Over 10.000 scientific papers have been published by members of the Materials Chain since the foundation of the University Alliance Ruhr in 2010. This tremendous output is proof of the excellent environment the Ruhr Area provides for research in the field of materials science and technology.
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2021 • 223 -Hydrogenases: Maturation and reactivity of enzymatic systems and overview of biomimetic models
Kleinhaus, J.T. and Wittkamp, F. and Yadav, S. and Siegmund, D. and Apfel, U.-P.
Chemical Society Reviews 50 1668-1784 (2021)While hydrogen plays an ever-increasing role in modern society, nature has utilized hydrogen since a very long time as an energy carrier and storage molecule. Among the enzymatic systems that metabolise hydrogen, [FeFe]-hydrogenases are one of the most powerful systems to perform this conversion. In this light, we will herein present an overview on developments in [FeFe]-hydrogenase research with a strong focus on synthetic mimics and their application within the native enzymatic environment. This review spans from the biological assembly of the natural enzyme and the highly controversial discussed mechanism for the hydrogen generation to the synthesis of multiple mimic platforms as well as their electrochemical behaviour. © The Royal Society of Chemistry.
view abstract doi: 10.1039/d0cs01089h 2021 • 222 Proteomics: A Tool to Study Platelet Function
Shevchuk, O. and Begonja, A.J. and Gambaryan, S. and Totzeck, M. and Rassaf, T. and Huber, T.B. and Greinacher, A. and Renne, T. and Sickmann, A.
International journal of molecular sciences 22 (2021)Platelets are components of the blood that are highly reactive, and they quickly respond to multiple physiological and pathophysiological processes. In the last decade, it became clear that platelets are the key components of circulation, linking hemostasis, innate, and acquired immunity. Protein composition, localization, and activity are crucial for platelet function and regulation. The current state of mass spectrometry-based proteomics has tremendous potential to identify and quantify thousands of proteins from a minimal amount of material, unravel multiple post-translational modifications, and monitor platelet activity during drug treatments. This review focuses on the role of proteomics in understanding the molecular basics of the classical and newly emerging functions of platelets. including the recently described role of platelets in immunology and the development of COVID-19.The state-of-the-art proteomic technologies and their application in studying platelet biogenesis, signaling, and storage are described, and the potential of newly appeared trapped ion mobility spectrometry (TIMS) is highlighted. Additionally, implementing proteomic methods in platelet transfusion medicine, and as a diagnostic and prognostic tool, is discussed.
view abstract doi: 10.3390/ijms22094776 2021 • 221 Studying the mechanism of phase separation in aqueous solutions of globular proteins via molecular dynamics computer simulations
Brudar, S. and Gujt, J. and Spohr, E. and Hribar-Lee, B.
Physical chemistry chemical physics : PCCP 23 415-424 (2021)Proteins are the most abundant biomacromolecules in living cells, where they perform vital roles in virtually every biological process. To maintain their function, proteins need to remain in a stable (native) state. Inter- and intramolecular interactions in aqueous protein solutions govern the fate of proteins, as they can provoke their unfolding or association into aggregates. The initial steps of protein aggregation are difficult to capture experimentally, therefore we used molecular dynamics simulations in this study. We investigated the initial phase of aggregation of two different lysozymes, hen egg-white (HEWL) and T4 WT* lysozyme and also human lens γ-D crystallin by using atomistic simulations. We monitored the phase stability of their aqueous solutions by calculating time-dependent density fluctuations. We found that all proteins remained in their compact form despite aggregation. With an extensive analysis of intermolecular residue-residue interactions we discovered that arginine is of paramount importance in the initial stage of aggregation of HEWL and γ-D crystallin, meanwhile lysine was found to be the most involved amino acid in forming initial contacts between T4 WT* molecules.
view abstract doi: 10.1039/d0cp05160h 2021 • 220 Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins
Pech, S. and Rehberg, M. and Janke, R. and Benndorf, D. and Genzel, Y. and Muth, T. and Sickmann, A. and Rapp, E. and Reichl, U.
Applied Microbiology and Biotechnology (2021)Abstract: Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. Key points: • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells © 2021, The Author(s).
view abstract doi: 10.1007/s00253-021-11150-z 2020 • 219 A combined experimental and modelling approach for the Weimberg pathway optimisation
Shen, L. and Kohlhaas, M. and Enoki, J. and Meier, R. and Schönenberger, B. and Wohlgemuth, R. and Kourist, R. and Niemeyer, F. and van Niekerk, D. and Bräsen, C. and Niemeyer, J. and Snoep, J. and Siebers, B.
Nature Communications 11 (2020)The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts. © 2020, The Author(s).
view abstract doi: 10.1038/s41467-020-14830-y 2020 • 218 Effect of ozone stress on the intracellular metabolites from Cobetia marina
Li, J. and Rumancev, C. and Lutze, H.V. and Schmidt, T.C. and Rosenhahn, A. and Schmitz, O.J.
Analytical and Bioanalytical Chemistry 412 5853-5861 (2020)A GCxGC-MS system was employed with a non-polar × mid-polar column set for the metabolic non-target analysis of Cobetia marina, the model bacteria for marine biofouling. C. marina was treated with ozone to investigate the intracellular metabolic state change under oxidative stress. A minimal inhibitory concentration test was involved to guarantee that the applied ozone dosages were not lethal for the cells. In this study, non-target analyses were performed to identify the metabolites according to the NIST database. As a result, over 170 signals were detected under normal living conditions including 35 potential metabolites. By the comparison of ozone-treated and non-treated samples, five compounds were selected to describe observed trends of signals in the contour plots. Oleic acid exhibited a slight growth by increasing ozone dosage. In contrast, other metabolites such as the amino acid l-proline showed less abundance after ozone treatment, which was more evident once ozone dosage was raised. Thus, this work could provide a hint for searching for up/downregulating factors in such environmental stress conditions for C. marina. [Figure not available: see fulltext.]. © 2020, The Author(s).
view abstract doi: 10.1007/s00216-020-02810-6 2020 • 217 Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism
Muscolino, E. and Schmitz, R. and Loroch, S. and Caragliano, E. and Schneider, C. and Rizzato, M. and Kim, Y.-H. and Krause, E. and Juranić Lisnić, V. and Sickmann, A. and Reimer, R. and Ostermann, E. and Brune, W.
Nature Microbiology 5 331-342 (2020)Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin–proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed ‘induced protein aggregation motif’. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
view abstract doi: 10.1038/s41564-019-0624-1 2020 • 216 Hydration in aqueous osmolyte solutions: The case of TMAO and urea
Sahle, C.J. and Schroer, M.A. and Niskanen, J. and Elbers, M. and Jeffries, C.M. and Sternemann, C.
Physical Chemistry Chemical Physics 22 11614-11624 (2020)The hydration and hydrogen-bond topology of small water solvated molecules such as the naturally occurring organic osmolytes trimethylamine N-oxide (TMAO) and urea are under intense investigation. We aim at furthering the understanding of this complex hydration by combining experimental oxygen K-edge excitation spectra with results from spectra calculated via the Bethe-Salpeter equation based on structures obtained from ab initio molecular dynamics simulations. Comparison of experimental and calculated spectra allows us to extract detailed information about the immediate surrounding of the solute molecules in the solvated state. We quantify and localize the influence of the solute on the hydrogen bond network of the water solvent and find spectroscopic fingerprints of a clear directional asymmetry around TMAO with strong and local kosmotropic influence around TMAO's NO head group and slight chaotropic influence around the hydrophobic methyl groups. The influence of urea on the local water network is qualitatively similar to that of TMAO but weaker in magnitude. The strongest influence of both molecules on the shape of the oxygen K-edge spectra is found in the first hydration shells. This journal is © the Owner Societies.
view abstract doi: 10.1039/c9cp06785j 2020 • 215 Molecular aspects of lipid metabolism in the midgut gland of the brown shrimp Crangon crangon
Martínez-Alarcón, D. and Hagen, W. and Held, C. and Saborowski, R.
Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology 248-249 (2020)The brown shrimp, Crangon crangon, is well adapted to the variable environmental conditions in the southern North Sea. It is very abundant, has high reproduction rates, and holds a key position in coastal ecosystems. This species has very low lipid deposits in the midgut gland, suggesting that the main function of the midgut gland is metabolic turnover rather than energy storage. Based on seasonal gene expression studies and established transcriptome data, we investigated key components of lipid metabolic pathways. Gene expression of triacylglycerol lipase, phospholipase, and fatty acid desaturase were analyzed and compared with that of other digestive enzymes involved in lipid, carbohydrate, and protein catabolism. Our results suggest that gene expression of digestive enzymes involved in lipid metabolism is modulated by the lipid content in the midgut gland and is related to food availability. Brown shrimp seem to be capable of using cellular phospholipids during periods of food paucity but high energetic (lipid) requirements. Two of three isoforms of fatty acid binding proteins (FABPs) from the midgut gland involved in fatty acid transport showed specific mutations of the binding site. We hypothesize that the mutations in FABPs and deficiencies in anabolic pathways limit lipid storage capacities in the midgut gland of C. crangon. In turn, food utilization, including lipid catabolism, has to be efficient to fulfill the energetic requirements of brown shrimp. © 2020 The Authors
view abstract doi: 10.1016/j.cbpb.2020.110465 2020 • 214 Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics
Kopczynski, D. and Hentschel, A. and Coman, C. and Schebb, N.H. and Hornemann, T. and Mashek, D.G. and Hartung, N.M. and Shevchuk, O. and Schött, H.-F. and Lorenz, K. and Torta, F. and Burla, B. and Zahedi, R.P. and Sickmann, A. ...
Analytical Chemistry 92 13672-13676 (2020)We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms. © 2020 American Chemical Society.
view abstract doi: 10.1021/acs.analchem.0c02793 2020 • 213 Standard Gibbs energy of metabolic reactions: IV. Triosephosphate isomerase reaction
Greinert, T. and Baumhove, K. and Sadowski, G. and Held, C.
Biophysical Chemistry 258 (2020)The glycolytic pathway is present in most organisms and represents a central part of the energy production mechanism in a cell. For a general understanding of glycolysis, the investigation from a thermodynamic point of view is essential and allows realising thermodynamic feasibility analyses under in vivo conditions. However, available literature standard Gibbs energies of reaction, ΔRg′0, are calculated using equilibrium-molality ratios Km′, which might lead to a misinterpretation of the glycolytic pathway. It was the aim of this work to thermodynamically investigate the triosephosphate isomerase (TPI) reaction to provide new activity-based reaction data. In vitro equilibrium experiments were performed, and activity coefficients were predicted with the equation of state electrolyte PC-SAFT (ePC-SAFT). The combination of experimental concentrations and predicted activity coefficients yielded the thermodynamic equilibrium constant Ka and a new value for ΔRg′0(298.15 K, pH 7) = 7.1 ± 0.3 kJ mol‑1. The availability of the new ΔRg′0 value allowed predicting influences of the reaction medium on the reaction equilibrium of the TPI reaction. In this work, influences of the initial substrate concentration, pH and Mg2+ concentration on the reaction equilibrium were investigated and a method is presented to predict these influences. The higher the substrate concentration and the higher the temperature, the stronger the reaction equilibrium is shifted on the product side. While the pH did not have a significant influence on the reaction equilibrium, Mg2+ yielded a shift of the reaction equilibrium to the substrate side. All these effects were predicted correctly with ePC-SAFT. Based on the ePC-SAFT predictions we concluded that a charge-reduction of the product by complexation of the product with Mg2+ was responsible for the strong influence of Mg2+ on the reaction equilibrium. Finally, the standard enthalpy of reaction of ΔRh′0(pH 7) = 18 ± 7 kJ mol‑1 was determined with the equilibrium constants Ka at 298.15 K, 304.15 K and 310.15 K using the van ‘t Hoff equation. © 2020 Elsevier B.V.
view abstract doi: 10.1016/j.bpc.2020.106330 2020 • 212 Standard Gibbs energy of metabolic reactions: V. Enolase reaction
Greinert, T. and Vogel, K. and Seifert, A.I. and Siewert, R. and Andreeva, I.V. and Verevkin, S.P. and Maskow, T. and Sadowski, G. and Held, C.
Biochimica et Biophysica Acta - Proteins and Proteomics 1868 (2020)The glycolytic pathway is one of the most important pathways for living organisms, due to its role in energy production and as supplier of precursors for biosynthesis in living cells. This work focuses on determination of the standard Gibbs energy of reaction ΔRg′0 of the enolase reaction, the ninth reaction in the glycolysis pathway. Exact ΔRg′0 values are required to predict the thermodynamic feasibility of single metabolic reactions or even of metabolic reaction sequences under cytosolic conditions. So-called “apparent” standard data from literature are only valid at specific conditions. Nevertheless, such data are often used in pathway analyses, which might lead to misinterpretation of the results. In this work, equilibrium measurements were combined with activity coefficients in order to obtain new standard values ΔRg′0 for the enolase reaction that are independent of the cytosolic conditions. Reaction equilibria were measured at different initial substrate concentrations and temperatures of 298.15 K, 305.15 K and 310.15 K at pH 7. The activity coefficients were predicted using the equation of state electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The ePC-SAFT parameters were taken from literature or fitted to new experimentally determined osmotic coefficients and densities. At 298.15 K and pH 7, a ΔRg′0(298.15 K, pH 7) value of −2.8 ± 0.2 kJ mol− 1 was obtained. This value differs by up to 5 kJ mol− 1 from literature data. Reasons are the poorly defined “standard” conditions and partly undefined reaction conditions of literature works. Finally, using temperature-dependent equilibrium constants and the van ‘t Hoff equation, the standard enthalpy of reaction of ΔRh′0(298.15 K, pH 7) = 27 ± 10 kJ mol− 1 was determined, and a similar value was found by quantum-chemistry calculations. © 2020 Elsevier B.V.
view abstract doi: 10.1016/j.bbapap.2020.140365 2020 • 211 Standard Gibbs energy of metabolic reactions: VI. Glyceraldehyde 3-phosphate dehydrogenase reaction
Greinert, T. and Vogel, K. and Mühlenweg, J.-K. and Sadowski, G. and Maskow, T. and Held, C.
Fluid Phase Equilibria 517 (2020)Glycolysis is a very central metabolic pathway for many organisms because it represents a key component in their energy production. For this reason, it has always been an extensively studied pathway. The glyceraldehyde 3-phosphate dehydrogenase (GDH) reaction is an important reaction of glycolysis yielding nicotinamide adenine dinucleotide (NADH). The aim of this work is to investigate the thermodynamics of the GDH reaction and determine the standard Gibbs energy of reaction ΔRg'0 and standard enthalpy of reaction ΔRh'0. Currently, so-called ‘standard’ data exist in the literature that depend on the conditions they were measured at. In this work, ΔRg'0 and ΔRh'0 values were determined that are independent from reaction conditions by accounting for the activity coefficients of the reacting substances. Therefore, the equation of state electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) was used. The required ePC-SAFT parameters were taken from literature or fitted to new experimental osmotic coefficients. A value of ΔRg'0 = 51.5 ± 0.4 kJ mol−1 was determined at 298.15 K. This value deviates by up to 10 kJ mol−1 from existing literature values, caused by activity coefficients in the reaction medium. It can be used to determine the Gibbs energy of reaction ΔRg', which will allow statements concerning the feasibility of the GDH reaction. Further, a method is presented to predict influences of pH, initial substrate concentration and Mg2+ concentration on the reaction equilibrium. Finally, we measured the standard reaction enthalpy for the GDH reaction ΔRh'0 by titration calorimetric measurements (ΔRh'0 = 4.6 ± 0.1 kJ mol−1). This value was within van ’t Hoff evaluated ΔRh'0 (9 ± 16 kJ mol−1) using temperature-dependent equilibrium constants from equilibrium measurements corrected by ePC-SAFT predicted activity coefficients. © 2020 Elsevier B.V.
view abstract doi: 10.1016/j.fluid.2020.112597 2020 • 210 Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies
Xuan, Y. and Bateman, N.W. and Gallien, S. and Goetze, S. and Zhou, Y. and Navarro, P. and Hu, M. and Parikh, N. and Hood, B.L. and Conrads, K.A. and Loosse, C. and Kitata, R.B. and Piersma, S.R. and Chiasserini, D. and Zhu, H. an...
Nature Communications 11 (2020)Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality. © 2020, The Author(s).
view abstract doi: 10.1038/s41467-020-18904-9 2020 • 209 Structure Defines Function: Clinically Relevant Mutations in ErbB Kinases
Niggenaber, J. and Hardick, J. and Lategahn, J. and Rauh, D.
Journal of Medicinal Chemistry 63 40-51 (2020)The ErbB receptor tyrosine kinase family members EGFR (epidermal growth factor receptor) and Her2 are among the prominent mutated oncogenic drivers of non-small cell lung cancer (NSCLC). Their importance in proliferation, apoptosis, and cell death ultimately renders them hot targets in cancer therapy. Small-molecule tyrosine kinase inhibitors seem well suited to be tailor-made therapeutics for EGFR mutant NSCLC; however, drug resistance mutations limit their success. Against this background, the elucidation and visualization of the three-dimensional structure of cancer-related kinases provide valuable insights into their molecular functions. This field has undergone a revolution because X-ray crystal structure determinations aided structure-based drug design approaches and clarified the effect of activating and resistance-conferring mutations. Here, we present an overview of important mutations affecting EGFR and Her2 and highlight their influence on the kinase domain conformations and active site accessibility. © 2019 American Chemical Society.
view abstract doi: 10.1021/acs.jmedchem.9b00964 2020 • 208 The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes
Stein, V. and Blank-Landeshammer, B. and Müntjes, K. and Märker, R. and Teichert, I. and Feldbrügge, M. and Sickmann, A. and Kück, U.
PLoS Genetics 16 (2020)The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343. served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phosphodeficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phosphodeficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth. © 2020 Stein et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
view abstract doi: 10.1371/journal.pgen.1008819 2019 • 207 2-Azo-, 2-diazocine-thiazols and 2-azo-imidazoles as photoswitchable kinase inhibitors: Limitations and pitfalls of the photoswitchable inhibitor approach
Schehr, M. and Ianes, C. and Weisner, J. and Heintze, L. and Müller, M.P. and Pichlo, C. and Charl, J. and Brunstein, E. and Ewert, J. and Lehr, M. and Baumann, U. and Rauh, D. and Knippschild, U. and Peifer, C. and Herges, R.
Photochemical and Photobiological Sciences 18 1398-1407 (2019)In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors. © 2019 The Royal Society of Chemistry and Owner Societies.
view abstract doi: 10.1039/c9pp00010k 2019 • 206 A new class of supramolecular ligands stabilizes 14-3-3 protein-protein interactions by up to two orders of magnitude
Gigante, A. and Grad, J.-N. and Briels, J. and Bartel, M. and Hoffmann, D. and Ottmann, C. and Schmuck, C.
Chemical Communications 55 111-114 (2019)We report the first supramolecular stabilizers of the interaction between 14-3-3ζ and two of its effectors, Tau and C-Raf, which are involved in neurodegenerative diseases and proliferative signal transduction, respectively. These supramolecular ligands open up an opportunity to modulate functions of 14-3-3 with these effectors. © The Royal Society of Chemistry.
view abstract doi: 10.1039/c8cc07946c 2019 • 205 A multiscale and multiphase model for the description of function-perfusion processes in the human liver
Lambers, L. and Ricken, T. and König, M.
Advances in Engineering Materials, Structures and Systems: Innovations, Mechanics and Applications - Proceedings of the 7th International Conference on Structural Engineering, Mechanics and Computation, 2019 304-308 (2019)Numerical simulations of biological systems have become more and more important in recent years. In order to understand and predict hepatic function in health and disease, we developed a multiscale and multiphase model for the description of function-perfusion processes in the liver. With respect to the different scales of the hierarchically structured liver, processes like glucose homeostasis or detoxification of paracetamol as well as the influence of hepatic disease like the non-alcoholic fatty liver disease (NAFLD) can be investigated. On the tissue scale the liver consists of hexagonal liver lobules, containing anisotropically oriented capillaries, called sinusoids, which lead macroscopically to an anisotropic blood flow as well as a nonlinear, anisotropic and poro-elastic response. This structure is described using a homogenization method based on the Theory of Porous Media (TPM). This leads to a coupled set of partial differential equations (PDE) describing the tissue deformation as well as the transport of blood and metabolites like nutrients or xenobiotics. The lobule scale is coupled to the cellular scale where hepatic metabolism takes place. With the use of embedded ODE equations we can simulate metabolic processes depending on various nutrients or substances. The performance of the developed theory is demonstrated by a numerical example. The results provide an overview of possible applications of our approach using the NAFLD as a showcase. During the development of fatty liver, fat is stored in the liver cells resulting in swelling of the hepatocytes. This accumulation of fat drives tissue growth, which has an impact on the blood perfusion in the liver lobules. The results also clarify the spatial distribution of flow and fat accumulation since many hepatic processes proceed zonated. The processes in one single lobule are then expanded to a group of lobules to investigate the mutual liver lobe interaction. © 2019 Taylor & Francis Group, London, UK.
view abstract doi: 10.1201/9780429426506-52 2019 • 204 Biomechanical stability and osteogenesis in a tibial bone defect treated by autologous ovine cord blood cells—a pilot study
Herten, M. and Zilkens, C. and Thorey, F. and Tassemeier, T. and Lensing-Höhn, S. and Fischer, J.C. and Sager, M. and Krauspe, R. and Jäger, M.
Molecules 24 (2019)The aim of this study was to elucidate the impact of autologous umbilical cord blood cells (USSC) on bone regeneration and biomechanical stability in an ovine tibial bone defect. Ovine USSC were harvested and characterized. After 12 months, full-size 2.0 cm mid-diaphyseal bone defects were created and stabilized by an external fixateur containing a rigidity measuring device. Defects were filled with (i) autologous USSC on hydroxyapatite (HA) scaffold (test group), (ii) HA scaffold without cells (HA group), or (iii) left empty (control group). Biomechanical measures, standardized X-rays, and systemic response controls were performed regularly. After six months, bone regeneration was evaluated histomorphometrically and labeled USSC were tracked. In all groups, the torsion distance decreased over time, and radiographies showed comparable bone regeneration. The area of newly formed bone was 82.5 ± 5.5% in the control compared to 59.2 ± 13.0% in the test and 48.6 ± 2.9% in the HA group. Labeled cells could be detected in lymph nodes, liver and pancreas without any signs of tumor formation. Although biomechanical stability was reached earliest in the test group with autologous USSC on HA scaffold, the density of newly formed bone was superior in the control group without any bovine HA. © 2019 by the authors.
view abstract doi: 10.3390/molecules24020295 2019 • 203 Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes
Rodziewicz, P. and Loroch, S. and Marczak and Sickmann, A. and Kayser, O.
Plant Science 284 108-116 (2019)Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes. © 2019 Elsevier B.V.
view abstract doi: 10.1016/j.plantsci.2019.04.008 2019 • 202 Intrasurgical Protein Layer on Titanium Arthroplasty Explants: From the Big Twelve to the Implant Proteome
Jäger, M. and Jennissen, H.P. and Haversath, M. and Busch, A. and Grupp, T. and Sowislok, A. and Herten, M.
Proteomics - Clinical Applications 13 (2019)Purpose: Aseptic loosening in total joint replacement due to insufficient osteointegration is an unsolved problem in orthopaedics. The purpose of the study is to obtain a picture of the initial protein adsorption layer on femoral endoprosthetic surfaces as the key to the initiation of osseointegration. Experimental design: The paper describes the first study of femoral stem explants from patients for proteome analysis of the primary protein layer. After 2 min in situ, the stems are explanted and frozen in liquid nitrogen. Proteins are eluted under reducing conditions and analyzed by LC-MS/MS. Results: After exclusion of proteins identified by a single peptide, the implant proteome is found to consist of 2802 unique proteins. Of these, 77% are of intracellular origin, 9% are derived from the plasma proteome, 8% from the bone proteome, and four proteins with highest specificity score could be assigned to the bone marrow proteome (transcriptome). The most abundant protein in the adsorbed total protein layer is hemoglobin (8–11%) followed by serum albumin (3.6–6%). Conclusions: A detailed knowledge of the initial protein film deposited onto the implants, as demonstrated here for the first time, may help to understand and predict the response of the osseous microenvironment to implant surfaces. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract doi: 10.1002/prca.201800168 2019 • 201 Investigating the Role of Mitochondria in Type 2 Diabetes – Lessons from Lipidomics and Proteomics Studies of Skeletal Muscle and Liver
Kappler, L. and Kollipara, L. and Lehmann, R. and Sickmann, A.
Advances in Experimental Medicine and Biology 1158 143-182 (2019)Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondrial lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre-analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed. © 2019, Springer Nature Singapore Pte Ltd.
view abstract doi: 10.1007/978-981-13-8367-0_9 2019 • 200 Major Differences between the Self-Assembly and Seeding Behavior of Heparin-Induced and in Vitro Phosphorylated Tau and Their Modulation by Potential Inhibitors
Despres, C. and Di, J. and Cantrelle, F.-X. and Li, Z. and Huvent, I. and Chambraud, B. and Zhao, J. and Chen, J. and Chen, S. and Lippens, G. and Zhang, F. and Linhardt, R. and Wang, C. and Klärner, F.-G. and Schrader, T. and La...
ACS Chemical Biology 14 1363-1379 (2019)Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer's disease and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that, unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced (Despres et al. (2017) Proc. Natl. Acad. Sci. U.S.A., 114, 9080-9085). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure-activity relationship study demonstrates that heparin-induced tau behaves differently from in vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau. © 2019 American Chemical Society.
view abstract doi: 10.1021/acschembio.9b00325 2019 • 199 Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator
Nshanian, M. and Lantz, C. and Wongkongkathep, P. and Schrader, T. and Klärner, F.-G. and Blümke, A. and Despres, C. and Ehrmann, M. and Smet-Nocca, C. and Bitan, G. and Loo, J.A.
Journal of the American Society for Mass Spectrometry 30 16-23 (2019)Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS) were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer’s disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool used to determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. The ESI-MS study of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment in the presence of CLR01 and phosphorylated tau reveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism of the modulation of protein-protein interactions in tau by CLR01. [Figure not available: see fulltext.]. © 2018, American Society for Mass Spectrometry.
view abstract doi: 10.1007/s13361-018-2027-6 2019 • 198 NIR sensor for aqueous urea solution film thickness and concentration measurement using a broadband light source
Lubnow, M. and Dreier, T. and Schulz, C.
Applied Optics 58 4546-4552 (2019)We demonstrate a multi-wavelength near-infrared (NIR) broadband absorption sensor for the simultaneous monitoring of layer thickness and urea concentration of aqueous urea solutions. Samples were prepared in thin-layer quartz transmission cells. Film thickness and urea mass fraction (at constant temperature) were determined from measured transmittance ratios in characteristic wavelength bands selected by narrowband filters in front of the detector and converted to absorbance ratios. Suitable emission bands were selected depending on the sensitivity of the NIR absorption spectrum of the solution with respect to temperature and solute concentration. For this purpose, Fourier transform IR spectra of aqueous urea solutions were recorded in the 1250–2500 nm wavelength range for urea concentrations between 0 and 40 wt.% and temperatures between 298 K and 338 K. A prototype sensor was designed using a continuous-wave fiber-coupled incoherent tungsten lamp, subsequent intensity modulation, and lock-in detection of the transmitted radiation. The sensor concept was validated with measurements using a calibration cell providing liquid layers of variable thicknesses (7–1000 μm). © 2019 Optical Society of America
view abstract doi: 10.1364/AO.58.004546 2019 • 197 Piezoelectric 3-D Fibrous Poly(3-hydroxybutyrate)-Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for Bone Tissue Engineering: Inorganic Phase Formation, Osteoblast Cell Adhesion, and Proliferation
Chernozem, R.V. and Surmeneva, M.A. and Shkarina, S.N. and Loza, K. and Epple, M. and Ulbricht, M. and Cecilia, A. and Krause, B. and Baumbach, T. and Abalymov, A.A. and Parakhonskiy, B.V. and Skirtach, A.G. and Surmenev, R.A.
ACS Applied Materials and Interfaces (2019)Elaboration of novel biocomposites providing simultaneously both biodegradability and stimulated bone tissue repair is essential for regenerative medicine. In particular, piezoelectric biocomposites are attractive because of a possibility to electrically stimulate cell response. In the present study, novel CaCO3-mineralized piezoelectric biodegradable scaffolds based on two polymers, poly[(R)3-hydroxybutyrate] (PHB) and poly[3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV), are presented. Mineralization of the scaffold surface is carried out by the in situ synthesis of CaCO3 in the vaterite and calcite polymorphs using ultrasound (U/S). Comparative characterization of PHB and PHBV scaffolds demonstrated an impact of the porosity and surface charge on the mineralization in a dynamic mechanical system, as no essential distinction was observed in wettability, structure, and surface chemical compositions. A significantly higher (4.3 times) piezoelectric charge and a higher porosity (∼15%) lead to a more homogenous CaCO3 growth in 3-D fibrous structures and result in a two times higher relative mass increase for PHB scaffolds compared to that for PHBV. This also increases the local ion concentration incurred upon mineralization under U/S-generated dynamic mechanical conditions. The modification of the wettability for PHB and PHBV scaffolds from hydrophobic (nonmineralized fibers) to superhydrophilic (mineralized fibers) led to a pronounced apatite-forming behavior of scaffolds in a simulated body fluid. In turn, this results in the formation of a dense monolayer of well-distributed and proliferated osteoblast cells along the fibers. CaCO3-mineralized PHBV surfaces had a higher osteoblast cell adhesion and proliferation assigned to a higher amount of CaCO3 on the surface compared to that on PHB scaffolds, as incurred from micro-computed tomography (μCT). Importantly, a cell viability study confirmed biocompatibility of all the scaffolds. Thus, hybrid biocomposites based on the piezoelectric PHB polymers represent an effective scaffold platform functionalized by an inorganic phase and stimulating the growth of the bone tissue. © 2019 American Chemical Society.
view abstract doi: 10.1021/acsami.9b04936 2019 • 196 Sensitive and selective detection of Cu2+ ions based on fluorescent Ag nanoparticles synthesized by R-phycoerythrin from marine algae Porphyra yezoensis
Xu, Y. and Hou, Y. and Wang, Y. and Wang, Y. and Li, T. and Song, C. and Wei, N. and Wang, Q.
Ecotoxicology and Environmental Safety 168 356-362 (2019)In this study, using a natural and green protein R-phycoerythrin (R-PE) extracted from marine Porphyra yezoensis as the stabilizer and reducer, silver nanoparticles (AgNPs) were synthesized. Based on this, a highly sensitive and selective method for the detection of Cu2+ ions was developed using R-PE-AgNPs as fluorescent probe. The interactions between R-PE-AgNPs and Cu2+ ions were systematically characterized by fluorescence spectroscopy, transmission electron microscopy (TEM), elemental mapping and Fourier transform infrared (FTIR). It was found that Cu2+ ions could cause aggregation of the R-PE-AgNPs, accompanied by the greatly increased particle size. Importantly, the method offered a wide linear detection range from 0 μM to 100.0 μM with a detection limit of 0.0190 μM. Moreover, the proposed method was successfully applied to analyze Cu2+ ions in tap water and lake water samples, acquiring satisfactory recovery between 91.6% and 102.2%. Such a green, fast and cost-effective fluorimetric method of the R-PE-AgNPs probe has great potential for tracing Cu2+ ions in diverse aqueous media. © 2018 Elsevier Inc.
view abstract doi: 10.1016/j.ecoenv.2018.10.102 2019 • 195 The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS
Malik, R. and Meng, H. and Wongkongkathep, P. and Corrales, C.I. and Sepanj, N. and Atlasi, R.S. and Klärner, F.-G. and Schrader, T. and Spencer, M.J. and Loo, J.A. and Wiedau, M. and Bitan, G.
