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- 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; Gƶhler, Rebecca; Lindner, Tobias; Haenle, Maximilian; Finke, Birgit; Meichsner, JĆ¼rgen; 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 ĀµCT 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 ĀµCT (3D) and afterwards by histomorphometry (2D). In comparison to uncoated Ti6Al4V implants demonstrating low BIC of 32.4% (histomorphometry) and 51.3% (ĀµCT), PPAAm and PPEDA coated implants showed a nonsignificant increase in BIC (histomorphometry: 45.7% and 53.5% and ĀµCT: 51.8% and 62.0%, resp.). Mean BIC calculated by ĀµCT 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 Ī¼CT analysis seems to be suitable as a nondestructive and accurate 3D imaging method for the evaluation of the bone-implant interface.
- 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.; GroĆe, Christina; Neugebauer, Ute; Methling, Karen; Lƶffler, 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 āomicsā 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.
- ItemBioactive secondary metabolites with multiple activities from a fungal endophyte(Oxford : Wiley-Blackwell, 2016) Bogner, Catherine W.; Kamdem, Ramsay S.T.; Sichtermann, Gisela; MatthƤus, Christian; Hƶlscher, Dirk; Popp, JĆ¼rgen; Proksch, Peter; Grundler, Florian M.W.; Schouten, AlexanderIn order to replace particularly biohazardous nematocides, there is a strong drive to finding natural product-based alternatives with the aim of containing nematode pests in agriculture. The metabolites produced by the fungal endophyte Fusarium oxysporum 162 when cultivated on rice media were isolated and their structures elucidated. Eleven compounds were obtained, of which six were isolated from a Fusarium spp. for the first time. The three most potent nematode-antagonistic compounds, 4-hydroxybenzoic acid, indole-3-acetic acid (IAA) and gibepyrone D had LC50 values of 104, 117 and 134 Ī¼g mlā1, respectively, after 72 h. IAA is a well-known phytohormone that plays a role in triggering plant resistance, thus suggesting a dual activity, either directly, by killing or compromising nematodes, or indirectly, by inducing defence mechanisms against pathogens (nematodes) in plants. Such compounds may serve as important leads in the development of novel, environmental friendly, nematocides.
- ItemA size dependent evaluation of the cytotoxicity and uptake of nanographene oxide(London [u.a.] : RSC, 2015) Mendes, Rafael Gregorio; Koch, Britta; Bachmatiuk, Alicja; Ma, Xing; Sanchez, Samuel; Damm, Christine; Schmidt, Oliver G.; Gemming, Thomas; Eckert, JĆ¼rgen; RĆ¼mmeli, Mark H.Graphene oxide (GO) has attracted great interest due to its extraordinary potential for biomedical application. Although it is clear that the naturally occurring morphology of biological structures is crucial to their precise interactions and correct functioning, the geometrical aspects of nanoparticles are often ignored in the design of nanoparticles for biological applications. A few in vitro and in vivo studies have evaluated the cytotoxicity and biodistribution of GO, however very little is known about the influence of flake size and cytotoxicity. Herein, we aim at presenting an initial cytotoxicity evaluation of different nano-sized GO flakes for two different cell lines (HeLa (Kyoto) and macrophage (J7742)) when they are exposed to samples containing different sized nanographene oxide (NGO) flakes (mean diameter of 89 and 277 nm). The obtained data suggests that the larger NGO flakes reduce cell viability as compared to smaller flakes. In addition, the viability reduction correlates with the time and the concentration of the NGO nanoparticles to which the cells are exposed. Uptake studies were also conducted and the data suggests that both cell lines internalize the GO nanoparticles during the incubation periods studied.
- ItemThermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency(London : Sage, 2017) Rennert, Knut; Nitschke, Mirko; Wallert, Maria; Keune, Natalie; Raasch, Martin; Lorkowski, Stefan; Mosig, Alexander S.Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyteāderived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyteāderived macrophages. In summary, we observed similar functionality and viability of primary monocyteāderived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.
