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26 Publikationen filtern
Molecular Insights into the Heme-Binding Potential of Plant NCR247-Derived Peptides
ErscheinungsjahrErschienen in:ChemBioChem : a European journal of chemical biology
S. Vaidya, D. Rathod, A. Ramoji, U. Neugebauer, D. Imhof
Heme is involved in many critical processes in pathogenic bacteria as iron acquisition by these microorganisms is achieved by either direct uptake of heme or use of heme-binding proteins called hemophores. Exploring the underlying mechanisms on a molecular level can open new avenues in understanding the host-pathogen interactions. Any imbalance of the heme concentration has a direct impact on the bacterial growth and survival. Thus, heme-regulated proteins that are involved in heme homeostasis poise to be promising targets for research. Similarly, naturally occurring compounds, including cysteine-rich peptides from either plant secondary metabolites or venom toxins from vertebrates and invertebrates, have been studied for their therapeutic potential. NCR247 is such a cysteine-rich peptide, known to be crucial for nitrogenase activity in M. truncatula and its symbiotic relation with S. meliloti. NCR247-derived peptides were suggested to serve as high-affinity heme-binding molecules with remarkable heme-capturing properties. A comprehensive biochemical and computational analysis of NCR247-derived peptides, however, redefines their heme-binding capacity and consequently their potential therapeutic role.
Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis
ErscheinungsjahrErschienen in:Scientific Reports
S. Mandal, R. Motganhalli Ravikumar, A. Tannert, A. Urbanek, R. Guliev, M. Naumann, S. Coldewey, U. Dahmen, L. Carvalho, L. Bastião Silva, U. Neugebauer
Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. The search for a universal decalcification method that is suitable for a range of biophotonic analyses remains an ongoing challenge. In this study, we systematically assessed five standard bone decalcification protocols, encompassing strong mineralic acids (3% and 5% nitric acid), a commercially available formulation of hydrochloric and formic acid), as well as weak organic acids (5% trichloroacetic acid and 8% formic acid), and a chelating agent (25% ethylenediamine-tetraacetic acid) with varying decalcification durations, using mouse long bones as our experimental model. Our imaging analysis panel included classical histological staining (Hematoxylin and Eosin, H&E), immunofluorescence staining, and label-free Raman microspectroscopic imaging. We used cryosections instead of paraffin sections since paraffin interferes with tissue Raman signals. This approach is not as commonly used as it is more prone to handling artifacts, but is the preferred method for subsequent Raman analysis. Decalcification efficacy was evaluated based on various qualitative and some quantitative imaging parameters by 2–3 independent observers. Our systematic approach revealed that the chelating agent, when used for 24 h, optimally preserved bone features and, thus, would be the ideal decalcifying agent for comprehensive subsequent analysis. However, the choice of decalcifier and the ideal decalcification duration may vary depending on the type and thickness of bone, necessitating tailored adjustments to meet specific experimental requirements.
Characterizing Metabolic Shifts in Septic Murine Kidney Tissue Using 2P-FLIM for Early Sepsis Detection
ErscheinungsjahrErschienen in:Bioengineering
S. Greiner, M. Ebrahimi, M. Rodewald, A. Urbanek, T. Meyer-Zedler, M. Schmitt, U. Neugebauer, J. Popp
In this study, thin mouse kidney sections from healthy mice and those infected leading to acute and chronic sepsis were examined with two-photon excited fluorescence lifetime imaging (2P-FLIM) using the endogenous fluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The results presented show that this approach is a powerful tool for investigating cell metabolism in thin tissue sections. An adapted measurement routine was established for these samples by performing a spectral scan, identifying a combination of two excitation wavelengths and two detection ranges suitable for detailed scan images of NADH and FAD. Selected positions in thin slices of the renal cortex of nine mice (three healthy, three with chronic sepsis, and three with acute sepsis) were studied using 2P-FLIM. In addition, overview images were obtained using two-photon excited fluorescence (2PEF) intensity. This study shows that healthy kidney slices differ considerably from those with acute sepsis with regard to their fluorescence lifetime signatures. The latter shows a difference in metabolism between the inner and outer cortex, indicating that outer cortical tubular cells switch their metabolism from oxidative phosphorylation to glycolysis in kidneys from mice with acute sepsis and back in later stages, as seen for mice with chronic infections. These findings suggest that 2P-FLIM could serve as a powerful tool for early-stage sepsis diagnosis and monitoring metabolic recovery during treatment.
ErscheinungsjahrStatusPrüfung ausstehendErschienen in:Analyst
S. Guo, A. Ramoji, A. Pistiki, H. Yilmaz, U. Glaser, D. Vasquez-Pinzon, I. Schie, U. Neugebauer, A. Silge, J. Popp, T. Bocklitz
In-depth structure-function profiling of the complex formation between clotting factor VIII and heme
ErscheinungsjahrErschienen in:Thrombosis research : an international journal on vascular obstruction, hemorrhage and hemostasis
M. Hopp, D. Ugurlar, B. Pezeshkpoor, A. Biswas, A. Ramoji, U. Neugebauer, J. Oldenburg, D. Imhof
Background and aims: Blood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. Methods: We herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. Results: FVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. Conclusion: Our study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.
