Journal: Scientific Reports

Article Title: Physiological activity in calm thermal indoor environments

doi: 10.1038/s41598-017-11755-3

Figure Lengend Snippet: PMV and PPD values obtained by collapsing across the sessions in the cooling experiment (upper panels) and the heating experiment (lower panels). The asterisks showed the significant differences (***p < 0.001). The grey areas in the left panels indicate the thermal comfort zone defined by the ASHARE Standard.

Article Snippet: PMV, PPD, and related parameters were obtained using a PMV meter (AM-101, Kyoto Electronics Manufacturing Co. Ltd., Kyoto, Japan).

Techniques:

Journal: Medicinal research reviews

Article Title: Drug repurposing approach to combating coronavirus: Potential drugs and drug targets

doi: 10.1002/med.21763

Figure Lengend Snippet: The genome organization of SARS-CoV, MERS-CoV, and SARS-CoV-2. The ORFs 1a/b encode 16 nsps. Other ORFs encode structural proteins including S, E, M, and N proteins as well as accessory proteins. E, envelop; M, membrane; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; nsp, nonstructural protein; ORF, open reading frame; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus

Article Snippet: 114 Both prophylactic and early therapeutic administration of remdesivir reduced lung viral load and improved clinical signs of disease as well as respiratory functions in a SARS-CoV infected mouse model. 112 Similar efficacy was also observed for prophylactic and therapeutic remdesivir treatment in a mouse model and a nonhuman primate (rhesus macaque) model of MERS-CoV infection, respectively.

Techniques:

Journal: Medicinal research reviews

Article Title: Drug repurposing approach to combating coronavirus: Potential drugs and drug targets

doi: 10.1002/med.21763

Figure Lengend Snippet: The determination of SARS-CoV-2 3CLpro and RdRp crystal structures facilitates the design and development of SARS-CoV-2 inhibitors. (A) The crystal structure of SARS-CoV-2 3CLpro in complex with a peptide-aldehyde inhibitor (PDB: 6M0K). (B) The cryo-EM structure of SARS-CoV-2 RdRp in complex with a template-primer RNA and remdesivir (PDB: 7BV2). 3CLpro, 3C-like protease; cryo-EM, cryogenic electron microscopy; RdRp, RNA-dependent RNA polymerase; SARS-CoV, severe acute respiratory syndrome coronavirus

Article Snippet: 114 Both prophylactic and early therapeutic administration of remdesivir reduced lung viral load and improved clinical signs of disease as well as respiratory functions in a SARS-CoV infected mouse model. 112 Similar efficacy was also observed for prophylactic and therapeutic remdesivir treatment in a mouse model and a nonhuman primate (rhesus macaque) model of MERS-CoV infection, respectively.

Techniques: Cryo-EM Sample Prep, Electron Microscopy

Journal: Medicinal research reviews

Article Title: Drug repurposing approach to combating coronavirus: Potential drugs and drug targets

doi: 10.1002/med.21763

Figure Lengend Snippet: The virus-based small molecule drugs with therapeutic potentials for CoVs

Article Snippet: 114 Both prophylactic and early therapeutic administration of remdesivir reduced lung viral load and improved clinical signs of disease as well as respiratory functions in a SARS-CoV infected mouse model. 112 Similar efficacy was also observed for prophylactic and therapeutic remdesivir treatment in a mouse model and a nonhuman primate (rhesus macaque) model of MERS-CoV infection, respectively.

Techniques: Activity Assay, Infection

Journal: Medicinal research reviews

Article Title: Drug repurposing approach to combating coronavirus: Potential drugs and drug targets

doi: 10.1002/med.21763

Figure Lengend Snippet: The host-based small molecule drugs with therapeutic potentials for CoVs

Article Snippet: 114 Both prophylactic and early therapeutic administration of remdesivir reduced lung viral load and improved clinical signs of disease as well as respiratory functions in a SARS-CoV infected mouse model. 112 Similar efficacy was also observed for prophylactic and therapeutic remdesivir treatment in a mouse model and a nonhuman primate (rhesus macaque) model of MERS-CoV infection, respectively.

Techniques: Activity Assay, Infection, In Vivo

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet:

Article Snippet: For SARS-CoV-2 spike binding ELISAs in B–7E, assays were performed as above, except for coating with the following proteins where indicated: SARS-CoV-2 wild type spike RBD (Cat# 40592-V08H, Sino Biologicals), SARS-CoV-2 D614G spike (Cat# 40589-V08H8, Sino Biologicals), SARS-CoV-2 B.1.617.2 spike (Cat# 40589-V08H10, Sino Biologicals), SARS-CoV-2 B.1.1.529 spike (Cat# 40589-V08H26), or SARS-CoV-2 BA.2 spike (Cat# 40589-V08H28, Sino Biologicals).

