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- ItemDetection of Protein Glycosylation Using Tip-Enhanced Raman Scattering(Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, RoystonThe correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
- ItemPH-Responsive Biohybrid Carrier Material for Phenol Decontamination in Wastewater(Columbus, Ohio : American Chemical Soc., 2018) Pretscher, Martin; Pineda-Contreras, Beatriz A.; Kaiser, Patrick; Reich, Steffen; Schöbel, Judith; Kuttner, Christian; Freitag, Ruth; Fery, Andreas; Schmalz, Holger; Agarwal, SeemaSmart polymers are a valuable platform to protect and control the activity of biological agents over a wide range of conditions, such as low pH, by proper encapsulation. Such conditions are present in olive oil mill wastewater with phenol as one of the most problematic constituents. We show that elastic and pH-responsive diblock copolymer fibers are a suitable carrier for Corynebacterium glutamicum, i.e., bacteria which are known for their ability to degrade phenol. Free C. glutamicum does not survive low pH conditions and fails to degrade phenol at low pH conditions. Our tea-bag like biohybrid system, where the pH-responsive diblock copolymer acts as a protecting outer shell for the embedded bacteria, allows phenol degradation even at low pH. Utilizing a two-step encapsulation process, planktonic cells were first encapsulated in poly(vinyl alcohol) to protect the bacteria against the organic solvents used in the second step employing coaxial electrospinning.
- ItemHigh-throughput screening Raman microspectroscopy for assessment of drug-induced changes in diatom cells(Cambridge : Royal Society of Chemistry, 2019) Rüger J.; Mondol A.S.; Schie I.W.; Popp J.; Krafft C.High-throughput screening Raman spectroscopy (HTS-RS) with automated localization algorithms offers unsurpassed speed and sensitivity to investigate the effect of dithiothreitol on the diatom Phaedactylum tricornutum. The HTS-RS capability that was demonstrated for this model system can be transferred to unmet analytical applications such as kinetic in vivo studies of microalgal assemblages. © 2019 The Royal Society of Chemistry.
- ItemElectron beam-induced immobilization of laccase on porous supports for waste water treatment applications(Basel : MDPI AG, 2014) Jahangiri, E.; Reichelt, S.; Thomas, I.; Hausmann, K.; Schlosser, D.; Schulze, A.The versatile oxidase enzyme laccase was immobilized on porous supports such as polymer membranes and cryogels with a view of using such biocatalysts in bioreactors aiming at the degradation of environmental pollutants in wastewater. Besides a large surface area for supporting the biocatalyst, the aforementioned porous systems also offer the possibility for simultaneous filtration applications in wastewater treatment. Herein a "green" water-based, initiator-free, and straightforward route to highly reactive membrane and cryogel-based bioreactors is presented, where laccase was immobilized onto the porous polymer supports using a water-based electron beam-initiated grafting reaction. In a second approach, the laccase redox mediators 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and syringaldehyde were cross-linked instead of the enzyme via electron irradiation in a frozen aqueous poly(acrylate) mixture in a one pot set-up, yielding a mechanical stable macroporous cryogel with interconnected pores ranging from 10 to 50 μm in size. The membranes as well as the cryogels were characterized regarding their morphology, chemical composition, and catalytic activity. The reactivity towards waste-water pollutants was demonstrated by the degradation of the model compound bisphenol A (BPA). Both membrane- and cryogel-immobilized laccase remained highly active after electron beam irradiation. Apparent specific BPA removal rates were higher for cryogel-than for membrane-immobilized and free laccase, whereas membrane-immobilized laccase was more stable with respect to maintenance of enzymatic activity and prevention of enzyme leakage from the carrier than cryogel-immobilized laccase. Cryogel-immobilized redox mediators remained functional in accelerating the laccase-catalyzed BPA degradation, and especially ABTS was found to act more efficiently in immobilized than in freely dissolved state.
- 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).
- ItemKey knowledge and data gaps in modelling the influence of CO2 concentration on the terrestrial carbon sink(München : Elsevier, 2016) Pugh, T.A.M.; Müller, C.; Arneth, A.; Haverd, V.; Smith, B.Primary productivity of terrestrial vegetation is expected to increase under the influence of increasing atmospheric carbon dioxide concentrations ([CO2]). Depending on the fate of such additionally fixed carbon, this could lead to an increase in terrestrial carbon storage, and thus a net terrestrial sink of atmospheric carbon. Such a mechanism is generally believed to be the primary global driver behind the observed large net uptake of anthropogenic CO2 emissions by the biosphere. Mechanisms driving CO2 uptake in the Terrestrial Biosphere Models (TBMs) used to attribute and project terrestrial carbon sinks, including that from increased [CO2], remain in large parts unchanged since those models were conceived two decades ago. However, there exists a large body of new data and understanding providing an opportunity to update these models, and directing towards important topics for further research. In this review we highlight recent developments in understanding of the effects of elevated [CO2] on photosynthesis, and in particular on the fate of additionally fixed carbon within the plant with its implications for carbon turnover rates, on the regulation of photosynthesis in response to environmental limitations on in-plant carbon sinks, and on emergent ecosystem responses. We recommend possible avenues for model improvement and identify requirements for better data on core processes relevant to the understanding and modelling of the effect of increasing [CO2] on the global terrestrial carbon sink.
- ItemProduction of highly concentrated and hyperpolarized metabolites within seconds in high and low magnetic fields(Cambridge : RSC Publ., 2019) Korchak, Sergey; Emondts, Meike; Mamone, Salvatore; Blümich, Bernhard; Glöggler, StefanHyperpolarized metabolites are very attractive contrast agents for in vivo magnetic resonance imaging studies enabling early diagnosis of cancer, for example. Real-time production of concentrated solutions of metabolites is a desired goal that will enable new applications such as the continuous investigation of metabolic changes. To this end, we are introducing two NMR experiments that allow us to deliver high levels of polarization at high concentrations (50 mM) of an acetate precursor (55% 13C polarization) and acetate (17% 13C polarization) utilizing 83% para-state enriched hydrogen within seconds at high magnetic field (7 T). Furthermore, we have translated these experiments to a portable low-field spectrometer with a permanent magnet operating at 1 T. The presented developments pave the way for a rapid and affordable production of hyperpolarized metabolites that can be implemented in e.g. metabolomics labs and for medical diagnosis.
- 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.
- ItemCharge isomers of myelin basic protein: Structure and interactions with membranes, nucleotide analogues, and calmodulin(San Francisco, CA : Public Library of Science, 2011) Wang, C.; Neugebauer, U.; Bürck, J.; Myllykoski, M.; Baumgärtel, P.; Popp, J.; Kursula, P.As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.
- 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.