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

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: 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, Scratch Wound Assay Assay, Fluorescence, MANN-WHITNEY, Software

Journal: Biology

Article Title: ACE2 Protein Landscape in the Head and Neck Region: The Conundrum of SARS-CoV-2 Infection

doi: 10.3390/biology9080235

Figure Lengend Snippet: Negative and positive controls for ACE2 used for both antibodies. Kidney tissues were used as negative controls and kidney or small intestine tissues for positive controls. Scale bar = 100 µm.

Article Snippet: The polyclonal rabbit anti-ACE2 (ab15348, Abcam, Cambridge, UK) and the monoclonal mouse anti-ACE2 (MAB933, R&D systems, Minneapolis, MN, USA) were diluted (1:1500 and 1:50, respectively) in PBS with 0.05% casein, were applied on sections and incubated overnight at 4 °C.

Techniques:

Journal: Biology

Article Title: ACE2 Protein Landscape in the Head and Neck Region: The Conundrum of SARS-CoV-2 Infection

doi: 10.3390/biology9080235

Figure Lengend Snippet: Immunohistochemical labeling of ACE2 in different head and neck tissues. ( A ) Normal salivary gland with ACE2 positive acini and ducts. ( B ) Whartin tumor with positive oncocytic columnar cells; a few inflammatory cells exhibit high expression of ACE2 (arrows). ( E ) Sinus: positive ciliated epithelium, red circle indicated the positivity in inflammatory cells within the stroma. ( F ) Sinus: positive seromucous glands. ( I ) Positive squamous stratified epithelium of lingua (buccal cavity). ( J ) Positive squamous stratified epithelium (weak intensity) of tonsil with some positive inflammatory cells with strong intensity (arrows). ( M ) Positive ciliated epithelium of vocal cord. ( N ) Positive squamous stratified epithelium of vocal cord, arrow indicates positivity in fibroblast. ( Q ) Positive squamous stratified epithelium of larynx. ( R ) Positive seromucous glands of larynx. ( U ) Positive squamous stratified epithelium of oropharynx. ( V) Faint positivity in squamous stratified epithelium of hypopharynx. ( C , D , G , H , K , L , O , P , S , T , W and X ) represent the same area than ( A , B , E , F , I , J , M , N , Q , R , U and V ) but using the monoclonal antibody; there is no positivity in all these locations using this antibody. For all photographs, scale bar = 100 µm.

Article Snippet: The polyclonal rabbit anti-ACE2 (ab15348, Abcam, Cambridge, UK) and the monoclonal mouse anti-ACE2 (MAB933, R&D systems, Minneapolis, MN, USA) were diluted (1:1500 and 1:50, respectively) in PBS with 0.05% casein, were applied on sections and incubated overnight at 4 °C.

Techniques: Immunohistochemical staining, Labeling, Expressing

Journal: Biology

Article Title: ACE2 Protein Landscape in the Head and Neck Region: The Conundrum of SARS-CoV-2 Infection

doi: 10.3390/biology9080235

Figure Lengend Snippet: Correlation between patient clinical data and ACE2 expression score in epithelial cells. Clinical data have been obtained for 52 patients.

Article Snippet: The polyclonal rabbit anti-ACE2 (ab15348, Abcam, Cambridge, UK) and the monoclonal mouse anti-ACE2 (MAB933, R&D systems, Minneapolis, MN, USA) were diluted (1:1500 and 1:50, respectively) in PBS with 0.05% casein, were applied on sections and incubated overnight at 4 °C.

Techniques: Expressing

Journal: Biology

Article Title: ACE2 Protein Landscape in the Head and Neck Region: The Conundrum of SARS-CoV-2 Infection

doi: 10.3390/biology9080235

Figure Lengend Snippet: The domain structure of ACE2 and collectrin. Both proteins are type I transmembrane glycoprotein with a signal peptide sequence (purple), a transmembrane domain (black) and a short C-terminal cytoplasmic domain. ACE2 has an extracellular amino-terminal catalytic domain with one zinc binding motif (HEXXH) (orange) corresponding to the active site of the enzyme. The carboxy-terminal cytoplasmic domain of ACE2 share 48% sequence homology with collectrin. The monoclonal anti-ACE2 antibody recognizes a predicted sequence in the N-terminal extracellular domain between Gln18 and Ser740, while the polyclonal antibody targets a precise sequence in the intracellular C-terminal domain corresponding to the 788-805 amino acids located within the homology region denoted by the green box.

Article Snippet: The polyclonal rabbit anti-ACE2 (ab15348, Abcam, Cambridge, UK) and the monoclonal mouse anti-ACE2 (MAB933, R&D systems, Minneapolis, MN, USA) were diluted (1:1500 and 1:50, respectively) in PBS with 0.05% casein, were applied on sections and incubated overnight at 4 °C.

Techniques: Sequencing, Binding Assay

Journal: Biology

Article Title: ACE2 Protein Landscape in the Head and Neck Region: The Conundrum of SARS-CoV-2 Infection

doi: 10.3390/biology9080235

Figure Lengend Snippet: Semi-quantitative evaluation of ACE2 expression assessed with the polyclonal antibody in different cell types of seven head and neck sites. The number value corresponds to the median obtained with the Kruskal-Wallis test. The ACE2 expression in inflammatory cells and fibroblasts was reported as positive (+), negative (-), or positive with a strong intensity (++).

Article Snippet: The polyclonal rabbit anti-ACE2 (ab15348, Abcam, Cambridge, UK) and the monoclonal mouse anti-ACE2 (MAB933, R&D systems, Minneapolis, MN, USA) were diluted (1:1500 and 1:50, respectively) in PBS with 0.05% casein, were applied on sections and incubated overnight at 4 °C.

