Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: a , Flow cytometry measurement of uptake by MFI of mNG + EVs (Fc-EVs versus ctrl-EVs versus no EVs) in HER2 positive breast cancer cells (SKBR-3 cells) when incubated with HER2-Ab (trastuzumab), control-Ab (IgG ctrl) or no Ab, showing significant increase in uptake of Fc-EVs by trastuzumab. b , Flow cytometry measurement of uptake (by MFI) of mNG + Fc-EVs decorated with the PD-L1-Ab atezolizumab in PD-L1 expression stimulated (IFNγ) or unstimulated malignant melanoma (B16F10) cells. c , Fluorescence microscopy images of B16F10 cells stained with DAPI (blue), untreated (UT) or treated with mNG + (green) Fc-EVs alone or with the PD-L1-Ab atezolizumab, showing increased uptake of the Fc-EVs when decorated with PD-L1-Ab. Panels a and b are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using one-way ( b ) or two-way ( a ) ANOVA with Tukey’s post-test compared with each value; P values are indicated above the plots throughout.

Article Snippet: The tissues were processed as described in ref. , with lysed tissue and nLuc + EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Techniques: Flow Cytometry, Incubation, Control, Expressing, Fluorescence, Microscopy, Staining

Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: IV injection of Fc-EVs with HER2-Ab (trastuzumab, Fc-EV + HER2-Ab) compared to control-Ab (Fc-EV + IgG-ctrl) in HER2 + breast cancer (SKBR-3) tumour-bearing Swiss nude mice. a , The experimental set-up with inoculation of SKBR-3 cells followed by tumour formation for 2 months before IV injection of nLuc + Fc-EVs with Abs, followed by tissue collection 30 min post injection. b , Fold change in detected EVs (based on luminescence) per gram tumour tissue compared to Fc-EV + IgG-ctrl. c , d , Accumulation of Fc-EV + HER2-Ab (based on luminescence) compared to Fc-EV + IgG-ctrl per gram spleen ( c ) and liver ( d ). All data are shown as mean ± s.d. n = 10 mice. Statistical significance was calculated using two-tailed unpaired t -test analysis compared with each value; P values are indicated above the plots throughout.

Article Snippet: The tissues were processed as described in ref. , with lysed tissue and nLuc + EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Techniques: IV Injection, Control, Injection, Two Tailed Test

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 1 | Engineering and characterization of receptor-based Axl antagonists. (a) Axl’s extracellular domain consists of two Ig-like domains containing high- and low-affinity Gas6 binding sites, followed by two fibronectin type III domains. Binding of Gas6 to Axl leads to receptor dimerization and activation of downstream signaling. Axl decoy receptors sequester Gas6, preventing activation of the Axl signaling cascade. (b) Overlaid flow cytometry dot plots representing binding of yeast-displayed wild-type Axl Ig1 (red) and unsorted Axl Ig1 library (blue) to 10 nM Gas6 (y axis) and expression levels on the yeast cell surface (x axis). (c) Flow cytometry histograms of the initial Axl library and intermediate sort products compared to wild-type Axl Ig1 (gray), measuring binding to 0.5 nM Gas6 (top row) and persistent Gas6 binding after a 30-h incubation with excess competitor (bottom row). MYD1 is also included for comparison. For clarity, only the gated population of yeast expressing Axl is shown. AU, arbitrary units. (d) Binding affinities of wild-type Axl Ig1, MYD1 and Axlnb to Gas6 as determined by KinExA. (e) Binding affinities to Gas6 of every permutation of the four mutations found in MYD1. Raw KinExA data and associated error values can be found in Supplementary Figures 2 and 3.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Activation Assay, Flow Cytometry, Expressing, Incubation, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 2 | Structural basis for high-affinity binding. (a) Gas6–MYD1 co-complex showing overall architecture and 2:2 stoichiometry. (b) MYD1 Ig1 (orange) and Gas6 LG1 (gray) domains showing the location of the four mutations in MYD1 with respect to the major binding site, which lies at the interface of these two domains. (c) Analysis of the wild-type structure (PDB code 2C5D) reveals steric crowding between the side chains of T457Gas6 and V92Axl. The V92A mutation alleviates this crowding in the MYD1 co-complex and facilitates local reorganization of side chains around V92A, exemplified by R48 and Q94. This in turn creates an elongated groove on MYD1 at the binding interface that allows reorientation of T457 on Gas6. (d) Reorientation of T457 results in capping of the N terminus of helix A. The wild-type (WT, green) and MYD1 (gray) structures are overlaid for comparison. (e) Capping stabilizes helix A, as seen by B-factor analysis (Online Methods).

