Journal: Cell reports

Article Title: Genetic- and diet-induced ω -3 fatty acid enrichment enhances TRPV4-mediated vasodilation in mice

doi: 10.1016/j.celrep.2022.111306

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Endothelial Cell Growth Medium MV 2 , Promo Cell , C-22121.

Techniques: Recombinant, Protease Inhibitor, Staining, Western Blot, Membrane, Protein Extraction, Plasmid Preparation, Software, Imaging, Microscopy

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Graphical representation detailing the placental cell culture models for in vitro SARS-CoV-2 ORF8 treatment. ( B ) Boxplots representing the fold change values of gene expression for individual genes, obtained from standardization of in vitro SARS-CoV-2 ORF8 treatment with 10 ng/mL, 20 ng/mL, or 100 ng/mL for 8 h from eight independent experiments ( n = 8) and 16 h from eleven independent experiments ( n = 11) on immortalized trophoblasts (HTR8/SVneo cells). Fold change calculated by DDCt method, relative to mock. Data are presented in boxplot where the middle line represent means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values in ng/mL, using one-way ANOVA Kruskal–Wallis with uncorrected Dunn’s test. ( C – E ) Relative expression of genes associated with complement activation in the placental cell culture models treated with 20 ng/mL of SARS-CoV-2 ORF8 for 8 h. The models included ( C ) HTR8/SVneo cells (mock, n = 6–8; and ORF8, n = 7–8) ( D ) primary human villous trophoblasts (HVT) (mock, n = 4–5; and ORF8, n = 4–5), and ( E ) iPSC-derived trophoblasts (mock, n = 5–6; and ORF8, n = 6). Relative expression was calculated by DCt method using GAPDH as the normalizing gene. Mann–Whitney U test was used for analysis. ( F ) Boxplots representing the percentage of C3b + HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 30 min (mock, n = 5; and ORF8, n = 6), 8 h (mock, n = 4; and ORF8, n = 4) or 16 h (mock, n = 4; and ORF8, n = 4). Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values. ( G ) Violin plots representing the levels of complement proteins in the supernatant of HTR8/SVneo cells treated with SARS-CoV-2 ORF8 for 8 h (mock, n = 5–6; and ORF8, n = 6) or 16 h (mock, n = 4–5; and ORF8, n = 6). Mann–Whitney U test was used for analysis ( p < 0.05). Immortalized pluripotent stem cells (iPSC). .

Article Snippet: Cells were pre-incubated for 2 h with serum-free RPMI followed by incubation with 10, 20, or 100 ng/mL of HEK293T cells-derived recombinant SARS-CoV-2 ORF8 in RPMI supplemented with 10% FBS and 1% P/S for 8 or 16 h. Experimental conditions optimized with the HTR8/SVneo cells were applied to iPSC-derived trophoblasts described above and the primary human villous trophoblasts (HVT) (10HU-214-1M, iXCells) cultured in Trophoblast Growth Medium (MD-0058, iXCells) at 37 °C in 5% CO 2 .

Techniques: Cell Culture, In Vitro, Gene Expression, Expressing, Activation Assay, Derivative Assay, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Boxplot showing the levels of human chorionic gonadotropin (hCG) in the supernatant of iPSC-derived trophoblasts cultures at day 0 and day 7 of differentiation. Data are presented in boxplot, middle line represents means, the bound of box represent interquartile range, and whiskers represent maximum and minimum values in mUI/mL from six independent experiments ( n = 6). ( B – E ) Bar plots representing expression of genes associated as phenotypic markers in the iPSC-derived trophoblasts cultures ( n = 6) at day 0 and day 7 of differentiation. ( B ) iPSC-associated transcripts— POU5F1 , ( C ) CTB-associated transcripts— KRT7 , CDX2 , and TP63 , ( D ) STB-associated transcripts— CGA , CGB , and PSG4 , and ( E ) EVT-associated transcripts— HTRA4 , and HLAG . Data are presented in scatter dot plot, line represent means, and whiskers standard derivation. Relative expression was calculated by DCt method using GAPDH as the normalizing gene. Data are presented as means ± SDs. Immortalized pluripotent stem cells (iPSC). Cytotrophoblast (CTB). Syncytiotrophoblasts (STB). Extravillous trophoblasts (EVT). POU Domain, Class 5, Transcription Factor 1 ( POU5F1 ). Kerantin 7 ( KRT7 ). Caudal Type Homeobox 2 ( CDX2 ), Tumor Protein P63 ( TP63 ). Glycoprotein Hormones, Alpha Polypeptide ( CGA ). Chorionic Gonadotropin Subunit Beta ( CGB ). Pregnancy Specific Beta-1-Glycoprotein 4 ( PSG4 ). High-Temperature Requirement Factor A4 ( HTRA4 ). Major Histocompatibility Complex, Class I, G ( HLAG ). Mann–Whitney U test was used for all analysis ( p < 0.05).

