Journal: Journal of the neurological sciences

Article Title: Role of Vitamin A Metabolism in IIH: Results from the Idiopathic Intracranial Hypertension Treatment Trial

doi: 10.1016/j.jns.2016.11.014

Figure Lengend Snippet: Dietary retinyl esters, retinol, and pro-vitamin A carotenoids are ingested. Vitamin A may be esterified into retinyl esters and stored primarily in the liver. In times of dietary retinoid insufficiency, retinyl ester stores are hydrolyzed to retinol for delivery to peripheral tissues through the circulation bound to RBP. Both all-trans-retinol (typically referred to as retinol) and beta-carotene may be converted enzymatically to all-trans-retinal. However, the visual chromophore 11-cis-retinal, owing to energetic considerations, is formed via the coupled enzymatic hydrolysis of all-trans-retinyl ester with the isomerization of the all-trans-retinoid to the 11-cis-isomer. All-trans-retinal either can be enzymatically oxidized to all-trans-retinoic acid, which regulates transcription of over 500 retinoid-responsive genes, or reduced enzymatically to all-trans-retinol. When all-trans-retinoic acid is no longer needed, it is oxidatively metabolized and eliminated from the body.

Article Snippet: Penta-deuterated all- trans -retinoic acid (atRA-d5) was employed as an internal standard and was purchased from Toronto Research Chemicals (North York, Ontario).

Techniques:

Journal: Journal of the neurological sciences

Article Title: Role of Vitamin A Metabolism in IIH: Results from the Idiopathic Intracranial Hypertension Treatment Trial

doi: 10.1016/j.jns.2016.11.014

Figure Lengend Snippet: Boxplot (horizontal bar at median value) of cerebrospinal fluid (CSF) all-trans retinoic acid (ATRA) by treatment group, acetazolamide (ACZ) and placebo, at baseline (Mo. 0) and at six months (Mo. 6). A significant difference is seen between the ACZ and placebo groups with respect to the change in CSF ATRA at six months.

Article Snippet: Penta-deuterated all- trans -retinoic acid (atRA-d5) was employed as an internal standard and was purchased from Toronto Research Chemicals (North York, Ontario).

Techniques:

Journal: Neurotherapeutics

Article Title: Calcitriol ameliorates motor deficits and prolongs survival of Chrne -deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2

doi: 10.1016/j.neurot.2024.e00318

Figure Lengend Snippet: Calcitriol enhances AChR clustering in C2C12 myotubes co-cultured with NSC34 ​cells (A) Culturing protocol of C2C12 myoblasts/myotubes with 10 ​ng/ml agrin and 10 −10 or 10 −8 ​M calcitriol for 24 ​h ​for ( B and C ). (B, E) Representative AChR clusters stained with α-bungarotoxin (red) on C2C12 myotubes with the indicated concentrations of calcitriol. Bar ​= ​10 ​μm. (C, F) Total area and total intensity of AChR clusters were blindly measured using MetaMorph software, and values were normalized to that without calcitriol. Mean and SD are indicated ( n ​= ​6 visual fields per well ​× ​4 wells). ∗ p ​< ​0.05, ∗∗∗ p ​< ​0.001, and ∗∗∗∗ p ​< ​0.0001 by one-way ANOVA followed by Tukey’s posthoc test. (D) Co-culturing protocol of C2C12 myoblasts/myotubes and NSC34 motor neurons with 10 −10 or 10 −8 ​M calcitriol for ( E , F , and G ). (G) Ratio of twitching myotubes in four wells was blindly counted. Mean and SD are plotted. ∗ p ​< ​0.05 by one-way ANOVA followed by Dunnett’s posthoc test compared to 0 ​M calcitriol.

Article Snippet: Total area, total intensity, and total length of AChR clusters were blindly measured using MetaMorph software, and values were normalized to that without calcitriol.

Techniques: Cell Culture, Staining, Software

Journal: Neurotherapeutics

Article Title: Calcitriol ameliorates motor deficits and prolongs survival of Chrne -deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2

doi: 10.1016/j.neurot.2024.e00318

Figure Lengend Snippet: RNA-seq analysis of co-cultured C2C12 myotubes and NSC34 motor neurons with or without calcitriol ( n ​= ​3 wells each) (A) Top 30 genes increased by 10 −10 ​M calcitriol. Mean and SD are plotted. (B) Heatmap of genes in the Reactome pathway “striated muscle contraction”. The heatmap was automatically generated by GSEA. For each gene, the lowest and the highest values are shown in dark blue and dark red, respectively. See Suppl. Fig. S2B for the enrichment plot of “striated muscle contraction”. (C) Representative upregulated genes that are mutated in CMS or highly expressed at the NMJ [ , ]. Mean and SD are plotted. P -values by two-way ANOVA followed by Sidak’s posthoc test are indicted for the top four genes. Two-way ANOVA compared gene expressions between control- and calcitriol-treated co-cultured cells.