Journal of Biological Chemistry 294 3501-3513 (2019)Mutations in superoxide dismutase 1 (SOD1) cause 15–20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1’s protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01–a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins–as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro. Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach. © 2019 Malik et al.
view abstract doi: 10.1074/jbc.RA118.005940 2019 • 194 The molecular tweezer CLR01 reduces aggregated, pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy
Herrera-Vaquero, M. and Bouquio, D. and Kallab, M. and Biggs, K. and Nair, G. and Ochoa, J. and Heras-Garvin, A. and Heid, C. and Hadrovic, I. and Poewe, W. and Wenning, G.K. and Klärner, F.-G. and Schrader, T. and Bitan, G. and ...
Biochimica et Biophysica Acta - Molecular Basis of Disease 1865 (2019)Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development. © 2019 Elsevier B.V.
view abstract doi: 10.1016/j.bbadis.2019.07.007 2018 • 193 Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy
Lu, T. and Schubert, C. and Cummings, M.D. and Bignan, G. and Connolly, P.J. and Smans, K. and Ludovici, D. and Parker, M.H. and Meyer, C. and Rocaboy, C. and Alexander, R. and Grasberger, B. and De Breucker, S. and Esser, N. and ...
Bioorganic and Medicinal Chemistry Letters 28 2159-2164 (2018)We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement. © 2018 Elsevier Ltd
view abstract doi: 10.1016/j.bmcl.2018.05.014 2018 • 192 Direct monitoring of the conformational equilibria of the activation loop in the mitogen-activated protein kinase p38α
Roser, P. and Weisner, J. and Simard, J.R. and Rauh, D. and Drescher, M.
Chemical Communications 54 12057-12060 (2018)Conformational transitions in protein kinases are crucial for the biological function of these enzymes. Here, we characterize and assess conformational equilibria of the activation loop and the effect of small molecule inhibitors in the MAP kinase p38α. Our work experimentally revealed the existence of a two-state equilibrium for p38α while the addition of inhibitors shifts the equilibrium between these two states. © 2018 The Royal Society of Chemistry.
view abstract doi: 10.1039/c8cc06128a 2018 • 191 Entropically driven Polymeric Enzyme Inhibitors by End-Group directed Conjugation
Hijazi, M. and Krumm, C. and Cinar, S. and Arns, L. and Alachraf, W. and Hiller, W. and Schrader, W. and Winter, R. and Tiller, J.C.
Chemistry - A European Journal 24 4523-4527 (2018)A new generic concept for polymeric enzyme inhibitors is presented using the example of poly(2-methyl-2-oxazoline) (PMOx) terminated with an iminodiacetate (IDA) function. These polymers are shown to be non-competitive inhibitors for horseradish peroxidase (HRP). Mechanistic investigations revealed that the polymer is directed to the protein by its end group and collapses at the surface in an entropy-driven process as shown by isothermal titration calorimetry. The dissociation constant of the complex was determined as the inhibition constant Ki using HRP kinetic activity measurements. Additional experiments suggest that the polymer does not form a diffusion layer around the protein, but might inhibit by inducing minor conformational changes in the protein. This kind of inhibitor offers new avenues towards designing bioactive compounds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract doi: 10.1002/chem.201800168 2018 • 190 Evaluation and benchmarking of an EC-QCL-based mid-infrared spectrometer for monitoring metabolic blood parameters in critical care units
Grafen, M. and Delbeck, S. and Busch, H. and Heise, H.M. and Ostendorf, A.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE 10501 (2018)Mid-infrared spectroscopy hyphenated with micro-dialysis is an excellent method for monitoring metabolic blood parameters as it enables the concurrent, reagent-free and precise measurement of multiple clinically relevant substances such as glucose, lactate and urea in micro-dialysates of blood or interstitial fluid. For a marketable implementation, quantum cascade lasers (QCL) seem to represent a favourable technology due to their high degree of miniaturization and potentially low production costs. In this work, an external cavity (EC)-QCL-based spectrometer and two Fourier-transform infrared (FTIR) spectrometers were benchmarked with regard to the precision, accuracy and long-term stability needed for the monitoring of critically ill patients. For the tests, ternary aqueous solutions of glucose, lactate and mannitol (the latter for dialysis recovery determination) were measured in custom-made flow-through transmission cells of different pathlengths and analyzed by Partial Least Squares calibration models. It was revealed, that the wavenumber tuning speed of the QCL had a severe impact on the EC-mirror trajectory due to matching the digital-Analog-converter step frequency with the mechanical resonance frequency of the mirror actuation. By selecting an appropriate tuning speed, the mirror oscillations acted as a hardware smoothing filter for the significant intensity variations caused by mode hopping. Besides the tuning speed, the effects of averaging over multiple spectra and software smoothing parameters (Savitzky-Golay-filters and FT-smoothing) were investigated. The final settings led to a performance of the QCL-system, which was comparable with a research FTIR-spectrometer and even surpassed the performance of a small FTIR-mini-spectrometer. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.
view abstract doi: 10.1117/12.2289625 2018 • 189 Hydrogenases: Recent developments and future perspectives
Wittkamp, F. and Senger, M. and Stripp, S.T. and Apfel, U.-P.
Chemical Communications 54 5934-5942 (2018)[FeFe]-Hydrogenases are the most efficient enzymes for catalytic hydrogen turnover. Their H2 production efficiency is hitherto unrivalled. However, functional details of the catalytic machinery and possible modes of application are discussed controversially. The incorporation of synthetically modified cofactors and utilization of semi-artificial enzymes only recently allowed us to shed light on key steps of the catalytic cycle. Herein, we summarize the essential findings regarding the redox chemistry of [FeFe]-hydrogenases and discuss their catalytic hydrogen turnover. We furthermore will give an outlook on potential research activities and exploit the utilization of synthetic cofactor mimics. © 2018 The Royal Society of Chemistry.
view abstract doi: 10.1039/c8cc01275j 2018 • 188 Molecular recognition of carboxylates in the protein leucine zipper by a multivalent supramolecular ligand: Residue-specific, sensitive and label-free probing by UV resonance Raman spectroscopy
Zakeri, B. and Niebling, S. and Martinéz, A.G. and Sokkar, P. and Sanchez-Garcia, E. and Schmuck, C. and Schlücker, S.
Physical Chemistry Chemical Physics 20 1817-1820 (2018)Ultraviolet resonance Raman (UVRR) spectroscopy is a selective, sensitive and label-free vibrational spectroscopic technique. Here, we demonstrate as proof of concept that UVRR can be used for probing the recognition between a multivalent supramolecular ligand and acidic residues in leucine zipper, an α-helical structural motif of many proteins. © 2017 Owner Societies.
view abstract doi: 10.1039/c7cp04971d 2018 • 187 On-surface nickel porphyrin mimics the reactive center of an enzyme cofactor
Zamborlini, G. and Jugovac, M. and Cossaro, A. and Verdini, A. and Floreano, L. and Lüftner, D. and Puschnig, P. and Feyer, V. and Schneider, C.M.
Chemical Communications 54 13423-13426 (2018)Metal-containing enzyme cofactors achieve their unusual reactivity by stabilizing uncommon metal oxidation states with structurally complex ligands. In particular, the specific cofactor promoting both methanogenesis and anaerobic methane oxidation is a porphyrinoid chelated to a nickel(i) atom via a multi-step biosynthetic path, where nickel reduction is achieved through extensive molecular hydrogenation. Here, we demonstrate an alternative route to porphyrin reduction by charge transfer from a selected copper substrate to commercially available 5,10,15,20-tetraphenyl-porphyrin nickel(ii). X-ray absorption measurements at the Ni L3-edge unequivocally show that NiTPP species adsorbed on Cu(100) are stabilized in the highly reactive Ni(i) oxidation state by electron transfer to the molecular orbitals. Our approach highlights how some fundamental properties of synthetically inaccessible biological cofactors may be reproduced by hybridization of simple metalloporphyrins with metal surfaces, with implications towards novel approaches to heterogenous catalysis. © 2018 The Royal Society of Chemistry.
view abstract doi: 10.1039/c8cc06739b 2017 • 186 A Systematic Structure–Activity Study of a New Type of Small Peptidic Transfection Vector Reveals the Importance of a Special Oxo-Anion-Binding Motif for Gene Delivery
Junghänel, S. and Karczewski, S. and Bäcker, S. and Knauer, S.K. and Schmuck, C.
ChemBioChem 18 2268-2279 (2017)We discovered a new class of artificial peptidic transfection vectors based on an artificial anion-binding motif, the guanidiniocarbonylpyrrole (GCP) cation. This new type of vector is surprisingly smaller than traditional systems, and our previous work suggested that the GCP group was important for promoting critical endosomal escape. We now present here a systematic comparison of similar DNA ligands featuring our GCP oxo-anion-binding motif with DNA ligands only consisting of naturally occurring amino acids. Structure–activity studies showed that the artificial binding motif clearly outperformed natural amino acids such as histidine, lysine, and arginine. It improved the ability to shuttle foreign genetic material into cells, yet successfully mediated endosomal escape. Also, plasmids that were complexed by our artificial ligands were stabilized against cytosolic degradation to some extent. This resulted in the successful expression of plasmid information (comparable to gold standards such as polyethyleneimine). Hence, our study clearly demonstrates the importance of the tailor-made GCP anion-binding site for efficient gene transfection. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract doi: 10.1002/cbic.201700433 2017 • 185 Characterization of methane oxidation in a simulated landfill cover system by comparing molecular and stable isotope mass balances
Schulte, M. and Jochmann, M.A. and Gehrke, T. and Thom, A. and Ricken, T. and Denecke, M. and Schmidt, T.C.
Waste Management 69 281-288 (2017)Biological methane oxidation may be regarded as a method of aftercare treatment for landfills to reduce climate relevant methane emissions. It is of social and economic interest to estimate the behavior of bacterial methane oxidation in aged landfill covers due to an adequate long-term treatment of the gas emissions. Different approaches assessing methane oxidation in laboratory column studies have been investigated by other authors recently. However, this work represents the first study in which three independent approaches, ((i) mass balance, (ii) stable isotope analysis, and (iii) stoichiometric balance of product (CO2) and reactant (CH4) by CO2/CH4-ratio) have been compared for the estimation of the biodegradation by a robust statistical validation on a rectangular, wide soil column. Additionally, an evaluation by thermal imaging as a potential technique for the localization of the active zone of bacterial methane oxidation has been addressed in connection with stable isotope analysis and CO2/CH4-ratios. Although landfills can be considered as open systems the results for stable isotope analysis based on a closed system correlated better with the mass balance than calculations based on an open system. CO2/CH4-ratios were also in good agreement with mass balance. In general, highest values for biodegradation were determined from mass balance, followed by CO2/CH4-ratio, and stable isotope analysis. The investigated topsoil proved to be very suitable as a potential cover layer by removing up to 99% of methane for CH4 loads of 35–65 g m–2 d–1 that are typical in the aftercare phase of landfills. Finally, data from stable isotope analysis and the CO2/CH4-ratios were used to trace microbial activity within the reactor system. It was shown that methane consumption and temperature increase, as a cause of high microbial activity, correlated very well. © 2017 Elsevier Ltd
view abstract doi: 10.1016/j.wasman.2017.07.032 2017 • 184 Conversion of an instantaneous activating K+ channel into a slow activating inward rectifier
Baumeister, D. and Hertel, B. and Schroeder, I. and Gazzarrini, S. and Kast, S.M. and Van Etten, J.L. and Moroni, A. and Thiel, G.
FEBS Letters 591 295-303 (2017)The miniature channel, Kcv, is a structural equivalent of the pore of all K+ channels. Here, we follow up on a previous observation that a largely voltage-insensitive channel can be converted into a slow activating inward rectifier after extending the outer transmembrane domain by one Ala. This gain of rectification can be rationalized by dynamic salt bridges at the cytosolic entrance to the channel; opening is favored by voltage-sensitive formation of salt bridges and counteracted by their disruption. Such latent voltage sensitivity in the pore could be relevant for the understanding of voltage gating in complex Kv channels. © 2016 Federation of European Biochemical Societies
view abstract doi: 10.1002/1873-3468.12536 2017 • 183 Deciphering lymphoma pathogenesis via state-of-the-art mass spectrometry-based quantitative proteomics
Psatha, K. and Kollipara, L. and Voutyraki, C. and Divanach, P. and Sickmann, A. and Rassidakis, G.Z. and Drakos, E. and Aivaliotis, M.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 1047 2-14 (2017)Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenesis of lymphoma has incremental value in deciphering the heterogeneity in complex deregulated molecular mechanisms/pathways of the lymphoma entities, implementing the current diagnostic and therapeutic strategies. Essential global, targeted and functional differential proteomics analyses although still evolving, have been successfully implemented to shed light on lymphoma pathogenesis to discover and explore the role of potential lymphoma biomarkers and drug targets. This review aims to outline and appraise the present status of MS-based quantitative proteomic approaches in lymphoma research, introducing the current state-of-the-art MS-based proteomic technologies, the opportunities they offer in biological discovery in human lymphomas and the related limitation issues arising from sample preparation to data evaluation. It is a synopsis containing information obtained from recent research articles, reviews and public proteomics repositories (PRIDE). We hope that this review article will aid, assimilate and assess all the information aiming to accelerate the development and validation of diagnostic, prognostic or therapeutic targets for an improved and empowered clinical proteomics application in lymphomas in the nearby future. © 2016 Elsevier B.V.
view abstract doi: 10.1016/j.jchromb.2016.11.005 2017 • 182 Design, Synthesis, and Biological Evaluation of Novel Type I1/2 p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R-Spine
Walter, N.M. and Wentsch, H.K. and Bührmann, M. and Bauer, S.M. and Döring, E. and Mayer-Wrangowski, S. and Sievers-Engler, A. and Willemsen-Seegers, N. and Zaman, G. and Buijsman, R. and Lämmerhofer, M. and Rauh, D. and Laufer, S.A.
Journal of Medicinal Chemistry 60 8027-8054 (2017)We recently reported 1a (skepinone-L) as a type I p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, as a type I inhibitor, it is entirely ATP-competitive and shows just a moderate residence time. Thus, the scope was to develop a new class of advanced compounds maintaining the structural binding features of skepinone-L scaffold like inducing a glycine flip at the hinge region and occupying both hydrophobic regions I and II. Extending this scaffold with suitable residues resulted in an interference with the kinase's R-Spine. By synthesizing 69 compounds, we could significantly prolong the target residence time with one example to 3663 s, along with an excellent selectivity score of 0.006 and an outstanding potency of 1.0 nM. This new binding mode was validated by cocrystallization, showing all binding interactions typifying type I1/2 binding. Moreover, microsomal studies showed convenient metabolic stability of the most potent, herein reported representatives. © 2017 American Chemical Society.
view abstract doi: 10.1021/acs.jmedchem.7b00745 2017 • 181 Enzymatic mineralization generates ultrastiff and tough hydrogels with tunable mechanics
Rauner, N. and Meuris, M. and Zoric, M. and Tiller, J.C.
Nature 543 407-410 (2017)The cartilage and skin of animals, which are made up of more than fifty per cent water, are rather stiff (having elastic moduli of up to 100 megapascals) as well as tough and hard to break (with fracture energies of up to 9,000 joules per square metre). Such features make these biological materials mechanically superior to existing synthetic hydrogels. Lately, progress has been made in synthesizing tough hydrogels, with double-network hydrogels achieving the toughness of skin and inorganic-organic composites showing even better performance. However, these materials owe their toughness to high stretchability; in terms of stiffness, synthetic hydrogels cannot compete with their natural counterparts, with the best examples having elastic moduli of just 10 megapascals or less. Previously, we described the enzyme-induced precipitation and crystallization of hydrogels containing calcium carbonate, but the resulting materials were brittle. Here we report the enzyme-induced formation of amorphous calcium phosphate nanostructures that are homogenously distributed within polymer hydrogels. Our best materials have fracture energies of 1,300 joules per square metre even in their fully water-swollen state - a value superior to that of most known water-swollen synthetic materials. We are also able to modulate their stiffness up to 440 megapascals, well beyond that of cartilage and skin. Furthermore, the highly filled composite materials can be designed to be optically transparent and to retain most of their stretchability even when notched. We show that percolation drives the mechanical properties, particularly the high stiffness, of our uniformly mineralized hydrogels. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
view abstract doi: 10.1038/nature21392 2017 • 180 Functionality of albumin-derived perfluorocarbon-based artificial oxygen carriers in the Langendorff-heart
Wrobeln, A. and Schlüter, K.D. and Linders, J. and Zähres, M. and Mayer, C. and Kirsch, M. and Ferenz, K.B.
Artificial Cells, Nanomedicine and Biotechnology 45 723-730 (2017)The aim of this study was to prove whether albumin-derived perfluorocarbon-based nanoparticles (capsules) can operate as a novel artificial oxygen carrier in a rat Langendorff-heart perfusion model. Hearts perfused with capsules showed increased left ventricular pressure and rate pressure product compared to hearts perfused with pure Krebs–Henseleit (KH)-buffer. The capsules prevented the myocardium from functional fail when in their absence a noxious ischemia was observed. Capsules did not change rheological properties of KH-buffer and could repeatedly reload with oxygen. This albumin-derived perfluorocarbon-based artificial oxygen carrier preserved the function of rat hearts due to the transport of oxygen in a satisfactory manner. Because of these positive results, the functionality of the applied capsules should be verified in living animals. © 2017 Informa UK Limited, trading as Taylor & Francis Group
view abstract doi: 10.1080/21691401.2017.1284858 2017 • 179 In Vivo Detoxification of Lipopolysaccharide by Antimicrobial Peptides
Zhang, W. and He, J. and Wu, J. and Schmuck, C.
Bioconjugate Chemistry 28 319-324 (2017)Abundant lipopolysaccharide (LPS) can result in sepsis and septic shock, indicating a serious Gram-negative bacterial contamination. We have developed a novel strategy based on dendritic antimicrobial peptides that can detoxify LPS. The dendritic antimicrobial peptides bind to LPS at the surface of Gram-negative bacteria, killing the bacteria but removing the LPS from the cell wall of dead Gram-negative bacteria, hence detoxifying pathogenic bacteria in its host cells and effectively improving survival of animals infected with Pseudomonas aeruginosa. Our findings provide a way to detoxify bacterial contamination. © 2016 American Chemical Society.
view abstract doi: 10.1021/acs.bioconjchem.6b00664 2017 • 178 Indazole-Based Covalent Inhibitors To Target Drug-Resistant Epidermal Growth Factor Receptor
Tomassi, S. and Lategahn, J. and Engel, J. and Keul, M. and Tumbrink, H.L. and Ketzer, J. and Mühlenberg, T. and Baumann, M. and Schultz-Fademrecht, C. and Bauer, S. and Rauh, D.
Journal of Medicinal Chemistry 60 2361-2372 (2017)The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder. © 2017 American Chemical Society.
view abstract doi: 10.1021/acs.jmedchem.6b01626 2017 • 177 Mechanical induction of bi-directional orientation of primary porcine bladder smooth muscle cells in tubular fibrin-poly(vinylidene fluoride) scaffolds for ureteral and urethral repair using cyclic and focal balloon catheter stimulation
Seifarth, V. and Grosse, J.O. and Gossmann, M. and Janke, H.P. and Arndt, P. and Koch, S. and Epple, M. and Artmann, G.M. and Artmann, A.T.
Journal of Biomaterials Applications 32 321-330 (2017)To restore damaged organ function or to investigate organ mechanisms, it is necessary to prepare replicates that follow the biological role model as faithfully as possible. The interdisciplinary field of tissue engineering has great potential in regenerative medicine and might overcome negative side effects in the replacement of damaged organs. In particular, tubular organ structures of the genitourinary tract, such as the ureter and urethra, are challenging because of their complexity and special milieu that gives rise to incrustation, inflammation and stricture formation. Tubular biohybrids were prepared from primary porcine smooth muscle cells embedded in a fibrin gel with a stabilising poly(vinylidene fluoride) mesh. A mechanotransduction was performed automatically with a balloon kyphoplasty catheter. Diffusion of urea and creatinine, as well as the bursting pressure, were measured. Light and electron microscopy were used to visualise cellular distribution and orientation. Histological evaluation revealed a uniform cellular distribution in the fibrin gel. Mechanical stimulation with a stretch of 20% leads to a circumferential orientation of smooth muscle cells inside the matrix and a longitudinal alignment on the outer surface of the tubular structure. Urea and creatinine permeability and bursting pressure showed a non-statistically significant trend towards stimulated tissue constructs. In this proof of concept study, an innovative technique of intraluminal pressure for mechanical stimulation of tubular biohybrids prepared from autologous cells and a composite material induce bi-directional orientation of smooth muscle cells by locally and cyclically applied mechanical tension. Such geometrically driven patterns of cell growth within a scaffold may represent a key stage in the future tissue engineering of implantable ureter replacements that will allow the active transportation of urine from the renal pelvis into the bladder. © The Author(s) 2017.
view abstract doi: 10.1177/0885328217723178 2017 • 176 Nanoparticulate versus ionic silver: Behavior in the tank water, bioaccumulation, elimination and subcellular distribution in the freshwater mussel Dreissena polymorpha
Zimmermann, S. and Ruchter, N. and Loza, K. and Epple, M. and Sures, B.
Environmental Pollution 222 251-260 (2017)Zebra mussels (Dreissena polymorpha) were exposed to polyvinylpyrrolidone (PVP)-coated silver nanoparticles (AgNP; hydrodynamic diameter 80 nm; solid diameter 50 nm) to investigate the behavior of Ag in the tank water with respect to its uptake, bioaccumulation, elimination and subcellular distribution in the mussel soft tissue. Parallel experiments were performed with ionic Ag (AgNO3) to unravel possible differences between the metal forms. The recovery of the applied Ag concentration (500 μg/L) in the tank water was clearly affected by the metal source (AgNP < AgNO3) and water type (reconstituted water < tap water). Filtration (< 0.45 μm) of water samples showed different effects on the quantified metal concentration depending on the water type and Ag form. Ag accumulation in the mussel soft tissue was neither influenced by the metal source nor by the water type. Ag concentrations in the mussel soft tissue did not decrease during 14 days of depuration. For both metal forms the Ag distribution within different subcellular fractions, i.e. metal-rich granules (MRG), cellular debris, organelles, heat-sensitive proteins (HSP) and metallothionein-like proteins (MTLP), revealed time-dependent changes which can be referred to intracellular Ag translocation processes. The results provide clear evidence for the uptake of Ag by the mussel soft tissue in nanoparticulate as well as in ionic form. Thus, zebra mussels could be used as effective accumulation indicators for environmental monitoring of both Ag forms. © 2016 Elsevier Ltd.
view abstract doi: 10.1016/j.envpol.2016.12.048 2017 • 175 Optimized expression-based microdissection of formalin-fixed lung cancer tissue
Grafen, M. and Hofmann, T.R. and Scheel, A.H. and Beck, J. and Emmert, A. and Küffer, S. and Danner, B.C. and Schütz, E. and Büttner, R. and Ostendorf, A. and Ströbel, P. and Bohnenberger, H.
Laboratory Investigation 97 863-872 (2017)Analysis of specific DNA alterations in precision medicine of tumors is crucially important for molecular targeted treatments. Lung cancer is a prototypic example and one of the leading causes of cancer-related deaths worldwide. One major technical problem of detecting DNA alterations in tissue samples is cellular heterogeneity, that is, mixture of tumor and normal cells. Microdissection is an important tool to enrich tumor cells from heterogeneous tissue samples. However, conventional laser capture microdissection has several disadvantages like user-dependent selection of regions of interest (ROI), high costs for dissection systems and long processing times. ROI selection in expression-based microdissection (xMD) directly relies on cancer cell-specific immunostaining. Whole-slide irradiation leads to localized energy absorption at the sites of most intensive staining and melting of a membrane covering the slide, so that tumor cells can be isolated by removing the complete membrane. In this study, we optimized xMD of lung cancer tissue by enhancing staining intensity of tumor cell-specific immunostaining and processing of the stained samples. This optimized procedure did not alter DNA quality and resulted in enrichment of mutated EGFR DNA from lung adenocarcinoma specimens after xMD. We here also introduce a quality control protocol based on digital whole-slide scanning and image analysis before and after xMD to quantify selectivity and efficiency of the procedure. In summary, this study provides a workflow for xMD, adapted and tested for lung cancer tissue that can be used for lung tumor cell dissection before diagnostic or investigatory analyses. © 2017 USCAP, Inc All rights reserved.
view abstract doi: 10.1038/labinvest.2017.31 2017 • 174 Poly(2-oxazoline)-Antibiotic Conjugates with Penicillins
Schmidt, M. and Bast, L.K. and Lanfer, F. and Richter, L. and Hennes, E. and Seymen, R. and Krumm, C. and Tiller, J.C.
Bioconjugate Chemistry 28 2440-2451 (2017)The conjugation of antibiotics with polymers is rarely done, but it might be a promising alternative to low-molecular-weight derivatization. The two penicillins penicillin G (PenG) and penicillin V (PenV) were attached to the end groups of different water-soluble poly(2-oxazoline)s (POx) via their carboxylic acid function. This ester group was shown to be more stable against hydrolysis than the β-lactam ring of the penicillins. The conjugates are still antimicrobially active and up to 20 times more stable against penicillinase catalyzed hydrolysis. The antibiotic activity of the conjugates against Staphylococcus aureus in the presence of penicillinase is up to 350 times higher compared with the free antibiotics. Conjugates with a second antimicrobial function, a dodecyltrimethylammonium group (DDA-X), at the starting end of the PenG and PenV POx conjugates are more antimicrobially active than the conjugates without DDA-X and show high activity in the presence of penicillinase. For example, the conjugates DDA-X-PEtOx-PenG and DDA-X-PEtOx-PenV are 200 to 350 times more active against S. aureus in the presence of penicillinase and almost as effective as the penicillinase stable cloxacollin (Clox) under these conditions. These conjugates show even greater activity compared to cloxacollin without this enzyme present. Further, both conjugates kill Escherichia coli more effectively than PenG and Clox. © 2017 American Chemical Society.
view abstract doi: 10.1021/acs.bioconjchem.7b00424 2017 • 173 Preparation and physicochemical characterization of matrix pellets containing APIs with different solubility via extrusion process
Hegyesi, D. and Thommes, M. and Kleinebudde, P. and Sovány, T. and Kása, P., Jr. and Kelemen, A. and Pintye-Hódi, K. and Regdon, G., Jr.
Drug Development and Industrial Pharmacy 43 458-464 (2017)In this study, a multiparticulate matrix system was produced, containing two different active pharmaceutical ingredients (APIs): enalapril-maleate and hydrochlorothiazide. The critical control points of the process were investigated by means of factorial design. Beside the generally used microcrystalline cellulose, ethylcellulose was used as matrix former to achieve modified drug release ensured by diffusion. The matrix pellets were made by extrusion-spheronization using a twin-screw extruder. Some pellet properties (aspect ratio, 10% interval fraction, hardness, deformation process) were determined. The aim of our study was to investigate how the two different APIs with different solubility and particle size influence the process. The amount of the granulation liquid plays a key role in the pellet shaping. A higher liquid feed rate is preferred in the pelletization process. © 2016 Informa UK Limited, trading as Taylor & Francis Group.
view abstract doi: 10.1080/03639045.2016.1261150 2017 • 172 Standard Gibbs energy of metabolic reactions: II. Glucose-6-phosphatase reaction and ATP hydrolysis
Meurer, F. and Do, H. T. and Sadowski, G. and Held, C.
Biophysical Chemistry 223 30--38 (2017)ATP (adenosine triphosphate) is a key reaction for metabolism. Tools from systems biology require standard reaction data in order to predict metabolic pathways accurately. However, literature values for standard Gibbs energy of ATP hydrolysis are highly uncertain and differ strongly from each other. Further, such data usually neglect the activity coefficients of reacting agents, and published data like this is apparent (condition-dependent) data instead of activity-based standard data. In this work a consistent value for the standard Gibbs energy of ATP hydrolysis was determined. The activity coefficients of reacting agents were modeled with electrolyte Perturbed Chain Statistical Associating Fluid Theory (ePC-SAFT). The Gibbs energy of ATP hydrolysis was calculated by combining the standard Gibbs energies of hexokinase reaction and of glucose-6-phosphate hydrolysis. While the standard Gibbs energy of hexokinase reaction was taken from previous work, standard Gibbs energy of glucose-6-phosphate hydrolysis reaction was determined in this work. For this purpose, reaction equilibrium molalities of reacting agents were measured at pH 7 and pH 8 at 298.15 K at varying initial reacting agent molalities. The corresponding activity coefficients at experimental equilibrium molalities were predicted with ePC-SAFT yielding the Gibbs energy of glucose-6-phosphate hydrolysis of -13.72 +/- 0.75 kJ. mol(-1). Combined with the value for hexokinase, the standard Gibbs energy of ATP hydrolysis was finally found to be - 31.55 +/- 127 kJ. mol(-1). For both, ATP hydrolysis and glucose-6-phosphate hydrolysis, a good agreement with own and literature values were obtained when influences of pH, temperature, and activity coefficients were explicitly taken into account in order to calculate standard Gibbs energy at pH 7, 298.15 K and standard state. (C) 2017 Elsevier B.V. All rights reserved.
view abstract doi: 10.1016/j.bpc.2017.02.005 2017 • 171 Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK
Bührmann, M. and Wiedemann, B.M. and Müller, M.P. and Hardick, J. and Ecke, M. and Rauh, D.
PLoS ONE 12 (2017)In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography. © 2017 Bührmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
view abstract doi: 10.1371/journal.pone.0184627 2017 • 170 Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor
Engel, J. and Smith, S. and Lategahn, J. and Tumbrink, H.L. and Goebel, L. and Becker, C. and Hennes, E. and Keul, M. and Unger, A. and Müller, H. and Baumann, M. and Schultz-Fademrecht, C. and Günther, G. and Hengstler, J.G. and Rauh, D.
Journal of Medicinal Chemistry 60 7725-7744 (2017)Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile. © 2017 American Chemical Society.
view abstract doi: 10.1021/acs.jmedchem.7b00515 2017 • 169 Sunlight-Dependent Hydrogen Production by Photosensitizer/Hydrogenase Systems
Adam, D. and Bösche, L. and Castañeda-Losada, L. and Winkler, M. and Apfel, U.-P. and Happe, T.
ChemSusChem 10 894-902 (2017)We report a sustainable in vitro system for enzyme-based photohydrogen production. The [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii was tested for photohydrogen production as a proton-reducing catalyst in combination with eight different photosensitizers. Using the organic dye 5-carboxyeosin as a photosensitizer and plant-type ferredoxin PetF as an electron mediator, HydA1 achieves the highest light-driven turnover number (TONHydA1) yet reported for an enzyme-based in vitro system (2.9×106 mol(H2) mol(cat)−1) and a maximum turnover frequency (TOFHydA1) of 550 mol(H2) mol(HydA1)−1 s−1. The system is fueled very effectively by ambient daylight and can be further simplified by using 5-carboxyeosin and HydA1 as a two-component photosensitizer/biocatalyst system without an additional redox mediator. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
view abstract doi: 10.1002/cssc.201601523 2017 • 168 Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition
Beck, F. and Geiger, J. and Gambaryan, S. and Solari, F.A. and Dell'aica, M. and Loroch, S. and Mattheij, N.J. and Mindukshev, I. and Pötz, O. and Jurk, K. and Burkhart, J.M. and Fufezan, C. and Heemskerk, J.W.M. and Walter, U. a...