Findaureus: An open-source application for locating Staphylococcus aureus in fluorescence-labelled infected bone tissue slices
ErscheinungsjahrErschienen in:PLoS ONE
S. Mandal, A. Tannert, B. Löffler, U. Neugebauer, L. Silva
Staphylococcus aureus (S. aureus) is a facultative pathogenic bacterium that can cause infections in various tissue types in humans. Fluorescence imaging techniques have been employed to visualize the bacteria in ex-vivo samples mostly in research, aiding in the understanding of the etiology of the pathogen. However, the multispectral images generated from fluorescence microscopes are complex, making it difficult to locate bacteria across image files, especially in consecutive planes with different imaging depths. To address this issue, we present Findaureus, an open-source application specifically designed to locate and extract critical information about bacteria, especially S. aureus. Due to the limited availability of datasets, we tested the application on a dataset comprising fluorescence-labelled infected mouse bone tissue images, achieving an accuracy of 95%. We compared Findaureus with other state-of-the-art image analysis tools and found that it performed better, given its specificity toward bacteria localization. The proposed approach has the potential to aid in medical research of bone infections and can be extended to other tissue and bacteria types in the future.
Charakterisierung der Hämabbau-induzierten Mikrozirkulationsstörung und Neuroinflammation im Mausmodell
Erscheinungsjahr
I. Knüpfer
Die Mortalität von Subarachnoidalblutungen (SAB) beträgt rund 40% und wird maßgeblich durch das Auftreten sekundärer Komplikationen wie dem zerebralen Vasospasmus bestimmt. Als wichtiger Faktor in der Ätiologie des selbigen werden oxidative Abbauprodukte des Hämoglobins, sogenannte Hämabbauprodukte (HDPs) gesehen, für die im Tiermodell eine gefäßverengende Wirkung nachgewiesen werden konnte. Das erste Teilprojekt beschäftigte sich mit der Vasoaktivität der HDPs in Abhängigkeit von der NO-Verfügbarkeit. Mit Hilfe eines in-vitro-Modells sollte der Thromboxan-Agonisten U46619 eine NO-unabhängige Vorspannung von zerebralen Arteriolen von Mäusen bewirken. Hierdurch konnte nachgewiesen werden, dass die HDPs unabhängig von der NO-Verfügbarkeit eine gefäßverengende Wirkung haben. Inwiefern HDPs eine Entzündungsreaktion auslösen können, war die zentrale Frage des zweiten Teilprojektes. Im Falle einer Läsion im Gehrin proliferieren Mikrogliazellen und migrieren in Richtung des Läsionsherds. An immunhistochemisch gefärbten Hirnschnitten von Mäusen, sollte detektiert werden, ob und inwiefern sich die Morphologie und Lokalisation von Mikrogliazellen nach experimenteller SAB oder HDP-Applikation ändern. Dabei ergaben sich zwischen allen Behandlungen signifikante Unterschiede bezüglich der Verzweigung der Mikroglia-Zellen, jedoch nicht hinsichtlich ihrer Lokalisation. Im dritten Teilprojekt erfolgten funktionelle Messungen der kortikalen und pialen Mikrozirkulation mittels Laser-Speckle-Contrast-Imaging. Diese nicht-invasive Bildgebung ermöglicht die Quantifizierung des Blutflusses an der Hirnoberfläche in Echt-Zeit. Dabei erfolgte die Messung bei Kontrolltieren und behandelten Mäusen (HDP-Injektion) in räumlicher (Hemisphären) und zeitlicher Abhängigkeit (Zeitpunkt nach Intervention). Hierbei zeigten sich signifikante Unterschiede bezüglich der Lokalisation sowie des Messzeitpunktes, jedoch nicht bezüglich der Interventionsgruppe.
