Gerald Brenner-Weiss

Karlsruhe Institute of Technology, Carlsruhe, Baden-Württemberg, Germany

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Publications (33)93.38 Total impact

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    ABSTRACT: Actinomycetales are known to produce various secondary metabolites including products with surface-active and emulsifying properties known as biosurfactants. In this study, the nonpathogenic actinomycetes Tsukamurella spumae and Tsukamurella pseudospumae are described as producers of extracellular trehalose lipid biosurfactants when grown on sunflower oil or its main component glyceryltrioleate. Crude extracts of the trehalose lipids were purified using silica gel chromatography. The structure of the two trehalose lipid components (TL A and TL B) was elucidated using a combination of matrix-assisted laser desorption/ionization time-of-flight/time-of-flight/tandem mass spectroscopy (MALDI-ToF-ToF/MS/MS) and multidimensional NMR experiments. The biosurfactants were identified as 1-α-glucopyranosyl-1-α-glucopyranosid carrying two acyl chains varying of C4 to C6 and C16 to C18 at the 2' and 3' carbon atom of one sugar unit. The trehalose lipids produced demonstrate surface-active behavior and emulsifying capacity. Classified as risk group 1 organisms, T. spumae and T. pseudospumae hold potential for the production of environmentally friendly surfactants.
    Applied microbiology and biotechnology. 08/2014;
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    ABSTRACT: In the fields of surgery and regenerative medicine, it is crucial to understand the interactions of proteins with the biomaterials used as implants. Protein adsorption directly influences cell-material interactions in vivo and, as a result, regulates e.g. cell adhesion on the surface of the implant. Therefore, the development of suitable analytical techniques together with well-defined model-systems allowing for the detection, characterization and quantification of protein adsorbates is essential. In this study, a protocol for the deposition of highly stable, thin gelatin-based films on various substrates has been developed. The hydrogel films were characterized morphologically and chemically. Due to the obtained low thickness of the hydrogel layer, this setup allowed for a quantitative study on the interaction of human proteins (albumin and fibrinogen) with the hydrogel by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins. Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer. This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.
    Biomacromolecules 06/2014; · 5.37 Impact Factor
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    ABSTRACT: Cross-talk between bacteria and mammalian cells is increasingly recognized as an important factor, especially during chronic infections. In particular, the interaction of extracellular bacterial signaling molecules with cells of the innate immune response is of special interest. In this context, we investigated whether the Pseudomonas quinolone signal (PQS) which is a quorum sensing molecule produced by bacteria and participates in biofilm formation and virulence has any influence on polymorphonuclear neutrophils (PMN), the cells of the "first line defense" against bacterial infections. We found that PQS did not enhance the bactericidal activity of PMN and did not induce apoptosis at concentrations up to 100 µM. However, PQS stimulated chemotaxis of PMN in doses of 10-100 µM. This PQS-dependent chemotaxis could be inhibited with SB203580 which blocks MAPkinase p38, suggesting a signaling pathway similar to AHL-12 induction. Using bacterial cell culture supernantants of Pseudomonas aeruginosa wild-type cells and a PQS-deficient mutant strain support the in vivo relevance of these findings. Since PQS is produced in the early phase of biofilm formation, PMN infiltration could be timely enough to eradicate bacteria before biofilm formation is completed, which confers the bacteria with a relative resistance to host defense mechanisms.
    Journal of applied biomaterials & functional materials. 05/2014;
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    ABSTRACT: The autoantigen U1-68/70 K is the dominant diagnostic marker in Mixed Connective Tissue Disease (MCTD) that until recently could not be expressed in its full-length form [Northemann et al., 1995]. Using cell-free expression screening, we successfully produced the snRNP protein U1-68/70 K in a soluble full-length form in Escherichia coli cells. The protein length and identity was determined by Western Blot and MS/MS analysis. Additionally, its reactivity in the autoimmune diagnostic was confirmed. Establishment of a cell-free expression system for this protein was important for further elucidation of protein expression properties such as the cDNA construct, expression temperature and folding properties; these parameters can now be determined in a fast and resource-conserving manner.
