in the 5’-untranslated region of genes. Being remnants of the RNA world, riboswitches are still working in today´s organisms and also offer various opportunities for current research. They consist of an aptamer domain that is capable of binding even small ligands with high affinity and specificity and an expression platform, for example a transcriptional terminator. Once a ligand binds to the aptamer, this expression platform is structurally rearranged, usually resulting in gene activation. As aptamers can be selected for binding nearly every molecule of interest, there is great concern to establish new expression systems based on riboswitch function. However, most aptamers binding ligands in vitro are not capable of functioning as gene regulating riboswitches in vivo due to a lack of applicable selection methods. This study therefore aims on the selection of RNA riboswitches in an in silico approach. In a secondary structure prediction we focus on transcriptional terminators that change their structure when a ligand is bound to an adjacent theophylline aptamer. Here, we present a theophylline riboswitch derived from an in silico selection which is able to regulate transcription in vivo.

Manja Malchau Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry Biochemistry / Molecular Biology [email protected] www.biochemie.uni-leipzig.de/agmoerl

146

9.6

Development of a prokaryotic expression system for in vivo protein biotinylation of dengue virus proteins for serological diagnostics Awadalkareem Mohammed Eljamal, Alexandros Hadjilaou, Sven Reiche, Christian Jassoy

Background: Dengue is a mosquito-borne infection caused by four distinct serotypes of dengue viruses (flavivirus). The humoral immune response induced during a dengue virus infection exhibits significant cross reactivity with other members of the flaviviruses in currently available serological tests. A specific biotinylation of dengue virus proteins may increase sensitivity and specificity of serological dengue virus diagnostics. Methods: In the present work, the prokaryotic pMAL c2X expression vector was modified by cloning a specific binding sequence for the biotin ligase BirA C-terminally of the open reading frame of the maltose binding protein gene. Full length dengue virus protein sequences of isolates from different strains were cloned in the open reading frame of the modified expression vector. Proteins were expressed in E. coli BL21DE3 cells together with biotin ligase in the presence of biotin. After immunoaffinity purification, the proteins were analyzed for purity, biotinylation and reactivity with human sera by SDS-PAGE and Western blot. Result: Our results demonstrate that the non-structural protein 1 (NS1) of all four serotypes and the structural envelope (Env) protein for subtype 1, 3 and 4 are stably expressed in E. coli. All proteins were detectable with an anti-biotin antibody indicating successful in vivo bio­ tinylation. Conclusion: The bacterially expressed recombinant dengue virus proteins carry a biotin moiety which can be used for purification and detection of the proteins. Use of these proteins in diagnostic tests may increase specificity and sensitivity for the detection of dengue virus infections.

Awadalkareem Mohammed Eljamal Universität Leipzig Faculty of Veterinary Medicine Institute of Virology [email protected] www.uni-leipzig.de/~virology

147

GENOME AND PROTEIN ENGINEERING

9.7

Role of the H3K4 methyltransferase MLL4 in mouse embryogenesis Christian Much

Subtoxic concentrations of Benzene and Toluene affect cellular metabolism and induce oxidative stress by Nrf2 pathway in lung epithelial cells (A549) Kalaimathi Murugesan, Stefanie Kliemt, Sven Baumann, Iljana Mögel, Irina Lehmann, Martin von Bergen-Tomm, Janina M. Tomm

Methylation of histone 3 on lysine 4 (H3K4) is a post-translational epigenetic modification that is associated with actively transcribed genes and catalyzed by a family of histone methyltransferases called Trithorax-group proteins. Mammals carry at least six, functionally non-redundant yeast Set1-related proteins. Arisen from gene duplication and characterized by a common SET domain, these proteins are Setd1a, Setd1b, MLL1, MLL2 , MLL3, and MLL4. A FRT flanked lacZ trapping cassette was inserted into the first intron of Mll4. Removal of the FRT flanked stop cassette in the germline by Flpe-mediated recombination fully restored wild type function (termed F allele). Removal of critical exons by Cre recombination in­ vokes a frameshift in the mRNA resulting in a premature stop codon (termed FC allele). No homozygous mutant Mll4 - /- animals of heterozygous matings were recovered. Consti­ tutive Mll4 - /- embryos are non-viable before 10.5 dpc. Surprisingly, the loss of only one copy of Mll4 leads to an embryonic phenotype in a parent-of-origin dependent manner. Embryos that inherit the null allele from the mother frequently show an incomplete closure of the neural tube at 9.5 and 10.5 dpc, whereas embryos carrying the null allele from the father can survive to term, but die perinatally in most of the cases. However, using the conditional approach we could demonstrate that Mll4 FC / FC embryos are viable, but die perinatally, whereas Mll4 FC / + embryos are phenotypically indistinguishable from their wild type littermates. The reason for this discrepancy between the two knockout strategies is on the way to be unrevealed.

Christian Much

148

9.8

In the developed countries people spend most of their time indoors and are thereby constantly exposed to volatile organic compounds that in extreme cases can cause severe adverse health effects like asthma and ‘‘Sick Building Syndrome’’. The underlying mechanisms on how volatile organic compounds like the predominant indoor pollutants benzene and toluene can affect the cellular equilibrium most often at non-acute toxic concentrations is largely unknown. Based on the indoor air concentration and previous studies on the toxicity of benzene and toluene, we analyzed two relevant non-acute toxic concentrations (0.1 mg / m3, 1 mg / m3) on human lung epithelial cell A549. The differentially expressed proteins on exposure to benzene and toluene were detected by DIGE approach and identified by MALDIand OrbiTrap-MS analysis. The pathway analysis by String databases and Ingenuity analysis revealed an enrichment of regulated proteins involved in oxidative stress, metabolism, and cell death signaling and inflammatory processes. The results were validated by immunoblot. These results demonstrate the detrimental effects of benzene and toluene already at nonacute toxic concentrations and will contribute to the judgement of toxicity of volatile organic compounds.

Kalaimathi Murugesan

Technische Universität Dresden Biotechnology Center (BIOTEC)

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry

[email protected] www.biotec.tu-dresden.de

[email protected] www.ufz.de/index.php?de=19388

149

GENOME AND PROTEIN ENGINEERING

9.9

Characterization of the interaction between interleukin-8 and glycosaminoglycans Karoline Nordsieck, Annelie Pichert, Daniel Huster, Annette G. Beck-Sickinger

Despite modern techniques, implant rejection and wound healing are major problems in regenerative medicine. Improved biomaterials have been reported to be advantageous to conservative materials. However, it is necessary to control and limit the inflammatory potential of implants coated with biomaterial layers like extracellular matrix (ECM). The chemokine interleukin-8 (IL-8) is a mediator in inflammatory processes and hence, we want to use this protein as tool to follow and estimate the inflammatory potential of biomaterials by studying the interactions with glycosaminoglycans (GAG s), which are part of the ECM.1 Previously, it could be shown that the C-terminal helix of IL-8 is not directly involved in the binding of the chemokine to hyaluronic acid.2 It has to be investigated whether this holds true for different biomaterials or ECM components. Therefore, a selectively 15 N-labeled IL-8 analog was generated by Expressed Protein Ligation (EPL). The binding to GAG s was analyzed by nuclear magnetic resonance (NMR) and fluorescence spectroscopy. NMR spectroscopy data revealed that an exchange of one amino acid induces improved affinity for GAG s compared to wild type. This data could be confirmed by fluorescence spectroscopy. 1

Proudfoot, A. E. I. et al. (2003) Proc. Natl. Acad. Sci. U. S. A 100, 1885 – 1890.

