Journal: Microorganisms
Article Title: Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum
doi: 10.3390/microorganisms11122968
Figure Lengend Snippet: Rabbit polyclonal antibodies to recombinant OMPs inhibit adhesion of F. necrophorum to bovine endothelial (EJG) cells. Adhesion inhibition assays with F. necrophorum and EJG cells using rabbit (1:100) polyclonal antibodies raised against ( A ) r43 kDa OMP; ( B ) rOmpH and rOmpA; ( C ) rOmpH, rOmpA, and rCSP; and ( D ) rOmpH, rOmpA, rCSP, and r43 kDa OMP. Significance was determined by unpaired two-tailed Student’s t -test, n = 3; * p < 0.05, ** p <0.01, *** p < 0.001, and **** p < 0.0001. The bars indicate the mean ± standard error of the mean (SEM) of data pooled from triplicate experiments. ( E ) Giemsa staining of EJG cells following adhesion inhibition assays: ( i ) uninfected (negative control), ( ii ) infected with F. necrophorum (positive control), and ( iii ) infected with F. necrophorum pre-treated with a combination of four polyclonal antibodies, as in panel ( D ).
Article Snippet: OMP proteins were identified by Western blot using a polyclonal antibody raised against F. necrophorum subsp. necrophorum OMPs as the primary antibody [ ] (Cocalico Biologicals, Stevens, PA, USA) and an alkaline phosphatase-conjugated anti-rabbit secondary antibody (12-448, EMD Millipore Corp, Billerica, MA, USA).
Techniques: Recombinant, Inhibition, Two Tailed Test, Staining, Negative Control, Infection, Positive Control
Figure 1 (A) Dot plot showing expression of cell markers across clusters. Cell types are listed on y axis showing expression of 45 selected genes identified by log fold change; genes are listed along x axis. Dot size reflects percentage of cells in a cluster expressing each gene, and dot color represents expression level. The plot shows clusters from 68,951 combined cells extracted from eight OEs with two biological replicates per condition. (B) Feature plot depicting expression of S SARS-CooV-2 transcript in hamster olfactory epithelium. Cell types are same as in
Figure 1 A (n = 2 biological replicates for each sample). (C) Representative confocal micrograph of IF-FISH experiment labeling RNA-FISH SARS-CoV-2 (magenta) and OMP protein (green) in hamster OE at 4 dpi. Rarely OMP-positive cells colocalize with SARS-CoV-2. No viral particles are detected in the axon bundles (asterisk). The line intensity scan drawn at the center of the OE section shows a discrete distribution of the pixel intensity of the two channels. (D) Top, IF-FISH confocal micrograph of OE tissue section showing colocalization of RNA FISH signal of SARS-CoV-2 (magenta) and antibody staining for OMP (green), suggesting that a small percentage of OSNs is infected. Bottom, IF-FISH in hamster OB section at 4 dpi shows rare RNA-FISH SARS-CoV-2 (magenta) detected in proximity to glomeruli labeled by OMP protein (green). (E) RNA-FISH SARS-CoV-2 signal (magenta) in the OE and lamina propria strongly correlates with the microglia marker AIF1/Iba1 (yellow), as highlighted by the enlarged picture in the white box. The line intensity scan of one single cell shows that SARS-CoV-2 signal is correlated with AIF1/Iba1 suggesting engulfment of viral particles by microglia. " width="100%" height="100%">
Journal: Cell
Article Title: Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia
doi: 10.1016/j.cell.2022.01.024
Figure Lengend Snippet: SARS-CoV-2 infects hamster OSNs very infrequently, related to Figure 1 (A) Dot plot showing expression of cell markers across clusters. Cell types are listed on y axis showing expression of 45 selected genes identified by log fold change; genes are listed along x axis. Dot size reflects percentage of cells in a cluster expressing each gene, and dot color represents expression level. The plot shows clusters from 68,951 combined cells extracted from eight OEs with two biological replicates per condition. (B) Feature plot depicting expression of S SARS-CooV-2 transcript in hamster olfactory epithelium. Cell types are same as in Figure 1 A (n = 2 biological replicates for each sample). (C) Representative confocal micrograph of IF-FISH experiment labeling RNA-FISH SARS-CoV-2 (magenta) and OMP protein (green) in hamster OE at 4 dpi. Rarely OMP-positive cells colocalize with SARS-CoV-2. No viral particles are detected in the axon bundles (asterisk). The line intensity scan drawn at the center of the OE section shows a discrete distribution of the pixel intensity of the two channels. (D) Top, IF-FISH confocal micrograph of OE tissue section showing colocalization of RNA FISH signal of SARS-CoV-2 (magenta) and antibody staining for OMP (green), suggesting that a small percentage of OSNs is infected. Bottom, IF-FISH in hamster OB section at 4 dpi shows rare RNA-FISH SARS-CoV-2 (magenta) detected in proximity to glomeruli labeled by OMP protein (green). (E) RNA-FISH SARS-CoV-2 signal (magenta) in the OE and lamina propria strongly correlates with the microglia marker AIF1/Iba1 (yellow), as highlighted by the enlarged picture in the white box. The line intensity scan of one single cell shows that SARS-CoV-2 signal is correlated with AIF1/Iba1 suggesting engulfment of viral particles by microglia.