    Protein Expression and Purification 01/2014; · 1.43 Impact Factor
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    ABSTRACT: Despite intense research on biological and biomedical applications of nanoparticles, our understanding of their basic interactions with the biological environment is still incomplete. Systematic variation of the physicochemical properties of the nanoparticles is widely seen as a promising strategy to obtain further insights. In view of the key role of the protein adsorption layer forming on nanoparticles in contact with biofluids, we systematically varied the surface charge of proteins adsorbing onto nanoparticles by chemical modification so as to examine the effect of Coulomb forces in modulating nano-bio interactions. We chose human serum albumin (HSA) as a model protein and ultra-small, negatively charged fluorescent gold nanoclusters (AuNCs) as model nanoparticles. By using fluorescence and CD spectroscopies, we measured binding affinities and structural changes upon binding of the HSA variants. The strengths of the protein-nanoparticle interactions were found to change substantially upon modifying the surface charge of HSA. Furthermore, by using inductively coupled plasma optical emission spectroscopy, confocal fluorescence microscopy, scanning transmission electron microscopy and cell viability assays, we observed that cellular interactions of the AuNCs, including their adherence to cell membranes, uptake efficiency and cytotoxicity, depended markedly on the different surface charges of the HSA variants adsorbed onto the nanoparticles. These results illustrate vividly that the cellular responses to nanoparticle exposure depend on the specific properties of the proteins that adsorb onto nanoparticles from biofluids.
    Advanced Materials Interfaces 12/2013;
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    ABSTRACT: Proteinaceous conditioning films (pCFs) are thought to play a key role in microbial adhesion, leading to the fouling of technical and biomedical devices and biofilm formation, which in turn causes material damage or persistent infections, respectively. However, little is definitively known about the process of surface conditioning via proteins. Herein, we demonstrate the potential of quartz crystal microbalance with dissipation coupled to MALDI-ToF mass spectrometry (QCM-D-MALDI) to investigate protein adsorption on different surfaces, enabling both the monitoring of CF formation and the determination of the molecular composition of CFs. After running QCM-D experiments, a subsequent tryptic on chip digestion step allows the identification of the proteins deposited on the sensor chip surface via MALDI-ToF mass spectrometry. Prominent blood plasma proteins, i.e., human serum albumin (HSA), fibrinogen (FG) and fibronectin (FN), were used. Chemically well defined sensor surfaces were prepared, among others, via self-assembled monolayer (SAM) technology. In cases where protein adsorption was observed by QCM-D, the adsorbed proteins were clearly detected and identified using MALDI-ToF/MS for both single-protein solutions of HSA, FG and FN as well as for protein mixtures. However, for equimolar protein mixtures on TiO2 surfaces, only signals attributed to FG and FN were observed in the mass spectra. No signals indicating the presence of HSA could be detected. This finding leads to the assumption that only FG and FN attach to the TiO2 sensor surface under the given experimental conditions.
    Analytica chimica acta 11/2013; 802:95-102. · 4.31 Impact Factor
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    ABSTRACT: A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor.
    PLoS ONE 01/2013; 8(12):e82240. · 3.53 Impact Factor
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    ABSTRACT: Covalent attachment of PEG to proteins, known as PEGylation, is currently one of the main approaches for improving the pharmacokinetics of biopharmaceuticals. However, the separation and characterization especially of positional isoforms of PEGylated proteins are still challenging tasks. A common purification strategy uses ion exchange chromatography with increasing ionic strength by shallow salt gradients. This paper presents a method which applies a linear pH gradient chromatography to separate five of six possible isoforms of mono-PEGylated lysozyme, modified with 5kDa and 10kDa mPEG-aldehyde. To identify the corresponding PEGylation sites a comparison of elution pH values and calculated isoelectric points of each isoform, was used. The resulting correlation showed an R(2)>0.99. Fractionation, tryptic digestion and subsequent MALDI-MS analysis of each peak, verified the predicted elution order. Based on UV areas the N-terminal amine at lysine 1 exhibited the highest reactivity, followed by the lysine 33 residue.
    Journal of Chromatography A 10/2012; · 4.61 Impact Factor
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    ABSTRACT: Bacteria, organized in biofilms, are a common cause of relapsing or persistent infections and the ultimate cause of implant-associated osteomyelitis. Bacterial biofilms initiate a prominent local inflammatory response with infiltration of polymorphonuclear neutrophils (PMN), the main protagonists of the local innate host defense against bacteria. In our previous work we found that PMN recognize and adhere to biofilms, and that phagocytosis and degranulation of bactericidal substances, such as lactoferrin, were initiated. In contrast to the situation with planktonic bacteria, opsonization of biofilms with immunoglobulin and complement was not required for PMN activation, suggesting that biofilms contain signaling components for PMN. In the present study we identified in the bacteria-free extracellular substance of Staphylococcus epidermidis biofilms protein fractions that activated PMN in vitro.
    The International journal of artificial organs 10/2012; · 1.76 Impact Factor
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    ABSTRACT: The biodegradable polyester poly-(3-hydroxybutyrate) (PHB), produced by Ralstonia eutropha in batch and fed-batch processes, was purified by electrofiltration. The protein film on PHB granules determines their high negative zeta potential, enabling the application of electrofiltration as an integrated technology in the downstream processing of PHB. In order to determine the optimal purification parameters, various pressure and electric field strength conditions were tested. Electrofiltration of PHB at 4bars and 4V/mm provided an up to four times higher concentration factor than conventional filtration. FT-Raman spectroscopy demonstrated that electrofiltration did not result in structural changes to the products. The study demonstrates the efficiency and practical advantages of electrofiltration as a promising downstream step in the PHB production technology.
