@article{LorsonRuoppNadernezhadetal.2020, author = {Lorson, Thomas and Ruopp, Matthias and Nadernezhad, Ali and Eiber, Julia and Vogel, Ulrich and Jungst, Tomasz and L{\"u}hmann, Tessa}, title = {Sterilization Methods and Their Influence on Physicochemical Properties and Bioprinting of Alginate as a Bioink Component}, series = {ACS Omega}, volume = {5}, journal = {ACS Omega}, number = {12}, doi = {10.1021/acsomega.9b04096}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229460}, pages = {6481-6486}, year = {2020}, abstract = {Bioprinting has emerged as a valuable threedimensional (3D) biomanufacturing method to fabricate complex hierarchical cell-containing constructs. Spanning from basic research to clinical translation, sterile starting materials are crucial. In this study, we present pharmacopeia compendial sterilization methods for the commonly used bioink component alginate. Autoclaving (sterilization in saturated steam) and sterile filtration followed by lyophilization as well as the pharmacopeia non-compendial method, ultraviolet (UV)-irradiation for disinfection, were assessed. The impact of the sterilization methods and their effects on physicochemical and rheological properties, bioprinting outcome, and sterilization efficiency of alginate were detailed. Only sterile filtration followed by lyophilization as the sterilization method retained alginate's physicochemical properties and bioprinting behavior while resulting in a sterile outcome. This set of methods provides a blueprint for the analysis of sterilization effects on the rheological and physicochemical pattern of bioink components and is easily adjustable for other polymers used in the field of biofabrication in the future.}, language = {en} } @article{WandreyWurzelHoffmannetal.2016, author = {Wandrey, Georg and Wurzel, Joel and Hoffmann, Kyra and Ladner, Tobias and B{\"u}chs, Jochen and Meinel, Lorenz and L{\"u}hmann, Tessa}, title = {Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform}, series = {Journal of Biological Engineering}, volume = {10}, journal = {Journal of Biological Engineering}, number = {11}, doi = {10.1186/s13036-016-0031-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166304}, year = {2016}, abstract = {Background Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation. Results We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNAPyl CUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures. Conclusion The developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNAPyl CUA pairs as well as further optimization of culture conditions.}, language = {en} } @article{FirdessaGoodAmstaldenetal.2015, author = {Firdessa, Rebuma and Good, Liam and Amstalden, Maria Cecilia and Chindera, Kantaraja and Kamaruzzaman, Nor Fadhilah and Schultheis, Martina and R{\"o}ger, Bianca and Hecht, Nina and Oelschlaeger, Tobias A. and Meinel, Lorenz and L{\"u}hmann, Tessa and Moll, Heidrun}, title = {Pathogen- and host-directed antileishmanial effects mediated by polyhexanide (PHMB)}, series = {PLoS Neglected Tropical Diseases}, volume = {9}, journal = {PLoS Neglected Tropical Diseases}, number = {10}, doi = {10.1371/journal.pntd.0004041}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148162}, pages = {e0004041}, year = {2015}, abstract = {Background Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. Methodology/Principal Findings Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. Conclusions Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.}, language = {en} } @article{HornBaumannPereiraetal.2012, author = {Horn, Michael and Baumann, Reto and Pereira, Jorge A. and Sidiropoulos, P{\´a}ris N. M. and Somandin, Christian and Welzl, Hans and Stendel, Claudia and L{\"u}hmann, Tessa and Wessig, Carsten and Toyka, Klaus V. and Relvas, Jo{\~a}o B. and Senderek, Jan and Suter, Ueli}, title = {Myelin is dependent on the Charcot-Marie-Tooth Type 4H disease culprit protein FRABIN/FGD4 in Schwann cells}, series = {Brain}, volume = {135}, journal = {Brain}, doi = {10.1093/brain/aws275}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125390}, pages = {3567-3583}, year = {2012}, abstract = {Studying the function and malfunction of genes and proteins associated with inherited forms of peripheral neuropathies has provided multiple clues to our understanding of myelinated nerves in health and disease. Here, we have generated a mouse model for the peripheral neuropathy Charcot-Marie-Tooth disease type 4H by constitutively disrupting the mouse orthologue of the suspected culprit gene FGD4 that encodes the small RhoGTPase Cdc42-guanine nucleotide exchange factor Frabin. Lack of Frabin/Fgd4 causes dysmyelination in mice in early peripheral nerve development, followed by profound myelin abnormalities and demyelination at later stages. At the age of 60 weeks, this was accompanied by electrophysiological deficits. By crossing mice