Blood 129 e1-e12 (2017)Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin aIIbb3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determinedhow Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPaseactivating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. © 2017 by The American Society of Hematology.
view abstract doi: 10.1182/blood-2016-05-714048 2017 • 167 Trisubstituted Pyridinylimidazoles as Potent Inhibitors of the Clinically Resistant L858R/T790M/C797S EGFR Mutant: Targeting of Both Hydrophobic Regions and the Phosphate Binding Site
Günther, M. and Lategahn, J. and Juchum, M. and Döring, E. and Keul, M. and Engel, J. and Tumbrink, H.L. and Rauh, D. and Laufer, S.
Journal of Medicinal Chemistry 60 5613-5637 (2017)Inhibition of the epidermal growth factor receptor represents one of the most promising strategies in the treatment of lung cancer. Acquired resistance compromises the clinical efficacy of EGFR inhibitors during long-term treatment. The recently discovered EGFR-C797S mutation causes resistance against third-generation EGFR inhibitors. Here we present a rational approach based on extending the inhibition profile of a p38 MAP kinase inhibitor toward mutant EGFR inhibition. We used a privileged scaffold with proven cellular potency as well as in vivo efficacy and low toxicity. Guided by molecular modeling, we synthesized and studied the structure-activity relationship of 40 compounds against clinically relevant EGFR mutants. We successfully improved the cellular EGFR inhibition down to the low nanomolar range with covalently binding inhibitors against a gefitinib resistant T790M mutant cell line. We identified additional noncovalent interactions, which allowed us to develop metabolically stable inhibitors with high activities against the osimertinib resistant L858R/T790M/C797S mutant. © 2017 American Chemical Society.
view abstract doi: 10.1021/acs.jmedchem.7b00316 2017 • 166 Uncertainty quantification and design-of-experiment in absorption-based aqueous film parameter measurements using Bayesian inference
Pan, R. and Daun, K.J. and Dreier, T. and Schulz, C.
Applied Optics 56 E1-E7 (2017)Diode laser-based multi-wavelength near-infrared (NIR) absorption in aqueous films is a promising diagnostic for making temporally resolved, simultaneous measurements of film thickness, temperature, and concentration of a solute. Our previous work in aqueous urea solutions aimed at determining simultaneously two of these system parameters, while the third one must be fixed or specified by additional measurements. The current work presents a simultaneous NIR absorption-based multi-parameter measurement of thickness, temperature, and solute concentration coupled with the Bayesian methodology that is used to infer probability densities for the obtained data. The Bayesian analysis is based on a temperature- and concentration-dependent spectral database generated with a Fourier transform infrared spectrometer in the range 5500-8000 cm-1 for water with variable temperature and urea concentration. The concept was first validated with measurements using a calibration cell. Probability densities in the measured parameters were quantified using a Markov chain Monte Carlo algorithm, which were used to derive credibility intervals. As a practical demonstration, the temporal variation of film thickness, urea concentration, and liquid temperature were recorded during evaporation of a liquid film deposited on a transparent heated quartz plate. © 2017 Optical Society of America.
view abstract doi: 10.1364/AO.56.0000E1 2016 • 165 A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii
Lefebvre-Legendre, L. and Reifschneider, O. and Kollipara, L. and Sickmann, A. and Wolters, D. and Kück, U. and Goldschmidt-Clermont, M.
Plant Journal 85 57-69 (2016)Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity. © 2015 The Authors The Plant Journal.
view abstract doi: 10.1111/tpj.13089 2016 • 164 A redox proteomics approach to investigate the mode of action of nanomaterials
Riebeling, C. and Wiemann, M. and Schnekenburger, J. and Kuhlbusch, T.A.J. and Wohlleben, W. and Luch, A. and Haase, A.
Toxicology and Applied Pharmacology 299 24-29 (2016)Numbers of engineered nanomaterials (ENMs) are steadily increasing. Therefore, alternative testing approaches with reduced costs and high predictivity suitable for high throughput screening and prioritization are urgently needed to ensure a fast and effective development of safe products. In parallel, extensive research efforts are targeted to understanding modes of action of ENMs, which may also support the development of new predictive assays. Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of ENMs. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Fluorescent dye based read-outs are most frequently used for cell testing in vitro but may be limited due to possible interference of the ENMs. Recently, other assays have been put forward such as acellular determination of ROS production potential using methods like electron spin resonance, antioxidant quantification or the use of specific sensors. In addition, Omics based approaches have gained increasing attention. In particular, redox proteomics can combine the assessment of oxidative stress with the advantage of getting more detailed mechanistic information. Here we propose a comprehensive testing strategy for assessing the oxidative stress potential of ENMs, which combines acellular methods and fast in vitro screening approaches, as well as a more involved detailed redox proteomics approach. This allows for screening and prioritization in a first tier and, if required, also for unraveling mechanistic details down to compromised signaling pathways. © 2016 Published by Elsevier Inc.
view abstract doi: 10.1016/j.taap.2016.01.019 2016 • 163 A Single Dose of the Anti-Resorptive Peptide Human Calcitonin Paradoxically Augments Particle- and Endotoxin-Mediated Pro-Inflammatory Cytokine Production in Vitro
Jablonski, H. and Wedemeyer, C. and Bachmann, H.S. and Schlagkamp, M. and Bernstein, A. and Jäger, M. and Kauther, M.D.
Hormone and Metabolic Research 48 607-612 (2016)The peptide hormone calcitonin (CT) is known to inhibit bone resorption and has previously been shown also to prevent particle-induced osteolysis, the leading cause of revision arthroplasty. In the present study, the influence of human CT on the initial inflammatory response to particulate wear debris or bacterial endotoxins, ultimately leading to osteoclast-mediated bone resorption, was analysed in human THP-1 macrophage-like cells. The cells were activated with either ultra-high molecular weight polyethylene (UHMWPE) particles or bacterial lipopolysaccharides (LPS) in order to simulate an osteolysis-associated inflammatory response. The cells were simultaneously treated with human CT (10-9 M). Cytokine production of tumour necrosis factor (TNF)-α was quantified on both RNA and protein levels while interleukins (IL)-1β and IL-6 were measured as secreted protein only. Stimulation of the cells with either particles or LPS led to a dose- and time-dependent increase of TNF-α mRNA production and protein secretion of TNF-α, IL-1β, and IL-6. Application of CT mostly enhanced cytokine production as elicited by UHMWPE particles while a pronounced transient inhibitory effect on LPS-induced inflammation became evident at 24 h of incubation. Human CT displayed ambivalent effects on the wear- and LPS-induced production of pro-inflammatory cytokines. Thereby, the peptide primarily upregulated particle-induced inflammation while LPS-induced cytokine secretion was temporarily attenuated in a distinct manner. It needs to be evaluated whether the pro- or anti-inflammatory action of CT contributes to its known anti-resorptive effects. Thus, the therapeutic potential of the peptide in the treatment of either particle- or endotoxin-mediated bone resorption could be determined.
view abstract doi: 10.1055/s-0042-108338 2016 • 162 Antifouling and Antibacterial Multifunctional Polyzwitterion/Enzyme Coating on Silicone Catheter Material Prepared by Electrostatic Layer-by-Layer Assembly
Vaterrodt, A. and Thallinger, B. and Daumann, K. and Koch, D. and Guebitz, G.M. and Ulbricht, M.
Langmuir 32 1347-1359 (2016)The formation of bacterial biofilms on indwelling medical devices generally causes high risks for adverse complications such as catheter-associated urinary tract infections. In this work, a strategy for synthesizing innovative coatings of poly(dimethylsiloxane) (PDMS) catheter material, using layer-by-layer assembly with three novel functional polymeric building blocks, is reported, i.e., an antifouling copolymer with zwitterionic and quaternary ammonium side groups, a contact biocidal derivative of that polymer with octyl groups, and the antibacterial hydrogen peroxide (H2O2) producing enzyme cellobiose dehydrogenase (CDH). CDH oxidizes oligosaccharides by transferring electrons to oxygen, resulting in the production of H2O2. The design and synthesis of random copolymers which combine segments that have antifouling properties by zwitterionic groups and can be used for electrostatically driven layer-by-layer (LbL) assembly at the same time were based on the atom-transfer radical polymerization of dimethylaminoethyl methacrylate and subsequent partial sulfobetainization with 1,3-propane sultone followed by quaternization with methyl iodide only or octyl bromide and thereafter methyl iodide. The alternating multilayer systems were formed by consecutive adsorption of the novel polycations with up to 50% zwitterionic groups and of poly(styrenesulfonate) as the polyanion. Due to its negative charge, enzyme CDH was also firmly embedded as a polyanionic layer in the multilayer system. This LbL coating procedure was first performed on prefunctionalized silicon wafers and studied in detail with ellipsometry as well as contact angle (CA) and zetapotential (ZP) measurements before it was transferred to prefunctionalized PDMS and analyzed by CA and ZP measurements as well as atomic force microscopy. The coatings comprising six layers were stable and yielded a more neutral and hydrophilic surface than did PDMS, the polycation with 50% zwitterionic groups having the largest effect. Enzyme activity was found to be dependent on the depth of embedment in the multilayer coating. Depending on the used polymeric building block, up to a 60% reduction in the amount of adhering bacteria and clear evidence for killed bacteria due to the antimicrobial functionality of the coating could be confirmed. Overall, this work demonstrates the feasibility of an easy to perform and shape-independent method for preparing an antifouling and antimicrobial coating for the significant reduction of biofilm formation and thus reducing the risk of acquiring infections by using urinary catheters. © 2016 American Chemical Society.
view abstract doi: 10.1021/acs.langmuir.5b04303 2016 • 161 Barium sulfate micro- and nanoparticles as bioinert reference material in particle toxicology
Loza, K. and Föhring, I. and Bünger, J. and Westphal, G.A. and Köller, M. and Epple, M. and Sengstock, C.
Nanotoxicology 10 1492-1502 (2016)The inhalation of particles and their exposure to the bronchi and alveoli constitute a major public health risk. Chemical as well as particle-related properties are important factors for the biological response but are difficult to separate from each other. Barium sulfate is a completely inert chemical compound, therefore it is ideally suited to separate these two factors. The biological response of rat alveolar macrophages (NR8383) was analyzed after exposure to barium sulfate particles with three different diameters (40 nm, 270 nm, and 1.3 μm, respectively) for 24 h in vitro (particle concentrations from 12.5 to 200 μg mL− 1). The particles were colloidally stabilized as well as fluorescently-labeled by carboxymethylcellulose, conjugated with 6-aminofluorescein. All kinds of barium sulfate particles were efficiently taken up by NR8383 cells and found inside endo-lysosomes, but never in the cell nucleus. Neither an inflammatory nor a cytotoxic response was detected by the ability of dHL-60 and NR8383 cells to migrate towards a chemotactic gradient (conditioned media of NR8383 cells) and by the release of inflammatory mediators (CCL2, TNF-α, IL-6). The particles neither caused apoptosis (up to 200 μg mL− 1) nor necrosis (up to 100 μg mL− 1). As only adverse reaction, necrosis was found at a concentration of 200 μg mL− 1 of the largest barium sulfate particles (1.3 μm). Barium sulfate particles are ideally suited as bioinert control to study size-dependent effects such as uptake mechanisms of intracellular distributions of pure particles, especially in nanotoxicology. © 2016 Informa UK Limited, trading as Taylor & Francis Group.
view abstract doi: 10.1080/17435390.2016.1235740 2016 • 160 Combined quantification of the global proteome, phosphoproteome, and proteolytic cleavage to characterize altered platelet functions in the human scott syndrome
Solari, F.A. and Mattheij, N.J.A. and Burkhart, J.M. and Swieringa, F. and Collins, P.W. and Cosemans, J.M.E.M. and Sickmann, A. and Heemskerk, J.W.M. and Zahedi, R.P.
Molecular and Cellular Proteomics 15 3154-3169 (2016)The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multipronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspaseregulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
view abstract doi: 10.1074/mcp.M116.060368 2016 • 159 Compatible solutes: Thermodynamic properties relevant for effective protection against osmotic stress
Held, C. and Sadowski, G.
Fluid Phase Equilibria 407 224-235 (2016)Organisms developed very different strategies to protect themselves against osmotic stress. To sustain high salt concentrations of their surrounding some organisms accumulate so-called compatible solutes (CSs), which increase the internal osmotic pressure without disturbing the organism's metabolism. At constant temperature, osmotic pressure is mainly determined by the concentration of the compatible solute and the osmotic coefficient of the aqueous solution, and to a minor extent also by solution densities. Thus, osmotic coefficients and densities were measured for aqueous CS solutions in a broad range of concentration and at three temperatures (273. K, 310. K, 323. K) at atmospheric pressure. Further, the solubility of CSs in water was measured as function of temperature to determine the maximum CS concentration that can be applied in aqueous solutions. CSs under investigation were trimethylamine N-oxide (TMAO), trehalose, citrulline, N,. N-dimethylglycine, DMSO, glycerol, methylglycine, and ectoine. The data was used to calculate real osmotic pressures induced by these CSs. PC-SAFT was applied to model thermodynamic properties and phase equilibria of aqueous CS solutions in quantitative agreement to experimental data. Among the CSs investigated in this work, TMAO induced the highest osmotic pressure and thus can be considered the best protector against osmotic stress. The data was finally analyzed concerning the influence of CSs molecular size, charge, and hydrophobicity on osmotic pressure. This included also the comparison to incompatible solutes (urea, glycine). © 2015 Elsevier B.V.
view abstract doi: 10.1016/j.fluid.2015.07.004 2016 • 158 Functional dissection of an alternatively spliced herpesvirus gene by splice site mutagenesis
Schommartz, T. and Loroch, S. and Alawi, M. and Grundhoff, A. and Sickmann, A. and Brune, W.
Journal of Virology 90 4626-4636 (2016)Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. © 2016, American Society for Microbiology. All Rights Reserved.
view abstract doi: 10.1128/JVI.02987-15 2016 • 157 Ibandronate in stable renal transplant recipients with low bone mineral density on long-term follow-up
Tillmann, F.P. and Schmitz, M. and Jäger, M. and Krauspe, R. and Rump, L.C.
International Urology and Nephrology 48 279-286 (2016)Background: Bone mineral density (BMD) has been reported to increase without specific treatment in long-term renal transplant recipients. The aim of this study was to evaluate the effect of ibandronate on BMD and kidney function in long-term renal transplant recipients as compared to a control group. Furthermore, we searched for a gender-specific treatment effect of ibandronate on BMD. Methods: In a retrospective, matched case-control study 60 stable renal transplant recipients were included on long-term follow-up. The patient cohort was divided into two groups. The control group (n = 30) comprised patients with close-to-normal bone mineral density who did not receive ibandronate treatment and the treatment group (n = 30) comprised patients with reduced bone mineral density who received ibandronate treatment. The groups were matched for sex, age at the time of renal transplantation, use of steroids, renal transplant function and time lag between the dual-energy X-ray absorptiometry (DEXA) measurements and renal transplantation. Patients of the treatment group were treated with 12.0 ± 6.7 g ibandronate. Treatment cycles lasted 19.3 ± 11.0 months. The first bone mineral density testing was performed 55.3 ± 60.2 months after renal transplantation followed by a second measurement 26.8 ± 12.1 months later. Results: Both groups did not differ in absolute (g/cm2) or relative (%) changes in BMD at the lumbar spine (0.033 ± 0.079 vs. 0.055 ± 0.066 g/cm2, p = 0.217 and 3.6 ± 7.8 vs. 6.4 ± 8.1 %, p = 0.124) or femoral neck (0.013 ± 0.106 vs. 0.025 ± 0.077 g/cm2, p = 0.647 and 3.2 ± 13.6 vs. 5.0 ± 13.1 %, p = 0.544) over the study period. There was no correlation of ibandronate dosages with changes in BMD (LS: r = −0.089; p = 0.639 and FN: r =+0.288; p = 0.445). We could neither determine a negative effect of ibandronate on renal transplant function over the study period, estimated via the CKD-EPI formula (−2.9 ± 7.6 vs. −2.7 ± 10.6 mL/min/1.73 m2, p = 0.900) nor a gender-specific action of ibandronate on bone mass changes. Conclusions: Ibandronate treatment was safe with respect to renal transplant function but did not result in a significant additive improvement in bone mineral density as compared to the untreated control group. A gender-specific action of ibandronate on BMD at the LS or FN could not be determined either. © 2015, Springer Science+Business Media Dordrecht.
view abstract doi: 10.1007/s11255-015-1133-7 2016 • 156 Inclusion of mPRISM potential for polymer-induced protein interactions enables modeling of second osmotic virial coefficients in aqueous polymer-salt solutions
Herhut, M. and Brandenbusch, C. and Sadowski, G.
Biotechnology Journal 11 146-154 (2016)The downstream processing of therapeutic proteins is a challenging task. Key information needed to estimate applicable workup strategies (e.g. crystallization) are the interactions of the proteins with other components in solution. This information can be deduced from the second osmotic virial coefficient B22, measurable by static light scattering. Thermodynamic models are very valuable for predicting B22 data for different process conditions and thus decrease the experimental effort. Available B22 models consider aqueous salt solutions but fail for the prediction of B22 if an additional polymer is present in solution. This is due to the fact that depending on the polymer concentration protein-protein interactions are not rectified as assumed within these models. In this work, we developed an extension of the xDLVO model to predict B22 data of proteins in aqueous polymer-salt solutions. To show the broad applicability of the model, lysozyme, γ-globulin and D-xylose ketol isomerase in aqueous salt solution containing polyethylene glycol were considered. For all proteins considered, the modified xDLVO model was able to predict the experimentally observed non-monotonical course in B22 data with high accuracy. When used in an early stage in process development, the model will contribute to an efficient and cost effective downstream processing development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/biot.201500086 2016 • 155 Influence of agglomeration and specific lung lining lipid/protein interaction on short-term inhalation toxicity
Wohlleben, W. and Driessen, M.D. and Raesch, S. and Schaefer, U.F. and Schulze, C. and Vacano, B.V. and Vennemann, A. and Wiemann, M. and Ruge, C.A. and Platsch, H. and Mues, S. and Ossig, R. and Tomm, J.M. and Schnekenburger, J. ...
Nanotoxicology 10 970-980 (2016)Abstract: Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurfTM and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurfTM promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs. © 2016 Informa UK Limited, trading as Taylor & Francis Group.
view abstract doi: 10.3109/17435390.2016.1155671 2016 • 154 Ions and solvation at biointerfaces
Valtiner, M. and Erbe, A. and Rosenhahn, A.
Biointerphases 11 (2016)
doi: 10.1116/1.4942207 2016 • 153 Kinetics of chemotaxis, cytokine, and chemokine release of NR8383 macrophages after exposure to inflammatory and inert granular insoluble particles
Schremmer, I. and Brik, A. and Weber, D.G. and Rosenkranz, N. and Rostek, A. and Loza, K. and Brüning, T. and Johnen, G. and Epple, M. and Bünger, J. and Westphal, G.A.
Toxicology Letters 263 68-75 (2016)Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16 h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16 h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles. © 2016 Elsevier Ireland Ltd
view abstract doi: 10.1016/j.toxlet.2016.08.014 2016 • 152 Measurements of liquid film thickness, concentration, and temperature of aqueous urea solution by NIR absorption spectroscopy
Pan, R. and Jeffries, J.B. and Dreier, T. and Schulz, C.
Applied Physics B: Lasers and Optics 122 (2016)A multi-wavelength near-infrared (NIR) diode laser absorption sensor has been developed and demonstrated for real-time monitoring of the thickness, solute concentration, and temperature of thin films of urea–water solutions. The sensor monitors the transmittance of three near-infrared diode lasers through the thin liquid film. Film thickness, urea mass fraction, and liquid temperature were determined from measured transmittance ratios of suitable combinations of lasers. Available laser wavelengths were selected depending on the variation of the NIR absorption spectrum of the solution with temperature and solute concentration. The spectral database was measured by a Fourier transform infrared spectrometer in the range 5500–8000 cm−1 for urea solutions between 5 and 40 wt% and temperatures between 298 and 338 K. A prototype sensor was constructed, and the sensor concept was first validated with measurements using a calibration cell providing liquid layers of variable thickness (200–1500 μm), urea mass fraction (5–40 wt%) and temperature (298–318 K). Temporal variations of film thickness and urea concentration were captured during the constant-temperature evaporation of a liquid film deposited on an optically polished heated quartz flat. © Springer-Verlag Berlin Heidelberg 2016.
view abstract doi: 10.1007/s00340-015-6290-y 2016 • 151 Monitoring Conformational Changes in the Receptor Tyrosine Kinase EGFR
Becker, C. and Öcal, S. and Nguyen, H.D. and Phan, T. and Keul, M. and Simard, J.R. and Rauh, D.
ChemBioChem 17 990-994 (2016)The receptor tyrosine kinase EGFR is regulated by complex conformational changes, and this conformational control is disturbed in certain types of cancer. Many ligands are known to bind EGFR in its active conformation, thereby preventing ATP from binding. Only a few ligands are known to stabilize EGFR in its inactive conformation, thus providing novel strategies for perturbing EGFR activity. We report a direct binding assay that enables the identification of novel ligands that bind to and stabilize the inactive conformation of EGFR. Too active? Stabilize the inactive conformation. EGFR is a target for tumor therapy, with many small molecules known to bind its kinase domain in the active conformation. A binding assay was developed to search for ligands stabilizing its inactive conformation, thus providing a tool for new approaches to target its dynamic regulatory mechanisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/cbic.201600115 2016 • 150 Neurotoxicity of the parkinson disease-associated pesticide ziram is synuclein-dependent in zebrafish embryos
Lulla, A. and Barnhill, L. and Bitan, G. and Ivanova, M.I. and Nguyen, B. and O’Donnell, K. and Stahl, M.C. and Yamashiro, C. and Klärner, F.-G. and Schrader, T. and Sagasti, A. and Bronstein, J.M.
Environmental Health Perspectives 124 1766-1775 (2016)Background: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood. Objective: We studied the mechanisms of ziram’s (a DTC fungicide) neurotoxicity in vivo. Methods: Zebrafish (ZF) embryos were utilized to determine ziram’s effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity. Results: Nanomolar-range concentrations of ziram caused selective loss of dopaminergic (DA) neurons and impaired swimming behavior. Because ziram increases α-synuclein (α-syn) concentrations in rat primary neuronal cultures, we investigated the effect of ziram on ZF γ-synuclein 1 (γ1). ZF express 3 synuclein isoforms, and ZF γ1 appears to be the closest functional homologue to α-syn. We found that recombinant ZF γ1 formed fibrils in vitro, and overexpression of ZF γ1 in ZF embryos led to the formation of neuronal aggregates and neurotoxicity in a manner similar to that of α-syn. Importantly, knockdown of ZF γ1 with morpholinos and disruption of oligomers with the molecular tweezer CLR01 prevented ziram’s DA toxicity. Conclusions: These data show that ziram is selectively toxic to DA neurons in vivo, and this toxicity is synuclein-dependent. These findings have important implications for understanding the mechanisms by which pesticides may cause PD. © 2016, Public Health Services, US Dept of Health and Human Services. All rights reserved.
view abstract doi: 10.1289/EHP141 2016 • 149 Non-monotonic course of protein solubility in aqueous polymer-salt solutions can be modeled using the sol-mxDLVO model
Herhut, M. and Brandenbusch, C. and Sadowski, G.
Biotechnology Journal 11 282-289 (2016)Protein purification is often performed using cost-intensive chromatographic steps. To discover economic alternatives (e.g., crystallization), knowledge on protein solubility as a function of temperature, pH, and additives in solution as well as their concentration is required. State-of-the-art models for predicting protein solubility almost exclusively consider aqueous salt systems, whereas "salting-in" and "salting-out" effects induced by the presence of an additional polymer are not considered. Thus, we developed the sol-mxDLVO model. Using this newly developed model, protein solubility in the presence of one salt and one polymer, especially the non-monotonic course of protein solubility, could be predicted. Systems considered included salts (NaCl, Na-p-Ts, (NH4)2SO4) and the polymer polyethylene glycol (MW: 2000 g/mol, 12000 g/mol) and proteins lysozyme from chicken egg white (pH 4 to 5.5) and D-xylose ketol-isomerase (pH 7) at 298.15 K. The results show that by using the sol-mxDLVO model, protein solubility in polymer-salt solutions can be modeled in good agreement with the experimental data for both proteins considered. The sol-mxDLVO model can describe the non-monotonic course of protein solubility as a function of polymer concentration and salt concentration, previously not covered by state-of-the-art models. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/biot.201500123 2016 • 148 Optical and electron microscopy study of laser-based intracellular molecule delivery using peptide-conjugated photodispersible gold nanoparticle agglomerates
Krawinkel, J. and Richter, U. and Torres-Mapa, M.L. and Westermann, M. and Gamrad, L. and Rehbock, C. and Barcikowski, S. and Heisterkamp, A.
Journal of Nanobiotechnology 14 (2016)Background: Cell-penetrating peptides (CPPs) can act as carriers for therapeutic molecules such as drugs and genetic constructs for medical applications. The triggered release of the molecule into the cytoplasm can be crucial to its effective delivery. Hence, we implemented and characterized laser interaction with defined gold nanoparticle agglomerates conjugated to CPPs which enables efficient endosomal rupture and intracellular release of molecules transported. Results: Gold nanoparticles generated by pulsed laser ablation in liquid were conjugated with CPPs forming agglomerates and the intracellular release of molecules was triggered via pulsed laser irradiation (λ = 532 nm, τpulse = 1 ns). The CPPs enhance the uptake of the agglomerates along with the cargo which can be co-incubated with the agglomerates. The interaction of incident laser light with gold nanoparticle agglomerates leads to heat deposition and field enhancement in the vicinity of the particles. This highly precise effect deagglomerates the nanoparticles and disrupts the enclosing endosomal membrane. Transmission electron microscopy images confirmed this rupture for radiant exposures of 25 mJ/cm2 and above. Successful intracellular release was shown using the fluorescent dye calcein. For a radiant exposure of 35 mJ/cm2 we found calcein delivery in 81 % of the treated cells while maintaining a high percentage of cell viability. Furthermore, cell proliferation and metabolic activity were not reduced 72 h after the treatment. Conclusion: CPPs trigger the uptake of the gold nanoparticle agglomerates via endocytosis and co-resident molecules in the endosomes are released by applying laser irradiation, preventing their intraendosomal degradation. Due to the highly localized effect, the cell membrane integrity is not affected. Therefore, this technique can be an efficient tool for spatially and temporally confined intracellular release. The utilization of specifically designed photodispersible gold nanoparticle agglomerates (65 nm) can open novel avenues in imaging and molecule delivery. Due to the induced deagglomeration the primary, small particles (~5 nm) are more likely to be removed from the body. © 2016 Krawinkel et al.
view abstract doi: 10.1186/s12951-015-0155-8 2016 • 147 Osteogenic differentiation and proliferation of bone marrow-derived mesenchymal stromal cells on PDLLA + BMP-2-coated titanium alloy surfaces
Haversath, M. and Hülsen, T. and Böge, C. and Tassemeier, T. and Landgraeber, S. and Herten, M. and Warwas, S. and Krauspe, R. and Jäger, M.
Journal of Biomedical Materials Research - Part A 104 145-154 (2016)RhBMP-2 is clinically applied to enhance bone healing and used in combination with titanium fixation implants. The purpose of this in vitro study was to compare the osteogenic differentiation and proliferation of hMSC on native polished versus sandblasted titanium surfaces (TS) and to test their behavior on pure poly-D,L-lactide (PDLLA) coated as well as PDLLA + rhBMP-2 coated TS. Furthermore, the release kinetics of PDLLA + rhBMP-2-coated TS was investigated. Human bone marrow cells were obtained from three different donors (A: male, 16 yrs; B: male, 27 yrs, C: male, 49 yrs) followed by density gradient centrifugation and flow cytometry with defined antigens. The cells were seeded on native polished and sandblasted TS, PDLLA-coated TS and PDLLA + rhBMP-2-coated TS. Osteogenic differentiation (ALP specific activity via ALP and BCA assay) and proliferation (LDH cytotoxicity assay) was examined on day 7 and 14 and release kinetics of rhBMP-2 was investigated on day 3, 7, 10, and 14. We found significant higher ALP specific activity and LDH activity on native polished compared to native sandblasted surfaces. PDLLA led to decreased ALP specific and LDH activity on both surface finishes. Additional rhBMP-2 slightly diminished this effect. RhBMP-2-release from coated TS decreased nearly exponentially with highest concentrations at the beginning of the cultivation period. The results of this in vitro study suggest that native TS stimulate hMSC significantly stronger toward osteogenic differentiation and proliferation than rhBMP-2 + PDLLA-layered TS in the first 14 days of cultivation. The PDLLA-layer seems to inhibit local hMSC differentiation and proliferation. © 2015 Wiley Periodicals, Inc.
view abstract doi: 10.1002/jbm.a.35550 2016 • 146 Reducing synuclein accumulation improves neuronal survival after spinal cord injury
Fogerson, S.M. and van Brummen, A.J. and Busch, D.J. and Allen, S.R. and Roychaudhuri, R. and Banks, S.M.L. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Morgan, J.R.
Experimental Neurology 278 105-115 (2016)Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury. © 2016 Elsevier Inc.
view abstract doi: 10.1016/j.expneurol.2016.02.004 2016 • 145 Simultaneous measurements of photocurrents and H2O2 evolution from solvent exposed photosystem 2 complexes
Vöpel, T. and Saw, E.N. and Hartmann, V. and Williams, R. and Müller, F. and Schuhmann, W. and Plumeré, N. and Nowaczyk, M. and Ebbinghaus, S. and Rögner, M.
Biointerphases 11 019001 (2016)In plants, algae, and cyanobacteria, photosystem 2 (PS2) catalyzes the light driven oxidation of water. The main products of this reaction are protons and molecular oxygen. In vitro, however, it was demonstrated that reactive oxygen species like hydrogen peroxide are obtained as partially reduced side products. The transition from oxygen to hydrogen peroxide evolution might be induced by light triggered degradation of PS2’s active center. Herein, the authors propose an analytical approach to investigate light induced bioelectrocatalytic processes such as PS2 catalyzed water splitting. By combining chronoamperometry and fluorescence microscopy, the authors can simultaneously monitor the photocurrent and the hydrogen peroxide evolution of light activated, solvent exposed PS2 complexes, which have been immobilized on a functionalized gold electrode. The authors show that under limited electron mediation PS2 displays a lower photostability that correlates with an enhanced H2O2 generation as a side product of the light induced water oxidation. © 2015 American Vacuum Society.
view abstract doi: 10.1116/1.4938090 2016 • 144 Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis
Zhang, Y. and Clausmeyer, J. and Babakinejad, B. and López Córdoba, A. and Ali, T. and Shevchuk, A. and Takahashi, Y. and Novak, P. and Edwards, C. and Lab, M. and Gopal, S. and Chiappini, C. and Anand, U. and Magnani, L. and Co...
ACS Nano 10 3214-3221 (2016)Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements. © 2016 American Chemical Society.
view abstract doi: 10.1021/acsnano.5b05211 2016 • 143 Standard Gibbs Energy of Metabolic Reactions: I. Hexokinase Reaction
Meurer, F. and Bobrownik, M. and Sadowski, G. and Held, C.
