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public/MTBLS13259/FILES/Metabolights/FB/B25-FB.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13259/FILES/Metabolights/FB/B23-FB.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13259/FILES/Metabolights/Acidic/A14-ABA.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13259/FILES/Metabolights/FB/B7-FB.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13259/FILES/Metabolights/FB/B27-FB.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13259/FILES/Metabolights/FB/B5-FB.rawprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13259<p>Fragmentation patterns and the product ion structures for the CKs and acidic hormones were predicted according to the available PRM spectrum. Genesis peak integration employed three smoothing points and a default S/N threshold of 0.5. Levels of CKs were quantified via a standard isotope dilution method.</p>MetaboLightsPublicLiquid Chromatography MS - positive - reverse phase<p>A 25 µL injection volume was used. The mobile phase consisted by ddH2O with 0.08% acetic acid (Solvent A) and acetonitrile with 0.08% acetic acid (Solvent B). Elution was performed at 0.5 mL·min-1 with the following gradient: 5% B for 0.5 min, linearly increasing to 45% B over 4.5 min, then to 95% B over 6.5 min, held for 1 min, and returned to initial conditions for 2 min, resulting in a total run time of 8.2 minutes. Chromeleon 6.8 (ThermoScientific, Ottawa, ON, Canada) was used for instrument control and data acquisition.</p>Integrated transcriptomic and hormonomic insights into cadmium tolerance of a Euglena mutabilis Fungal-Algal-Bacterial (FAB) consortium.Zeynab AzimychetabiTrent UniversityMixturemass spectrometry assay<p>A sequential extraction strategy, adapted from previously described protocols [38,39], was used to isolate distinct hormone classes from a single sample. This approach allowed for the recovery of 39 cytokinins (CKs) and acidic hormones, including abscisic acid (ABA), indole-3-acetic acid (IAA), salicylic acid (SA), jasmonic acid (JA), and five key gibberellins (GA1, GA4, GA7, GA9, GA20) (Supplementary Table 1).</p><p>To enable isotope dilution-based quantification, each sample was spiked with deuterated internal standards (IS) in 2 mL of ice-cold 50% acetonitrile (ACN). These included 2H4-ABA (60.9 ng; PBI, Saskatoon, Canada), 10 ng each of 2H5-IAA and 2H6-SA (OLChemim, Czech Republic), and 10 ng each of the 2H2-gibberellins GA1, GA4, GA7, GA9, and GA20. An additional 10 ng of 2H-labeled CK standards, encompassing aromatic, methylthiolated, and glucoside derivatives, were also added. The CKs scanned for comprised benzyladenine (BA), benzyladenine riboside (BAR), kinetin (KIN), cis-Zeatin (cZ) and its derivatives (cZR, cZ9G, cZNT, cZOG, cZROG), dihydroZeatin (DZ) and derivatives (DZNT, DZOG, DZR, DZROG, DZ9G), isopentenyladenine (iP) and its forms (iPNT, iP9G, iPR), along with methylthiolated and trans-Zeatin forms (2MeSiP, 2MeSiPR, 2MeSZ, 2MeSZR, tZ, tZR, tZ9G, tZNT, tZOG, tZROG) (Supplementary Table 1). Although a specific JA standard was not included, 2H4-ABA was used for JA quantification.</p><p>Supernatants and media controls were resuspended in 1.5 mL 50% acetonitrile and vortexed to ensure homogeneity. Samples were centrifuged (maximum speed for 10 minutes), and the resulting supernatant was split equally: 750 µL for acidic hormone analysis and 750 µL for CK analysis. Seven method blanks were processed concurrently. Acidic hormone samples were purified using HLB solid-phase extraction (SPE) cartridges (Canadian Life Sciences, Cat. No. VO34445) and CKs using 1 mL of 1M formic acid and MCX SPE cartridges (Canadian Life Sciences, Cat. No. VO54445).</p>Euglena mutabilishttps://www.ebi.ac.uk/metabolights/MTBLS13259Zeynab Azimychetabi. zeynazimuychetabi@trentu.ca.RJ Emery. Trent University. nemery@trentu.ca.Barry Saville. Trent University. barrysaville@trentu.ca.Emma Kaszecki. Trent University. emmakaszecki@trentu.ca.