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MB Sample ID: SA247777
Local Sample ID: | xdhA_Azathioprine_R4 |
Subject ID: | SU002563 |
Subject Type: | Bacteria |
Subject Species: | Veillonella parvula |
Taxonomy ID: | 29466 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002563 |
Subject Type: | Bacteria |
Subject Species: | Veillonella parvula |
Taxonomy ID: | 29466 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
xdhA_Azathioprine_R4 | SA247777 | FL031156 | xdhA | Strain |
xdhA_Azathioprine_R4 | SA247777 | FL031156 | Azathioprine | Drug |
Collection:
Collection ID: | CO002556 |
Collection Summary: | All strain experiments were done in the Xavier lab. Strains and growth conditions: we utilized the following strains of Veillonella for growth and metabolic analysis; Veillonella parvula SKV38 [DR071], Veillonella parvula SKV38 xdh::cat* [DR214], Veillonella parvula SKV38 pucD::cat* [DR213]. All strains were first streaked on an agar plate with SK media (composition: yeast extract 10 gL-1, casitone 10 gL-1, NaCl 2 gL-1, K2HPO4 0.4 gL-1) supplemented with 50 mM lactate and 40 mM KNO3 (SKLN medium) and antibiotics if required. From this agar plate, a single colony was selected and inoculated in 5 mL SKLN media and grown for 24 hours. Next, overnight cells from this inoculum were grown using a 1/50 inoculum of the overnight culture, on either SK, SK + 50 mM lactate (SKL), SK + 40 mM nitrate (SKN), and SKLN. Supernatants were collected at the mid-exponential phase (OD600~0.4) and collected for metabolomic analyses (HILIC-pos metabolite profiling method described above). For analysis of the metabolism of the IBD drugs, cells were prepared as described above, and mid-exponential phase cells were collected, washed twice with sterile SK, and resuspended in 100 µl of SK. A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Additionally, sterile SK was prepared in an identical fashion to serve as a control for the experiment. Cells were incubated in a plate reader (4 wells/treatment) with low shaking and 37ºC for about ~5 h, and when the OD600 reached 0.4 cells were collected. For collection, 700 µl of the suspensions were centrifuged at 21,000 g for 2 min, and 400 µl of the supernatant harvested and stored at -20 ºC for analysis. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day. |
Sample Type: | Culture Media |
Treatment:
Treatment ID: | TR002575 |
Treatment Summary: | A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day. |
Sample Preparation:
Sampleprep ID: | SP002569 |
Sampleprep Summary: | Bacterial supernatant (media) metabolites were profiled using the HILIC-pos and HILIC-neg methods in order to estimate purines and thiopurines metabolism. Media samples were prepared as follows: mid-exponential Veillonella cultures (OD600 = 0.3-0.4) were harvested by centrifugation at 20,000g at 4°C for 1 minute, supernatants (spent media) were aliquoted and stored at -80°C until metabolite profiling was conducted. For the HILIC-pos method, media samples (10 µL) were extracted using 90 µL HILIC extraction solution (74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid) with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8) and extracts were cleared by centrifugation (10 min, 9,000 x g, Room Temperature). For media profiled in the HILIC-neg mode, 30 µL of media and metabolites extracted using 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis. |
Combined analysis:
Analysis ID | AN004039 | AN004040 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
Column | Waters Atlantis HILIC (150 x 2 mm, 3 μm) | Phenomenex Luna NH2 (150 x 2.1mm,3um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | Abundance | Abundance |
Chromatography:
Chromatography ID: | CH002987 |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Waters Atlantis HILIC (150 x 2 mm, 3 μm) |
Column Temperature: | 30C |
Flow Gradient: | Isocratically with 5% mobile phase A for 1 minute followed by a linear gradient to 40% mobile phase B over 10 minutes |
Flow Rate: | 250 µL/min |
Solvent A: | 100% water; 10 mM ammonium formate; 0.1% formic acid |
Solvent B: | 100% acetonitrile; 0.1% formic acid |
Chromatography Type: | HILIC |
Chromatography ID: | CH002988 |
Instrument Name: | Shimadzu Nexera X2 |
Column Name: | Phenomenex Luna NH2 (150 x 2.1mm,3um) |
Column Temperature: | 30C |
Flow Gradient: | The column was eluted with initial conditions of 10% mobile phase A and 90% mobile phase B followed by a 10 min linear gradient to 100% mobile phase A. |
Flow Rate: | 400 µL/min |
Solvent A: | 100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide |
Solvent B: | 75% acetonitrile/25% methanol; 10 mM ammonium hydroxide |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003786 |
Analysis ID: | AN004039 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
Ion Mode: | POSITIVE |
MS ID: | MS003787 |
Analysis ID: | AN004040 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
Ion Mode: | NEGATIVE |