Biochemistry 55 5665-5674 (2016)The standard Gibbs energy of reaction enables calculation of the driving force of a (bio)chemical reaction. Gibbs energies of reaction are required in thermodynamic approaches to determine fluxes as well as single reaction conversions of metabolic bioreactions. The hexokinase reaction (phosphorylation of glucose) is the entrance step of glycolysis, and thus its standard Gibbs energy of reaction (ΔRg°) is of great impact. ΔRg° is accessible from equilibrium measurements, and the very small concentrations of the reacting agents cause usually high error bars in data reduction steps. Even worse, works from literature do not account for the nonideal behavior of the reacting agents (activity coefficients were assumed to be unity); thus published ΔRg° values are not standard data. Consistent treatment of activity coefficients of reacting agents is crucial for the accurate determination of standard Gibbs energy from equilibrium measurements. In this work, equilibrium molalities of hexokinase reaction were measured with an enzyme kit. These results were combined with reacting agents' activity coefficients obtained with the thermodynamic model ePC-SAFT. Pure-component parameters for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were fitted to experimental osmotic coefficients (water + Na2ATP, water + NaADP). ΔRg° of the hexokinase reaction at 298.15 K and pH 7 was found to be -17.83 ± 0.52 kJ·mol-1. This value was compared with experimental literature data; very good agreement between the different ΔRg° values was obtained by accounting for pH, pMg, and the activity coefficients of the reacting agents. © 2016 American Chemical Society.
view abstract doi: 10.1021/acs.biochem.6b00471 2016 • 142 Thermodynamics of Bioreactions
Held, C. and Sadowski, G.
Annual Review of Chemical and Biomolecular Engineering 7 395-414 (2016)Thermodynamic principles have been applied to enzyme-catalyzed reactions since the beginning of the 1930s in an attempt to understand metabolic pathways. Currently, thermodynamics is also applied to the design and analysis of biotechnological processes. The key thermodynamic quantity is the Gibbs energy of reaction, which must be negative for a reaction to occur spontaneously. However, the application of thermodynamic feasibility studies sometimes yields positive Gibbs energies of reaction even for reactions that are known to occur spontaneously, such as glycolysis. This article reviews the application of thermodynamics in enzyme-catalyzed reactions. It summarizes the basic thermodynamic relationships used for describing the Gibbs energy of reaction and also refers to the nonuniform application of these relationships in the literature. The review summarizes state-of-the-art approaches that describe the influence of temperature, pH, electrolytes, solvents, and concentrations of reacting agents on the Gibbs energy of reaction and, therefore, on the feasibility and yield of biological reactions. Copyright © 2016 by Annual Reviews. All rights reserved.
view abstract doi: 10.1146/annurev-chembioeng-080615-034704 2016 • 141 Two birds with one stone: Parallel quantifi cation of proteome and phosphoproteome using iTRAQ
Solari, F.A. and Kollipara, L. and Sickmann, A. and Zahedi, R.P.
Methods in Molecular Biology 1394 25-41 (2016)Altered and abnormal levels of proteins and their phosphorylation states are associated with many disorders. Detection and quantification of such perturbations may provide a better understanding of pathological conditions and help finding candidates for treatment or biomarkers. Over the years, isobaric mass tags for relative quantification of proteins and protein phosphorylation by mass spectrometry have become increasingly popular. One of the most commonly used isobaric chemical tags is iTRAQ (isobaric tag for relative and absolute quantitation). In a typical iTRAQ-8plex experiment, a multiplexed sample amounts for up to 800 μg of peptides. Using state-of-the-art LC-MS approaches, only a fraction (~5 %) of such a sample is required to generate comprehensive quantitative data on the global proteome level, so that the bulk of the sample can be simultaneously used for quantitative phosphoproteomics. Here, we provide a simple and straightforward protocol to perform quantitative analyses of both proteome and phosphoproteome from the same sample using iTRAQ. © Springer Science+Business Media New York 2016.
view abstract doi: 10.1007/978-1-4939-3341-9_3 2016 • 140 Wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces via entrapment in low potential phenothiazine-modified redox polymers
Pinyou, P. and Ruff, A. and Pöller, S. and Alsaoub, S. and Leimkühler, S. and Wollenberger, U. and Schuhmann, W.
Bioelectrochemistry 109 24-30 (2016)Phenothiazine-modified redox hydrogels were synthesized and used for the wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces. The effects of the pH value and electrode surface modification on the biocatalytic activity of the layers were studied in the presence of vanillin as the substrate. The enzyme electrodes were successfully employed as bioanodes in vanillin/O2 biofuel cells in combination with a high potential bilirubin oxidase biocathode. Open circuit voltages of around 700mV could be obtained in a two compartment biofuel cell setup. Moreover, the use of a rather hydrophobic polymer with a high degree of crosslinking sites ensures the formation of stable polymer/enzyme films which were successfully used as bioanode in membrane-less biofuel cells. © 2015 Elsevier B.V.
view abstract doi: 10.1016/j.bioelechem.2015.12.005 2015 • 139 A Redox Hydrogel Protects the O2-Sensitive [FeFe]-Hydrogenase from Chlamydomonas reinhardtii from Oxidative Damage
Oughli, A.A. and Conzuelo, F. and Winkler, M. and Happe, T. and Lubitz, W. and Schuhmann, W. and Rüdiger, O. and Plumeré, N.
Angewandte Chemie - International Edition 54 12329-12333 (2015)The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2. [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2. Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2, demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2/O2 mixed feed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/anie.201502776 2015 • 138 A Synergistic Interaction between Chk1- and MK2 Inhibitors in KRAS-Mutant Cancer
Dietlein, F. and Kalb, B. and Jokic, M. and Noll, E.M. and Strong, A. and Tharun, L. and Ozretić, L. and Künstlinger, H. and Kambartel, K. and Randerath, W.J. and Jüngst, C. and Schmitt, A. and Torgovnick, A. and Richters, A. a...
Cell 162 146-159 (2015)Summary KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers. © 2015 Elsevier Inc.
view abstract doi: 10.1016/j.cell.2015.05.053 2015 • 137 Amyloid β-Protein Assembly: The Effect of Molecular Tweezers CLR01 and CLR03
Zheng, X. and Liu, D. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Bowers, M.T.
Journal of Physical Chemistry B 119 4831-4841 (2015)The early oligomerization of amyloid β-protein (Aβ) has been shown to be an important event in the pathology of Alzheimer's disease (AD). Designing small molecule inhibitors targeting Aβ oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on Aβ self-assembly. CLR01 was found to bind to Aβ directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of Aβ42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early Aβ oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and Aβ oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment. (Graph Presented). © 2015 American Chemical Society.
view abstract doi: 10.1021/acs.jpcb.5b00692 2015 • 136 Associations between three specific a-cellular measures of the oxidative potential of particulate matter and markers of acute airway and nasal inflammation in healthy volunteers
Janssen, N.A.H. and Strak, M. and Yang, A. and Hellack, B. and Kelly, F.J. and Kuhlbusch, T.A.J. and Harrison, R.M. and Brunekreef, B. and Cassee, F.R. and Steenhof, M. and Hoek, G.
Occupational and Environmental Medicine 72 49-56 (2015)Introduction We evaluated associations between three a-cellular measures of the oxidative potential (OP) of particulate matter (PM) and acute health effects. Methods We exposed 31 volunteers for 5 h to ambient air pollution at five locations: an underground train station, two traffic sites, a farm and an urban background site. Each volunteer visited at least three sites. We conducted health measurements before exposure, 2 h after exposure and the next morning. We measured air pollution on site and characterised the OP of PM2.5 and PM10 using three a-cellular assays; dithiotreitol (OPDTT), electron spin resonance (OPESR) and ascorbic acid depletion (OPAA). Results In single-pollutant models, all measures of OP were significantly associated with increases in fractional exhaled nitric oxide and increases in interleukin-6 in nasal lavage 2 h after exposure. These OP associations remained significant after adjustment for co-pollutants when only the four outdoor sites were included, but lost significance when measurements at the underground site were included. Other health end points including lung function and vascular inflammatory and coagulation parameters in blood were not consistently associated with OP. Conclusions We found significant associations between three a-cellular measures of OP of PM and markers of airway and nasal inflammation. However, consistency of these effects in two-pollutant models depended on how measurements at the underground site were considered. Lung function and vascular inflammatory and coagulation parameters in blood were not consistently associated with OP. Our study, therefore, provides limited support for a role of OP in predicting acute health effects of PM in healthy young adults.
view abstract doi: 10.1136/oemed-2014-102303 2015 • 135 Calcitonin gene-related peptide modulates the production of pro-inflammatory cytokines associated with periprosthetic osteolysis by THP-1 macrophage-like cells
Jablonski, H. and Kauther, M.D. and Bachmann, H.S. and Jäger, M. and Wedemeyer, C.
NeuroImmunoModulation 22 152-165 (2015)Objective: An anti-resorptive impact of the neuropeptide calcitonin gene-related peptide (CGRP) on periprosthetic osteolysis, the leading cause of early prosthesis loosening, has been shown previously. In this study, the impact of CGRP on pro-inflammatory cytokine production associated with periprosthetic osteolysis was analysed using THP-1 macrophage-like cells. Methods: Cells were stimulated with ultra-high-molecular-weight polyethylene (UHMWPE) particles (cell-to-particle ratios of 1:100 and 1:500) and lipopolysaccharides (LPS; 1 μg/ml) to establish osteolytic conditions, and simultaneously treated with CGRP (10-8M). Receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and tumour necrosis factor (TNF)-α mRNA expression were measured by quantitative RT-PCR. RANK protein was detected by Western blot. Secreted protein levels of TNF-α as well as interleukin (IL)-1β and IL-6 were quantified in cell culture supernatants by ELISA and Bio-Plex cytokine assay, respectively. Results: Activation of macrophage-like cells failed to enhance the production of RANK but led to a dose- and time-dependent increase of TNF-α mRNA and secreted protein levels of TNF-α, IL-1β and IL-6. Application of CGRP time-dependently suppressed TNF-α mRNA expression induced by low-particle concentrations and LPS, while both particle- and LPS-induced secretion of TNF-α was inhibited. A pronounced inhibitory effect of CGRP on LPS-induced cytokine production at 24 h of incubation was also observed with IL-1β and IL-6. Conclusions: CGRP shows a time-dependent inhibitory effect on the secretion of osteolysis-associated pro-inflammatory cytokines, indicating an indirect anti-resorptive influence of the neuropeptide on both aseptic prosthesis loosening and bacterially induced bone resorption which might enhance the life time of total joint replacements. © 2014 S. Karger AG, Basel.
view abstract doi: 10.1159/000360988 2015 • 134 Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts
Hadjicharalambous, C. and Kozlova, D. and Sokolova, V. and Epple, M. and Chatzinikolaidou, M.
Journal of Biomedical Materials Research - Part A 103 3834-3842 (2015)Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells. © 2015 Wiley Periodicals, Inc.
view abstract doi: 10.1002/jbm.a.35527 2015 • 133 Combining X-ray crystallography and molecular modeling toward the optimization of pyrazolo[3,4-d ]pyrimidines as potent c-Src inhibitors active in vivo against neuroblastoma
Tintori, C. and Fallacara, A.L. and Radi, M. and Zamperini, C. and Dreassi, E. and Crespan, E. and Maga, G. and Schenone, S. and Musumeci, F. and Brullo, C. and Richters, A. and Gasparrini, F. and Angelucci, A. and Festuccia, C. a...
Journal of Medicinal Chemistry 58 347-361 (2015)c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells. © 2014 American Chemical Society.
view abstract doi: 10.1021/jm5013159 2015 • 132 Controlling the charge of pH-responsive redox hydrogels by means of redox-silent biocatalytic processes. A biocatalytic off/on switch
Contin, A. and Plumeré, N. and Schuhmann, W.
Electrochemistry Communications 51 50-53 (2015)Coupling of redox-silent biocatalytic processes for analyte detection with enzyme-catalyzed redox reactions for signal generation is proposed by the modulation of electrostatic interactions between a pH-responsive polymer and a redox enzyme to control the off-on transition for electrochemical signal generation. Glassy carbon electrodes are modified with a poly(vinyl)imidazole Os(bipyridine)2Cl redox hydrogel film entrapping urease and PQQ-dependent glucose dehydrogenase, while glucose is present in the solution. The off-on transition is based on the detection of urea as model analyte which is hydrolyzed to ammonia by urease within the hydrogel film concomitantly increasing the local pH value thus invoking deprotonation of the imidazole groups at the polymer backbone. The decrease of positive charges at the polymer decreases electrostatic repulsion between the polymer and the positively charged PQQ-dependent glucose dehydrogenase. Hence, electron transfer rates between polymer-bound Os complexes and PQQ inside the enzyme are enhanced activating electrocatalytic oxidation of glucose. This process generates the electrochemical signal for urea detection. © 2014 Elsevier B.V.
view abstract doi: 10.1016/j.elecom.2014.12.001 2015 • 131 Coupling of an enzymatic biofuel cell to an electrochemical cell for self-powered glucose sensing with optical readout
Pinyou, P. and Conzuelo, F. and Sliozberg, K. and Vivekananthan, J. and Contin, A. and Pöller, S. and Plumeré, N. and Schuhmann, W.
Bioelectrochemistry 106 22-27 (2015)A miniaturized biofuel cell (BFC) is powering an electrolyser invoking a glucose concentration dependent formation of a dye which can be determined spectrophotometrically. This strategy enables instrument free analyte detection using the analyte-dependent BFC current for triggering an optical read-out system. A screen-printed electrode (SPE) was used for the immobilization of the enzymes glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) for the biocatalytic oxidation of glucose and reduction of molecular oxygen, respectively. The miniaturized BFC was switched-on using small sample volumes (ca. 60μL) leading to an open-circuit voltage of 567mV and a maximal power density of (6.8±0.6) μWcm-2. The BFC power was proportional to the glucose concentration in a range from 0.1 to 1.0mM (R2=0.991). In order to verify the potential instrument-free analyte detection the BFC was directly connected to an electrochemical cell comprised of an optically-transparent SPE modified with methylene green (MG). The reduction of the electrochromic reporter compound invoked by the voltage and current flow applied by the BFC let to MG discoloration, thus allowing the detection of glucose. © 2015 Elsevier B.V..
view abstract doi: 10.1016/j.bioelechem.2015.04.003 2015 • 130 Covalent-Allosteric Kinase Inhibitors
Weisner, J. and Gontla, R. and Van der westhuizen, L. and Oeck, S. and Ketzer, J. and Janning, P. and Richters, A. and Mühlenberg, T. and Fang, Z. and Taher, A. and Jendrossek, V. and Pelly, S.C. and Bauer, S. and Van otterlo, W....
Angewandte Chemie - International Edition 54 10313-10316 (2015)Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/anie.201502142 2015 • 129 Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III
Björkqvist, J. and De Maat, S. and Lewandrowski, U. and Di Gennaro, A. and Oschatz, C. and Schönig, K. and Nöthen, M.M. and Drouet, C. and Braley, H. and Nolte, M.W. and Sickmann, A. and Panousis, C. and Maas, C. and Renné, T.
Journal of Clinical Investigation 125 3132-3146 (2015)Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes. © 2015, American Society for Clinical Investigation. All rights reserved.
view abstract doi: 10.1172/JCI77139 2015 • 128 Electrochemical detection of single E. coli bacteria labeled with silver nanoparticles
Sepunaru, L. and Tschulik, K. and Batchelor-McAuley, C. and Gavish, R. and Compton, R.G.
Biomaterials Science 3 816-820 (2015)A proof-of-concept for the electrochemical detection of single Escherichia coli bacteria decorated with silver nanoparticles is reported. Impacts of bacteria with an electrode - held at a suitably oxidizing potential - lead to an accompanying burst of current with each collision event. The frequency of impacts scales with the concentration of bacteria and the charge indicates the extent of decoration. © The Royal Society of Chemistry.
view abstract doi: 10.1039/c5bm00114e 2015 • 127 Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label
Zimdars, A. and Gebala, M. and Hartwich, G. and Neugebauer, S. and Schuhmann, W.
Talanta 143 19-26 (2015)Abstract The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. © 2015 Elsevier B.V.
view abstract doi: 10.1016/j.talanta.2015.04.041 2015 • 126 Extracellular Matrix Glycoprotein-Derived Synthetic Peptides Differentially Modulate Glioma and Sarcoma Cell Migration
Brösicke, N. and Sallouh, M. and Prior, L.-M. and Job, A. and Weberskirch, R. and Faissner, A.
Cellular and Molecular Neurobiology 35 741-753 (2015)Glycoproteins of the extracellular matrix (ECM) regulate proliferation, migration, and differentiation in numerous cell lineages. ECM functions are initiated by small peptide sequences embedded in large constituents that are recognized by specific cellular receptors. In this study, we have investigated the biological effects of peptides derived from collagen type IV and tenascin-C compared to the well-known RGD peptide originally discovered in fibronectin. The influence of glycoproteins and corresponding peptides on the migration of the glioma cell lines U-251-MG and U-373-MG and the sarcoma line S-117 was studied. When the cell lines were tested in a modified Boyden chamber assay on filters coated with the ECM glycoproteins, glioma cells showed a strong migration response on tenascin-C and the basal lamina constituent collagen IV, in contrast to S-117 cells. In order to identify relevant stimulatory motifs, peptides derived from fibronectin (6NHX-GRGDSF), tenascin-C (TN-C, VSWRAPTA), and collagen type IV (MNYYSNS) were compared, either applied in solution in combination with ECM glycoprotein substrates, in solution in the presence of untreated membranes, or coated on the filters of the Boyden chambers. Using this strategy, we could identify the novel tenascin-C-derived peptide motif VSWRAPTA as a migration stimulus for glioma cells. Furthermore, while kin peptides generally blocked the effects of the respective homologous ECM proteins, unexpected effects were observed in heterologous situations. There, in several cases, addition of soluble peptides strongly boosted the response to the coated ECM proteins. We propose that peptides may synergize or antagonize each other by stimulating different signaling pathways. © 2015, Springer Science+Business Media New York.
view abstract doi: 10.1007/s10571-015-0170-1 2015 • 125 Identification and further development of potent TBK1 inhibitors
Richters, A. and Basu, D. and Engel, J. and Ercanoglu, M.S. and Balke-Want, H. and Tesch, R. and Thomas, R.K. and Rauh, D.
ACS Chemical Biology 10 289-298 (2015)The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules. © 2014 American Chemical Society.
view abstract doi: 10.1021/cb500908d 2015 • 124 Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro
Mahli, A. and Thasler, W.E. and Patsenker, E. and Müller, S. and Stickel, F. and Müller, M. and Seitz, H.K. and Cederbaum, A.I. and Hellerbrand, C.
Oncotarget 6 41464-41478 (2015)Clinical studies propose a causative link between the consumption of alcohol and the development and progression of liver disease in obese individuals. However, it is incompletely understood how alcohol and obesity interact and whether the combined effects are additive or synergistic. In this study, we developed an in vitro model to address this question. Lipid accumulation in primary human hepatocytes was induced by incubation with oleic acid. Subsequently, steatotic and control hepatocytes were incubated with up to 50 mM alcohol. This alcohol concentration on its own revealed only minimal effects but significantly enhanced oleate-induced lipogenesis and cellular triglyceride content compared to control cells. Similarly, lipid peroxidation, oxidative stress and pro-inflammatory gene expression as well as CYP2E1 levels and activity were synergistically induced by alcohol and steatosis. CYP2E1 inhibition blunted these synergistic pathological effects. Notably, alcohol and cellular steatosis also induced autophagy in a synergistic manner, and also this was mediated via CYP2E1. Further induction of autophagy ameliorated the joint effects of alcohol and oleic acid on hepatocellular lipid accumulation and inflammatory gene expression while inhibition of autophagy further enhanced the dual pathological effects. Further analyses revealed that the joint synergistic effect of alcohol and steatosis on autophagy was mediated via activation of the JNK-pathway. In summary, our data indicate that alcohol induces not only pathological but also protective mechanisms in steatotic hepatocytes via CYP2E1. These findings may have important implications on the prognosis and treatment of alcoholic liver disease particularly in obese individuals.
view abstract doi: 10.18632/oncotarget.6203 2015 • 123 Identification of proteins involved in inhibition of spheroid formation under microgravity
Riwaldt, S. and Pietsch, J. and Sickmann, A. and Bauer, J. and Braun, M. and Segerer, J. and Schwarzwälder, A. and Aleshcheva, G. and Corydon, T.J. and Infanger, M. and Grimm, D.
Proteomics 15 2945-2952 (2015)Many types of cells transit in vitro from a two- to a three-dimensional growth, when they are exposed to microgravity. The underlying mechanisms are not yet understood. Hence, we investigated the impact of microgravity on protein content and growth behavior. For this purpose, the human thyroid cancer cells FTC-133 were seeded either in recently developed cell containers that can endure enhanced physical forces and perform media changes and cell harvesting automatically or in T-25 culture flasks. All cells were cultured for five days at 1g. Afterwards, a part of the cell containers were flown to the International Space Station, while another part was kept on the ground. T-25 flasks were mounted on and next to a Random Positioning Machine. The cells were cultured for 12 days under the various conditions, before they were fixed with RNAlater. All fixed cultures showed monolayers, but three-dimensional aggregates were not detected. In a subsequent protein analysis, 180 proteins were identified by mass spectrometry. These proteins did not indicate significant differences between cells exposed to microgravity and their 1g controls. However, they suggest that an enhanced production of proteins related to the extracellular matrix could detain the cells from spheroid formation, while profilin-1 is phosphorylated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/pmic.201500067 2015 • 122 Intermittent high-dose treatment with erlotinib enhances therapeutic efficacy in EGFR-mutant lung cancer
Schöttle, J. and Chatterjee, S. and Volz, C. and Siobal, M. and Florin, A. and Rokitta, D. and Hinze, Y. and Dietlein, F. and Plenker, D. and König, K. and Albus, K. and Heuckmann, J.M. and Rauh, D. and Franz, T. and Neumaier, B...
Oncotarget 6 38458-38468 (2015)Treatment with EGFR kinase inhibitors improves progression-free survival of patients with EGFR-mutant lung cancer. However, all patients with initial response will eventually acquire resistance and die from tumor recurrence. We found that intermittent high-dose treatment with erlotinib induced apoptosis more potently and improved tumor shrinkage significantly than the established low doses. In mice carrying EGFR-mutant xenografts intermittent high-dose treatment (200 mg/kg every other day) was tolerable and prolonged progression-free survival and reduced the frequency of acquired resistance. Intermittent EGFR-targeted high-dose schedules induce more profound as well as sustained target inhibition and may afford enhanced therapeutic efficacy.
view abstract doi: 10.18632/oncotarget.6276 2015 • 121 Light Induced H2 Evolution from a Biophotocathode Based on Photosystem 1 - Pt Nanoparticles Complexes Integrated in Solvated Redox Polymers Films
Zhao, F. and Conzuelo, F. and Hartmann, V. and Li, H. and Nowaczyk, M.M. and Plumeré, N. and Rögner, M. and Schuhmann, W.
Journal of Physical Chemistry B 119 13726-13731 (2015)We report on a biophotocathode based on photosystem 1 (PS1)-Pt nanoparticle complexes integrated in a redox hydrogel for photoelectrocatalytic H2 evolution at low overpotential. A poly(vinyl)imidazole Os(bispyridine)2Cl polymer serves as conducting matrix to shuttle the electrons from the electrode to the PS1-Pt complexes embedded within the hydrogel. Light induced charge separation at the PS1-Pt complexes results in the generation of photocurrents (4.8 ± 0.4 μA cm-2) when the biophotocathodes are exposed to anaerobic buffer solutions. Under these conditions, the protons are the sole possible electron acceptors, suggesting that the photocurrent generation is associated with H2 evolution. Direct evidence for the latter process is provided by monitoring the H2 production with a Pt microelectrode in scanning electrochemical microscopy configuration over the redox hydrogel film containing the PS1-Pt complexes under illumination. © 2015 American Chemical Society.
view abstract doi: 10.1021/acs.jpcb.5b03511 2015 • 120 Mass spectrometry-based proteomics for relative protein quantification and biomarker identification in primary human hepatocytes
Dietz, L. and Sickmann, A.
Methods in Molecular Biology 1250 251-265 (2015)Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The twodimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results. © Springer Science+Business Media New York 2015.
view abstract doi: 10.1007/978-1-4939-2074-7_18 2015 • 119 Mechanism of protection of catalysts supported in redox hydrogel films
Fourmond, V. and Stapf, S. and Li, H. and Buesen, D. and Birrell, J. and Rüdiger, O. and Lubitz, W. and Schuhmann, W. and Plumeré, N. and Léger, C.
Journal of the American Chemical Society 137 5494-5505 (2015)The use of synthetic inorganic complexes as supported catalysts is a key route in energy production and in industrial synthesis. However, their intrinsic oxygen sensitivity is sometimes an issue. Some of us have recently demonstrated that hydrogenases, the fragile but very efficient biological catalysts of H<inf>2</inf> oxidation, can be protected from O<inf>2</inf> damage upon integration into a film of a specifically designed redox polymer. Catalytic oxidation of H<inf>2</inf> produces electrons which reduce oxygen near the film/solution interface, thus providing a self-activated protection from oxygen [Plumeré et al., Nat Chem. 2014, 6, 822-827]. Here, we rationalize this protection mechanism by examining the time-dependent distribution of species in the hydrogenase/polymer film, using measured or estimated values of all relevant parameters and the numerical and analytical solutions of a realistic reaction-diffusion scheme. Our investigation sets the stage for optimizing the design of hydrogenase-polymer films, and for expanding this strategy to other fragile catalysts. © 2015 American Chemical Society.
view abstract doi: 10.1021/jacs.5b01194 2015 • 118 Mesoscopic simulation of phospholipid membranes, peptides, and proteins with molecular fragment dynamics
Truszkowski, A. and Van Den Broek, K. and Kuhn, H. and Zielesny, A. and Epple, M.
Journal of Chemical Information and Modeling 55 983-997 (2015)Molecular fragment dynamics (MFD) is a variant of dissipative particle dynamics (DPD), a coarse-grained mesoscopic simulation technique for isothermal complex fuids and soft matter systems with particles that are chosen to be adequate fluid elements. MFD choses its particles to be small molecules which may be connected by harmonic springs to represent larger molecular entities in order to maintain a comparatively accurate representation of covalent bonding and molecular characteristics. For this study the MFD approach is extended to accomplish long-term simulations (up to the microsecond scale) of large molecular ensembles (representing millions of atoms) containing phospholipid membranes, peptides, and proteins. For peptides and proteins a generally applicable fragmentation scheme is introduced in combination with specific backbone forces that keep native spatial shapes with adequate levels of flexibility or rigidity. The new approach is demonstrated by MFD simulations of the formation and characteristics of phospholipid membranes and vesicles, vesicle-membrane fusion, the backbone force dependency of the overall structural flexibility of dumbbell-shaped Calmodulin, the stability of subunit-aggregation of tetrameric hemoglobin, and the collaborative interaction of Kalata B1 cyclotides with a phospholipid membrane. All findings are in reasonable agreement with experimental as well as alternative simulation results. Thus, the extended MFD approach may become a new tool for biomolecular system studies to allow for comparatively fast simulative investigations in combination with a comparatively high chemical granularity. © 2015 American Chemical Society.
view abstract doi: 10.1021/ci5006096 2015 • 117 Modeling function–perfusion behavior in liver lobules including tissue, blood, glucose, lactate and glycogen by use of a coupled two-scale PDE–ODE approach
Ricken, T. and Werner, D. and Holzhütter, H.G. and König, M. and Dahmen, U. and Dirsch, O.
Biomechanics and Modeling in Mechanobiology 14 515-536 (2015)This study focuses on a two-scale, continuum multicomponent model for the description of blood perfusion and cell metabolism in the liver. The model accounts for a spatial and time depending hydro-diffusion–advection–reaction description. We consider a solid-phase (tissue) containing glycogen and a fluid-phase (blood) containing glucose as well as lactate. The five-component model is enhanced by a two-scale approach including a macroscale (sinusoidal level) and a microscale (cell level). The perfusion on the macroscale within the lobules is described by a homogenized multiphasic approach based on the theory of porous media (mixture theory combined with the concept of volume fraction). On macro level, we recall the basic mixture model, the governing equations as well as the constitutive framework including the solid (tissue) stress, blood pressure and solutes chemical potential. In view of the transport phenomena, we discuss the blood flow including transverse isotropic permeability, as well as the transport of solute concentrations including diffusion and advection. The continuum multicomponent model on the macroscale finally leads to a coupled system of partial differential equations (PDE). In contrast, the hepatic metabolism on the microscale (cell level) was modeled via a coupled system of ordinary differential equations (ODE). Again, we recall the constitutive relations for cell metabolism level. A finite element implementation of this framework is used to provide an illustrative example, describing the spatial and time-depending perfusion–metabolism processes in liver lobules that integrates perfusion and metabolism of the liver. © 2014, Springer-Verlag Berlin Heidelberg.
view abstract doi: 10.1007/s10237-014-0619-z 2015 • 116 Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc
Anderson, C.T. and Radford, R.J. and Zastrow, M.L. and Zhang, D.Y. and Apfel, U.-P. and Lippard, S.J. and Tzounopoulos, T.
Proceedings of the National Academy of Sciences of the United States of America 112 E2705-E2714 (2015)Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling. © 2015, National Academy of Sciences. All rights reserved.
view abstract doi: 10.1073/pnas.1503348112 2015 • 115 Molecular Tweezers Inhibit Islet Amyloid Polypeptide Assembly and Toxicity by a New Mechanism
Lopes, D.H.J. and Attar, A. and Nair, G. and Hayden, E.Y. and Du, Z. and McDaniel, K. and Dutt, S. and Bandmann, H. and Bravo-Rodriguez, K. and Mittal, S. and Klärner, F.-G. and Wang, C. and Sanchez-Garcia, E. and Schrader, T. an...
ACS Chemical Biology 10 1555-1569 (2015)In type-2 diabetes (T2D), islet amyloid polypeptide (IAPP) self-associates into toxic assemblies causing islet β-cell death. Therefore, preventing IAPP toxicity is a promising therapeutic strategy for T2D. The molecular tweezer CLR01 is a supramolecular tool for selective complexation of K residues in (poly)peptides. Surprisingly, it inhibits IAPP aggregation at substoichiometric concentrations even though IAPP has only one K residue at position 1, whereas efficient inhibition of IAPP toxicity requires excess CLR01. The basis for this peculiar behavior is not clear. Here, a combination of biochemical, biophysical, spectroscopic, and computational methods reveals a detailed mechanistic picture of the unique dual inhibition mechanism for CLR01. At low concentrations, CLR01 binds to K1, presumably nucleating nonamyloidogenic, yet toxic, structures, whereas excess CLR01 binds also to R11, leading to nontoxic structures. Encouragingly, the CLR01 concentrations needed for inhibition of IAPP toxicity are safe in vivo, supporting its development toward disease-modifying therapy for T2D. © 2015 American Chemical Society.
view abstract doi: 10.1021/acschembio.5b00146 2015 • 114 Nano-hydroxyapatite-coated metal-ceramic composite of iron-tricalcium phosphate: Improving the surface wettability, adhesion and proliferation of mesenchymal stem cells in vitro
Surmeneva, M.A. and Kleinhans, C. and Vacun, G. and Kluger, P.J. and Schönhaar, V. and Müller, M. and Hein, S.B. and Wittmar, A. and Ulbricht, M. and Prymak, O. and Oehr, C. and Surmenev, R.A.
