Journal: Frontiers in Immunology

Article Title: Endothelial Immunity Trained by Coronavirus Infections, DAMP Stimulations and Regulated by Anti-Oxidant NRF2 May Contribute to Inflammations, Myelopoiesis, COVID-19 Cytokine Storms and Thromboembolism

doi: 10.3389/fimmu.2021.653110

Figure Lengend Snippet: (A–E) The mRNA transcripts of four types of coronavirus receptors such as membrane alanyl aminopeptidase (ANPEP, CD13, receptor for human coronavirus-229E), carcinoembryonic antigen family cell adhesion molecule 1 (CEACAM1, receptor for mouse hepatitis virus), angiotensin-converting enzyme 2 (ACE, receptor for SARS-CoV, SARS-CoV2), and dipeptidyl peptidase-4 (DPP4, receptor for MERS-CoV) as well as TMPRSS2 are found in two clusters of human heart endothelial cells. The data mining analyses were performed on the Single Cell RNA-Seq database of the Broad Institute of MIT and Harvard (Single CellBeta Portal; https://singlecell.broadinstitute.org/single_cell/study/SCP498/transcriptional-and-cellular-diversity-of-the-human-heart#study-summary ). (F–J) The mRNA transcripts of four types of coronavirus receptors such as ANPEP, CEACAM1, ACE, and DPP4 are found in mouse aortic endothelial cell clusters. The data mining analyses were performed on the Single Cell RNA-Seq database of the Broad Institute of MIT and Harvard (Single CellBeta Portal; https://singlecell.broadinstitute.org/single_cell/study/SCP289/single-cell-analysis-of-the-normal-mouse-aorta-reveals-functionally-distinct-endothelial-cell-populations#study-summay , PMID: 31146585). (F) ANPEP expressions in three endothelial cell clusters were circled in red in the Scatter; (G) ANPEP expressions in three endothelial cell clusters were boxed in red in the Distribution; (H) CEACAM1 expressions in three endothelial cell clusters were also boxed in read in the Distribution; (I) ACE expressions in three endothelial cell clusters were also boxed in read in the Distribution; (J) DPP4 expressions in three endothelial cell clusters were also boxed in read in the Distribution. (K) A new working model: Infection of vascular endothelial cells by SARS-CoV2/MERS-CoV causes innate immune responses in endothelial cells, which may induces cytokine storm, and triggers thromboembolism. The part of figure was created with BioRender.com .

Article Snippet: To test this hypothesis, we searched the expression of these four receptors and coronavirus S protein priming serine protease TMPRSS2 ( ) in human EC in the MIT-Harvard Broad Institute Single Cell Beta Porter single-cell RNA-Seq database, Study: Transcriptional and Cellular Diversity of the Human Heart ( https://singlecell.broadinstitute.org/single_cell/study/SCP498 ).

Techniques: Membrane, Virus, RNA Sequencing, Single-cell Analysis, Infection

Journal: bioRxiv

Article Title: The furin cleavage site is required for pathogenesis, but not transmission of SARS-CoV-2

doi: 10.1101/2025.03.10.642264

Figure Lengend Snippet: (A) Alignment of the S1/S2 cleavage site of SARS-CoV-2 WA1 and series of mutant viruses generated for evaluation including deletion of the furin cleavage site (ΔFCS), truncation of the extended loop (ΔQTQTN), and disruption of the furin cleavage site motif (PQQAR). (B) SARS-CoV-2 spike trimer structure (gray) highlighting the S1/S2 cleavage loop. WT (left) and PQQAR mutant (right) are zoomed with mutated residues (Q682, Q683) in orange to disrupt the furin cleavage site. (C) Schematic of SARS-CoV-2 spike with PQQAR substitutions identified. (D) Viral titer from Vero E6 infected with WT (black) or PQQAR (orange) SARS-CoV-2 at an MOI of 0.01 (n = 3). (E) Viral titer from Calu-3 2B4 infected with WT or PQQAR SARS-CoV-2 at an MOI of 0.01 (n = 3). Data are mean ± SD. Statistical analysis measured by two-tailed Student’s t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Article Snippet: Vero E6 cells and Vero E6 cells expressing TMPRSS2 (Sekisui XenoTech) were grown in Dulbecco modified Eagle medium (DMEM; Gibco #11965–092) supplemented with 10% fetal bovine serum (FBS) (HyClone #SH30071.03) and 1% antibiotic-antimycotic (Gibco #5240062).

Techniques: Mutagenesis, Generated, Disruption, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: The furin cleavage site is required for pathogenesis, but not transmission of SARS-CoV-2

doi: 10.1101/2025.03.10.642264

Figure Lengend Snippet: (A) Schematic of SARS-Cov-2 virion sucrose cushion purification approach. (B) Lysates from sucrose cushion purified WT, PQQAR, and ΔFCS virions grown in Vero E6 were probed with α-Spike and α-Nucleocapsid (N) antibodies by Western blot. Full-length spike (FL) and S1/S2 cleavage product are indicated. (C) Quantification of densitometry of the proportion between FL (black) and S1/S2 (red) of the total spike shown (lower). (D) Schematic of SARS-CoV-2 entry and protease usage including knockout of TMPRSS2 mediated entry. (E) Viral titer from Calu-3 TMPRSS2 knock-out cells infected with WT (black) or PQQAR (orange) SARS-CoV-2 at an MOI of 0.01 (n = 3). (F) Viral titer at 48hpi from Calu3 WT and Calu3 TMPRSS2-/- cells. Statistical analysis measured by two-tailed Student’s t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Entry schematic made in Biorender.

Article Snippet: Vero E6 cells and Vero E6 cells expressing TMPRSS2 (Sekisui XenoTech) were grown in Dulbecco modified Eagle medium (DMEM; Gibco #11965–092) supplemented with 10% fetal bovine serum (FBS) (HyClone #SH30071.03) and 1% antibiotic-antimycotic (Gibco #5240062).

Techniques: Purification, Western Blot, Knock-Out, Infection, Two Tailed Test