Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 1 | SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Binding Assay, Clone Assay, Marker, Magnetic Beads

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 5 | SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B), except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase If, a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: SDS Page, Western Blot, Activity Assay, Magnetic Beads, Labeling, Positive Control

Journal: Frontiers in microbiology

Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

doi: 10.3389/fmicb.2021.660149

Figure Lengend Snippet: FIGURE 6 | Expression of hRNase A-ACE2NTD150 in T7 Express LysY/lacIq (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

Article Snippet: Ab Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 expression in human fetal hippocampus. A Image of a coronal section of the temporal lobe showing the hippocampal formation. Insert represents the picture showed in ( B ). B Picture showing the Hippocampus Fimbrial Angle (HFA) between the fimbria and the Ammon’s horn (CA) ventricular surfaces. Inserts represent the areas that are showed in C, D, and F. ACE2 immunopositive (ACE2 +) migratory cells were observed from the HFA ventricular epithelium (VE) to the subpial region (pial surface: ps), through a radial migratory stream of cells toward the dentate gyrus (DGMS). C High power photomicrograph of the HFA showing the distribution of ACE2 + cells in the ventricular epithelium (VE) and the subventricular zone (SVZ). D High power photomicrograph of ventricular surface showing ACE2 + cells in VE and the subventricular zone (SVZ) of proximal ( D ) side of the HFA. E Ventricular surface showing ACE2 immunopositive particles accumulate on the basal pole membrane of VE progenitors (arrows). F Picture showing ACE2 + migrating cells reaching the developing Dentate Gyrus (DG) at the pial surface (ps) of FDS. G High power photomicrographs of migratory ACE2 + cells reaching he ps of the DGMS. H – J High power pictures showing ACE2 expression in the advance processes of migrating cells: (arrows in J and H ), and cells around blood vessels (arrows in I ). K – M ACE2 expression in choroidal plexus cells. Both epithelial cells (ec) in the ventricular surface, and stromal pericytes (pc) around blood vessels (bv) were immunopositive (arrows). N ACE2 expression in DG ventricular epithelium and DGMS at the caudal pole of hippocampus (FC and IG). Scale bar: A 500 µm; B 300 µm; C , D 50 µm; E 10 µm; F 150 µm; G , N 100 µm; H–K 15 µm; L , M 5 µm. bv blood vessel, CA cornu ammonis, DGMS dentate gyrus migratory stream, ec epithelial cell, FDS fimbrio-dentate sulcus, Fi fimbria, HFA hippocampus fimbrial angle, pc pericytes, ps pial surface, SVZ sub-ventricular zone, st choroidal plexus stroma, VE ventricular Epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Membrane

