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- ItemTowards Bacteria Counting in DI Water of Several Microliters or Growing Suspension Using Impedance Biochips(Basel : MDPI, 2020) Kiani, Mahdi; Tannert, Astrid; Du, Nan; Hübner, Uwe; Skorupa, Ilona; Bürger, Danilo; Zhao, Xianyue; Blaschke, Daniel; Rebohle, Lars; Cherkouk, Charaf; Neugebauer, Ute; Schmidt, Oliver G.; Schmidt, HeidemarieWe counted bacterial cells of E. coli strain K12 in several-microliter DI water or in several-microliter PBS in the low optical density (OD) range (OD = 0.05–1.08) in contact with the surface of Si-based impedance biochips with ring electrodes by impedance measurements. The multiparameter fit of the impedance data allowed calibration of the impedance data with the concentration cb of the E. coli cells in the range of cb = 0.06 to 1.26 × 109 cells/mL. The results showed that for E. coli in DI water and in PBS, the modelled impedance parameters depend linearly on the concentration of cells in the range of cb = 0.06 to 1.26 × 109 cells/mL, whereas the OD, which was independently measured with a spectrophotometer, was only linearly dependent on the concentration of the E. coli cells in the range of cb = 0.06 to 0.50 × 109 cells/mL.
- ItemCharacterization and prediction of the mechanism of action of antibiotics through NMR metabolomics(London : BioMed Central, 2016) Hoerr, Verena; Duggan, Gavin E.; Zbytnuik, Lori; Poon, Karen K.H.; Große, Christina; Neugebauer, Ute; Methling, Karen; Löffler, Bettina; Vogel, Hans J.Background: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. Results: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. Conclusion: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.
- ItemLight-responsive paper strips as CO-releasing material with a colourimetric response(Cambridge : RSC Publishing, 2017) Reddy G., Upendar; Liu, Jingjing; Hoffmann, Patrick; Steinmetzer, Johannes; Görls, Helmar; Kupfer, Stephan; Askes, Sven H. C.; Neugebauer, Ute; Gräfe, Stefanie; Schiller, AlexanderCarbon monoxide (CO) is known for its multifaceted role in human physiology, and molecules that release CO in a controlled way have been proposed as therapeutic drugs. In this work, a light-responsive CO-releasing molecule (CORM-Dabsyl) showed a strong colourimetric response upon photochemical CO-release, owing to the tight conjugation of a Mn(i) tricarbonyl centre to a dabsyl chromophoric ligand (L). Whereas the complex was very stable in the dark in nitrogen-purged aqueous media, CO-release was effectively triggered using 405 nm irradiation. CORM-Dabsyl, L and the inactive product iCORM-Dabsyl have been investigated by DFT and TD-DFT calculations. Only mild toxicity of CORM-Dabsyl was observed against LX-2 and HepaRG® human cell lines (IC50 ∼ 30 μM). Finally, to develop a CO storage and release material that is readily applicable to therapeutic situations, CORM-Dabsyl was loaded on low-cost and easily disposable paper strips, from which the light triggered CO-release was conveniently visible with the naked eye.
- ItemMicrofluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress(Basel : MDPI, 2018) Pilát, Zdeněk; Bernatová, Silvie; Ježek, Jan; Kirchhoff, Johanna; Tannert, Astrid; Neugebauer, Ute; Samek, Ota; Zemánek, PavelAnalyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.
