Journal: World Journal of Gastrointestinal Oncology

Article Title: Taurine suppresses gastric intestinal metaplasia in patient-derived organoids and Atp4a -/- mice

doi: 10.4251/wjgo.v18.i2.114161

Figure Lengend Snippet: Taurine increases the mucin 5AC (MUC5AC) level and inhibits the expression of caudal type homeobox 2, MUC2, and Trefoil factor family 3 in Atp4a -/- mice. A: Expression of caudal type homeobox 2 (CDX2), mucin 2 (MUC2), Trefoil factor family 3 (TFF3) and MUC5AC in Atp4a -/- mouse gastric tissues was assessed via immunohistochemistry (IHC) staining ( n = 6); B: IHC scores of CDX2, MUC2, and TFF3 ( n = 6); C: IHC score of MUC5AC ( n = 6). a P < 0.01 vs Atp4a -/- ; b P < 0.01 vs wild-type (WT).

Article Snippet: The gastric tissue sections were dewaxed, rehydrated, and incubated with primary antibodies against MUC5AC (1:200, GTX11335; GeneTex, Irvine, CA, United States), CDX2 (1:100, ab101532; Abcam, Cambridge, United Kingdom), MUC2 (1:2000, 27675-1-AP; Proteintech, Wuhan, China), and TFF3 (1:800, 23277-1-AP; Proteintech).

Techniques: Expressing, Immunohistochemistry

Journal: World Journal of Gastrointestinal Oncology

Article Title: Taurine suppresses gastric intestinal metaplasia in patient-derived organoids and Atp4a -/- mice

doi: 10.4251/wjgo.v18.i2.114161

Figure Lengend Snippet: Taurine downregulates the protein expression of caudal type homeobox 2 and Trefoil factor family 3 in gastric intestinal metaplasia control organoids. A-C: Western blotting was conducted to assess the protein levels of caudal type homeobox 2 (CDX2) and Trefoil factor family 3 (TFF3); D-I: Relative protein levels were quantified. a P < 0.01 vs normal control; b P < 0.05 vs gastric intestinal metaplasia (GIM) control (CON); c P < 0.01 vs GIM CON.

Article Snippet: The gastric tissue sections were dewaxed, rehydrated, and incubated with primary antibodies against MUC5AC (1:200, GTX11335; GeneTex, Irvine, CA, United States), CDX2 (1:100, ab101532; Abcam, Cambridge, United Kingdom), MUC2 (1:2000, 27675-1-AP; Proteintech, Wuhan, China), and TFF3 (1:800, 23277-1-AP; Proteintech).

Techniques: Expressing, Control, Western Blot

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: CYP2C9 and CYP2C19 variants cDNA and encoded amino acid changes

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques:

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: Data search results for CYP2C9 and CYP2C19 variants in the gnomAD data bank The database search results were obtained from gnomAD ( https://gnomad.broadinstitute.org/ ) and dbSNP ( https://www.ncbi.nlm.nih.gov/projects/SNP/ ) in August 2018.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques:

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Quantitative western blot analysis of newly identified variants in CYP2C9 and CYP2C19 (*P ≤ 0.05; ***P < 0.001 vs. WT). (B) Effect of MG132 treatment on the protein levels of CYP2C9 and CYP2C19. All values are mean ± S.E.M. for three separate independent assays. ANOVA was performed to compare gene expression, followed by multiple comparisons tests for individual comparisons when significant effects were detected. *P ≤ 0.05; ***P ≤ 0.001.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Western Blot, Expressing