Article Snippet: Cells were pre-incubated for 2 h with serum-free RPMI followed by incubation with 10, 20, or 100 ng/mL of HEK293T cells-derived recombinant SARS-CoV-2 ORF8 in RPMI supplemented with 10% FBS and 1% P/S for 8 or 16 h. Experimental conditions optimized with the HTR8/SVneo cells were applied to iPSC-derived trophoblasts described above and the primary human villous trophoblasts (HVT) (10HU-214-1M, iXCells) cultured in Trophoblast Growth Medium (MD-0058, iXCells) at 37 °C in 5% CO 2 .

Techniques: Derivative Assay, Expressing, Immunopeptidomics, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: ( A ) Line chart of absorbance values obtained in the SARS-CoV-2 ORF8 peptides - C1q binding assay using different molarities of C1q (10, 20, and 50 nM) and 1 mM of 28 different peptides spanning SARS-CoV-2 ORF8 region. Absorbance calculated by discounting the blank (0 nM C1q). ( B ) The ORF8 peptides presenting dose-dependent binding with C1q (#4 red, #10 purple, #19 blue, and #25 yellow) in molecular binding model between SARS-CoV-2 ORF8 dimers and C1qA-Globular (model 1). ( C ) Bar charts representing docking and confidence scores for in silico docking models SARS-CoV-2 ORF8 and C1qA-G. The ORF8 peptides #4, #10, #19, and #25 represented on amino acid (aa) sequence alignment of docking models. ( D ) Representative docking poses of the globular domain of C1q (PDB ID: 1PK6) with SARS-CoV-2 ORF8 protein (PDB ID:7JTL) in 2D interaction diagrams. The ORF8 and C1q residues with significant interactions from each protein are highlighted, labeled, and properly annotated. The hydrogen bonds, pi-pi, and salt bridges formed between ORF8 and C1q are also shown by colored dash or arrow lines. The polar (turquoise), hydrophobic (green), positively charged (purple), and negatively charged (orange) residues are represented in colored spheres. Shown below each of the interaction diagrams is a table of interacting or contact residues between the SARS-CoV-2 ORF8 protein and the globular C1q domain, including their aa positions, aa distance measured in Angstrom (Å), and the corresponding specific binding interactions. ( E ) Boxplots representing the relative expression of genes analyzed in HTR8/SVneo trophoblast treated with ORF8 peptides #4, #10, #19, and #25 using 0.1 nM and 1 nM ( n = 5–6) or mock ( n = 7–10). ( F ) Boxplots representing the relative expression of genes analyzed in primary human villous trophoblast (HVT) treated with 0.1 nM and 1 nM of ORF8 peptide #10 or Mock. Relative expression was calculated by DCt method using GAPDH as the normalizing gene. ( G ) Boxplots representing the percentage of live C3b+ cells in mock ( n = 4) and ORF8 peptides #4, #10, #19, and #25 0.1 nM and 1 nM treated ( n = 4–6) HTR8/SVneo trophoblasts. Data are presented in boxplot, middle line represents means, the bound of box represents interquartile range, and whiskers represent maximum and minimum values. ( H ) Graphical representation of main results. Comparisons using one-way ANOVA Kruskal–Wallis with Fisher test ( p < 0.05). .

Article Snippet: Cells were pre-incubated for 2 h with serum-free RPMI followed by incubation with 10, 20, or 100 ng/mL of HEK293T cells-derived recombinant SARS-CoV-2 ORF8 in RPMI supplemented with 10% FBS and 1% P/S for 8 or 16 h. Experimental conditions optimized with the HTR8/SVneo cells were applied to iPSC-derived trophoblasts described above and the primary human villous trophoblasts (HVT) (10HU-214-1M, iXCells) cultured in Trophoblast Growth Medium (MD-0058, iXCells) at 37 °C in 5% CO 2 .

Techniques: Binding Assay, In Silico, Sequencing, Labeling, Expressing

Journal: The EMBO Journal

Article Title: Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

doi: 10.1038/s44318-024-00260-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Cells were pre-incubated for 2 h with serum-free RPMI followed by incubation with 10, 20, or 100 ng/mL of HEK293T cells-derived recombinant SARS-CoV-2 ORF8 in RPMI supplemented with 10% FBS and 1% P/S for 8 or 16 h. Experimental conditions optimized with the HTR8/SVneo cells were applied to iPSC-derived trophoblasts described above and the primary human villous trophoblasts (HVT) (10HU-214-1M, iXCells) cultured in Trophoblast Growth Medium (MD-0058, iXCells) at 37 °C in 5% CO 2 .

Techniques: Recombinant, Variant Assay, Generated, Plasmid Preparation, FLAG-tag, Sequencing, Digital PCR, cDNA Synthesis, SYBR Green Assay, Blocking Assay, Software, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Spectrophotometry, Imaging