- ItemStructural insights into heme binding to IL-36α proinflammatory cytokine(Berlin : Nature Publishing, 2019) Wißbrock, Amelie; Goradia, Nishit; Kumar, Amit; Paul George, Ajay Abisheck; Kühl, Toni; Bellstedt, Peter; Ramachandran, Ramadurai; Hoffmann, Patrick; Galler, Kerstin; Popp, Jürgen; Neugebauer, Ute; Hampel, Kornelia; Zimmermann, Bastian; Adam, Susanne; Wiendl, Maximilian; Krönke, Gerhard; Hamza, Iqbal; Heinemann, Stefan H.; Frey, Silke; Hueber, Axel J.; Ohlenschläger, Oliver; Imhof, DianaCytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
- ItemNew perspectives for viability studies with high-content analysis Raman spectroscopy (HCA-RS)(Berlin : Nature Publishing, 2019) Mondol, Abdullah S.; Töpfer, Natalie; Rüger, Jan; Neugebauer, Ute; Popp, Jürgen; Schie, Iwan W.Raman spectroscopy has been widely used in clinical and molecular biological studies, providing high chemical specificity without the necessity of labels and with little-to-no sample preparation. However, currently performed Raman-based studies of eukaryotic cells are still very laborious and time-consuming, resulting in a low number of sampled cells and questionable statistical validations. Furthermore, the approach requires a trained specialist to perform and analyze the experiments, rendering the method less attractive for most laboratories. In this work, we present a new high-content analysis Raman spectroscopy (HCA-RS) platform that overcomes the current challenges of conventional Raman spectroscopy implementations. HCA-RS allows sampling of a large number of cells under different physiological conditions without any user interaction. The performance of the approach is successfully demonstrated by the development of a Raman-based cell viability assay, i.e., the effect of doxorubicin concentration on monocytic THP-1 cells. A statistical model, principal component analysis combined with support vector machine (PCA-SVM), was found to successfully predict the percentage of viable cells in a mixed population and is in good agreement to results obtained by a standard cell viability assay. This study demonstrates the potential of Raman spectroscopy as a standard high-throughput tool for clinical and biological applications.
- ItemSingle cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, UteHepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.
- ItemCorrelation of crystal violet biofilm test results of Staphylococcus aureus clinical isolates with Raman spectroscopic read-out(Chichester [u.a.] : Wiley, 2021) Ebert, Christina; Tuchscherr, Lorena; Unger, Nancy; Pöllath, Christine; Gladigau, Frederike; Popp, Jürgen; Löffler, Bettina; Neugebauer, UteBiofilm-related infections occur quite frequently in hospital settings and require rapid diagnostic identification as they are recalcitrant to antibiotic therapy and make special treatment necessary. One of the standard microbiological in vitro tests is the crystal violet test. It indirectly determines the amount of biofilm by measuring the optical density (OD) of the crystal violet-stained biofilm matrix and cells. However, this test is quite time-consuming, as it requires bacterial cultivation up to several days. In this study, we correlate fast Raman spectroscopic read-out of clinical Staphylococcus aureus isolates from 47 patients with different disease background with their biofilm-forming characteristics. Included were low (OD < 10), medium (OD ≥ 10 and ≤20), and high (OD > 20) biofilm performers as determined by the crystal violet test. Raman spectroscopic analysis of the bacteria revealed most spectral differences between high and low biofilm performers in the fingerprint region between 750 and 1150 cm−1. Using partial least square regression (PLSR) analysis on the Raman spectra involving the three categories of biofilm formation, it was possible to obtain a slight linear correlation of the Raman spectra with the biofilm OD values. The PLSR loading coefficient highlighted spectral differences between high and low biofilm performers for Raman bands that represent nucleic acids, carbohydrates, and proteins. Our results point to a possible application of Raman spectroscopy as a fast prediction tool for biofilm formation of bacterial strains directly after isolation from the infected patient. This could help clinicians make timely and adapted therapeutic decision in future.
- ItemAutomated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands(Weinheim : Wiley-VCH-Verl., 2020) Götz, Theresa; Dahms, Marcel; Kirchhoff, Johanna; Beleites, Claudia; Glaser, Uwe; Bohnert, Jürgen A.; Pletz, Mathias W.; Popp, Jürgen; Schlattmann, Peter; Neugebauer, UteA Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemLabel-Free Characterization of Macrophage Polarization Using Raman Spectroscopy(Basel : Molecular Diversity Preservation International (MDPI), 2023) Naumann, Max; Arend, Natalie; Guliev, Rustam R.; Kretzer, Christian; Rubio, Ignacio; Werz, Oliver; Neugebauer, UteMacrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes.