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End date: 2024 × Start date: 2020 × Has files: Yes × WGL Institute: ISAS ×

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    Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry
    (Columbus, Ohio : American Chemical Society, 2023) Kale, Dipali; Kikul, Frauke; Phapale, Prasad; Beedgen, Lars; Thiel, Christian; Brügger, Britta
    Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.
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    Surface Plasmon Resonance Sensitivity Enhancement Based on Protonated Polyaniline Films Doped by Aluminum Nitrate
    (Basel : MDPI, 2022) Al-Bataineh, Qais M.; Shpacovitch, Victoria; Sadiq, Diyar; Telfah, Ahmad; Hergenröder, Roland
    Complex composite films based on polyaniline (PANI) doped hydrochloric acid (HCl) incorporated with aluminum nitrate (Al(NO3)3) on Au-layer were designed and synthesized as a surface plasmon resonance (SPR) sensing device. The physicochemical properties of (PANI-HCl)/Al(NO3)3 complex composite films were studied for various Al(NO3)3 concentrations (0, 2, 4, 8, 16, and 32 wt.%). The refractive index of the (PANI-HCl)/Al(NO3)3 complex composite films increased continuously as Al(NO3)3 concentrations increased. The electrical conductivity values increased from 5.10 µS/cm to 10.00 µS/cm as Al(NO3)3 concentration increased to 32 wt.%. The sensitivity of the SPR sensing device was investigated using a theoretical approach and experimental measurements. The theoretical system of SPR measurement confirmed that increasing Al(NO3)3 in (PANI-HCl)/Al(NO3)3 complex composite films enhanced the sensitivity from about 114.5 [Deg/RIU] for Au-layer to 159.0 [Deg/RIU] for Au-((PANI-HCl)/Al(NO3)3 (32 wt.%)). In addition, the signal-to-noise ratio for Au-layer was 3.95, which increased after coating by (PANI-HCl)/Al(NO3)3 (32 wt.%) complex composite layer to 8.82. Finally, we conclude that coating Au-layer by (PANI-HCl)/Al(NO3)3 complex composite films enhances the sensitivity of the SPR sensing device.
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    IR-MALDI Mass Spectrometry Imaging with Plasma Post-Ionization of Nonpolar Metabolites
    (Columbus, Ohio : American Chemical Society, 2022) Schneemann, Julian; Schäfer, Karl-Christian; Spengler, Bernhard; Heiles, Sven
    Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus.
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    During early stages of cancer, neutrophils initiate anti-tumor immune responses in tumor-draining lymph nodes
    (Maryland Heights, MO : Cell Press, 2022) Pylaeva, Ekaterina; Korschunow, Georg; Spyra, Ilona; Bordbari, Sharareh; Siakaeva, Elena; Ozel, Irem; Domnich, Maksim; Squire, Anthony; Hasenberg, Anja; Thangavelu, Kruthika; Hussain, Timon; Goetz, Moritz; Lang, Karl S; Gunzer, Matthias; Hansen, Wiebke; Buer, Jan; Bankfalvi, Agnes; Lang, Stephan; Jablonska, Jadwiga
    Tumor-draining lymph nodes (LNs) play a crucial role during cancer spread and in initiation of anti-cancer adaptive immunity. Neutrophils form a substantial population of cells in LNs with poorly understood functions. Here, we demonstrate that, during head and neck cancer (HNC) progression, tumor-associated neutrophils transmigrate to LNs and shape anti-tumor responses in a stage-dependent manner. In metastasis-free stages (N0), neutrophils develop an antigen-presenting phenotype (HLA-DR+CD80+CD86+ICAM1+PD-L1-) and stimulate T cells (CD27+Ki67highPD-1-). LN metastases release GM-CSF and via STAT3 trigger development of PD-L1+ immunosuppressive neutrophils, which repress T cell responses. The accumulation of neutrophils in T cell-rich zones of LNs in N0 constitutes a positive predictor for 5-year survival, while increased numbers of neutrophils in LNs of N1-3 stages predict poor prognosis in HNC. These results suggest a dual role of neutrophils as essential regulators of anti-cancer immunity in LNs and argue for approaches fostering immunostimulatory activity of these cells during cancer therapy.
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    Azapeptide activity-based probes for the SARS-CoV-2 main protease enable visualization of inhibition in infected cells
    (Cambridge : RSC, 2023) Vanhoutte, Roeland; Barniol-Xicota, Marta; Chiu, Winston; Vangeel, Laura; Jochmans, Dirk; De Jonghe, Steven; Zidane, Hadeer; Barr, Haim M.; London, Nir; Neyts, Johan; Verhelst, Steven H. L.