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: Migration routes of DG progenitors. A Picture of a coronal section of the temporal lobe showing GFAP expression in radial glia cells and fibers of the Sub-ventricular Zone (SVZ) and Migratory Stream (DGMS) of the Dentate Gyrus (DG), from the Hippocampus Fimbrial Angle (HFA) to the pial surface (ps). DGMS has been delimited by arrows. B High power picture of the DG Ventricular Epithelium (VE), Sub-ventricular Zone (SVZ) and intermediate zone (IZ). Arrows label GFAP positive fibers. C High power picture of ACE2-expressing cells (cells with DAB-nickel precipitate; arrows) following GFAP radial glia fibers of DG ventricular epithelium (VE; DAB precipitate; arrows head). D , E Cresyl violet staining showing the DG ventricular (VE) and sub-ventricular (SVZ) zones with perivascular accumulation of cells crossing the sub-ventricular withe matter (alveus) in the DG migratory stream (DGMS; arrows). F ACE2 expression in perivascular migrating cells (arrowheads) and pericytes (arrows) in the alveus. G Doublecortin expression in DG migrating neuronal precursors and neurons in DG molecular layer (DGML). ACE2 is expressed in migratory precursors of the DGMS (DAB-nickel precipitate; arrows) but not in DGML, single doublecortin expressing cells (DAB precipitate). Scale bar: A 200 µm; B 100 µm; C – E 50 µm; F 25 µm. bv blood vessel, DGH Dentate Gyrus Hilux, DGMS Dentate Gyrus Migratory Stream, DGML Dentate Gyrus Molecular Layer, HFA Hippocampus Fimbrial Angle, ps pial surface, VE ventricular epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Migration, Expressing, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 + cells in DGMS are TBR2 + neural progenitors. A High power picture showing DG ventricular epithelium (VE) expressing ACE2 and TBR2 in the Hippocampal Fimbrial Angle (HFA). Double immunopositive, ACE2 and TBR2, cells are mainly localized in the basal surface of the VE (arrows) and in cells that are migrating into the subventricular zone (arrowhead). B Microphotograph showing the Dentate Gyrus Migratory Stream (DGMS) from the HFA to the pial surface (ps), where DGMS migratory cells follow dorsally to the Dentate Gyrus Hilux (DGH) or ventrally to Dentate Gyrus Molecular Layer (DGML). Insets localized the areas showed in ( A , C , and D ). C High power microphotograph showing ACE2 (DAB precipitate in the cytoplasm; arrowheads) and TBR2 (DAB-nickel precipitate in the nucleus; arrows) expression in ventricular and migratory cells. D High power of the zone represented in the inset of B showing double labeled ACE2 and TBR immunopositive cells in the DGMS (arrows). E Picture showing DG ventricular epithelium (VE) expressing ACE2 and TBR2 at the Hippocampal Fimbrial Angle (HFA) (small arrows) and in migrating cells in the SVZ and DGMS (arrowheads). Large arrows show ACE2 and TBR immunopositive cells migrating near to a blood vessel (bv). F Low power picture of an immunofluorescence processed section, showing the DGMS at the caudal pole of the hippocampus, where the DG turns dorsally to became the fasciola cinerea. Ventral and dorsal DGMS are delimited by arrows. G , H High power picture of immunofluorescence showing ACE2 immunolabeling in the cytoplasm (green) and TBR2 immunolabeling in the nucleus (reed) in DGMS cells. Scale bar: A , C , E , G 10 µm: B 200 µm; D , H 7 µm; F 300 µm. bv blood vessel, CA Ammon’s horn, DGH Dentate Gyrus Hilux, DGML Dentate Gyrus Molecular Layer, DGMS Dentate Gyrus Migratory Stream, HFA Hippocampus Fimbrial Angle, ps pial surface, SVZ sub-ventricular Zone, VE ventricular epithelium

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Labeling, Immunofluorescence, Immunolabeling

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 and neuropilin 1 (NP1) co-localize in the membrane of DGMS cells. A ACE2 immunofluorescent cells in the DGMS between sub-ventricular zone of the HFA and the ps (arrows). B High power confocal photomicrograph sowing the co-expression of ACE2 and NP1 in the membrane of migrating cells (arrows). The arrowhead shows the point where Z-stack projection have been reconstructed (view in the x and z plane), to demonstrate co-localization of ACE2 (reed dots) and NP1 (green/yellow dots) in the cellular membrane. C – E Microphotographs showing ACE2 and NP1 co-expression in DGMS cells. Inserts show the same cells with clear co-expression of ACE2 and NP1 in the membrane of leading processes (arrows). F – H DGMS cell showing strong co-expression of ACE2 and NP1 in the cellular membrane of leading processes (arrows). Scale bar: A : 250 µm; B 10 µ; C – E 20 µm; F – H 15 µm

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Membrane, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: Neuronal progenitors of the dentate gyrus express the SARS-CoV-2 cell receptor during migration in the developing human hippocampus

