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: Pathogens and Immunity

Article Title: Characteristics and Functions of Infection-enhancing Antibodies to the N-terminal Domain of SARS-CoV-2

doi: 10.20411/pai.v9i2.679

Figure Lengend Snippet: Mechanism of infection-enhancement of SARS-CoV-2. (A) Infection-enhancing activity of IgG and purified Fab generated from a panel of NTD-binding, infection-enhancing mAbs. Enhancement was measured in VSV-SARS-CoV-2 S pseudovirus (PV) assays using PV bearing S from Wuhan, Delta, or Omicron variants. Mean values for percentage enhancement by IgG and Fab against each PV were compared using a 2-tailed Student's t test, *** P <0.0001, NS=not significant. (B) Flow cytometry of cell-surface ACE2 expression on 293T-ACE2, intestinal (Caco-2), and respiratory (Calu-3) epithelial cell lines as compared to an isotype matched IgG2A control antibody. MFI=Mean Fluorescent Intensity. (C) Infection of 293T-ACE2, Caco-2 and Calu-3 epithelial cell lines with a Wuhan-PV. Luciferase activity was measured in cell lysates 24 hours after infection and expressed as the median of relative light units (RLU) for 5 replicate wells. (D) Percentage enhancement of Wuhan-PV infection by NTD-binding mAbs in 293T-ACE2, Calu-3, and Caco-2 cells. Mean values for percentage enhancement of infection between 2 cell types were compared using a 2-tailed Student's t test, *** P <0.0001.

Article Snippet: Cells were then stained with either anti-ACE2-Alexa Fluor® 647-conjugated antibody (R&D Systems) or an isotope-matched control mouse IgG2A antibody (R&D systems) for 30 minutes at room temperature.

Techniques: Infection, Activity Assay, Purification, Generated, Binding Assay, Flow Cytometry, Expressing, Control, Luciferase

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: ACE2 screening in human cells and the characterization of ACE2-NPs. (a) Western blotting detection of ACE2 in five cell lines. β-actin was used as the reference. (b) Immunofluorescence microscopy showing the location of ACE2 (red) in HEK-293T-ACE2 cells. Nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. (c) Immunofluorescence microscopy showing the location of ACE2 (red) and the adherence of S1 (green) on ACE2-NPs. The scale bar indicates 500 nm. (d) TEM image of ACE2-NPs. The scale bar indicates 200 nm. (e) Hydrodynamic diameters and surface charges of NPs. The results are shown as the means ± standard deviations (SDs). (f) ELISA results showing the ACE2 levels in NPs and cell lysates. ***, P < 0.001, relative to the total cell lysate group. (g) Comparison of the amounts of ACE2 antibody bound to ACE2-NPs and HEK-293T-ACE2 cells containing equal amounts of membrane content. n.s., not significant.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Inhibitory effect of ACE2-NPs on S1 recruitment. (a) Binding kinetics for NPs and SARS-CoV-2 RBD loaded on SA biosensors. (b) Western blotting detection of S1 and (e) D614G-S1 binding to HK-2 in the absence and presence of NPs. β-actin was used as the reference. (c) Immunofluorescence microscopy revealing the protective effect of NPs on cells exposed to S1 (green). The region of interest in the S1-treated group is magnified in the inset graph. Nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. (d) Binding kinetics for increasing concentrations of ACE2 and D614G-S1 loaded on SA biosensors. The fitted curves are colored red. The fitting coefficient ( R 2 ) is 0.96.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Binding Assay, Western Blot, Immunofluorescence, Microscopy, Staining

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Inhibitory effect of ACE2-NPs on S1-induced apoptosis. (a) Top nine enriched biological processes in GO analysis. (b) Protein bands of OPA1, cytochrome c, and cleaved caspase (c-caspase) 3. β-actin was used as the reference. (c) OPA1 and (d) cytochrome c mRNA expression relative to β-actin expression. The results are shown as as the means ± SDs. ***, P < 0.001. (e) Apoptosis of HK-2 cells exposed to S1 and D614G-S1. Representative flow cytometry scatter plots of cells stimulated by S1 in the absence and presence of 100 μg mL –1 ACE2-NPs are shown. The overall results are presented in a bar graph. ***, P < 0.001.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Expressing, Flow Cytometry

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Evaluation of the antiviral activity of ACE2-NPs. (a) Immunofluorescence microscopy showing the invasion of S pseudovirions into HK-2 cells. S pseudovirions were traced by S staining using a FITC-labeled antibody (green). Nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. (b) TEM images of S pseudovirions adsorbed onto NPs. The regions of interest are magnified in the inset graphs, in which pseudovirions and NPs are colored red and blue, respectively. The scale bar indicates 200 nm. (c) Dose-dependent antiviral activity of ACE2-NPs and (d) 293T-NPs. The fitted curves obtained by linear regression are in burgundy. The fitting coefficients of ACE2- and 293T-NPs are 0.98 and 0.99, respectively.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Activity Assay, Immunofluorescence, Microscopy, Staining, Labeling

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Inhibitory effect of ACE2-NPs on S pseudovirion infection in mice. (a) Diagram depicting the establishment of the pseudovirion-based mouse infection model. Adenovirus, Adv; pseudovirions, Pv. (b) Protein bands of Flag-tag and His-tag in mouse lungs. β-actin was used as the reference. (c) EGFP mRNA expression relative to β-actin expression in mouse livers, (d) lungs, and (e) kidneys. The results are shown as the means ± SDs. ***, P < 0.001. (f) Immunofluorescence microscopy revealing the inhibitory effect of ACE2-NPs on pseudovirion infection in mouse lungs. Flag-tag and His-tag are shown in red and green fluorescence, respectively. The scale bar indicates 20 μm.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Infection, FLAG-tag, Expressing, Immunofluorescence, Microscopy, Fluorescence

Journal: ACS Nano

Article Title: Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection

doi: 10.1021/acsnano.0c06836

Figure Lengend Snippet: Distribution and toxicity analysis of ACE2-NPs. (a) ELISA results showing the ACE2 content in mouse serum at 1, 3, 6, and 12 h post injection of ACE2-NPs. (b) In vitro imaging of DiO-labeled ACE2-NPs in mouse kidneys, lungs, spleens, livers, and hearts, at 0.5, 1, 3, 6, 12, and 24 h post intravenous administration. (c) Counts of RBCs, WBCs, and PLTs in mouse blood at 1, 3, and 5 days post injection of ACE2-NPs. The results are presented as the means ± SDs. (d) HE staining of the organs of mice treated with sterile PBS and ACE2-NPs. The scale bar indicates 200 μm.

Article Snippet: The ACE2 orientation was analyzed by determining the extents of binding of a FITC-labeled anti-ACE2 antibody (10108-MM36-F, Sino Biological) to ACE2-NPs and HEK-293T-ACE2 cells, as recently described.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, In Vitro, Imaging, Labeling, Staining