Journal: Annals of Neurology

Article Title: Clinical and Molecular Genetic Characterization of Landau Kleffner Syndrome: An Observational Cohort and Experimental Study

doi: 10.1002/ana.27306

Figure Lengend Snippet: Molecular modelling and effects of GluN2A missense variants on recombinant mRNA protein levels and cell‐surface trafficking. (A–C) R518 is a key agonist binding residue. (A) NMDAR agonist‐binding domain from PDB 7EU7 showing L‐glutamate making contacts (green) with GluA2 H485, S511, L512, T513, T690, Y730, D731, and H‐bonds (blue) with T690, T513, and R518. The R518 makes contacts with I497 and N515, and H‐bonds with G486 and T513. (B) The shorter side chain of C518 loses contacts with L‐glutamate/I497/N515 and H‐bonds with G486/T513, but makes one new contact with F524. (C) H518 loses all contacts with L‐glutamate/N515 and H‐bonds with G486/T513, and has significant clashes (purple) with I497, V522, F524, and A764. (D) Relative gene expression (fold‐change) for GluN2A variants R518C and R518H normalized to wild‐type GluN2A (data derived from triplicate measurements). (E) Representative Western blot of HEK293 whole‐cell lysates probed with anti‐GluN2A antibody (top) and anti‐GAPDH antibody as a loading control (bottom), with (F) plot of protein expression for GluN2A R518C and GluN2A R518H variants relative to wild‐type GluN2A, normalized to GAPDH (average results from 3 independent transfections). (G) Representative confocal microscopy image of HEK293 cells co‐transfected with wild‐type GluN1 and wild‐type GluN2A (×63 magnification) enlarged to show co‐localization of surface GluN2A with membrane marker WGA (arrow). (H) Comparison of confocal microscopy images from HEK293 cells transfected with wild‐type GluN1 and either wild‐type GluN2A, or GluN2A R518C and GluN2A R518H variants (×63). (I) Quantification of relative fluorescence intensity of surface GluN2A R518C and GluN2A R518H compared to wild‐type GluN2A (average measurements from 3 independent transfections). (J) Properties of EPSCs mediated by di‐heteromeric GluN1/GluN2A, GluN1/GluN2A R518H and GluN1/GluN2A R518C NMDARs expressed in artificial synapses. (K) Mean amplitude of EPSCs from cells expressing the indicated subunit combinations. The n values for each observation were GluN1/GluN2A (n = 22), GluN1/GluN2A R518H (n = 12), and GluN1/GluN2A R518C (n = 18). Statistics undertaken using one‐way ANOVA in Prism 7.0 (GraphPad). ANOVA = analysis of variance; Con = non‐transfected HEK293 cells; EPSC = excitatory post‐synaptic currents; ns = not significant; NMDAR = N‐Methyl‐D‐Aspartate receptor; PDB = protein structure databank; WGA = wheat germ agglutinin; WT = wild‐type. ** p = < 0.005, **** p = < 0.0001. [Color figure can be viewed at www.annalsofneurology.org ]

Article Snippet: Molecular modeling was performed using the crystal structure of the human GluN1‐GluN2A NMDAR or γ‐aminobutyric acid B receptor (GABA B R) obtained from the protein structure databank (PDB), 7EU7 (GluN1‐GluN2A NMDA receptor in complex with S‐ketamine, glycine, and glutamate) and 4PAS (heterodimeric coiled‐coil structure of human GABA B R).

Techniques: Recombinant, Binding Assay, Residue, Gene Expression, Derivative Assay, Western Blot, Control, Expressing, Transfection, Confocal Microscopy, Membrane, Marker, Comparison, Fluorescence

