Journal: bioRxiv

Article Title: Gut sulfide metabolism modulates behavior and brain bioenergetics

doi: 10.1101/2025.04.09.647962

Figure Lengend Snippet: (A) Scheme showing experimental setup. ( B,C) Western blot analysis (B) and quantitation (C) of MT-CO1 in murine brain on 1.5% MRD. n=3 independent mice for each conditions. (D) Brain CoQ redox status in mice maintained on 1.5% MRD (n=3 independent mice). (E) Representative axial T2-weighted MRI showing asymmetric or complete loss of lateral ventricles (red arrowheads) in Villin Cre Sqor fl/fl mice compared to controls, n=4 independent mice. (F-I) Western blot (F, H) and quantitation (G, I) of the choroid plexus markers, transthyretin (TTR), and aquaporin 1 (AQP1), n=3 independent mice. (J) Scheme showing long-range modulation of brain bioenergetics and function by diet and gut SQOR oxidation capacity.

Article Snippet: MT-CO1 (1D6E1A8) and MT-CO2 (ab110258) were from Abcam and ATOH1 (21215-1-AP), transthyretin (11891-1-AP), aquaporin 1 (20333-1-AP), IBA 1 (10904-1-AP), GFAP (60190-1-Ig), CD31 (28083-1-AP), SQOR (17256-1-AP) were from Proteintech.

Techniques: Western Blot, Quantitation Assay

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Isoforms and functional domains of the chromodomain-helicase-DNA-binding protein 8 (CHD8) protein. The three isoforms of CHD8 protein, including (1) CHD8-S, a short isoform; (2) CHD8-L1, a long isoform; and (3) CHD8-L2, a long isoform . CHD8-L1 and CHD8-L2 are composed of two histone-binding chromodomains (C1 and C2, yellow), a chromatin-remodeling helicase domain (helicase, cyan), multiple protein-interacting chromatin organization modifier domains (CR, magenta), and a DNA-binding brahma and kismet domain (BRK, pink) . The position of the identified variant relative to CHD8-L1 and CHD8-L2 isoforms is indicated in red.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Functional Assay, Binding Assay, Variant Assay

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Variant validation using Sanger sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR). (a) The Sanger chromatograms indicate the absence of the variant in the maternal DNA and presence in the proband fibroblasts and blood DNA, indicating a nonmaternal inheritance of the variant. (b) Two sets of cDNA primers, including a set of primers upstream of the variant and another downstream of the variant (Table and Figure ), were used to conduct qRT-PCR on CHD8 cDNA in the proband line versus the control lines. (c) CHD8 transcripts captured by both upstream and downstream cDNA primers showed significant reduction (upstream primers: ~42%, downstream primers: ~33%) in the CHX− proband samples relative to that of controls (Wilcoxon test: p = 0.0313 for both primer sets). CHX+ samples of both the proband and the controls showed equivalent levels of CHD8 transcripts.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Variant Assay, Biomarker Discovery, Sequencing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Human Mutation

Article Title: CHD8 Variant and Rett Syndrome: Overlapping Phenotypes, Molecular Convergence, and Expanding the Genetic Spectrum

doi: 10.1155/humu/5485987

Figure Lengend Snippet: Immunoblotting and mass spectrometry–based proteomic analysis. (a) Western blots indicating the level of CHD8 protein detected from controls (C1, C2) and proband (P) samples. Three technical repeats ( n = 3) of Western blotting using the CHD8 C-terminal antibody (Cell Signaling Technologies #11891, 1:1000) showed the relative quantities of CHD8-L1 and CHD8-L2 against GAPDH (loading control). (b) Protein band quantification of the Western blots showed a significant reduction of the CHD8-L1 and CHD8-L2 isoform levels in the proband (P) (L1: ~51%, L2: ~48%) compared to those of the controls (C) (Mann–Whitney test: p = 0.0089, p = 0.0238, respectively). (c) The abundance of CHD8 is ranked significantly lower in the proteome of the proband compared to the controls. (d) The abundance of CHD8 is significantly lower in proband fibroblasts (70%, red dot) and lies outside of the control range (80%–104%, n = 5). (e) Volcano plot showed the relative amount of proteins in the proband line compared to the controls, with vertical lines indicating +/−1.5 log 2 -fold change and the horizontal line indicating statistical significance. CHD8 is reduced significantly by ~30% ( p < 0.001) in the proband line compared to the controls. MeCP2 (green) is significantly reduced by ~43% ( p < 0.01), whereas bromodomain adjacent to zinc finger domain 1A ( BAZ1A ) encoding the accessory subunit of the ATP-dependent chromatin assembly factor (ACF) (orange) is significantly increased by ~72% ( p < 0.001). CHD8-regulated proteins (purple), including acylglycerol kinase (AGK), CDC42-binding protein kinase (CDC42BPB), phosphatase and tensin homolog (PTEN), and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), showed a reduction in their corresponding protein abundance, with AGK being the highest at ~55% ( p < 0.001). Transportin 3 (TNPO3), nuclear receptor corepressor 1 (NCOR1), and proteasome assembly chaperone 2 (PSMG2) showed an increase of abundance with TNPO3 being the highest at ~39% ( p < 0.001). (f) STRING network analysis revealed coexpression (black), interactions (magenta), and comentions in literature (lime green) between CHD8, MeCP2, CDKL5, FOXG1, and ACF.

Article Snippet: A C-terminal primary antibody raised against CHD8 (Cell Signaling Technologies #11891, 1:1000) was used to quantify CHD8 protein relative to the housekeeping protein GAPDH (Sigma, #G9545, 1:5000).

Techniques: Western Blot, Mass Spectrometry, Control, MANN-WHITNEY, Binding Assay, Phospho-proteomics, Quantitative Proteomics