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he-S4-P2-D9.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/highpH/Blasche-S3_P5-E3_highpH.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox035.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox051.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox161.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox271.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev030.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/stdA_13_AA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev220.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/highpH/Blasche-S3_P5-E1_highpH.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox220.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev077.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/Blasche-S3_P3-H12_AA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev204.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox204.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/highpH/Blasche-S3_P5-G5_highpH.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/SCFA/Blasche-S4-P2-D4.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/community_positive_LCMS/dulox006_community.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox056.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/Blasche-S3_P3-A12_AA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev051.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox030.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/highpH/Blasche-S3_P5-F3_highpH.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/Blasche-S3_P3-D7_AA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev161.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev225.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/highpH/Blasche-S3_P5-H7_highpH.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox225.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/highpH/Blasche-S3_P5-A11_highpH.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox292.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/Blasche-S3_P3-D2_AA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/SCFA/std2_7_SCFA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/SCFA/Blasche-S4-P1-C11.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev292.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/SCFA/Blasche-S4-P1-D6.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/Blasche-S3_P3-A4_AA.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/negative_LCMS/isostev187.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/positive_LCMS/dulox187.d.zipftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480/FILES/RAW_FILES/AA/Blasche-S3_P3-G5_AA.d.zipprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11480<p><strong>Untargeted: </strong>Raw data files were converted to .mzML format using msconvert (ProteoWizard) with peakPicking and zeroSamples functions applied. The .mzML files were uploaded to R and XCMS used to perform peak picking, refinement, correspondence, and peak filling with the parameters listed in supplementary table. Statistical analysis was performed using MetaboAnalyst 6.0. Near-constant variables were filtered by interquartile range so 25&nbsp;% of features were removed and the data were then normalized by sum, log transformed and pareto scaled. Correlation analysis was performed using the PatternHunter tool to highlight features more significantly altered by combination treatment with both duloxetine and isosteviol than by either treatment alone. The correspondence of feature intensity to this pattern was determined using Pearson correlation. A significance threshold of correlation coefficient &gt; ± 0.8 and p-value &lt; 0.05 was employed. Metabolite identification was performed by comparison of accurate mass to theoretical mass with an error tolerance of ±10 ppm and where possible by matching of MS2 spectra to database MS2 spectra using the PEP Search tool.</p>MetaboLightsPublicLiquid Chromatography MS - negative - reverse phaseLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - positive - reverse phase<p><strong>Untargeted: </strong>Analysis was performed using an Agilent 1290 Infinity II LC system. Separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 2.1 × 100 mm, 1.8 μm; Agilent 858700-902) with a ZOBRAX Eclipse Plus guard column (C18, 2.1 × 5 mm, 1.8 μm; Agilent 821725-901) at 40ºC. The multisampler was kept at a temperature of 4 ºC. The injection volume was 1 μL and the flow rate was 0.4 mL/min. The mobile phases consisted of A: water + 0.1% formic acid + 5 mM ammonium formate; B: methanol + 0.1% formic acid + 5 mM ammonium formate. The 10 min gradient started with 5% solvent B, which was increased to 90 % by 5&nbsp;min and then further increased to 100 % by 7 min, before returning to 5&nbsp;% solvent B for a 3&nbsp;min re-equilibration.