doi: 10.1007/s00018-023-04787-8

Figure Lengend Snippet: ACE2 expression in migrating neural crest progenitors. A , B Anti-ACE2 immunofluorescence showed expression of ACE2 in the cytoplasm of cultured dental derived NCP, with perinuclear accumulation. A’ Dental derived NCP express Nestin protein (red immunofluorescence). B’ Cells with polarized morphology ACE2 expression is stronger and accumulated in the cellular membrane of the progression pole (arrows). C – I Scratch-wound assay in confluent cultures. D 10 min after the scratch (t0h) cells at the wound edge strongly expressed ACE2 (arrows). E – I At 12–24 h (t12h, t24h) after the scratch, polarized elongated cells migrated into the wound with strong expression of ACE2 in the cellular membrane of advance processes (arrows) including in intercellular nanotubes (arrows head in H ). I Quantification of ACE2 expression by immunofluorescence intensity and intracellular distribution with the ImageJ tool for measuring corrected total cell fluorescence (CTCF). Data shown as mean ± S.D. The number of cells analyzed were: T0h (cytoplasm or membrane) n = 26, T12h (cytosol or membrane) n = 22. Comparisons between cytoplasm and cell membrane at T0h and at T12h were made with tests for paired samples (Wilcoxon signed-rank test) and comparisons between cytoplasm at T0h and T12h or cell membrane at T0h and T12h were made with test for independent samples (Mann–Whitney test for T0h test), using the software GraphPad Prism. a.u arbitrary units. Asterisks indicate p -value: **** p < 0.0001 ns not significant. J At 48 h after the scratch the wound was completely cellularized with increased expression of ACE2 in some cell membranes of neighboring cells at the place where the wound was performed (arrows)

Article Snippet: In addition to the rabbit polyclonal anti-ACE2 from Abcam, we have processed parallel sections using another rabbit polyclonal anti-ACE2 from Sigma-Aldrich and a mouse monoclonal anti-ACE2 from R&D Systems.

Techniques: Expressing, Immunofluorescence, Cell Culture, Derivative Assay, Membrane, Scratch Wound Assay Assay, Fluorescence, MANN-WHITNEY, Software

Journal: Gut Microbes

Article Title: Gut microbiota-derived metabolites confer protection against SARS-CoV-2 infection

doi: 10.1080/19490976.2022.2105609

Figure Lengend Snippet: Commensal Clostridia species reduce Ace2 expression via SCFA production. (A) Ace2 mRNA expression in male SPF and GF mice. (B) Ace2 mRNA expression in SPF and GF mice treated with antibiotics for two weeks. (C–D) 16S rRNA sequencing was performed. (C) Relative abundance is shown at the family level; (D) non-metric multidimensional scaling analysis is shown. (E) The relative abundance of Clostridia species in fecal pellets of antibiotic-treated mice was measured by qPCR. (F–G) Ace2 mRNA expression (F) and fecal SCFA concentrations (G) in SPF, GF, and GF mice colonized with either bulk fecal bacteria from SPF mice (CONV, conventionalized) or fecal bacteria enriched for Clostridia . (H) Ace2 mRNA expression in SPF, GF, or SCFA-treated GF mice. (I–J) ACE2 protein expression was measured via Western blot (i; densitometry relative to the Ctrl condition) and immunofluorescence (j; images are representative of 2 independent experiments) in kidneys from GF mice given control or SCFA water (butyrate alone or a cocktail of acetate, butyrate, and propionate). (K) Ace2 mRNA expression in the colons of control- or SCFA-treated wild type or Gpr41 −/− Gpr43 −/− mice. Error bars indicate mean±SEM. Significance was determined using one-way ANOVA with Tukey’s test for multiple comparisons. (A–H) represent 2-3 independent experiments; images in (I–K) are representative of 2 independent experiments. SI = small intestine; FB = fecal bacteria. * p <0.05; ** p <0.01. See also Figures S1 and S2A.

Article Snippet: The membrane was blocked using 3% non-fat milk for 1 h at room temperature and then probed with a rat anti-mouse ACE2 antibody (1:500; R&D Systems MAB3437) at 4°C overnight.

