Journal: Frontiers in Cardiovascular Medicine

Article Title: SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity

doi: 10.3389/fcvm.2022.1013262

Figure Lengend Snippet: Spike enhances platelet activation. (A) Histopathological evidence of platelet aggregates in the thrombotic microvasculature of SARS-COV-2-infected lungs from four COVID-19 patients. Numeric codes identify patients. Platelets were stained using an anti-CD61 glycoprotein antibody. Scale bar: 100 μm. (B) Experimental scheme to study platelet activation and aggregate formation. Vero cells transfected to express either Green Fluorescent Protein (GFP) or SARS-CoV-2 Spike were incubated with pre-labeled washed platelets and shaken at 200 rpm for 10 min at 37°C. The plate was centrifuged, fixed, and stained with Cell Mask and antibodies recognizing either GFP or Spike. (C) Representative images showing platelet aggregates. Vero cells stained with Cell Mask are in blue; labeled platelets are in red; GFP or Spike are in green. Scale bar, 20 μm. (D) Number of aggregates larger than 40,000 px 2 . Results are from n = 3 independent experiments. Data are mean ± SEM, statistical significance is indicated (paired Student’s t -test). (E) Violin plot showing the size of the aggregates found in all the experiments performed. Statistical significance is indicated (unpaired Student’s t -test). (F) Experimental scheme for platelet activation in suspension. (G–I) Percentage of platelet aggregation when incubated with vehicle (G) , stimulated with collagen (H) or CRP (I) . Results are from N = 6 independent experiments. Data are mean ± SEM statistical significance is indicated (paired Student’s t -test). (J) Number of adherent platelets per field. Results are from n = 3 independent experiments each performed in duplicate. Each dot represents the mean of six images quantified. Data are mean ± SEM. Statistical significance is indicated (paired Student’s t -test). (K) Percentage of the area covered by adherent platelets. Results are from n = 3 independent experiments performed in duplicate; each dot represents the mean of six images quantified. (L) Representative images of platelets adhering on collagen. Images were acquired using a high content fluorescent microscope followed by analysis using the ImageJ software (Fiji). Platelets were stained with F-actin (in red). Scale bar, 5 μm.

Article Snippet: PRP was incubated with VSV-G (1:10), Spike pseudoparticles (1:10), His-tag recombinant SARS-CoV-2 Spike Protein S1/S2 (S-ECD) (1 ng/ml; Thermo Fisher Scientific, Waltham, MA, United States; aa11-1208; RP-87680) or Flag-tag recombinant SARS-CoV-2 Spike RBD (1 ng/ml; Bio-Techne, Minneapolis, MN, United States, 10689-CV-100) for 10 min at 37°C, followed by stimulation with thrombin (0.5 units).

Techniques: Activation Assay, Infection, Staining, Transfection, Incubation, Labeling, Suspension, Microscopy, Software

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a ELISA screen for identification of potential SARS-CoV-2 RBD binders. Negative control wells containing expression media, and positive control wells containing VNAR E06 (anti-serum albumin) and rabbit monoclonal CR3022-RB (anti-SARS-CoV-2 Spike) are indicated. b Primary screen for identification of neutralizing VNAR domains. Concentration-dependent neutralization of pseudotyped SARS-CoV-2 (black) or SARS-CoV-1 (red) in HEK293T cells transiently expressing ACE2. Data represents mean ± s.e.m. relative luminescence units (RLUs) from n = 3 independent biological experiments. c Left , rank-ordered IC 50 values for neutralizing VNARs from panel ( b ). VNARs with IC 50 < 10 nM (dashed line) were selected for further characterization. Upper right, depiction of primary sequences of selected VNARs, relative length of complementarity determining regions (CDR1, CDR3) and location of cysteine residues (teal) are shown. d Phylogenetic tree of selected virus taxa, divergent lineages of betacoronaviruses are shown. Glycoproteins encoded by the indicated viruses (*) were used to generate pseudoviruses. e Heatmap summarizing IC 50 values for neutralization of the indicated pseudovirus with the indicated VNAR antibody. Values are derived from experiments described in ( f ). f Secondary validation of selected neutralizing VNAR domains. Concentration-dependent neutralization of viral particles pseudotyped with glycoproteins natively encoded by either SARS-CoV-2 (black), SARS-CoV-1 (red), WIV1-CoV (blue). MERS-CoV (green), or VSV (purple) in Calu-3 cells. Cell viability was also assessed in the presence of increasing concentrations of VNARs (yellow). Data represents mean ± s.e.m. RLU values from n = 3 independent biological experiments. g Concentration-dependent neutralization of replication-competent authentic SARS-CoV-2, strain USA_WA1/2020 in Vero E6 cells. Data represents mean ± s.e.m. RFU values from n = 3 independent biological experiments.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Expressing, Positive Control, Concentration Assay, Neutralization, Derivative Assay

