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- ItemEnhanced calcium ion mobilization in osteoblasts on amino group containing plasma polymer nanolayer(London : BioMed Central, 2018-3-21) Staehlke, Susanne; Rebl, Henrike; Finke, Birgit; Mueller, Petra; Gruening, Martina; Nebe, J. BarbaraBackground: Biomaterial modifications—chemical and topographical—are of particular importance for the integration of materials in biosystems. Cells are known to sense these biomaterial characteristics, but it has remained unclear which physiological processes bio modifications trigger. Hence, the question arises of whether the dynamic of intracellular calcium ions is important for the characterization of the cell–material interaction. In our prior research we could demonstrate that a defined geometrical surface topography affects the cell physiology; this was finally detectable in a reduced intracellular calcium mobilization after the addition of adenosine triphosphate (ATP). Results: This new contribution examines the cell physiology of human osteoblasts concerning the relative cell viability and the calcium ion dynamic on different chemical modifications of silicon–titanium (Ti) substrates. Chemical modifications comprising the coating of Ti surfaces with a plasma polymerized allylamine (PPAAm)-layer or with a thin layer of collagen type-I were compared with a bare Ti substrate as well as tissue culture plastic. For this purpose, the human osteoblasts (MG-63 and primary osteoblasts) were seeded onto the surfaces for 24 h. The relative cell viability was determined by colorimetric measurements of the cell metabolism and relativized to the density of cells quantified using crystal violet staining. The calcium ion dynamic of osteoblasts was evaluated by the calcium imaging analysis of fluo-3 stained vital cells using a confocal laser scanning microscope. The positively charged nano PPAAm-layer resulted in enhanced intracellular calcium ion mobilization after ATP-stimulus and cell viability. This study underlines the importance of the calcium signaling for the manifestation of the cell physiology. Conclusions: Our current work provides new insights into the intracellular calcium dynamic caused by diverse chemical surface compositions. The calcium ion dynamic appears to be a sensitive parameter for the cell physiology and, thus, may represent a useful approach for evaluating a new biomaterial. In this regard, reliable in vitro-tests of cell behavior at the interface to a material are crucial steps in securing the success of a new biomaterial in medicine.
- ItemMulti-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures(London : BioMed Central, 2016) Viell, Jörn; Inouye, Hideyo; Szekely, Noemi K.; Frielinghaus, Henrich; Marks, Caroline; Wang, Yumei; Anders, Nico; Spiess, Antje C.; Makowski, LeeBackground: The valorization of biomass for chemicals and fuels requires efficient pretreatment. One effective strategy involves the pretreatment with ionic liquids which enables enzymatic saccharification of wood within a few hours under mild conditions. This pretreatment strategy is, however, limited by water and the ionic liquids are rather expensive. The scarce understanding of the involved effects, however, challenges the design of alternative pretreatment concepts. This work investigates the multi length-scale effects of pretreatment of wood in 1-ethyl-3-methylimidazolium acetate (EMIMAc) in mixtures with water using spectroscopy, X-ray and neutron scattering. Results: The structure of beech wood is disintegrated in EMIMAc/water mixtures with a water content up to 8.6 wt%. Above 10.7 wt%, the pretreated wood is not disintegrated, but still much better digested enzymatically compared to native wood. In both regimes, component analysis of the solid after pretreatment shows an extraction of few percent of lignin and hemicellulose. In concentrated EMIMAc, xylan is extracted more efficiently and lignin is defunctionalized. Corresponding to the disintegration at macroscopic scale, SANS and XRD show isotropy and a loss of crystallinity in the pretreated wood, but without distinct reflections of type II cellulose. Hence, the microfibril assembly is decrystallized into rather amorphous cellulose within the cell wall. Conclusions: The molecular and structural changes elucidate the processes of wood pretreatment in EMIMAc/water mixtures. In the aqueous regime with >10.7 wt% water in EMIMAc, xyloglucan and lignin moieties are extracted, which leads to coalescence of fibrillary cellulose structures. Dilute EMIMAc/water mixtures thus resemble established aqueous pretreatment concepts. In concentrated EMIMAc, the swelling due to decrystallinization of cellulose, dissolution of cross-linking xylan, and defunctionalization of lignin releases the mechanical stress to result in macroscopic disintegration of cells. The remaining cell wall constituents of lignin and hemicellulose, however, limit a recrystallization of the solvated cellulose. These pretreatment mechanisms are beyond common pretreatment concepts and pave the way for a formulation of mechanistic requirements of pretreatment with simpler pretreatment liquors. © 2016 Viell et al.
- 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.
- ItemPseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool(London : BioMed Central, 2016) Bocklitz, Thomas W.; Salah, Firas Subhi; Vogler, Nadine; Heuke, Sandro; Chernavskaia, Olga; Schmidt, Carsten; Waldner, Maximilian J.; Greten, Florian R.; Bräuer, Rolf; Schmitt, Michael; Stallmach, Andreas; Petersen, Iver; Popp, JürgenDue to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making.
- ItemDevelopment of a flow-fluorescence in situhybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor(London : BioMed Central, 2013) Nettmann, Edith; Fröhling, Antje; Heeg, Kathrin; Klocke, Michael; Schlüter, Oliver; Mumme, JanBackground: The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable. Results: Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively. Conclusions: The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.
- ItemCharacterization and prediction of the mechanism of action of antibiotics through NMR metabolomics(London : BioMed Central, 2016) Hoerr, Verena; Duggan, Gavin E.; Zbytnuik, Lori; Poon, Karen K.H.; Große, Christina; Neugebauer, Ute; Methling, Karen; Löffler, Bettina; Vogel, Hans J.Background: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. Results: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. Conclusion: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.
- ItemFunctional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells(London : BioMed Central, 2013) Kinzebach, Sven; Dietz, Lisa; Klüter, Harald; Thierse, Hermann-Josef; Bieback, KarenBackground: Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions: The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.
- ItemMAC and baseband processors for RF-MIMO WLAN(London : BioMed Central, 2011) Stamenkovic, Zoran; Tittelbach-Helmrich, Klaus; Krstic, Milos; Ibanez, Jesus; Elvira, Victor; Santamaria, IgnacioThe article describes hardware solutions for the IEEE 802.11 medium access control (MAC) layer and IEEE 802.11a digital baseband in an RF-MIMO WLAN transceiver that performs the signal combining in the analogue domain. Architecture and implementation details of the MAC processor including a hardware accelerator and a 16-bit MAC-physical layer (PHY) interface are presented. The proposed hardware solution is tested and verified using a PHY link emulator. Architecture, design, implementation, and test of a reconfigurable digital baseband processor are described too. Description includes the baseband algorithms (the main blocks being MIMO channel estimation and Tx-Rx analogue beamforming), their FPGA-based implementation, baseband printed-circuit-board, and real-time tests.
- ItemThe carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats(London : BioMed Central, 2014) Rittinghausen, Susanne; Hackbarth, Anja; Creutzenberg, Otto; Ernst, Heinrich; Heinrich, Uwe; Leonhardt, Albrecht; Schaudien, DirkBackground: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods. Methods: A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 109 or 5 � 109 WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (108 WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans. Results: Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D - the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis. Conclusion: We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs.
- ItemPreventing carbon nanoparticle-induced lung inflammation reduces antigen-specific sensitization and subsequent allergic reactions in a mouse model(London : BioMed Central, 2015) Kroker, Matthias; Sydlik, Ulrich; Autengruber, Andrea; Cavelius, Christian; Weighardt, Heike; Kraegeloh, Annette; Unfried, KlausBackground Exposure of the airways to carbonaceous nanoparticles can contribute to the development of immune diseases both via the aggravation of the allergic immune response in sensitized individuals and by adjuvant mechanisms during the sensitization against allergens. The cellular and molecular mechanisms involved in these adverse pathways are not completely understood. We recently described that the reduction of carbon nanoparticle-induced lung inflammation by the application of the compatible solute ectoine reduced the aggravation of the allergic response in an animal system. In the current study we investigated the influence of carbon nanoparticles on the sensitization of animals to ovalbumin via the airways. Ectoine was used as a preventive strategy against nanoparticle-induced neutrophilic lung inflammation. Methods Balb/c mice were repetitively exposed to the antigen ovalbumin after induction of airway inflammation by carbon nanoparticles, either in the presence or in the absence of ectoine. Allergic sensitization was monitored by measurement of immunoglobulin levels and immune responses in lung and lung draining lymph nodes after challenge. Furthermore the role of dendritic cells in the effect of carbon nanoparticles was studied in vivo in the lymph nodes but also in vitro using bone marrow derived dendritic cells. Results Animals exposed to antigen in the presence of carbon nanoparticles showed increased effects with respect to ovalbumin sensitization, to the allergic airway inflammation after challenge, and to the specific TH2 response in the lymph nodes. The presence of ectoine during the sensitization significantly reduced these parameters. The number of antigen-loaded dendritic cells in the draining lymph nodes was identified as a possible cause for the adjuvant effect of the nanoparticles. In vitro assays indicate that the direct interaction of the particles with dendritic cells is not able to trigger CCR7 expression, while this endpoint is achieved by lung lavage fluid from nanoparticle-exposed animals. Conclusions Using the intervention strategy of applying ectoine into the airways of animals we were able to demonstrate the relevance of neutrophilic lung inflammation for the adjuvant effect of carbon nanoparticles on allergic sensitization.n.