</p><p><br></p><p><strong>Amino acids: </strong>Analysis was performed using an Agilent 1290 Infinity II LC system. Amino acids were measured using a low pH HILIC method. 0.25 μl of sample was injected and analytes were separated on a Waters Acquity BEH Amide column (1.7 µm, 2.1 mm x 100 mm) maintained at 35ºC. The gradient used a constant flow rate of 0.9 ml/min and proceeded as follows: 0 min: 15% buffer A (1:1 acetonitrile:water, 10 mM ammonium formate, 0.176% formic acid) and 85% buffer B (95:5:5 acetonitrile:methanol:water, 10 mM ammonium formate, 0.176% formic acid), 0.7 min: 85% B, 2.55 min: 5% B, 2.9 min: 5% B, 2.91 min: 85% B, 3.5 min: stop time.&nbsp;</p><p><br></p><p><strong>Organic acids: </strong>Analysis was performed using an Agilent 1290 Infinity II LC system. Organic acids were measured using a high pH HILIC method. 2 μl of sample was injected and analytes were separated on a Waters Atlantis Premier BEH Z-HILIC (1.7 µm, 2.1 mm x 100 mm) column maintained at 35ºC. The gradient used a constant flow rate of 0.5 ml/min and proceeded as follows: 0 min: 15% buffer A (15mM ammonium bicarbonate in water, pH 9) and 85% buffer B (15mM ammonium bicarbonate in 9:1 acetonitrile:water, pH 9), 1 min: 85% B, 3 min: 25% B, 4 min: 25% B, 4.01 min: 85% B, 5&nbsp;min: stop time.&nbsp;</p><p><br></p><p><strong>SCFA: </strong>Analysis was performed using an Agilent 1290 Infinity II LC system. SCFA were measured using a porous graphitic carbon column. 1 μl of sample was injected and analytes were separated on a Thermo Scientific Hypercarb PGC column (3 µm, 50 mm × 2.1 mm) maintained at 40ºC. The gradient used a constant flow rate of 0.15 ml/min and proceeded as follows: 0 min: 100% buffer A (water, 0.1% formic acid) and 0% buffer B (acetonitrile, 0.1% formic acid), 4 min: 60% B, 4.1 min: 100% B, 6 min: 100% B, 6.01 min: 0% B, 9 min: stop time.</p>Impact of low-calorie sweeteners on gut bacteria is modulated by common xenobiotics. 10.1101/2025.03.28.645995.University of CambridgeRob BradleyWhole culture extractmixtureWater blankCulture mediaProcedural blankSupernatant extractmass spectrometry<p>LC-MS samples, both whole cultures and supernatants, were extracted by adding 4x the amount of ice cold (-20 °C) extraction buffer (1:1 ratio of methanol and acetonitrile with internal standards (75µM caffeine, 60µM ibuprofen) spiked in) followed by an incubation at 4 ºC for 15&nbsp;min. Samples were then centrifuged at 4 °C and 4000 rpm for 25 min and supernatants were transferred to 96-well plates for LC-MS analysis. Samples for concentration calibration (1 in 2 dilution of compounds) and bacteria-free compound controls were processed in the same way.</p>Roseburia intestinalisCalibration curveCulture MediaWater blankPooled QCParabacteroides merdaeStandard mixProcedural blankSegatella copriCommunity mixhttps://www.ebi.ac.uk/metabolights/MTBLS11480Bini Ramachandran. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. bini.r8@gmail.com.Vinita Periwal. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. vp406@cantab.ac.uk.Kiran Patil. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. kp533@mrc-tox.cam.ac.uk.Rob Bradley. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. rb2085@cam.ac.uk.Hilal Ozgur. European Molecular Biology Laboratory (EMBL), Heidelberg. Meyerhofstr. 1, Heidelberg, Germany. hilal.ozgur@embl.de.Indra Roux. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. ir340@cam.ac.uk.Nonantzin Beristain Covarrubias. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. nab74@mrc-tox.cam.ac.uk.Rui Guan. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. rg684@mrc-tox.cam.ac.uk.Sonja Blasche. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. sb2487@cam.ac.uk.Stephan Kamrad. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. sk727@mrc-tox.cam.ac.uk.Anna Lindell. MRC Toxicology, University of Cambridge. Gleeson Building, Tennis Court Road, Cambridge, CB21QR. ali@cambiotics.com.<p><strong>Untargeted: </strong>Analysis was performed using the Agilent MassHunter Workstation Profinder 10.0 software. Area under the curve was extracted, using the Batch Recursive Feature Extraction function for small molecules and peptides. Retention time was restricted from 0.5 to 8.0 minutes and a tolerance was set to 0.05 minutes. Peaks above a raw hight of 4000 in at least one sample were extracted and peaks below 4000 in all samples were regarded as noise and discarded.</p><p><br></p><p><strong>Targeted: </strong>Data were analysed using MassHunter Workstation Quantitative Analysis for QQQ v10.1.