2

David, R. et al. (2008) J. Am. Chem. Soc. 130, 15311-15.

9.10 Microbial induced mineralization for the development of a sustainable self-healing mortar Karen Stumm, Andreas Hecker, Markus Gläser, Horst-Michael Ludwig

Bacteria are able to precipitate biominerales. The application of this ability was one factor for the project: Development of new methods and material for the creation of sculptures on the basis of self-healing mineral mortar for the sustainable preservation and maintenance of historical and artistic cultural monuments, funded by the AIF-ZIM. Cement based mortars are an alternative to costly natural stones for the construction and preservation of artistic monuments. One drawback of cement is the durability, estimated with 30a, much shorter than of natural stones with around 80a. The occurrence of cracks is one main cause for the reduced durability. Another drawback is the emission of the CO 2 during the manufacture of cement. The increase of emission of CO 2 affects everyone. In fact the cement production contributes up to 7 % of the global anthropogenic CO 2. It is essential to optimize the use of resources. Long lasting self-healing materials could be a future contribution. Therefore, the aim of the project is to develop a long-lasting mineral mortar with the ability to self-heal cracks in an early stage. The ability of bacteria to biomineralize and the requirement to develop a sustainable selfhealing mortar connected applied microbiology, construction material science and art and craft to a promising interdisciplinary project. The idea is to mix bacterial spores and nutrients to the mortar matrix. In case of a crack the bacteria germinate and start their activity which leads to C aCO 3 precipitation, sealing the crack. First in situ experiments with bacteria isolated from the environment showed encour­ aging results. Worrell E., Price L., Martin N., Hendriks C., Ozawa Meida L. (2001) Carbon dioxide emissions from the global cement industry. Annu. Rev. Energy Environ. 26, 303 – 329 Castanier S., Métayer-Levrel G. L., Perthuisot J.- P. (1999) Ca-carbonates precipitation and limestone genesis – the microbiogeologist point of view. Sedimentary Geology 126, 9 – 23

150

Karoline Nordsieck

Dr. Karen Stumm

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry Biochemistry and Bioorganic Chemistry

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry White Biotechnology

[email protected] www.biochemie.uni-leipzig.de/agbs

[email protected] www.biochemie.uni-leipzig.de

151

GENOME AND PROTEIN ENGINEERING

9.11 Regulated intramembrane proteolysis in the ECF sigma factor mediated envelope stress response in Bacillus subtilis Thomas Wiegert

The Gram-positive bacterium Bacillus subtilis represents an important organism in biotechnology. It is highly amenable to genetic manipulation and widely used as a model to investigate gene regulation and cell differentiation in prokaryotes. In the industry, production strains are grown in large scale fermentors to produce enzymes and vitamins. The ability of bacteria to detect, respond to and resist environmental stresses is a key element in their survival and productivity. The aim of our studies is to analyze signaling pathways responding to envelope stress in B. subtilis which are characterized by the proteolytic cleavage of a regulatory protein in the plane of the membrane. Such mechanisms, summarized by the term ‘regulated intramembrane proteolysis’ (RIP), have been well investigated for eukaryotes. However, in the past decade emerging evidence revealed that RIP also plays a prominent role in a variety of important and highly controlled bacterial transmembrane signaling processes.1 The extracytoplasmic function (ECF) sigma factor σW controls genes constituting an anti­ biosis regulon. It is inactivated by a specific transmembrane anti sigma factor (RsiW) that sequesters σW from interaction with RNA polymerase. We could show that upon an envelope stress signal RsiW undergoes RIP under participation of at least four different proteases that are organized in two modules, each consisting of a site-specific peptidase (site-1: PrsW, site-2: RasP) which prepares RsiW for further degradation by downstream proteases (site-1: unknown, site-2: Clp). As a result, σW is released and induces about 30 operons that confer intrinsic immunity against different antimicrobial agents. RasP belongs to the S2P-family of intramembrane cleaving proteases.2 PrsW is the first member of an orthologous group of bacterial membrane embedded proteins to which a function could be assigned.3 It cleaves near the C-terminal end of RsiW in a site-specific manner.4 1 2 3 4

Heinrich, J., Wiegert, T. (2009) Res. Microbiol. 160: 696 – 703. Schöbel, S., Zellmeier, S., Schumann, W., Wiegert, T. (2004). Mol. Microbiol. 52:1091 – 1105. Heinrich, J., Wiegert, T. (2006). Mol. Microbiol. 62: 566 – 579. Heinrich, J., Hein, K., Wiegert, T. (2009). Mol. Microbiol. 74: 1412 – 1426.

Prof.  Dr. Thomas Wiegert Hochschule Zittau / Görlitz Faculty of Mathematics / Sciences Biotechnology / Microbiology [email protected] http://f-n.hszg.de

152

POSTERS

10.

Biophysics

BIOPHYSICS

10.1 Resolving length changes of single DNA molecules with angstrom accuracy in real time Hergen Brutzer, Alexander Huhle, Daniel Klaue, Ralf Seidel

The understanding of many molecular processes inside the cell requires highly accurate sensors to probe the mechanisms of individual biomolecules. For instance, subnanometer resolution is required to resolve the opening of single base pairs of a DNA molecule by a proceeding helicase. We show here how the single molecule technique magnetic tweezers can be used to detect length changes of a few angstroms of a double-stranded DNA molecule in real time. In our assay the DNA is tethered to a micrometer-sized microsphere, whose position is recorded. This tracking is realized with a camera and wide-field illumination, which is very simple to implement and offers the attractive possibility to determine the positions of many particles in parallel. Real-time tracking rates have so far been limited to several tens of frames per second due to the high computational effort of the employed software routines. Here, we demonstrate three dimensional real-time tracking at 2500 frames per second using a fast CMOS camera for image acquisition and employing GPU based computing. Computationally demanding parts of the tracking algorithm are performed in the GPU that is specialized for highly parallelized execution. High tracking rates are crucial to overcome the shot-noise limitations of camera-based detection and to improve the spatial resolution for events occurring on the second time scale. Thus we can use magnetic tweezers to resolve fast, dynamic processes of biomolecules in real time.

