MetaboLights

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of metabolites was based on fragmentation spectra, retention time and mass compared to authentic standards as previously outlined by the metabolomics standards initiative (MSI)[1]. Metabolites that matched an authentic standard with or without a fragmentation spectrum were classified as identified (MSI level 1). Metabolites which did not match to any authentic standards but had spectral similarities with spectral libraries https://www.genome.jp/kegg/pathway.html, were classified as putatively annotated and analysed further based on fragmentation spectra if available. Ref:[1] Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, Fan TW, Fiehn O, Goodacre R, Griffin JL, Hankemeier T, Hardy N, Harnly J, Higashi R, Kopka J, Lane AN, Lindon JC, Marriott P, Nicholls AW, Reily MD, Thaden JJ, Viant MR. Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics. 2007 Sep;3(3):211-221. doi:10.1007/s11306-007-0082-2. PMID:24039616.MetaboLightsPublicLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - negative - hilicSamples were analyzed via Hydrophilic interaction liquid chromatography (HILIC) on a Dionex UltiMate 3000 RSLC system (Thermo Fisher Scientific, Hemel Hempstead, UK) using a ZIC-pHILIC column (150 mm × 4.6 mm, 5 μm column, Merck Sequant). The column was maintained at 25°C and samples were eluted over 26 min at a flow rate of 0.3 mL/ min with a linear gradient over 15 min from an initial ratio of 80% acetonitrile (B) and 20% 20 mM ammonium carbonate in water (A) to 20% B and 80% A, followed by 95% A and 5% B for 2 min followed by re-equilibration at 80% B and 20% A for 9 min. The injection volume was 10 μL and samples were maintained at 5°C prior to injection.Investigation of the threonine metabolism of Echinococcus multilocularis: EmTDH as a potential drug target in alveolar echinococcosis.Clement RegnaultUniversity of GlasgowTissueCulture SupernatantmixtureFluidsample preparation blankpure substancemass spectrometry assayMedium samples of CM and assay medium in which metacestode vesicles were incubated (VM), metacestode vesicle fluid (VF) and metacestode vesicle tissue (VT) were extracted in a buffer consisting of HPLC grade chloroform:methanol:water (1:3:1 ratio[CR1] ) and stored at -80°C until further use. [CR1] Echinococcus multilocularisreference compoundGrowth Mediumsample preparation blankhttps://www.ebi.ac.uk/metabolights/MTBLS10738Anissa Bartezko. Institute of Parasitology, Vetsuisse Faculty, University of Bern, Switzerland.Martin Lochner. Institute of Biochemistry and Molecular Medicine, University of Bern, Switzerland.Marc Kaethner. Institute of Parasitology, Vetsuisse Faculty, University of Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland.Pascal Zumstein. Institute of Parasitology, Vetsuisse Faculty, University of Bern, Switzerland.Stefan Schürch. Department of Chemistry and Biochemistry, University of Bern, Switzerland.Matías Preza. Institute of Parasitology, Vetsuisse Faculty, University of Bern, Switzerland.Philipp Grossenbacher. Institute of Biochemistry and Molecular Medicine, University of Bern, Switzerland.Clement Regnault. Integrated Protein Analysis - Mass Spectrometry unit, MVLS Shared Research Facilities, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom. clement.regnault@glasgow.ac.uk.Britta Lundström-Stadelmann. Institute of Parasitology, Vetsuisse Faculty, University of Bern, Switzerland; Multidisciplinary Center for Infectious Diseases, University of Bern, Bern, Switzerland. britta.lundstroem@unibe.ch.Daniel Villalobos Ramírez. Department of Bioinformatics, University of Würzburg, Germany.For each sample subset (medium, VF and VT), LC-MS raw data was processed with IDEOM (Creek et al., 2012) which uses the XCMS (Smith et al., 2006) and mzMatch software (Scheltema et al., 2011) in the R environment. A list of putatively annotated metabolites was generated and the abundances of all [13C]- isotopologues for these were obtained using the software mzMatch-ISO (Chokkathukalam et al., 2013).Spent mediumReplicateLabellingclement.regnault@glasgow.ac.uk3 mL of two- to three-months-old E. multilocularis metacestode vesicles were cultured in 3 mL DMEM without threonine or glucose, supplemented with 0.2% FBS, 110 mg/ L sodium pyruvate, 4.5 g/ L glucose, 4 mM L-glutamine and 5 mM unlabeled