Colloids and Surfaces B: Biointerfaces 135 386-393 (2015)Thin radio-frequency magnetron sputter deposited nano-hydroxyapatite (HA) films were prepared on the surface of a Fe-tricalcium phosphate (Fe-TCP) bioceramic composite, which was obtained using a conventional powder injection moulding technique. The obtained nano-hydroxyapatite coated Fe-TCP biocomposites (nano-HA-Fe-TCP) were studied with respect to their chemical and phase composition, surface morphology, water contact angle, surface free energy and hysteresis. The deposition process resulted in a homogeneous, single-phase HA coating. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells (hMSCs) was studied using biological short-term tests in vitro. The surface of the uncoated Fe-TCP bioceramic composite showed an initial cell attachment after 24 h of seeding, but adhesion, proliferation and growth did not persist during 14 days of culture. However, the HA-Fe-TCP surfaces allowed cell adhesion, and proliferation during 14 days. The deposition of the nano-HA films on the Fe-TCP surface resulted in higher surface energy, improved hydrophilicity and biocompatibility compared with the surface of the uncoated Fe-TCP. Furthermore, it is suggested that an increase in the polar component of the surface energy was responsible for the enhanced cell adhesion and proliferation in the case of the nano-HA-Fe-TCP biocomposites. © 2015 Elsevier B.V.
view abstract doi: 10.1016/j.colsurfb.2015.07.057 2015 • 113 Neuritogenic Militarinone-Inspired 4-Hydroxypyridones Target the Stress Pathway Kinase MAP4K4
Schröder, P. and Förster, T. and Kleine, S. and Becker, C. and Richters, A. and Ziegler, S. and Rauh, D. and Kumar, K. and Waldmann, H.
Angewandte Chemie - International Edition 54 12398-12403 (2015)Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4-hydroxy-2-pyridone core structure induce pronounced neurite outgrowth, but their protein target has not been identified. Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The most potent 4-hydroxy-2-pyridone is a selective ATP-competitive inhibitor of MAP4K4 but not of the other stress pathway related kinases, as proven by biochemical analysis and by a crystal structure of the inhibitor in complex with MAP4K4. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases. No stress: Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/anie.201501515 2015 • 112 Non-Thermal Dielectric Barrier Discharge (DBD) effects on proliferation and differentiation of human fibroblasts are primary mediated by hydrogen peroxide
Balzer, J. and Heuer, K. and Demir, E. and Hoffmanns, M.A. and Baldus, S. and Fuchs, P.C. and Awakowicz, P. and Suschek, C.V. and Opländer, C.
PLoS ONE 10 (2015)The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H2O2) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H2O2 (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H2O2 was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H2O2. Therefore, a clinical DBD treatment must be well-balanced in order to avoid possible unwanted side effects such as a delayed healing process.
view abstract doi: 10.1371/journal.pone.0144968 2015 • 111 Peptide self-assembly triggered by metal ions
Zou, R. and Wang, Q. and Wu, J. and Wu, J. and Schmuck, C. and Tian, H.
Chemical Society Reviews 44 5200-5219 (2015)Through their unique and specific interactions with various metal ions, naturally occurring proteins control structures and functions of many biological processes and functions in organisms. Inspired by natural metallopeptides, chemists have developed artificial peptides which coordinate with metal ions through their functional groups either for introducing a special reactivity or for constructing nanostructures. However, the design of new coordination peptides requires a deep understanding of the structures, assembly properties, and dynamic behaviours of such peptides. This review briefly discusses strategies of peptide self-assembly induced by metal coordination to different natural and non-natural binding sites in the peptide. The structures and functions of the obtained aggregates are described as well. We also highlight some examples of a metal-induced peptide self-assembly with relevance to biotechnology applications. © The Royal Society of Chemistry.
view abstract doi: 10.1039/c5cs00234f 2015 • 110 Perfluorodecalin-filled Poly(n-butyl-cyanoacrylate) nanocapsules as potential artificial oxygen carriers: Preclinical safety and biocompatibility
Laudien, J. and Groß-Heitfeld, C. and Mayer, C. and De Groot, H. and Kirsch, M. and Ferenz, K.B.
Journal of Nanoscience and Nanotechnology 15 5637-5648 (2015)With regard to the development of artificial blood substitutes, perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules are already discussed for the use as artificial oxygen carriers. The aim of the present study was to thoroughly investigate the preclinical safety and biocompatibility of the perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules prepared by interfacial polymerization. Nanocapsules were assessed for physical and microbial stability. Subsequent to intravenous infusion to anesthetized rats, effects on systemic parameters, microcirculation, circulatory in vivo half-life, acid base/metabolic status, organ damage and biodistribution were evaluated using inter alia 19F-NMR spectroscopy and in vivo microscopy. Perfluorodecalin-filled poly (n-butyl-cyanoacrylate) nanocapsules displayed physical and microbianl stability over a period of 4 weeks and the circulatory in vivo half-life was t1/2 = 30 min. In general, all animals tolerated intravenous infusion of the prepared nanocapsules, even though several side-effects occurred. As a consequence of nanocapsule infusion, a transient decrease in mean arterial blood pressure, impairment of hepatic microcirculation, organ/tissue damage of liver, spleen and small intestine, as well as an elevation of plasma enzyme activities such as lactate dehydrogenase, creatine kinase and aspartate aminotransferase could be observed. The assessment of the distribution pattern revealed nanocapsule accumulation in spleen, kidney and small intestine. Perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules conformed to basic requirements of drugs under preclinical development but further improvement is needed to establish these nanocapsules as novel artificial oxygen carriers. Copyright © 2015 American Scientific Publishers All rights reserved.
view abstract doi: 10.1166/jnn.2015.10044 2015 • 109 Post-polymerization of urease-induced calcified, polymer hydrogels
Rauner, N. and Buenger, L. and Schuller, S. and Tiller, J.C.
Macromolecular Rapid Communications 36 224-230 (2015)Urease-induced calcification is an innovative method to artificially produce highly filled CaCO3-based composite materials by intrinsic mineralization of hydrogels. The mechanical properties of these hybrid materials based on poly(2-hydroxyethylacrylate) cross-linked by triethylene glycol dimethacrylate are poor. Increasing the degree of calcification to up to 94 wt% improves the Young's moduli (YM) of the materials from some 40 MPa to more than 300 MPa. The introduction of calcium carbonate affine groups to the hydrogel matrix by copolymerizing acrylic acid and [2-(methacryloyloxy) ethyl]trimethylammonium chloride, respectively, does not increase the stiffness of the composites. A Young's modulus of more than 1 GPa is achieved by post-polymerization (PP) of the calcified hydrogels, which proves that the size of the contact area between the matrix and calcium carbonate crystals is the most crucial parameter for controlling the stiffness of hybrid materials. Switching from low Tg to high Tg hydrogel matrices (based on poly(N,N-dimethyl acrylamide)) results in a YM of up to 3.5 GPa after PP. (Chemical Equation Presented). © 2014 Wiley-VCH Verlag GmbH & Co. KGaA.
view abstract doi: 10.1002/marc.201400426 2015 • 108 Proteomic analysis of protein carbonylation: A useful tool to unravel nanoparticle toxicity mechanisms
Driessen, M.D. and Mues, S. and Vennemann, A. and Hellack, B. and Bannuscher, A. and Vimalakanthan, V. and Riebeling, C. and Ossig, R. and Wiemann, M. and Schnekenburger, J. and Kuhlbusch, T.A.J. and Renard, B. and Luch, A. and Haase, A.
Particle and Fibre Toxicology 12 (2015)Background: Oxidative stress, a commonly used paradigm to explain nanoparticle (NP)-induced toxicity, results from an imbalance between reactive oxygen species (ROS) generation and detoxification. As one consequence, protein carbonyl levels may become enhanced. Thus, the qualitative and quantitative description of protein carbonylation may be used to characterize how biological systems respond to oxidative stress induced by NPs. Methods: We investigated a representative panel of 24 NPs including functionalized amorphous silica (6), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and boehmite. Surface reactivities of all NPs were studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with all NPs, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D and/or 2D immunoblotting and identified by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF). In parallel, tissue homogenates from rat lungs intratracheally instilled with silver NPs were studied. Results: Eleven NPs induced elevated levels of carbonylated proteins. This was in good agreement with the surface reactivity of the NPs as obtained by ESR and the reduction in cell viability as assessed by WST-1 assay. By contrast, results obtained by DCFDA assay were deviating. Each NP induced an individual pattern of protein carbonyls on 2D immunoblots. Affected proteins comprised cytoskeletal components, proteins being involved in stress response, or cytoplasmic enzymes of central metabolic pathways such as glycolysis and gluconeogenesis. Furthermore, induction of carbonyls upon silver NP treatment was also verified in rat lung tissue homogenates. Conclusions: Analysis of protein carbonylation is a versatile and sensitive method to describe NP-induced oxidative stress and, therefore, can be used to identify NPs of concern. Furthermore, detailed information about compromised proteins may aid in classifying NPs according to their mode of action. © 2015 Driessen et al.
view abstract doi: 10.1186/s12989-015-0108-2 2015 • 107 Selective enzymatic removal of elastin and collagen from human abdominal aortas: Uniaxial mechanical response and constitutive modeling
Schriefl, A.J. and Schmidt, T. and Balzani, D. and Sommer, G. and Holzapfel, G.A.
Acta Biomaterialia 17 125-136 (2015)The ability to selectively remove the structurally most relevant components of arterial wall tissues such as collagen and elastin enables ex vivo biomechanical testing of the remaining tissues, with the aim of assessing their individual mechanical contributions. Resulting passive material parameters can be utilized in mathematical models of the cardiovascular system. Using eighteen wall specimens fromnon-atherosclerotic human abdominal aortas (55±11 years; 9 female, 9 male), we tested enzymatic approaches for the selective digestion of collagen and elastin, focusing on their application to human abdominal aortic wall tissues from different patients with varying sample morphologies. The study resulted in an improved protocol for elastin removal, showing how the enzymatic process is affected by inadequate addition of trypsin inhibitor. We applied the resulting protocol to circumferential and axial specimens from the media and the adventitia, and performed cyclic uniaxial extension tests in the physiological and supra-physiological loading domain. The collagenase-treated samples showed a (linear) response without distinct softening behavior, while the elastase-treated samples exhibited a nonlinear, anisotropic response with pronounced remanent deformations (continuous softening), presumably caused by some sliding of collagen fibers within the damaged regions of the collagen network. In addition, our data showed that the stiffness in the initial linear stress-stretch regime at low loads is lower in elastin-free tissue compared to control samples (i.e. collagen uncrimping requires less force than the stretching of elastin), experimentally confirming that elastin is responsible for the initial stiffness in elastic arteries. Utilizing a continuum mechanical description to mathematically capture the experimental results we concluded that the inclusion of a damage model for the non-collagenous matrix material is, in general, not necessary. To model the softening behavior, continuous damage was included in the fibers by adding a damage variable which led to remanent strains through the consideration of damage. © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
view abstract doi: 10.1016/j.actbio.2015.01.003 2015 • 106 Solvent free production of porous PDLLA/calcium carbonate composite scaffolds improves the release of bone growth factors
Schliephake, H. and Vucak, M. and Boven, J. and Backhaus, S. and Annen, T. and Epple, M.
Oral and Maxillofacial Surgery 19 133-141 (2015)Purpose: Incorporation of alkaline nano-/microparticles for neutralization of acidic degradation products into degradable polymer foams requires the use of organic solvents, which may compromise biocompatibility and may be associated with biological hazards. The aim of the present study was to develop and validate a solvent-free method to produce porous poly (DL-lactic acid)/calcium carbonate composite scaffolds (PDLLA/CaCO<inf>3</inf>) for controlled release of incorporated osteogenic growth factors. Methods: Composite PDLLA/CaCO<inf>3</inf> granules were produced using a milling process and compared to composite material fabricated through a solution precipitation process using organic solvents. Particle size and mineral content were comparable in both groups. Supercritical carbon dioxide pressure was used to incorporate rhBMP2 into both composites. Results: Gas foaming resulted in comparable pore structures in both groups exhibiting a homogenous distribution of CaCO<inf>3</inf> microparticles in the polymer scaffolds. The elasticity modulus of both types of scaffolds was not significantly different whereas the bending strength of the solvent-free produced scaffolds was significantly lower. The pH values remained constant between 6.90 and 7.25 during degradation of both composites. Release of BMP2 was significantly higher and the induction of alkaline phosphatase was more reliable in the group of scaffolds produced without organic solvents. Conclusion: Solvent-free fabrication of composite PDLLA/CaCO<inf>3</inf> scaffolds for controlled release of bone growth factors through gas foaming significantly enhances the release of growth factors and improves the biological efficacy of the incorporated growth factors. © 2014, Springer-Verlag Berlin Heidelberg.
view abstract doi: 10.1007/s10006-014-0463-4 2015 • 105 Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates
Franken, L. and Klein, M. and Spasova, M. and Elsukova, A. and Wiedwald, U. and Welz, M. and Knolle, P. and Farle, M. and Limmer, A. and Kurts, C.
Scientific Reports 5 (2015)A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.
view abstract doi: 10.1038/srep12940 2015 • 104 Structure-based design and synthesis of covalent-reversible inhibitors to overcome drug resistance in EGFR
Basu, D. and Richters, A. and Rauh, D.
Bioorganic and Medicinal Chemistry 23 2767-2780 (2015)The clinical success of covalent kinase inhibitors in the treatment of EGFR-dependent non-small cell lung cancer (NSCLC) has rejuvenated the appreciation of reactive small molecules. Acquired drug resistance against first-line EGFR inhibitors remains the major bottleneck in NSCLC and is currently addressed by the application of fine-tuned covalent drugs. Here we report the design, synthesis and biochemical evaluation of a novel class of EGFR inhibitors with a covalent yet reversible warhead. A series of WZ4002 analogs, derived from anilinopyrimidine and 3-substituted-2-cyanoacrylamide scaffolds, exhibit strong and selective inhibitory activity against clinically relevant EGFRL858R and EGFRL858R/T790M. © 2015 Elsevier Ltd. All rights reserved.
view abstract doi: 10.1016/j.bmc.2015.04.038 2015 • 103 The dynamic influence of cells on the formation of stable emulsions in organic–aqueous biotransformations
Collins, J. and Grund, M. and Brandenbusch, C. and Sadowski, G. and Schmid, A. and Bühler, B.
Journal of Industrial Microbiology and Biotechnology 42 1011-1026 (2015)Emulsion stability plays a crucial role for mass transfer and downstream processing in organic–aqueous bioprocesses based on whole microbial cells. In this study, emulsion stability dynamics and the factors determining them during two-liquid phase biotransformation were investigated for stereoselective styrene epoxidation catalyzed by recombinant Escherichia coli. Upon organic phase addition, emulsion stability rapidly increased correlating with a loss of solubilized protein from the aqueous cultivation broth and the emergence of a hydrophobic cell fraction associated with the organic–aqueous interface. A novel phase inversion-based method was developed to isolate and analyze cellular material from the interface. In cell-free experiments, a similar loss of aqueous protein did not correlate with high emulsion stability, indicating that the observed particle-based emulsions arise from a convergence of factors related to cell density, protein adsorption, and bioreactor conditions. During styrene epoxidation, emulsion destabilization occurred correlating with product-induced cell toxification. For biphasic whole-cell biotransformations, this study indicates that control of aqueous protein concentrations and selective toxification of cells enables emulsion destabilization and emphasizes that biological factors and related dynamics must be considered in the design and modeling of respective upstream and especially downstream processes. © 2015, Society for Industrial Microbiology and Biotechnology.
view abstract doi: 10.1007/s10295-015-1621-x 2015 • 102 The Lys-Specific Molecular Tweezer, CLR01, Modulates Aggregation of the Mutant p53 DNA Binding Domain and Inhibits Its Toxicity
Herzog, G. and Shmueli, M.D. and Levy, L. and Engel, L. and Gazit, E. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Segal, D.
Biochemistry 54 3729-3738 (2015)The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic β-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of β-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates. © 2015 American Chemical Society.
view abstract doi: 10.1021/bi501092p 2015 • 101 The role of calcitonin receptor signalling in polyethylene particle-induced osteolysis
Neuerburg, C. and Wedemeyer, C. and Goedel, J. and Schlepper, R. and Hilken, G. and Schwindenhammer, B. and Schilling, A.F. and Jäger, M. and Kauther, M.D.
Acta Biomaterialia 14 125-132 (2015)The detection of peptides from the calcitonin (CT) family in the periarticular tissue of loosened implants has raised hopes of opening new regenerative therapies in the process of aseptic loosening, which remains the major cause of early implant failure in endoprosthetic surgery. We have previously shown the roles of α-calcitonin gene-related peptide (α-CGRP) and the CALCA gene which encodes α-CGRP/CT in this process. To uncover the role of direct calcitonin receptor (CTR) mediated signalling, we studied particle-induced osteolysis (PIO) in a murine calvaria model with a global deletion of the CTR (CTR-KO) using μCT analysis and histomorphometry. As expected, CTR-KO mice revealed reduced bone volume compared to wild-type (WT) controls (p < 0.05). In CTR-KO mice we found significantly higher RANKL (receptor activator of NF-κB ligand) expression in the particle group than in the control group. The increase in osteoclast numbers by the particles was twice as high as the increase of osteoclasts in the WT mice (400 vs. 200%). Changes in the eroded surface and actual osteolysis due to ultrahigh-molecular-weight polyethylene particles were similar in WTs and CTR-KOs. Taken together, our findings strengthen the relevance of the OPG/RANK/RANKL system in the PIO process. CTR seems to have an effect on osteoclast differentiation in this context. As there were no obvious changes of the amount of PIO in CTR deficiency, regenerative strategies in aseptic loosening of endoprosthetic implants based on peptides arising from the CT family should rather focus on the impact of α-CGRP. © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
view abstract doi: 10.1016/j.actbio.2014.11.051 2015 • 100 The topical use of non-thermal dielectric barrier discharge (DBD): Nitric oxide related effects on human skin
Heuer, K. and Hoffmanns, M.A. and Demir, E. and Baldus, S. and Volkmar, C.M. and Röhle, M. and Fuchs, P.C. and Awakowicz, P. and Suschek, C.V. and Opländer, C.
Nitric Oxide - Biology and Chemistry 44 52-60 (2015)Dielectric barrier discharge (DBD) devices generate air plasma above the skin containing active and reactive species including nitric oxide (NO). Since NO plays an essential role in skin physiology, a topical application of NO by plasma may be useful in the treatment of skin infections, impaired microcirculation and wound healing. Thus, after safety assessments of plasma treatment using human skin specimen and substitutes, NO-penetration through the epidermis, the loading of skin tissue with NO-derivates in vitro and the effects on human skin in vivo were determined. After the plasma treatment (0-60 min) of skin specimen or reconstructed epidermis no damaging effects were found (TUNEL/MTT). By Franz diffusion cell experiments plasma-induced NO penetration through epidermis and dermal enrichment with NO related species (nitrite 6-fold, nitrate 7-fold, nitrosothiols 30-fold) were observed. Furthermore, skin surface was acidified ( ~ pH 2.7) by plasma treatment (90 s). Plasma application on the forearms of volunteers increased microcirculation fourfold in 1-2 mm and twofold in 6-8 mm depth in the treated skin areas. Regarding the NO-loading effects, skin acidification and increase in dermal microcirculation, plasma devices represent promising tools against chronic/infected wounds. However, efficacy of plasma treatment needs to be quantified in further studies and clinical trials. © 2014 Elsevier Inc. All rights reserved.
view abstract doi: 10.1016/j.niox.2014.11.015 2015 • 99 Toxicity Inhibitors Protect Lipid Membranes from Disruption by Aβ42
Malishev, R. and Nandi, S. and Kolusheva, S. and Levi-Kalisman, Y. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Jelinek, R.
ACS Chemical Neuroscience 6 1860-1869 (2015)Although the precise molecular factors linking amyloid β-protein (Aβ) to Alzheimer's disease (AD) have not been deciphered, interaction of Aβ with cellular membranes has an important role in the disease. However, most therapeutic strategies targeting Aβ have focused on interfering with Aβ self-assembly rather than with its membrane interactions. Here, we studied the impact of three toxicity inhibitors on membrane interactions of Aβ42, the longer form of Aβ, which is associated most strongly with AD. The inhibitors included the four-residue C-terminal fragment Aβ(39-42), the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and the lysine-specific molecular tweezer, CLR01, all of which previously were shown to disrupt different steps in Aβ42 self-assembly. Biophysical experiments revealed that incubation of Aβ42 with each of the three modulators affected membrane interactions in a distinct manner. Interestingly, EGCG and CLR01 were found to have significant interaction with membranes themselves. However, membrane bilayer disruption was reduced when the compounds were preincubated with Aβ42, suggesting that binding of the assembly modulators to the peptide attenuated their membrane interactions. Importantly, our study reveals that even though the three tested compounds affect Aβ42 assembly differently, membrane interactions were significantly inhibited upon incubation of each compound with Aβ42, suggesting that preventing the interaction of Aβ42 with the membrane contributes substantially to inhibition of its toxicity by each compound. The data suggest that interference with membrane interactions is an important factor for Aβ42 toxicity inhibitors and should be taken into account in potential therapeutic strategies, in addition to disruption or remodeling of amyloid assembly. © 2015 American Chemical Society.
view abstract doi: 10.1021/acschemneuro.5b00200 2015 • 98 Unraveling the interactions between cold atmospheric plasma and skin-components with vibrational microspectroscopy
Kartaschew, K. and Mischo, M. and Baldus, S. and Bründermann, E. and Awakowicz, P. and Havenith, M.
Biointerphases 10 (2015)Using infrared and Raman microspectroscopy, the authors examined the interaction of cold atmospheric plasma with the skin's built-in protective cushion, the outermost skin layer stratum corneum. Following a spectroscopic analysis, the authors could identify four prominent chemical alterations caused by plasma treatment: (1) oxidation of disulfide bonds in keratin leading to a generation of cysteic acid; (2) formation of organic nitrates as well as (3) of new carbonyl groups like ketones, aldehydes and acids; and (4) reduction of double bonds in the lipid matter lanolin, which resembles human sebum. The authors suggest that these generated acidic and NO-containing functional groups are the source of an antibacterial and regenerative environment at the treatment location of the stratum corneum. Based upon the author's results, the authors propose a mechanistic view of how cold atmospheric plasmas could modulate the skin chemistry to produce positive long-term effects on wound healing: Briefly, cold atmospheric plasmas have the potential to transform the skin itself into a therapeutic resource. © 2015 American Vacuum Society.
view abstract doi: 10.1116/1.4919610 2014 • 97 3D-electrode architectures for enhanced direct bioelectrocatalysis of pyrroloquinoline quinone-dependent glucose dehydrogenase
Sarauli, D. and Peters, K. and Xu, C. and Schulz, B. and Fattakhova-Rohlfing, D. and Lisdat, F.
ACS Applied Materials and Interfaces 6 17887-17893 (2014)We report on the fabrication of a complex electrode architecture for efficient direct bioelectrocatalysis. In the developed procedure, the redox enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase entrapped in a sulfonated polyaniline [poly(2-methoxyaniline-5-sulfonic acid)-co-aniline] was immobilized on macroporous indium tin oxide (macroITO) electrodes. The use of the 3D-conducting scaffold with a large surface area in combination with the conductive polymer enables immobilization of large amounts of enzyme and its efficient communication with the electrode, leading to enhanced direct bioelectrocatalysis. In the presence of glucose, the fabricated bioelectrodes show an exceptionally high direct bioelectrocatalytical response without any additional mediator. The catalytic current is increased more than 200-fold compared to planar ITO electrodes. Together with a high long-term stability (the current response is maintained for >90% of the initial value even after 2 weeks of storage), the transparent 3D macroITO structure with a conductive polymer represents a valuable basis for the construction of highly efficient bioelectronic units, which are useful as indicators for processes liberating glucose and allowing optical and electrochemical transduction. © 2014 American Chemical Society.
view abstract doi: 10.1021/am5046026 2014 • 96 A fluorescent light-up probe as an inhibitor of intracellular beta-tryptase
Wang, Q. and Shi, X. Y. and Zhu, X. X. and Ehlers, M. and Wu, J. C. and Schmuck, C.
Chemical Communications 50 6120--6122 (2014)A pyrene-functionalized peptidic inhibitor 1 binds to and inhibits beta-tryptase in a non-competitive and reversible manner even in cells. Upon protein binding a fluorescence increase of the two pyrene fluorophores is observed which allows using 1 as a fluorescent light-up probe for this enzyme.
view abstract doi: 10.1039/c4cc02208d 2014 • 95 A switchable peptide sensor for real-time lysosomal tracking
Chen, L. and Wu, J. C. and Schmuck, C. and Tian, H.
Chemical Communications 50 6443--6446 (2014)A bis-spiropyran functionalized peptide 1, which exhibits good cellpermeability, excellent biocompatibility and low cytotoxicity, has been developed for reversible and real-time lysosomal tracking.
view abstract doi: 10.1039/c4cc00670d 2014 • 94 Amyloid-β Peptide Induces Mitochondrial Dysfunction by Inhibition of Preprotein Maturation
Mossmann, D. and Vögtle, F.-N. and Taskin, A. and Teixeira, P. and Ring, J. and Burkhart, JuliaM. and Burger, N. and Pinho, C. and Tadic, J. and Loreth, D. and Graff, C. and Metzger, F. and Sickmann, A. and Kretz, O. and Wiedeman...
Cell Metabolism 20 662-669 (2014)Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aβ). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aβ inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aβ peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease. © 2014 Elsevier Inc. All rights reserved.
view abstract doi: 10.1016/j.cmet.2014.07.024 2014 • 93 Apoptotic, inflammatory, and fibrogenic effects of two different types of multi-walled carbon nanotubes in mouse lung
Van Berlo, D. and Wilhelmi, V. and Boots, A.W. and Hullmann, M. and Kuhlbusch, T.A.J. and Bast, A. and Schins, R.P.F. and Albrecht, C.
Archives of Toxicology 88 1725-1737 (2014)There is increasing concern about the toxicity of inhaled multi-walled carbon nanotubes (MWCNTs). Pulmonary macrophages represent the primary cell type involved in the clearance of inhaled particulate materials, and induction of apoptosis in these cells has been considered to contribute to the development of lung fibrosis. We have investigated the apoptotic, inflammogenic, and fibrogenic potential of two types of MWCNTs, characterised by a contrasting average tube length and entanglement/agglomeration. Both nanotube types triggered H2O2 formation by RAW 264.7 macrophages, but in vitro toxicity was exclusively seen with the longer MWCNT. Both types of nanotubes caused granuloma in the mouse lungs. However, the long MWCNT induced a more pronounced pro-fibrotic (mRNA expression of matrix metalloproteinase-8 and tissue inhibitor of metalloproteinase-1) and inflammatory (serum level of monocyte chemotactic protein-1) response. Masson trichrome staining also revealed epithelial cell hyperplasia for this type of MWCNT. Enhanced apoptosis was detected by cleaved caspase 3 immunohistochemistry in lungs of mice treated with the long and rigid MWCNT and, to a lesser extent, with the shorter, highly agglomerated MWCNT. However, staining was merely localised to granulomatous foci, and neither of the MWCNTs induced apoptosis in vitro, evaluated by caspase 3/7 activity in RAW 264.7 cells. In addition, our study reveals that the inflammatory and pro-fibrotic effects of MWCNTs in the mouse lung can vary considerably depending on their composition. The in vitro analysis of macrophage apoptosis appears to be a poor predictor of their pulmonary hazard. © 2014 Springer-Verlag.
view abstract doi: 10.1007/s00204-014-1220-z 2014 • 92 Biotribology of a vitamin E-stabilized polyethylene for hip arthroplasty - Influence of artificial ageing and third-body particles on wear
Grupp, T.M. and Holderied, M. and Mulliez, M.A. and Streller, R. and Jäger, M. and Blömer, W. and Utzschneider, S.
Acta Biomaterialia 10 3068-3078 (2014)The objective of our study was to evaluate the influence of prolonged artificial ageing on oxidation resistance and the subsequent wear behaviour of vitamin E-stabilized, in comparison to standard and highly cross-linked remelted polyethylene (XLPE), and the degradation effect of third-body particles on highly cross-linked remelted polyethylene inlays in total hip arthroplasty. Hip wear simulation was performed with three different polyethylene inlay materials (standard: γ-irradiation 30 kGy, N2; highly cross-linked and remelted: γ-irradiation 75 kGy, EO; highly cross-linked and vitamin E (0.1%) blended: electron beam 80 kGy, EO) machined from GUR 1020 in articulation with ceramic and cobalt-chromium heads. All polyethylene inserts beneath the virgin references were subjected to prolonged artificial ageing (70 °C, pure oxygen at 5 bar) with a duration of 2, 4, 5 or 6 weeks. In conclusion, after 2 weeks of artificial ageing, standard polyethylene shows substantially increased wear due to oxidative degradation, whereas highly cross-linked remelted polyethylene has a higher oxidation resistance. However, after enhanced artificial ageing for 5 weeks, remelted XLPE also starts oxidate, in correlation with increased wear. Vitamin E-stabilized polyethylene is effective in preventing oxidation after irradiation cross-linking even under prolonged artificial ageing for up to 6 weeks, resulting in a constant wear behaviour. © 2014 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.
view abstract doi: 10.1016/j.actbio.2014.02.052 2014 • 91 Catch me if you can: Challenges and applications of cross-linking approaches
Tinnefeld, V. and Sickmann, A. and Ahrends, R.
European Journal of Mass Spectrometry 20 99-116 (2014)Biomolecular complexes are the groundwork of life and the basis for cell signaling, energy transfer, motion, stability and cellular metabolism. Understanding the underlying complex interactions on the molecular level is an essential step to obtain a comprehensive insight into cellular and systems biology. For the investigation of molecular interactions, various methods, including Förster resonance energy transfer, nuclear magnetic resonance spectroscopy, X-ray crystallography and yeast two-hybrid screening, can be utilized. Nevertheless, the most reliable approach for structural proteomics and the identification of novel protein-binding partners is chemical cross-linking. The rationale is that upon forming a covalent bond between a protein and its interaction partner (protein, lipid, RNA/DNA, carbohydrate) the native complex state is "frozen" and accessible for detailed mass spectrometric analysis. In this review we provide a synopsis on crosslinker design, chemistry, pitfalls, limitations and novel applications in the field, and feature an overview of current software applications. © 2014 IM Publications LLP.
view abstract doi: 10.1255/ejms.1259 2014 • 90 Comparison of silver nanoparticles stored under air or argon with respect to the induction of intracellular free radicals and toxic effects toward keratinocytes
Ahlberg, S. and Meinke, M.C. and Werner, L. and Epple, M. and Diendorf, J. and Blume-Peytavi, U. and Lademann, J. and Vogt, A. and Rancan, F.
European Journal of Pharmaceutics and Biopharmaceutics 88 651-657 (2014)Bacterial infections decreased considerably after the discovery of antibiotics. Nevertheless, because of the rising rate of infections caused by antibiotic-resistant bacteria strains, the search for new bactericidal agents has again become a crucial topic in clinical medicine. Silver nanoparticles (AgNP) have a huge potential in dermatology and wound care management because of their ability to release silver ions (Ag+ ions) in a prolonged and sustained way. However, negative effects of silver on the patient's cells should not be underestimated. Furthermore, it has been controversially discussed whether AgNP are responsible for nanoparticle-specific outcomes or not. In this study, we investigated the effects of AgNP on human skin keratinocytes (HaCaT) in order to better understand the mechanisms of cytotoxicity and to improve the use of this highly reactive biocide in wound healing. We found that most of the cells with internalized AgNP displayed the typical morphological signs of apoptosis. The cell viability assay (XTT) showed concentration-dependent toxic effects of the AgNP toward HaCaT cells. The generation of reactive oxygen species (ROS) induced by AgNP was investigated in cell suspensions by means of electron paramagnetic resonance (EPR) spectroscopy. In order to distinguish between the effects of Ag+ ions released during AgNP storage and those of Ag+ ions released after nanoparticle application, we compared AgNP stored under air (O2) with AgNP stored under argon (Ar). Dispersions of AgNP stored under Ar have a low content of Ag+ ions because of the absence of oxygen which is needed for oxidative dissolution. The results show that Ag+ ions released during particle storage are responsible for most of the ROS produced during 1 h incubation with the cells. AgNP (Ar) also induced intracellular ROS but to a much smaller extent compared to AgNP (O2). These findings highlight the complexity of experiments to assess the toxicity of AgNP and suggest the possibility of reducing AgNP toxic effects by storing AgNP formulations and even silver-containing wound dressing under an inert gas atmosphere. © 2014 Elsevier B.V. All rights reserved.
view abstract doi: 10.1016/j.ejpb.2014.07.012 2014 • 89 Correlating structure and ligand affinity in drug discovery: A cautionary tale involving second shell residues
Tziridis, A. and Rauh, D. and Neumann, P. and Kolenko, P. and Menzel, A. and Bräuer, U. and Ursel, C. and Steinmetzer, P. and Stürzebecher, J. and Schweinitz, A. and Steinmetzer, T. and Stubbs, M.T.