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A–C) CCK8 assay of HaCaT and BEAS-2B cells post 1–3 months of chronic arsenic exposure (100 nM). (D) Immunoblot analysis of ALKBH1, ALKBH5, FTO, METTL3, and METTL14 protein expression in control HaCaT cells and HaCaT cells exposed to 100 nM arsenic for 3 months. (E–G) Immunoblot analysis of ALKBH1 expression in HaCaT and BEAS-2B cells post 1–3 months of chronic arsenic exposure (100 nM). (H) Immunoblot analysis of ALKBH1 expression in HaCaT cells upon arsenic treatment for 72 h. (I) Immunoblot analysis of ALKBH1 expression in BEAS-2B cells upon arsenic treatment for 72 h. (J) Immunoblot analysis of ALKBH1 expression in HaCaT cells upon cadmium treatment for 72 h. (K) Immunoblot analysis of ALKBH1 expression in HaCaT cells pre-treated with N-acetylcysteine (NAC) and further treated with arsenic for 72 h. * p < 0.05; ** p < 0.01; Student’s t test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: CCK-8 Assay, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) Experimental design schematic detailing the development of arsenic transformed cells. The schematic was made using BioRender. (B and C) RT-qPCR of ALKBH1 mRNA in no As control (HaCaT), chronic arsenic-exposed (Chr As), As-T, and As-Tr cells. As-T #1 and As-T #2 represent two distinct cell lines derived from two distinct tumors. (D) Immunoblot analysis of ALKBH1 expression in normal human epidermal keratinocytes (NHEK), HaCaT, As-T, and As-Tr cells. As-T #1 and As-T #2 represent two distinct cell lines derived from two distinct tumors. As-Tr and As-Tr-2 represent two distinct cell lines derived from two distinct clones from soft agar. (E) Immunoblot analysis of ALKBH1 protein stability in As-Tr cells compared to HaCaT cells using a cycloheximide (CHX) chase assay. (F) Immunoblot analysis of ALKBH1 expression in HaCaT, As-T, and As-Tr cells treated with MG-132 (10 μM) and Bafilomycin A1 (BafnA1, 50 nM) for 6 h. * p < 0.05; *** p < 0.001; Student’s t test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: Transformation Assay, Quantitative RT-PCR, Control, Derivative Assay, Western Blot, Expressing, Clone Assay

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A and B) Immunoblot analysis of ALKBH1 expression in As-Tr (A) and As-T (B) cells with ALKBH1 knockdown. shALKBH1 #1 and #2 represent the two unique shRNAs targeting ALKBH1. As-T #1 and As-T #2 represent two distinct cell lines derived from two distinct tumors. (C) CCK8 assay of As-Tr cells with ALKBH1 knockdown. (D and E) Colony forming assay of As-T cells with ALKBH1 knockdown. (F and G) Soft agar cloning assay of As-T cells with ALKBH1 knockdown. (H) Tumor growth of As-T cells with ALKBH1 knockdown. (I) Tumor weight as in (H). * p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: Western Blot, Expressing, Knockdown, Derivative Assay, CCK-8 Assay, Cloning

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) Quantification of the m 6 A/A ratios in polyadenylated mRNA by UHPLC-MS/MS in As-Tr cells with ALKBH1 knockdown. shALKBH1 #1 and #2 represent the two unique shRNAs targeting ALKBH1. (B) m 6 A mRNA dot blot in As-Tr cells with ALKBH1 knockdown. (C) Immunoblot analysis of METTL3 expression in shALKBH1 #1 As-Tr cells transfected with control or METTL3 siRNA. (D) CCK8 data of shALKBH1 #1 As-Tr cells with METTL3 knockdown. (E) Immunoblot analysis of m 6 A writers and erasers in As-Tr cells with ALKBH1 knockdown. (F) In vitro demethylation assay and m 6 A dot blot demonstrating that the pre-incubation of the purified ALKBH1 protein plus 10 μM As leads to demethylation. Demethylation conditions were used as described previously. (G) In vitro demethylation assay and m 6 A dot blot without ALKBH1 purified protein demonstrating that arsenic alone does not lead to demethylation. Demethylation conditions were used as described previously. * p < 0.05; *** p < 0.001; Student’s t test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: Tandem Mass Spectroscopy, Knockdown, Dot Blot, Western Blot, Expressing, Transfection, Control, In Vitro, Demethylation Assay, Incubation, Purification

