ampr richard murray lab cidar moclo extension unpublished 120956 c51m superfolder yellow fluorescent protein sfyfp  (Addgene inc)

Journal: CNS Neuroscience & Therapeutics

Article Title: Triptolide Inhibits Epileptic Seizures by Rescuing the Neuroinflammation‐Related GABAergic Dysfunction in Mice

doi: 10.1111/cns.70586

Figure Lengend Snippet: TPL enhanced GABAergic “braking effect” in KA mouse model of epilepsy. (A) Schematic diagram of optogenetical stimulation and microelectrode recording on KA model; (B) Clusters view and waveform of recorded neurons (left); autocorrelation of recorded neurons (middle); histological image showing the placement of electrode and optic fiber in dorsal hippocampal DG for extracellular recording (right, Blue‐DAPI); (C) Peri‐event raster histograms of the event‐related response of neurons to optogenetical stimulation; (D) The duration of the light‐induced “braking effect” (Normal, n = 11 cells; Control, n = 10 cells; TPL, n = 11 cells). (E) Example images showing co‐localization of VGAT‐EYFP with astrocytes (GFAP) in hippocampus (scale bar = 200 μm), CA1 and DG region (scale bar = 50 μm); (F) The mean fluorescence intensity of GFAP in CA1 and DG region (Normal, n = 3 mice; Control, n = 3 mice; TPL, n = 3 mice); (G) The number of GFAP + astrocytes CA1 and DG region (Normal, n = 3 mice; Control, n = 3 mice; TPL, n = 3 mice); (H) The mean fluorescence intensity of VGAT in CA1 and DG region (Normal, n = 3 mice; Control, n = 3 mice; TPL, n = 3 mice). Normal means sham operated VGAT‐CHR2‐EYFP mice administrated with solvent at 0.1 mL/10 g; Control means VGAT‐CHR2‐EYFP epileptic mice administrated with solvent at 0.1 mL/10 g; TPL means VGAT‐CHR2‐EYFP epileptic mice administrated with TPL at 50 μg/kg. Data are presented as mean ± SEM. Kruskal‐Wallis test was used for D. * p < 0.05, **** p < 0.0001 compared with Normal. # p < 0.05 compared with Control.

Article Snippet: C57BL/6 mice and vesicular GABA transporter (VGAT)‐Channelrhodopsin 2 (ChR2)‐enhanced Yellow Fluorescent Protein (eYFP) mice (VGAT‐ChR2‐eYFP mice; No. 014548) were reared as previously described and were genotyped according to the protocols promulgated by the Jackson Laboratory.

Techniques: Control, Fluorescence, Solvent

Journal: CNS Neuroscience & Therapeutics

Article Title: Triptolide Inhibits Epileptic Seizures by Rescuing the Neuroinflammation‐Related GABAergic Dysfunction in Mice

doi: 10.1111/cns.70586

Figure Lengend Snippet: TPL reverses astrocytic and GABAergic neuronal dysregulation in the KA model. (A) Representative WB results of GFAP expression and related quantitative statistical plots. (B) Representative WB results of GAD65/67 expression and related quantitative statistical plots. (C) 3D reconstructions of GFAP + astrocytes and VGAT + GABAergic neurons (scale bar = 10 μm); (D) The total volume of GFAP + astrocytes (Normal, n = 6 fields; Control, n = 5 fields; TPL, n = 5 fields). (E) The volume of single GFAP + astrocytes (Normal, n = 6 cells; Control, n = 5 cells; TPL, n = 5 cells). (F) The total volume of VGAT + GABAergic neurons (Normal, n = 6 fields; Control, n = 6 fields; TPL, n = 6 fields). Normal means sham‐operated VGAT‐CHR2‐EYFP mice administrated with solvent at 0.1 mL/10 g; Control means VGAT‐CHR2‐EYFP epileptic mice administrated with solvent at 0.1 mL/10 g; TPL means VGAT‐CHR2‐EYFP epileptic mice administrated with TPL at 50 μg/kg. Data are presented as mean ± SEM. One‐way ANOVA (with Tukey test for post hoc comparison) was used for A‐B and D‐F. * p < 0.05, *** p < 0.001 compared with Normal. # p < 0.05, ## p < 0.01, #### p < 0.0001 compared with Control.

