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- ItemIn-vivo Raman spectroscopy: from basics to applications(Bellingham, Wash. : SPIE, 2018) Cordero, Eliana; Latka, Ines; Matthäus, Christian; Schie, Iwan W.; Popp, JürgenFor more than two decades, Raman spectroscopy has found widespread use in biological and medical applications. The instrumentation and the statistical evaluation procedures have matured, enabling the lengthy transition from ex-vivo demonstration to in-vivo examinations. This transition goes hand-in-hand with many technological developments and tightly bound requirements for a successful implementation in a clinical environment, which are often difficult to assess for novice scientists in the field. This review outlines the required instrumentation and instrumentation parameters, designs, and developments of fiber optic probes for the in-vivo applications in a clinical setting. It aims at providing an overview of contemporary technology and clinical trials and attempts to identify future developments necessary to bring the emerging technology to the clinical end users. A comprehensive overview of in-vivo applications of fiber optic Raman probes to characterize different tissue and disease types is also given.
- ItemIn Vitro Selection of Specific DNA Aptamers Against the Anti-Coagulant Dabigatran Etexilate(Berlin : Nature Publishing, 2018) Aljohani, Maher M; Chinnappan, Raja; Eissa, Shimaa; Alsager, Omar A; Weber, Karina; Cialla-May, Dana; Popp, Jürgen; Zourob, MohammedDabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.Dabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.
- ItemNonresonant Raman spectroscopy of isolated human retina samples complying with laser safety regulations for in vivo measurements(Bellingham, Wash. : SPIE, 2019) Stiebing, Clara; Schie, Iwan W.; Knorr, Florian; Schmitt, Michael; Keijzer, Nanda; Kleemann, Robert; Jahn, Izabella J.; Jahn, Martin; Kiliaan, Amanda J.; Ginner, Laurin; Lichtenegger, Antonia; Drexler, Wolfgang; Leitgeb, Rainer A.; Popp, JürgenRetinal diseases, such as age-related macular degeneration, are leading causes of vision impairment, increasing in incidence worldwide due to an aging society. If diagnosed early, most cases could be prevented. In contrast to standard ophthalmic diagnostic tools, Raman spectroscopy can provide a comprehensive overview of the biochemical composition of the retina in a label-free manner. A proof of concept study of the applicability of nonresonant Raman spectroscopy for retinal investigations is presented. Raman imaging provides valuable insights into the molecular composition of an isolated ex vivo human retina sample by probing the entire molecular fingerprint, i.e., the lipid, protein, carotenoid, and nucleic acid content. The results are compared to morphological information obtained by optical coherence tomography of the sample. The challenges of in vivo Raman studies due to laser safety limitations and predefined optical parameters given by the eye itself are explored. An in-house built setup simulating the optical pathway in the human eye was developed and used to demonstrate that even under laser safety regulations and the above-mentioned optical restrictions, Raman spectra of isolated ex vivo human retinas can be recorded. The results strongly support that in vivo studies using nonresonant Raman spectroscopy are feasible and that these studies provide comprehensive molecular information of the human retina. © The Authors. Published by SPIE.
- 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.
- ItemRaman spectroscopy-based identification of toxoid vaccine products(Berlin : Nature Publishing, 2018) Silge, Anja; Bocklitz, Thomas W.; Becker, Bjoern; Matheis, Walter; Popp, Jürgen; Bekeredjian-Ding, IsabelleVaccines are complex biomedicines. Manufacturing is time consuming and requires a high level of quality control (QC) to guarantee consistent safety and potency. An increasing global demand has led to the need to reduce time and cost of manufacturing. The evolving concepts for QC and the upcoming threat of falsification of biomedicines define a new need for methods that allow the fast and reliable identification of vaccines. Raman spectroscopy is a non-destructive technology already established in QC of classical medicines. We hypothesized that Raman spectroscopy could be used for identification and differentiation of vaccine products. Raman maps obtained from air-dried samples of combination vaccines containing antigens from tetanus, diphtheria and pertussis (DTaP vaccines) were summarized to compile product-specific Raman signatures. Sources of technical variance were emphasized to evaluate the robustness and sensitivity in downstream data analysis. The data management approach corrects for spatial inhomogeneities in the dried sample while offering a proper representation of the original samples inherent chemical signature. Reproducibility of the identification was validated by a leave-one-replicate-out cross-validation. The results highlighted the high specificity and sensitivity of Raman measurements in identifying DTaP vaccine products. The results pave the way for further exploitation of the Raman technology for identification of vaccines in batch release and cases of suspected falsification.
- ItemBioactive secondary metabolites with multiple activities from a fungal endophyte(Oxford : Wiley-Blackwell, 2016) Bogner, Catherine W.; Kamdem, Ramsay S.T.; Sichtermann, Gisela; Matthäus, Christian; Hölscher, Dirk; Popp, Jürgen; Proksch, Peter; Grundler, Florian M.W.; Schouten, AlexanderIn order to replace particularly biohazardous nematocides, there is a strong drive to finding natural product-based alternatives with the aim of containing nematode pests in agriculture. The metabolites produced by the fungal endophyte Fusarium oxysporum 162 when cultivated on rice media were isolated and their structures elucidated. Eleven compounds were obtained, of which six were isolated from a Fusarium spp. for the first time. The three most potent nematode-antagonistic compounds, 4-hydroxybenzoic acid, indole-3-acetic acid (IAA) and gibepyrone D had LC50 values of 104, 117 and 134 μg ml−1, respectively, after 72 h. IAA is a well-known phytohormone that plays a role in triggering plant resistance, thus suggesting a dual activity, either directly, by killing or compromising nematodes, or indirectly, by inducing defence mechanisms against pathogens (nematodes) in plants. Such compounds may serve as important leads in the development of novel, environmental friendly, nematocides.
- ItemAutofluorescence lifetime augmented reality as a means for real-time robotic surgery guidance in human patients(Berlin : Nature Publishing, 2019) Gorpas, Dimitris; Phipps, Jennifer E.; Bec, Julien; Ma, Dinglong; Dochow, Sebastian; Yankelevich, Diego R.; Sorger, Jonathan; Popp, Jürgen; Bewley, Arnaud Fassett; Gandour-Edwards, Regina F.; Marcu, Laura; Farwell, D. GregoryDue to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols.
- ItemErarbeitung eines mobilen mikrosystemtechnischen Gesamtsystems für Anreicherung, Nachweis und Charakterisierung zirkulierender Tumorzellen, Teilvorhaben IPHT: Entwicklung eines chipbasierten elektrischen Nachweises zur parallelen Identifikation von Tumorspezifischen Markern : Abschlussbericht ; Laufzeit des Vorhabens: 01.01.2011 - 30.06.2014(Hannover : Technische Informationsbibliothek (TIB), 2014) Weber, Karina; Popp, JürgenDie Charakterisierung von zirkulierenden Tumorzellen stellt ein wichtiges diagnostisches Hilfsmittel für das Therapiemonitoring und die Therapieoptimierung bei Krebspatienten dar. Im Fokus des Verbundvorhabens „MiNa-CTC“ stand daher die Erforschung und Realisierung eines miniaturisierten, kostengünstigen, zuverlässigen, effizienten und mobilen Gesamtsystems für die Anreicherung, den Nachweis und die Charakterisierung zirkulierender Tumorzellen. Das Teilvorhaben des IPHT befasst sich dabei insbesondere mit der Erforschung einer chipbasierten Nachweismethode zur Detektion verschiedener Tumor-spezifischer Marker. Hierbei wurden insbesondere die Biomarker KRAS, PIK3CA und BRAF sowie im Hinblick auf eine mögliche Real-Time Amplifikation die Referenzgene RPL13A und GusBeta betrachtet. Im Ergebnis steht eine optisch auslesbare und miniaturisierte Chip-Plattform zum spezifischen Nachweis von Punktmutationen, die als Teillösung in ein Gesamtsystem integriert werden konnte. Das modular aufgebaute Gesamtsystem konnte erfolgreich an verschiedenen Probenmatrices validiert werden und stellt die Basis für ein zukünftig dezentral einsetzbares System zum Therapiemonitoring dar.