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Author: Striesow, Johanna × DDC: 610 ×

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    An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
    (Lausanne : Frontiers Media, 2021) Graf, Alexander C.; Striesow, Johanna; Pané-Farré, Jan; Sura, Thomas; Wurster, Martina; Lalk, Michael; Pieper, Dietmar H.; Becher, Dörte; Kahl, Barbara C.; Riedel, Katharina
    Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.
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    The molecular and physiological consequences of cold plasma treatment in murine skin and its barrier function
    (New York, NY [u.a.] : Elsevier, 2020) Schmidt, Anke; Liebelt, Grit; Striesow, Johanna; Freund, Eric; Woedtke, Thomas von; Wende, Kristian; Bekeschus, Sander
    Cold plasma technology is an emerging tool facilitating the spatially controlled delivery of a multitude of reactive species (ROS) to the skin. While the therapeutic efficacy of plasma treatment has been observed in several types of diseases, the fundamental consequences of plasma-derived ROS on skin physiology remain unknown. We aimed to bridge this gap since the epidermal skin barrier and perfusion plays a vital role in health and disease by maintaining homeostasis and protecting from environmental damage. The intact skin of SKH1 mice was plasma-treated in vivo. Gene and protein expression was analyzed utilizing transcriptomics, qPCR, and Western blot. Immunofluorescence aided the analysis of percutaneous skin penetration of curcumin. Tissue oxygenation, perfusion, hemoglobin, and water index was investigated using hyperspectral imaging. Reversed-phase liquid-chromatography/mass spectrometry was performed for the identification of changes in the lipid composition and oxidation. Transcriptomic analysis of plasma-treated skin revealed modulation of genes involved in regulating the junctional network (tight, adherence, and gap junctions), which was confirmed using qPCR, Western blot, and immunofluorescence imaging. Plasma treatment increased the disaggregation of cells in the stratum corneum (SC) concomitant with increased tissue oxygenation, gap junctional intercellular communication, and penetration of the model drug curcumin into the SC preceded by altered oxidation of skin lipids and their composition in vivo. In summary, plasma-derived ROS modify the junctional network, which promoted tissue oxygenation, oxidation of SC-lipids, and restricted penetration of the model drug curcumin, implicating that plasma may provide a novel and sensitive tool of skin barrier regulation. © 2020 The Author(s)