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a Sequence alignment between SARS-CoV-1, SARS-CoV-2, and the MERS RBDs. Residues different from SARS-CoV-2 are highlighted in red and the interaction interface for VNAR-3B4 is marked with a blue box. Bolded letters indicate residues critical for the interaction between the RBD and 3B4 with arrows indicating the residues that form a backbone beta-sheet. The sequence alignment is numbered above according to SARS-CoV-2. b Surface representations of SARS-CoV-1 (pink, above) and MERS (green, below) with variant residues colored red. The ACE2 binding interface is highlighted in purple for SARS-CoV-1 and the DPP4 binding interface in orange for MERS. The homology-modeled interaction interface for 3B4 is colored blue for both structures. c Zoomed in view of the modeled interaction interface between 3B4 (blue) and SARS-CoV-1 (pink, above) and MERS (green, below), with 3B4 colored blue in both pictures. Interacting residues are highlighted as in Fig. , showing the backbone interactions in black dashes and sidechain to backbone or sidechain to sidechain interactions shown as yellow dashes. Insets show the orientation of the zoomed in view. d Overlays of the 3B4 interface from modeled RBDs and their matching RBDs obtained by x-ray crystallography. The panel above shows the modeled SARS-CoV-1 RBD, colored pink, aligned with the crystal structure of SARS-CoV-1 RBD bound to ACE2 (PDB id: 2AJF), colored magenta. The panel below shows the modeled MERS RBD, colored light green, aligned with the crystal structure of MERS RBD bound to DPP4 (PDB id: 4L72), colored dark green.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Sequencing, Variant Assay, Binding Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Wheat germ-based in vitro ubiquitination analysis of RING proteins showed high molecular smears. a At4g11680 and its corresponding RING mutant were expressed with biotin tag and analysed as a representative protein in the presence or absence of FLAG-Ub, E1, and/or AtUBC8 without tag for the ubiquitination activity. Replacement of the third metal ligand, Cys to Ser, caused reduced smear upon blotting against FLAG-Ub with anti-FLAG-HRP antibody. Absence of the E1 or AtUBC8 from the reaction did not abolish the high molecular smear. b Three other RING proteins At1g80400, At2g22120, and At1g11020, and their RING mutants together with At4g11680, were analysed in the presence of FLAG-Ub. The four proteins with RING mutants showed significantly reduced activity upon blotting with anti-FLAG-HRP antibody, whereas the proteins with intact RING domains showed activity without the addition of E1 or E2. The side arrow refers to the free FLAG-Ub that migrated to the bottom of the gel

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: In Vitro, Mutagenesis, Activity Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Wheat germ-based in vitro ubiquitination assays of various types of RING proteins. 23 FLAG-tagged RING proteins of various RING types were mixed with FLAG-Ub in the presence or absence of N-bio-UBC10 as indicated by plus (+) or minus (–) above each lane. E3 activity was determined by the presence or absence of a smear when FLAG-Ub was detected by anti-FLAG-HRP antibody. This is indicated by plus (+) or minus (–), respectively, below each lane respectively

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: In Vitro, Activity Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Thioester assay of 35 Arabidopsis E2s. The crude proteins for each of the 35 bio-E2s were incubated with FLAG-Ub for 5 min at 37 °C and treated with DTT or 8 M urea (-DTT). Immunoblot analysis against FLAG-Ub using anti-FLAG-HRP antibodies shows the presence of DTT-sensitive Ub conjugation activity for all E2s tested. Arrows show the expected E2-Ub adduct for each E2 in the absence of DTT. The side arrows show the free FLAG-Ub and expected FLAG-Ub adducts with WE2 and WE1 as arranged from bottom to top

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: Incubation, Western Blot, Conjugation Assay, Activity Assay

Journal: BMC Plant Biology

Article Title: Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes

doi: 10.1186/s12870-015-0660-9

Figure Lengend Snippet: Immunoblot analysis of the N-terminal FLAG-tagged RING protein library expressed by the wheat germ cell-free system. For analysis, 2 μL of crude recombinant RING proteins with N-terminus FLAG tag was loaded onto SDS-PAGE and detected by anti-FLAG-HRP antibody. A total of 204 out of 208 RING proteins analysed were detected. Arrows show the expected signal for each RING protein. Blue asterisks refer to proteins with high molecular smears, while red asterisks refer to RING proteins did not show high molecular smears and were subsequently used in the in vitro ubiquitination analysis (Fig. , Fig. )

Article Snippet: In a total reaction volume of 25 μL, we mixed 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 3 mM ATP, 1 mg/ml BSA, 400 nM human recombinant FLAG-Ub (Boston Biochem, http://www.bostonbiochem.com ), 3 μL recombinant E2s.

Techniques: Western Blot, Recombinant, FLAG-tag, SDS Page, In Vitro

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet:

Article Snippet: C57BL/6J mice were tail-vein injected with pCMV3- CDH1 (Sino Biological, mouse/MG50671-CF, ) or EGFP (Sino Biological, Aequorea Victoria/AG13105-CF) using PEI Max transfection reagent (PEI, Polysciences) according to a method modified from .

Techniques: Recombinant, Chromatin Immunoprecipitation, AST Assay, Immunoprecipitation, shRNA, Luciferase, Software