Hergen Brutzer

156

10.2 Cell traction force measurements of malignant and benign breast cell lines Anya Burkart, Thomas Fuhs, Mareike Zink, Josef A. Käs

Cells in the human body must attach to and spread on surfaces, enabling them to live and grow. In order to attach to a substrate or to the extracellular matrix, stress fibers and actin polymerization in the cell generate internal tensile forces and exert traction on the surface via focal adhesions. These traction forces are essential for cell migration, cell shape main­ tenance, and mechanical signal generation. Moreover, traction forces may play a crucial role during cancer progression and metastasis. For this reason, we intend to perform traction force measurements for breast cancer cell lines MCF-7, MDA-MB-436, and MDA-MB-231 in comparison to the benign breast cell line MCF-10A on fibronectin-coated polyacrylamide gels. This may help to clarify how traction forces are involved in the mechanisms underlying cancer development and progression.

Anya Burkart

Technische Universität Dresden Biotechnology Center (BIOTEC)

Universität Leipzig Institute of Experimental Physics I Soft Matter Physics

[email protected] www.biotec.tu-dresden.de

[email protected] www.uni-leipzig.de/~pwm

157

BIOPHYSICS

10.3 On the biomechanics of stem cell niche formation in the gut: Modelling growing organoids Peter Buske, Jens Przybilla, Markus Löffler, Jörg Galle

10.4 Development of a hES derived cardiomyocyte screening platform for the electrophysiological and impedimetric detection of active pharmaceutical ingredients adverse effects Stephan Fleischer, Heinz-Georg Jahnke, Daniella Steel, Peter Sartipy, Andrea A. Robitzki

In vitro culture of intestinal tissue has been tried for decades. Only recently Sato and coworkers succeeded in establishing long term intestinal culture demonstrating that cells expressing the Lgr5 gene can give rise to organoids with crypt-like domains similar to those found in vivo. In these cultures, Paneth cells provide essential signals in supporting stem cell function. We have recently developed an individual cell-based computational model of the intestinal tissue. The model is capable of quantitatively reproducing a comprehensive set of experi­ mental data on intestinal cell organization. Here, based on this model, we present a computa­ tional model of intestinal organoid formation. For this purpose, we introduced a flexible basal membrane which assigns a bending modulus to the organoid surface. This membrane can be re-organized by cells attached to it depending on the cells’ differentiation status. Accordingly, the morphology of the epithelium is self-organized. We hypothesize that local tissue curvature is a key regulatory factor regarding stem cell organization in the intestinal tissue by controlling Paneth cell specification. In simulation studies our model does closely resemble the spatio-temporal organization of intestinal organoids. According to our results, stem cell expansion in an organoid sensitively depends on its biomechanics. We encourage a number of experiments that will enable new insights into mechanotransduction in the intestine and suggest model extensions in the field of gland formation.

Before active pharmaceutical ingredients (API) can be applied to humans, extensive safety testing has to be done to prevent negative side effects. Especially, the undesired influence of novel API candidates on the electrophysiological properties of cardiomyocytes like arrhythmias caused by an QT prolongation are in the focus of pharmaceutical safety assessment and an important exclusion criterion in the API approval. Actual safety testing comprises hERG -channel screenings, in vitro and in vivo animal models. Their major limitations are the low complexity especially for the channel screenings and the poor extrapolation from animal to human cardiomyocytes. In this context human stem cell derived cardiomyocytes offers a great opportunity to overcome these limitations. Therefore, we used an in vivo like innovative three-dimensional cardiomyocytes cluster (CMC) culture model derived from human embyonic stem cells (hES). To detect API effects in a non-invasive, label-free and quantitative manner we developed a microcavity-array screening platform that can be upscaled for standardized HTS / HCS systems. For a comprehensive detection of adverse effects we combined the two biosensoric measurement methods of electrochemical impedance spectroscopy (EIS) and electric field potential recording (FPR) in a novel hybrid measurement platform. Using reference compounds, we successfully detected QT prolongation as well as toxic effects. ES cell derived 3D cardiomyocytes clusters in combination with the MCA based screening technology are a promising tool and versatile tool for in vitro direct detection and quantification of API side effects.

Stephan Fleischer Peter Buske

158

Universität Leipzig Interdisciplinary Centre for Bioinformatics (IZBI )

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology

[email protected] www.izbi.uni-leipzig.de

[email protected] www.uni-leipzig.de/~dmpt

159

BIOPHYSICS

10.5 Lipidlayer and antibody functionalized LbL-microcarriers as novel drug delivery systems Martin Göse, Jacqueline Leßig, Daniel Huster, Uta Reibetanz

Targeted, local, time controlled and low dose applications of active agents are necessary for effective treatment of numerous diseases and to reduce side effects caused by many common systemically applied therapeutics. Due to the modular design, LbL polymer coated microcarriers offer the opportunity to combine those features in one delivery system. The principle of the stepwise adsorption of oppositely charged biopolymers onto a colloidal template allows the integration of a defined amount of active agents into the multilayer and / or core. The release within the cell can be controlled by the biodegradable multilayer. Additional surface modifications allow a high specific functionality such as for fast or targeted uptake by cells. Here, we present recent investigations regarding the development of a functionalized car­ rier aiming at specific cell interaction and uptake. A lipidlayer, assembled on top of the biopolymer system, provides biocompatibility and can be modified to allow further binding of specific antibodies. As templates, CaCO3-microparticles with an average diameter of 5 µm have been used for the coating with protamine sulfate and dextran sodium sulfate to build up a stable biopolymer multilayer. This polymer system induces only low cytotoxicity. Outermost, a PEG and biotin-functionalized phospholipidlayer was applied by liposome spreading to reduce unspecific cell binding and to initialize the further binding of specific antibodies via biotin-streptavidin coupling. Finally, those microcarriers will help us to address desired cells in a highly specific way facilitating the release of the transported agents.

160

10.6 Probing the mechanosensing of neurons with magnetic tweezers Tina Händler, Bernd Kohlstrunk, Josef A. Käs

During its lifetime, a cell has to manage many activities, ranging from the organisation of intracellular structures over migration to multicellular challenges such as tissue formation. The regulation of these functions is not only ensured by biochemical pathways, but also by mechanical cues the cell receives from its environment – a phenomenon known as mechanosensitivity. Cellular responses to mechanical stimuli are, for example, observed as changes in differentiation or spreading behaviour. However, a general mechanism explaining how cells actually translate mechanical parameters of their environment into reactions has not been identified yet. To explore the principles of mechanosensitivity experimentally, controlled mechanical stresses are applied to cells. In the presented work, a magnetic tweezers setup is utilised to induce highly localised mechanical stimuli to the growth cone of neurons. By observation of the immediate cell reaction, characteristics and parameters influencing the mechanosensing of neuronal cells shall be determined.

