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Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Targeted metabolic profiling of CHKA patient fibroblasts . A , schematic presentation of the CDP-choline branch of the Kennedy pathway. Levels of p-choline ( B ), choline ( C ), and GPC-choline ( D ) in skin fibroblasts from controls and patients carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp). Control data were obtained from skin fibroblasts isolated from three healthy individuals. Data are shown as individual points representing technical replicates from three independent experiments. Statistical significance was determined by one-Way ANOVA followed by Dunnett’s test for multiple comparisons; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Control, Isolation
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Biallelic CHKA variants are associated with altered lipid profile . Expression levels of major lipids ( A ) and summary of fold change in major lipid species ( B ), as well as PC and PE subspecies ( C ) and their median fold change vs control ( D ) in patient fibroblasts carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp). Control data were obtained from skin fibroblasts isolated from three healthy individuals. The bounds of the boxplots correspond to the 25th and 75th percentiles of data with the center line corresponding to the median value and each dot representing an individual lipid species from three independent experiments. The upper / lower whiskers extend from the hinges to the largest/smallest value no more than 1.5 times the interquartile range away from the hinges ( A and C ). The significance of a median pair-wise fold-change in lipid amounts was determined using Pairwise Wilcoxon signed rank test with Bonferroni correction; ∗ p < 0.05, ∗∗∗ p < 0.001. PC, Phosphatidylcholine; PE, phosphatidylethanolamine, PI, phosphatidylinositol, PG, phosphatidylglycerol, SM, sphingomyelin, TG, triacylglycerol.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Expressing, Control, Isolation
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Biallelic CHKA variants are associated with increased nuclear envelope translocation of CCTα . A , representative images of patient-derived fibroblasts carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp) and fibroblasts heterozygous for the p.Pro194Ser variant, co-immunostained with antibodies against PCYT1A (CCTα) and lamin A/C. Control fibroblasts were derived from skin biopsies of three healthy individuals. B , quantification of CCTα enrichment at the nuclear envelope. Data represent the mean ± SD from 15 to 26 nuclei per condition, collected from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test; ∗∗∗∗ p < 0.0001. C , Western blot analysis of CCTα expression in control and CHKA variant fibroblasts; β-actin was used as a loading control. D , quantification of the ratio of the lower to upper CCTα bands, reflecting the relative abundance of dephosphorylated versus phosphorylated forms ( , ). Bars represent mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test; ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Translocation Assay, Derivative Assay, Variant Assay, Control, Western Blot, Expressing
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Biallelic CHKA variants (p.Pro194Ser or P.Arg141Trp) are associated with altered expression of key lipid metabolism regulators . A–B , quantitative PCR analysis of CHKA , CHKB , PPARA , PPARB and CPT1B mRNA levels in fibroblasts carrying biallelic CHKA variants. Control is the mean of three different normal human skin fibroblast lines. C , Western blot analysis of PPARA, PPARB, and CPT1B protein expression. D , quantification of PPARA, PPARB, and CPT1B western blots relative to GAPDH. Results are the mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Analysis of mitochondrial and glycolytic functions in patient fibroblasts carrying biallelic CHKA variants . Oxygen consumption rate (OCR) in fibroblasts carrying biallelic CHKA variants and controls (mean of three different normal human skin fibroblast lines) normalized to DNA content ( A–B ) or mitochondrial content ( C–D ). Sequential injections of oligomycin (1 μM), FCCP (1 μM), and a mixture of rotenone (1 μM) and antimycin A (1 μM) were used to assess mitochondrial respiration, respectively. Data are shown as individual points representing technical replicates (individual wells) from three independent experiments; bars represent mean ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ∗ p < 0.001, ∗∗ p < 0.001, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques:
