Journal: bioRxiv

Article Title: SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

doi: 10.1101/2020.10.27.357558

Figure Lengend Snippet: (a) Affinity between S1 and hACE2 determined by Bio-layer interferometry. Biotinylated S1 protein (S1–614D or S1–614G) was loaded onto surface of streptavidin biosensors. Association was conducted using hACE2 protein followed by dissociation. (b) Binding of Fc-tagged or polyhistidine-tagged S1 to BHK-hACE2 cells is shown as peaks of fluorescence detected by flow cytometry. (c) Replication kinetics of recombinant viruses in (left) Vero E6 at 37°C and (right) hNE at 33°C. Supernatant was collected at indicated time points and titrated by plaque assay. Data represent the mean ± s.d. of three replicates (Vero E6) and four replicates (hNE). (d) Replication kinetics of recombinant viruses in NhBE at 33°C (left), 37°C (middle) and 39°C (right). NhBE were infected with 100,000 PFU of each virus. Supernatants were collected daily and titrated by TCID50 assay. Data represent the mean ± s.d. of four replicates. ( c-d ) Statistical significance was determined by two-sided unpaired Student’s t -test without adjustments for multiple comparisons. ( c ) P values (left to right): left, NS, P =0.9132; NS P =0.0604; NS P =0.2394; NS P =0.2389; NS P =0.2778; NS P =0.2781; right, NS P =0.1520; NS P =0.3891; NS P =0.9110; NS P =0.8985; NS P =0.1464. ( d ) P values (left to right): left, NS P =0.7943; NS P =0.5025; NS P =0.6683; NS P =0.8985; * P =0.0220; middle, ** P =0.0065; NS P =0.4660; NS P =0.3134; * P =0.0159; right, ** P =0.0094; **** P <10 −4 ; ** P =0.0028; *** P =0.0009. ( e-f ) Competition assay of recombinant viruses in hNE at 33°C and NhBE at 33°C, 37°C and 39°C. The inoculum was prepared by mixing two viruses at 1:1 ratio based on PFU ml −1 and used for infection of hNE and NhBE. Apical wash and supernatant were collected daily, and extracted RNA was used for sequencing. ( e-f ) Bar graph shows proportion of sequencing reads encoding either S-614D or S-614G, and square dots represent individual data points.

Article Snippet: Recombinant hACE2 protein (Cat: RP01266) was purchased from ABclonal.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Recombinant, Plaque Assay, Infection, Virus, TCID50 Assay, Competitive Binding Assay, Sequencing

Journal: bioRxiv

Article Title: SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

doi: 10.1101/2020.10.27.357558

Figure Lengend Snippet: ( a ) Experimental scheme for infection of hACE2-KI mice intranasally infected recombinant SARS-CoV-2 S−614D and SARS-CoV-2 S−614G viruses. Oropharyngeal swabs were sampled daily and tissue samples were analyzed in sub-groups of 4 mice at 2 and 4 days post infection (dpi) in two independent experiments. ( b ) Quantitative RT-PCR analysis of oropharyngeal swabs of inoculated hACE2-KI and wild-type mice. ( c,d ) Pie chart representation of mean frequencies of A or G nucleotide at position 23,403 corresponding to SARS-CoV-2 S−614D and SARS-CoV-2 S−614G , respectively. Each pie chart illustrates the ratio of A/G detected from individual oropharyngeal swab samples (c) and tissues (d) at indicated time post infection. OB, olfactory bulb; ND, not detected.

Article Snippet: Recombinant hACE2 protein (Cat: RP01266) was purchased from ABclonal.