Application of biophotonics in bone infection research
Erscheinungsjahr
S. Mandal
This thesis addresses the complex challenge of bone infections, a critical global health issue that is difficult to treat effectively. Utilizing advanced techniques, including fluorescence microscopy and Raman spectroscopy, this research provides insights into the molecular mechanisms of these infections. A validated decalcification protocol was developed to maintain tissue integrity, allowing for a comprehensive analysis across multiple imaging methods. The "Introduction" provides context on bone infections, detailing the challenges in preclinical research, including tissue decalcification and imaging, and highlights fluorescence microscopy and Raman spectroscopy as key tools for molecular investigation. In "Results and Discussion," various decalcification methods are compared, identifying EDTA-based protocols as the most effective for preserving tissue quality and compatibility with biophotonic analyses. This section also reports findings from fluorescence imaging and Raman spectroscopy of infected bone tissues, focusing on bacterial distribution and tissue interactions in a hematogenous osteomyelitis mouse model, with new insights into pelvic bone infections that inform future therapeutic approaches. The thesis also introduces Findaureus, a user-friendly tool for analyzing multispectral fluorescence images. Designed without AI due to data constraints, Findaureus achieves high accuracy (95%) in bacterial localization, facilitating efficient interpretation of complex images. In "Conclusion and Outlooks," the thesis summarizes its contributions—highlighting the decalcification protocol, imaging advancements, and the Findaureus application. It reflects on the limitations and potential for future work, underscoring the impact of these findings on patient care and bone infection management. An "Appendix" includes data, supporting publications, and records of research dissemination activities that enriched this work through collaboration.
Confocal microscopy is well known for its capability to produce 3D images with increased optical resolution and contrast in living multicellular thick samples. Concurrently, it can be used as a powerful spectroscopic tool, facilitating fluorescence methods such as fluorescence correlation spectroscopy (FCS), fluorescence-lifetime imaging microscopy (FLIM), and single-molecule förster resonance energy transfer (FRET). However, the optical resolution attained is limited by the diffraction limit of light. On the other hand, stimulated emission depletion (STED) microscopy has become a powerful tool that provides a superior spatial resolution compared to a confocal system while retaining all spectroscopic capabilities. However, the powers of STED lasers far exceed those used in conventional laser scanning microscopy, which can damage live samples and prevent the possibility of long-term imaging. The entire doctoral thesis comprises of three projects. The first part of the thesis work focuses on optimizing long-term live-cell imaging techniques for confocal and STED imaging. The work highlights a comparative study of long-term imaging possibilities by new Nile red-derivative dyes, one of which is an exchangeable membrane dye that only temporarily resides in the membrane. The second part of this thesis work investigates the different fluorescence parameters of photoconverted species of commonly used fluorophores in confocal and STED microscopy. The work explores into the extent of photoconversion of fluorophores and also highlights ways to circumvent the effect. The last part of the thesis focuses on the development of a custom-built lightsheet and point confocal setup. The lightsheet module is gentle in terms of light dose and also efficient is capturing live-specimen dynamics with good spatio-temporal resolution. The confocal beam path enables spectroscopic measurements and it can be easily evolved into a super-resolution STED microscopy beam path.
Quantification of Polystyrene Uptake by Different Cell Lines Using Fluorescence Microscopy and Label-Free Visualization of Intracellular Polystyrene Particles by Raman Microspectroscopic Imaging
ErscheinungsjahrErschienen in:Cells: open access journal
A. Roth, A. Tannert, N. Ziller, S. Eiserloh, B. Göhrig, R. Guliev, M. Gonzalez Vazquez, M. Naumann, A. Mosig, S. Stengel, A. Heutelbeck, U. Neugebauer
Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells.
Application of raman spectroscopy for studying bacteria of the family enterobacteriaceae
Erscheinungsjahr
A. Nakar
This thesis investigated using Raman spectroscopy for identifying Enterobacteriaceae and their antibiotic resistances. The study aimed to develop a rapid, accurate diagnostic tool, as current methods are costly and inefficient, leading to increased antibiotic resistance due to inappropriate antibiotic use. The research involved collecting a dataset of Enterobacteriaceae, encompassing 8 genera. Measurements were taken using Single-Cell Raman Microspectroscopy (SC-RMS) and Fiber Probe-Based Raman Spectroscopy (FP-RS). Machine learning models were trained to classify the bacteria based on spectral data. SC-RMS showed limited success (47% accuracy), while FP-RS achieved 100% accuracy on the training dataset and 94% on a validation dataset, demonstrating its potential in differentiating Enterobacteriaceae. Subsequently, Raman spectra of 24 clinical isolates were analyzed using UV-Resonance Raman Spectroscopy (UVRRS). The goal was to discriminate between E. coli, K. pneumoniae, and K. oxytoca. While SC-RMS had limited effectiveness, UVRRS achieved high accuracy (92% at the genus level and 90% at the species level), marking the first successful differentiation of K. oxytoca using Raman spectroscopy. The study also explored differentiating antibiotic-resistant and sensitive Enterobacteriaceae. Both SC-RMS and UVRRS correctly classified 70-75% of the isolates, with UVRRS demonstrating a 90% sensitivity for resistant strains. This differentiation may be related to a higher DNA/Protein ratio in resistant bacteria. Overall, the findings enhance the application of Raman spectroscopy in microbiology, particularly for Enterobacteriaceae. The research supports its use in identifying pathogens, determining their genus and species, and assessing antibiotic resistance. The study contributes significantly to the development of Raman-based microbial diagnostics, promising to improve infectious disease diagnostics, patient care, and the fight against antimicrobial resistance.