    The COVID-19 pandemic has revealed the vulnerability of the modern, global society. With expected waves of future infections by SARS-CoV-2, treatment options for infected individuals will be crucial in order to decrease mortality and hospitalizations. The SARS-CoV-2 main protease is a validated drug target, for which the first inhibitor has been approved for use in patients. To facilitate future work on this drug target, we designed a solid-phase synthesis route towards azapeptide activity-based probes that are capped with a cysteine-reactive electrophile for covalent modification of the active site of Mpro. This design led to the most potent ABP for Mpro and one of the most potent inhibitors reported thus far. We demonstrate that this ABP can be used to visualize Mpro activity and target engagement by drugs in infected cells.
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    Metabolic Profiling of Thymic Epithelial Tumors Hints to a Strong Warburg Effect, Glutaminolysis and Precarious Redox Homeostasis as Potential Therapeutic Targets
    (Basel : MDPI, 2022) Alwahsh, Mohammad; Knitsch, Robert; Marchan, Rosemarie; Lambert, Jörg; Hoerner, Christian; Zhang, Xiaonan; Schalke, Berthold; Lee, De-Hyung; Bulut, Elena; Graeter, Thomas; Ott, German; Kurz, Katrin S.; Preissler, Gerhard; Schölch, Sebastian; Farhat, Joviana; Yao, Zhihan; Sticht, Carsten; Ströbel, Philipp; Hergenröder, Roland; Marx, Alexander; Belharazem, Djeda
    Thymomas and thymic carcinomas (TC) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. Metabolic profiles of snap-frozen thymomas (WHO types A, AB, B1, B2, B3, n = 12) and TCs (n = 3) were determined by high resolution magic angle spinning 1H nuclear magnetic resonance (HRMAS 1H-NMR) spectroscopy. Metabolite-based prediction of active KEGG metabolic pathways was achieved with MetPA. In relation to metabolite-based metabolic pathways, gene expression signatures of TETs (n = 115) were investigated in the public “The Cancer Genome Atlas” (TCGA) dataset using gene set enrichment analysis. Overall, thirty-seven metabolites were quantified in TETs, including acetylcholine that was not previously detected in other nonendocrine cancers. Metabolite-based cluster analysis distinguished clinically indolent (A, AB, B1) and aggressive TETs (B2, B3, TCs). Using MetPA, six KEGG metabolic pathways were predicted to be activated, including proline/arginine, glycolysis and glutathione pathways. The activated pathways as predicted by metabolite-profiling were generally enriched transcriptionally in the independent TCGA dataset. Shared high lactic acid and glutamine levels, together with associated gene expression signatures suggested a strong “Warburg effect”, glutaminolysis and redox homeostasis as potential vulnerabilities that need validation in a large, independent cohort of aggressive TETs. If confirmed, targeting metabolic pathways may eventually prove as adjunct therapeutic options in TETs, since the metabolic features identified here are known to confer resistance to cisplatin-based chemotherapy, kinase inhibitors and immune checkpoint blockers, i.e., currently used therapies for non-resectable TETs.
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    Identification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro
    (Heidelberg : Springer, 2022) Le-Trilling, Vu Thuy Khanh; Mennerich, Denise; Schuler, Corinna; Sakson, Roman; Lill, Julia K.; Kasarla, Siva Swapna; Kopczynski, Dominik; Loroch, Stefan; Flores-Martinez, Yulia; Katschinski, Benjamin; Wohlgemuth, Kerstin; Gunzer, Matthias; Meyer, Folker; Phapale, Prasad; Dittmer, Ulf; Sickmann, Albert; Trilling, Mirko
    Background: The SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Results: Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. Conclusions: In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology.
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    Inhibiting the glycerophosphodiesterase EDI3 in ER-HER2+ breast cancer cells resistant to HER2-targeted therapy reduces viability and tumour growth
    (Berlin, Heidelberg : Springer, 2023) Keller, Magdalena; Rohlf, Katharina; Glotzbach, Annika; Leonhardt, Gregor; Lüke, Simon; Derksen, Katharina; Demirci, Özlem; Göçener, Defne; AlWahsh, Mohammad; Lambert, Jörg; Lindskog, Cecilia; Schmidt, Marcus; Brenner, Walburgis; Baumann, Matthias; Zent, Eldar; Zischinsky, Mia-Lisa; Hellwig, Birte; Madjar, Katrin; Rahnenführer, Jörg; Overbeck, Nina; Reinders, Jörg; Cadenas, Cristina; Hengstler, Jan G.; Edlund, Karolina; Marchan, Rosemarie