Martin Göse

Tina Händler

Universität Leipzig Faculty of Medicine Institute of Medical Physics and Biophysics

Universität Leipzig Institute of Experimental Physics I Soft Matter Physics

[email protected] www.uni-leipzig.de/~biophys

[email protected] www.uni-leipzig.de/~pwm

161

BIOPHYSICS

10.7 TWO in ONE: The development of a microcontrollerbased hybrid bioelectronic measurement system

10.8 Active multi-electrode arrays based on zinc oxide Fabian Klüpfel, Bernd Ulrich Sebastian Schmidt, Alexander Lajn, Holger von Wenckstern, Josef A. Käs, Marius Grundmann

Heinz-Georg Jahnke, Ronny Azendorf, Andrea A. Robitzki

Label-free detection and real-time monitoring of cellular alterations by the use of bioelectronic sensors and sensor-arrays is an emerging technology. In this field it is a major goal to integrate complementary detection methods to obtain the most comprehensive data from the same cell or tissue sample. Impedance spectroscopy (EIS) and electrophysiological recording (EPR) perfectly matches the demands for label-free detection and real-time monitoring of cellular alterations and complement each other making them perfect candidates for combination in high content screening systems. Due to the oppositional nature of both techniques with EIS as an active and EPR as a passive measurement method until today, there were no combined systems available. To overcome these limitations, we developed a hybrid measurement system that uses microelectrodes where both measurement circuit paths are connected to the same measurement electrode. To prevent interferences between both measurement paths we developed a µC controlled switching circuit to separate the EPR and EIS circuit paths by low noise switches. This assures synchronous switching of all paths in the low microsecond range. Especially, to avoid an overcharge of the EPR amplifier circuits, the amplifier inputs are switched to the ground just right before the reference electrode is connected to the counter electrode connection of the impedance analyser. With our developed 60 channel hybrid measurement system prototype we could successfully demonstrate the capabilities of our patented technology.

Dr. Heinz-Georg Jahnke Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology [email protected] www.uni-leipzig.de/~dmpt

162

Multi-electrode arrays are integrated devices used to monitor the communication of nerve cell networks. Besides commerically available passive arrays, silicon-based chips were developed to increase the electrode number and the lateral measurement resolution. However, the noise level of such devices is too high to detect signals of primary neurons reliably, and the opacity of silicon prohibits optical transmission microscopy.1 Electronic devices based on oxide semiconductors as ZnO-related compounds were undergoing rapid progress during the last years, partly driven by the needs of the display industry. Besides the optical transparency, properties like high mobility, low material costs and the possibility to fabricate electronic devices on glass or flexible polymer substrates add to the attractiveness of these materials. With ZnO-related materials several different types of field-effect transistors have been dem­ on­strated.2-5 We chose ZnO-based metal-semiconductor field-effect transistors (MESFETs) as a starting point to evaluate the usability of oxide semiconductors for active MEA s, as MESFETs are generally considered to have lower noise than MOSFETs. So far, we fabricated test chips with such devices, compared several different gate materials and evaluated the coupling between electrolyte and chip. 1 2 3 4 5

Lambacher et al., Applied Physics A, 79, 1607-1611 (2004) Nomura et al., Science, 300, 1269-1272 (2003) Fortunato et al., Solid-State Electronics, 52, 443 – 448 (2008) Frenzel et al., Thin Solid Films, 518, 1119-1123 (2009) Schein et al., IEEE Electron Device Letters, published online (2012)

Fabian Klüpfel Universität Leipzig Institute of Experimental Physics II Semiconductor Physics [email protected] www.uni-leipzig.de/~hlp

163

BIOPHYSICS

10.9 Domain motion of 5´-nucleotidase Ulrike Krug, Antje Keim, Nathan S. Alexander, Richard A. Stein, Hassane Mchaourab, Jens Meiler, Norbert Sträter

In vertebrates, ecto-5’-nucleotidase (CD73, 5NT) hydrolyzes extracellular AMP to adenosine as part of extracellular purinergic signaling pathways. The structure of a related nucleotidase from E. coli has been characterized in open and closed conformations, which differ in the relative orientation of the two domains by a rotation of up to 96°. The domain movement can be described as a rotation of the C-terminal domain around an axis which passes through the center of the C-terminal domain. The resulting hinge-bending domain movement is unique in that the cleft between the domains does not open up, but the residues of the domain interface slide along the interface. The conformational change is necessary for the catalytic action of the enzyme, presumably to allow for substrate binding and product release.1,2 To study the relation between protein structure, function and the domain motion of E. coli 5NT we use NMR , FRET and EPR spectroscopy. EPR studies revealed that the open and closed conformations are also present in solution. Addition of inhibitor can shift the equilibrium to the closed conformation, although the enzyme still adopts other conformations. With computational methods we evaluate the EPR data and try to characterize the population of different conformations in the apo state as well as the inhibitor bound state of the enzyme. 1 Knöfel, Sträter, J. Mol. Biol. 2001, 309, 255-266. 2 Schultz-Heienbrok et al., Biochemistry 2005; 44, 2244-2252.

164

10.10 Control of cell adhesion via non-covalently bound adhesion ligands Christina Müller, Andreas Müller, Rayk Hassert, Annette G. Beck-Sickinger, Tilo Pompe

Cells actively sense material properties and respond to its biochemical composition and mechanical characteristics, like substrate stiffness. In most studies, cells are connected to the underlying substrate by means of covalently attached adhesion ligands. However, experimental evidence indicates that ligand affinity and mobility have a pivotal role in controlling cell adhesion as well. We study the impact of ligand affinity and substrate stiffness on the biophysical and biochemical cell response using natural adhesion proteins (i. e. fibronectin) and synthetic peptide ligands by means of time-resolved cell traction force microscopy (CTFM). Stiffness and surface chemistry of polyacrylamide hydrogels were controlled by crosslinker density and coating of thin films of maleic acid copolymers, respectively. Spreading and simultaneous adaption of force levels of human umbilical vein endothelial cells (HUVEC) was monitored during the first two hours after cell seeding. Computational analysis of traction forces, deformation energies and net dipole moments reveals correlations to substrate stiffness and ligand affinity. Furthermore, RGD -containing peptides with different sequences were used to systematically alter ligand affinity. Cell culture experiments showed only weak non-covalent binding of the peptides, in contrast to a support of cell adhesion in case of covalent attachment. Currently, several peptides are under investigation to increase peptide affinity for non-covalent attachment schemes. Ultimately, we aspire to demonstrate the essential role of non-covalently bound ligands in cell adhesion signalling.

Ulrike Krug

Christina Müller

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers

Universität Leipzig Faculty of Biosciences, Pharmacy and Psychology Institute of Biochemistry Biophysical Chemistry

[email protected] www.uni-leipzig.de/~straeter

[email protected] www.biochemie.uni-leipzig.de/agpompe/home.php

165

BIOPHYSICS

10.11 Cellular adhesion – A key mechanism for compartmentalization and tumor spreading? Steve Pawlizak, Anatol Fritsch, Mareike Zink, Josef A. Käs

