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- ItemQuantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry(Columbus, Ohio : American Chemical Society, 2023) Kale, Dipali; Kikul, Frauke; Phapale, Prasad; Beedgen, Lars; Thiel, Christian; Brügger, BrittaDolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.
- ItemGlycerylphytate as an ionic crosslinker for 3D printing of multi-layered scaffolds with improved shape fidelity and biological features(London : Royal Society of Chemistry, 2020) Mora-Boza, A.; Włodarczyk-Biegun, M.K.; Del Campo, A.; Vázquez-Lasa, B.; Román, J.S.The fabrication of intricate and long-term stable 3D polymeric scaffolds by a 3D printing technique is still a challenge. In the biomedical field, hydrogel materials are very frequently used because of their excellent biocompatibility and biodegradability, however the improvement of their processability and mechanical properties is still required. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt%) ink of gelatin methacryloyl (GelMA)/chitosan and a novel crosslinking agent, glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of a straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision and shape fidelity (resolution ≈150 μm), and prevented the collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable under physiological conditions for at least one month. Preliminary in vitro assays using L929 fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.
- ItemTime-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe(Cambridge : RSC, 2020) Breen, Colum; Pal, Robert; Elsegood, Mark R.J.; Teat, Simon J.; Iza, Felipe; Wende, Kristian; Buckley, Benjamin R.; Butler, StephenPeroxynitrite (ONOO-) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO- in cell biology, improved methods for the selective detection and real-time analysis of ONOO- are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO- in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO- in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment. This journal is © The Royal Society of Chemistry.
- ItemA non-cytotoxic resin for micro-stereolithography for cell cultures of HUVECs(Basel : MDPI, 2020) Männel, Max J.; Fischer, Carolin; Thiele, JulianThree-dimensional (3D) printing of microfluidic devices continuously replaces conventional fabrication methods. A versatile tool for achieving microscopic feature sizes and short process times is micro-stereolithography (µSL). However, common resins for µSL lack biocompatibility and are cytotoxic. This work focuses on developing new photo-curable resins as a basis for µSL fabrication of polymer materials and surfaces for cell culture. Different acrylate-and methacrylate-based compositions are screened for material characteristics including wettability, surface roughness, and swelling behavior. For further understanding, the impact of photo-absorber and photo-initiator on the cytotoxicity of 3D-printed substrates is studied. Cell culture experiments with human umbilical vein endothelial cells (HUVECs) in standard polystyrene vessels are compared to 3D-printed parts made from our library of homemade resins. Among these, after optimizing material composition and post-processing, we identify selected mixtures of poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) methyl ethyl methacrylate (PEGMEMA) as most suitable to allow for fabricating cell culture platforms that retain both the viability and proliferation of HUVECs. Next, our PEGDA/PEGMEMA resins will be further optimized regarding minimal feature size and cell adhesion to fabricate microscopic (microfluidic) cell culture platforms, e.g., for studying vascularization of HUVECs in vitro. © 2020 by the authors.
- ItemGas-discharge plasma-assisted functionalization of titanium implant surfaces(Baech : Trans Tech Publications Ltd., 2010) Schröder, Karsten; Finke, Birgit; Polak, Martin; Lüthen, Frank; Nebe, Barbara; Rychly, Joachim; Bader, Rainer; Lukowski, Gerold; Walschus, Uwe; Schlosser, Michael; Ohl, Andreas; Weltmann, Klaus DieterA crucial factor for in-growth of metallic implants in the bone stock is the rapid cellular acceptance whilst prevention of bacterial adhesion on the surface. Such contradictorily adhesion events could be triggered by surface properties. There already exists fundamental knowledge about the influence of physicochemical surface properties like roughness, titanium dioxide modifications, cleanness, and (mainly ceramic) coatings on cell and microbial behavior in vitro and in vivo. The titanium surface can be equipped with antimicrobial properties by plasma-based copper implantation, which allows the release and generation of small concentrations of copper ions during contact with water-based biological liquids. Additionally, the titanium surface was equipped with amino groups by the deposition of an ultrathin plasma polymer. This coating on the one hand does not significantly reduce the generation of copper ions, and on the other hand improves the adhesion and spreading of osteoblast cells. The process development was accompanied by physicochemical surface analyses like XPS, FTIR, contact angle, SEM, and AFM. Very thin modified layers were created, which are resistant to hydrolysis and delamination. These titanium surface functionalizations were found to have either an antimicrobial activity or cell-adhesive properties. Intramuscular implantation of titanium samples coated with the cell-adhesive plasma polymer in rats revealed a reduced inflammation reaction compared to uncoated titanium. © (2010) Trans Tech Publications.
- 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, Jürgen; Rümmeli, 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.