carrying alleles of Frabin/Fgd4 flanked by loxP sequences with animals expressing Cre recombinase in a cell type-specific manner, we show that Schwann cell-autonomous Frabin/Fgd4 function is essential for proper myelination without detectable primary contributions from neurons. Deletion of Frabin/Fgd4 in Schwann cells of fully myelinated nerve fibres revealed that this protein is not only required for correct nerve development but also for accurate myelin maintenance. Moreover, we established that correct activation of Cdc42 is dependent on Frabin/Fgd4 function in healthy peripheral nerves. Genetic disruption of Cdc42 in Schwann cells of adult myelinated nerves resulted in myelin alterations similar to those observed in Frabin/Fgd4-deficient mice, indicating that Cdc42 and the Frabin/Fgd4-Cdc42 axis are critical for myelin homeostasis. In line with known regulatory roles of Cdc42, we found that Frabin/Fgd4 regulates Schwann cell endocytosis, a process that is increasingly recognized as a relevant mechanism in peripheral nerve pathophysiology. Taken together, our results indicate that regulation of Cdc42 by Frabin/Fgd4 in Schwann cells is critical for the structure and function of the peripheral nervous system. In particular, this regulatory link is continuously required in adult fully myelinated nerve fibres. Thus, mechanisms regulated by Frabin/Fgd4-Cdc42 are promising targets that can help to identify additional regulators of myelin development and homeostasis, which may crucially contribute also to malfunctions in different types of peripheral neuropathies.}, language = {en} } @article{AltmannMutWolfetal.2021, author = {Altmann, Stephan and Mut, J{\"u}rgen and Wolf, Natalia and Meißner-Weigl, Jutta and Rudert, Maximilian and Jakob, Franz and Gutmann, Marcus and L{\"u}hmann, Tessa and Seibel, J{\"u}rgen and Ebert, Regina}, title = {Metabolic glycoengineering in hMSC-TERT as a model for skeletal precursors by using modified azide/alkyne monosaccharides}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {6}, issn = {1422-0067}, doi = {10.3390/ijms22062820}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259247}, year = {2021}, abstract = {Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac\(_4\)ManNAz) and N-alkyneacetylmannosamine (Ac\(_4\)ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac\(_4\)ManNAz was detectable for up to six days while Ac\(_4\)ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.}, language = {en} } @article{HahnBeudertGutmannetal.2021, author = {Hahn, Lukas and Beudert, Matthias and Gutmann, Marcus and Keßler, Larissa and Stahlhut, Philipp and Fischer, Lena and Karakaya, Emine and Lorson, Thomas and Thievessen, Ingo and Detsch, Rainer and L{\"u}hmann, Tessa and Luxenhofer, Robert}, title = {From Thermogelling Hydrogels toward Functional Bioinks: Controlled Modification and Cytocompatible Crosslinking}, series = {Macromolecular Bioscience}, volume = {21}, journal = {Macromolecular Bioscience}, number = {10}, doi = {10.1002/mabi.202100122}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257542}, year = {2021}, abstract = {Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study, a well-known Diels-Alder two-step post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). The diblock copolymers are partially hydrolyzed and subsequently modified by acid/amine coupling with furan and maleimide moieties. While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel is functionalized with RGD-peptides using thiol-maleimide chemistry and cell proliferation as well as morphology of fibroblasts seeded on top of the hydrogels confirm the cell adhesion facilitated by the peptides. Finally, bioink formulations are tested for biocompatibility by incorporating fibroblasts homogenously inside the polymer solution pre-printing. After the printing and crosslinking process good cytocompatibility is confirmed. The established bioink system combines a two-step approach by physical precursor gelation followed by an additional chemical stabilization, offering a broad versatility for further biomechanical adaptation or bioresponsive peptide modification.}, language = {en} } @article{CataldiRaschigGutmannetal.2023, author = {Cataldi, Eleonora and Raschig, Martina and Gutmann, Marcus and Geppert, Patrick T. and Ruopp, Matthias and Schock, Marvin and Gerwe, Hubert and Bertermann, R{\"u}diger and Meinel, Lorenz and Finze, Maik and Nowak-Kr{\´o}l, Agnieszka and Decker, Michael and L{\"u}hmann, Tessa}, title = {Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch}, series = {ChemBioChem}, volume = {24}, journal = {ChemBioChem}, number = {5}, doi = {10.1002/cbic.202200570}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312480}, year = {2023}, abstract = {The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site-specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.}, language = {en} }