Biological Chemistry 395 891-903 (2014)A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.
view abstract doi: 10.1515/hsz-2014-0158 2014 • 88 D-lactate-selective amperometric biosensor based on the cell debris of the recombinant yeast Hansenula polymorpha
Smutok, O.V. and Dmytruk, K.V. and Karkovska, M.I. and Schuhmann, W. and Gonchar, M.V. and Sibirny, A.A.
Talanta 125 227-232 (2014)A d-lactate-selective biosensor has been developed using cellsdebris of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha, overproducing d-lactate: cytochrome c-oxidoreductase (EC 22.214.171.124, d-lactate dehydrogenase (cytochrome), DlDH). The H. polymorpha DlDH-producer was constructed in two steps. First, the gene CYB2 was deleted on the background of the C-105 (gcr1 catX) strain of H. polymorpha impaired in glucose repression and devoid of catalase activity to avoid specific l-lactate-cytochrome c oxidoreductase activity. Second, the homologous gene DLD1 coding for DlDH was overexpressed under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration in the Δcyb2 strain. The selected recombinant strain possesses 6-fold increased DlDH activity as compared to the initial strain. The cellsdebris was used as a biorecognition element of a biosensor, since DlDH is strongly bound to mitochondrial membranes. The cellsdebris, prepared by mechanic disintegration of recombinant cells, was immobilized on a graphite working electrode in an electrochemically generated layer using an Os-complex modified cathodic electrodeposition polymer. Cytochrome c was used as additional native electron mediator to improve electron transfer from reduced DlDH to the working electrode. The constructed d-lactate-selective biosensors are characterized by a high sensitivity (46.3-61.6 A M-1 m-2), high selectivity and sufficient storage stability. © 2014 Elsevier B.V.
view abstract doi: 10.1016/j.talanta.2014.02.041 2014 • 87 Effects of microtubule mechanics on hydrolysis and catastrophes
Müller, N. and Kierfeld, J.
Physical Biology 11 (2014)We introduce a model for microtubule (MT) mechanics containing lateral bonds between dimmers in neighboring protofilaments, bending rigidity of dimers, and repulsive interactions between protofilaments modeling steric constraints to investigate the influence of mechanical forces on hydrolysis and catastrophes. We use the allosteric dimer model, where tubulin dimers are characterized by an equilibrium bending angle, which changes from 0°to 22°by hydrolysis of a dimer. This also affects the lateral interaction and bending energies and, thus, the mechanical equilibrium state of the MT. As hydrolysis gives rise to conformational changes in dimers, mechanical forces also influence the hydrolysis rates by mechanical energy changes modulating the hydrolysis rate. The interaction via the MT mechanics then gives rise to correlation effects in the hydrolysis dynamics, which have not been taken into account before. Assuming a dominant influence of mechanical energies on hydrolysis rates, we investigate the most probable hydrolysis pathways both for vectorial and random hydrolysis. Investigating the stability with respect to lateral bond rupture, we identify initiation configurations for catastrophes along the hydrolysis pathways and values for a lateral bond rupture force. If we allow for rupturing of lateral bonds between dimers in neighboring protofilaments above this threshold force, our model exhibits avalanche-like catastrophe events. © 2014 IOP Publishing Ltd Printed in the UK.
view abstract doi: 10.1088/1478-3975/11/4/046001 2014 • 86 Feedback mechanism for microtubule length regulation by stathmin gradients
Zeitz, M. and Kierfeld, J.
Biophysical Journal 107 2860-2871 (2014)We formulate and analyze a theoretical model for the regulation of microtubule (MT) polymerization dynamics by the signaling proteins Rac1 and stathmin. In cells, the MT growth rate is inhibited by cytosolic stathmin, which, in turn, is inactivated by Rac1. Growing MTs activate Rac1 at the cell edge, which closes a positive feedback loop. We investigate both tubulin sequestering and catastrophe promotion as mechanisms for MT growth inhibition by stathmin. For a homogeneous stathmin concentration in the absence of Rac1, we find a switchlike regulation of the MT mean length by stathmin. For constitutively active Rac1 at the cell edge, stathmin is deactivated locally, which establishes a spatial gradient of active stathmin. In this gradient, we find a stationary bimodal MT-length distribution for both mechanisms of MT growth inhibition by stathmin. One subpopulation of the bimodal length distribution can be identified with fast-growing and long pioneering MTs in the region near the cell edge, which have been observed experimentally. The feedback loop is closed through Rac1 activation by MTs. For tubulin sequestering by stathmin, this establishes a bistable switch with two stable states: one stable state corresponds to upregulated MT mean length and bimodal MT length distributions, i.e., pioneering MTs; the other stable state corresponds to an interrupted feedback with short MTs. Stochastic effects as well as external perturbations can trigger switching events. For catastrophe-promoting stathmin, we do not find bistability. © 2014 Biophysical Society.
view abstract doi: 10.1016/j.bpj.2014.10.056 2014 • 85 Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells
Dautova, Y. and Kozlova, D. and Skepper, J.N. and Epple, M. and Bootman, M.D. and Proudfoot, D.
PLoS ONE 9 (2014)Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥1 μM) reduced intracellular Ca2+ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms. © 2014 Dautova et al.
view abstract doi: 10.1371/journal.pone.0097565 2014 • 84 FLiK: A direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations
Simard, J.R. and Rauh, D.
Methods in Enzymology 548 147-171 (2014)Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the Kd of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput. © 2014 Elsevier Inc. All rights reserved.
view abstract doi: 10.1016/B978-0-12-397918-6.00006-9 2014 • 83 Gold nanoparticles interfere with sperm functionality by membrane adsorption without penetration
Taylor, U. and Barchanski, A. and Petersen, S. and Kues, W.A. and Baulain, U. and Gamrad, L. and Sajti, L. and Barcikowski, S. and Rath, D.
Nanotoxicology 8 118-127 (2014)To examine gold nanoparticle reprotoxicity, bovine spermatozoa were challenged with ligand-free or oligonucleotide-conjugated gold nanoparticles synthesized purely without any surfactants by laser ablation. Sperm motility declined at nanoparticle mass dose of 10 μg/ml (corresponding to ∼14 000 nanoparticles per sperm cell) regardless of surface modification. Sperm morphology and viability remained unimpaired at all concentrations. Transmission electron microscopy showed an modification dependant attachment of nanoparticles to the cell membrane of spermatozoa, but provided no evidence for nanoparticle entrance into sperm cells. A molecular examination revealed a reduction of free thiol residues on the cell membrane after nanoparticle exposure, which could explain the decrease in sperm motility. Sperm fertilising ability decreased after exposure to 10 μg/ml of ligand-free nanoparticles indicating that agglomerated ligand-free nanoparticles interfere with membrane properties necessary for fertilisation. In conclusion, nanoparticles may impair key sperm functions solely by interacting with the sperm surface membrane. © 2014 Informa UK Ltd. All rights reserved.
view abstract doi: 10.3109/17435390.2013.859321 2014 • 82 Immobilization of proteins in their physiological active state at functionalized thiol monolayers on ATR-germanium crystals
Schartner, J. and Gavriljuk, K. and Nabers, A. and Weide, P. and Muhler, M. and Gerwert, K. and Kötting, C.
ChemBioChem 15 2529-2534 (2014)Protein immobilization on solid surfaces has become a powerful tool for the investigation of protein function. Physiologically relevant molecular reaction mechanisms and interactions of proteins can be revealed with excellent signal-to-noise ratio by vibrational spectroscopy (ATR-FTIR) on germanium crystals. Protein immobilization by thiol chemistry is well-established on gold surfaces, for example, for surface plasmon resonance. Here, we combine features of both approaches: a germanium surface functionalized with different thiols to allow specific immobilization of various histidine-tagged proteins with over 99% specific binding. In addition to FTIR, the surfaces were characterized by XPS and fluorescence microscopy. Secondary-structure analysis and stimulus-induced difference spectroscopy confirmed protein activity at the atomic level, for example, physiological cation channel formation of Channelrhodopsin 2. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/cbic.201402478 2014 • 81 Impact of the configuration of a chiral, activating carrier on the enantioselectivity of entrapped lipase from Candida rugosa in cyclohexane
Tobis, J. and Tiller, J.C.
Biotechnology Letters 36 1661-1667 (2014)Lipase from Candida rugosa was loaded into an amphiphilic polymer co-network (APCN) composed of the chiral poly[(R)-N-(1-hydroxybutan-2-yl) acrylamide] [P-(R)-HBA] and P-(S)-HBA, respectively, linked by poly(dimethylsiloxane). The nanophase-separated amphiphilic morphology affords a 38,000-fold activation of the enzyme in the esterification of 1-phenylethanol with vinyl acetate. Further, the enantioselectivity of the entrapped lipase was influenced by the configuration of the chiral, hydrophilic polymer matrix. While the APCN with the (S)-configuration of the APCN affords 5.4 faster conversion of the (R)-phenylethanol compared to the respective (S)-enantiomer, the (R)-APCN allows an only a 2.8 faster conversion of the (R)-enantiomer of the alcohol. Permeation-experiments reveal that the enantioselectivity of the reaction is at least partially caused by specific interactions between the substrates and the APCN. © 2014 Springer Science+Business Media Dordrecht.
view abstract doi: 10.1007/s10529-014-1519-0 2014 • 80 Interaction with mesenchymal stem cells provokes natural killer cells for enhanced IL-12/IL-18-induced interferon-gamma secretion
Thomas, H. and Jäger, M. and Mauel, K. and Brandau, S. and Lask, S. and Flohé, S.B.
Mediators of Inflammation 2014 (2014)Tissue injury induces an inflammatory response accompanied by the recruitment of immune cells and of mesenchymal stem cells (MSC) that contribute to tissue regeneration. After stimulation with interleukin- (IL-) 12 and IL-18 natural killer (NK) cells secrete the proinflammatory cytokine interferon- (IFN-) γ. IFN-γ plays a crucial role in the defense against infections and modulates tissue regeneration. In consideration of close proximity of NK cells and MSC at the site of injury we investigated if MSC could influence the ability of NK-cells to produce IFN-γ. Coculture experiments were performed with bone marrow-derived human MSC and human NK cells. MSC enhanced the ability of IL-12/IL-18-stimulated NK cells to secrete IFN-γ in a dose-dependent manner. This activation of NK cells was dependent on cell-cell contact as well as on soluble factors. The increased IFN-γ secretion from NK cells after contact with MSC correlated with an increased level of intracellular IFN-γ. Alterations in the IL-12 signaling pathway including an increased expression of the IL-12 β 1 receptor subunit and an increased phosphorylation of signal transducer and activator of transcription 4 (STAT4) could be observed. In conclusion, MSC enhance the IFN-γ release from NK cells which might improve the defense against infections at the site of injury but additionally might affect tissue regeneration. © 2014 Heike Thomas et al.
view abstract doi: 10.1155/2014/143463 2014 • 79 Investigations on the activity of poly(2-oxazoline) enzyme conjugates dissolved in organic solvents
Konieczny, S. and Krumm, C. and Doert, D. and Neufeld, K. and Tiller, J.C.
Journal of Biotechnology 181 55-63 (2014)The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-oxazoline) (PHeptOx). As reported previously on the examples of model enzymes POXylation mediated by pyromellitic acid dianhydride results in highly modified, organosoluble protein conjugates. This procedure is here extended to a larger number of proteins and optimized for the different polyoxazolines. The resulting polymer-enzyme conjugates (PEC) became soluble in different organic solvents ranging from hydrophilic DMF to even toluene. These conjugates were characterized regarding their solubility and especially their activity in organic solvents and in some cases the PECs showed significantly (up to 153,000 fold) higher activities than the respective native enzymes. © 2014 Elsevier B.V.
view abstract doi: 10.1016/j.jbiotec.2014.03.035 2014 • 78 Is the antioxidative effectiveness of a bilberry extract influenced by encapsulation?
Baum, M. and Schantz, M. and Leick, S. and Berg, S. and Betz, M. and Frank, K. and Rehage, H. and Schwarz, K. and Kulozik, U. and Schuchmann, H. and Richling, E.
Journal of the Science of Food and Agriculture 94 2301-2307 (2014)BACKGROUND: Bilberries (Vaccinium myrtillus L.) have been suggested to have preventive properties against diseases associated with oxidative stress such as colon cancer or inflammatory bowel diseases. Therefore the gastrointestinal tract is regarded as a potential target for prevention. In this study the antioxidative properties of a commercially available anthocyanin-rich bilberry extract (BE) were investigated in comparison with four different BE-loaded microcapsule systems. As markers to describe the antioxidant status in this cellular system, intracellular reactive oxygen species (ROS) levels, oxidative DNA damage and total glutathione (tGSH) levels were monitored. RESULTS: Incubations with the BE-loaded capsule systems showed an increase in cellular glutathione levels and reduction of ROS levels at high BE concentrations (100-500 μg mL-1) and a positive effect on the formation of DNA strand breaks (5-10 μg mL-1 BE). The biological properties of BE-loaded pectin amide core-shell capsules, whey protein matrix capsules and coated apple pectin matrix capsules were comparable to those of the non-encapsulated BE. CONCLUSION: Overall, the BE and the encapsulated BE types tested have antioxidative activity under the studied assay conditions in terms of the prevention of oxidative DNA damage, the reduction of intracellular ROS and the enhancement of cellular tGSH. © 2014 Society of Chemical Industry.
view abstract doi: 10.1002/jsfa.6558 2014 • 77 Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes
Brandau, S. and Jakob, M. and Bruderek, K. and Bootz, F. and Giebel, B. and Radtke, S. and Mauel, K. and Jäger, M. and Flohé, S.B. and Lang, S.
PLoS ONE 9 (2014)Methodology/Principal Findings: We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN. Conclusions/Significance: This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue. Copyright: © 2014 Brandau et al. Background: Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs. © 2014 Brandau et al.
view abstract doi: 10.1371/journal.pone.0106903 2014 • 76 Molecular Tweezers Targeting Transthyretin Amyloidosis
Ferreira, N. and Pereira-Henriques, A. and Attar, A. and Klärner, F.-G. and Schrader, T. and Bitan, G. and Gales, L. and Saraiva, M.J. and Almeida, M.R.
Neurotherapeutics 11 450-461 (2014)Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the "molecular tweezers", CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer's disease and Parkinson's disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis. © 2014 The Author(s).
view abstract doi: 10.1007/s13311-013-0256-8 2014 • 75 Nano-scale morphology of melanosomes revealed by small-angle X-ray scattering
Gorniak, T. and Haraszti, T. and Garamus, V.M. and Buck, A.R. and Senkbeil, T. and Priebe, M. and Hedberg-Buenz, A. and Koehn, D. and Salditt, T. and Grunze, M. and Anderson, M.G. and Rosenhahn, A.
PLoS ONE 9 (2014)Melanosomes are highly specialized organelles that produce and store the pigment melanin, thereby fulfilling essential functions within their host organism. Besides having obvious cosmetic consequences - determining the color of skin, hair and the iris - they contribute to photochemical protection from ultraviolet radiation, as well as to vision (by defining how much light enters the eye). Though melanosomes can be beneficial for health, abnormalities in their structure can lead to adverse effects. Knowledge of their ultrastructure will be crucial to gaining insight into the mechanisms that ultimately lead to melanosome-related diseases. However, due to their small size and electron-dense content, physiologically intact melanosomes are recalcitrant to study by common imaging techniques such as light and transmission electron microscopy. In contrast, X-ray-based methodologies offer both high spatial resolution and powerful penetrating capabilities, and thus are well suited to study the ultrastructure of electron-dense organelles in their natural, hydrated form. Here, we report on the application of small-angle X-ray scattering - a method effective in determining the three-dimensional structures of biomolecules - to whole, hydrated murine melanosomes. The use of complementary information from the scattering signal of a large ensemble of suspended organelles and from single, vitrified specimens revealed a melanosomal sub-structure whose surface and bulk properties differ in two commonly used inbred strains of laboratory mice. Whereas melanosomes in C57BL/6J mice have a well-defined surface and are densely packed with 40-nm units, their counterparts in DBA/2J mice feature a rough surface, are more granular and consist of 60-nm building blocks. The fact that these strains have different coat colors and distinct susceptibilities to pigment-related eye disease suggest that these differences in size and packing are of biological significance.
view abstract doi: 10.1371/journal.pone.0090884 2014 • 74 Prostacyclin suppresses twist expression in the presence of indomethacin in bone marrow- derived mesenchymal stromal cells
Kemper, O. and Herten, M. and Fischer, J. and Haversath, M. and Beck, S. and Classen, T. and Warwas, S. and Tassemeier, T. and Landgraeber, S. and Lensing-Höhn, S. and Krauspe, R. and Jäger, M.
Medical Science Monitor 20 2219-2227 (2014)Background: Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods: Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45–/14–). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10–7 M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10–6 M (Group 1, A), 10–5 M (Group 2, B), 10–4 M (Group 3, A/B), and together with 10–7 M iloprost (Groups 4–6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34–, CD45–, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity). Results: Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently. Conclusions: Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin, it completely suppressed Twist. Thus, in the treatment of avascular osteonecrosis or painful bone marrow edema, the undesirable effects of indomethacin might be counterbalanced by iloprost. © Med Sci Monit, 2014.
view abstract doi: 10.12659/MSM.890953 2014 • 73 Safety and pharmacological characterization of the molecular tweezer CLR01-A broad-spectrum inhibitor of amyloid proteins' toxicity
Attar, A. and Chan, W.C. and Klärner, F.-G. and Schrader, T. and Bitan, G.
BMC Pharmacology and Toxicology 15 (2014)Background: The "molecular tweezer" CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer's disease and other amyloidoses, here we studied its safety and pharmacokinetics. Methods: Toxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer's disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5-72 h post administration. Results: Examination of CLR01's effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound's putative "processspecific" mechanism of action. A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin. The brain penetration of CLR01 was found to be 1-3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h. Conclusion: Estimation of brain levels compared to amyloid ß-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01. The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses. © 2014 Attar et al.
view abstract doi: 10.1186/2050-6511-15-23 2014 • 72 Specific anion effects on the pressure dependence of the protein-protein interaction potential
Möller, J. and Grobelny, S. and Schulze, J. and Steffen, A. and Bieder, S. and Paulus, M. and Tolan, M. and Winter, R.
Physical Chemistry Chemical Physics 16 7423-7429 (2014)We present a study on ion specific effects on the intermolecular interaction potential V(r) of dense protein solutions under high hydrostatic pressure conditions. Small-angle X-ray scattering in combination with a liquid-state theoretical approach was used to determine the effect of structure breaking/making salt anions (Cl-, SO4 2-, PO4 3-) on the intermolecular interaction of lysozyme molecules. It was found that besides the Debye-Hückel charge screening effect, reducing the repulsiveness of the interaction potential V(r) at low salt concentrations, a specific ion effect is observed at high salt concentrations for the multivalent kosmotropic anions, which modulates also the pressure dependence of the protein-protein interaction potential. Whereas sulfate and phosphate strongly influence the pressure dependence of V(r), chloride anions do not. The strong structure-making effect of the multivalent anions, dominating for the triply charged PO4 3-, renders the solution structure less bulk-water-like at high salt concentrations, which leads to an altered behavior of the pressure dependence of V(r). Hence, the particular structural properties of the salt solutions are able to influence the spatial organization and the intermolecular interactions of the proteins, in particular upon compression. These results are of interest for exploring the combined effects of ionic strength, temperature and pressure on the phase behavior of protein solutions, but may also be of relevance for understanding pressure effects on the hydration behavior of biological matter under extreme environmental conditions. This journal is © the Partner Organisations 2014.
view abstract doi: 10.1039/c3cp55278k 2014 • 71 Support and challenges to the melanosomal casing model based on nanoscale distribution of metals within iris melanosomes detected by X-ray fluorescence analysis
Gorniak, T. and Haraszti, T. and Suhonen, H. and Yang, Y. and Hedberg-Buenz, A. and Koehn, D. and Heine, R. and Grunze, M. and Rosenhahn, A. and Anderson, M.G.
Pigment Cell and Melanoma Research 27 831-834 (2014)Melanin within melanosomes exists as eumelanin or pheomelanin. Distributions of these melanins have been studied extensively within tissues, but less often within individual melanosomes. Here, we apply X-ray fluorescence analysis with synchrotron radiation to survey the nanoscale distribution of metals within purified melanosomes of mice. The study allows a discovery-based characterization of melanosomal metals, and, because Cu is specifically associated with eumelanin, a hypothesis-based test of the 'casing model' predicting that melanosomes contain a pheomelanin core surrounded by a eumelanin shell. Analysis of Cu, Ca, and Zn shows variable concentrations and distributions, with Ca/Zn highly correlated, and at least three discrete patterns for the distribution of Cu vs. Ca/Zn in different melanosomes - including one with a Cu-rich shell surrounding a Ca/Zn-rich core. Thus, the results support predictions of the casing model, but also suggest that in at least some tissues and genetic contexts, other arrangements of melanin may co-exist. © 2014 John Wiley & Sons A/S.
view abstract doi: 10.1111/pcmr.12278 2014 • 70 Telechelic Poly(2-oxazoline)s with a biocidal and a polymerizable terminal as collagenase inhibiting additive for long-term active antimicrobial dental materials
Fik, C.P. and Konieczny, S. and Pashley, D.H. and Waschinski, C.J. and Ladisch, R.S. and Salz, U. and Bock, T. and Tiller, J.C.
Macromolecular Bioscience 14 1569-1579 (2014)Dental repair materials face the problem that the dentin below the composite fillings is actively decomposed by secondary caries and extracellular proteases. To address this problem, poly(2-methyloxazoline) with a biocidal and a polymerizable terminal was explored as additive for a commercial dental adhesive. 2.5 wt% of the additive rendered the adhesive contact-active against Streptococcus mutans and washing with water for 101 d did not diminish this effect. The adhesive with 5 wt% additive kills S. mutans cells in the tubuli of bovine dentin. Further, the additive inhibits bacterial collagenase at 0.5 wt% and reduces activity of MMP-9. Human MMPs bound to dentin are inhibited by 96% in a medium with 5 wt% additive. Moreover, no adverse effect on the enamel/dentine shear bond strength was detected. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/mabi.201400220 2014 • 69 The pathology of orthopedic implant failure is mediated by innate immune system cytokines
Landgraeber, S. and Jäger, M. and Jacobs, J.J. and Hallab, N.J.
Mediators of Inflammation 2014 (2014)All of the over 1 million total joint replacements implanted in the US each year are expected to eventually fail after 15-25 years of use, due to slow progressive subtle inflammation at the bone implant interface. This inflammatory disease state is caused by implant debris acting, primarily, on innate immune cells, that is, macrophages. This slow progressive pathological bone loss or "aseptic loosening" is a potentially life-threatening condition due to the serious complications in older people (>75 yrs) of total joint replacement revision surgery. In some people implant debris (particles and ions from metals) can influence the adaptive immune system as well, giving rise to the concept of metal sensitivity. However, a consensus of studies agrees that the dominant form of this response is due to innate reactivity by macrophages to implant debris where both danger (DAMP) and pathogen (PAMP) signalling elicit cytokine-based inflammatory responses. This paper discusses implant debris induced release of the cytokines and chemokines due to activation of the innate (and the adaptive) immune system and the subsequent formation of osteolysis. Different mechanisms of implant-debris reactivity related to the innate immune system are detailed, for example, danger signalling (e.g., IL-1 β, IL-18, IL-33, etc.), toll-like receptor activation (e.g., IL-6, TNF- α, etc.), apoptosis (e.g., caspases 3-9), bone catabolism (e.g., TRAP5b), and hypoxia responses (Hif1- α). Cytokine-based clinical and basic science studies are in progress to provide diagnosis and therapeutic intervention strategies. © 2014 Stefan Landgraeber et al.
view abstract doi: 10.1155/2014/185150 2014 • 68 Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways
Beck, F. and Geiger, J. and Gambaryan, S. and Veit, J. and Vaudel, M. and Nollau, P. and Kohlbacher, O. and Martens, L. and Walter, U. and Sickmann, A. and Zahedi, R.P.
Blood 123 e1-e10 (2014)One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-signaling cascade and inhibits virtually all platelet-activating key mechanisms. Using quantitative mass spectrometry, we analyzed time-resolved phosphorylation patterns in human platelets after treatment with iloprost, a stable prostacyclin analog, for 0, 10, 30, and 60 seconds to characterize key mediators of platelet inhibition and activation in 3 independent biological replicates. We quantified over 2700 different phosphorylated peptides of which 360 were significantly regulated upon stimulation. This comprehensive and time-resolved analysis indicates that platelet inhibition is a multipronged process involving different kinases and phosphatases aswell asmany previously unanticipated proteins and pathways. © 2014 by The American Society of Hematology.
view abstract doi: 10.1182/blood-2013-07-512384 2014 • 67 Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates
Salehi, M. and Schneider, L. and Ströbel, P. and Marx, A. and Packeisen, J. and Schlücker, S.
Nanoscale 6 2361-2367 (2014)SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates. © 2014 The Royal Society of Chemistry.
view abstract doi: 10.1039/c3nr05890e 2013 • 66 BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis
Sreeramulu, S. and Mostizky, Y. and Sunitha, S. and Shani, E. and Nahum, H. and Salomon, D. and Hayun, L.B. and Gruetter, C. and Rauh, D. and Ori, N. and Sessa, G.
Plant Journal 74 905-919 (2013)Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor-like cytoplasmic kinase sub-family (RLCK-XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T-DNA insertion lines for eight BSK genes (BSK1-BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24-epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR-responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3-like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3-like kinases. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.
view abstract doi: 10.1111/tpj.12175 2013 • 65 Combination of a photosystem 1-based photocathode and a photosystem 2-based photoanode to a z-scheme mimic for biophotovoltaic applications
Kothe, T. and Plumeré, N. and Badura, A. and Nowaczyk, M.M. and Guschin, D.A. and Rögner, M. and Schuhmann, W.
Angewandte Chemie - International Edition 52 14233-14236 (2013)Z-Scheme on wires: The two photosystems of the natural photosynthetic Z-scheme have been connected by immobilizing them within redox hydrogels on individual electrodes. Upon irradiation, this biophotovoltaic device produced photocurrents as a closed and autonomous system. The open-circuit voltage of the cell corresponds to the potential difference between the two redox hydrogels and indicates the coupling of the two charge separation steps. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
view abstract doi: 10.1002/anie.201303671 2013 • 64 Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response
Stahl, S. and Burkhart, J.M. and Hinte, F. and Tirosh, B. and Mohr, H. and Zahedi, R.P. and Sickmann, A. and Ruzsics, Z. and Budt, M. and Brune, W.
PLoS Pathogens 9 (2013)During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR. © 2013 Stahl et al.
view abstract doi: 10.1371/journal.ppat.1003544 2013 • 63 De Novo design of protein kinase inhibitors by in silico identification of hinge region-binding fragments
Urich, R. and Wishart, G. and Kiczun, M. and Richters, A. and Tidten-Luksch, N. and Rauh, D. and Sherborne, B. and Wyatt, P.G. and Brenk, R.
ACS Chemical Biology 8 1044-1052 (2013)Protein kinases constitute an attractive family of enzyme targets with high relevance to cell and disease biology. Small molecule inhibitors are powerful tools to dissect and elucidate the function of kinases in chemical biology research and to serve as potential starting points for drug discovery. However, the discovery and development of novel inhibitors remains challenging. Here, we describe a structure-based de novo design approach that generates novel, hinge-binding fragments that are synthetically feasible and can be elaborated to small molecule libraries. Starting from commercially available compounds, core fragments were extracted, filtered for pharmacophoric properties compatible with hinge-region binding, and docked into a panel of protein kinases. Fragments with a high consensus score were subsequently short-listed for synthesis. Application of this strategy led to a number of core fragments with no previously reported activity against kinases. Small libraries around the core fragments were synthesized, and representative compounds were tested against a large panel of protein kinases and subjected to co-crystallization experiments. Each of the tested compounds was active against at least one kinase, but not all kinases in the panel were inhibited. A number of compounds showed high ligand efficiencies for therapeutically relevant kinases; among them were MAPKAP-K3, SRPK1, SGK1, TAK1, and GCK for which only few inhibitors are reported in the literature. © 2013 American Chemical Society.
view abstract doi: 10.1021/cb300729y 2013 • 62 Electrochemical detection of chloride levels in sweat using silver nanoparticles: A basis for the preliminary screening for cystic fibrosis
Toh, H.S. and Batchelor-Mcauley, C. and Tschulik, K. and Compton, R.G.
Analyst 138 4292-4297 (2013)Cystic fibrosis is a common disease which has an associated characteristic symptom of high sweat chloride content. Thus, chloride ion quantification in sweat is important towards the screening of cystic fibrosis. Electrochemical methods, being cost effective and convenient, can be exploited for this. The electrochemical oxidation of silver nanoparticles in the absence of chloride ions gives one voltammetric signal related to the oxidation of silver to silver ions. The presence of chloride ions in the solution causes the appearance of an additional signal at a lower potential which is related to the oxidation of silver to silver chloride. This signal has a peak height which correlates linearly with the concentration of chloride ions from 2 mM to 40 mM when the electrochemical experiments are performed on silver nanoparticle modified screen printed electrodes. Thus, reliable quantification was found to be achievable. Furthermore, chloride ion levels of diluted synthetic sweat samples are measured accurately with the modified electrodes. Thus, the detection of the chloride ion concentration with a silver nanoparticle modified electrode provides a proof-of-concept for a point-of-care system for preliminary screening of cystic fibrosis. © 2013 The Royal Society of Chemistry.
view abstract doi: 10.1039/c3an00843f 2013 • 61 Interaction of antitumor flavonoids with dsDNA in the absence and presence of Cu(II)
Temerk, Y.M. and Ibrahim, M.S. and Kotb, M. and Schuhmann, W.
Analytical and Bioanalytical Chemistry 405 3839-3846 (2013)The binding of antitumor flavonoids, namely 3-hydroxyflavone (3HF) and hesperidin (Hesp) with dsDNA was investigated in the absence and presence of Cu(II) using cyclic voltammetry and square wave voltammetry at the hanging mercury drop electrode. The reduction currents of 3HF, 3HF-Cu complex, and the 3HF-β-cyclodextrin inclusion complex decreased after intercalation into dsDNA. The intercalation of Hesp into dsDNA is weak. dsDNA is reduced at a potential of -1.48 V overlaying the reduction of Hesp. In contrast, in the presence of Cu(II), the interaction of Hesp with dsDNA leads to a much stronger intercalation. The binding constants of the flavonoid-Cu complex with dsDNA were evaluated and calibration graphs for the determination of dsDNA were obtained from the decrease in the peak current in the cyclic voltammograms of 3HF in the presence of dsDNA. The proposed method exhibited good recovery and reproducibility for indirect determination of dsDNA. © 2013 Springer-Verlag Berlin Heidelberg.
view abstract doi: 10.1007/s00216-012-6675-2 2013 • 60 Interaction of proteins identified in human thyroid cells
Pietsch, J. and Riwaldt, S. and Bauer, J. and Sickmann, A. and Weber, G. and Grosse, J. and Infanger, M. and Eilles, C. and Grimm, D.
