Institut für Chemie und Bioanalytik

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  • Publikation
    Nanobiocatalysts with inbuilt cofactor recycling for oxidoreductase catalysis in organic solvents
    (Royal Society of Chemistry, 2023) Sahlin, Jenny; Wu, Congyu; Buscemi, Andrea; Schärer, Claude; Nazemi, Seyed Amirabbas; S. K., Rejaul; Herrera-Reinoza, Nataly; Jung, Thomas A.; Shahgaldian, Patrick [in: Nanoscale Advances]
    The major stumbling block in the implementation of oxidoreductase enzymes in continuous processes is their stark dependence on costly cofactors that are insoluble in organic solvents. We describe a chemical strategy that allows producing nanobiocatalysts, based on an oxidoreductase enzyme, that performs biocatalytic reactions in hydrophobic organic solvents without external cofactors. The chemical design relies on the use of a silica-based carrier nanoparticle, of which the porosity can be exploited to create an aqueous reservoir containing the cofactor. The nanoparticle core, possessing radial-centred pore channels, serves as a cofactor reservoir. It is further covered with a layer of reduced porosity. This layer serves as a support for the immobilisation of the selected enzyme yet allowing the diffusion of the cofactor from the nanoparticle core. The immobilised enzyme is, in turn, shielded by an organosilica layer of controlled thickness fully covering the enzyme. Such produced nanobiocatalysts are shown to catalyse the reduction of a series of relevant ketones into the corresponding secondary alcohols, also in a continuous flow fashion. © 2023 RSC.
    01A - Beitrag in wissenschaftlicher Zeitschrift
  • Publikation
    Stochastic chain termination in bacterial pilus assembly
    (Nature, 2023) Giese, Christoph; Puorger, Chasper; Ignatov, Oleksandr; Bečárová, Zuzana; Weber, Marco E.; Schärer, Martin A.; Capitani, Guido; Glockshuber, Rudi [in: Nature Communications]
    Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction. In addition, we demonstrate that FimI contributes to anchoring the pilus to the outer membrane and report the crystal structures of (i) FimI in complex with the assembly chaperone FimC, (ii) the FimI-FimC complex bound to the N-terminal domain of FimD, and (iii) a ternary complex between FimI, FimA and FimC that provides structural insights on pilus assembly termination and pilus anchoring by FimI.
    01A - Beitrag in wissenschaftlicher Zeitschrift
  • Publikation
    An origami like 3D patterned cellulose-based scaffold for bioengineering cardiovascular applications
    (Springer, 2023) Rodriguez, Gabriela Melo; Trueb, Donata; Köser, Joachim; Schoelkopf, Joachim; Gullo, Maurizio [in: Cellulose]
    In this work we describe the manufacturing of cellulosic, cell compatible scaffolds with an inherent 3D origami crease pattern for applications in cardiac tissue engineering. Different cellulosic materials were studied, among them cotton linters, fibers obtained from eucalyptus, pine, spruce and lyocell. Formed sheets made of cotton linters were chosen for further study due to the highest biocompatibility and mechanical properties best suited for cardiomyocytes in wet and dry conditions: E - modulus of 0.8 GPa, tensile strength of 4.7 MPa and tensile strength in wet environment of 2.28 MPa. Cell alignment is desired to achieve directional contraction of the cardiac tissue, and several options were investigated to achieve fiber alignment, e.g. a dynamic sheet former and Rapid Köthen sheet former. Although the orientation was minimal, cells cultured on the cellulose fibers grew and aligned along the fibers. Origami inspired crease patterns were applied to the cellulose scaffolds in order to introduce directional flexibility beneficial for cardiac contraction. The transfer of a Miura-ori crease pattern was successfully applied in two ways: folding of the dried sheet between PET foils pre-formed in a 3D printed mold, and in situ wet fiber molding on a 3D-patterned mesh mounted in the sheet former’s sieve section. The latter approach enables upscaling for potential mass production.
    01A - Beitrag in wissenschaftlicher Zeitschrift
  • Publikation
    A perfused in vitro human iPSC-derived blood–brain barrier faithfully mimics transferrin receptor-mediated transcytosis of therapeutic antibodies
    (Springer, 2023) Burgio, Floriana; Gaiser, Carine; Brady, Kevin; Gatta, Viviana; Class, Reiner; Schrage, Ramona; Suter-Dick, Laura [in: Cellular and Molecular Neurobiology]
    Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application. A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com.
    01A - Beitrag in wissenschaftlicher Zeitschrift
  • Publikation
    SARS-CoV-2 variants of concern and clinical severity in the mexican pediatric population
    (MDPI, 2023) Maldonado-Cabrera, Anahí; Colin-Vilchis, Jesus Alejandro; Haque, Ubydul; Velazquez, Carlos; Alvarez Villaseñor, Andrea Socorro; Magdaleno-Márquez, Luis Eduardo; Calleros-Muñoz, Carlos Iván; Figueroa-Enríquez, Karen Fernanda; Angulo-Molina, Aracely; Gallego-Hernández, Ana Lucía [in: Infectious Disease Reports]
    The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) presents global heterogeneity, and their relative effect on pediatric severity is still limited. In this study, we associate VOCs with pediatric clinical severity outcomes in Mexico. Bioinformatics methods were used to characterize VOCs and single amino acid (aa) mutations in 75,348 SARS-CoV-2 genetic sequences from February 2020 to October 2022. High-predominance VOCs groups were calculated and subsequently associated with 372,989 COVID-19 clinical pediatric outcomes. We identified 21 high-frequency mutations related to Omicron lineages with an increased prevalence in pediatric sequences compared to adults. Alpha and the other lineages had a significant increase in case fatality rate (CFR), intensive critical unit (ICU) admission, and automated mechanical ventilation (AMV). Furthermore, a logistic model with age-adjusted variables estimated an increased risk of hospitalization, ICU/AMV, and death in Gamma and Alpha, in contrast to the other lineages. We found that, regardless of the VOCs lineage, infant patients presented the worst severity prognoses. Our findings improve the understanding of the impact of VOCs on pediatric patients across time, regions, and clinical outcomes. Enhanced understanding of the pediatric severity for VOCs would enable the development and improvement of public health strategies worldwide.
    01A - Beitrag in wissenschaftlicher Zeitschrift