Article Snippet: Total area, total intensity, and total length of AChR clusters were blindly measured using MetaMorph software, and values were normalized to that without calcitriol.

Techniques: RNA Sequencing, Cell Culture, Generated, Control

Journal: Neurotherapeutics

Article Title: Calcitriol ameliorates motor deficits and prolongs survival of Chrne -deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2

doi: 10.1016/j.neurot.2024.e00318

Figure Lengend Snippet: Calcitriol enhances Rspo2 expression via distal promoter region of Rspo2 and enhances MuSK phosphorylation (A) Culturing protocol of C2C12 myotubes with cycloheximide for 18 ​h and 10 −10 ​M calcitriol for 16 ​h. ( B, E ) qRT-PCR of indicated genes. Gene expression was normalized to that of B2m and then to the ratio of DMSO without cycloheximide (B) or DMSO-treated WT C2C12 ​cells (E) . Mean and SD are indicated ( n ​= ​3 wells each). ∗ p ​< ​0.05, ∗∗ p ​< ​0.01, ∗∗∗ p ​< ​0.001, and ∗∗∗∗ p ​< ​0.0001 by one-way ANOVA followed by Tukey’s posthoc test. (C) ChIP-seq tracks of VDR for Rspo2 normalized to 10 7 tags in control- and calcitriol-treated IDG-SW3 mouse osteocytic cells that were differentiated for 35 days . An arrowhead points to the VDR-binding site ∼15 ​kbp upstream to Rspo2 . (D) Culturing protocol of wild-type (WT) and genetically engineered C2C12 ​cells lacking the VDR-binding site at the distal promoter region of Rspo2 (R2-VDR KO) with 10 −10 ​M calcitriol for 16 ​h. (F) Culturing protocol of WT and R2-VDR KO C2C12 myotubes with 5 ​ng/ml agrin for 1 ​h and 10 −10 ​M calcitriol for 16 ​h. ( G ) Total MuSK was immunoprecipitated (IP) with an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. Note that the concentration of agrin was reduced to 5 ​ng/ml so that MuSK phosphorylation was marginally induced by agrin alone. ( H ) Signal intensities of phosphorylated MuSK were normalized for total MuSK in agrin- and calcitriol-treated cells, and then for the ratio of wild-type (WT) C2C12 ​cells. Mean and SD are indicated ( n ​= ​3 lanes each). Statistical significance was calculated by unpaired Student’s t -test.

Article Snippet: Total area, total intensity, and total length of AChR clusters were blindly measured using MetaMorph software, and values were normalized to that without calcitriol.

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Gene Expression, ChIP-sequencing, Control, Binding Assay, Immunoprecipitation, Concentration Assay

Journal: Neurotherapeutics

Article Title: Calcitriol ameliorates motor deficits and prolongs survival of Chrne -deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2

doi: 10.1016/j.neurot.2024.e00318

Figure Lengend Snippet: Calcitriol improves motor deficits and increases gene expressions of Rspo2 , Rapsn , and Dusp6 in tibialis anterior muscle of Chrne KO mouse. (A) Protocols of intraperitoneal administration of 1 ​% ethanol (vehicle) and 0.016 ​μg/kg/day calcitriol for Chrne KO mice starting from 6 weeks of age. For the analysis of mouse survival, calcitriol was administered every other day until the mouse died. For the analysis of the skeletal muscle, calcitriol was administered every day for 2 weeks. (B) Survival rate of Chrne KO male and female mice with or without calcitriol treatment (0.016 ​μg/kg/day). The survival rate of calcitriol-treated mice was significantly longer than that of vehicle-treated mice in both males ( p ​= ​0.043) and females ( p ​= ​0.026) by log-rank test. (C) Endurance time on a rota-rod of wild-type (WT) and Chrne KO mice with or without calcitriol for 2 weeks. Mean and SD are indicated. ∗ p ​< ​0.05 and ∗∗∗∗ p ​< ​0.0001 by one-way ANOVA followed by Tukey’ s posthoc test. The mice were observed up to 500 ​s on the rotarod. The actual endurance time of each mouse is indicated in Suppl. Fig. S4DF . (D) Voluntary motor activities measured by an IR Actinometer of wild-type (WT) and Chrne KO mice with or without calcitriol for 2 weeks. Mean and SD are indicated. ∗∗ p ​< ​0.01 and ∗∗∗∗ p ​< ​0.0001 by one-way ANOVA followed by Tukey’ s posthoc test. The actual number of crossing beams per hour of each mouse is indicated in Suppl. Fig. S4EG . (E) qRT-PCR of genes related to NMJ and muscle differentiation. Gene expression was normalized to that of B2m and then to the ratio of vehicle-treated wild-type (WT) mice. Mean and SD are indicated ( n ​= ​12 wells each). ∗ p ​< ​0.05, ∗∗ p ​< ​0.01, ∗∗∗ p ​< ​0.001, and ∗∗∗∗ p ​< ​0.0001 by one-way ANOVA followed by Tukey’s posthoc test.

