Fig 1: Gene array strategy to monitor BH4-regulating genes in RNA samples of healthy controls (HC) and ME/CFS subjects with orthostatic intolerance (ME + OI). (A) Blood samples were collected in a PAXgene Tube, processed for mRNA, and utilized for mRNA-based gene array study as described under the method section. An mRNA-based gene array was performed with 42 glucose metabolic genes and 4 housekeeping genes in ME + OI and age-/gender-matched healthy controls (n = 3 out of 4). Normalized Ct values were displayed in a representative heatmap (drawn by Morpheus software in an online server of Broad Institute) followed by clustering analysis. The color gradient was selected from green to red indicating low to high expression of genes. A cluster of blue arrows on the dendrogram indicated the upregulated genes in ME + OI subjects (n = 3). These genes encode for 6GPD or G6PDH (glucose 6 phosphate dehydrogenase), DHPR (dihydropteridine reductase), lactate dehydrogenase (LDH), CR (carbonyl reductase), phenylalanine hydroxylase (PAH), GTPCH1 (GTP cyclohydrolase1), DHFR (dihydrofolate reductase), NOS3 (nitric oxide synthase1or eNOS). (B) Expressions of several housekeeping genes such as ahsp (encodes for α hemoglobin stabilizing protein or AHSP), hprt1 (encodes for Hypoxanthine phosphoribosyl transferase 1or HPRT1), hbs1(encodes for HBS1-like protein), and bactin (encodes for β-actin) were monitored by real-time PCR analysis in both HC and ME + OI samples to nullify the loading error and confirm the quality of mRNA samples. Results are mean ± SEM of 3 different experiments. Ns = not significant. (C) The Venn diagram summarizes the numbers of downregulated (green circle; 5 genes), upregulated (red circle; 20 genes), and unaltered (shared circle; 17 genes) genes. Expression of the tryptophan hydroxylase (tph) gene was not included by the software due to its detection being much lower than the cut-off value. Volcano plot analyses represent the realtime mRNA validations of (D) G6PDH (*P < 0.05 by unpaired t-test), (E) TALDO1(**P < .01 by unpaired t-test), (F) 6PGL or 6-phosphogluconolactonase (no significance), (G) LDH (**P < .01 by unpaired t-test), (H) HK or hexokinase(***P < .001 by unpaired t-test), (I) GAPDH or glyceraldehyde-3-phosphate dehydrogenase (*P < 0.05 by unpaired t-test), (J) GTPCH1 (*P < 0.05 by unpaired t-test), in ME + OI and age-/gender-matched HC (n = 10 per group). (K) Semi-quantitative RT-PCR amplifications followed by resolving PCR products with 1.5% agarose gel and imaging in SmartDoc imaging system (Accuris Instruments) using smart glow dye demonstrated mRNA expressions of genes encoding G6PDH and GTPCH1 in HC and ME + OI subjects. The expression β-actin was visualized as a housekeeping gene. Results are confirmed after 3 different experiments.
Fig 2: Anaerobic PPP upregulated the biosynthesis of BH4. (A) BH4 was quantified in the plasma samples of ME + OI (n = 10) and age-/gender-matched HC subjects. The BH4 levels in plasma were normalized with total protein concentration and further adjusted with the molecular weight of BH4 (241.25 g/mole). The final result was expressed by the pmol/mg unit. (B) The level of GTPCH1 or GCH1 enzyme was quantified in PBMCs of ME + OI (n = 10) and HC (n = 10) healthy subjects as described under the method section. (C) Dihydrobiopterin (BH2), the BH4 metabolite and also a precursor of BH4 via the regenerative pathway, was quantified by ELISA in the plasma samples of ME + OI (n = 10) and HC (n = 10) subjects as described under method section. (D) Plasma levels of dihydropteridine reductase (DHPR), the critical enzyme of BH4 biosynthesis via the regenerative pathway, had been quantified by ELISA in ME + OI (n = 10) and HC (n = 10) subjects. Results are mean ±SEM of 3 independent experiments. An unpaired t-test was performed to test the significance of the mean between groups that resulted in *P < 0.05 and ***P < 0.001 vs HC groups. (E) An ELISA study was performed to quantify the expression of GTPCH1 in C20 microglial cells treated with increasing doses of R5P under regular and hypoxic conditions. A two-way ANOVA [2 effectors are condition (regular/hypoxia) and treatment (control/R5P)] was performed to test the significance of the mean between groups. ***P < 0.001 and *P < 0.05 vs regular and hypoxia controls respectively. Ns = no significance. (F) Immunofluorescence study of GTPCH1 was performed in C20 microglia treated with 10 µM R5P under regular and hypoxic conditions. Nuclei were stained with DAPI (blue). ELISA-based quantifications of (G) BH4 and (H) BH2 levels were performed in C20 microglial cells treated with 2,5, and 10 μM of R5P under regular(green bars) and hypoxic (red bars) conditions. A two-way ANOVA was performed to test the significance of the mean between groups. *P < .05 and ***P < .001 vs controls. Ns = no significance. Results were confirmed after 3 different experiments.
Fig 3: Knocking down of anaerobic PPP by taldo1 siRNA ameliorated the expression of BH4 via inhibition of direct biosynthetic pathway. C20 microglial cells were treated with 0.25 μg of taldo1 siRNA (validated and inventoried by Thermofisher) for 24 hrs followed by the treatment with 10 μM R5P and immediate placement under hypoxic conditions. (A) After 24 hrs, cells were immunoblotted with TALDO1 antibody to see the taldo1 knock-down efficiency. The result was normalized with the expression β-actin. (B) Quantification of PRPP was performed in whole cell lysate of C20 microglial after treatment with 5 and 10 μM R5P under regular (green) and hypoxic conditions as discussed in the method section. *P < .05, **P < .001, and ***P < .0001 vs control. The significance of means between groups was analyzed by two-way ANOVA. (C) Dual IF analysis of β-actin (green) and IMPDH2 (red) in C20 microglia under hypoxic control, 10 μM R5P, and 10 μM R5P + taldo1 siRNA treatments. (D) Quantification analyses of mean fluorescence intensities (MFI) for IMPDH2 and corresponding β-actin were performed on 20 randomly selected cells, normalized, and then plotted as a histogram. One-way ANOVA was performed to test the significance of the mean between groups (***P < .0001). (E) Dual IF analyses of β-actin and GMPS in C20 microglia under similar treatment conditions. Nuclei were stained with DAPI. (F) Quantification studies followed by statistical analyses were performed as described under 6D. (G) BH4 ELISA was performed in C20 microglia under similar conditions. The significance of the mean was measured by a one-way ANOVA followed by the derivation of descriptive statistics F3,16 = 58.248 (>Fc = 2.65); **P < .01. The significance of the mean between groups resulted in **P < 0.01 between control and R5P treatment and **P < .01 between R5P and R5P + siRNA groups. The result was confirmed after 3 different experiments of 5 different repeats. (H) GTPCH1 ELISA was performed in C20 microglial cells under similar conditions. The significance of the mean was measured by a one-way ANOVA and the resultant descriptive statistics demonstrate F3,16 = 8.19 (>Fc = 2.65); ***P < .001. The significance of the mean between groups resulted in **P < .01 between control and R5P treatment and ***P < .001 between R5P and R5P + siRNA groups. (I) Immunofluorescence analysis of TALDO1 (green) and (B) GTPCH1 (red) was performed in taldo1 siRNA-treated and R5P-stimulated C20 microglial cells and imaged in a fluorescence microscope. DAPI staining was adopted to stain nuclei. Results were confirmed after 3 different experiments.
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