    Bioresource Technology 08/2012; 123:272-8. · 5.04 Impact Factor
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    ABSTRACT: N-Acyl-L-homoserine lactones (AHLs) are small cell-to-cell signaling molecules involved in the regulation of population density and local gene expression in microbial communities. Recent evidence shows that contact of this signaling system, usually referred to as quorum sensing, to living eukaryotes results in interactions of AHL with host cells in a process termed "interkingdom signaling". So far details of this process and the binding site of the AHLs remain unknown; both an intracellular and a membrane-bound receptor seem possible, the first of which requires passage through the cell membrane. Here, we used sum-frequency-generation (SFG) spectroscopy to investigate the integration, conformation, orientation, and translocation of deuterated N-acyl-L-homoserine lactones (AHL-d(n)) with varying chain length (8, 12, and 14 C atoms) in lipid bilayers consisting of a 1:1 mixture of POPC:POPG supported on SiO(2) substrates (prepared by vesicle fusion). We found that all AHL-d(n) derivatives are well-ordered within the supported lipid bilayer (SLB) in a preferentially all-trans conformation of the deuterated alkyl chain and integrated into the upper leaflet of the SLB with the methyl terminal groups pointing downward. For the bilayer system described above, no flip-flop of AHL-d(n) from the upper leaflet to the lower one could be observed. Spectral assignments and interpretations were further supported by Fourier transform infrared and Raman spectroscopy.
    Langmuir 05/2012; 28(22):8456-62. · 4.38 Impact Factor
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    ABSTRACT: Glucocorticoids, steroid hormones of the adrenal gland, are an integral part of the stress response and regulate glucose metabolism. Natural and synthetic glucocorticoids are widely used in anti-inflammatory therapy but can have severe side effects. In vivo tests are needed to identify novel glucocorticoids and to screen compounds for unwanted effects on glucocorticoid signaling. We created the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY assay to monitor glucocorticoid signaling in vivo. The GRIZLY assay detects stress-induced glucocorticoid production in single zebrafish larvae, measures disruption of glucocorticoid signaling by an organotin pollutant metabolite, and specifically identifies a compound stimulating endogenous glucocorticoid production in a chemical screen. Our assay has broad applications in stress research, environmental monitoring, and drug discovery.
    ACS Chemical Biology 04/2012; 7(7):1178-83. · 5.44 Impact Factor
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    ABSTRACT: N-Acyl-L-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-L-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum's acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein.
    Analytical and Bioanalytical Chemistry 02/2012; 403(2):473-82. · 3.66 Impact Factor
  • N A Kahle, G Brenner-Weiss, J Overhage, U Obst, G M Hänsch
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    ABSTRACT: When bacteria colonize surfaces, they socialize and form biofilms. This process is well regulated and relies on the communication among the bacteria via so-called "quorum sensing molecules". Among those, N-(3-oxododecanoyl)-l-homoserine lactone (AHL-12), generated by Pseudomonas aeruginosa and other Gram-negative bacteria, activates not only bacteria but also interacts with mammalian cells. Among others, it activates phagocytic cells and - as we had shown previously - it is chemotactic for human polymorphonuclear neutrophils (PMN) in vitro. In the present study, we analyzed the signalling pathway of AHL-12 in PMN. We focused on the mitogen activated protein (MAP) kinase p38, because SB203580, an inhibitor of p38, prevented the AHL-12 induced chemotaxis. We found that in response to AHL-12, p38 was phosphorylated within minutes, as was its downstream target, the MAPKAP-Kinase-2 (MK2). In PMN, the major substrate of MK2 is the leukocyte specific protein 1 (LSP1), which binds to F-actin and participates directly in actin polymerization and cell migration. In response to AHL-12, LSP1 was phosphorylated and co-localized with F-actin in polarized PMN, suggesting that AHL-12-induced migration depended on p38 and LSP1 activation.
    Immunobiology 02/2012; · 2.81 Impact Factor
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    ABSTRACT: Nanoparticles (NPs) are decorated with proteins and other biomolecules when they get into contact with biological systems. The presence of proteins in cell culture medium can therefore have effects on the biological outcome in cell-based tests. In this study, the manufactured nanomaterials silicon dioxide (SiO(2)), titanium dioxide (TiO(2)), iron-III-oxide (Fe(2)O(3)), and carbon black (CB) were used to study their interaction with single proteins from bovine and human plasma (albumin, fibrinogen and IgG) as well as with complete human serum. The protein binding capacity of the material was investigated and 1D gel electrophoresis was used to separate the bound proteins and to identify the bands by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry. We found that the NP surface chemistry had a great impact on the amount of bound protein with distinct ligands for each of the tested particles. The hydrophobic CB NPs bound much more protein than the hydrophilic metal oxide NPs. Among the single proteins investigated, fibrinogen showed the strongest affinity for SiO(2), TiO(2) and CB NPs. The identified proteins from human serum adsorbed to these NPs were very different. Only apolipoprotein A1 was found to be adsorbed to all NPs. These studies will help to explain the different degree of biological responses observed after in vitro exposure of cells in the absence or presence of serum and might also support the interpretation of in vivo experiments were NPs come directly into contact with blood plasma.