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) Protein docking using AutoDock highlighting potential cysteine residues that arsenic (pink) may bind to in the mouse ALKBH1 protein (green). (B) Schematic of DARTS assay using arsenic. The schematic was made using BioRender. (C) Immunoblot analysis of DARTS assay conducted in HaCaT cell lysates and incubated with 10 μM As or vehicle control for 1 h and immunoblotted for ALKBH1 and GAPDH. (D) Schematic of arsenic-biotin conjugate experimental setup. The schematic was made using BioRender. (E) Pull-down assay of ALKBH1 binding to the arsenic-biotin conjugate. HaCaT cells were incubated with 10 μM of the arsenic-biotin conjugate for 2 h at room temperature (RT). Biotin controls were processed in parallel. (F) Pull-down assay of ALKBH1 binding to the arsenic-biotin conjugate. As-Tr cells were incubated with 10 μM of the arsenic-biotin conjugate for 2 h at RT. Biotin controls were processed in parallel. (G) Pull-down assay of recombinant ALKBH1 binding to the arsenic-biotin conjugate. Purified protein was incubated with 10 μM of the arsenic-biotin conjugate for 2 h at RT. Biotin controls were processed in parallel. (H) Immunoblot analysis of DARTS assay conducted using lysates from shALKBH1 #1 As-Tr cells expressing C118A and C207A mutant ALKBH1. Lysates were incubated with 10 μM As or vehicle control for 1 h and blotted for ALKBH1 and GAPDH. (I) Pull-down assay of recombinant WT, C118A, and C207A ALKBH1 binding to the arsenic-biotin conjugate. Purified proteins were incubated with 10 μM of the arsenic-biotin conjugate for 2 h at RT. Biotin controls were incubated and processed in parallel. (J) Immunoblot analysis of HA and m 6 A mRNA dot blot in shALKBH1 #1 As-Tr cells expressing empty vector (EV), WT ALKBH1, catalytically inactive mutant ALKBH1 (D233A), and C118A and C207A mutant ALKBH1. 40 ng of mRNA was loaded per sample. (K) CCK8 assay of shALKBH1 #1 As-Tr cells expressing empty vector (EV), WT ALKBH1, catalytically inactive mutant ALKBH1 (D233A), and C118A and C207A mutant ALKBH1. ** p < 0.01; Student’s t-test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: Western Blot, Incubation, Control, Pull Down Assay, Binding Assay, Recombinant, Purification, Expressing, Mutagenesis, Dot Blot, Plasmid Preparation, CCK-8 Assay

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) Volcano plot of m 6 A-IP-seq targets in shALKBH1 #1 As-Tr cells with NR2C2 as a top target labeled (black arrow). (B) HOMER motif analysis demonstrating the canonical m 6 A GGACU motif is enriched across experimental samples. (C) IGV visualization of increased m 6 A enrichment on representative site on the NR2C2 transcript. (D) qPCR analysis of the m 6 A levels on the NR2C2 transcript following m 6 A-IP in shNC and shALKBH1 #1 As-Tr cells. (E) qPCR analysis of the m 6 A levels on the NR2C2 transcript following m 6 A-IP of HaCaT control cells without or without 200 nM arsenic for 72 h and As-Tr cells. (F) CLIP-qPCR analysis of FLAG (ALKBH1) binding to sites on the NR2C2 transcript ( NM_001291694.2 , site 1: regions 1508243–1504892, and site 2: 150486–15048792, respectively) with an anti-FLAG or immunoglobulin G (IgG) antibody in shALKBH1 #1 As-Tr cells transfected with a construct expressing ALKBH1 (FLAG). (G) Immunoblot analysis of NR2C2 in shNC and shALKBH1 #1 As-Tr cells. (H) RT-qPCR of NR2C2 mRNA levels in shNC and shALKBH1 #1 As-Tr cells. (I) Immunoblot analysis of NR2C2 expression in HaCaT and A431 SCC cells. (J) Immunoblot analysis of NR2C2 expression in control (no As) and As-Tr cells. (K) Immunoblot analysis of NR2C2 expression in HaCaT cells treated with 200 nM arsenic for 72 h. (L) Immunoblot analysis of HA and NR2C2 expression in shALKBH1 #1 As-Tr cells expressing empty vector (EV), WT ALKBH1, catalytically inactive mutant ALKBH1 (D233A), and C118A and C207A mutant ALKBH1. (M) Immunoblot analysis of METTL3, METTL14, and NR2C2 expression in shALKBH1 #1 As-Tr cells transfected with control or METTL3/METTL14 siRNA. (N) Immunoblot analysis of YTHDF1 and NR2C2 expression in shALKBH1 #1 As-Tr cells transfected with control or YTHDF1 siRNA. (O) RNA immunoprecipitation (RIP) and qPCR analysis showing the binding of YTHDF1 to the NR2C2 transcript in As-Tr cells. (P) CCK8 assay of shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. (Q) Soft agar cloning assay of shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. (R) CCK8 assay of As-Tr cells with or without NR2C2 overexpression. (S) Soft agar cloning assay of As-Tr cells with or without NR2C2 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: Labeling, Control, Binding Assay, Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Mutagenesis, RNA Immunoprecipitation, CCK-8 Assay, Knock-Out, Cloning, Over Expression

