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- ItemAssessment of Subsampling Strategies in Microspectroscopy of Environmental Microplastic Samples(Lausanne : Frontiers Media, 2021) Brandt, Josef; Fischer, Franziska; Kanaki, Elisavet; Enders, Kristina; Labrenz, Matthias; Fischer, DieterThe analysis of environmental occurrence of microplastic (MP) particles has gained notable attention within the past decade. An effective risk assessment of MP litter requires elucidating sources of MP particles, their pathways of distribution and, ultimately, sinks. Therefore, sampling has to be done in high frequency, both spatially and temporally, resulting in a high number of samples to analyze. Microspectroscopy techniques, such as FTIR imaging or Raman particle measurements allow an accurate analysis of MP particles regarding their chemical classification and size. However, these methods are time-consuming, which gives motivation to establish subsampling protocols that require measuring less particles, while still obtaining reliable results. The challenge regarding the subsampling of environmental MP samples lies in the heterogeneity of MP types and the relatively low numbers of target particles. Herein, we present a comprehensive assessment of different proposed subsampling methods on a selection of real-world samples from different environmental compartments. The methods are analyzed and compared with respect to resulting MP count errors, which eventually allows giving recommendations for staying within acceptable error margins. Our results are based on measurements with Raman microspectroscopy, but are applicable to any other analysis technique. We show that the subsampling-errors are mainly due to statistical counting errors (i.e., extrapolation from low numbers) and only in edge cases additionally impacted by inhomogeneous distribution of particles on the filters. Keeping the subsampling-errors low can mainly be realized by increasing the fraction of MP particles in the samples.
- ItemA Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain(Lausanne : Frontiers Media, 2020) Rust, Ruslan; Kirabali, Tunahan; Grönnert, Lisa; Dogancay, Berre; Limasale, Yanuar D.P.; Meinhardt, Andrea; Werner, Carsten; Laviña, Bàrbara; Kulic, Luka; Nitsch, Roger M.; Tackenberg, Christian; Schwab, Martin E.The distinct organization of the brain’s vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain’s vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models. © Copyright © 2020 Rust, Kirabali, Grönnert, Dogancay, Limasale, Meinhardt, Werner, Laviña, Kulic, Nitsch, Tackenberg and Schwab.
- ItemBone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes(Lausanne : Frontiers Media, 2022) Bains, Amanpreet Kaur; Behrens Wu, Lena; Rivière, Jennifer; Rother, Sandra; Magno, Valentina; Friedrichs, Jens; Werner, Carsten; Bornhäuser, Martin; Götze, Katharina S.; Cross, Michael; Platzbecker, Uwe; Wobus, ManjaMyelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.
- ItemUnderstanding Beta-Lactam-Induced Lysis at the Single-Cell Level(Lausanne : Frontiers Media, 2021) Wong, Felix; Wilson, Sean; Helbig, Ralf; Hegde, Smitha; Aftenieva, Olha; Zheng, Hai; Liu, Chenli; Pilizota, Teuta; Garner, Ethan C.; Amir, Ariel; Renner, Lars D.Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to β-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating β-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features-turgor pressure, mechanosensitive channels, and cell shape changes-that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to β-lactams aimed at targeting cellular turgor.
- ItemElectron-to Hole Transport Change Induced by Solvent Vapor Annealing of Naphthalene Diimide Doped with Poly(3-Hexylthiophene)(Lausanne : Frontiers Media, 2021) Janus, Krzysztof; Danielewicz, Kinga; Chlebosz, Dorota; Goldeman, Waldemar; Kiersnowski, AdamHerein we report on fabrication and properties of organic field-effect transistors (OFETs) based on the spray-coated films of N,N′-dioctyl naphthalene diimide (NDIC8) doped with 2.4 wt% of poly (3-hexylthiophene) (P3HT). OFETs with the untreated NDIC8:P3HT films revealed electron conductivity [μe* = 5 × 10–4 cm2×(Vs)−1]. After the annealing in chloroform vapor the NDIC8:P3HT films revealed the hole transport only [μh* = 0.9 × 10–4 cm2×(Vs)−1]. Due to the chemical nature and energy levels, the hole transport was not expected for NDIC8-based system. Polarized optical- and scanning electron microscopies indicated that the solvent vapor annealing of the NDIC8:P3HT films caused a transition of their fine-grained morphology to the network of branched, dendritic crystallites. Grazing incidence wide-angle X-ray scattering studies indicated that the above transition was accompanied by a change in the crystal structure of NDIC8. The isotropic crystal structure of NDIC8 in the untreated film was identical to the known crystal structure of the bulk NDIC8. After the solvent annealing the crystal structure of NDIC8 changed to a not-yet-reported polymorph, that, unlike in the untreated film, was partially oriented with respect to the OFET substrate.