Journal: Nanomaterials

Article Title: In Vitro Genotoxicity Evaluation of an Antiseptic Formulation Containing Kaolin and Silver Nanoparticles

doi: 10.3390/nano12060914

Figure Lengend Snippet: Results of the MLA after 3 h ( a ) and 24 h ( b ) treatment of L5178Y TK+/− cells. Each figure shows the induction of gene mutations, represented as mutant frequency ×10 −6 , by silver-kaolin formulation (black bars), its corresponding silver-kaolin release (grey bars), and for the negative (C−) and positive controls (C+) (white bars with black edge). It also shows the cytotoxicity of silver-kaolin formulation (continuous line) and its corresponding silver-kaolin release (dotted line), represented as % RTG values compared to the negative control for each testing condition. C+: cells treated with 100 µM MMS. • : difference from negative control based on OECD TG 490 global evaluation factor (GEF).

Article Snippet: The mouse lymphoma assay (MLA) was conducted using mouse lymphoma L5178Y TK +/− cells, obtained from the American Type Culture Collection [L5178Y TK+/(clone3.7.2C)] (ATCC ® CRL9518TM)).

Techniques: Mutagenesis, Formulation, Negative Control

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: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor.

doi: 10.1038/mt.2012.267

Figure Lengend Snippet: Figure 4 Bispecific γδ T cells produce proinflammatory cytokines when endogenous TCR and introduced CAR are stimulated. (a) CAR+γδ T cells at day 35 of co-culture on aAPC were stimulated for 4 hours with a mock cocktail (media alone) or leukocyte activation cocktail (LAC, PMA/ ionomycin) to induce TCR stimulation and then analyzed by flow cytometry. CAR+ T cells were gated and tumor necrosis factor-α (TNF-α, top) and interferon-γ (IFN-γ, bottom) production is shown. (b) Luminex array (27-Plex) of cytokines secreted by CAR+γδ T cells in conditions described in a. (c) Similar to a except that EL4-CD19neg and EL4-CD19+ were used instead of mock/LAC. (d) Same as b but with EL4-CD19neg and EL4-CD19+ tar- gets. Student’s t-test for statistical analysis between mock and LAC (in b) and EL4-CD19neg and EL4-CD19+ (in d) where *P < 0.05, **P < 0.01, and ***P < 0.001. Data are representative of four donors for a and c and mean ± SD (n = 3) for b and d. aAPC, artificial antigen-presenting cell; CAR, chimeric antigen receptor; FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; PDGFβ, platelet-derived growth factor-β; PMA, phorbol 12-myristate 13-acetate; RANTES, regulated and normal T cell expressed and secreted; TCR, T-cell receptor; VEGF, vascular endothelial growth factor.

Article Snippet: Codon-optimized DNA plasmids for SB transposase (SB11) and a second generation CD19-specific CAR (designated CD19RCD28) transposon are described elsewhere.5 NALM-6 and EL4 cell lines were acquired from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and American Type Culture Collection (Manassas, VA), respectively.

Techniques: Co-Culture Assay, Activation Assay, Flow Cytometry, Luminex, Derivative Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor.

doi: 10.1038/mt.2012.267

Figure Lengend Snippet: Figure 5 Specific lysis of CD19+ tumor cell lines by CAR+γδ T cells. (a) Standard 4-hour CRA of (a) CARnegγδ T cells against CD19+ B-ALL cell lines (REH, Kasumi-2, and Daudi-β2M) or primary CD19+ B cells from autologous (Auto) or allogeneic (Allo) donors, (b) CAR+γδ T cells against EL4-CD19neg (open squares) and EL4-CD19+ (closed squares) tumor cells, and (c) CARnegγδ T cells (open squares) and CAR+γδ T cells (closed squares) against CD19+ NALM-6 tumor cells. Data are mean ± SD from four healthy donors (average of triplicate measurements for each donor) that were pooled from two independent experiments. B-ALL, B-cell acute lymphoblastic leukemia; CAR, chimeric antigen receptor; CRA, chromium release assay; E:T, effector to target ratio.

Article Snippet: Codon-optimized DNA plasmids for SB transposase (SB11) and a second generation CD19-specific CAR (designated CD19RCD28) transposon are described elsewhere.5 NALM-6 and EL4 cell lines were acquired from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and American Type Culture Collection (Manassas, VA), respectively.

Techniques: Lysis, Release Assay