Article Snippet: Tissue sections were stained with primary antibodies against OMP( ) (1:50 dilution) and NP (1:200 dilution, MyBiosource cat no. MBS8574840), Anti-Iba1 (1:250 dilution, Abcam cat no. ab178846).
Techniques: Expressing, Labeling, Staining, Infection, Marker
Figure 6 (A) (Top) En bloc resection of the cribriform plate along with underlying mucosa from the olfactory cleft, which contains OE more superiorly and respiratory epithelium below. (Bottom) Section of this human olfactory epithelium stained for OMP (green) and LDB1 (red), OSN-specific and OSN-enriched markers, respectively. Nuclei are labeled with DAPI (blue). (B) Confocal micrograph of RNA FISH for SARS-CoV-2 gRNA (magenta) and OSN/OSN progenitor marker ATF-5 (green) in COVID-19 + human OE. The SARS-CoV-2 probe targets the antisense strand of the S gene, detecting replicating virus. Nuclei are stained with DAPI. SARS-CoV-2 signal is detected in the apical layers of the epithelium (asterisk), proximal to SUS cells, and in the basal layer where HBCs reside. Correlation of the RNA-FISH SARS-CoV-2 signal and markers for OSNs (ATF5), sustentacular cells (Krt-18), and microglia (AIF1/Iba1) is measured by Pearson’s correlation coefficient (R) in COVID-19 (red) and control (blue) human OE autopsies (top right panel). Quantification of RNA-FISH signal was measured as local maxima at the apical, neuronal, and basal layers for a total of 2,140 cells in infected and 1,819 cells in control OEs. Only apical and basal layers have significantly enriched signal in infected OEs. (C) RNA-FISH SARS-CoV-2 signal (magenta), detected in the neuronal layer (OE), marked in between the two white lines, and lamina propria. S probe signal (magenta) strongly correlates with microglia marker AIF1/Iba1 (yellow) immunofluorescence (IF) signal, as indicated by arrows. The number of AIF1/Iba1-positive cells is measured over the number of total cells counted (right panel). In COVID-19 patients, a significantly enrichment of microglia is observed in the lamina propria, while in the neuronal layer (OE), more variability between images is observed. (D) Bar plot of the percentage of Atf5 RNA + cells over total number of DAPI-positive cells on sections of COVID-19 (146) and control (189) human OEs. No significant difference between samples is detected (n = 508 for 146, n = 458 for 189). Cells with more than two RNA puncta were counted as positive. On the right, a representative image of sample 146 for Atf5 RNA-FISH (red) and OMP protein (green). (E) Percentage of total counts of OE, RE, and immune-cell-type genes. Pairwise Wilcoxon test shows no significant difference between samples. Distribution of normalized counts for OE and RE genes for each sample (bottom panels). (F) SARS-CoV-2 counts plotted in decreasing order (red) together with normalized counts for OE (blue), RE (black), and immune cells (gray). (G) Correlation of house-keeping genes (ACTB, GAPDH, PPIA, and PGK1) and post-mortem times were used as a proxy of RNA integrity. Pearson’s test shows no significant correlation. (H) Coverage map for hCoV-OC43 in sample 2,186. (I) Surrogate variable analysis reveals sample 205 as an extreme outlier. SV1 does not correlate with known variables (COVID-19, processing batch, age, sex, post-mortem time, or days after symptoms onset), but rather in all cases distinguishes sample 205 from other samples. (J) Box plot depicting normalized counts between pooled control and infected samples for antiviral genes. (K) GO analysis for upregulated genes depicting significant enrichment for genes involved in immune/antiviral responses. " width="100%" height="100%">
Journal: Cell
Article Title: Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia
doi: 10.1016/j.cell.2022.01.024