Compartmentalization is a fundamental process of cellular organization that occurs in particular during embryonic development. A simple model system demonstrating compartmentalization involves mixing together two different populations of suspended cells. After a certain time, this mixture will eventually segregate into two phases, whereas mixtures of the same cell type will not. The differential adhesion hypothesis by Malcom  S. Steinberg explains this organization behavior by differences in surface tension and adhesiveness of the interacting cells.1 To understand to which extent the same physical principles affect tumor growth and spreading between compartments,2 we investigate cellular mechanical properties and interactions of various cell types, such as healthy and cancerous breast cell lines of different malignancy as well as primary cells from human cervix carcinoma. To this end, a set of techniques is applied: The Optical Stretcher is used for whole cell rheology. Cell-cell-adhesion forces are directly measured with a modified atomic force microscope. 3D segregation experiments are employed with a newly developed setup for long-term observation of droplet cultures. The combination of these techniques will help to clarify the role of cellular adhesion for compartmentalization and tumor spreading. 1 R. A. Foty, M. S. Steinberg: The differential adhesion hypothesis: a direct evaluation, Dev. Biol. 278 (1): 255–263 (2005) 2 A. Fritsch, M. Höckel, T. Kießling, K. D. Nnetu, F. Wetzel, M. Zink, J. A. Käs: Are biomechanical changes necessary for tumour progression?, Nature Physics 6 (10): 730–732 (2010)

Steve Pawlizak

166

10.12 The trans-dienelactone hydrolase from Cupriavidus necator: a novel metalloenzyme Christian Roth, Silke Wegener, Michael Schlömann, Norbert Sträter

Fluoroaromatic compounds are very persistent exclusively man made environmental pollutants. Their unique properties render them as potent inhibitors for enzymes or as com­ pounds that interfere with membrane transport processes or cell-cell communication. Due to the extraordinary stability of a fluor-carbon bond these compounds are very recalcitrant against degradation. Nevertheless, a few bacteria are able to metabolize these compounds and use them as the sole energy and carbon source. Cupriavidus necator is able to degrade 4-fluorobenzoate to intermediates of the citric acid cycle. During bacterial growth, enzymes of the metabolic pathway of unsubstituted aromatic compounds are induced as well as a dienelactone hydrolase (t DLH) and a maleylacetate reductase crucial for complete meta­ bolization. A sequence alignment shows no relationship of t DLH to enzymes of the well known dienelactone hydrolase family and t DLH does not contain the catalytic triad that is essential for activity of these enzymes. Furthermore, activity of t DLH depends on the presence of metal ions with a preference for hard metal ions. However, the alignment shows that t DLH can be classified in an uncharacterized protein family of putative metal dependent hydrolases and cyclases. In contrast to previously characterized pathways, the gene of t DLH is not part of a gene cluster inducible in response to available substituted or unsubstituted aromatic compounds. To get insight in the structure and function of this so far uncharacterized enzyme family we expressed and characterized the enzyme for subsequent structural studies.

Christian Roth

Universität Leipzig Institute of Experimental Physics I Soft Matter Physics

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Bioanalytical Chemistry Structural Analysis of Biopolymers

[email protected] www.uni-leipzig.de/~pwm

[email protected] www.uni-leipzig.de/~straeter

167

BIOPHYSICS

10.13 Dynamic force spectroscopy on fluorescence labeled tau-peptides and monoclonal antibodies measured by using Optical Tweezers Tim Stangner, Carolin Wagner, David Singer, Christof Gutsche, Olaf Ueberschär, Ralf Hoffmann, Friedrich Kremer Since humans become older and older with the fast evolution of medicine, degenerative diseases edge ever closer to focus of research. Especially Alzheimer’s disease is the most common form of dementia. Each Alzheimer patient shows two commonly known changes in the brain: senile plaques of β-amyloid-peptide and tangles of hyper-phosphorylated tau proteins. Dynamic force spectroscopy (DFS) is performed by using optical tweezers on the level of single receptor-ligand-interactions. Here, we report about the specific binding of two anti-human tau-monoclonal antibodies (mAbs), HPT-104 and HPT-110, interacting with synthetic (non-) fluorescence-labeled tau-peptides with different phosphorylation pattern. The fluorescent tagged tau-peptides, anchored on Melamin-resin beads, are presorted with the fluorescence activated cell sorting (FACS) method in order to achieve homogenous surface coverage. Specific binding events between peptide and mAbs are described according to the Dudko-Hummer-Szabo-model.1 A comparison between labeled and non-labeled tau-peptide and their interactions with mAbs shall show the influence of the fluorescein-molecule on the parameters, obtained by the Dudko-Hummer-Szabo-model.1 1 Dudko et al.; PNAS October 14, 2008 vol. 105 no. 41 15755-15760

Tim Stangner

168

10.14 Novel electroactive polymer-based nanopore membranes for the controlled size selective biomolecule filtration Marek Staude, Christo Tchernev, Christoph Prönnecke, Sabine Schmidt, Henning Ebert, Heinz-Georg Jahnke, Andrea A. Robitzki The electrical controlled passing of specific biomolecules as an important device in sensoric and actuatoric modules is in focus of today’s microsystem technology and nanotechnology research. Especially in the context of novel theragnostic concepts the controlled release of therapeutics or the filtered diffusion of biomarkers into biosensoric units play a crucial role for sensitivity, feasibility and stability of intelligent implants. To overcome the limitations of passive filtration membranes and polymer-based release systems, we developed a novel electroactive polymer-based nanopore membrane, where the nanopores can be opened and closed by an electrical switched oxidation and reduction, respectively. Based on a highly ordered aluminium oxide nanoporous substrate, we were able to develop a highly controlled electrochemical deposition of a polypyrol (PP y) layer, resulting in membrane with defined nanopores depending on the size of molecules e. g. proteins that should pass the membrane. The incorporation of dodecyl benzene sulfonate (DBS) anions increases the electrical conductivity and enables the feasible electrical controlled oxidation and reduction of the PP y. Thus, opening and closing of the nanopores is achieved by the osmotic swelling of the reduced or shrinking of the oxidized polymer layer. In first proof of principle experiments we were able to demonstrate the reversible opening and closing of nanopores that were optimized for the passing of methylene blue.

Marek Staude

Universität Leipzig Institute of Experimental Physics I Molecular Physics

Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology

[email protected] www.uni-leipzig.de/~mop

[email protected] www.uni-leipzig.de/~dmpt

169

BIOPHYSICS

10.15 Embryotoxicity testing with 3D stem cell structures Silvia Vinz, Randy Kurz, Andrea A. Robitzki

Embryonic development comprises a sequence of very fast and complex changes. Accord­ ingly, the exposure of the developing organism to chemicals and pharmaceuticals involves an elevated risk of interference with these vital processes, resulting in embryo lethality, growth retardation or delayed organ growth. Therefore, fast and reliable embryotoxicity tests are needed. Besides in vivo testing in animals, there are established standard in vitro tests for embryotoxicity, but most of them still require the killing of animals. The more recently developed embryonic stem cell test uses established embryonic stem cell lines instead. Thereby, drug effects on the viability and differentiation of embryonic stem cells are analysed. Both assays are done in adherent two-dimensional culture systems, but usually three-dimensional structures are thought to be more close to the in vivo situation. Embryotoxic effects on three-dimensional stem cell structures can be investigated via impedance measurement as a non-invasive label-free real-time detection system for cellular alterations with the use of a microcavity array developed in our group. For that purpose, ES ‑D3 cells are used to generate embryoid bodies in 96-well plates using a horizontal shaking device. With these three-dimensional stem cell structures, in combination with the impedance measurement on the microcavitiy array, time and concentration-dependent toxic effects of drugs and chemicals on embryonic cells can be detected. This could be demonstrated by using known embryotoxic and non-embryotoxic substances in our test system.