Article Snippet: C57BL/6 mice and vesicular GABA transporter (VGAT)‐Channelrhodopsin 2 (ChR2)‐enhanced Yellow Fluorescent Protein (eYFP) mice (VGAT‐ChR2‐eYFP mice; No. 014548) were reared as previously described and were genotyped according to the protocols promulgated by the Jackson Laboratory.

Techniques: Expressing, Control, Solvent, Comparison

Journal: Advanced Science

Article Title: A Serum Resistant Polymer with Exceptional Endosomal Escape and mRNA Delivery Efficacy for CRISPR Gene Therapy

doi: 10.1002/advs.202413006

Figure Lengend Snippet: Endosomal escape behaviors of FD17. a) Schematic representation of Gal8‐YFP recruitment to disrupted endosomes and the mechanism of endosomal membrane disruption by FD17. b) Fluorescent microscopy was used to capture images of Gal8‐YFP‐HeLa cells exposed to FD17/luciferase mRNA complexes at various time points ranging from 0 to 6 h. The scale bar is 20 µm. c) Average number and d) area of Gal8‐YFP spots per cell treated with FD17/luciferase mRNA complexes over the same time course. The N/P ratio of the FD17/mRNA complex was 2.5. e) Fluorescence microscopy images showcasing Gal8‐YFP‐HeLa cells subjected to treatment with FD17/luciferase mRNA complexes at varying N/P ratios. The scale bar is 20 µm. f) Average number and area of Gal8‐YFP spots per cell treated with FD17/luciferase mRNA complexes at varying N/P ratios. The mRNA delivery experiments were conducted for 6 h. g) Correlation analysis between the level of luciferase expression and the extent of endosomal membrane disruption in cells treated with FD17/luciferase mRNA complexes. n.s. p > 0.05 were subjected to analysis using one‐way ANOVA, which was then followed by the Newman‐Keuls Multiple Comparison Test.

Article Snippet: To quantify the ability of FD17/mRNA complexes to overcome this barrier, we utilized a HeLa cell line stably expressing Galectin 8 (Gal8) fused with yellow fluorescent protein (YFP).

Techniques: Membrane, Disruption, Microscopy, Luciferase, Fluorescence, Expressing, Comparison

Journal: Nucleic Acids Research

Article Title: Transcription factors induce differential splicing of duplicated ribosomal protein genes during meiosis

doi: 10.1093/nar/gkae1321

Figure Lengend Snippet: Splicing of the small subunit protein S9/uS4 dRPGs is differentially regulated by transcription-associated proteins. ( A ) Pipeline for the identification of paralog-specific repressors of RPS9 splicing. The intron of RPS9A was tagged with Mango I aptamer and its associated protein complex was affinity purified on streptavidin beads linked to TO1-Biotin. Sixty-four nonessential nuclear proteins identified by mass spectrometry were deleted in yeast and their effect on splicing examined using YFP splicing reporters containing either the RPS9A or RPS9B introns. ( B ) Enrichment of RPS9A pre-mRNA after pull-down on streptavidin beads relative to input was measured for strain expressing the Mango I-tagged intron (RPS9A-Mango I) and normalized to that measured with the control untagged strain ( RPS9A) for three experiments. ( C ) The relative effect on splicing of the reporters for the introns of RPS9A (iA) or RPS9B (iB) was measured in a strain with a deletion for a known negative regulator of RPS9A splicing ( rps9b Δ) compared to WT in three experiments. ( D ) Deletion of RPS9A interacting protein genes differentially regulates the splicing of RPS9 paralogs. The heatmap indicates the effects on splicing via either the RPS9A or RPS9B intron as indicated; the 21 gene knockouts coding for the RPS9A intron-interacting proteins resulting in a >40% (log 2 0.5) change in the signal of YFP splicing reporters are shown. As indicated by the scale on the right, five gene deletions increased the splicing of the RPS9A reporter by at least 2-fold (log 2 1) and show a difference of 40% (log 2 0.5) or more in splicing of the A reporter over the B reporter, resulting in the selection of these five genes for further analysis. Gene ontology analysis highlighted their common role in transcription regulation, and using a GO term mapper, two more genes were associated with that process and identified with gray dots.

Article Snippet: The RNA Mango I tag of the RPS9 intron and plasmids containing the YFP (yellow fluorescent protein)gene (pYFP) or introns of RPS9A or RPS9B inserted in the YFP gene (pY-iA-FP or pY-iB-FP) were generated by gene synthesis (Bio Basic Canada).

Techniques: Affinity Purification, Mass Spectrometry, Expressing, Control, Selection