Journal: bioRxiv

Article Title: Eukaryotic initiation factor 3d Regulates Context-Dependent Pain Hypersensitivity Through the Integrated Stress Response

doi: 10.64898/2025.12.21.695844

Figure Lengend Snippet: (A) Transcriptomic profiling shows EIF3D expression across multiple human tissue types with highest expression in the human DRG. (B, C) Spatial and single nucleus transcriptomic data illustrates EIF3D localization within human DRG neurons, neuronal subtypes (B), and non-neuronal cell types (C). (D–D′) Immunohistochemical labeling of healthy human DRG sections with DAPI (nuclei, blue), Peripherin (neuronal marker, green), and EIF3D (red) confirms neuronal expression. (E) Scatter plot depicts EIF3D protein levels do not change relative to neuronal soma size. (F) Pearson’s (linear) and Spearman’s (non-parametric) correlation analyses quantitatively show no association between EIF3D protein expression and soma size (p>0.05). (Data from UTD CAPS Sensoryomics database) .

Article Snippet: This manuscript utilized eIF3d +/+ and eIF3d +/- mouse lines obtained from Cyagen which were subsequently backcrossed with wild-type C57BL/6 x 129S4/SvJae mice sourced from Charles River.

Techniques: Expressing, Immunohistochemical staining, Labeling, Marker

Journal: bioRxiv

Article Title: Eukaryotic initiation factor 3d Regulates Context-Dependent Pain Hypersensitivity Through the Integrated Stress Response

doi: 10.64898/2025.12.21.695844

Figure Lengend Snippet: (A) Proteomic expression of EIF3D, quantified by arbitrary units (AU), showed no difference between human males and females (p>0.05). (Data from UTD CAPS Sensoryomics database)

Article Snippet: This manuscript utilized eIF3d +/+ and eIF3d +/- mouse lines obtained from Cyagen which were subsequently backcrossed with wild-type C57BL/6 x 129S4/SvJae mice sourced from Charles River.

Techniques: Expressing

Journal: bioRxiv

Article Title: Eukaryotic initiation factor 3d Regulates Context-Dependent Pain Hypersensitivity Through the Integrated Stress Response

doi: 10.64898/2025.12.21.695844

Figure Lengend Snippet: (A) Neuronal cell-type cluster annotations from the spatial and single nucleus transcriptomic dataset. (B) Non-neuronal cell-type cluster annotations from the spatial and single nucleus transcriptomic dataset. (C) eIF3d expression in DRG neuronal cells in rhesus macaque. (D) eIF3d expression in DRG non-neuronal cells in rhesus macaque. (E) eIF3d expression in DRG neuronal cells in cynomolgus macaque. (F) eIF3d expression in DRG non-neuronal cells in cynomolgus macaque. (G) eIF3d expression in DRG neuronal cells in guinea pig. (H) eIF3d expression in DRG non-neuronal cells in guinea pig. (I) eIF3d expression in DRG neuronal cells in mouse. (J) eIF3d expression in DRG non-neuronal cells in mouse. (K) Transcriptomic profiling of the eIF3d gene expression across tissue types in mice, with the highest being in the DRG. (Data from UTD CAPS Sensoryomics database)

Article Snippet: This manuscript utilized eIF3d +/+ and eIF3d +/- mouse lines obtained from Cyagen which were subsequently backcrossed with wild-type C57BL/6 x 129S4/SvJae mice sourced from Charles River.

Techniques: Expressing, Gene Expression

Journal: bioRxiv

Article Title: Eukaryotic initiation factor 3d Regulates Context-Dependent Pain Hypersensitivity Through the Integrated Stress Response

doi: 10.64898/2025.12.21.695844

Figure Lengend Snippet: (A) Schematic representation of the transgenic strategy for eIF3d knockout generation, based on Cyagen’s design specifications, illustrating the targeted genomic locus, excised exons, and positions of gRNA sequences. (B) Primer sequences sizes used for genotyping PCR assays. (C) Representative PCR-based genotyping confirming eIF3d allele status. (D) Validation of heterozygous model via quantitative RT-qPCR show decrease eIF3d expression in HET mice (p ≤ 0.001). (E) ICC analysis of primary neuronal cultures from WT and HET mice corroborate decreased eIF3d expression in cultured cells (p<0.0001). (F, F’, G, G’) Representative images of ICC validation of the eIF3d mouse model in cultured primary mouse neurons via intensity observation. DAPI (nuclear marker, blue), Peripherin (neuronal marker, green), and eIF3d (red). (H) IHC validation of eIF3d heterozygous knockout mouse model in cryosectioned mouse DRG tissue corroborate decreased expression of eIF3d (p<0.0001). (I, I’, J, J’) Representative images for IHC validation of eIF3d expression in the mouse model via intensity observation. DAPI (nuclear marker, blue), Peripherin (neuronal marker, green), and eIF3d (red).

Article Snippet: This manuscript utilized eIF3d +/+ and eIF3d +/- mouse lines obtained from Cyagen which were subsequently backcrossed with wild-type C57BL/6 x 129S4/SvJae mice sourced from Charles River.

Techniques: Transgenic Assay, Knock-Out, Biomarker Discovery, Quantitative RT-PCR, Expressing, Cell Culture, Marker

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