Journal: Cell reports

Article Title: Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity

doi: 10.1016/j.celrep.2025.116311

Figure Lengend Snippet: (A) SUnSET analysis of global translation levels across no As, Chr As, and As-Tr cells. (B) SUnSET analysis of global translation levels across HaCaT cells with ALKBH1 knockdown with or without arsenic treatment (200 nM, 72 h). (C) SUnSET analysis of global translation levels in As-Tr cells with ALKBH1 knockdown. (D) SUnSET analysis of global translation levels in shALKBH1 #1 As-Tr cells transfected with control or NR2C2 siRNA. (E) Immunoblot analysis of p-4EBP1 and 4EBP1 (total) in As-Tr cells with ALKBH1 knockdown. (F) Log2 fold change of SESN1 , SESN2 , and SESN3 mRNA expression in shALKBH1 #1 As-Tr cells as compared to shNC. Data taken from RNA-sequencing used as input for m 6 A-IP-sequencing. (G–I) IGV visualization of NR2C2 binding on SESN1 , SESN2 , and SESN3 transcripts taken from NR2C2 ChIP-seq data (ENCSR454GVT, ENCSR750LYM, ENCSR559ZKI) from ENCODE. (J) Immunoblot analysis of SESN1 and ALKBH1 expression in shNC and shALKBH1 #1 As-Tr cells. (K) Immunoblot analysis of p-4EBP1, 4EBP1 (total), SESN1, and NR2C2 expression in shALKBH1 #1 As-Tr cells with or without NR2C2 knockout. **** p < 0.0001; Student’s t test.

Article Snippet: Of note, we used the murine ALKBH1 protein for this analysis as the human ALKBH1 protein was unusable for this analysis in the Protein DataBank (PDB).

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, RNA Sequencing, Sequencing, Binding Assay, ChIP-sequencing, Knock-Out

Journal: Frontiers in Molecular Biosciences

Article Title: Local sequence context at KRAS codons modulates DNA repair efficiency: insights from molecular dynamics simulations

doi: 10.3389/fmolb.2025.1654434

Figure Lengend Snippet: A representation of Rad4 with a KRAS DNA sequence. Domains are visualised in colours corresponding and table with hairpin’s represented with sticks. The right hand table contains domain amino acids number information.

Article Snippet: This DNA structure was combined with the Rad4 protein, Protein Databank (PDB ID: 2QSG) with our DNA replacing the thymine adducted DNA present in this PDB file ( ).

Techniques: Sequencing

Journal: Communications Chemistry

Article Title: Human O- GlcNAcase catalytic-stalk dimer anchors flexible histone binding domains

doi: 10.1038/s42004-025-01813-7

Figure Lengend Snippet: Potential binding sites of the pHAT domains to nucleosomes, highlighting proximity and spacing of the H3K36 residues ( a ) as well as the H3K36 and H4K 5,8,12, and 16 residues ( b ) in the nucleosome (PDBID:1kx5: gray). OGA is colored by domain: catalytic domain, dark blue; stalk, yellow; linker, and HAT-like domain: green. The distance between K36 residues (red) is 73.5 Å, and the total distance between pHAT densities is 107 Å (green). The distance between the K36 residue and the H4K 5,8,12,16 is 72.8 Å (pink). c OGA-L pHAT binding to histone modifications such as H3K36 Me and acetylated H4 tails would facilitate recruitment to sites of active transcription and DNA repair. The structural features identified for OGA-L are likely to increase the local concentration of tihe OGA-L and allow flexible movement of the catalytic domain to facilitate O- GlcNAc removal from proteins in proximity. The OGA model is shown with unstructured linkers added from the Alphafold2 colored by domain: catalytic domain, dark blue; stalk, yellow; linker, and pHAT domain, green.

Article Snippet: Additional data compared in this study from the protein databank: Alphafold2 Multimer, PDB ID: 5M7R, PDB ID: 5VVO, PDB ID:5UHK, PDB ID: 5M7S, PDBID:5UN9, PDB ID:5UHL, PDB ID: 5M7T, PDB ID: 5UHO, PDB ID: 8P0L, PDB ID: 9BA8, 9BA9, PDB ID: 1KX5 .

Techniques: Binding Assay, Residue, Concentration Assay