Figure Lengend Snippet: Quality control analyses of human OE autopsies, related to Figure 6 (A) (Top) En bloc resection of the cribriform plate along with underlying mucosa from the olfactory cleft, which contains OE more superiorly and respiratory epithelium below. (Bottom) Section of this human olfactory epithelium stained for OMP (green) and LDB1 (red), OSN-specific and OSN-enriched markers, respectively. Nuclei are labeled with DAPI (blue). (B) Confocal micrograph of RNA FISH for SARS-CoV-2 gRNA (magenta) and OSN/OSN progenitor marker ATF-5 (green) in COVID-19 + human OE. The SARS-CoV-2 probe targets the antisense strand of the S gene, detecting replicating virus. Nuclei are stained with DAPI. SARS-CoV-2 signal is detected in the apical layers of the epithelium (asterisk), proximal to SUS cells, and in the basal layer where HBCs reside. Correlation of the RNA-FISH SARS-CoV-2 signal and markers for OSNs (ATF5), sustentacular cells (Krt-18), and microglia (AIF1/Iba1) is measured by Pearson’s correlation coefficient (R) in COVID-19 (red) and control (blue) human OE autopsies (top right panel). Quantification of RNA-FISH signal was measured as local maxima at the apical, neuronal, and basal layers for a total of 2,140 cells in infected and 1,819 cells in control OEs. Only apical and basal layers have significantly enriched signal in infected OEs. (C) RNA-FISH SARS-CoV-2 signal (magenta), detected in the neuronal layer (OE), marked in between the two white lines, and lamina propria. S probe signal (magenta) strongly correlates with microglia marker AIF1/Iba1 (yellow) immunofluorescence (IF) signal, as indicated by arrows. The number of AIF1/Iba1-positive cells is measured over the number of total cells counted (right panel). In COVID-19 patients, a significantly enrichment of microglia is observed in the lamina propria, while in the neuronal layer (OE), more variability between images is observed. (D) Bar plot of the percentage of Atf5 RNA + cells over total number of DAPI-positive cells on sections of COVID-19 (146) and control (189) human OEs. No significant difference between samples is detected (n = 508 for 146, n = 458 for 189). Cells with more than two RNA puncta were counted as positive. On the right, a representative image of sample 146 for Atf5 RNA-FISH (red) and OMP protein (green). (E) Percentage of total counts of OE, RE, and immune-cell-type genes. Pairwise Wilcoxon test shows no significant difference between samples. Distribution of normalized counts for OE and RE genes for each sample (bottom panels). (F) SARS-CoV-2 counts plotted in decreasing order (red) together with normalized counts for OE (blue), RE (black), and immune cells (gray). (G) Correlation of house-keeping genes (ACTB, GAPDH, PPIA, and PGK1) and post-mortem times were used as a proxy of RNA integrity. Pearson’s test shows no significant correlation. (H) Coverage map for hCoV-OC43 in sample 2,186. (I) Surrogate variable analysis reveals sample 205 as an extreme outlier. SV1 does not correlate with known variables (COVID-19, processing batch, age, sex, post-mortem time, or days after symptoms onset), but rather in all cases distinguishes sample 205 from other samples. (J) Box plot depicting normalized counts between pooled control and infected samples for antiviral genes. (K) GO analysis for upregulated genes depicting significant enrichment for genes involved in immune/antiviral responses.
Article Snippet: Tissue sections were stained with primary antibodies against OMP( ) (1:50 dilution) and NP (1:200 dilution, MyBiosource cat no. MBS8574840), Anti-Iba1 (1:250 dilution, Abcam cat no. ab178846).
Techniques: Control, Staining, Labeling, Marker, Virus, Infection, Immunofluorescence
Journal: AIDS
Article Title: HIV-1 detection in the olfactory mucosa of HIV-1-infected participants
doi: 10.1097/qad.0000000000002102
Figure Lengend Snippet: Fig. 1. Immunohistochemistry of the olfactory mucosa sample from subject #12. (a) Low power H&E image showing olfactory mucosa cells intermixed with brush bristles (arrows). (b) Anti-OMP immunohistochemistry showing a strong (þþþ) and diffuse staining. (c and d) Anti-CD4þ and anti-CD8þ immunohistochemistry, respectively, showing few positive cells (arrows) mixed with olfactory mucosa cells. H&E, hemotoxylin and eosin; OMP, olfactory marker protein.
Article Snippet: Hematoxylin and eosin slides were examined, and immunohistochemistry was performed using antibodies against olfactory marker protein (OMP) (clone B-6) (Santa Cruz Biotechnology Inc., Dallas, Texas, USA), CD4þ (clone SP35), CD8þ (clone SP57) (Ventana Medical Systems Inc., Tucson, Arizona, USA), and TAT [clone 1 (02–001)] (Thermo Fisher Scientific, Rockford, Illinois, USA).
Techniques: Immunohistochemistry, Staining, Marker
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