International Journal of Molecular Sciences 14 1164-1178 (2013)Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. © 2013 by the authors; licensee MDPI, Basel, Switzerland.
view abstract doi: 10.3390/ijms14011164 2013 • 59 Measurement of the silver ion concentration in wound fluids after implantation of silver-coated megaprostheses: Correlation with the clinical outcome
Hussmann, B. and Johann, I. and Kauther, M.D. and Landgraeber, S. and Jäger, M. and Lendemans, S.
BioMed Research International 2013 (2013)Background. Tumor patients and patients after traumas are endangered by a reduced immune defense, and a silver coating on their megaprostheses may reduce their risks of infection. The aim of this study was to determine the silver ion concentration directly measured from the periprosthetic tissue and the influence on the clinical outcome. Material and Methods. Silver ions were evaluated in 5 mL wound fluids two days postoperatively and in blood patients 7 and 14 days after surgery using inductively coupled plasma emission spectrometry in 18 patients who underwent total joint replacement with a silver-coated megaendoprosthesis. Results. The concentration of silver ions averaged 0.08 parts per million. Patients who showed an increased silver concentration in the blood postoperatively presented a lower silver concentration in the wound fluids and a delayed decrease in C-reactive protein levels. There were significantly fewer reinfections and shorter hospitalization in comparison with a group that did not receive a silver-coated megaprosthesis. Conclusion. An increased concentration of silver in the immediate surroundings of silver-coated prostheses was demonstrated for the first time in cohorts of patients with trauma or tumors. An elevated concentration of silver ions in the direct periprosthetic tissue may have reduced the infection rate. © 2013 B. Hussmann et al.
view abstract doi: 10.1155/2013/763096 2013 • 58 Medical applications of surface-enhanced Raman scattering
Xie, W. and Schlücker, S.
Physical Chemistry Chemical Physics 15 5329-5344 (2013)This perspective article provides an overview of selected medical applications of surface-enhanced Raman scattering (SERS), highlighting recent developments and trends. The use of SERS for detection, analysis and imaging has attracted great interest in the past decade owing to its high sensitivity and molecular fingerprint specificity. SERS can deliver chemical and structural information from analytes rapidly and nondestructively in a label-free manner. Alternatively, SERS labels or nanotags, when conjugated to target-specific ligands, can be employed for the selective detection and localization of the corresponding target molecule. Biomedical applications based on both approaches are highlighted. © 2013 the Owner Societies.
view abstract doi: 10.1039/c3cp43858a 2013 • 57 Molecular tweezers modulate 14-3-3 protein-protein interactions
Bier, D. and Rose, R. and Bravo-Rodriguez, K. and Bartel, M. and Ramirez-Anguita, J.M. and Dutt, S. and Wilch, C. and Klärner, F.-G. and Sanchez-Garcia, E. and Schrader, T. and Ottmann, C.
Nature Chemistry 5 234-239 (2013)Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins-a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)-in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. © 2013 Macmillan Publishers Limited. All rights reserved.
view abstract doi: 10.1038/nchem.1570 2013 • 56 Response: platelet transcriptome and proteome--relation rather than correlation.
Geiger, J. and Burkhart, J.M. and Gambaryan, S. and Walter, U. and Sickmann, A. and Zahedi, R.P.
Blood 121 5257-5258 (2013)
doi: 10.1182/blood-2013-04-493403 2013 • 55 Solid crystal suspensions containing griseofulvin-Preparation and bioavailability testing
Reitz, E. and Vervaet, C. and Neubert, R.H.H. and Thommes, M.
European Journal of Pharmaceutics and Biopharmaceutics 83 193-202 (2013)The improvement of the bioavailability of poorly soluble drugs has been an important issue in pharmaceutical research for many years. Despite the suggestion of several other technologies in the past, drug particle size reduction is still an appropriate strategy to guarantee high bioavailability of various drugs. A few years ago, the Solid Crystal Suspension (SCS) technology was suggested, in which crystalline drug particles are ground and dispersed in a highly soluble crystalline carrier by a hot melt extrusion process. The current study demonstrates the scale-up of the SCS technology to standard, lab-scale extrusion equipment - a change from previous investigations, which used small batch sizes. A twin-screw extruder was modified to account for the rapid crystallization of the carrier. The screw speed and the barrel temperature were identified as critical process parameters and were varied systematically in several experimental designs. Finally, parameters were identified that produced extrudates with rapid dissolution rates. After extrusion, the extrudates were milled to granules and then tableted. These tablets were investigated with respect to their bioavailability in beagle dogs. It was found that drug particle size reduction in the hot melt extrusion led to 3.5-fold higher bioavailability in these dogs than occurred with the physical mixture of the used substances. The solid crystal suspension formulation had a slightly higher bioavailability than the marked product. In conclusion, the SCS technology was successfully scaled up to lab-scale equipment, and the concept was confirmed by a bioavailability study. © 2012 Elsevier B.V. All rights reserved.
view abstract doi: 10.1016/j.ejpb.2012.09.012 2013 • 54 The biocompatibility of dense and porous Nickel-Titanium produced by selective laser melting
Habijan, T. and Haberland, C. and Meier, H. and Frenzel, J. and Wittsiepe, J. and Wuwer, C. and Greulich, C. and Schildhauer, T.A. and Köller, M.
Materials Science and Engineering C 33 419-426 (2013)Nickel-Titanium shape memory alloys (NiTi-SMA) are of biomedical interest due to their unusual range of pure elastic deformability and their elastic modulus, which is closer to that of bone than any other metallic or ceramic material. Newly developed porous NiTi, produced by Selective Laser Melting (SLM), is currently under investigation as a potential carrier material for human mesenchymal stem cells (hMSC). SLM enables the production of highly complex and tailor-made implants for patients on the basis of CT data. Such implants could be used for the reconstruction of the skull, face, or pelvis. hMSC are a promising cell type for regenerative medicine and tissue engineering due to their ability to support the regeneration of critical size bone defects. Loading porous SLM-NiTi implants with autologous hMSC may enhance bone growth and healing for critical bone defects. The purpose of this study was to assess whether porous SLM-NiTi is a suitable carrier for hMSC. Specimens of varying porosity and surface structure were fabricated via SLM. hMSC were cultured for 8 days on NiTi specimens, and cell viability was analyzed using two-color fluorescence staining. Viable cells were detected on all specimens after 8 days of cell culture. Cell morphology and surface topography were analyzed by scanning electron microscopy (SEM). Cell morphology and surface topology were dependent on the orientation of the specimens during SLM production. The Nickel ion release can be reduced significantly by aligned laser processing conditions. The presented results clearly attest that both dense SLM-NiTi and porous SLM-NiTi are suitable carriers for hMSC. Nevertheless, before carrying out in vivo studies, some work on optimization of the manufacturing process and post-processing is required. © 2012 Elsevier B.V.
view abstract doi: 10.1016/j.msec.2012.09.008 2013 • 53 Zinc Oxide Nanoparticles Induce Necrosis and Apoptosis in Macrophages in a p47phox- and Nrf2-Independent Manner
Wilhelmi, V. and Fischer, U. and Weighardt, H. and Schulze-Osthoff, K. and Nickel, C. and Stahlmecke, B. and Kuhlbusch, T.A.J. and Scherbart, A.M. and Esser, C. and Schins, R.P.F. and Albrecht, C.
PLoS ONE 8 (2013)In view of the steadily increasing use of zinc oxide nanoparticles in various industrial and consumer applications, toxicological investigations to evaluate their safety are highly justified. We have investigated mechanisms of ZnO nanoparticle-induced apoptosis and necrosis in macrophages in relation to their important role in the clearance of inhaled particulates and the regulation of immune responses during inflammation. In the murine macrophage RAW 264.7 cell line, ZnO treatment caused a rapid induction of nuclear condensation, DNA fragmentation, and the formation of hypodiploid DNA nuclei and apoptotic bodies. The involvement of the essential effector caspase-3 in ZnO-mediated apoptosis could be demonstrated by immunocytochemical detection of activated caspase-3 in RAW 264.7 cells. ZnO specifically triggered the intrinsic apoptotic pathway, because Jurkat T lymphocytes deficient in the key mediator caspase-9 were protected against ZnO-mediated toxicity whereas reconstituted cells were not. ZnO also caused DNA strand breakage and oxidative DNA damage in the RAW 264.7 cells as well as p47phox NADPH oxidase-dependent superoxide generation in bone marrow-derived macrophages. However, ZnO-induced cell death was not affected in bone marrow-derived macrophages of mice deficient in p47phox or the oxidant responsive transcription factor Nrf2. Taken together, our data demonstrate that ZnO nanoparticles trigger p47phox NADPH oxidase-mediated ROS formation in macrophages, but that this is dispensable for caspase-9/3-mediated apoptosis. Execution of apoptotic cell death by ZnO nanoparticles appears to be NADPH oxidase and Nrf2-independent but rather triggered by alternative routes. © 2013 Wilhelmi et al.
view abstract doi: 10.1371/journal.pone.0065704 2012 • 52 Characterizing permeability and stability of microcapsules for controlled drug delivery by dynamic NMR microscopy
Henning, S. and Edelhoff, D. and Ernst, B. and Leick, S. and Rehage, H. and Suter, D.
Journal of Magnetic Resonance 221 11-18 (2012)Microscopic capsules made from polysaccharides are used as carriers for drugs and food additives. Here, we use NMR microscopy to assess the permeability of capsule membranes and their stability under different environmental conditions. The results allow us to determine the suitability of different capsules for controlled drug delivery. As a measure of the membrane permeability, we monitor the diffusion of paramagnetic molecules into the microcapsules by dynamic NMR microimaging. We obtained the diffusion coefficients of the probe molecules in the membranes and in the capsule core by comparing the measured time dependent concentration maps with numerical solutions of the diffusion equation. The results reveal that external coatings strongly decrease the permeability of the capsules. In addition, we also visualized that the capsules are stable under gastric conditions but dissolve under simulated colonic conditions, as required for targeted drug delivery. Depending on the capsule, the timescales for these processes range from 1 to 28 h. © 2012 Elsevier Inc. All rights reserved.
view abstract doi: 10.1016/j.jmr.2012.05.009 2012 • 51 Cross-amyloid interaction of Aβ and IAPP at lipid membranes
Seeliger, J. and Evers, F. and Jeworrek, C. and Kapoor, S. and Weise, K. and Andreetto, E. and Tolan, M. and Kapurniotu, A. and Winter, R.
Angewandte Chemie - International Edition 51 679-683 (2012)Membrane controlled protein assembly: A study of the amyloid interaction of the islet amyloid polypeptide (IAPP), β-amyloid (Aβ), and a mixture of both with an anionic model raft membrane showed the dominant effect of IAPP on the aggregation process and on the hydrogen-bonding pattern of the assemblies present in the mixture (see picture). The analysis of the interaction of Aβ with IAPP-GI-a non-amyloidogenic IAPP mimic-confirmed these findings. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/anie.201105877 2012 • 50 Determination of pre-steady-state rate constants on the escherichia coli pyruvate dehydrogenase complex reveals that loop movement controls the rate-limiting step
Balakrishnan, A. and Nemeria, N.S. and Chakraborty, S. and Kakalis, L. and Jordan, F.
Journal of the American Chemical Society 134 18644-18655 (2012)Spectroscopic identification and characterization of covalent and noncovalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. The Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), consisting of multiple copies of enzymic components and coenzymes, performs the oxidative decarboxylation of pyruvate to acetyl-CoA and is central to carbon metabolism linking glycolysis to the Krebs cycle. On the basis of earlier studies, we hypothesized that the dynamic regions of the E1p component, which undergo a disorder-order transition upon substrate binding to thiamin diphosphate (ThDP), play a critical role in modulation of the catalytic cycle of PDHc. To test our hypothesis, we kinetically characterized ThDP-bound covalent intermediates on the E1p component, and the lipoamide-bound covalent intermediate on the E2p component in PDHc and in its variants with disrupted active-site loops. Our results suggest that formation of the first covalent predecarboxylation intermediate, C2α-lactylthiamin diphosphate (LThDP), is rate limiting for the series of steps culminating in acetyl-CoA formation. Substitutions in the active center loops produced variants with up to 900-fold lower rates of formation of the LThDP, demonstrating that these perturbations directly affected covalent catalysis. This rate was rescued by up to 5-fold upon assembly to PDHc of the E401K variant. The E1p loop dynamics control covalent catalysis with ThDP and are modulated by PDHc assembly, presumably by selection of catalytically competent loop conformations. This mechanism could be a general feature of 2-oxoacid dehydrogenase complexes because such interfacial dynamic regions are highly conserved. © 2012 American Chemical Society.
view abstract doi: 10.1021/ja3062375 2012 • 49 DFT calculations suggest a new type of self-protection and self-inhibition mechanism in the mammalian heme enzyme myeloperoxidase: Nucleophilic addition of a functional water rather than one-electron reduction
Sicking, W. and Somnitz, H. and Schmuck, C.
Chemistry - A European Journal 18 10937-10948 (2012)The mammalian heme enzyme myeloperoxidase (MPO) catalyzes the reaction of Cl- to the antimicrobial-effective molecule HOCl. During the catalytic cycle, a reactive intermediate "Compound I" (Cpd I) is generated. Cpd I has the ability to destroy the enzyme. Indeed, in the absence of any substrate, Cpd I decays with a half-life of 100 ms to an intermediate called Compound II (Cpd II), which is typically the one-electron reduced Cpd I. However, the nature of Cpd II, its spectroscopic properties, and the source of the additional electron are only poorly understood. On the basis of DFT and time-dependent (TD)-DFT quantum chemical calculations at the PBE0/6-31G* level, we propose an extended mechanism involving a new intermediate, which allows MPO to protect itself from self-oxidation or self-destruction during the catalytic cycle. Because of its similarity in electronic structure to Cpd II, we named this intermediate Cpd IIa'. However, the suggested mechanism and our proposed functional structure of Cpd IIa' are based on the hypothesis that the heme is reduced by charge separation caused by reaction with a water molecule, and not, as is normally assumed, by the transfer of an electron. In the course of this investigation, we found a second intermediate, the reduced enzyme, towards which the new mechanism is equally transferable. In analogy to Cpd II′, we named it FeIIa'. The proposed new intermediates Cpd IIa' and FeIIa' allow the experimental findings, which have been well documented in the literature for decades but not so far understood, to be explained for the first time. These encompass a) the spontaneous decay of Cpd I, b) the unusual (chlorin-like) UV/Vis, circular dichroism (CD), and resonance Raman spectra, c) the inability of reduced MPO to bind CO, d) the fact that MPO-Cpd II reduces SCN- but not Cl-, and e) the experimentally observed auto-oxidation/auto-reduction features of the enzyme. Our new mechanism is also transferable to cytochromes, and could well be viable for heme enzymes in general. Heme mechanisms explained: Direct visual comparison with Cpd II demonstrates that Cpd II′ is a one-electron reduced intermediate with respect to the heme system. In both cases an electron is transferred: in Cpd II from an external donor, and in Cpd II′ through charge separation caused by reaction with a water molecule (see figure). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/chem.201103477 2012 • 48 DipA, a pore-forming protein in the outer membrane of lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates
Thein, M. and Bonde, M. and Bunikis, I. and Denker, K. and Sickmann, A. and Bergström, S. and Benz, R.
PLoS ONE 7 (2012)Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species. © 2012 Thein et al.
view abstract doi: 10.1371/journal.pone.0036523 2012 • 47 E. coli LoiP (YggG), a metalloprotease hydrolyzing Phe-Phe bonds
Lütticke, C. and Hauske, P. and Lewandrowski, U. and Sickmann, A. and Kaiser, M. and Ehrmann, M.
Molecular BioSystems 8 1775-1782 (2012)YggG is a conserved lipoprotein localized to the outer membrane of Gram negative bacteria. Even though the expressed open reading frame has been identified previously, the Escherichia coli protein remained uncharacterized. We report that YggG of E. coli is a metalloprotease that cleaves its targets preferentially between Phe-Phe residues. Since the yggG promoter is upregulated when bacteria are subjected to media of low osmolarity, YggG was named LoiP (low osmolarity induced protease). LoiP has an intramolecular disulfide (S-S) bond that is formed even in the absence of the periplasmic oxido-reductase DsbA and proper membrane localization of LoiP can depend on another putative metalloprotease, YfgC. © 2012 The Royal Society of Chemistry.
view abstract doi: 10.1039/c2mb05506f 2012 • 46 Fluorescence labels in kinases: A high-throughput kinase binding assay for the identification of DFG-out binding ligands
Simard, J.R. and Rauh, D.
Methods in Molecular Biology 800 95-117 (2012)Despite the hundreds of kinase inhibitors currently in discovery and pre-clinical phases, the number of kinase inhibitors which have been approved and are on the market remains low by comparison. This discrepancy reflects the challenges which accompany the development of kinase inhibitors which are relatively specific and less toxic. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibiting kinase activity, but the highly conserved nature of the ATP-binding site contributes to poor inhibitor selectivity, issues which have particularly hampered the development of novel kinase inhibitors. We developed a high-throughput screening technology that can discriminate for inhibitors which stabilize the inactive "DFG-out" kinase conformation by binding within an allosteric pocket adjacent to the ATP-binding site. Here, we describe how to use this approach to measure the K d of ligands, as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries at high throughput. © 2012 Springer Science+Business Media, LLC.
view abstract doi: 10.1007/978-1-61779-349-3_8 2012 • 45 Fluorophore labeled kinase detects ligands that bind within the MAPK insert of p38α kinase
Getlik, M. and Simard, J.R. and Termathe, M. and Grütter, C. and Rabiller, M. and van Otterlo, W.A.L. and Rauh, D.
PLoS ONE 7 (2012)The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. © 2012 Getlik et al.
view abstract doi: 10.1371/journal.pone.0039713 2012 • 44 Interactions of multicationic bis(guanidiniocarbonylpyrrole) receptors with double-stranded nucleic acids: Syntheses, binding studies, and atomic force microscopy imaging
Klemm, K. and Stojković, M.R. and Horvat, G. and Tomišić, V. and Piantanida, I. and Schmuck, C.
Chemistry - A European Journal 18 1352-1363 (2012)Compounds 1-3, composed of two guanidiniocarbonylpyrrole moieties linked by oligoamide bridges and differing in number and type of basic groups, were prepared. The sites and degree of protonation of 1-3 depend strongly on the pH value. The interactions of these compounds with several double-stranded (ds) DNA and dsRNA were investigated by means of UV/Vis and CD spectroscopy as well as isothermal titration microcalorimetry (ITC). These studies revealed that the binding of 1-3 to the polynucleotides is driven by three factors, the presence of aliphatic amino groups, the protonation state of the compounds, and the steric properties of the polynucleotide binding site, that is, the shape and structure of their grooves. The results obtained by all applied methods consistently indicated that receptors 1-3 bind to the minor groove of DNA, but, by contrast, to the major groove of RNA. Additionally, it was shown by atomic force microscopy (AFM) imaging that upon interaction of compound 2 with calf thymus (ct) DNA induced aggregation of the DNA occurs, leading to pronounced changes in its secondary structure. © 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.
view abstract doi: 10.1002/chem.201101544 2012 • 43 Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): Sequence characterisation, expression pattern and characterisation of proteolytic activity
Balczun, C. and Siemanowski, J. and Pausch, J.K. and Helling, S. and Marcus, K. and Stephan, C. and Meyer, H.E. and Schneider, T. and Cizmowski, C. and Oldenburg, M. and Höhn, S. and Meiser, C.K. and Schuhmann, W. and Schaub, G.A.
Insect Biochemistry and Molecular Biology 42 240-250 (2012)Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2. © 2011 Elsevier Ltd.
view abstract doi: 10.1016/j.ibmb.2011.12.006 2012 • 42 Mass transport controlled oxygen reduction at anthraquinone modified 3D-CNT electrodes with immobilized Trametes hirsuta laccase
Sosna, M. and Stoica, L. and Wright, E. and Kilburn, J.D. and Schuhmann, W. and Bartlett, P.N.
Physical Chemistry Chemical Physics 14 11882-11885 (2012)Carbon nanotubes covalently modified with anthraquinone were used as an electrode for the immobilization of Trametes hirsuta laccase. The adsorbed laccase is capable of oxygen reduction at a mass transport controlled rate (up to 3.5 mA cm-2) in the absence of a soluble mediator. The storage and operational stability of the electrode are excellent. This journal is © 2012 the Owner Societies.
view abstract doi: 10.1039/c2cp41588g 2012 • 41 Preparation and comparative release characteristics of three anthocyanin encapsulation systems
Oidtmann, J. and Schantz, M. and Mäder, K. and Baum, M. and Berg, S. and Betz, M. and Kulozik, U. and Leick, S. and Rehage, H. and Schwarz, K. and Richling, E.
Journal of Agricultural and Food Chemistry 60 844-851 (2012)Bilberries (Vaccinium myrtillus L.) and their major polyphenolic constituents, anthocyanins, have preventive activities inter alia against colon cancer and inflammatory bowel diseases. However, anthocyanins are sensitive to environmental conditions; thus their bioavailability in the gastrointestinal tract is an important determinant of their in vivo activity. In the study reported here, the potential benefits of encapsulating an anthocyanin rich bilberry extract (BE) on anthocyanin stability were investigated. Nonencapsulated BE and three different BE loaded microcapsule systems were incubated in simulated gastric fluid (SGF) and fed state simulated intestinal fluid (FeSSIF). After exposure to these media, released anthocyanins were identified and quantified by HPLC with UV/Vis detection. Although a rapid release of anthocyanins was observed within the first 20 min, encapsulation of anthocyanins doubled the amount of available anthocyanins after 150 min of incubation. These results illustrate the ability of encapsulation to inhibit early degradation of anthocyanins in the intestinal system. © 2011 American Chemical Society.
view abstract doi: 10.1021/jf2047515 2012 • 40 Protein recognition: Calixarene connection
Nature Chemistry 4 519-520 (2012)
doi: 10.1038/nchem.1386 2012 • 39 The biocompatibility of metal-organic framework coatings: An investigation on the stability of SURMOFs with regard to water and selected cell culture media
Hanke, M. and Arslan, H.K. and Bauer, S. and Zybaylo, O. and Christophis, C. and Gliemann, H. and Rosenhahn, A. and Wöll, C.
Langmuir 28 6877-6884 (2012)Highly porous thin films based on a [Cu(bdc) 2] n (bdc = benzene-1,4-dicarboxylic acid) metal-organic framework, MOF, grown using liquid-phase epitaxy (LPE) show remarkable stability in pure water as well as in artificial seawater. This opens the possibility to use these highly porous coatings for environmental and life science applications. Here we characterize in detail the stability of these SURMOF 2 thin films under aqueous and cell culture conditions. We find that the material degrades only very slowly in water and artificial seawater (ASW) whereas in typical cell culture media (PBS and DMEM) a rapid dissolution is observed. The release of Cu 2+ ions resulting from the dissolution of the SURMOF 2 in the liquids exhibits no adverse effect on the adhesion of fibroblasts, prototype eukaryotic cells, to the substrate and their subsequent proliferation, thus demonstrating the biocompatibility of SURMOF 2 surface coatings. Thus, the results are an important step toward application of these porous materials as a slow release matrix, for example, for pharmaceuticals and growth factors. © 2012 American Chemical Society.
view abstract doi: 10.1021/la300457z 2012 • 38 The effect of ionic strength, temperature, and pressure on the interaction potential of dense protein solutions: From nonlinear pressure response to protein crystallization
Möller, J. and Schroer, M.A. and Erlkamp, M. and Grobelny, S. and Paulus, M. and Tiemeyer, S. and Wirkert, F.J. and Tolan, M. and Winter, R.
Biophysical Journal 102 2641-2648 (2012)Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b 2 values for controlled protein crystallization. © 2012 Biophysical Society.
view abstract doi: 10.1016/j.bpj.2012.04.043 2012 • 37 Thermodynamic properties of aqueous salt containing urea solutions
Sadeghi, M. and Held, C. and Samieenasab, A. and Ghotbi, C. and Abdekhodaie, M.J. and Taghikhani, V. and Sadowski, G.
Fluid Phase Equilibria 325 71-79 (2012)Urea and inorganic ions are present in some of the physiological systems, e.g. urine. Understanding the interactions in urea/salt/water is a preliminary step to shed light on more complicated behavior of multi-component physiological systems. State-of-the-art models as well as thermophysical properties can be applied to understand the interactions in these systems. In order to determine such interactions densities, mean ionic activity coefficients (MIACs), osmotic coefficients, and solubility were measured in aqueous solutions of urea and different salts. Densities were determined at temperatures 293.15, 303.15, and 313.15K for urea concentrations up to 3molal and up to 1molal for NaCl. Osmotic coefficients and MIACs were obtained at 310.15K by using the vapor-pressure osmometry and potentiometry methods, respectively. Ternary aqueous urea solutions containing NaCl, KCl, NaBr, KBr, LiBr, NaNO 3, and LiNO 3 at two different concentrations of urea (0.3 and 1molal) as well as at three salt concentrations (0.25, 0.5, and 0.75molal) were considered. Moreover, urea solubility was also measured at 310.15K in 3 and 5molal NaCl solutions in the present work. Experimental data obtained in this work showed that the salt primarily dictates the volumetric properties and the MIAC while the solute with higher concentration determines the behavior of osmotic coefficients in these solutions. The ePC-SAFT model (without any adjustable mixture parameter) was used to accurately predict the experimental densities, activity and osmotic coefficients, and solubilities of the studied mixtures. © 2012 Elsevier B.V.
view abstract doi: 10.1016/j.fluid.2012.04.003 2012 • 36 Turing instabilities in a mathematical model for signaling networks
Rätz, A. and Röger, M.
Journal of Mathematical Biology 65 1215-1244 (2012)GTPase molecules are important regulators in cells that continuously run through an activation/deactivation and membrane-attachment/membrane-detachment cycle. Activated GTPase is able to localize in parts of the membranes and to induce cell polarity. As feedback loops contribute to the GTPase cycle and as the coupling between membrane-bound and cytoplasmic processes introduces different diffusion coefficients a Turing mechanism is a natural candidate for this symmetry breaking. We formulate a mathematical model that couples a reaction-diffusion system in the inner volume to a reaction-diffusion system on the membrane via a flux condition and an attachment/detachment law at the membrane. We present a reduction to a simpler non-local reaction-diffusion model and perform a stability analysis and numerical simulations for this reduction. Our model in principle does support Turing instabilities but only if the lateral diffusion of inactivated GTPase is much faster than the diffusion of activated GTPase. © 2011 Springer-Verlag.
view abstract doi: 10.1007/s00285-011-0495-4 2011 • 35 A minimalist model for ion partitioning and competition in a K + channel selectivity filter
Kast, S.M. and Kloss, T. and Tayefeh, S. and Thiel, G.
Journal of General Physiology 138 371-373 (2011)
doi: 10.1085/jgp.201110694 2011 • 34 Accumulation of silver nanoparticles by cultured primary brain astrocytes
Luther, E.M. and Koehler, Y. and Diendorf, J. and Epple, M. and Dringen, R.
Nanotechnology 22 (2011)Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24h resulted in a time-and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4h with identical amounts of silver as AgNO 3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways. © 2011 IOP Publishing Ltd.
view abstract doi: 10.1088/0957-4484/22/37/375101 2011 • 33 Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: Efficiency and cytotoxicity
Durán, M.C. and Willenbrock, S. and Barchanski, A. and Müller, J.-M.V. and Maiolini, A. and Soller, J.T. and Barcikowski, S. and Nolte, I. and Feige, K. and Murua Escobar, H.
Journal of Nanobiotechnology 9 (2011)Background: Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence.Results: The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect.Conclusions: This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered. © 2011 Durán et al; licensee BioMed Central Ltd.
view abstract doi: 10.1186/1477-3155-9-47 2011 • 32 Construction of uricase-overproducing strains of Hansenula polymorpha and its application as biological recognition element in microbial urate biosensor
Dmytruk, K.V. and Smutok, O.V. and Dmytruk, O.V. and Schuhmann, W. and Sibirny, A.A.
BMC Biotechnology 11 (2011)Background: The detection and quantification of uric acid in human physiological fluids is of great importance in the diagnosis and therapy of patients suffering from a range of disorders associated with altered purine metabolism, most notably gout and hyperuricaemia. The fabrication of cheap and reliable urate-selective amperometric biosensors is a challenging task.Results: A urate-selective microbial biosensor was developed using cells of the recombinant thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. The construction of uricase (UOX) producing yeast by over-expression of the uricase gene of H. polymorpha is described. Following a preliminary screening of the transformants with increased UOX activity in permeabilized yeast cells the optimal cultivation conditions for maximal UOX yield namely a 40-fold increase in UOX activity were determined.The UOX producing cells were coupled to horseradish peroxidase and immobilized on graphite electrodes by physical entrapment behind a dialysis membrane. A high urate selectivity with a detection limit of about 8 μM was found.Conclusion: A strain of H. polymorpha overproducing UOX was constructed. A cheap urate selective microbial biosensor was developed. © 2011 Dmytruk et al; licensee BioMed Central Ltd.
view abstract doi: 10.1186/1472-6750-11-58 2011 • 31 Electron transfer between genetically modified Hansenula polymorpha yeast cells and electrode surfaces via os-complex modified redox polymers
Shkil, H. and Schulte, A. and Guschin, D.A. and Schuhmann, W.
ChemPhysChem 12 806-813 (2011)Graphite electrodes modified with redox-polymer-entrapped yeast cells were investigated with respect to possible electron-transfer pathways between cytosolic redox enzymes and the electrode surface. Either wild-type or genetically modified Hansenula polymorpha yeast cells over-expressing flavocytochrome b2 (FC b2) were integrated into Os-complex modified electrodeposition polymers. Upon increasing the L-lactate concentration, an increase in the current was only detected in the case of the genetically modified cells. The overexpression of FC b2 and the related amplification of the FC b2/L-lactate reaction cycle was found to be necessary to provide sufficient charge to the electron-exchange network in order to facilitate sufficient electrochemical coupling between the cells, via the redox polymer, to the electrode. The close contact of the Os-complex modified polymer to the cell wall appeared to be a prerequisite for electrically wiring the cytosolic FC b2/L-lactate redox activity and suggests the critical involvement of a plasma membrane redox system. Insights in the functioning of whole-cell-based bioelectrochemical systems have to be considered for the successful design of whole-cell biosensors or microbial biofuel cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/cphc.201000889 2011 • 30 Exploring the piezophilic behavior of natural cosolvent mixtures
Schroer, M.A. and Zhai, Y. and Wieland, D.C.F. and Sahle, C.J. and Nase, J. and Paulus, M. and Tolan, M. and Winter, R.
Angewandte Chemie - International Edition 50 11413-11416 (2011)Marine organisms have evolved a surprising mechanism to counteract the deleterious effects of urea by trimethylammonium N-oxide (TMAO). The effect of pressure on the structure and intermolecular interactions of lysozyme in urea and TMAO solutions was studied (see picture). These findings help to understand the compensatory effect of urea-TMAO mixtures in deep-sea organisms. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/anie.201104380 2011 • 29 Functionalization of DNA nanostructures with proteins
Saccà, B. and Niemeyer, C.M.
