Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Chloroplast expression vector and PCR analysis. (A) pLD-BD Cry2Aa2 operon (9.8 kb) with PCR primer binding sites and expected fragment sizes. PCR analysis of untransformed and putative chloroplast transformants using two primer sets: (B) 1P1M and (C) 3P3M. Lane 1, 1 kb ladder; lane 2, untransformed; lanes 3–7, pLD-BD Cry2Aa2 operon putative transformants; lane 8, pLD-BD Cry2Aa2 operon plasmid DNA.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Expressing, Plasmid Preparation, Binding Assay

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: 10% SDS–PAGE gel stained with R-250 Coomassie blue. Loaded protein concentrations are provided in parentheses. Lane 1, prestained protein standard; lane 2, partially purified Cry2Aa2 protein from E. coli (5 μg); lane 3, single gene-derived Cry2Aa2 pellet extract solubilized in 50 mM NaOH (22.4 μg); lane 4, single gene-derived Cry2Aa2 supernatant (66.5 μg); lane 5, operon-derived Cry2Aa2 pellet extract solubilized in 50 mM NaOH (22.9 μg); lane 6, operon-derived Cry2Aa2 supernatant (58.6 μg); lane 7, untransformed tobacco pellet extract solubilized in 50 mM NaOH (29.8 μg); lane 8, untransformed tobacco supernatant (30.4 μg). Colored compounds observed in the supernatant of transgenic plants interfered with the DC Bio-Rad protein assays.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: SDS Page, Staining, Purification, Derivative Assay, Transgenic Assay

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Transmission electron micrographs. Operon-derived Cry2Aa2 leaf sections in young (A), mature (B, D), and old, bleached leaf (C). (E) Single gene-derived Cry2Aa2 mature leaf; (F) mature untransformed leaf.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Transmission Assay, Derivative Assay

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Protein quantification by ELISA in young, mature, and old transgenic leaves. (A) Single gene-derived Cry2Aa2 expression shown as a percentage of total soluble protein. (B) Operon-derived Cry2Aa2 expression shown as a percentage of total soluble protein.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Enzyme-linked Immunosorbent Assay, Transgenic Assay, Derivative Assay, Expressing

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Insect bioassays. (A, D, G) Untransformed tobacco leaves; (B, E, H) single gene-derived Cry2Aa2 transformed leaves; (C, F, I) operon-derived Cry2Aa2 transformed leaves. (A–C) Bioassays with Heliothis virescens; (D–F) bioassays with Helicoverpa zea; (G–I) bioassays with Spodoptera exigua. All leaf samples for each replicate were from the same leaf. Two samples were evaluated per treatment, and observed daily for mortality and leaf damage for five days. Treatments were replicated three times. Insects were tested at 5 or 10 days old (see text for details).

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Derivative Assay, Transformation Assay

Journal: Neurotherapeutics

Article Title: Fosgonimeton, a Novel Positive Modulator of the HGF/MET System, Promotes Neurotrophic and Procognitive Effects in Models of Dementia

doi: 10.1007/s13311-022-01325-5

Figure Lengend Snippet: Fosgonimeton prevents LPS-induced cognitive deficits in vivo. a Percentage of spontaneous alternations in the T-maze are shown. Mice were injected with lipopolysaccharide (LPS; 0.25 mg/kg, I.P.) at day 1, then received daily treatment with fosgonimeton, vehicle, or positive control memantine for 14 days. On day 14, mice were assessed for performance in the spontaneous alternation T-maze task. Memantine (0.1 mg/kg, I.P.), as well as fosgonimeton at 0.25, 0.5, 1, and 1.25 mg/kg S.C., significantly prevented LPS-induced cognitive deficits. Data presented as mean ± SEM; n = 10. b Percentage recovery in spontaneous alternations is shown by group. Percentage recovery was significantly increased with memantine, as well as with fosgonimeton at 0.25, 0.5, 1, and 1.25 mg/kg. Data presented as mean ± SEM; n = 10. c – e Bar graphs showing the levels of LPS-induced interleukin 1 beta (IL-1β), tumor necrosis factor α (TNF-α), and IL-6 release in THP-1 differentiated macrophages in the presence and absence of fosgo-AM at 0.01 pM, 1 pM, 100 pM, 10 nM, or 1 µM. Compared with their respective vehicle-treated group, THP-1 differentiated macrophages treated with fosgo-AM (1 µM) exhibited a significant reduction in release of c IL-1β, d TNF-α, and e IL-6 Data for each treatment were averaged and presented as mean ± SEM; Number of experimental replicates was as follows: n = 6 for LPS + vehicle for all 3 cytokine assays, n = 3 for LPS + fosgo-AM for all 3 cytokine assays; for vehicle groups, n = 3 (IL-1β assay), 5 (TNF-α assay), and 6 (IL-6 assay). Statistical differences in percentage of spontaneous alterations were determined by one-way ANOVA with Dunnett’s post hoc test. Statistical differences in percentage of recovery were determined by Kruskal–Wallis test with Dunn’s post hoc test. Statistical differences in IL-1β, TNF-α, and IL-6 were determined by one-way ANOVA followed by Dunnett’s multiple comparison post hoc test * p < 0.05, ** p < 0.01, *** p < 0.001 compared with LPS only

Article Snippet: Before treatment, the complete media were exchanged for serum-free media and incubated at 37 °C, 5% CO 2 for 3 h. The media were aspirated, and cells were treated with 50 µL vehicle (0.05% DMSO, 0.05% FBS, 0.5 ng/ml HGF) or fosgo-AM (1 µM, 10 nM, 100 pM, 1 pM or 0.01 pM in vehicle) in triplicate and incubated at 37 °C, 5% CO 2 for 20 min. After 20 min, the treatment was aspirated and 100 µL LPS (50 ng/mL) was applied to the culture and incubated at 37 °C, 5% CO 2 for 24 h. For cytokine quantification, cell culture supernatant was assayed by HTRF kits to assess levels of IL-1β (Human IL-1β kit, #62HIL1BPEG, Cisbio, Codolet, France) and IL-6 (Human IL-6 kit, 62HIL06PET, Cisbio, Codolet, France), and by ELISA (Human TNF-α ELISA kit, KHC3011, ThermoFisher, Waltham, Massachusetts) to determine the levels of TNF-α.

Techniques: In Vivo, Injection, Positive Control