Techniques: Expressing, Sequencing, Bacteria, Western Blot, Immunofluorescence, Control

Journal: medRxiv

Article Title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

doi: 10.1101/2021.04.08.21255150

Figure Lengend Snippet: ( a ) Schematic outline of the S 3 /ACE2 neutralization assay. Anti-SARS-CoV-2 serum antibodies are monitored for their capacity in blocking the S 3 /ACE2 interaction. ACE2 binding to S protein was detected though use of a fluorescently labeled secondary antibody and the signal intensities are inversely proportional to the neutralizing potential of the anti-S protein antibodies. ( b ) Serum dilution IC 50 values were calculated for 104 healthy adult donor samples collected prior to November 2019 (pre-COVID-19 pandemic). The mean IC50 values and SD were used to establish a lower limit cutoff of 50 indicated by the dashed line (mean IC 50 of 12.5 + 4 × 9.0 SD = 50 serum dilution). ( c ) Representative concentration response curves for ten healthy donor serum samples and ( d ) ten SARS-CoV-2 seropositive donors with varying levels of anti-S protein IgG antibody. Mean ± s.d. shown in graph b . S protein / ACE2 structure was generated with PDB 7a98.

Article Snippet: Plates were agitated on a plate shaker for 60 minutes then the ACE2 mouse Fc fusion protein (Creative Biomart or produced by EPFL Protein Production and Structure Core Facility) was then added to each well at a final concentration of 1 μg/ml and agitated for a further 60 minutes.

Techniques: Neutralization, Blocking Assay, Binding Assay, Labeling, Concentration Assay, Generated

Journal: medRxiv

Article Title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

doi: 10.1101/2021.04.08.21255150

Figure Lengend Snippet: ( a ) Cross-validation studies between S 3 /ACE2 surrogate neutralization assay and the “gold standard” SARS-CoV-2 cytopathic effect (CPE) neutralization assay. Seropositive donors (n=206) with varying levels of anti-S protein antibodies were selected for comparison of the assays. Donors consisted of COVID-19 hospitalized patients (n=31), symptomatic infected donors with RT-PCR-documentation (n=64) and other seropositive donors identified through random sampling, volunteers or contact with a confirmed SARS-CoV-2 infected individual in a Swiss population serological survey (n=111). ( b ) Correlation between the live SARS-CoV-2 virus cytopathic effect and S protein pseudotyped neutralization assays. A group of 74 samples from S protein seropositive donors were compared in the live virus CPE and S protein pseudotyped virus cell based neutralization assays. Correlation between the two neutralization assays is represented by the black dashed line and the 20 serum dilution IC 50 cutoff for positivity in the CPE assay is shown with the blue dashed line.

Article Snippet: Plates were agitated on a plate shaker for 60 minutes then the ACE2 mouse Fc fusion protein (Creative Biomart or produced by EPFL Protein Production and Structure Core Facility) was then added to each well at a final concentration of 1 μg/ml and agitated for a further 60 minutes.

Techniques: Biomarker Discovery, Neutralization, Comparison, Infection, Reverse Transcription Polymerase Chain Reaction, Sampling, Virus

Journal: medRxiv

Article Title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

doi: 10.1101/2021.04.08.21255150

Figure Lengend Snippet: The surrogate neutralization assay was performed with two panels of S 3 coupled beads consisting of the 2019-nCov S protein and S mutations produced with one or more amino acid substitutions or deletions. ( a ) Concentration response curves of the REGN10933 therapeutic antibody evaluated against S protein mutations from three viral variants in the S 3 /ACE2 assay. ( b-c ) Serum dilutions from RT-PCR positive donors 3506 and 9504 ( b ) or 1034 and 5537 ( c ) in the S 3 -ACE2 assay with two separate panels of S 3 coupled beads. ( c ) Spider plot and heatmap showing IC 50 serum dilutions for donors 1034 and 5537 serums samples against the indicated S protein mutations found in variants of concern including B.1.1.7 and P1. In these graphs, green background corresponds to an IC 50 dilution >100 for strong blocking of the S 3 /ACE2 interaction, yellow background for moderate blocking with IC 50 dilutions from 50 to 100 and red background for low to no blocking activity against the indicated S protein mutant.

Article Snippet: Plates were agitated on a plate shaker for 60 minutes then the ACE2 mouse Fc fusion protein (Creative Biomart or produced by EPFL Protein Production and Structure Core Facility) was then added to each well at a final concentration of 1 μg/ml and agitated for a further 60 minutes.

Techniques: Neutralization, Produced, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Activity Assay, Mutagenesis