</p>Catalogue IDTreatmentPhylumDoserobbradley410@gmail.com<p>Bacteria were inoculated from second passage at a starting OD600 of 0.05 and grown anaerobically at 37 °C in the presence of the compounds and the combination. Controls, the solvent control (bacteria plus DMSO) and the incubation control (bacteria-free mGAM with the compounds), were treated the same way. After 24 hours the sampled were taken out and whole culture (wc, no further treatment), supernatant (sup, centrifuged at 4000g for 25 min) and compound control were collected and stored at -80 °C until extraction.</p>Metabolomicsmicrobial communityNutrition, Artificial Sweetenerssweetening agentuntargeted metabolitesCompound combinationtargeted metabolitesHuman gut bacteriamicrobial communityNutrition, Artificial Sweetenerssweetening agentuntargeted metabolitesCompound combinationtargeted metabolitesHuman gut bacteria<p><strong>Untargeted: </strong>Analysis was performed on an Agilent 6546 LC/Q-TOF. The instrument was operated in TOF MS acquisition mode in either positive or negative polarity. (30-1500&nbsp;m/z). The source parameters were as follows: gas temperature: 200ºC, drying gas: 9&nbsp;L/min, nebulizer: 20&nbsp;psi, sheath gas temperature: 400&nbsp;ºC, sheath gas flow: 12&nbsp;L/min, VCap: 3000&nbsp;V, nozzle voltage: 0&nbsp;V, fragmentor: 110&nbsp;V, skimmer 45&nbsp;V, Oct RF Vpp: 750&nbsp;V. The online mass calibration was performed using a reference solution (positive: 121.05 and 922.01 m/z; negative: 112.99 and 1033.99&nbsp;m/z). Collision energies used were 0&nbsp;V, 10&nbsp;V, 20&nbsp;V, 40&nbsp;V. Compounds of interest were identified based on their retention time, accurate mass, and fragmentation patterns, using pure standards for method development, compound identification and calibration.</p><p><br></p><p><strong>Targeted: </strong>Analysis was performed on an Agilent 6470 triple quadrupole mass spectrometer with JetStream ESI source operated in dynamic multiple reaction monitoring (dMRM) mode. A pooled QC sample, blanks and serial dilutions of a mixed analytical standard were injected throughout each run. Concentrations were obtained via external calibration using standard curves.&nbsp;</p>NADalpha-ketoglutaric acidSuccinic acidFADAcetyl-CoALactic acidcAMPL-malic acidCitric acidaconitic acidfalseImpact of low-calorie sweeteners on gut bacteria is modulated by commonly used xenobiotics<p>The gut microbiota is implicated in adverse effects associated with low-calorie sweeteners. Yet, the direct impact of sweeteners on gut bacteria has not been systematically assessed. Here we report interactions between 25 phylogenetically diverse gut bacterial strains and 39 commercially used sweeteners. We tested these sweeteners individually and in combination with four commonly co-consumed compounds, viz., advantame, caffeine, vanillin, and duloxetine. Three quarters of the sweeteners individually impacted growth of at least one bacterial strain. Further, &gt;100 interactions were found between sweeteners and the four co-consumed compounds. Isosteviol, a commonly used sweetener, and duloxetine, an antidepressant, synergistically inhibited Roseburia intestinalis, a bacterium linked to glucose homeostasis, and Parabacteroides merdae, a prevalent commensal linked to healthy microbiota. Proteomic, metabolomic, and genetic analyses indicate altered small molecule transport underpinning this sweetener-drug synergy. The isosteviol-duloxetine combination also modulated metabolism of a synthetic gut bacterial community leading to increased toxicity to HeLa cells and altered secretion of inflammation modulatory cytokines IL-6 and IL-8 by Caco-2 cells. Our data provides a mechanistic basis for sweetener-gut bacteria interaction and warrants considering their interaction with drugs in side-effect assessments.</p>2025-06-112024-10-25MTBLS11480MTBLC30772MTBLC16135MTBLC28484MTBLC30768MTBLC189936MTBLC36796MTBLC35621MTBLC17431MTBLC16449MTBLC29016MTBLC22653MTBLC22660MTBLC18127MTBLC18211MTBLC18243MTBLC16865MTBLC18237MTBLC28300MTBLC15428MTBLC27570MTBLC30653MTBLC28683MTBLC25017MTBLC17191MTBLC16857MTBLC25094MTBLC16811MTBLC15940MTBLC18257MTBLC28044MTBLC26271MTBLC17310MTBLC16410MTBLC16709MTBLC17822MTBLC27897MTBLC18186MTBLC27266MTBLC30797MTBLC15351MTBLC15741MTBLC16238MTBLC42111MTBLC15846MTBLC17489MTBLC30769MTBLC30915MTBLC22211CHEBI:30772CHEBI:16135CHEBI:28484CHEBI:30768CHEBI:189936CHEBI:36796CHEBI:35621CHEBI:17431CHEBI:16449CHEBI:29016CHEBI:22653CHEBI:22660CHEBI:18127CHEBI:18211CHEBI:18243CHEBI:16865CHEBI:18237CHEBI:28300CHEBI:15428CHEBI:27570CHEBI:30653CHEBI:28683CHEBI:25017CHEBI:17191CHEBI:16857CHEBI:25094CHEBI:16811CHEBI:15940CHEBI:18257CHEBI:28044CHEBI:26271CHEBI:17310CHEBI:16410CHEBI:16709CHEBI:17822CHEBI:27897CHEBI:18186CHEBI:27266CHEBI:30797CHEBI:15351CHEBI:15741CHEBI:16238CHEBI:42111CHEBI:15846CHEBI:17489CHEBI:30769CHEBI:30915CHEBI:22211