threonine or 5 mM [U-13C]-L-threonine, respectively, at 37 °C for 24 h under humid, microaerobic atmosphere. Control medium (CM) without metacestode vesicles was incubated under the same conditions. Each condition was set up in four biological replicates.MetabolomicsTargeted screeningL-threonine 3-dehydrogenasethreonineDisulfiramsanguinarineTargeted screeningL-threonine 3-dehydrogenasethreonineDisulfiramsanguinarineFor the MS analysis, a Thermo Orbitrap QExactive (Thermo Fisher Scientific) was operated in polarity switching mode and the MS settings were Resolution 70,000, AGC 1e6, m/ z range 70–1050, sheath gas 40, auxiliary gas 5, sweep gas 1, probe temperature 150°C and capillary temperature 320°C. The samples were analyzed in positive mode ionization (source voltage +3.8 kV, S-Lens RF Level 30.00, SLens Voltage 25.00 V, Skimmer Voltage 15.00 V, Inject Flatopole Offset 8.00 V, Bent Flatapole DC 6.00 V) and negative mode ionization (source voltage -3.8 kV). The calibration mass range was extended to cover small metabolites by inclusion of low-mass calibrants with the standard Thermo calmix masses (below m/ z 138), butylamine (C4H11N1) for positive ion electrospray ionisation (PIESI) mode (m/z 74.096426) and COF3 for negative ion electrospray ionisation (NIESI) mode (m/z 84.9906726).Malatealpha-ketoglutarate2-HydroxyglutarateSuccinateCitrateL-AspartatefalseInvestigation of the threonine metabolism of Echinococcus multilocularis: EmTDH as a potential drug target in alveolar echinococcosisAlveolar echinococcosis is a severe zoonotic disease caused by the fox tapeworm E. multilocularis. New treatment options are urgently needed. Whole-organism-based screening allows the discovery of potential new anti-echinococcal compounds, but suffers from low- to medium-throughput. An alternative approach to identify novel compounds is target-based screening, which requires in-depth knowledge of the basic biology of the pathogen of interest. We recently showed that E. multilocularis metacestode vesicles scavenge large amounts of L-threonine from the culture medium that was neither stored nor overused for protein synthesis. This motivated us to study the effect of L-threonine on the parasite and how it is metabolized. We showed that L-threonine significantly increased growth of E. multilocularis metacestode vesicles and the formation of novel metacestode vesicles from germinal layer cell cultures. The non-proteinogenic threonine analog 3-hydroxynorvaline had the contrary effect. We traced [U-13C]-L-threonine and metabolites in metacestode vesicles and culture medium and detected [13C]-labeling in aminoacetone and glycine, indicating that L-threonine was metabolized by threonine dehydrogenase (TDH) and 2-amino-3-ketobutyrate coenzyme A ligase (KBL), but not by threonine deaminase. We further detected [13C2]-glutathione, which suggested that E. multilocularis metacestode vesicles synthesize glutathione via L-threonine-derived glycine in vitro. EmTDH mediated threonine metabolism in E. multilocularis metacestode vesicles was further confirmed by quantitative real-time PCR which showed high expression of emtdh. EmTDH was enzymatically active in metacestode vesicle extracts and thus we established an enzymatic assay to evaluate the activity of compounds against recombinantly expressed EmTDH. We tested 11 compounds (disulfiram, myricetin, seven quinazoline carboxamides, quercetin and sanguinarine) and of these, disulfiram, myricetin and sanguinarine were highly active against recEmTDH. Upon testing of these three compounds against E. multilocularis metacestode vesicles and GL cells, sanguinarine showed high in vitro activity and was thus further characterized by calculating IC50 values against metacestode vesicles, GL cells, as well as mammalian cells. Our results suggested that sanguinarine should be further followed for its potential activity against E. multilocularis in mouse models of AE. Furthermore, the here-established TDH assay could serve as high-throughput target-based discovery platform for novel anti-echinococcal compounds in the future.2026-09-142024-07-25MTBLS10738MTBLC16857MTBLC17906MTBLC15428MTBLC16015MTBLC16643MTBLC30769MTBLC30915MTBLC17084MTBLC15741MTBLC6650MTBLC17053CHEBI:16857CHEBI:17906CHEBI:15428CHEBI:16015CHEBI:16643CHEBI:30769CHEBI:30915CHEBI:17084CHEBI:15741CHEBI:6650CHEBI:17053