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Biallelic CHKA variants (p.Pro194Ser or P.Arg141Trp) are associated with increased mitochondrial fragmentation and ROS production . A , representative images of fibroblasts carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp) and with controls (mean of three different normal human skin fibroblast lines), stained with MitoTracker Deep Red to visualize mitochondrial morphology. B–E , Confocal z-stack images of patient fibroblasts were processed to calculate mitochondrial mass, area, aspect ratio, and form factor using ImageJ 1.54p software. Data are shown as individual points representing separate image fields from three independent experiments; bars represent mean ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s test for multiple comparisons; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. F , representative images of fibroblasts carrying biallelic CHKA variants (p.Pro194Ser and p.Arg141Trp) and fibroblasts heterozygous for the p.Pro194Ser variant stained with MitoSOX to measure mitochondrial ROS production. G , quantification of MitoSOX fluorescence intensity normalized to cell number. Data are shown as individual points representing separate image fields from three independent experiments; bars represent mean ± SD. Statistical significance was determined using one-way ANOVA followed by Dunnett’s test for multiple comparisons; ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Staining, Software, Variant Assay, Fluorescence
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: Biallelic CHKA variants (p.Pro194Ser or P.Arg141Trp) are associated with increased lipid peroxidation . A , representative images of fibroblasts carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp), fibroblasts heterozygous for the p.Pro194Ser variant and controls (mean of three different normal human skin fibroblast lines), stained with C11-BODIPY to determine lipid peroxidation. B , quantification of oxidized C11- BODIPY fluorescence intensity to cell number ratio. Data are shown as individual points representing separate image fields from three independent experiments; Bars represent mean ± SD. Statistical significance was determined using one-way ANOVA followed by Dunnett’s test for multiple comparisons; ∗ p < 0.05, ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Variant Assay, Staining, Fluorescence
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: FCCP reverses the increase in ROS production in fibroblasts carrying biallelic CHKA variants . A–B , representative images of fibroblasts carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp) and control fibroblasts derived from three independent healthy skin fibroblast lines, stained with MitoSOX to assess ROS production following 72 h of FCCP treatment. Quantification of MitoSOX mean fluorescence intensity is shown below. Data are presented as individual points representing independent image fields from three independent experiments; bars represent mean ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Control, Derivative Assay, Staining, Fluorescence
Journal: The Journal of Biological Chemistry
Article Title: Neurodevelopmental disease-causing variants in choline kinase CHKA gene couple phosphatidylcholine synthesis to oxidative stress damage and disease etiology
doi: 10.1016/j.jbc.2025.110983
Figure Lengend Snippet: FCCP reverses the increase in lipid peroxidation observed in fibroblasts carrying biallelic CHKA variants . A , Representative images of fibroblasts carrying biallelic CHKA variants (p.Pro194Ser or p.Arg141Trp) stained with C11-BODIPY to measure lipid peroxidation levels after treatment with FCCP for 72 h. B , quantification of oxidized to reduced C11-BODIPY fluorescence intensity ratio. Data are shown as individual points representing separate image fields from three independent experiments; bars represent mean ± SD. Statistical significance was determined using one-Way ANOVA followed by Dunnett’s test for multiple comparisons; ∗∗∗∗ p < 0.0001.
Article Snippet: Quantitative real-time PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Cat. no. 4444557,
Techniques: Staining, Fluorescence
Journal: FASEB BioAdvances
Article Title: Folic Acid Supplementation Attenuates Hepatic Steatosis by Enhancing Choline Availability and Remodeling Fatty Acid Profiles in Mice Fed a High‐Fat Diet
doi: 10.1096/fba.2025-00251
Figure Lengend Snippet: Relative mRNA expression levels of (A) phosphatidylethanolamine N‐methyltransferase ( Pemt ), (B) phospholipase D1 ( Pld1 ), (C) choline dehydrogenase ( Chdh ), (D) choline kinase a ( Chka ), and (E) choline‐phosphate cytidylyltransferase A ( Pcyt1a ) in the liver. Different letters indicate statistically significant differences between the means of groups by one‐way ANOVA with Tukey–Kramer post hoc test. Data presented as means ± SEM, n = 5–8/group.
Article Snippet: An equivalent volume of cDNA was then added to TaqMan Master Mix (Thermo Fisher Scientific) along with gene‐specific probes for phosphatidylethanolamine N‐methyltransferase ( Pemt ; Mm04933134_m1), phospholipase D1 ( Pld1 ; Mm01289339_m1), choline dehydrogenase ( Chdh ; Mm00549261_m1), choline kinase alpha ( Chka ;
Techniques: Expressing