Silvia Vinz Universität Leipzig Center for Biotechnology and Biomedicine (BBZ) Institute of Biochemistry Molecular Biological-Biochemical Processing Technology [email protected] www.uni-leipzig.de/~dmpt

170

10.16 The binding of monoclonal antibodies and tau peptides investigated on a single-molecule level Carolin Wagner, David Singer, Tim Stangner, Ralf Hoffmann, Friedrich Kremer

Optical tweezers-assisted dynamic force spectroscopy (DFS) is employed to investigate specific receptor / ligand bindings on the level of single binding events. Here, the spe­ cific binding of the anti-human tau monoclonal antibody (mAb), HPT-101, to synthetic tau-­ peptides with 2 potential phosphorylation sites (Thr231 and Ser235) is analyzed. According to ELISA-measurements, the antibody binds only specifically to the doublephosphorylated tau-peptide. It is shown by DFS that HPT-101 binds also to each sort of the mono-phosphorylated peptides. By analyzing the measured rupture-force distributions, characteristic parameters like the lifetime of the bond without force τ0, the characteristic length xts and the free energy of activation ΔG are determined for all interactions.1 Thereby, it can be shown how the attachments with the mono-phosphorylated peptides add up in the case of the double-phosphorylated peptide in order to form the strong specific binding. 1 C. Wagner et al., Soft Matter, 2011, 7 (9), 4370–4378

Carolin Wagner Universität Leipzig Institute of Experimental Physics I Molecular Physics [email protected] www.uni-leipzig.de/~mop

171

BIOPHYSICS

10.17 Mathematical modeling of liver tumor growth William Weens, Dirk Drasdo, Stefan Höhme, Jan G. Hengstler

As recently demonstrated for liver regeneration after drug-induced damage, organization and growth processes can be systematically analyzed by a process chain of experiments, image analysis and modeling.1 The authors of [1] were able to quantitatively characterize the architecture of liver lobules, the repetitive functional building blocks of liver, and turn this into a quantitative mathematical model capable to predict a previously unrecognized order mechanism. The model prediction could subsequently be experimentally validated. Here, we extend this model to the multi-lobular scale, guided by experimental findings on carcinogenesis in liver. We explore the possible scenarios leading to the different tumor phenotypes experimentally observed in mouse. Our model considers the hepatocytes, the main cell type in liver, as individual units with a single cell based model and the blood vessel system as a network of extensible objects. Model motion is computed based on explicit discretized Langevin equation and cell interactions are either Hertz or JKR forces. The model is parameterized by measurable values on the cell and tissue scale and its results are directly compared to the experimental findings. We show here an example of a model prediction. Effects that are detectible for small tumor nodules and reflect properties of the tumor cells are not reflected in the tumor shape or phenotype at tumor sizes exceeding half of the lobule size. 1 Stefan Höhme et al., Proceedings of the National Academy of Sciences of the United States of America, 107(23):10371– 10376, 2010.

William Weens INRIA BANG Project Team

[email protected] www.rocq.inria.fr/bang/ww/index.html

172

INDEX

Index A Abdel-Aziz, Heba 91 Adolph, Stephanie 44, 71 Aigner, Achim 45 Akhavuz, Ömir 132 Alber, Gottfried 53, 68, 95, 143 Alexander, Nathan S. 164 Altenburger, Rolf 101 Anastassiadis, Konstantinos 131, 138 Anderegg, Ulf 137 Annibal, Andrea 82 Ansorge, Michael 126 Antos, Christopher 46 Arcangeli, Annarosa 47 Arendt, Thomas 73, 136 Argenton, Francesco 46 Arkona, Christoph 49 Arnhold, Jürgen 67 Arnold, Antje 130 Askar, Ghadir Barbar 62 Aust, Gabriela 129 Azendorf, Ronny 162 B Basiouni, Shereen 48 Bauer, Alexander 115 Baumann, Sven 110, 149 Becchetti, Andrea 47 Becker, Daniel 49 Becker, Holger 124 Becker, Susen 89 Beck-Sickinger, Annette G. 52, 57, 63, 150, 165

Bedawy, Essam 115 Belder, Detlev 92 Beligny, Samuel 75 Belkin, Shimshon 21 Benz, Karin 124 Bergen-Tomm, Martin von 94,110,149

Berger-Hoffmann, Renate 103 Berthold, Nicole 78, 84, 143 Beyer, Andreas 110 Bhuiyan, Abul Fateh Md. Khaled 104 Binder, Hans 111, 121 Bloßfeld, Marcus 50, 67 Blüher, Matthias 87 Bockamp, Ernesto 115 Bollineni, Ravi Chand 82, 83, 86 Böttger, Jan 124 Brand, Michael 46, 145 Brandtner, Eva-Maria 99 Brenner, Sebastian 138 Briel, Detlef 62 Bringmann, Andreas 51, 74 Brulport, Marc 115 Brüser, Antje 142 Brüstle, Oliver 134 Brust, Peter 62 Brutzer, Hergen 156 Buchholz, Frank 36 Burkart, Anya 157 Buske, Peter 158 Buttlar, Heiner von 143 C Ceglarek, Uta 89, 96 Chatzinotas, Antonis 23, 104 Chen, Rui 51 Chiglintseva, Maria N. 93 Chollet, Constance 52 Cicuta, Pietro 28, 40 Crociani, Olivia 47 D D’Alessandro, Lorenza A. 114 D’Amico, Massimo 47 Daminelli, Simone 109 Dautel, Franziska 110 Dhein, Stefan 132, 133 Dobslaff, Kristin 84

Döhler, Christoph 85 Dohmen, Pascal Maria 132, 133 Dooley, Steven 114 Dorow, Juliane 96 Drasdo, Dirk 114, 115, 116, 117, 172 Dwai, Yamen 66 E Ebert, Henning 169 Eichler, Franka 119 Elad, Tal 21 Els, Sylvia 52 Enard, Wolfgang 77 Endes, Carola 104 Eschke, Maria 53, 68 Ewe, Alexander 45 F Fabian, Claire 125 Fasold, Mario 111 Fedorova, Maria 82, 83, 86, 87, 100 Findeiß, Sven 146 Fleischer, Stephan 159 Flemmig, Jörg 67 Frank, René 57 Franke, Katja 126 Freudigmann, Christian 124 Friebel, Adrian 114 Fritsch, Anatol 166 Fritsche, Stefanie 95, 143 Frolov, Andrej 87, 88 Frolova, Nadezhda 54 Frömberg, Anja 45 Fuhrmann, Herbert 44, 48, 70, 71, 135 Fuhs, Thomas 157 G Galle, Jörg 129, 158 Gallego, Beatriz 59 Gasparoli, Luca 47 Gather, Malte C. 24