Chemical Society Reviews 40 5910-5921 (2011)Proteins possess intrinsic functionalities, which have been optimized in billions of years of natural evolution. The conjugation of proteins with artificial nucleic acids allows one to further functionalize proteins with a synthetically accessible, physicochemically robust tag, which is addressable in a highly specific manner by Watson-Crick hybridization. The resulting DNA-protein conjugates can be advantageously used in a variety of applications, ranging from biomedical diagnostics to DNA-based nanofabrication. This critical review provides an overview on chemical approaches to the synthesis of DNA-protein conjugates and their applications in biomolecular nanosciences (96 references). © The Royal Society of Chemistry 2011.
view abstract doi: 10.1039/c1cs15212b 2011 • 28 Identification of Eps15 as antigen recognized by the monoclonal antibodies aa2 and ab52 of the wuerzburg hybridoma library against Drosophila brain
Halder, P. and Chen, Y.-c. and Brauckhoff, J. and Hofbauer, A. and Dabauvalle, M.-C. and Lewandrowski, U. and Winkler, C. and Sickmann, A. and Buchner, E.
PLoS ONE 6 (2011)The Wuerzburg Hybridoma Library against the Drosophilabrain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies. © 2011 Halder et al.
view abstract doi: 10.1371/journal.pone.0029352 2011 • 27 Immuno-surface-enhanced coherent anti-Stokes Raman scattering microscopy: Immunohistochemistry with target-specific metallic nanoprobes and nonlinear Raman microscopy
Schlücker, S. and Salehi, M. and Bergner, G. and Schütz, M. and Ströbel, P. and Marx, A. and Petersen, I. and Dietzek, B. and Popp, J.
Analytical Chemistry 83 7081-7085 (2011)Immunohistochemistry (IHC) is one of the most widely used staining techniques for diagnostic purposes. The selective localization of target proteins in tissue specimens by conventional IHC is achieved with dye- or enzyme-labeled antibodies in combination with light microscopy. In this contribution, we demonstrate the proof-of-principle for IHC based on surface-enhanced coherent Raman scattering for contrast generation. Specifically, antibody-labeled metallic nanoshells in conjunction with surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy are employed for the selective, sensitive, and rapid localization of the basal cell protein p63 in normal prostate tissue. Negative control experiments were performed in order to confirm the selective binding of the target-specific metal nanoprobes and to disentangle the role of plasmonic (metal) and molecular (Raman reporter) resonances in this plasmon-assisted four-wave mixing technique. © 2011 American Chemical Society.
view abstract doi: 10.1021/ac201284d 2011 • 26 Mechanistic studies of Fc-PNA(·DNA) surface dynamics based on the kinetics of electron-transfer processes
Hüsken, N. and Gȩbala, M. and La Mantia, F. and Schuhmann, W. and Metzler-Nolte, N.
Chemistry - A European Journal 17 9678-9690 (2011)N-Terminally ferrocenylated and C-terminally gold-surface-grafted peptide nucleic acid (PNA) strands were exploited as unique tools for the electrochemical investigation of the strand dynamics of short PNA(·DNA) duplexes. On the basis of the quantitative analysis of the kinetics and the diffusional characteristics of the electron-transfer process, a nanoscopic view of the Fc-PNA(·DNA) surface dynamics was obtained. Loosely packed, surface-confined Fc-PNA single strands were found to render the charge-transfer process of the tethered Fc moiety diffusion-limited, whereas surfaces modified with Fc-PNA·DNA duplexes exhibited a charge-transfer process with characteristics between the two extremes of diffusion and surface limitation. The interplay between the inherent strand elasticity and effects exerted by the electric field are supposed to dictate the probability of a sufficient approach of the Fc head group to the electrode surface, as reflected in the measured values of the electron-transfer rate constant, k 0. An in-depth understanding of the dynamics of surface-bound PNA and PNA·DNA strands is of utmost importance for the development of DNA biosensors using (Fc-)PNA recognition layers. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/chem.201003764 2011 • 25 Nucleotide recognition in water by a guanidinium-based artificial tweezer receptor
Kuchelmeister, H.Y. and Schmuck, C.
Chemistry - A European Journal 17 5311-5318 (2011)The water-soluble tweezer receptor 1 with two symmetric peptidic arms, which are connected by an aromatic scaffold and contain lysine, phenylalanine, and a guanidinium-based anion-binding site as headgroup, has been synthesized. UV/Vis-derived Job plots show that the receptor forms 1:1 complexes with nucleotides and phosphate in buffered water at neutral pH. Binding constants have been determined by fluorescence and UV/Vis spectroscopy. All nucleotides tested were bound very efficiently, even in pure water, with binding constants between 104 and 105 M-1. Interestingly, all mononucleotides were bound much stronger than phosphate by a factor of at least 5 to 10. Furthermore 1 favors the binding of adenosine monophosphate (AMP) over adenosine diphosphate (ADP) and adenosine triphosphate (ATP), which is unprecedented for artificial nucleotide receptors reported so far. According to NMR spectroscopy and molecular modeling studies, the efficient binding is a result of strong electrostatic contacts supported by π-π interactions with the nucleobase within the cavity-shaped receptor. Nucleotides preferred: The symmetric peptide-based tweezer receptor 1 forms stable 1:1 complexes with nucleotides in water (pH 7). The host prefers nucleotides over phosphate and mononucleotides over di- or trinucleotides (see scheme). Hence, complex formation is due also to π-stacking interactions with the nucleobase. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/chem.201003393 2011 • 24 Phosphoproteome analysis of the platelet plasma membrane
Premsler, T. and Lewandrowski, U. and Sickmann, A. and Zahedi, R.P.
Methods in Molecular Biology 728 279-290 (2011)Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context. In addition, since platelet activation and aggregation usually require surface contact with the surrounding tissue, special interest focuses on this contacting region, and hence on the subproteome of the platelet plasma membrane. In this chapter, we present a mass spectrometry-driven approach capable of dealing with the task of platelet plasma membrane proteomics and phosphoproteomics. The outlined protocols include strategies for the isolation and purification of plasma membrane proteins by aqueous two-phase partitioning and subsequent enrichment of phosphopeptides via titanium dioxide chromatography. © 2011 Springer Science+Business Media, LLC.
view abstract doi: 10.1007/978-1-61779-068-3_19 2011 • 23 Physiology and pathophysiology of nitrosative and oxidative stress in osteoarthritic joint destruction
Ziskoven, C. and Jäger, M. and Kircher, J. and Patzer, T. and Bloch, W. and Brixius, K. and Krauspe, R.
Canadian Journal of Physiology and Pharmacology 89 455-466 (2011)Osteoarthritis (OA) is one of the most common chronic diseases, with increasing importance due to increased life expectancy. On a cellular level, the pathophysiology of joint function impairment and ultimate destruction associated with OA remains poorly understood. Free radicals are highly reactive molecules involved in both normal intracellular signal transduction and degenerative cellular processes. An imbalance between the free radical burden and cellular scavenging mechanisms, defined as oxidative stress, has been identified as a relevant factor in OA pathogenesis. This literature review elucidates the involvement of nitrosative and oxidative stress in cellular ageing in joints, cell senescence, and apoptosis. Free radical exposure is known to promote cellular senescence and apoptosis, and the involvement of radical oxygen species (ROS) in inflammation, fibrosis control, and pain nociception has been proven. A relatively novel approach to OA pathophysiology considers the joint to be a dynamic system consisting of 3, continuously interacting compartments, cartilage, synoviaZl tissue, and subchondral bone. Current knowledge concerning free radical involvement in paracrine signalling in OA is reviewed. The interrelationship between oxidative imbalances and OA pathophysiology may provide a novel approach to the comprehension, and therefore modification, of OA disease progression and symptom control.
view abstract doi: 10.1139/Y11-055 2011 • 22 Structure and phase behavior of archaeal lipid monolayers
Jeworrek, C. and Evers, F. and Erlkamp, M. and Grobelny, S. and Tolan, M. and Chong, P.L.-G. and Winter, R.
Langmuir 27 13113-13121 (2011)We report X-ray reflectivity (XRR) and grazing incidence X-ray diffraction (GIXD) measurements of archaeal bipolar tetraether lipid monolayers at the air-water interface. Specifically, Langmuir films made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at three different temperatures, i.e., 68, 76, and 81 °C, were examined. The dependence of the structure and packing properties of PLFE monolayers on surface pressure were analyzed in a temperature range between 10 and 50 °C at different pH values. Additionally, the interaction of PLFE monolayers (using lipids derived from cells grown at 76 °C) with the ion channel peptide gramicidin was investigated as a function of surface pressure. A total monolayer thickness of approximately 30 Å was found for all monolayers, hinting at a U-shaped conformation of the molecules with both head groups in contact with the interface. The monolayer thickness increased with rising film pressure and decreased with increasing temperature. At 10 and 20 °C, large, highly crystalline domains were observed by GIXD, whereas at higher temperatures no distinct crystallinity could be observed. For lipids derived from cells grown at higher temperatures, a slightly more rigid structure in the lipid dibiphytanyl chains was observed. A change in the pH of the subphase had an influence only on the structure of the lipid head groups. The addition of gramicidin to an PLFE monolayer led to a more disordered state as observed by XRR. In GIXD measurements, no major changes in lateral organization could be observed, except for a decrease of the size of crystalline domains, indicating that gramicidin resides mainly in the disordered areas of the monolayer and causes local membrane perturbation, only. © 2011 American Chemical Society.
view abstract doi: 10.1021/la202027s 2010 • 21 Accurate prediction of neuroblastoma outcome based on miRNA expression profiles
Schulte, J.H. and Schowe, B. and Mestdagh, P. and Kaderali, L. and Kalaghatgi, P. and Schlierf, S. and Vermeulen, J. and Brockmeyer, B. and Pajtler, K. and Thor, T. and De Preter, K. and Speleman, F. and Morik, K. and Eggert, A. a...
International Journal of Cancer 127 2374-2385 (2010)For neuroblastoma, the most common extracranial tumour of childhood, identification of new biomarkers and potential therapeutic targets is mandatory to improve risk stratification and survival rates. MicroRNAs are deregulated in most cancers, including neuroblastoma. In this study, we analysed 430 miRNAs in 69 neuroblastomas by stem-loop RT-qPCR. Prediction of event-free survival (EFS) with support vector machines (SVM) and actual survival times with Cox regression-based models (CASPAR) were highly accurate and were independently validated. SVM-accuracy for prediction of EFS was 88.7% (95% CI: 88.5-88.8%). For CASPAR-based predictions, 5y-EFS probability was 0.19% (95% CI: 0-38%) in the CASPAR-predicted short survival group compared with 0.78% (95%CI: 64-93%) in the CASPAR-predicted long survival group. Both classifiers were validated on an independent test set yielding accuracies of 94.74% (SVM) and 5y-EFS probabilities as 0.25 (95% CI: 0.0-0.55) for short versus 1 ± 0.0 for long survival (CASPAR), respectively. Amplification of the MYCN oncogene was highly correlated with deregulation of miRNA expression. In addition, 37 miRNAs correlated with TrkA expression, a marker of excellent outcome, and 6 miRNAs further analysed in vitro were regulated upon TrkA transfection, suggesting a functional relationship. Expression of the most significant TrkA-correlated miRNA, miR-542-5p, also discriminated between local and metastatic disease and was inversely correlated with MYCN amplification and event-free survival. We conclude that neuroblastoma patient outcome prediction using miRNA expression is feasible and effective. Studies testing miRNA-based predictors in comparison to and in combination with mRNA and aCGH information should be initiated. Specific miRNAs (e.g., miR-542-5p) might be important in neuroblastoma tumour biology, and qualify as potential therapeutic targets. © 2010 UICC.
view abstract doi: 10.1002/ijc.25436 2010 • 20 Active transport of amino acids by a guanidiniocarbonyl-pyrrole receptor
Urban, C. and Schmuck, C.
Chemistry - A European Journal 16 9502-9510 (2010)Herein we report the synthesis and characterization of a transporter 9 for N-acetylated amino acids. Transporter 9 is a conjugate of a guanidiniocarbonyl pyrrole cation, one of the most efficient carboxylate binding motifs reported so far, and a hydrophobictris (dodecylbenzyl) group, which ensures solubility in organic solvents. In its protonated form, 9 binds N-acetylated amino acid carboxylates in wetorganic solvents with association constantsin the range of 104m-1 as estimated by extraction experiments. Aromatic amino acids are preferred due to additional cation-α-interactions of the amino acid side chain with the guanidiniocarbonyl pyrrole moiety. U-tube experiments established efficient transport across a bulk liquid chloroform phase with fluxes approaching 10-6 molm-2s-1. In experiments with single substrates, the release rate of the amino acid from the receptor-substrate complex at the interface with the receiving phase is rate determining. In contrast to this, in competition experiments with several substrates, the therm-odynamic affinity to 9 becomes decisive. As 9 can only transport anions in its protonated form and has a pKa value of approximately 7, pH-driven active transport of amino acids is also possible. Transport occurs as a symport of the amino acid carboxylate and a proton. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/chem.201000509 2010 • 19 An integrated catalytic approach to fermentable sugars from cellulose
Rinaldi, R. and Engel, P. and Büchs, J. and Spiess, A.C. and Schüth, F.
ChemSusChem 3 1151-1153 (2010)The production of fermentable sugars from cellulose in almost quantitative yield is accelerated. Starting from cello-oligomers obtained by acid hydrolysis of cellulose in an ionic liquid, the catalytic approach described herein, integrating acid and enzymatic catalysis, quantitatively converts cellulose to fermentable sugars (glucose and cellobiose) within only a few hours. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/cssc.201000153 2010 • 18 Characterization of a novel interaction between vasodilator-stimulated phosphoprotein and abelson interactor 1 in human platelets: A concerted computational and experimental approach
Dittrich, M. and Strassberger, V. and Fackler, M. and Tas, P. and Lewandrowski, U. and Sickmann, A. and Walter, U. and Dandekar, T. and Birschmann, I.
Arteriosclerosis, Thrombosis, and Vascular Biology 30 843-850 (2010)Objective: The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. Methods and results: Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum. uni-wuerzburg.de/PlateletWeb.php), we performed a motif search of all sequences and identified potential target sites of class I EVH1 domains in human platelet proteins. Performing affinity purification with VASP-EVH1 domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 domain-specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. Conclusion: The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, as they offer the potential to analyze signal transduction beyond the scope of established pathways. © 2010 American Heart Association, Inc.
view abstract doi: 10.1161/ATVBAHA.109.200683 2010 • 17 Effect of surface charge distribution on the adsorption orientation of proteins to lipid monolayers
Tiemeyer, S. and Paulus, M. and Tolan, M.
Langmuir 26 14064-14067 (2010)The adsorption orientation of the proteins lysozyme and ribonuclease A (RNase A) to a neutral 1,2-dipalmitoyl-snglycero- 3-phosphocholine (DPPC) and a negatively charged stearic acid lipid film was investigated by means of X-ray reflectivity. Both proteins adsorbed to the negatively charged lipid monolayer, whereas at the neutral monolayer, no adsorption was observed. For acquiring comprehensive information on the proteins' adsorption, X-ray reflectivity data were combined with electron densities obtained from crystallographic data. With this method, it is possible to determine the orientation of adsorbed proteins in solution underneath lipid monolayers. While RNase A specifically coupled with its positively charged active site to the negatively charged lipid monolayer, lysozyme prefers an orientation with its long axis parallel to the Langmuir film. In comparison to the electrostatic maps of the proteins, our results can be explained by the discriminative surface charge distribution of lysozyme and RNase A. © 2010 American Chemical Society.
view abstract doi: 10.1021/la102616h 2010 • 16 Effect of urea and glycerol on the adsorption of ribonuclease a at the air-water interface
Hüsecken, A.K. and Evers, F. and Czeslik, C. and Tolan, M.
Langmuir 26 13429-13435 (2010)This study reports on the influence of nonionic cosolvents on the interfacial structure of ribonuclease A (RNase) adsorbed at the air-water interface. We applied X-ray reflectometry to obtain detailed volume fraction profiles of the adsorbed layers and to follow the effect of glycerol and urea on the adsorbate structure as a function of cosolvent concentration. Under all conditions studied, the adsorbed RNase layer maintains its compact shape, and the adsorbed RNase molecules adopt a flat-on orientation at the interface. Both kosmotropic glycerol and chaotropic urea exert profound effects on the adsorbate: The surface excess decreases linearly with glycerol content and is also reduced at low urea concentration. However, at high urea concentration, parts of the adsorbed layer are dehydrated and become exposed to air. The electron density and volume fraction profiles of the adsorbed protein provide clear evidence that these effects are ruled by different mechanisms. © 2010 American Chemical Society.
view abstract doi: 10.1021/la102222z 2010 • 15 Guanidiniocarbonylpyrrole-aryl derivatives: Structure tuning for spectrophotometry; recognition of specific DNA and RNA sequences and for antiproliferative activity
Hernandez-Folgado, L. and Baretić, D. and Piantanida, I. and Marjanović, M. and Kralj, M. and Rehm, T. and Schmuck, C.
Chemistry - A European Journal 16 3036-3056 (2010)We present a systematic study of different guanidiniocarbonylpyrrole-aryl derivatives designed to interact with DNA or RNA both through intercalation of an aromatic moiety into the base stack of the nucleotide and through groove binding of a guanidiniocarbonylpyrrole cation. We varied 1 ) the size of the aromatic ring (benzene, naphthalene, pyrene and acridine), 2) the length and flexibility of the linker connecting the two binding groups, and 3) the total number of positive charges present at different pH values. The compounds and their interactions with DNA and RNA were studied by UV/Vis, fluorescence and CD spectroscopy. Antiproliferative activities against human tumour cell lines were also determined. Our studies show that efficient interaction with, for example, DNA requires a significantly large aromatic ring (pyrene) connected through a flexible linker to the pyrrole moiety. However, a positive charge, as in 12, is also needed. Compound 12 allows for base-pair-selective recognition of ds-DNA at physiological pH values. The antiproliferative activities of these compounds correlate with their binding affinities towards DNA, suggesting that their biological effects are most probably due to DNA binding. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
view abstract doi: 10.1002/chem.200901999 2010 • 14 High-pressure SAXS study of folded and unfolded ensembles of proteins
Schroer, M.A. and Paulus, M. and Jeworrek, C. and Krywka, C. and Schmacke, S. and Zhai, Y. and Wieland, D.C.F. and Sahle, C.J. and Chimenti, M. and Royer, C.A. and Garcia-Moreno, B. and Tolan, M. and Winter, R.
Biophysical Journal 99 3430-3437 (2010)A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil. © 2010 by the Biophysical Society.
view abstract doi: 10.1016/j.bpj.2010.09.046 2010 • 13 Identification of Thiazolidinones Spiro-Fused to IndoIin-2-ones as Potent and Selective Inhibitors of the Mycobacterium tuberculosis Protein Tyrosine Phosphatase B
Vintonyak, V.V. and Warburg, K. and Kruse, H. and Grimme, S. and Hübet, K. and Rauh, D. and Waldmann, H.
Angewandte Chemie - International Edition 49 5902-5905 (2010)(Figure Presented) The best of 40000: Detailed structureactivity- relationship studies revealed key structural elements of indolin-2-on-3- spirothiazolidinones (see example) and their appropriate configuration for strong inhibitory activity against the pathophysiologically relevant title protein. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
view abstract doi: 10.1002/anie.201002138 2010 • 12 Kynurenine inhibits chondrocyte proliferation and is increased in synovial fluid of patients with septic arthritis
Lögters, T.T. and Laryea, M.D. and Jäger, M. and Schädel-Höpfner, M. and Windolf, J. and Flohé, S. and Altrichter, J. and Scholz, M. and Paunel-Görgülü, A.N.
Journal of Orthopaedic Research 28 1490-1496 (2010)Kynurenine, the major degradation product of tryptophan has been shown to directly damage various tissues. Its potential contribution to septic arthritis is unknown. In this study, we analyzed the putative diagnostic value of kynurenine for bacterial joint infection and its potential harmful effects on cartilage. In a prospective study 41 patients with a joint effusion who had undergone arthrocentesis were included. Tryptophan and kynurenine levels from synovial fluid were quantified by HPLC. Diagnostic value of kynurenine was evaluated and its effects on the proliferation of the chondrocyte cell line ATDC5 were determined. Synovial fluid kynurenine values from patients with septic arthritis (4.1 ± 0.8 μmol/L, n = 9) were significantly increased compared to patients with non-infectious inflammatory arthropathy (1.8 ± 0.2 μmol/L, n = 17) or osteoarthritis (1.2 ± 0.1 μmol/L, n = 15, p < 0.01). At a cut-off value of 2.28 μmol/L kynurenine had a sensitivity of 0.89 and a specificity of 0.87. Further, kynurenine inhibited chondrocyte (ATDC5) cell proliferation in a dose-dependent manner. Septic arthritis is associated with significantly increased values of synovial kynurenine. Furthermore kynurenine inhibits proliferation of chondrocytes, which strongly suggests a pathophysiological effect of kynurenine on cartilage in inflammatory arthropathies. © 2010 Orthopaedic Research Society.
view abstract doi: 10.1002/jor.21158 2010 • 11 Malignant transformation in a defined genetic background: Proteome changes displayed by 2D-PAGE
Pütz, S.M. and Vogiatzi, F. and Stiewe, T. and Sickmann, A.
Molecular Cancer 9 (2010)Background: Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).Results: The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot.Conclusion: These findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis. © 2010 Pütz et al; licensee BioMed Central Ltd.
view abstract doi: 10.1186/1476-4598-9-254 2010 • 10 Nonendosomal cellular uptake of ligand-free, positively charged gold nanoparticles
Taylor, U. and Klein, S. and Petersen, S. and Kues, W. and Barcikowski, S. and Rath, D.
Cytometry Part A 77 439-446 (2010)Gold nanoparticles (GNPs) have interesting optical properties, such as exceptionally high quantum yields and virtually limitless photostability. Therefore, they show the potential for applications as biomarkers especially suitable for in vivo and long-term studies. The generation of GNPs using pulsed laser light rather than chemical means provides nanoparticles, which are remarkably stable in a variety of media without the need of stabilizing agents or ligands. This stabilization is achieved by partial oxidation of the gold surface resulting in positively charged GNPs. However, little is known about cellular uptake of such ligand-free nanoparticles, their intracellular fate, or cell viability after nanoparticle contact. The current work is aimed to explore the response of a bovine cell line to GNP exposure mainly using laser scanning confocal microscopy (LSCM) supported by other techniques. Cultured bovine immortalized cells (GM7373) were coincubated with GNP (average diameter 15 nm, 50 μM Au) for 2, 24, and 48 h. The detection of GNP-associated light scattering by the LSCM facilitated a clear distinction between GNP-containing cells and the negative controls. After 48 h, 75% of cells had visibly incorporated nanoparticles. No colocalization was detected with either Rab5a or Lamp1-positive structures, i.e., endosomes or lysosomes, respectivley. However, transmission electron microscope analysis of GNP-coincubated cells indicated the nanoparticles to be positioned within electron-dense structures. Coincubation at 4°C did not inhibit nanoparticle uptake, suggesting diffusion as possible entrance mechanism. Although the assessment of cell morphology, membrane integrity, and apoptosis revealed no GNP-related loss of cell viability at a gold concentration of 25 μM or below, a cytotoxic effect was observed in a proliferation assay after exposing low cell numbers to 50 μM Au and above. In conclusion, this study confirmed the cellular uptake of ligand-free gold nanoparticles during coincubation apparently without using endocytic pathways. © 2010 International Society for Advancement of Cytometry.
view abstract doi: 10.1002/cyto.a.20846 2010 • 9 Positively charged calcium phosphate/polymer nanoparticles for photodynamic therapy
Klesing, J. and Wiehe, A. and Gitter, B. and Gräfe, S. and Epple, M.
Journal of Materials Science: Materials in Medicine 21 887-892 (2010)The charge of nanoparticles influences their ability to pass through the cellular membrane, and a positive charge should be beneficial. The negative charge of calcium phosphate nanoparticles with an inner shell of carboxymethyl cellulose (CMC) was reversed by adding an outer shell of poly(ethyleneimine) (PEI) into which the photoactive dye 5,10,15,20-tetrakis(3-hydroxyphenyl)- porphyrin (mTHPP) was loaded. The aqueous dispersion of the nanoparticles was used for photodynamic therapy with HT29 cells (human colon adenocarcinoma cells), HIG-82 cells (rabbit synoviocytes), and J774A.1 cells (murine macrophages). A high photodynamic activity (killing) together with a very low dark toxicity was observed for HIG-82 and for J774.1 cells at 2 μM dye concentration. The killing efficiency was equivalent to the pure photoactive dye that, however, needs to be administered in alcoholic solution. © 2009 Springer Science+Business Media, LLC.
view abstract doi: 10.1007/s10856-009-3934-7 2010 • 8 Proapoptotic influenza A virus protein PB1-F2 forms a nonselective ion channel
Henkel, M. and Mitzner, D. and Henklein, P. and Meyer-Almes, F.-J. and Moroni, A. and DiFrancesco, M.L. and Henkes, L.M. and Kreim, M. and Kast, S.M. and Schubert, U. and Thiel, G.
PLoS ONE 5 (2010)Background: PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. Methodology/Principal Findings: Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAVPR8), from A/Brevig Mission/1/1918(H1N1) (IAVSF2) or the H5N1 consensus sequence (IAVBF2) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. Conclusion/Significance: The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development. © 2010 Henkel et al.
view abstract doi: 10.1371/journal.pone.0011112 2010 • 7 Reversible and noncompetitive inhibition of β-tryptase by protein surface binding of tetravalent peptide ligands identified from a combinatorial split-mix library
Wich, P.R. and Schmuck, C.
Angewandte Chemie - International Edition 49 4113-4116 (2010)(Figure Presented) Molecular plug: On-bead screening of a combinatorial library of 216 tetravalent oligopeptides reveals highly specific, noncompetitive inhibitors of the serine protease β-tryptase with nanomolar affinity. The ligands most likely bind to the protein surface and act as a molecular plug that blocks access to the active sites, which are buried inside a central cavity (see picture). © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
view abstract doi: 10.1002/anie.200907221 2010 • 6 Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer
Cadenas, C. and Franckenstein, D. and Schmidt, M. and Gehrmann, M. and Hermes, M. and Geppert, B. and Schormann, W. and Maccoux, L.J. and Schug, M. and Schumann, A. and Wilhelm, C. and Freis, E. and Ickstadt, K. and Rahnenführer,...
Breast Cancer Research 12 (2010)Introduction: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets.Methods: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription.Results: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species.Conclusions: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner. © 2010 Cadenas et al.; licensee BioMed Central Ltd.
view abstract doi: 10.1186/bcr2599 2010 • 5 Salt bridges in the miniature viral channel Kcv are important for function
Hertel, B. and Tayefeh, S. and Kloss, T. and Hewing, J. and Gebhardt, M. and Baumeister, D. and Moroni, A. and Thiel, G. and Kast, S.M.
European Biophysics Journal 39 1057-1068 (2010)The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K+channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the Wrst transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intrasubunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K+ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing. © European Biophysical Societies' Association 2009.
view abstract doi: 10.1007/s00249-009-0451-z 2010 • 4 Scanning electrochemical microscopy in neuroscience
Schulte, A. and Nebel, M. and Schuhmann, W.
Annual Review of Analytical Chemistry 3 299-318 (2010)This article reviews recent work involving the application of scanning electrochemical microscopy (SECM) to the study of individual cultured living cells, with an emphasis on topographical and functional imaging of neuronal and secretory cells of the nervous and endocrine system. The basic principles of biological SECM and associated negative amperometric-feedback and generator/collector-mode SECM imaging are discussed, and successful use of the methodology for screening soft and fragile membranous objects is outlined. The drawbacks of the constant-height mode of probe movement and the benefits of the constant-distance mode of SECM operation are described. Finally, representative examples of constant-height and constant-distance mode SECM on a variety of live cells are highlighted to demonstrate the current status of single-cell SECM in general and of SECM in neuroscience in particular. Copyright © 2010 by Annual Reviews. All rights reserved.
view abstract doi: 10.1146/annurev.anchem.111808.073651 2010 • 3 SiRNA-loaded multi-shell nanoparticles incorporated into a multilayered film as a reservoir for gene silencing
Zhang, X. and Kovtun, A. and Mendoza-Palomares, C. and Oulad-Abdelghani, M. and Fioretti, F. and Rinckenbach, S. and Mainard, D. and Epple, M. and Benkirane-Jessel, N.
Biomaterials 31 6013-6018 (2010)In this study, we presented a new type of coating based on polyelectrolyte multilayers containing sequentially adsorbed active shRNA calcium phosphate nanoparticles for locally defined and temporarily variable gene silencing. Therefore, we investigated multi-shell calcium phosphate-shRNA nanoparticles embedded into a polyelectrolyte multilayer for gene silencing. As model system, we synthesized triple-shell calcium phosphate-shRNA nanoparticles (NP) and prepared polyelectrolyte multilayers films made of nanoparticles and poly-(l-lysine) (PLL). The biological activities of these polyelectrolyte multilayers films were tested by the production of osteopontin and osteocalcin in the human osteoblasts (HOb) which were cultivated on the PEM films. This new strategy can be used to efficiently control the bone formation and could be applicable in tissue engineering. © 2010 Elsevier Ltd.
view abstract doi: 10.1016/j.biomaterials.2010.04.024 2010 • 2 The resorption of nanocrystalline calcium phosphates by osteoclast-like cells
Detsch, R. and Hagmeyer, D. and Neumann, M. and Schaefer, S. and Vortkamp, A. and Wuelling, M. and Ziegler, G. and Epple, M.
Acta Biomaterialia 6 3223-3233 (2010)Nanocrystalline calcium phosphates containing carbonate have a high similarity to bone mineral. The reactions of bone cells (primary osteoblasts and osteoclast-like cells) on these materials as well as on sintered β-tricalcium phosphate and hydroxyapatite (HA) confirmed a good biocompatibility of the nanocrystalline samples. However, osteoclastic differentiation was constrained on the carbonate-rich samples, leading to a small number of osteoclast-like cells on the materials and few resorption pits. The grain size of the calcium phosphate ceramics (nano vs. micro) was less important than expected from to physico-chemical considerations. When comparing the nanocrystalline samples, the highest resorption rate was found for nano-HA with a low carbonate content, which strongly stimulated the differentiation of osteoclast-like cells on its surface. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
view abstract doi: 10.1016/j.actbio.2010.03.003 2010 • 1 Towards a high potential biocathode based on direct bioelectrochemistry between horseradish peroxidase and hierarchically structured carbon nanotubes
Jia, W. and Schwamborn, S. and Jin, C. and Xia, W. and Muhler, M. and Schuhmann, W. and Stoica, L.
Physical Chemistry Chemical Physics 12 10088-10092 (2010)Adsorption of horseradish peroxidase (HRP) on graphite rod electrodes sequentially modified with carbon microfibers (CMF) carrying carbon nanotubes in a hierarchically structured arrangement and finally pyrene hexanoic acid (PHA) for improving hydrophilicity of the electrode surface is the basis for the direct bioelectrocatalytic reduction of H 2O 2 at potentials as high as about +600 mV. The high-potential direct bioelectrocatalytic reduction of H 2O 2 is implying a direct bioelectrochemical communication between the Fe IVO,P + redox state known as compound I. The HRP loading was optimized leading to a current of 800 μA at a potential of 300 mV. © 2010 the Owner Societies.
view abstract doi: 10.1039/c0cp00349b