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- ItemFemtosecond stimulated Raman spectroscopy of the cyclobutane thymine dimer repair mechanism: A computational study(Washington, DC : American Chemical Society, 2014) Ando, H.; Fingerhut, B.P.; Dorfman, K.E.; Biggs, J.D.; Mukamel, S.Cyclobutane thymine dimer, one of the major lesions in DNA formed by exposure to UV sunlight, is repaired in a photoreactivation process, which is essential to maintain life. The molecular mechanism of the central step, i.e., intradimer C-C bond splitting, still remains an open question. In a simulation study, we demonstrate how the time evolution of characteristic marker bands (C=O and C=C/C-C stretch vibrations) of cyclobutane thymine dimer and thymine dinucleotide radical anion, thymidylyl(3′→5′)-thymidine, can be directly probed with femtosecond stimulated Raman spectroscopy (FSRS). We construct a DFT(M05-2X) potential energy surface with two minor barriers for the intradimer C5-C′5 splitting and a main barrier for the C6-C′6 splitting, and identify the appearance of two C5=C6 stretch vibrations due to the C6-C′6 splitting as a spectroscopic signature of the underlying bond splitting mechanism. The sequential mechanism shows only absorptive features in the simulated FSRS signals, whereas the fast concerted mechanism shows characteristic dispersive line shapes. (Figure Presented).
- ItemMonoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)(San Francisco, California, US : PLOS, 2016) Thamm, Kristina; Graupner, Sylvi; Werner, Carsten; Huttner, Wieland B.; Corbeil, Denis; Nabi, Ivan RThe pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.
- ItemInteraction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells(Auckland : DOVE Medical Press, 2014) Woltmann, B.; Torger, B.; Müller, M.; Hempel, U.Background: Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis). For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles' composition and surface net charge. Materials and methods: Polyelectrolyte complex nanoparticles (PECNPs) composed of the polycations poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) in combination with the polyanions dextran sulfate (DS) or cellulose sulfate (CS) were prepared. PECNPs' physicochemical properties (size, net charge) were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs) cultured on immobilized PECNP films (5-50 nmol·cm-2) by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay, as well as cell morphology (phase contrast microscopy). Results: PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC+NP) or negative (PEC-NP) net charge were obtained. The PECNP composition significantly affected cell morphology and metabolic activity, whereas the net charge had a negligible influence. Therefore, we classified PECNPs into "variant systems" featuring a significant dose dependency of metabolic activity (DEAE/CS, PEI/DS) and "invariant systems" lacking such a dependency (DEAE/DS, PEI/CS). Immunofluorescence imaging of fluorescein isothiocyanate isomer I (FITC)-labeled PECNPs suggested internalization into hMSCs remaining stable for 8 days. Conclusion: Our study demonstrated that PECNP composition affects hMSC behavior. In particular, the PEI/CS system showed biocompatibility in a wide concentration range, representing a suitable system for local drug delivery from PECNP-functionalized bone substitute materials.
- ItemPersistent effectivity of gas plasma-treated, long time-stored liquid on epithelial cell adhesion capacity and membrane morphology(San Francisco, CA : Public Library of Science, 2014) Hoentsch, M.; Bussiahn, R.; Rebl, H.; Bergemann, C.; Eggert, M.; Frank, M.; Von Woedtke, T.; Nebe, B.Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbecco's Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first timedependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.
- ItemGuidance of mesenchymal stem cells on fibronectin structured hydrogel films(San Francisco, California, US : PLOS, 2014) Kasten, Annika; Naser, Tamara; Brüllhoff, Kristina; Fiedler, Jörg; Müller, Petra; Möller, Martin; Rychly, Joachim; Groll, Jürgen; Brenner, Rolf E.; Engler, Adam J.Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.
- ItemDiscovery of chitin in skeletons of non-verongiid Red Sea demosponges(San Francisco, California, US : PLOS, 2018) Ehrlich, Hermann; Shaala, Lamiaa A.; Youssef, Diaa T. A.; Żółtowska- Aksamitowska, Sonia; Tsurkan, Mikhail; Galli, Roberta; Meissner, Heike; Wysokowski, Marcin; Petrenko, Iaroslav; Tabachnick, Konstantin R.; Ivanenko, Viatcheslav N.; Bechmann, Nicole; Joseph, Yvonne; Jesionowski, TeofilMarine demosponges (Porifera: Demospongiae) are recognized as first metazoans which have developed over millions of years of evolution effective survival strategies based on unique metabolic pathways to produce both biologically active secondary metabolites and biopolymer-based stiff skeletons with 3D architecture. Up to date, among marine demosponges, only representatives of the Verongiida order have been known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within skeletons of non-verongiid demosponges Acarnus wolffgangi and Echinoclathria gibbosa collected in the Red Sea. Calcofluor white staining, FTIR and Raman analysis, ESI-MS, SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm, with strong evidence, the finding of α-chitin in the skeleton of both species. We suggest that, the finding of chitin within these representatives of Poecilosclerida order is a promising step in the evaluation of these sponges as novel renewable sources for both biologically active metabolites and chitin, which are of prospective application for pharmacology and biomedicine.
- ItemEffects of new beta-type Ti-40Nb implant materials, brain-derived neurotrophic factor, acetylcholine and nicotine on human mesenchymal stem cells of osteoporotic and non osteoporotic donors(San Francisco, CA : Public Library of Science (PLoS), 2018) Kauschke, V.; Gebert, A.; Calin, M.; Eckert, J.; Scheich, S.; Heiss, C.; Lips, K.S.Introduction Treatment of osteoporotic fractures is still challenging and an urgent need exists for new materials, better adapted to osteoporotic bone by adjusted Young’s modulus, appropriate surface modification and pharmaceuticals. Materials and methods Titanium-40-niobium alloys, mechanically ground or additionally etched and titanium-6-alu-minium-4-vanadium were analyzed in combination with brain-derived neurotrophic factor, acetylcholine and nicotine to determine their effects on human mesenchymal stem cells in vitro over 21 days using lactate dehydrogenase and alkaline phosphatase assays, live cell imaging and immunofluorescence microscopy. Results Cell number of human mesenchymal stem cells of osteoporotic donors was increased after 14 d in presence of ground titanium-40-niobium or titanium-6-aluminium-4-vanadium, together with brain-derived neurotrophic factor. Cell number of human mesenchymal stem cells of non osteoporotic donors increased after 21 d in presence of titanium-6-aluminium-4-vanadium without pharmaceuticals. No significant increase was measured for ground or etched titanium-40-niobium after 21 d. Osteoblast differentiation of osteoporotic donors was significantly higher than in non osteoporotic donors after 21 d in presence of etched, ground titanium-40-niobium or titanium-6-aluminium-4-vanadium accompanied by all pharmaceuticals tested. In presence of all alloys tested brain-derived neurotrophic factor, acetylcholine and nicotine increased differentiation of cells of osteoporotic donors and accelerated it in non osteoporotic donors. Conclusion We conclude that ground titanium-40-niobium and brain-derived neurotrophic factor might be most suitable for subsequent in vivo testing.
- ItemNonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength(San Francisco, California, US : PLOS, 2016) Lu-Walther, Hui-Wen; Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, RainerStructured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.