Techniques: Virus, Recombinant, Lysis, Reporter Gene Assay, Cell Stimulation, Electron Microscopy, Luminex, Enzyme-linked Immunospot, Luciferase, Plasmid Preparation, Software, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Schematic of the screening for candidate membrane proteins involved in SARS-CoV-2 entry. Schematic illustration of the labeling procedure according to EMARS. After EMARS reaction, the fluorescein-labeled proteins were purified and then analyzed using mass spectrometry.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Purification, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: SARS-CoV-2 spike protein-based EMARS probes. ( A ) ACE2 expression in Caco-2 and A549 cells. Western blot analysis of Caco-2 and A549 cell lysates; 10 μg protein samples were subjected to SDS-PAGE (on 10% gels) and stained with anti-ACE2 antibody. Arrows indicate bands of the ACE2 protein. ( B ) Immunocytochemical staining of ACE2 in Caco-2 and A549 cells. Staining with the anti-ACE2 antibody (ACE2+2 nd 568) was performed as described in Experimental procedure . Negative control samples (2 nd 568) were also prepared simultaneously. White bar: 100 μm. ( C ) Immunocytochemical staining of SARS-CoV-2 spike proteins in Caco-2 and A549 cells. Staining of monovalent Alexa Fluor 488-labeled spike proteins (spike-488) and the two-step staining (spike protein followed by Alexa Fluor 488 secondary antibody; spike+2 nd 488) were performed with DIC images. Negative control samples (cAb-488 or 2 nd 488) were also prepared simultaneously. White bar: 100 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Expressing, Western Blot, SDS Page, Staining, Negative Control, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Proximity labeling near the cell membrane-bound SARS-CoV-2 spike protein. ( A, B ) Fluorescein-labeled proximal proteins around cell membrane-bound SARS-CoV-2 spike proteins. The EMARS reaction described in the “Experimental procedure” was performed in Caco-2 ( A ) and A549 ( B ) cells using a spike protein ( Spike (RBD) ) and HRP-conjugated anti-mouse IgG ( mouse HRP ). The EMARS products were subsequently subjected to Western blot analysis to detect fluorescein-labeled proteins as candidate proximal proteins. In Caco-2 cells, HRP-conjugated Cholera Toxin B Subunit B ( CTxB-HRP ) was used for EMARS reaction as the positive control for membrane protein labeling. For loading controls, the PVDF membrane was stained with Coomassie Brilliant Blue after western blot analysis (right column)

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Labeling, Western Blot, Positive Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Co-localization of the identified proteins with cell membrane-bound SARS-CoV-2 spike proteins. Representative images of co-localization with SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells were co-stained for SARS-CoV-2 spike protein (green) and the antibodies recognizing ACE2, CD133, Cadherin 17, DPP4, and VAPA (Red). The resulting specimens were subsequently stained with appropriate secondary antibodies and DAPI (Blue), then observed using confocal microscopy (20× objective). Co-localization is indicated in yellow in the “Merge” images. White bar: 10 μm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Candidate proteins located near SARS-CoV-2 spike proteins. ( A to D ) Morphological observation of SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells observed using electron microscopy. Cultured Caco-2 cells were fixed and co-stained with the SARS-CoV-2 spike protein (indicated as 20 nm particles), and candidate molecules identified. CD133 ( A ), DPP4 ( B ), CDH17 ( C ), and VAPA ( D ) are indicated as 10 nm particles. Red arrows indicate the locations of SARS-CoV-2 spike proteins. Yellow arrow heads indicate the location of each candidate protein. Scale bar; 200 or 500 nm.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: Electron Microscopy, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: In vitro infection assay of SARS-CoV-2 pseudovirus. ( A ) Expression of ACE2 and candidate membrane proteins in transfectant HEK293 cells. Western blot analysis of transfectant cell lysates; Each cell lysates were subjected to SDS-PAGE (on 6 to 10% gels) and stained with antibodies recognizing ACE2 or candidate membrane proteins. Arrows indicate bands of the target proteins. The CBB staining image indicates load control. Asterisks indicate predicted nonspecific bands. ( B ) Schematic illustration of the assay procedure using HEK293T transfectant host cells. ( C ) Representative images of GFP-positive P-ACE2 cells after pSARS-CoV-2 infection. ACE2-expressing HEK293T cells were treated (pSARS-CoV-2 (+)) or not treated (pSARS-CoV-2 (-)) with pSARS-CoV-2, followed by fluorescein microscopic observation. Two independent experiments were carried out. White bar: 100 μm. ( D-F ) Flow cytometric analysis of pSARS-CoV-2-infected cells. P-ACE2 cells ( D ), candidate protein-single expressing cells ( E ), and candidate protein-coexpressing P-ACE2 cells ( F ) were analyzed using BD FACS Canto II. GFP-positive cells were defined as the infected cells with a GFP fluorescence intensity of 10 3 or higher (P3 area). Two ( E ) or five ( D and F ) independent replications were carried out in each experiment. ( G ) Increase in pSARS-CoV-2 infection in candidate protein-coexpressing P-ACE2 cells. The number of GFP-positive cells in each cell was quantified using flow cytometry. The number of infected cells (GFP-positive) in P-ACE2–CD133, –CDH17, and –VAPA was significantly higher than that in P-ACE2 cells ( P < 0.05 or P < 0.005; Dunnett's test), but not in P-ACE2-GPC3 (N.D.) as the negative control.

