Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 2: Bulk RNA-seq using iPSC-derived forebrain neurons treated with FGF21, PF-05231023 and vehicle. (A) Volcano plots indicate differentially expressed genes with FDR 0.05. (B) Venn diagram showing expression profiles for the 974 genes with expression that could be affected by both FGF21 and PF-05231023 as determined by RNA-seq (FDR < 0.05). The middle and bottom Venn diagrams illustrate overlapping up-regulated and down-regulated genes in response to FGF21 and the long-acting FGF21 molecule. Those results indicate that those overlapping genes displayed identical directionality. (C) Heatmap showing expression profiles for the 974 genes affected by FGF21 and PF-05231023.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: RNA Sequencing, Derivative Assay, Expressing

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 3: Plasma FGF21 concentrations were positively correlated with plasma GABA concentrations. (A) Plasma FGF21 concentrations were associated with plasma GABA levels in patients with alcohol use disorder. (B) both FGF21, and PF-05231023 could substantially increase the level of GABA in the culture medium of iPSC-derived forebrain neurons.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Clinical Proteomics, Derivative Assay

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 4: FGF21 and PF-05231023 induced GABA concentrations in iPSC-derived forebrain organoids. (A) A schematic outline of procedures used during the differentiation of iPSC-derived forebrain organoids. The middle panel displays images of iPSC-derived forebrain organoids during differentiation. The bottom panel of the schematic displays representative examples of staining for neural progenitors (SOX2) and neurons (TUJ1). (B) FGF21 and PF-05231023 substantially induced the concentrations of GABA in the culture medium of iPSC-derived forebrain organoids.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Derivative Assay, Staining

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 5: REST, EZH2 and H3K27me3 co-occupied the promoter regions of genes associated with GABA catabolism. (A) Heatmap showing expression profiles for the 15 genes involved in the GABAergic synapse pathway. (B) ChIP assays showing that REST, EZH2 and H3K27me3 co-occupied the promoter regions of the 13 selected genes as shown in Figure 5A that were associated with the GABAergic synapse pathway in response to FGF21 and PF-05231023 treatment of iPSC-derived forebrain neurons (n ¼ 4). Percentages of ChIP DNA/input were determined by qPCR. Data are represented as % input, (enrichment relative to IgG control) ¼ % input (antibody against REST, EZH2 and H3K27me3) e % input (IgG). ChIP-qPCR was performed in triplicate. Three independent experiments were performed. This figure represents results from one of the three experiments. (C) Co- Immunoprecipitation (IP) was used to determine whether REST protein could interact with EZH2 in iPSC-derived forebrain neurons. Whole cell lysates from iPSC-derived fore- brain neurons were immunoprecipitated with anti-REST antibodies or anti-IgG antibodies and protein samples were immunoblotted and probed with antibodies against REST or EZH2.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Expressing, Derivative Assay, Control, ChIP-qPCR, Immunoprecipitation

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 6: REST-EZH2-H3K37me3 complex-mediated gene repression in a drug dependent fashion. ChIP assays were performed in iPSC-derived forebrain neurons treated with FGF21, PF-05231023 and vehicle. We confirmed that changes in REST binding were correlated with changes in mRNA expression for all 13 genes. Specifically, in the presence of FGF21, all 13 binding sites except GNAI1 displayed significantly increased REST binding. In parallel, the recruitment of EZH2 and H3K27me3 also increased substantially, thus confirming that changes in REST-EZH2-H3K37me3 occupancy were highly correlated with transcription.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Derivative Assay, Binding Assay, Expressing

Journal: Molecular metabolism

Article Title: Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

doi: 10.1016/j.molmet.2023.101798

Figure Lengend Snippet: Figure 7: Schematic diagram illustrating the effects of FGF21 on GABA catabolism. Specifically, FGF21 and PF-05231023 significantly induced GABA levels in iPSC-derived forebrain neurons. We identified a drug-dependent occupancy of REST, EZH2, H3K27me3 in the promoter regions of genes associated with GABA catabolism i.e., ABAT (a key enzyme responsible for GABA catabolism), GRBBR1 (GABA B receptor 1), and SLC32A1 (GABA transporter), which resulted in transcriptional repression.

Article Snippet: Concentrations of the naïve form of FGF21 (Peprotech, cat#100-42) [22,23] and PF-05231023 (MedChemExpress, cat: HY-113697) [24,25] were selected to fall within the range of blood concentrations for these drugs observed during preclinical and clinical studies.