    Background: Intrinsic or acquired resistance to HER2-targeted therapy is often a problem when small molecule tyrosine kinase inhibitors or antibodies are used to treat patients with HER2 positive breast cancer. Therefore, the identification of new targets and therapies for this patient group is warranted. Activated choline metabolism, characterized by elevated levels of choline-containing compounds, has been previously reported in breast cancer. The glycerophosphodiesterase EDI3 (GPCPD1), which hydrolyses glycerophosphocholine to choline and glycerol-3-phosphate, directly influences choline and phospholipid metabolism, and has been linked to cancer-relevant phenotypes in vitro. While the importance of choline metabolism has been addressed in breast cancer, the role of EDI3 in this cancer type has not been explored. Methods: EDI3 mRNA and protein expression in human breast cancer tissue were investigated using publicly-available Affymetrix gene expression microarray datasets (n = 540) and with immunohistochemistry on a tissue microarray (n = 265), respectively. A panel of breast cancer cell lines of different molecular subtypes were used to investigate expression and activity of EDI3 in vitro. To determine whether EDI3 expression is regulated by HER2 signalling, the effect of pharmacological inhibition and siRNA silencing of HER2, as well as the influence of inhibiting key components of signalling cascades downstream of HER2 were studied. Finally, the influence of silencing and pharmacologically inhibiting EDI3 on viability was investigated in vitro and on tumour growth in vivo. Results: In the present study, we show that EDI3 expression is highest in ER-HER2 + human breast tumours, and both expression and activity were also highest in ER-HER2 + breast cancer cell lines. Silencing HER2 using siRNA, as well as inhibiting HER2 signalling with lapatinib decreased EDI3 expression. Pathways downstream of PI3K/Akt/mTOR and GSK3β, and transcription factors, including HIF1α, CREB and STAT3 were identified as relevant in regulating EDI3 expression. Silencing EDI3 preferentially decreased cell viability in the ER-HER2 + cells. Furthermore, silencing or pharmacologically inhibiting EDI3 using dipyridamole in ER-HER2 + cells resistant to HER2-targeted therapy decreased cell viability in vitro and tumour growth in vivo. Conclusions: Our results indicate that EDI3 may be a potential novel therapeutic target in patients with HER2-targeted therapy-resistant ER-HER2 + breast cancer that should be further explored.
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    Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins
    (San Francisco, Calif. : Public Library of Science, 2022) Maqsood, Zahra; Clark, Joanne C.; Martin, Eleyna M.; Cheung, Yam Fung Hilaire; Morán, Luis A.; Watson, Sean E. T.; Pike, Jeremy A.; Di, Ying; Poulter, Natalie S.; Slater, Alexandre; Lange, Bodo M. H.; Nieswandt, Bernhard; Eble, Johannes A.; Tomlinson, Mike G.; Owen, Dylan M.; Stegner, David; Bridge, Lloyd J.; Wierling, Christoph; Watson, Steve P.
    The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.
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    The influence of Sb doping on the local structure and disorder in thermoelectric ZnO:Sb thin films
    (Lausanne : Elsevier, 2023) Ribeiro, Joana M.; Rodrigues, Frederico J.; Correia, Filipe C.; Pudza, Inga; Kuzmin, Alexei; Kalinko, Aleksandr; Welter, Edmund; Barradas, Nuno P.; Alves, Eduardo; LaGrow, Alec P.; Bondarchuk, Oleksandr; Welle, Alexander; Telfah, Ahmad; Tavares, Carlos J.
    Thermoelectric transparent ZnO:Sb thin films were deposited by magnetron sputtering, with Sb content varying between 2 and 14 at%. As evidenced by X-ray diffraction analysis, the films crystallize in the ZnO wurtzite structure for lower levels of Sb-doping, developing a degree of amorphization for higher levels of Sb-doping. Temperature-dependent (10–300 K) X-ray absorption spectroscopy studies of the produced thin films were performed at the Zn and Sb K-edges to shed light on the influence of Sb doping on the local atomic structure and disorder in the ZnO:Sb thin films. The analysis of the Zn K-edge EXAFS spectra by the reverse Monte Carlo method allowed to extract detailed and accurate structural information in terms of the radial and bond angle distribution functions. The obtained results suggest that the introduction of antimony to the ZnO matrix promotes static disorder, which leads to partial amorphization with very small crystallites (∼3 nm) for large (12–14 at%) Sb content. Rutherford backscattering spectrometry (RBS) experiments enabled the determination of the in-depth atomic composition profiles of the films. The film composition at the surfaces determined by X-ray photoelectron spectroscopy (XPS) matches that of the bulk determined by RBS, except for higher Sb-doping in ZnO films, where the concentration of oxygen determined by XPS is smaller near the surface, possibly due to the formation of oxygen vacancies that lead to an increase in electrical conductivity. Traces of Sb–Sb metal bonds were found by XPS for the sample with the highest level of Sb-doping. Time-of-flight secondary ion mass spectrometry obtained an Sb/Zn ratio that follows that of the film bulk determined by RBS, although Sb is not always homogeneous, with samples with smaller Sb content (2 and 4 at% of Sb) showing a larger Sb content closer to the film/substrate interface. From the optical transmittance and reflectance curves, it was determined that the films with the lower amount of Sb doping have larger optical band-gaps, in the range of 2.9–3.2 eV, while the partially amorphous films with higher Sb content have smaller band-gaps in the range of 1.6–2.1 eV. Albeit the short-range crystalline order (∼3 nm), the film with 12 at% of Sb has the highest absolute Seebeck coefficient (∼56 μV/K) and a corresponding thermoelectric power factor of ∼0.2 μW·K−2·m−1.