Journal: Microbial Cell Factories

Article Title: Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling

doi: 10.1186/s12934-016-0580-x

Figure Lengend Snippet: Yeast strains and plasmids used in the present study

Article Snippet: E. coli DH5α containing the M3499 plasmid (Addgene plasmid #51674) was purchased from Addgene (Cambridge, MA, US).

Techniques:

Journal: iScience

Article Title: Activation of limbal epithelial proliferation is partly controlled by the ACE2-LCN2 pathway

doi: 10.1016/j.isci.2024.110534

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Ace2 , Proteintech , Cat:# 21115-1-AP; RRID: AB_10732845.

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, RNA Sequencing, Software

Journal: Cell Research

Article Title: Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

doi: 10.1038/s41422-020-00450-0

Figure Lengend Snippet: a Workflow indicating identification of ACE2-binding peptides. After virtual screening, for each high-scoring candidate peptide, in vitro experiments were performed to validate its binding ability to ACE2. Picture represents 3D structure of ACE2 (PDB ID: 3D0G) and binding site (red sphere) for virtual screening. Key residue side chains are shown in blue. b Top 3D structure represents surface analysis of the binding site of ACE2 with SARS-COV-2 spike protein. Red circle marks binding site of ACE2 with SARS-COV-2 spike protein. Red and blue arrows show high charge areas in pocket by creating an interpolated charge surface; green and purple arrows point to porose area and hydrophobic domain, respectively. Middle 3D complex structure represents interaction between protein ACE2 and peptide drug dalbavancin after molecular dynamics simulation. Red structure represents dalbavancin, and green residues represent the four residues (Glu329, Gln325, Gln42, and Asp38) important for the binding of SARS-CoV-2 spike to ACE2. Lime, pale green, mint, and violet dashed lines in bottom picture represent hydrogen bonds, van der Waals bonds, Pi-Donor hydrogen bonds, and Pi-Alkyl bonds, respectively.

Article Snippet: Briefly, dalbavancin (10 μg, HY-17586, MedChemExpress), point mutated ACE2 (2 μg), anti-ACE2 antibody (1 μg, 10108-T60, Sino Biological), and protein A agarose (40 μl, 15918014, ThermoFisher) were incubated overnight at 4 °C.

Techniques: Binding Assay, In Vitro, Residue

Journal: Cell Research

Article Title: Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

doi: 10.1038/s41422-020-00450-0

Figure Lengend Snippet: a ACE2 (0.5 μg) and SARS-CoV-2 spike protein (0.5 μg) were mixed and treated with various candidate peptide drugs (10 μM). Co-precipitated proteins were identified by western blot analysis using anti-ACE2 antibody. Analyzed proteins are indicated on the right. For positive control (bottom), ACE2-His (0.5 μg) and SARS-CoV-2 spike protein (0.5 μg) were mixed and treated with various concentration of ACE2-hFc, and then co-precipitated proteins were identified by western blot analysis using anti-His-tag antibody. b ACE2 (2 μg/ml) was crosslinked to microplates by N-oxysuccinimide esters for ELISA. SARS-CoV-2 spike protein (10 ng/mL) and tested candidate drugs (1 μM) were incubated with ACE2, and extra un-crosslinked ACE2 protein (100 ng/mL) was used as a positive control. SARS-CoV-2 spike protein antibodies were used for chromogenic reaction. c Binding curves of immobilized human ACE2 with SARS-CoV-2 spike protein (left, positive control) and dalbavancin (right). Concentration-response SPR experiment showing binding of dalbavancin to ACE2 with an equilibrium dissociation constant (K D ) of ~147 nM.

Article Snippet: Briefly, dalbavancin (10 μg, HY-17586, MedChemExpress), point mutated ACE2 (2 μg), anti-ACE2 antibody (1 μg, 10108-T60, Sino Biological), and protein A agarose (40 μl, 15918014, ThermoFisher) were incubated overnight at 4 °C.

Techniques: Western Blot, Positive Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: Cell Research

Article Title: Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

doi: 10.1038/s41422-020-00450-0

Figure Lengend Snippet: a Vero E6 cells were pre-incubated with indicated concentrations of dalbavancin for 1 h and cells were infected with SARS-CoV-2 at an MOI of 0.01. After 72 h, viral yield in cell supernatant was quantified by qRT-PCR. Cytotoxicity of these drugs to Vero E6 cells was measured by CCK-8 assays. Left and right y-axis of graphs represent mean % inhibition of virus yield and cytotoxicity of drugs, respectively. Experiments were performed in triplicate. CC 50 , half-cytotoxic concentration; SI, selectivity index. b After 1 h pre-incubation with different doses of dalbavancin, Caco-2 cells were infected with SARS-CoV-2 at an MOI of 0.01 for 72 h. The viral yield in the cell supernatant was quantified by qRT-PCR. c Inhibition of SARS-CoV-2 spike pseudo-virion entry by dalbavancin. HEK293/hACE2 cells were pre-incubated with indicated concentrations of dalbavancin, and then SARS-CoV-2 pseudo-virions were added to HEK293/hACE2 cells. At 48 hpi, transduction efficiency was measured according to luciferase activities. Means ± SD from at least three independent experiments with technical triplicates are shown. d , e Time-of-addition experiment of dalbavancin. For “Full-time” treatment, Vero E6 cells were pre-treated with testing drug (1 μM) for 0.5 h, and virus was then added to allow attachment for 1 h. Afterwards, the virus–drug mixture was removed, and the cells were cultured with dalbavancin (1 μM) at 24 hpi. For “Entry” treatment, dalbavancin (1 μM) was added to the cells for 0.5 h before viral attachment, and at 1 hpi., the virus–drug mixture was replaced with fresh culture medium and maintained for 24 h. For “Post-entry” experiment, dalbavancin (1 μM) was added at 0.5 hpi, and maintained for 24 h. Cells were infected with SARS-CoV-2 at an MOI of 0.05, and virus yield in the infected cell supernatants was quantified by qRT-PCR ( d ) and NP expression in infected cells was analyzed by western blot ( e ). f ACE2 mutant (2 μg) was incubated with dalbavancin (10 μg), anti-ACE2 antibody (1 μg), and protein A agarose (40 μL) overnight. After co-IP treatment, dalbavancin was analyzed by LC-MS/MS. Wild-type ACE2 (WT) and PBS were used as positive and negative control, respectively. Statistical significance was measured by two-way analysis of variance (ANOVA) compared with control group. ns, not significant, ** P < 0.05, ** P < 0.01.

Article Snippet: Briefly, dalbavancin (10 μg, HY-17586, MedChemExpress), point mutated ACE2 (2 μg), anti-ACE2 antibody (1 μg, 10108-T60, Sino Biological), and protein A agarose (40 μl, 15918014, ThermoFisher) were incubated overnight at 4 °C.

Techniques: Incubation, Infection, Quantitative RT-PCR, CCK-8 Assay, Inhibition, Virus, Concentration Assay, Transduction, Luciferase, Cell Culture, Expressing, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Negative Control, Control