Techniques: Expressing

Journal: Viruses

Article Title: SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells

doi: 10.3390/v15030736

Figure Lengend Snippet: Distribution of ACE2 receptor and attachment of SARS-CoV-2 and human coronavirus (HCoV-NL63) to tissue sections of human trachea. These images show formalin-fixed paraffin-embedded cross-sections of human tracheal sections stained with ImmPRESS VR anti-mouse/rabbit IgG horseradish peroxidase (HRP) polymer detection kit. Dark brown represents the presence of a protein interacting with a specific antibody and is considered a positive expression. Pale brown background, and the nucleus counterstained with hematoxylin is blue. ( a ) Expression of ACE2 in human trachea revealed by immunostaining with mouse anti-ACE2 monoclonal antibody (4 μg/mL); and ( d ) corresponding negative control tissue sections stained with secondary anti-mouse HRP antibody only. ( b , c , e , f ) The tissues sections presented in this panel show virus binding in formalin-fixed paraffin-embedded cross-sections after overnight incubation with 250 μL ( b ) heat-inactivated SARS-Related Coronavirus 2, Isolate USA-WA1/2020 or ( c ) HCoV-NL63. ( e , f ) Virus-incubated sections that were stained with secondary anti-rabbit HRP antibody only. Scale bar—100 μm.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-ACE2 (4 μg/mL; E-11 Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-pan-cytokeratin (0.5 μg/mL; AE1/AE3; Bio-Rad Laboratories, Hercules, CA, USA); anti-HCoV-NL63 nucleocapsid (N) protein monoclonal antibody (2D4; Ingenasa-Eurofins, Madrid, Spain) (0.25 µg/mL) and a recombinant anti-SARS-CoV-2 nucleocapsid (N) protein rabbit monoclonal antibody (0.75 μg/mL) (BEI Resources) [The following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-SARS Coronavirus/SARS-Related Coronavirus 2 Nucleocapsid Protein (produced in vitro), NR-53791; SinoBio Cat: 40143-R001].

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Expressing, Immunostaining, Negative Control, Binding Assay, Incubation

Journal: Viruses

Article Title: SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells

doi: 10.3390/v15030736

Figure Lengend Snippet: Characterization of human respiratory epithelial cells (HRECs) for pan-cytokeratin and angiotensin-converting enzyme-2 (ACE2) using target-specific markers. Primary HRECs stained for mouse monoclonal to anti-pan-cytokeratin (epithelial cell marker; 0.5 μg/mL) showed strong expression both in ( a ) IHC and ( c ) flow cytometry analysis. ( b ) HRECs incubated with a secondary antibody only displayed minimum background. ( c ) HRECs were also quantified for the ACE2 expression using flow cytometry. Flow cytometry data was collected using an Attune NxT flow cytometer. A representative of 10,000 events were acquired and analyzed for each sample. Cells were gated for singlet population using forward (FSA) and side-scatter (SSA) properties, and the mean of percent live cell population was used to quantify the levels of ( c-i ) pan-cytokeratin and ( c-ii ) ACE2 ( n = 4). The bar graph represents the mean with the standard error of the mean (SEM).

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-ACE2 (4 μg/mL; E-11 Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-pan-cytokeratin (0.5 μg/mL; AE1/AE3; Bio-Rad Laboratories, Hercules, CA, USA); anti-HCoV-NL63 nucleocapsid (N) protein monoclonal antibody (2D4; Ingenasa-Eurofins, Madrid, Spain) (0.25 µg/mL) and a recombinant anti-SARS-CoV-2 nucleocapsid (N) protein rabbit monoclonal antibody (0.75 μg/mL) (BEI Resources) [The following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-SARS Coronavirus/SARS-Related Coronavirus 2 Nucleocapsid Protein (produced in vitro), NR-53791; SinoBio Cat: 40143-R001].

Techniques: Staining, Marker, Expressing, Flow Cytometry, Incubation

Journal: European Thyroid Journal

Article Title: Detection of SARS-CoV-2 infection in thyroid follicular cells from a COVID-19 autopsy series

doi: 10.1530/ETJ-22-0074

Figure Lengend Snippet: (A) ACE-2 expression in a thyroid specimen with extensive staining, and (B) in the lung, with staining in <10% of the cells (arrows) (scale bar: 100 μm); (C) TMPRSS2 expression in the thyrocytes with strong cytoplasmatic expression (scale bar: 100 μm) and (D) in the lung with strong cytoplasmatic/nuclear staining (scale bar: 100 μm); (E) Furin moderates cytoplasmatic expression in the thyrocytes (scale bar: 100 μm) and (F) in the lung with low cytoplasmatic staining (arrows) (scale bar: 100 μm).

Article Snippet: The antibodies used were: mouse monoclonal anti-SARS/SARS-CoV-2 coronavirus nucleocapsid antibody (Invitrogen, MA1-7404, 1:100); mouse monoclonal SARS-CoV/SARS-CoV-2 spike antibody ((1A9), GTX632604, Genetex (Irvine, CA, USA), 1:200); mouse monoclonal anti-ACE-2 (Invitrogen, MA5-31395, 1:2500); rabbit monoclonal anti-TMPRSS2 antibody ((EPR3862), Abcam, ab109131, 1:1000); rabbit polyclonal anti-furin (Invitrogen, PA5-96680; 1:200); and rabbit monoclonal anti-cleaved caspase-3 (Asp175, Cell Signalling, 9664, 1:1000).

Techniques: Expressing, Staining