Gebhardt, Rolf 115, 124 Genz, Maika 144 Ghallab, Ahmed 114 Gillissen, Adrian 72 Gläser, Markus 151 Godoy, Patricio 114 Godthardt, Kathrin 139 Gómez-Ruiz, Santiago 59 Goos, Karl-Heinz 65 Göse, Martin 160 Göttsch, Claudia 94 Grahnert, Andreas 53, 68 Greifenhagen, Uta 88 Greiner-Stöffele, Thomas 35 Gremse, Felix 114 Grosche, Antje 74 Grundmann, Marius 163 Grunert, Steffen 112, 113 Gunthorpe, Martin 55 Günzburg, Walter H. 99 Gutsche, Christof 168 H Hadjilaou, Alexandros 147 Hage, Thorsten 55 Hagmeyer, Britta 124 Hahn-Tomer, Sonja 23 Halwachs, Sandra 76 Hammad, Seddik 114 Händler, Tina 161 Hans, Stefan 145 Harms, Hauke 23, 104 Hartman, Melanie 125 Harwardt, Bernadette 58 Hassert, Rayk 63, 165 Haupt, Simone 134 Haupt, V. Joachim 109 Hecker, Andreas 151 Heinke, Florian 112, 113 Heinrich, Jan-Michael 54 Heller, Sandra 56, 60

Helmschrodt, Christin 89 Hengstler, Jan G. 114, 115, 172 Hensel, Andreas 67 Hermes, Matthias 115 Hettwer, Karina 90 Hey-Hawkins, Evamarie 57, 59, 61,144 Hintze, Vera 137 Hinze, Arnd 125 Hirrlinger, Johannes 31 Höbel, Sabrina 45 Hofbauer, Lorenz C. 94 Hoffmann, Ralf 78, 82, 83, 84, 86, 87, 88, 95, 98, 100, 103, 143, 144, 168, 171 Hoffmann, Steve 111 Hofmann, Sven 57 Höhme, Stefan 114, 115, 172 Hollborn, Margrit 51 Holldorf, Joscha 144 Holst, Birgitte 52 Honscha, Kerstin U. 76 Honscha, Walther 76 Hopp, Lydia 121 Höppner, Christoph 124 Horatscheck, André 75 Hoser, Stefanie 91 Huhle, Alexander 156 Huster, Daniel 150, 160

I Ilkavets, Iryna 114 Isik, Zerrin 120 J Jäger, Carsten 136 Jagiella, Nick 116, 117 Jähne, Martin 90 Jahnke, Heinz-Georg 64, 134, 159, 162, 169

Jassoy, Christian 66, 147 Jezierski, Stefan 92

Jung, Matthias 58 Jungke, Peggy 145 K Kalbitzer, Liv 126 Kalkhof, Stefan 94, 110 Kaluđerović, Goran N. 59 Kamzolova, Svetlana V. 93 Käs, Josef A. 157, 161, 163, 166 Keim, Antje 164 Kelber, Olaf 91 Kendler, Michael 64 Kiefer, Phillip 133 Kiessling, Fabian 114, 116 Kim, Boo Geun 75 Kind, Gabriel 108 Kirchberger, Jürgen 142 Kizil, Caghan 46 Klaue, Daniel 156 Klein, Anke 92 Kliemt, Stefanie 94, 149 Klingmüller, Ursula 114 Kloos, Marco 142 Klüpfel, Fabian 163 Knappe, Daniel 78, 95, 143 Kohen, Leon 51 Köhler, Gabriele 53, 68 Kohlstrunk, Bernd 161 Köhne, Martin 135 Köpke, Katja 118 Kortz, Linda 96 Krauß, Michel 97 Krehan, Mario 56, 60 Kreisig, Thomas 84, 98 Kremer, Friedrich 168, 171 Krügener, Sven 90 Krug, Ulrike 164 Küchler, Beate 46 Kuppardt, Anke 104 Kurz, Randy 170

L Labudde, Dirk 108, 112, 113, 118, 119 Lajn, Alexander 163 Langenberger, David 111 Laue, Hendrik 116 Laustriat, Delphine 134 Lehmann, Irina 110, 149 Lerchner, Johannes 99 Leßig, Jacqueline 160 Liebscher, Bianca 108, 118 Lindner, Stefan 76 Lisurek, Michael 75 Löffler, Markus 129, 158 Loguercio, Salvatore 110 Ludwig, Horst-Michael 151 Lunina, Julia N. 93 M Malchau, Manja 146 Marquass, Bastian 139 Maschke, Jan 64 Masselli, Marika 47 Mattusch, Jürgen 104 Mchaourab, Hassane 164 McKenna, Charles E. 40 Meer, Jan Roelof van der 104 Meiler, Jens 164 Melamed, Sahar 21 Merkenschlager, Andreas 50 Mertens, Florian 99 Milic, Ivana 86, 100 Mögel, Iljana 149 Mohammed Eljamal, Awadalkareem 147

Mohr, Christoph 54 Mohr, Friedrich-Wilhelm 132, 133 Möller, Stephanie 137 Morawski, Markus 136 Morgunov, Igor G. 93 Mörl, Mario 146 Moro, Enrico 46

Much, Christian 148 Mugridge, Kenneth 47 Müller, Andreas 165 Müller, Benedikt 117 Müller, Christa E. 102 Müller, Christina 165 Müller, Katrin 103 Müller, Margareta 117 Müller, Uwe 53, 68, 95, 143 Murugesan, Kalaimathi 149 Mussolino, Claudio 37 N Naaldijk, Yahaira 127 Nagl, Stefan 92 Neitsch, Johannes 114 Neumann, Katrin 131 Neumann, Wilma 61 Nieber, Karen 50, 62, 65, 67, 72, 91 Nordsieck, Karoline 150 O Oates, Andrew C. 46 Oelmann, Katja 133 Oelschlägel, Diana 58 Ortwein, Jutta 75 Otto, Wolfgang 110 Özhan-Kizil, Günes 46 P Pagel, Mareen 63 Pannicke, Thomas 74 Pannier, Angela 101 Papini, Anna Maria 34 Pawlizak, Steve 166 Pechanski, Marc 134 Peinemann, Frank 128 Petersen, Pia S. 52 Petters, Oliver 128, 139 Petzold, Tony 119 Pichert, Annelie 150

Piehler, Daniel 53, 68 Pillozzi, Serena 47 Pippel, Jan 102 Pompe, Tilo 126, 165 Pönick, Sarah 64 Prasse, Agneta 103 Preusser, Tobias 114 Prönnecke, Christoph 169 Przybilla, Jens 129, 158 Puppe, Verena 115 R Rademann, Jörg 49, 75 Rasser, Anne 54 Raszek, Mikolaj 65 Raue, Andreas 114 Recklinghausen, Iris von 114 Reibetanz, Uta 160 Reiche, Sven 66, 147 Reichenbach, Andreas 74 Reif, Raymond 114 Reimann, Matthias 109 Reitmajer, Franziska 67 Rho, Seong-Hwan 114 Richter, Tina 53, 68 Robitzki, Andrea A. 20, 64, 134, 159, 162, 169, 170