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Relative CYP2C9 Vivid enzyme activity. Bar graphs show normalized Vivid enzymatic activity of the CYP2C9 WT and variant proteins (nanomolar fluorescent product formed per minute per milligram of total protein); error bars depict S.D. of the mean in three independent activity assays. (B) Relative CYP2C19 Vivid enzyme activity. Bar graphs show normalized Vivid enzymatic activity of the CYP2C19 WT and variant proteins (nanomolar fluorescent product formed per minute per milligram of total protein); error bars depict S.D. of the mean in three experimental samples. Enzyme activity was analyzed using one-way ANOVA, with mean comparison for each variant genotype against the WT control performed using Dunnet’s test. Corresponding P values were multiplicity adjusted and reported as such. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Activity Assay, Variant Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) CYP2C9 Vivid enzyme activity vs. protein content: correlation of recombinant protein quantity vs. Vivid enzyme activity for CYP2C9 variant allozymes. The normalized protein quantities are plotted on the horizontal axis and the normalized Vivid enzyme activities are plotted on the vertical axis. R2 = 0.693 and P = 0.0015. (B) CYP2C19 Vivid enzyme activity vs. protein content: correlation of recombinant protein quantity vs. Vivid enzyme activity for CYP2C19 variant allozymes. The normalized protein quantities are plotted on the horizontal axis and the normalized Vivid enzyme activities are plotted on the vertical axis. R2 = 0.825 and P = 0.0003. Associations between protein content and enzyme activity were evaluated using Pearson correlations, with two-sided t test P values reported. The solid line illustrates the line of identity for the association between normalized activity and protein content.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Activity Assay, Recombinant, Variant Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Michaelis-Menten curves of the enzymatic activities of the recombinant WT and 9 variant CYP2C9 allozymes (including CYP2C9*3) toward tolbutamide (each point represents the mean ± S.D. of three separate experiments). (B) Michaelis-Menten curves of the enzymatic activities of the recombinant WT and eight variant CYP2C19 proteins (including CYP2C19*3) toward (S)-mephenytoin (each point represents the mean ± S.D. of three separate experiments). Enzyme kinetics was modeled using nonlinear regression based on the Michaelis-Menten equation.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Recombinant, Variant Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: Enzyme kinetic properties of recombinant WT and mutant CYP2C9 proteins for tolbutamide hydroxylation and in silico functional prediction of CYP2C9 variants These data represent the mean ± S.D. of three independently performed catalytic assays. Concordance is defined as agreement of the kinetic assay result with at least two of three in silico software results.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: Recombinant, Mutagenesis, In Silico, Functional Assay, Kinetic Assay, Software, Activity Assay

Journal: Drug Metabolism and Disposition

Article Title: Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19

doi: 10.1124/dmd.118.084269

Figure Lengend Snippet: (A) Correlation analysis between enzyme activities determined by high-throughput assay vs. velocity of product formation determined by mass spectrometric assay for CYP2C9 variant allozymes. Relative velocity values of hydroxy tolbutamide formation (nanomoles of product formed per milliliter per minute) at 50 μM tolbutamide concentration are plotted on the horizontal axis and relative enzyme activity values determined by Vivid assay (nanomolars of fluorescent product formed per minute per milligram of total protein) are plotted on the vertical axis. R2 = 0.907 and P = < 0.0001. (B) Correlation analysis between enzyme activities determined by high-throughput assay vs. velocity of product formation determined by mass spectrometric assay for CYP2C19 variants. Relative velocity values of (S)-4-hydroxy mephenytoin formation (nanomoles of product formed per milliliter per minute) at 1000 μM (S)-mephenytoin concentration are plotted on the horizontal axis and relative Vivid enzyme activity values determined by Vivid assay (nanomolars of fluorescent product formed per minute per milligram of total protein) are plotted on the vertical axis. R2 = 0.898 and P ≤ 0.0001. Associations between protein content and enzyme activity were evaluated using Pearson correlations, with two-sided t test P values reported. The solid line illustrates the line of identity for the association between enzyme activities determined by high-throughput assay and intrinsic clearance determined by mass spectrometric assay.

Article Snippet: Human CYP2C9 cDNA and human CYP2C19 cDNA clones in the eukaryotic expression vector pCMV6-XL5 were obtained from OriGene Technologies, Inc. (Rockville, MD).

Techniques: High Throughput Screening Assay, Variant Assay, Concentration Assay, Activity Assay