Techniques: Infection, Recombinant, Quantitative RT-PCR

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: The role of CTHRC1 in SG function. ( a ) Sweat test of WT and Cthrc1 −/− mice. Dashed boxes indicate sweaty areas of mice paw pads. ( b ) Quantification of sweat dots on the paw pads of WT and Cthrc1 −/− mice. Each dot in the statistical graph represents the number of dark dots on one hind paw from an individual mouse ( n = 10). ( c ) Light-sheet microscopy images showing the SGs and their surrounding microvascular networks of WT and Cthrc1 −/− mice. 3D reconstruction of light-sheet microscopy images was preformed using Imaris software (Scale bar: 100 μm). ( d ) Representative H&E images of SGs and their adjacent microvessels. Arrowheads highlight the location of microvessels (Scale bar: 100 μm, top row; Scale bar: 25 μm, bottom row). ( e ) Representative images of laser Doppler blood flow perfusion and corresponding BF of WT and Cthrc1 −/− mice. Dashed boxes are at the same location as the ROIs in BF and indicate the palm pads of the mice left hind paws. ( f ) Analysis of the blood flow of the ROIs of WT and Cthrc1 −/− mice ( n = 3). Results were normalized to the WT mice. Results are presented as the mean ± SD, * p < 0.05, * * * p < 0.001. CTHRC1 Collagen triple helix repeat containing-1 protein, Cthrc1 collagen triple helix repeat containing-1 gene, SG sweat gland, WT wild type, H&E hematoxylin and eosin, BF bright field, ROIs regions of interest, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Microscopy, Software, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: The role of CTHRC1 in the development of SGs and their adjacent vasculature. ( a ) Comparison of SG markers K14 and K18 and secretion-related marker ATP1a1 between WT and Cthrc1 −/− mice. K14, K18 and ATP1a1, green; CD31, red; DAPI, blue (Scale bar: 50 μm). ( b – d ) The relative expression values of K8 , K18 and Atp1a1 mRNA of SGs of WT and Cthrc1 −/− mice ( n = 3). ( e ) Comparison of vasculature formation around SGs by staining for endothelial adherens (VE-cadherin) and tight junctions’ marker (ZO-1). VE-Cadherin and ZO-1, green; K18 and K19, red; DAPI, blue (Scale bar: 50 μm). ( f ) Scatter plot showing the down-regulated genes in the Cthrc1 −/− mice compared to WT mice by GO enrichment analysis (top 20 GO terms). The rich factor represents the ratio of the down-regulated genes to all genes enriched in the corresponding GO term. Dot size represents the number of down-regulated genes enriched in a specific GO term. Dot color represents the P value obtained by GO analysis. P < 0.05 was used to indicate significant enrichment. ( g ) The relative expression values of Agtr1a, Flt4 and Vegfd mRNA in dermal tissue of WT and Cthrc1 −/− mice ( n = 3). Results are presented as the mean ± SD, * * * p < 0.001, * * * * p < 0.0001; ns, not significant. CTHRC1 Collagen triple helix repeat containing-1 protein, Cthrc1 collagen triple helix repeat containing-1 gene, SG sweat gland, K cytokeratin, ATP1a1 ATPase Na + /K + transporting subunit alpha 1, CD31 platelet and endothelial cell adhesion molecule 1, WT wild type, mRNA messenger RNA, VE-cadherin vascular endothelial cadherin, ZO-1 zonula occludens-1, DAPI 4′,6-diamidino-2-phenylindole, GO gene ontology, MAPK mitogen-activated protein kinase, Agtr1a , angiotensin II receptor type 1a, Flt4 fms related receptor tyrosine kinase 4, Vegfd vascular endothelial growth factor D, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Comparison, Marker, Expressing, Staining, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: Identification of WT and Cthrc1 −/− mice-derived DMECs. ( a ) Schematic overview of the procedure carried out to isolate and purify DMECs. ( b ) Morphological properties of DMECs at days 5 and 7 (Scale bar: 100 μm, top row; Scale bar: 500 μm, bottom row). ( c ) Factor VII flow cytometry analysis of the positive rate of DMECs. DMECs displaying positive staining for ( d ) CD31 (Scale bar: 50 μm), ( e ) factor VII (Scale bar: 50 μm) and ( f ) BSL (Scale bar: 50 μm). ( g ) The relative expression values of Cthrc1 mRNA in DMECs ( n = 3). ( h ) Western blot analysis showing the CTHRC1 expression level of DMECs ( n = 3). Results are presented as the mean ± SD, * * * p < 0.001. Cthrc1 Collagen triple helix repeat containing-1 gene, WT wild type, DMECs dermal microvascular endothelial cells, CD31 platelet and endothelial cell adhesion molecule 1, BSL Bandeiraea simplicifolia lectin, mRNA messenger RNA, CTHRC1 collagen triple helix repeat containing-1 protein, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Derivative Assay, Flow Cytometry, Staining, Expressing, Western Blot, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: Regulation of CTHRC1 of the angiogenic ability of DMECs derived from WT and Cthrc1 −/− mice. ( a ) Immunofluorescence analysis of Ki67 expression of DEMCs after treatment with different concentrations of rmCTHRC1. The addition of VEGF (50 ng/ml) was used as a positive control. Ki67, green; DAPI, blue (Scale bar: 50 μm). ( b ) Quantification of the rate of Ki67 + DMECs in randomly acquired confocal images ( n = 5). ( c ) Florescent imaging displaying DiI-Ac-LDL uptake by DMECs in response to different concentrations of rmCTHRC1 stimuli. The addition of VEGF (50 ng/ml) was used as a positive control. DiI-AC-LDL, red; DAPI, blue (Scale bar: 50 μm). ( d ) Quantify the geometric mean fluorescence intensity of DiI-AC-LDL uptake by DMECs in randomly acquired confocal images ( n = 5). ( e ) Phase-contrast images of the capillary-like structures of DMECs treated with rmCTHRC1 (300 ng/ml) after 10 h. The addition of VEGF (50 ng/ml) and PBS was used as a positive and negative controls, respectively (Scale bar: 200 μm). ( f , g ) Quantification of tube formation degree of DMECs using tube length and nodes number (n = 9). ( h ) Immunofluorescence analysis of intercellular junction formation of DMECs treated with rmCTHRC1 (300 ng/ml) after 3 days (Scale bar: 100 μm). Results are presented as the mean ± SD, * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001; ns, not significant. CTHRC1 Collagen triple helix repeat containing-1 protein, Cthrc1 collagen triple helix repeat containing-1 gene, WT wild type, DMECs dermal microvascular endothelial cells, Ki67 antigen identified by monoclonal antibody Ki 67, rmCTHRC1 mouse CTHRC1 recombinant protein, VEGF vascular endothelial growth factor, DAPI 4′,6-diamidino-2-phenylindole, DiI-Ac-LDL DiI-labeled acetylated low-density lipoprotein, PBS phosphate buffered saline, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Derivative Assay, Immunofluorescence, Expressing, Positive Control, Imaging, Fluorescence, Recombinant, Labeling, Saline, Standard Deviation