    Toxicology Letters 01/2012; 208(1):41-50. · 3.15 Impact Factor
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    ABSTRACT: Some bacterial life states are only difficult to describe and to detect because they are on the border of active metabolism. A prominent example is the so-called viable but non-culturable state, which is mainly characterized by the inability of bacteria to grow on synthetic media. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF/MS) in combination with multivariate data analysis represents a powerful tool for mass-spectrometric pattern recognition of biological samples. This method is already used for differentiation of bacterial strains. In this study we present a rapid readout method based on MALDI-ToF/MS in combination with principal component analysis to classify the bacterial non-culturable state using Enterococcus faecalis as a model organism. By applying this technique to samples of different physiological states, distinct clusters were calculated and all mass spectra were classified correctly into groups of similar type concerning their physiological state.
    Analytical and Bioanalytical Chemistry 09/2011; 401(5):1593-600. · 3.66 Impact Factor
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    ABSTRACT: Downstream processing of chitosan requires several technological steps that contribute to the total production costs. Precipitation and especially evaporation are energy-consuming processes, resulting in higher costs and limiting industrial scale production. This study investigated the filtration kinetics of chitosan derived from cell walls of fungi and from exoskeletons of arthropods by electrofiltration, an alternative method, thus reducing the downstream processing steps and costs. Experiments with different voltages and pressures were conducted in order to demonstrate the effect of both parameters on filtration kinetics. The concentration of the biopolymer was obtained by the average factor of 40 by applying an electric field of 4 V/mm and pressure of 4 bars. A series of analytical experiments demonstrated the lack of structural and functional changes in chitosan molecules after electrofiltration. These results, combined with the reduction of energy and processing time, define the investigated method as a promising downstream step in the chitosan production technology.
    Biotechnology Journal 04/2011; 7(2):262-74. · 3.71 Impact Factor
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    ABSTRACT: In vitro gut fermentation systems are relevant tools to study health benefits of foodstuffs. Most of them are commonly used to investigate the degradation of nutrients or the development of gut flora. Using these models, strong cytotoxic effects of the resulting samples on cultured cells were observed. Hence, the aim of the present study was to develop a modified in vitro fermentation model that simulates the whole digestive tract and generates fermented samples that are suitable for testing in cell culture experiments. Wholemeal wheat flour (wwf) was digested and fermented in vitro with a fermentation model using different ox gall concentrations (41·6 and 0·6 g/l). The resulting fermentation supernatants (fs) were characterised for metabolites and biological effects in HT29 cells. The fermentation of wwf increased chemopreventive SCFA and decreased carcinogenic deoxycholic acid (DCA). The strong cytotoxic effects of the fs, which were partly due to cholic acid and DCA, were diminished by lowering the ox gall concentration, allowing the use of the samples in cell culture experiments. In conclusion, an in vitro digestion model, which can be used to study the effects of foodstuffs on chemoprevention and gut health in colon cells, is introduced and its physiological relevance is demonstrated.
    The British journal of nutrition 03/2011; 105(5):678-87. · 3.45 Impact Factor
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    ABSTRACT: Alloantibodies are known to influence transplant outcomes. Apart from human leukocyte antigens (HLA), non-HLA targets have been suggested to play a significant role, but little is known about their nature. Here, we present a novel method for identification and characterization of cell surface antigens bound by alloreactive antibodies. Our method consists of 2 consecutive steps: first, immunoprecipitation of cell surface proteins is carried out with serum and, second, matrix-assisted laser desorption/ionization-time-of-flight is used to fingerprint the precipitated cell-surface proteins. As an example, we performed immunoprecipitation with peripheral blood lymphocytes, which had been incubated with an alloreactive serum; immune complexes were coupled to protein-G beads and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; differential protein fractions were then analyzed by matrix-assisted laser desorption/ionization-time-of-flight. The method was validated with serum as well as with plasmapheresis material, which contained antibodies of known HLA specificities, demonstrating its applicability for clinical use.
    Human immunology 02/2010; 71(5):462-7. · 2.55 Impact Factor
  • A. Heinold, B. Kuehl, G. Brenner-Weiss, G. Opelz, T. H. Tran
    Transplantation 01/2010; 90. · 3.78 Impact Factor