<p>Hormone quantification was performed using Xcalibur 3.0.63 (ThermoFisher Scientific). Peaks were identified based on accurate monoisotopic [M+H]+ masses and comparison of retention times with internal standards. Extracted ion chromatograms (XICs) were constructed using the precursor ion and the two most intense fragment ions, applying a mass tolerance of 3 ppm. Quantification was based on peak areas from XICs. Supernatant hormone concentrations were normalized to the number of E. mutabilis cells per 12 mL of media to establish the concentration per cell.</p>AgeHeavy metalzeynazimychetabi@trentu.ca<p>Supernatant from culture samples (12 mL) were harvested from each flask at 7-, 14-, and 21-days following inoculation and filtered through a 0.2 µm polyethersulfone (PES) syringe filter (Fisher Scientific, Cat. No. 13-1001-06) into 50 mL Falcon tubes. Media controls underwent the same processing. All processed samples were flash-frozen in liquid nitrogen, lyophilized using a LabConco FreeZone freeze dryer (Kansas City, MO, USA), and stored at −80°C until extraction.</p>MetabolomicsThermo Dionex Ultimate 3000 UHPLC systemThermo Q-Exactive quadrupole Orbitrap mass spectrometerCadmium- Day 14- Acidicuntargeted analysisCadmium Blank- Inoculation- CK NTExtraction Blank- CK FBCadmium- Day 7- AcidicHormonesCadmium- Day 7- CK NTliquid chromatography mass spectrumMixtureControl Blank-Inoculation- AcidicParallel Reaction MonitoringControl Blank-Inoculation- CK NTExtraction Blank- CK NTAgeControl-Day 7- CK NTCadmium- Day 21- AcidicCadmium- Day 21- CK NTControl Blank-Inoculation- CK FBControl-Day 7- CK FBControl- Day 14- CK FBCadmium- Day 21- CK FBControl Blank-Inoculation-Acidictargeted metabolitesHeavy metalCadmium Blank- Inoculation- CK FBCadmium- Day 14- CK- NTCadmium- Day 7- CK FBControl-Day 7- AcidicExtraction Blank- AcidicControl- Day 14- CK NTCadmium Blank- Inoculation- AcidicEuglena mutabilisCadmium- Day 14- CK FBControl- Day 14- AcidicThermo Dionex Ultimate 3000 UHPLC systemThermo Q-Exactive quadrupole Orbitrap mass spectrometerCadmium- Day 14- Acidicuntargeted analysisCadmium Blank- Inoculation- CK NTExtraction Blank- CK FBCadmium- Day 7- AcidicHormonesCadmium- Day 7- CK NTliquid chromatography mass spectrumMixtureControl Blank-Inoculation- AcidicParallel Reaction MonitoringControl Blank-Inoculation- CK NTExtraction Blank- CK NTAgeControl-Day 7- CK NTCadmium- Day 21- AcidicCadmium- Day 21- CK NTControl- Day 14- CK FBCadmium- Day 21- CK FBControl Blank-Inoculation- CK FBControl-Day 7- CK FBControl Blank-Inoculation-Acidictargeted metabolitesHeavy metalCadmium Blank- Inoculation- CK FBCadmium- Day 14- CK- NTCadmium- Day 7- CK FBControl-Day 7- AcidicExtraction Blank- AcidicControl- Day 14- CK NTCadmium Blank- Inoculation- AcidicEuglena mutabilisCadmium- Day 14- CK FBControl- Day 14- Acidic<p>Hormone fractions were analyzed using ultra high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) on a Thermo Q-Exactive quadrupole Orbitrap mass spectrometer coupled to a Thermo Dionex Ultimate 3000 UHPLC system, following a previously established method [38]. Separation was achieved on a Kinetex C18 column (2.1 mm × 50 mm, 2.6 µm particle size; Phenomenex, Torrance, CA, USA), with the column maintained near room temperature (22°C). A 25 µL injection volume was used. </p>(Z)-2-methyl-4-(7H-purin-6-ylamino)but-2-en-1-ol2-(1H-indol-3-yl)acetic acid(2R,3S,4R,5R)-2-(hydroxymethyl)-5-[6-[[(Z)-4-hydroxy-3-methylbut-2-enyl]amino]purin-9-yl]oxolane-3,4-diolN-(3-methylbut-2-enyl)-7H-purin-6-amine2-methylthio-trans/cis-zeatin ribosidefalseIntegrated transcriptomic and hormonomic insights into cadmium tolerance of a Euglena mutabilis Fungal-Algal-Bacterial (FAB) consortium<p>Coordinated stress response in<em> E. mutabilis</em> involving early Cd uptake, chloroplast-based detoxification, metabolic buffering via paramylon, suppression of growth-promoting hormones, and community-mediated resilience.</p>2026-07-142025-11-04MTBLS13259MTBLC16411MTBLC46570MTBLC17660MTBLC20838MTBLC19715CHEBI:16411CHEBI:46570CHEBI:17660CHEBI:20838CHEBI:19715