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- ItemSecondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies(Columbus, Ohio : American Chemical Society, 2016) Davies, Heather S.; Singh, Prabha; Deckert-Gaudig, Tanja; Deckert, Volker; Rousseau, Karine; Ridley, Caroline E.; Dowd, Sarah E.; Doig, Andrew J.; Pudney, Paul D. A.; Thornton, David J.; Blanch, Ewan W.The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
- ItemFiber-based SORS-SERDS system and chemometrics for the diagnostics and therapy monitoring of psoriasis inflammatory disease in vivo(Washington, DC : Optica, 2021-1-28) Schleusener, Johannes; Guo, Shuxia; Darvin, Maxim E.; Thiede, Gisela; Chernavskaia, Olga; Knorr, Florian; Lademann, Jürgen; Popp, Jürgen; Bocklitz, Thomas W.Psoriasis is considered a widespread dermatological disease that can strongly affect the quality of life. Currently, the treatment is continued until the skin surface appears clinically healed. However, lesions appearing normal may contain modifications in deeper layers. To terminate the treatment too early can highly increase the risk of relapses. Therefore, techniques are needed for a better knowledge of the treatment process, especially to detect the lesion modifications in deeper layers. In this study, we developed a fiber-based SORS-SERDS system in combination with machine learning algorithms to non-invasively determine the treatment efficiency of psoriasis. The system was designed to acquire Raman spectra from three different depths into the skin, which provide rich information about the skin modifications in deeper layers. This way, it is expected to prevent the occurrence of relapses in case of a too short treatment. The method was verified with a study of 24 patients upon their two visits: the data is acquired at the beginning of a standard treatment (visit 1) and four months afterwards (visit 2). A mean sensitivity of ≥85% was achieved to distinguish psoriasis from normal skin at visit 1. At visit 2, where the patients were healed according to the clinical appearance, the mean sensitivity was ≈65%.
- ItemMonitoring excited-state relaxation in a molecular marker in live cells–a case study on astaxanthin(London : Royal Society of Chemistry (RSC), 2021) Yang, Tingxiang; Chettri, Avinash; Radwan, Basseem; Matuszyk, Ewelina; Baranska, Malgorzata; Dietzek, BenjaminSmall molecules are frequently used as dyes, labels and markers to visualize and probe biophysical processes within cells. However, very little is generally known about the light-driven excited-state reactivity of such systems when placed in cells. Here an experimental approach to study ps time-resolved excited state dynamics of a benchmark molecular marker, astaxanthin, in live human cells is introduced. © The Royal Society of Chemistry 2021.
- ItemA new human adipocyte model with PTEN haploinsufficiency(Abingdon : Taylor and Francis Inc., 2020) Kässner F.; Kirstein A.; Händel N.; Schmid G.L.; Landgraf K.; Berthold A.; Tannert A.; Schaefer M.; Wabitsch M.; Kiess W.; Körner A.; Garten A.Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
- ItemThe role of risk communication in public health interventions. An analysis of risk communication for a community quarantine in Germany to curb the SARS-CoV-2 pandemic(San Francisco, California, US : PLOS, 2021) Scholz, Juliane; Wetzker, Wibke; Licht, Annika; Heintzmann, Rainer; Scherag, André; Weis, Sebastian; Pletz, Mathias; Betsch, Cornelia; Bauer, Michael; Dickmann, Petra; Frey, RosemaryBackground: Separating ill or possibly infectious people from their healthy community is one of the core principles of non-pharmaceutical interventions. However, there is scarce evidence on how to successfully implement quarantine orders. We investigated a community quarantine for an entire village in Germany (Neustadt am Rennsteig, March 2020) with the aim of better understanding the successful implementation of quarantine measures. Methods: This cross-sectional survey was conducted in Neustadt am Rennsteig six weeks after the end of a 14-day mandatory community quarantine. The sample size consisted of 562 adults (64% of the community), and the response rate was 295 adults, or 52% (33% of the community). Findings: National television was reported as the most important channel of information. Contact with local authorities was very limited, and partners or spouses played a more important role in sharing information. Generally, the self-reported information level was judged to be good (211/289 [73.0%]). The majority of participants (212/289 [73.4%]) approved of the quarantine, and the reported compliance was 217/289 (75.1%). A self-reported higher level of concern as well as a higher level of information correlated positively with both a greater acceptance of quarantine and self-reported compliant behaviour. Interpretation: The community quarantine presented a rare opportunity to investigate a public health intervention for an entire community. In order to improve the implementation of public health interventions, public health risk communication activities should be intensified to increase both the information level (potentially leading to better compliance with community quarantine) and the communication level (to facilitate rapport and trust between public health authorities and their communities). © 2021 Scholz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
- ItemNonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength(San Francisco, California, US : PLOS, 2016) Lu-Walther, Hui-Wen; Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, RainerStructured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.
- ItemFiber enhanced Raman spectroscopic analysis as a novel method for diagnosis and monitoring of diseases related to hyperbilirubinemia and hyperbiliverdinemia(Cambridge : Soc., 2016) Yan, Di; Domes, Christian; Domes, Robert; Frosch, Timea; Popp, Jürgen; Pletz, Mathias W.; Frosch, TorstenFiber enhanced resonance Raman spectroscopy (FERS) is introduced for chemically selective and ultrasensitive analysis of the biomolecules hematin, hemoglobin, biliverdin, and bilirubin. The abilities for analyzing whole intact, oxygenated erythrocytes are proven, demonstrating the potential for the diagnosis of red blood cell related diseases, such as different types of anemia and hemolytic disorders. The optical fiber enables an efficient light-guiding within a miniaturized sample volume of only a few micro-liters and provides a tremendously improved analytical sensitivity (LODs of 0.5 μM for bilirubin and 0.13 μM for biliverdin with proposed improvements down to the pico-molar range). FERS is a less invasive method than the standard ones and could be a new analytical method for monitoring neonatal jaundice, allowing a precise control of the unconjugated serum bilirubin levels, and therefore, providing a better prognosis for newborns. The potential for sensing very low concentrations of the bile pigments may also open up new opportunities for cancer research. The abilities of FERS as a diagnostic tool are explored for the elucidation of jaundice with different etiologies including the rare, not yet well understood diseases manifested in green jaundice. This is demonstrated by quantifying clinically relevant concentrations of bilirubin and biliverdin simultaneously in the micro-molar range: for the case of hyperbilirubinemia due to malignancy, infectious hepatitis, cirrhosis or stenosis of the common bile duct (1 μM biliverdin together with 50 μM bilirubin) and for hyperbiliverdinemia (25 μM biliverdin and 75 μM bilirubin). FERS has high potential as an ultrasensitive analytical technique for a wide range of biomolecules and in various life-science applications.
- ItemIntestinal epithelial barrier integrity investigated by label-free techniques in ulcerative colitis patients([London] : Macmillan Publishers Limited, part of Springer Nature, 2023) Quansah, Elsie; Gardey, Elena; Ramoji, Anuradha; Meyer-Zedler, Tobias; Goehrig, Bianca; Heutelbeck, Astrid; Hoeppener, Stephanie; Schmitt, Michael; Waldner, Maximillian; Stallmach, Andreas; Popp, JürgenThe intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM), to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential advantage of non-linear multimodal imaging is to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in vivo imaging.
- ItemObject detection networks and augmented reality for cellular detection in fluorescence microscopy(New York, NY : Rockefeller Univ. Press, 2020) Waithe, Dominic; Brown, Jill M.; Reglinski, Katharina; Diez-Sevilla, Isabel; Roberts, David; Eggeling, ChristianObject detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines. © 2020 Waithe et al.