Journal: Bioactive Materials

Article Title: Sulfated liposome-based artificial cell membrane glycocalyx nanodecoys for coronavirus inactivation by membrane fusion

doi: 10.1016/j.bioactmat.2023.10.021

Figure Lengend Snippet: Nanodecoy membrane fusion causes virus block. The cryo-TEM image of (A) SARS-CoV-2 or (B) SARS-CoV-2 incubated with 40S_Lip. The arrow pointing the spike protein. Scale bar: 50 nm. (C) The membrane fusion profile of the FRET lipid labeled nanodecoy 40S_Lip(FRET) incubated with no FRET lipid labeled nanodecoy 40S_Lip, SARS-CoV-2 pseudovirus, or 40S_Lip and SARS-CoV-2 pseudovirus. (D) The MD simulation result of the spike protein incubated with 40S_Lip for 0, 0.12, and 2 ns. (E) The potential of mean force curves for 40S_Lip and S protein. (F) The CD spectrum of the native spike protein or when incubated with 40S_Lip. (G) The percentage of the detected free spike protein after incubation with heparin, 40S_Lip(Large), or 40S_Lip. (H) The percentage of the detected spike protein on the SARS-CoV-2 pseudovirus after incubation with heparin, 40S_Lip(Large), or 40S_Lip.

Article Snippet: The SARS-CoV-2 spike protein S1 RBD ELISA kit was purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Membrane, Virus, Blocking Assay, Incubation, Labeling

Journal: Nature communications

Article Title: A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry.

doi: 10.1038/s41467-021-24156-y

Figure Lengend Snippet: Fig. 7 Agents that reduce TMPRSS2 expression markedly inhibit SARS-CoV-2 pseudoviral infection. A Schematic of pseudoviral construction and assay. The S protein of SARS-CoV-2 was C-terminally tagged with HiBiT. B Level of viral transduction in various cell lines plotted on a logarithmic scale. Calu-3 and Caco-2 cells had the highest observed rates of infection. Data are mean ± SEM (n = 4–6 biologically independent samples). C Effects of HHT or HFG on pseudoviral-mediated GFP expression, determined by immunostaining, scale bar = 500 µm. D, E Effects of increasing concentrations of homoharringtonine (HHT) (D) or halofuginone (HFG) (E) on SARS-CoV-2 pseudoviral infection. Data are mean ± SEM (n = 3–6 biologically independent samples). F, G SARS- CoV-2 pseudoviral infection of primary human bronchial epithelial cells in the presence of increasing concentrations of HHT (F) and HFG (G). Data are mean ± SEM (n = 3 biologically independent samples). H SARS-CoV-2 pseudoviral infection of Caco-2 cells transfected with DCAF1 siRNA along with HFG treatment (100 nM). Data are mean ± SEM (n = 6 biologically independent samples). I SARS-CoV-2 pseudoviral infection of Caco-2 cells transfected with TMPRSS2 WT or lysine mutant prior to HFG treatment (100 nM). All SARS-CoV-2 pseudoviral data is corrected to cell number as determined by CellTiter- Glo. Data are mean ± SEM (n = 6 biologically independent samples). P-values are shown for comparisons to 0 time point or control, or as indicated by one- way ANOVA with Dunnett’s test of multiple comparisons (F, G), or a two-way ANOVA with Tukey’s test of multiple comparisons (B, H, I).

Article Snippet: Pseudovirus with SARS-CoV-2 spike protein with a Cterminal HiBiT tag was generated by co-transfection of 293 T cells with psPAX2 (Addgene, MA), pLenti-c-mGFP (Origene, MD), and PFC37K-HIBIT-SARS-CoV-2-S (Backbone was from Promega, WI) by using lipofectamine 3000 (Invitrogen, CA).

Techniques: Expressing, Infection, Transduction, Immunostaining, Transfection, Mutagenesis, Control

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Schematic illustration of the workflow of the UCAD assay. b Sequence design of the dsDNA template for triggering the collateral cleavage activity of CRISPR-Cas12a, as well as the strategy to split the dsDNA template into TS and NTS probes for UCAD. c Proximity binding of the two UCAD probes to the same anti-RBD antibody through affinity motifs enhances the T m of the duplex domain from 10 to 46 °C and thus enables the stable binding between TS and NTS probes. d , e Kinetic curves for the detection of anti-RBD IgG ( d ) and IgM ( e ) in commercially purchased certified anti-SARS-CoV-2 IgG/IgM positive and negative serum samples using UCAD. f A calibration curve for the detection of anti-SARS-CoV-2 spike protein RBD human mAb (clone: OTIH401) in the range of 1 aM to 1 pM was established by plotting fluorescence intensity at 40 min of the CRISPR-Cas12a reaction as a function of target concentrations, which was also compared with UCAD without RPA amplification. Each concentration has replicated measurements ( n = 2). g , h Detection of anti-RBD IgG ( g ) and IgM ( h ) in certified anti-SARS-CoV-2 IgG/IgM positive serum diluted in negative human serum with dilution factors from 1 to 100,000 using both UCAD and commercial ELISA kits. Each concentration has replicated measurements ( n = 2). Source data are available in the Source Data file.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Sequencing, Activity Assay, CRISPR, Binding Assay, Fluorescence, Amplification, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Detection of the target anti-human RBD and closely related nontargeted anti-MERS-CoV spike protein RBD human mAb (clone: m336), anti-SARS-CoV-2 nucleocapsid (N) protein human mAb, and anti-SARS-CoV-2 spike protein RBD rabbit pAb at a concentration of 10 fM using UCAD. Each sample was measured twice in two independent experiments. b , c Evaluation of the specificity of UCAD for anti-RBD IgG ( b ) and IgM ( c ) against anti-SARS-CoV in clinical sera from three SARS patients in 2003. Each serum sample was measured twice in two independent experiments. The UCAD signals of anti-SARS-CoV positive sera were significantly lower than the anti-SARS-CoV-2 positive serum ( p = 0.0004 for IgG, p = 0.0025 for IgM). Ordinary one-way ANOVA were used to compare the difference between multiple groups. d Schematic illustration of the modularity of UCAD for varying mutants of RBD and the N protein of SARS-CoV-2 by switching the recognition motif on the TS probe. e Heatmap of the detection of 10 fM wild-type (WT), Delta specific, and Omicron specific anti-RBD human mAb by using WT RBD, Delta RBD, and Omicron RBD (B.1.1.529) modified TS probes. f Heatmap of the detection of 10 fM WT RBD human mAb and anti-N protein human mAb with WT RBD and N protein modified TS probes. Source data are available in the Source Data file. ns: p > 0.05, *** p ≤ 0.001.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Concentration Assay, Modification

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Schematic illustration of lateral flow readout for UCAD for the detection of anti-SARS-CoV-2 IgG and IgM. b The design of the lateral flow strip that captured all anti-FAM labeled AuNPs at C-line in the absence of the target antibody. In the presence of ant-SARS-CoV-2 IgG or IgM, the FAM-Dig dual-labeled reporters were cleaved by Cas12a-crRNA so that AuNPs could escape the capture at C-line and accumulated at T-line through the immobilized secondary antibody. c Visual detection of 15 positive clinical sera and 15 pre-pandemic sera by UCAD integrated with lateral flow strips.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Stripping Membranes, Labeling