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29 Publikationen filtern
A comparative study of robustness to noise and interpretability in U-Net-based denoising of Raman spectra
ErscheinungsjahrStatusPrüfung ausstehendErschienen in:Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
A. Mokari, S. Eiserloh, O. Ryabchykov, U. Neugebauer, T. Bocklitz
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.
ErscheinungsjahrErschienen in:The analyst: the analytical journal of the Royal Society of Chemistry
S. Guo, A. Ramoji, A. Pistiki, H. Yilmaz, U. Glaser, D. Vasquez-Pinzon, I. Schie, U. Neugebauer, A. Silge, J. Popp, T. Bocklitz
Raman Spectroscopy Can Identify Acute and Persistent Biochemical Changes in Leukocytes From Patients With COVID-19 and Non-COVID-19-Associated Sepsis
ErscheinungsjahrStatusPrüfung ausstehendErschienen in:Biotechnology journal : systems & synthetic biology, nanobiotech, medicine
A. Ramoji, P. Baumbach, O. Ryabchykov, A. Pistiki, J. Rueger, D. Pinzon, A. Silge, S. Deinhardt-Emmer, I. Schie, K. Weber, C. Neu, U. Neugebauer, M. Kiehntopf, T. Bocklitz, J. Popp, S. Coldewey
Sepsis remains a major clinical challenge, often resulting in long-term physiological and immunological disturbances. This study employed high-throughput single-cell Raman spectroscopy to analyze the biochemical profiles of peripheral blood leukocytes from patients with non-COVID-19 and COVID-19-associated sepsis. Leukocytes were assessed at multiple timepoints, including the acute phase (Days 3 and 7 after sepsis onset) and late recovery phase (6 and 12 months after sepsis onset). Raman spectroscopic profiles of leukocytes showed clear separation between healthy controls and sepsis patients during the acute phase with high balanced accuracy (BAcc: 95%–98%). Spectral differences between acute and recovery phases (BAcc: 84%–97%) and between recovery-phase leukocytes and those from healthy controls (BAcc: 81%–90%) were also observed, indicating long-lasting molecular alterations. Furthermore, distinct profiles were identified between non-COVID-19 and COVID-19-associated sepsis during the acute phase (BAcc: 65%–71%) and in the late-recovery phase (BAcc: 71%–83%). These findings demonstrate that Raman spectroscopy enables label-free, high-throughput profiling of leukocyte biochemistry across the sepsis trajectory. This suggests that Raman spectroscopy is a promising tool for high-throughput screening, offering insights into the biomolecular changes in sepsis and providing a diagnostic platform to differentiate between sepsis etiologies, a significant advancement in the field of sepsis diagnostics.
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.