- ItemBlood platelet enrichment in mass-producible surface acoustic wave (SAW) driven microfluidic chips(Cambridge : RSC, 2019) Richard, Cynthia; Fakhfouri, Armaghan; Colditz, Melanie; Striggow, Friedrich; Kronstein-Wiedemann, Romy; Tonn, Torsten; Medina-Sánchez, Mariana; Schmidt, Oliver G.; Gemming, Thomas; Winkler, AndreasThe ability to separate specific biological components from cell suspensions is indispensable for liquid biopsies, and for personalized diagnostics and therapy. This paper describes an advanced surface acoustic wave (SAW) based device designed for the enrichment of platelets (PLTs) from a dispersion of PLTs and red blood cells (RBCs) at whole blood concentrations, opening new possibilities for diverse applications involving cell manipulation with high throughput. The device is made of patterned SU-8 photoresist that is lithographically defined on the wafer scale with a new proposed methodology. The blood cells are initially focused and subsequently separated by an acoustic radiation force (ARF) applied through standing SAWs (SSAWs). By means of flow cytometric analysis, the PLT concentration factor was found to be 7.7, and it was proven that the PLTs maintain their initial state. A substantially higher cell throughput and considerably lower applied powers than comparable devices from literature were achieved. In addition, fully coupled 3D numerical simulations based on SAW wave field measurements were carried out to anticipate the coupling of the wave field into the fluid, and to obtain the resulting pressure field. A comparison to the acoustically simpler case of PDMS channel walls is given. The simulated results show an ideal match to the experimental observations and offer the first insights into the acoustic behavior of SU-8 as channel wall material. The proposed device is compatible with current (Lab-on-a-Chip) microfabrication techniques allowing for mass-scale, reproducible chip manufacturing which is crucial to push the technology from lab-based to real-world applications. © The Royal Society of Chemistry.
- ItemImpact of plasma jet vacuum ultraviolet radiation on reactive oxygen species generation in bio-relevant liquids([S.l.] : American Institute of Physics, 2015) Jablonowski, H.; Bussiahn, R.; Hammer, M.U.; Weltmann, K.-D.; von Woedtke, T.; Reuter, S.Plasma medicine utilizes the combined interaction of plasma produced reactive components. These are reactive atoms, molecules, ions, metastable species, and radiation. Here, ultraviolet (UV, 100–400 nm) and, in particular, vacuum ultraviolet (VUV, 10–200 nm) radiation generated by an atmospheric pressure argon plasma jet were investigated regarding plasma emission, absorption in a humidified atmosphere and in solutions relevant for plasma medicine. The energy absorption was obtained for simple solutions like distilled water (dH2O) or ultrapure water and sodium chloride (NaCl) solution as well as for more complex ones, for example, Rosewell Park Memorial Institute (RPMI 1640) cell culture media. As moderate stable reactive oxygen species, hydrogen peroxide (H2O2) was studied. Highly reactive oxygen radicals, namely, superoxide anion (O2•−) and hydroxyl radicals (•OH), were investigated by the use of electron paramagnetic resonance spectroscopy. All species amounts were detected for three different treatment cases: Plasma jet generated VUV and UV radiation, plasma jet generated UV radiation without VUV part, and complete plasma jet including all reactive components additionally to VUV and UV radiation. It was found that a considerable amount of radicals are generated by the plasma generated photoemission. From the experiments, estimation on the low hazard potential of plasma generated VUV radiation is discussed.
- ItemRedox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma(Austin, Tex. : Landes Bioscience, 2015) Bekeschus, Sander; Schmidt, Anke; Bethge, Lydia; Masur, Kai; von Woedtke, Thomas; Hasse, Sybille; Wende, KristianIn plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.
- ItemThe LEGATO cross-disciplinary integrated ecosystem service research framework: an example of integrating research results from the analysis of global change impacts and the social, cultural and economic system dynamics of irrigated rice production(Heidelberg : Springer Verlag, 2017) Spangenberg, J.H.; Beaurepaire, A.L.; Bergmeier, E.; Burkhard, B.; van Chien, H.; Cuong, L.Q.; Görg, C.; Grescho, V.; Hai, L.H.; Heong, K.L.; Horgan, F.G.; Hotes, S.; Klotzbücher, A.; Klotzbücher, T.; Kühn, I.; Langerwisch, F.; Marion, G.; Moritz, R.F.A.; Nguyen, Q.A.; Ott, J.; Sann, C.; Sattler, C.; Schädler, M.; Schmidt, A.; Tekken, V.; Thanh, T.D.; Thonicke, K.; Türke, M.; VáclavÃk, T.; Vetterlein, D.; Westphal, C.; Wiemers, M.; Settele, J.In a cross-disciplinary project (LEGATO) combining inter- and transdisciplinary methods, we quantify the dependency of rice-dominated socio-ecological systems on ecosystem functions (ESF) and the ecosystem services (ESS) the integrated system provides. In the collaboration of a large team including geo- and bioscientists, economists, political and cultural scientists, the mutual influences of the biological, climate and soil conditions of the agricultural area and its surrounding natural landscape have been analysed. One focus was on sociocultural and economic backgrounds, another on local as well as regional land use intensity and biodiversity, and the potential impacts of future climate and land use change. LEGATO analysed characteristic elements of three service strands defined by the Millennium Ecosystem Assessment (MA): (a) provisioning services: nutrient cycling and crop production; (b) regulating services: biocontrol and pollination; and (c) cultural services: cultural identity and aesthetics. However, in line with much of the current ESS literature, what the MA called supporting services is treated as ESF within LEGATO. As a core output, LEGATO developed generally applicable principles of ecological engineering (EE), suitable for application in the context of future climate and land use change. EE is an emerging discipline, concerned with the design, monitoring and construction of ecosystems and aims at developing strategies to optimise ecosystem services through exploiting natural regulation mechanisms instead of suppressing them. Along these lines LEGATO also aims to create the knowledge base for decision-making for sustainable land management and livelihoods, including the provision of the corresponding governance and management strategies, technologies and system solutions.