Techniques: Derivative Assay

Journal: Nature medicine

Article Title: Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

doi: 10.1038/nm.3585

Figure Lengend Snippet: (a) Schematic of human 11p15.5 CRC amplicon indicating the position of miR-483 within IGF2 (UCSC Genome Browser). (b) Functional analysis of miR-483 , Igf2 or both in Apc -null colon organoids. Tamoxifen-treated Apc flox/flox ; villin-CreER adult colon organoids were infected with appropriate combinations of control LMP retrovirus, Igf2 cDNA-IRES-GFP or lentivirus miR-483/GFP to generate Apc -null (A), Apc -null/ Igf2 (AI), Apc -null/ miR-483 (AM) or Apc -null/ miR-483 / Igf2 (AIM) organoids. Significant high-grade epithelial dysplasia with nuclear pleiomorphism was only observed with miR-483 -overexpressing organoids (AM and AIM) but not A or AI. H&E, day 50 post-infection. Scale bars, 50 μm. (c) Dysplasia index from blinded pathologic analysis reveals marked transformation of miR-483 -containing modules (AM, AIM) versus Apc -null or Apc/Igf2 (AI or AIM vs. A, ** = P < 0.0001 ) versus Igf2 (AI vs. A; AIM vs. AM, * = P < 0.05 ). Each individual data point represents n=4–6 microscopic fields containing viable organoids. Day 50 post-infection. Mean +/− SE. (d) AM but not A cells demonstrated robust in vivo tumorigenicity following s.c. transplantation into NSG mice (day 60 post-implantation, 500,000 cells, passage 5). H&E staining demonstrated poorly differentiated adenocarcinoma, scale bar, 50 μm. Tumors exhibited PCNA-positive high mitotic index and expressed epithelial markers villin and E-cadherin but not the stromal marker smooth-muscle actin (SMA), scale bars, 50 μm. (bottom, right), AM versus Apc -null (“A”) or other two gene combinations, Tumor weight examined at day 60 post s.c. organoid implantation, n=10 tumors/genotype. * = P < 0.0001 . (e) 96-well invasion and proliferation analyses. FACS-sorted EpCAM + disaggregated cells from day 14 organoids of the indicated genotypes were replated at 10,000 cells/well into 96-well air-liquid interface culture to form secondary organoids. After 14 days of additional culture, proliferation and invasion in A, AI, AM and AIM organoids was determined by Calcein Red-Orange, AM fluorescence signal. N=12 wells/genotype, mean+/−SE. For proliferation * = P < 0.00003 and for invasion * = P < 1 × 10 −7 , † = P <0.03 , all vs. the Apc-null “A” module. (f) MiR-483 represses Pdlim2 and Abtb1 RNA in FACS-sorted EpCAM + /GFP + cells from Apc -null colon organoids 10 days after infection with lentivirus encoding miR-483 or negative control miR . N=3 experiments, mean +/− SE, * = P<0.05 . (g) PDLIM2 rescue experiments. AM vs. A organoids were assayed after 14d of culture in 96-well format for invasion and proliferation with either PDLIM2 cDNA overexpression or Pdlim2 shRNA knockdown. * = P < 0.05 . N=12, mean +/− SE. (h) Schematic of mediation of miR-483 -induced dysplasia by Pdlim2 and other potential targets.

Article Snippet: Mouse Igf2 cDNA was obtained from OriGene (Plasmid # MG201606 ).

Techniques: Amplification, Functional Assay, Infection, Control, Transformation Assay, In Vivo, Transplantation Assay, Staining, Marker, Fluorescence, Negative Control, Over Expression, shRNA, Knockdown

Journal: Life Science Alliance

Article Title: Identification of new drugs to counteract anti-spike IgG-induced hyperinflammation in severe COVID-19

doi: 10.26508/lsa.202302106

Figure Lengend Snippet: (A, B, C) Representative data of macrophage activation assay for (A) the PI3Kα inhibitor alpelisib, (B) the PI3Kδ and PI3Kγ inhibitor duvelisib, and (C) the PI3Kδ inhibitor idelalisib.

Article Snippet: Aleplisib (BYL719) , Selleckchem.com , Cat#S1815.

Techniques: Activation Assay

Journal: Life Science Alliance

Article Title: Identification of new drugs to counteract anti-spike IgG-induced hyperinflammation in severe COVID-19

doi: 10.26508/lsa.202302106

Figure Lengend Snippet:

Article Snippet: Aleplisib (BYL719) , Selleckchem.com , Cat#S1815.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software