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 4 | MYD1 Fc inhibits Axl activation and downstream signaling in skov3.ip cells. (a) Wild- type (WT) Axl Fc and MYD1 Fc, but not Axlnb Fc, can inhibit Gas6-mediated Axl activation in vitro. (b) Inhibition of Axl activation leads to reduced levels of phosphorylated Akt and Erk1/2 and an increase in the epithelial marker e-cadherin. For full (uncut) blots, see Supplementary Figure 11.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Activation Assay, In Vitro, Inhibition, Marker

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 5 | Sequestration of Gas6 by MYD1 Fc inhibits metastasis. (a) Amount of free Gas6 in serum of mice 12 h after administration of a single dose of MYD1 Fc. (b) Kinetics of Gas6 sequestration (black) and MYD1 Fc clearance (red) following a 1 mg per kg body weight dose of MYD1 Fc. (c) Using the off-rates of the Gas6-Axl Fc interactions (Fig. 3b), dissociation of Gas6 bound to either wild-type Axl Fc (red) or MYD1 Fc (blue) is plotted over time. The in vivo clearance of the Axl decoy receptors as measured in c is overlaid in black. Two mice were analyzed for each data point in b and c. (d–f) Tumor burden in in vivo models of metastatic human ovarian cancer. The number of visible metastases in animals treated with Axlnb Fc, wild-type Axl Fc or MYD1 Fc was counted in the skov3.ip (d) and OVCAR (f) tumor models. Representative images of mice from each treatment group in the skov3.ip model are shown, and arrows indicate disease (e). In both models, animals were administered 10 mg per kg body weight of the indicated protein twice weekly. (g) Lung metastases in the 4T1 luciferase breast cancer model, as quantified by ex vivo bioluminescent imaging. Mice received intravenous injections of the indicated treatment twice weekly. (h) Representative bioluminescent images of lungs and spleens from each treatment group; scale bar, 1 cm. Error bars represent ± s.d., n = 6–12 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: In Vivo, Luciferase, Ex Vivo, Imaging

Journal: International Journal of Clinical and Experimental Medicine

Article Title: The diagnostic value of neutrophil gelatinase-associated lipocalin and hepcidin in bacteria translocation of liver cirrhosis

doi:

Figure Lengend Snippet: Concentrations of neutrophil gelatinase-associated lipocalin (NGAL) and hepcidin in serum of liver cirrhotic animal model and controls

Article Snippet: Quantitative measurements of ascitic fluid hepcidin and NGAL concentrations were determined using a polyclonal antibody-based enzyme-linked immunosorbent assay specific for rat and human hepcidin (Eiaab Science Co. Ltd, china) and NGAL (BioPorto Diagnostics A/S, Denmark).

Techniques: Animal Model, Translocation Assay

Journal: International Journal of Clinical and Experimental Medicine

Article Title: The diagnostic value of neutrophil gelatinase-associated lipocalin and hepcidin in bacteria translocation of liver cirrhosis

doi:

Figure Lengend Snippet: Diagnostic performance of serum NGAL, hepcidin or their combination in liver cirrhotic animal model

Article Snippet: Quantitative measurements of ascitic fluid hepcidin and NGAL concentrations were determined using a polyclonal antibody-based enzyme-linked immunosorbent assay specific for rat and human hepcidin (Eiaab Science Co. Ltd, china) and NGAL (BioPorto Diagnostics A/S, Denmark).

Techniques: Diagnostic Assay, Bacteria

Journal: International Journal of Clinical and Experimental Medicine

Article Title: The diagnostic value of neutrophil gelatinase-associated lipocalin and hepcidin in bacteria translocation of liver cirrhosis

doi:

Figure Lengend Snippet: Concentrations of NGAL and hepcidin in ascites of liver cirrhotic animal model

Article Snippet: Quantitative measurements of ascitic fluid hepcidin and NGAL concentrations were determined using a polyclonal antibody-based enzyme-linked immunosorbent assay specific for rat and human hepcidin (Eiaab Science Co. Ltd, china) and NGAL (BioPorto Diagnostics A/S, Denmark).

Techniques: Bacteria

Journal: International Journal of Clinical and Experimental Medicine

Article Title: The diagnostic value of neutrophil gelatinase-associated lipocalin and hepcidin in bacteria translocation of liver cirrhosis

doi:

Figure Lengend Snippet: Diagnostic performance of neutrophil gelatinase-associated lipocalin (NGAL), hepcidin or their combination in ascites of liver cirrhotic animal model

Article Snippet: Quantitative measurements of ascitic fluid hepcidin and NGAL concentrations were determined using a polyclonal antibody-based enzyme-linked immunosorbent assay specific for rat and human hepcidin (Eiaab Science Co. Ltd, china) and NGAL (BioPorto Diagnostics A/S, Denmark).

Techniques: Diagnostic Assay, Bacteria