Journal: Burns & Trauma

Article Title: Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo

doi: 10.1093/burnst/tkac035

Figure Lengend Snippet: rmCTHRC1 promotes the recovery of SG function by inducing adjacent microvascular network remodeling. ( a ) Workflow for rmCTHRC1 injection and evaluating the blood flow perfusion and SG function recovery of Cthrc1 −/− mice. ( b ) Representative images of laser Doppler blood flow and corresponding BF of Cthrc1 −/− mice treated with rmCTHRC1 (600 ng/ml) for 0, 3 and 7 days. The injection of PBS was used as a negative control. Dashed boxes are at the same location as the ROIs in BF and indicate the palm pads of the mice left hind paws. ( c ) Analysis of the blood flow of the ROIs ( n = 3). Results were normalized to the PBS group at 0 days. ( d ) Sweat test of Cthrc1 −/− mice treated with rmCTHRC1 for 7 days. The PBS group was used as a control. Dashed boxes indicate sweaty areas of mouse paw pads (at the same location as the ROIs in BF). ( e ) Quantification of sweat dots on the paw pads of Cthrc1 −/− mice. Each dot represents the number of dark dots on one hind paw from an individual mouse ( n = 5). ( f , g ) Representative H&E and immunofluorescence images of SGs of Cthrc1 −/− mice treated with rmCTHRC1 or PBS for 7 days (Scale bar: 100 μm, top rows; Scale bar: 50 μm, bottom rows). ( h , i ) Immunofluorescence analysis of the number (CD31), endothelium junction (VE-cadherin), and basement membrane (laminin) of SG adjacent microvessels. K18, ATP1a1, VE-cadherin, green; ZO-1, CD31, K19, Laminin, red; DAPI, blue (Scale bar: 100 μm, top rows; Scale bar: 50 μm, bottom rows). Results are presented as the mean ± SD, * p < 0.05. rmCTHRC1 Mouse CTHRC1 recombinant protein, SG sweat gland, Cthrc1 collagen triple helix repeat containing-1 gene, BF bright field, PBS phosphate-buffered saline, ROIs regions of interest, H&E hematoxylin and eosin, CD31 platelet and endothelial cell adhesion molecule 1, VE-cadherin vascular endothelial cadherin, K cytokeratin, ATP1a1 ATPase Na + /K + transporting subunit alpha 1, ZO-1 zonula occludens-1, DAPI 4′,6-diamidino-2-phenylindole, SD standard deviation

Article Snippet: DMECs were first harvested and resuspended in an EGM-2 medium containing 300 ng/ml mouse CTHRC1 recombinant protein (rmCTHRC1; AdipoGen, USA).

Techniques: Injection, Negative Control, Control, Immunofluorescence, Membrane, Recombinant, Saline, Standard Deviation