Article Snippet: Total area, total intensity, and total length of AChR clusters were blindly measured using MetaMorph software, and values were normalized to that without calcitriol.

Techniques: Quantitative RT-PCR, Gene Expression

Journal: Neurotherapeutics

Article Title: Calcitriol ameliorates motor deficits and prolongs survival of Chrne -deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2

doi: 10.1016/j.neurot.2024.e00318

Figure Lengend Snippet: Calcitriol improves the NMJ structure in diaphragm of Chrne KO mouse. Wild-type and Chrne KO mice were intraperitoneally administrated with 1 ​% ethanol (vehicle) or 0.016 ​μg/kg/day calcitriol in 1 ​% ethanol every day for 2 weeks from 6 to 8 weeks of age. (A) Representative confocal images of the NMJs of the right diaphragm at 8 weeks of age stained with anti-synaptophysin antibody (green) and α-bungarotoxin (red) to visualize the nerve terminals and acetylcholine receptors (AChRs), respectively. Scale bar ​= ​25 ​μm. (B) Morphometric analyses of the area, total signal intensity, and the number of α-bungarotoxin-positive signals for AChR clusters and synaptophysin-positive signals for synaptic vesicles. AChR areas and synaptophysin-positive areas were manually traced individually and measured by MetaMorph software. The measurement was blinded. Mean ​± ​SD ( n ​= ​8 NMJs per mouse ​× ​4 mice) are indicated. ∗ p ​< ​0.05, ∗∗ p ​< ​0.01, ∗∗∗ p ​< ​0.001, and ∗∗∗∗ p ​< ​0.0001 by one-way ANOVA followed by Tukey’s posthoc test. (C) Schematic summary of the effect of calcitriol on the NMJ. Calcitriol directly enhances the expression of Rspo2 via VDR. Rspo2 binds to Lgr5 and enhances agrin-LRP4-MuSK signaling pathway [ , ]. Calcitriol markedly increases synaptic vesicles likely via Rspo2. Solid and broken lines indicate direct and indirect effects, respectively.

Article Snippet: Total area, total intensity, and total length of AChR clusters were blindly measured using MetaMorph software, and values were normalized to that without calcitriol.

Techniques: Staining, Software, Expressing

Journal: Journal of experimental botany

Article Title: A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination.

doi: 10.1093/jxb/eri077

Figure Lengend Snippet: Fig. 2. (A) Bars of the histogram represent MtPPRD1 expression determined by quantitative real time RT-PCR in the Medicago truncatula embryo axis at various times throughout germination and post germina- tion. The percentage of germinated seeds (solid symbols) correspond to the mean of three replicates of 50 seeds. (B) SAMS expression determined by quantitative real time RT-PCR in the Medicago truncatula embryo axis throughout germination and post-germination. Expression of genes in arbitrary units (A.U.) corresponds to the ratio of the copy number of cDNA of the studied gene (MtPPRD1 or SAMS) on the copy number of the constitutive control MSC27 gene in 5 ll of first strands. Bars indicate standards errors (n=3).

Article Snippet: As expected the subtraction resulted in the complete extinction of the constitutively expressed MSC27 and in the appearance of a clone corresponding to S-adenosyl methionine synthetase (SAMS), whose expression increases during Medicago truncatula germination (Gallardo et al., 2003).

Techniques: Expressing, Quantitative RT-PCR, Control