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4 protocols using dl indole 3 lactic acid

1

Indole-3-Acetic Acid Assimilation in Caballeronia glathei

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All IAA derivatives used in this study (indole-3-acetic acid, 3-(2-hydroxyethyl)indole, ethyl-3-indole-acetate, 3-(3-hydroxypropyl)indole, 3-indoleacetonitrile, indole-3-acetamide, indole-3-butyric acid, DL-indole-3-lactic acid, indole-3-propionic acid, tryptamine, indole-3-acrycil acid and indole-3-carboxylic acid) and H218O were purchased from Sigma-Aldrich. All cloning and protein expression reagents were from ThermoFisher Scientific (Vilnius, Lithuania). All other reagents used in this study were of analytical or higher grade.
Caballeronia glathei strain DSM50014 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. This strain was routinely cultivated in M1 medium (5 g L−1 peptone, 3 g L−1 meat extract, pH 7). For the IAA assimilation experiments, C. glathei DSM50014 was cultivated in M9 medium (3.5 g L−1 Na2HPO4, 1.5 g L−1 KH2PO4, 2.5 g L−1 NaCl, 0.2 g L−1 MgSO4, 0.01 g L−1 CaCl2). Escherichia coli strains used in this study are listed in Supplementary Table S2. All E. coli strains were cultivated in LB medium. Ampicillin (50 μg mL−1) and streptomycin (30 μg mL−1) were added when necessary.
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2

Tryptophan Metabolism Metabolite Analysis

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Methanol (CH3OH), ammonium acetate (CH3COONH4), acetonitrile (CH3CN), picolinic acid, 3-hydroxykynurenine, quinolinic acid, serotonin, 5-hydroxytryptophan, kynurenine, 3-hydroxyanthranilic acid, tryptamine, L-tryptophan, 5-hydroxyindole, acetic acid, indoxyl sulfate, N-Acetylserotonin, xanthurenic acid, indole-3-acetamide, kynurenic acid, DL-indole-3-lactic acid, indole-3-carboxaldehyde, indole-3-acetic acid, tryptophol, melatonin, 5-hydroxyindole acetic acid-D5, serotonin-D4, indole-3-lactic acid-D4, kynurenic acid-D5, melatonin-D4, picolinic acid-D3, tryptamine-D4, xanthurenic acid-D4, 3-hydroxyanthranilic acid-D3, 3-hydroxykynurenine-13C2-15N, 5-hydroxytryptophan-D4, indole-3-acetamide-D5, kynurenine-D4, L-tryptophan-13C11,15N, and tryptophan-D5 were purchased from Sigma Aldrich, Merck KGaA (Darmstadt, Germany) and other reagents and chemicals used in the study were purchased from local suppliers. The reagents and chemicals were of analytical grade or higher purity. Additionally, the yeast strain S. cerevisiae STG S101 was purchased from Fermentis (Marcq-En-Baroeul, France).
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Inflammasome Activation in BMMs/BMDCs

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BMMs or BMDCs (1 × 106/ml) were incubated at 37°C in a humidified 5% CO2 atmosphere. Cells were stimulated with lipopolysaccharide (LPS; 500 ng/ml for 4 h, Sigma-Aldrich), providing the first signal for inflammasome activation. Additionally, cells were incubated for 18 h with or without potential inhibitors: CM35 (and its fractions), CMCAP, minimal medium, glycine (Sigma-Aldrich), glucuronoxylomannan (GXM), and aromatic metabolites: (Sigma) 3-Phenyllactic acid (PLA); DL-p-Hydroxyphenyllactic acid (HPLA) and DL-Indole-3-lactic acid (ILA), alone or in combination. After that, cells were treated with nigericin (20 μM for 40 or 90 minutes, InvivoGen), providing the second signal for inflammasome activation. Alternatively, cells stimulated with LPS were infected with the fungal strains opsonized with mAb 18B7 (a kind gift from A. Casadevall, Johns Hopkins Bloomberg School of Public Health) [60 (link)]. Controls included conditions without LPS or nigericin.
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4

Metabolic Profiling of Tryptophan Pathway

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Rac-metalaxyl (purity ≥ 98.5%) and metalaxyl-M (purity ≥ 98.0%) were obtained from Institute for Control of Agrochemicals, Ministry of Agriculture and Rural Affairs (Beijing, China). Deuterium oxide (D2O, 99.9% D) and sodium 3-trimethylsilyl [2,2,3,3-2H4] propionate (TSP) were bought from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Unlabled tryptophan, quinolinic acid (QA), xanthurenic acid (XA), serotonin hydrochloride, indole-3-acetic acid (IAA), tryptamine, indole-3-propionic acid (IPA), 3-hydroxyanthranilic acid (HAA), 5-hydroxyindole-3-acetic acid (HIAA), 3-hydroxykynurenine (HK), kynurenine (KYN), kynurenic acid (KA), DL-indole-3-lactic acid (ILA), melatonin, unlabeled amino acids, and [U-13C, U-15N] labeled cell free amino acid mixture were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2H5-tryptophan, 2H2-tryptamin, 2H4-melatonin, 2H2-IAA, 2H2-HIAA, 2H5-KA, and 2H4-serotonin were purchased from C/D/N Isotopes Inc. (Pointe-Claire, QC, Canada). 2H4-KYN and 2H2-HAA were obtained from Buchem (Apeldoorn, Netherlands). K2HPO4·3H2O, and NaH2PO4·2H2O were bought from Sinopharm Chemical Co., Ltd. (Beijing, China). Solvents for sample preparation and UPLC-MS/MS analysis were HPLC grade and purchased from Merck Chemicals (Shanghai, China). Ultrapure water was generated by a Milli-Q system from Millipore (Billerica, MA, USA).
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