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- ItemFormation mechanism for stable hybrid clusters of proteins and nanoparticles(Washington D.C. : American Chemical Society, 2015) Moerz, Sebastian T.; Kraegeloh, Annette; Chanana, Munish; Kraus, TobiasCitrate-stabilized gold nanoparticles (AuNP) agglomerate in the presence of hemoglobin (Hb) at acidic pH. The extent of agglomeration strongly depends on the concentration ratio [Hb]/[AuNP]. Negligible agglomeration occurs at very low and very high [Hb]/[AuNP]. Full agglomeration and precipitation occur at [Hb]/[AuNP] corresponding to an Hb monolayer on the AuNP. Ratios above and below this value lead to the formation of an unexpected phase: stable, microscopic AuNP–Hb agglomerates. We investigated the kinetics of agglomeration with dynamic light scattering and the adsorption kinetics of Hb on planar gold with surface-acoustic wave-phase measurements. Comparing agglomeration and adsorption kinetics leads to an explanation of the complex behavior of this nanoparticle–protein mixture. Agglomeration is initiated either when Hb bridges AuNP or when the electrostatic repulsion between AuNP is neutralized by Hb. It is terminated when Hb has been depleted or when Hb forms multilayers on the agglomerates that stabilize microscopic clusters indefinitely.
- ItemSurviving the surf: The tribomechanical properties of the periostracum of Mytilus sp(Amsterdam : Elsevier, 2014) Wählisch, Felix C.; Peter, Nicolas J.; Torrents Abad, Oscar; Oliveira, Mariana V.G.; Schneider, Andreas S.; Schmahl, Wolfgang; Griesshaber, Erika; Bennewitz, RolandWe investigated the friction and wear behavior as well as the mechanical properties of the periostracum of Mytilus sp. Tribological properties were determined with a reciprocal sliding microtribometer, while mechanical characterization was performed using a nanoindenter. Measurements were performed in dry and wet conditions. On the dry periostracum we found a low friction coefficient of 0.078 ± 0.007 on the young parts and a higher one of 0.63 ± 0.02 on the old parts of the shell. Under wet, saline, conditions we only observed one average coefficient of friction of 0.37 ± 0.01. Microscopic ex situ analysis indicated that dry periostracum wore rather rapidly by plowing and fatigue, while it exhibited a high wear resistance when immersed in salt water. The Young’s modulus and hardness of the periostracum were also investigated in both dry and wet conditions. Under dry conditions the Young’s modulus of the periostracum was 8 ± 3 GPa, while under wet conditions it was 0.21 ± 0.05 GPa. The hardness of dry periostracum samples was 353 ± 127 MPa, whereas the hardness of wet samples was 5 ± 2 MPa. It was found that, in the wet state, viscous behavior plays a significant role in the mechanical response of the periostracum. Our results strongly indicate that the periostracum can provide an important contribution to the overall wear resistance of Mytilus sp. shell.
- ItemA hyperbranched dopamine-containing PEG-based polymer for the inhibition of α-synuclein fibrillation(Orlando, Fla. : Academic Press, 2015) Breydo, Leonid; Newland, Ben; Zhang, Hong; Rosser, Anne; Werner, Carsten; Uversky, Vladimir N.; Wang, WenxinAggregation of α-synuclein is believed to play an important role in Parkinson's disease and in other neurodegenerative maladies. Small molecule inhibitors of this process are among the most promising drug candidates for neurodegenerative diseases. Dendrimers have also been studied for anti-fibrillation applications but they can be difficult and expensive to synthetize. Here we show that RAFT polymerization can be used to produce a hyperbranched polyethylene glycol structure via a one-pot reaction. This polymer included a dopamine moiety, a known inhibitor of α-synuclein fibril formation. Dopamine within the polymer structure was capable of aggregation inhibition, although not to the same degree as free dopamine. This result opens up new avenues for the use of controlled radical polymerizations as a means of preparing hyperbranched polymers for anti-fibrillation activity, but shows that the incorporation of functional groups from known small molecules within polymers may alter their biological activity.
- ItemReal-time monitoring of calcium carbonate and cationic peptide deposition on carboxylate-SAM using a microfluidic SAW biosensor(Frankfurt am Main : Beilstein-Institut, 2014) Pohl, Anna; Weiss, Ingrid M.A microfluidic biosensor with surface acoustic wave technology was used in this study to monitor the interaction of calcium carbonate with standard carboxylate self-assembled monolayer sensor chips. Different fluids, with and without biomolecular components, were investigated. The pH-dependent surface interactions of two bio-inspired cationic peptides, AS8 and ES9, which are similar to an extracellular domain of the chitin synthase involved in mollusc shell formation, were also investigated in a biological buffer system. A range of experimental conditions are described that are suitable to study non-covalent molecular interactions in the presence of ionic substances, such as, mineral precursors below the solubility equilibrium. The peptide ES9, equal to the mollusc chitin synthase epitope, is less sensitive to changes in pH than its counterpart AS8 with a penta-lysine core, which lacks the flanking acidic residues. This study demonstrates the extraordinary potential of microfluidic surface acoustic wave biosensors to significantly expand our experimental capabilities for studying the principles underlying biomineralization in vitro.