Rödel, Gerhard 22 Röder, Ingo 30 Rohani, Leili 130 Röhnert, Peter 124 Rohn, Susanne 136 Rostovskaya, Maria 138 Roth, Christian 167 Roy, Janine 120 Rückner, Antje 69 Rudzok, Susanne 110 S Sabat, Robert 68 Sack, Ulrich 54

Salameh, Aida 133 Sartipy, Peter 159 Saydaminova, Kamola 131 Schaefer, Michael 92 Schäfer, Ingo 56, 60 Scharnweber, Dieter 137 Scheunemann, Matthias 62 Schild, Enrico 52 Schlegel, Franziska 132, 133 Schlömann, Michael 167 Schmidt, Bernd Ulrich Sebastian 163 Schmidt, Rico 88 Schmidt, Sabine 169 Schmitt-Jansen, Mechthild 101 Schnabelrauch, Matthias 137 Schön, Astrid 60 Schönberg, Maria 67 Schöneberg, Torsten 142 Schöniger, Axel 70, 71 Schormann, Wiebke 115 Schroeder, Insa S. 58 Schroeder, Michael 109, 120 Schubert, Andreas 72 Schubert, Susanna 56, 60 Schulz, Ronny 128, 139 Schumann, Julia 44, 48, 70, 71 Schütte, Julia 124 Schütz, Anja 75 Schwartz, Thue W. 52 Schwarz, Michael 115 Schwen, Lars Ole 114 Seibel, Peter 56, 60 Seidel, Diana 134 Seidel, Ralf 156 Shacham-Diamand, Yosi 21 Siegert, Fritzi 62 Siegfried, Konrad 23, 104 Simasi, Jacinta 72 Simon, Jan C. 64 Simon, Kirsten 90 Singer, David 144, 168, 171

Sinningen, Kathrin 94 Smissen, Anja van der 137 Soltmann, Bettina 101 Soltmann, Ulrich 101 Stadler, Peter F. 111, 146 Stangner, Tim 168, 171 Staude, Marek 169 Steel, Daniella 159 Stein, Richard A. 164 Stelzle, Martin 124 Stenzel, Werner 53 Stewart, Francis 131 Stöckel, Katja 135 Stolzing, Alexandra 125, 127, 130 Sträter, Norbert 62, 78, 85, 95, 97, 102, 142, 144, 164, 167

Stumm, Karen 151 Suttkus, Anne 136

T Tchernev, Christo 169 Thiery, Joachim 89, 96 Tiedke, Wolfgang 47 Timmel, Tobias 115 Timmer, Jens 114 Tomm, Janina M. 149 Trump, Saskia 110 Turković, Igor 73 U Ueberham, Uwe 73 Ueberschär, Olaf 168 Uhlig, Steffen 90 V Vahlenkamp, Thomas W. 69 Velychko, Sergii 138 Vignon-Clementel, Irene 116, 117 Vinz, Silvia 170 Vogler, Stefanie 74

W Wagner, Carolin 168, 171 Wagner, Stefan 75 Warszawska, Katarzyna 68 Waßermann, Louise 76 Weens, William 172 Wegener, Silke 167 Weidinger, Gilbert 46 Weirauch, Ulrike 45 Weiser, Dieter 91 Weissheimer, Nadine 146 Wenckstern, Holger von 163 Werner, Simon 124 Weyer, Sven 77 Wiedemann, Peter 51, 74 Wiegert, Thomas 152 Wießler, Manfred 63 Winkelmann, Victoria 91 Winter, Christof 120 Wirth, Henry 121 Wüstneck, Nico 128 Y Yagur-Kroll, Sharon 21 Yan, Jia-Jiun 46 Yates, Karen 102 Z Zahn, Michael 62, 78, 95 Zauner, Thomas 103 Zebisch, Matthias 85, 97, 102 Zellmer, Sebastian 115 Zenker, Jessica 56, 60 Zink, Mareike 157, 166 Žižak, Željko 59 Zolghadr, Kourosh 29 Zscharnack, Matthias 139 Züchner, Thole 84, 98, 103

PROG R AM 9:00 AM OPENING

Jörg Geiger



Head of Department Research and Technology, Saxon State Ministry for Higher Education, Research and the Arts



Prof. Dr. Beate Schücking



Rector, Universität Leipzig



Prof. Dr. Gerhard Rödel



Vice-Rector, Technische Universität Dresden

9:30 AM SESSION 1 – CELLS AS ACTIVE SENSORS

Prof. Dr. Andrea A. Robitzki



Prof. Dr. Ingo Röder



Technische Universität Dresden



Automatic tracking and quantification of dynamic cellular characteristics



Dr. Johannes Hirrlinger



Max-Planck-Institut für Experimentelle Medizin Göttingen



Genetic tools to study the brain: from transgenic mice to metabolic imaging



Dr. Gerald Böhm



BIO-NET Leipzig Technologietransfergesellschaft mbH



Venture Office BIO CITY LEIPZIG



Universität Leipzig

3:30 PM POSTERSESSION & COFFEE BREAK



Real Time Monitoring of cellular inflammatory and degenerative processes on microarrays

4:00 PM SESSION 3 – MOLECULAR MEDICINE AND PROTEIN DESIGN



Prof. Dr. Shimshon Belkin



Prof. Dr. Anna Maria Papini



Hebrew University Jerusalem



Università degli Studi di Firenze



Towards functional microbial sensor cell arrays



Prof. Dr. Gerhard Rödel



Post-translationally modified peptides to develop efficient IVD to follow up disease activity of autoimmune diseases: a challenge for theragnostics



Technische Universität Dresden



Dr. Thomas Greiner-Stöffele



Recombinant yeast cells as a tool for biosensor applications



Prof. Dr. Hauke Harms



Helmholtz-Zentrum für Umweltforschung – UFZ Leipzig



Arsolux: cell-based detection system for arsenic contamination



Jun.-Prof. Dr. Malte Gather



Technische Universität Dresden



Living lasers – towards laser-based biosensors

12:00 AM POSTERSESSION & LUNCH BREAK 1:30 PM SESSION 2 – LIVE CELL ANALYSIS

Dr. Pietro Cicuta



University of Cambridge



Analysing infection of macrophages by Salmonella



Dr. Kourosh Zolghadr



ChromoTek GmbH München

® and the Fluorescent Chromobodies 



2-Hybrid (F2H ) assay: New technologies for real time analysis of cellular components in living cells



c-LEcta GmbH, Leipzig



Technologies for the development of tailored enzymes and strains



Prof. Dr. Frank Buchholz



Technische Universität Dresden



Designer recombinases reverse HIV infection



Dr. Claudio Mussolino



Medizinische Hochschule Hannover



Genome surgery using designer nucleases: ZFN s or TALEN s?

6:00 PM POSTER AWARDS 6:15 PM EVENING LECTURE

Prof. Dr. Charles McKenna



University of Southern California



Phosphonate chemistry in medicine: from bone actives to antiviral agents

7:00 PM GET TOGETHER ALL DAY Industrial exhibition and information desk of the National Contact Point Life Sciences