- ItemBenchmark datasets for 3D MALDI- and DESI-imaging mass spectrometry(Oxford : Oxford University Press, 2015) Oetjen, Janina; Veselkov, Kirill; Watrous, Jeramie; McKenzie, James S.; Becker, Michael; Hauberg-Lotte, Lena; Kobarg, Jan Hendrik; Strittmatter, Nicole; MrĆ³z, Anna K.; Hoffmann, Franziska; Trede, Dennis; Palmer, Andrew; Schiffler, Stefan; Steinhorst, Klaus; Aichler, Michaela; Goldin, Robert; Guntinas-Lichius, Orlando; von Eggeling, Ferdinand; Thiele, Herbert; Maedler, Kathrin; Walch, Axel; Maass, Peter; Dorrestein, Pieter C.; Takats, Zoltan; Alexandrov, TheodoreBackground: Three-dimensional (3D) imaging mass spectrometry (MS) is an analytical chemistry technique for the 3D molecular analysis of a tissue specimen, entire organ, or microbial colonies on an agar plate. 3D-imaging MS has unique advantages over existing 3D imaging techniques, offers novel perspectives for understanding the spatial organization of biological processes, and has growing potential to be introduced into routine use in both biology and medicine. Owing to the sheer quantity of data generated, the visualization, analysis, and interpretation of 3D imaging MS data remain a significant challenge. Bioinformatics research in this field is hampered by the lack of publicly available benchmark datasets needed to evaluate and compare algorithms. Findings: High-quality 3D imaging MS datasets from different biological systems at several labs were acquired, supplied with overview images and scripts demonstrating how to read them, and deposited into MetaboLights, an open repository for metabolomics data. 3D imaging MS data were collected from five samples using two types of 3D imaging MS. 3D matrix-assisted laser desorption/ionization imaging (MALDI) MS data were collected from murine pancreas, murine kidney, human oral squamous cell carcinoma, and interacting microbial colonies cultured in Petri dishes. 3D desorption electrospray ionization (DESI) imaging MS data were collected from a human colorectal adenocarcinoma. Conclusions: With the aim to stimulate computational research in the field of computational 3D imaging MS, selected high-quality 3D imaging MS datasets are provided that could be used by algorithm developers as benchmark datasets.
- ItemDecreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress(Washington, DC : Soc., 2019) Liu, Boqun; Hasrat, Zarief; Poolman, Bert; Boersma, Arnold J.; Mullineaux, Conrad W.Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.
- ItemNeutrophil extracellular trap formation is elicited in response to cold physical plasma(Hoboken, NJ : Wiley, 2016) Bekeschus, Sander; Winterbourn, VChristine C.; Kolata, Julia; Masur, Kai; Hasse, Sybille; Brƶker, Barbara M.; Parker, Heather A.Cold physical plasma is an ionized gas with a multitude of components, including hydrogen peroxide and other reactive oxygen and nitrogen species. Recent studies suggest that exposure of wounds to cold plasma may accelerate healing. Upon wounding, neutrophils are the first line of defense against invading microorganisms but have also been identified to play a role in delayed healing. In this study, we examined how plasma treatment affects the functions of peripheral blood neutrophils. Plasma treatment induced oxidative stress, as assessed by the oxidation of intracellular fluorescent redox probes; reduced metabolic activity; but did not induce early apoptosis. Neutrophil oxidative burst was only modestly affected after plasma treatment, and the killing of Pseudomonas aeruginosa and Staphylococcus aureus was not significantly affected. Intriguingly, we found that plasma induced profound extracellular trap formation. This was inhibited by the presence of catalase during plasma treatment but was not replicated by adding an equivalent concentration of hydrogen peroxide. Plasma-induced neutrophil extracellular trap formation was not dependent on the activity of myeloperoxidase or NADPH oxidase 2 but seemed to involve short-lived molecules. The amount of DNA release and the time course after plasma treatment were similar to that with the common neutrophil extracellular trap inducer PMA. After neutrophil extracellular traps had formed, concentrations of IL-8 were also significantly increased in supernatants of plasma-treated neutrophils. Both neutrophil extracellular traps and IL-8 release may aid antimicrobial activity and spur inflammation at the wound site. Whether this aids or exacerbates wound healing needs to be tested.
- ItemThe synergistic effect of chlorotoxin-mApoE in boosting drug-loaded liposomes across the BBB(London : BioMed Central, 2019) Formicola, Beatrice; Dal, Magro, Roberta; Montefusco-Pereira, Carlos V.; Lehr, ClausāMichael; Koch, Marcus; Russo, Laura; Grasso, Gianvito; Deriu, Marco A.; Danani, Andrea; Bourdoulous, Sandrine; Re, FrancescaWe designed liposomes dually functionalized with ApoE-derived peptide (mApoE) and chlorotoxin (ClTx) to improve their blood-brain barrier (BBB) crossing. Our results demonstrated the synergistic activity of ClTx-mApoE in boosting doxorubicin-loaded liposomes across the BBB, keeping the anti-tumour activity of the drug loaded: mApoE acts promoting cellular uptake, while ClTx promotes exocytosis of liposomes. Ā© 2019 The Author(s).
- ItemComplete Genome Sequence of a New Ruminococcaceae Bacterium Isolated from Anaerobic Biomass Hydrolysis(Washington, DC : American Soc. for Microbiology, 2018) Hahnke, Sarah; Abendroth, Christian; Langer, Thomas; CodoƱer, Francisco M.; Ramm, Patrice; Porcar, Manuel; Luschnig, Olaf; Klocke, MichaelA new Ruminococcaceae bacterium, strain HV4-5-B5C, participating in the anaerobic digestion of grass, was isolated from a mesophilic two-stage laboratoryscale leach bed biogas system. The draft annotated genome sequence presented in this study and 16S rRNA gene sequence analysis indicated the affiliation of HV4-5- B5C with the family Ruminococcaceae outside recently described genera. Ā© 2018 Hahnke et al.