Article Snippet: Recombinant SARS-CoV-2 spike protein (S1-RBD) was purchased from Sino Biological (40592-V05H; S1-RBD-mouse Fc, Beijing, China).

Techniques: In Vitro, Infection, Expressing, Transfection, Western Blot, SDS Page, Staining, Fluorescence, Flow Cytometry, Negative Control

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a Schematic illustrating the SARS-COV-2 genomic landscape and the deletions/substitutions in ΔE G /ΔE G 68, main structural and accessory proteins indicated. Four overlapping fragments covering the whole SARS-CoV-2 genome were amplified by PCR (Fragments A-D, see also Supplementary Fig. ). b Complementation efficiency of Vero-E2T cells, analyzed by FFU (focus forming units) quantification after infection with ΔE G 3* (ΔE G with an additional stop codon in ORF3a) at different multiplicities of infection (MOI) or medium-only control (ctrl) three and six days post-infection ( n = 2 individual cultures), for corresponding genome copies, see Supplementary Fig. . c Passaging of 1:10 and 1:100 (after p2) dilutions of cell-free supernatant (Input = Passage 0) of wild-type SARS-CoV-2 (Muc-1, B.1), ΔE G 3* and ΔE G 68 on non-complementing Vero E6 cells (initial infection MOI = 1). Data from one representative experiment are shown; analysis was performed in duplicates. d Transmission electron microscopy analysis of recombinant wild-type SARS-CoV-2 (rCoV2) or vaccine candidates ΔE G and ΔE G 68 showing the presence of the characteristic spike protein (indicated with arrows). e Immunoblot analysis of viral protein production in Vero E6-TMPRSS2 cells infected for 24 h with rCoV2, E**fs, ΔE G 3*, ΔE G 68 or medium only (ctrl), probed with anti-NSP2, anti-N, anti-S, anti-ORF3a (full-length [fl] and truncated [tr] forms indicated with arrows), anti-ORF6, anti-ORF7a, anti-ORF8, and anti-beta-actin (β-ACT) antibodies. f Detection of N and S (magenta), F-actin (green), nuclei (blue) and ORF6 or ORF8 in Vero E6-TMPRSS2 cells infected with rCoV2, E**fs, ΔE G 3* or ΔE G 68. Scale bar is 100 nm in ( d ), 50 µm and 20 µm in ( f ) (overview and ROI images, respectively).

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Amplification, Infection, Control, Passaging, Transmission Assay, Electron Microscopy, Recombinant, Western Blot

Journal: NPJ Vaccines

Article Title: High protection and transmission-blocking immunity elicited by single-cycle SARS-CoV-2 vaccine in hamsters

doi: 10.1038/s41541-024-00992-z

Figure Lengend Snippet: a – c Modulation after transfection: Flow cytometry staining of THP-1 cells for HLA-A/B/C, CD80, CD275, and HLA-DR surface expression 48 h after transfection with expression plasmids for ORF6 ( a ), ORF8 ( b ), or Envelope ( c ) proteins, compared with control transfection. d – i Modulation after infection: d A549-ACE2-TMPRSS2 cells were infected with recombinant wild-type (rCoV2), E**fs, ΔE G 68, or XBB.1.5 SARS-CoV-2 virus (MOI = 0.1) for 24 h and stained for HLA-A/B/C, CD44 and CD275. e Median fluorescence intensity (MFI) of HLA-A/B/C and CD275. The same infection was conducted on HEK293T-ACE2 and their respective supernatant was then applied on THP-1 for 48 h before surface staining and analysis. f Histogram showing the expression of CD44, HLA-A/B/C, CD80, CD275, and HLA-DR on THP-1 after 48 h. g Median fluorescence intensity of CD44, HLA-A/B/C, CD80, and CD275 markers on THP-1 after 48 h incubation. h Comparison of wild-type or ΔE G 68 conditions for their expression of CD80 and HLA-A/B/C. The frequency of cells inside the gate in ( h ) is shown in ( i ). Median is shown for ( e ) and ( g ), mean and S.D. for ( i ). The gating strategy is shown in Supplementary Fig. .

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche ), sheep polyclonal anti-SARS-CoV-2 ORF3a , rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto ), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).

Techniques: Transfection, Flow Cytometry, Staining, Expressing, Control, Infection, Recombinant, Virus, Fluorescence, Incubation, Comparison