- ItemTemperature sensitivity of decomposition in relation to soil organic matter pools: Critique and outlook(Göttingen : Copernicus GmbH, 2005) Reichstein, M.; Kätterer, T.; Andrén, O.; Ciais, P.; Schulze, E.-D.; Cramer, W.; Papale, D.; Valentini, R.Knorr et al. (2005) concluded that soil organic carbon pools with longer turnover times are more sensitive to temperature. We show that this conclusion is equivocal, largely dependent on their specific selection of data and does not persist when the data set of Kätterer et al. (1998) is analysed in a more appropriate way. Further, we analyse how statistical properties of the model parameters may interfere with correlative analyses that relate the Q 10 of soil respiration with the basal rate, where the latter is taken as a proxy for soil organic matter quality. We demonstrate that negative parameter correlations between Qio-values and base respiration rates are statistically expected and not necessarily provide evidence for a higher temperature sensitivity of low quality soil organic matter. Consequently, we propose it is premature to conclude that stable soil carbon is more sensitive to temperature than labile carbon.
- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
- ItemQuantification of internalized silica nanoparticles via STED microscopy(London : Hindawi, 2015) Peuschel, Henrike; Ruckelshausen, Thomas; Cavelius, Christian; Kraegeloh, AnnetteThe development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 x 10^10 particles mL^-1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27 of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 10^12 particles mL^-1) of the smaller particles induced cytotoxicity.
- ItemQuantification of osseointegration of plasma-polymer coated titanium alloyed implants by means of microcomputed tomography versus histomorphometry(New York [u.a.] : Hindawi, 2015) Gabler, Carolin; Zietz, Carmen; Bieck, Richard; Göhler, Rebecca; Lindner, Tobias; Haenle, Maximilian; Finke, Birgit; Meichsner, Jürgen; Testrich, Holger; Nowottnick, Mathias; Frerich, Bernhard; Bader, RainerA common method to derive both qualitative and quantitative data to evaluate osseointegration of implants is histomorphometry. The present study describes a new image reconstruction algorithm comparing the results of bone-to-implant contact (BIC) evaluated by means of µCT with histomorphometry data. Custom-made conical titanium alloyed (Ti6Al4V) implants were inserted in the distal tibial bone of female Sprague-Dawley rats. Different surface configurations were examined: Ti6Al4V implants with plasma-polymerized allylamine (PPAAm) coating and plasma-polymerized ethylenediamine (PPEDA) coating as well as implants without surface coating. After six weeks postoperatively, tibiae were explanted and BIC was determined by µCT (3D) and afterwards by histomorphometry (2D). In comparison to uncoated Ti6Al4V implants demonstrating low BIC of 32.4% (histomorphometry) and 51.3% (µCT), PPAAm and PPEDA coated implants showed a nonsignificant increase in BIC (histomorphometry: 45.7% and 53.5% and µCT: 51.8% and 62.0%, resp.). Mean BIC calculated by µCT was higher for all surface configurations compared to BIC detected by histomorphometry. Overall, a high correlation coefficient of 0.70 () was found between 3D and 2D quantification of BIC. The μCT analysis seems to be suitable as a nondestructive and accurate 3D imaging method for the evaluation of the bone-implant interface.
- ItemUnraveling gene regulatory networks from time-resolved gene expression data - a measures comparison study(London : BioMed Central, 2011) Hempel, Sabrina; Koseska, Aneta; Nikoloski, Zoran; Kurths, Jürgen; Walther, DirkBackground Inferring regulatory interactions between genes from transcriptomics time-resolved data, yielding reverse engineered gene regulatory networks, is of paramount importance to systems biology and bioinformatics studies. Accurate methods to address this problem can ultimately provide a deeper insight into the complexity, behavior, and functions of the underlying biological systems. However, the large number of interacting genes coupled with short and often noisy time-resolved read-outs of the system renders the reverse engineering a challenging task. Therefore, the development and assessment of methods which are computationally efficient, robust against noise, applicable to short time series data, and preferably capable of reconstructing the directionality of the regulatory interactions remains a pressing research problem with valuable applications. Results Here we perform the largest systematic analysis of a set of similarity measures and scoring schemes within the scope of the relevance network approach which are commonly used for gene regulatory network reconstruction from time series data. In addition, we define and analyze several novel measures and schemes which are particularly suitable for short transcriptomics time series. We also compare the considered 21 measures and 6 scoring schemes according to their ability to correctly reconstruct such networks from short time series data by calculating summary statistics based on the corresponding specificity and sensitivity. Our results demonstrate that rank and symbol based measures have the highest performance in inferring regulatory interactions. In addition, the proposed scoring scheme by asymmetric weighting has shown to be valuable in reducing the number of false positive interactions. On the other hand, Granger causality as well as information-theoretic measures, frequently used in inference of regulatory networks, show low performance on the short time series analyzed in this study. Conclusions Our study is intended to serve as a guide for choosing a particular combination of similarity measures and scoring schemes suitable for reconstruction of gene regulatory networks from short time series data. We show that further improvement of algorithms for reverse engineering can be obtained if one considers measures that are rooted in the study of symbolic dynamics or ranks, in contrast to the application of common similarity measures which do not consider the temporal character of the employed data. Moreover, we establish that the asymmetric weighting scoring scheme together with symbol based measures (for low noise level) and rank based measures (for high noise level) are the most suitable choices.
- ItemExpression stability of commonly used reference genes in canine articular connective tissues(London : BioMed Central, 2007) Ayers, Duncan; Clements, Dylan N.; Salway, Fiona; Day, Philip J.R.Background: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Realtime reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS). Results: The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were RPL13A and YWHAZ (M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were RPL13A and SDHA (M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were B2M and TBP (M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were SDHA and YWHAZ (K6) or SDHA and HMBS (DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression. Conclusion: The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies.