Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: (A) Targeting strategy for the expression of engineered hBChE. The targeting vector contains two Rosa26 homology arms, flanking the expression cassette for hBChE and a selection marker (puromycin resistant gene, Puro) driven by a constitutive promoter UbiC (Ubiquitin C promoter). hBChE and Puro are separated by a self-cleavable peptide T2A. gRNA: guide RNA. (B) Integration of the targeting vector into Rosa26 locus is verified by PCR (left panel) and southern blotting (right panel). Positive clones display an additional band of the expected size. Three independent experiments were performed with similar results. (C) Confirmation of hBChE expression in targeted cells by immunoblots with different antibodies. Numbers on left side indicate molecular weight markers. kD: kilodalton. Full scans of the western blots at . Three independent experiments were performed with similar results. (D) Confirmation of secretion of engineered hBChE in the culture media by ELISA. The box plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). N=number of independent experiments. (E) Cocaine hydrolysis activity in vitro . Cell culture supernatants were collected from cells targeted by hBChE or mBChE. Cocaine hydrolysis activity was examined by a clearance assay in vitro . N=3 (independent experiments), error bars represent standard deviation, and the measure of center represents the average. (F) Cell cycle profiles. FACS (fluorescence activated cell sorting) of control (Ctrl) and hBChE- expressing epidermal stem cells. PI: propidium iodine. Three independent experiments were performed with similar results. Text in this legend has been adapted from our previous work in ref. .

Article Snippet: Human BChE quantikine ELISA kit was obtained from R&D systems (Minneapolis, MN).

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Ubiquitin Proteomics, Southern Blot, Clone Assay, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Activity Assay, In Vitro, Cell Culture, Standard Deviation, Fluorescence, FACS, Control

Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: (A) Skin organoids are developed from control or hBChE -producing cells, and transplanted to the host mice. Cells were infected with lentivirus encoding firefly luciferase before engraftment to allow intravital imaging of the skin grafts. (B) Histological examination of grafted skin collected from mice grafted with control (GWT) or hBChE skin organoids (GhBChE). Scale bar=50 μm. (C) Sections of grafted skin were immunostained with different antibodies as indicated (Krt10: keratin 10, a marker for early epidermal differentiation, Lor: Loricrin, a marker for early epidermal differentiation, β4: β4-integrin, CD104, a marker for skin basement membrane). Dashed lines denote the basement of skin. Epi: epidermis, Der: dermis. Scale bar=50 μm. (D) Mice are grafted with control or hBChE skin organoids. Presence of hBChE in blood was determined by ELISA for 10 weeks after engraftment (n=5 mice in each group). (E) Cocaine pharmacokinetics in the NAc (nucleus accumbens) after an i.p. administration of 10 mg/kg cocaine in GhBChE and GWT mice (n = 6 for each group). Data are plotted as mean±SEM (standard error of the mean). A two-compartment in vivo pharmacokinetic model was built to represent the cocaine concentration-time profile in the NAc. (F) Changes in DA (dopamine) level in the NAc after an i.p. administration of 10 mg/kg cocaine in GhBChE and GWT mice (n = 6 for each group). Individual lines for each animal were plotted. Treatment×time interaction: F 6, 70 =6.549, P＜0.0001, two-way ANOVA. (G-H) Cocaine-induced locomotor activity in in GhBChE and GWT mice (n = 11 for GWT, n=8 for GhBChE). Non-linear dose versus response curve was simulated to represent cocaine-induced dose-response locomotor activity. (G) Total distance traveled after 0, 10, 20, 40 mg/kg cocaine i.p. injection. Treatment×doses interaction: F 3, 68 =11.83, P＜ 0.0001, two-way ANOVA. (H) Stereotypic counts after 0, 10, 20, 40 mg/kg cocaine i.p. injection. Treatment×doses interaction: F 3, 65 =6.223, P=0.0009, two-way ANOVA. (I) Lethality rates after injection of 40, 80, 120, 160 mg/kg cocaine in GhBChE and GWT mice (n=3 independent experiments, for each experiment, 8 animals were examined in each test group). Individual data point represents the result from each experiment. Error bars represent SEM, and the measure of center represents the average. Text in this legend has been adapted from our previous work in ref. .

Article Snippet: Human BChE quantikine ELISA kit was obtained from R&D systems (Minneapolis, MN).

Techniques: Control, Infection, Luciferase, Imaging, Marker, Membrane, Enzyme-linked Immunosorbent Assay, Drug discovery, In Vivo, Concentration Assay, Activity Assay, Injection

Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: (A) The AAVS1 targeting strategy for expression of engineered hBChE. The targeting vector contains two AAVS1 homology arms, flanking the expression cassette for hBChE and a selection marker (puromycin resistant gene, Puro) driven by a constitutive promoter UbiC (Ubiquitin C promoter). hBChE and Puro are separated by a self-cleavable peptide T2A. (B) Integration of the targeting vector into AAVS1 locus is verified by southern blotting. Positive clones display an additional band of the expected size. Three independent experiments were performed with similar results. (C) Expression of hBChE is confirmed in targeted cells by immunoblots with different antibodies as indicated. Full scans of the western blots at . Three independent experiments were performed with similar results. (D) Secretion of engineered hBChE in the culture media is confirmed by ELISA. N=3 (independent experiments). Box plots were generated with the same elements as described in . (E) Image of nude mouse grafted with organotypic human skin culture. Intravital imaging shows efficient incorporation of grafted cells expressing luciferase (right side) or control cells (left side) upon engraftment. (F) Sections of grafted skin were immunostained with different antibodies as indicated. Dashed lines denote the basement of skin. Scale bar=50 μm. (G) Mice are grafted with control or hBChE skin organoids. Presence of hBChE in blood was determined by ELISA for 8 weeks after engraftment (n=3 mice in each group). Text in this legend has been adapted from our previous work in ref. .

Article Snippet: Human BChE quantikine ELISA kit was obtained from R&D systems (Minneapolis, MN).

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Ubiquitin Proteomics, Southern Blot, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Imaging, Luciferase, Control

Journal: bioRxiv

Article Title: Gut microbiota gate host exposure to metabolites from dietary Solanums

doi: 10.1101/2024.03.20.584512

Figure Lengend Snippet: A) Total steroidal alkaloid concentration in the serum of C57BL/6J mice fed potato-supplemented diet and treated with antibiotics. Aglycone concentrations were estimated using a solanidine standard curve. Data shown are the mean±SD (n=5 mice). P values were determined using a two-sided Welch’s T test. B) In vitro inhibition of recombinant human AChE activity by potato steroidal alkaloids. Data shown are normalized to AChE treated with vehicle only (DMSO for solanine, chaconine and ethanol for solanidine) with a total vehicle concentration of 1% for all treatments. Values shown are the mean±SD (n=3 assays per concentration). 5 ng of AChE was used in each reaction. C) In vitro inhibition of cholinesterase activity by 50 μM potato SAs in human protein homogenates from normal colonic tissue (n=3 donors) and Caco-2 colonic epithelial cells. Activity was normalized to the activity of the vehicle-treated sample. Values shown are the mean±SD of three replicate assays per homogenate. 5 ng each of recombinant human AChE and BChE were used for pure protein reactions, while 35 μg total protein was used for each homogenate. Multiplicity-adjusted P values were calculated using Tukey’s multiple comparison tests. D) Inhibition of recombinant human AChE activity by spent media from human stool (n=11 donors) incubated with 20 μM solanine in SAAC minimal medium. Activity was normalized to the activity of AChE treated with spent media from the same donor sample incubated with vehicle only. Values shown are the mean±SD of three AChE activity assays for each donor and compound combination. The relative abundances of select metabolites following incubation with solanine are shown below for each stool sample. Values shown are EIC peak areas normalized to the maximum peak area across all samples. P values were determined using a two-sided Welch’s T test. Ns: not significant with P>0.05.

Article Snippet: BChE activity was analogously measured using 5 ng of recombinant human BChE (R&D Systems) and butyrylthiocholine iodide.

Techniques: Concentration Assay, In Vitro, Inhibition, Recombinant, Activity Assay, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

doi: 10.3390/ijms252011320

Figure Lengend Snippet: Validation analysis of HeLa cell clones with integrated rhBCHE. ( A ) Schematic diagram of the rhBCHE template. ( B ) Circularization system of the rhBCHE template. Yellow and blue arrows represent multimolecular and monomolecular products, respectively, in the 5 ng/μL circularization system. ( C ) Western blot analysis. After ultrafiltration, the cell supernatant was analyzed by Western blot using different amounts of rhBChE standards as control. ( D ) Deglycosylation assay of the supernatant protein. The molecular weight of the supernatant protein decreased following deglycosylation with rhBChE standard. ( E ) qPCR detection of BCHE transcription levels. ( F ) Analysis of supernatant protein activity. The activity of supernatants protein after ultrafiltration from different cells was determined using the Ellman assay. ( G ) Determination of anti-DDVP activity. A 50 μm concentration was determined as the median lethal concentration dose for WT HeLa cells, and cell viability in other clones was measured relative to this (N = 4). ( C , E , G ) Data are presented as the mean ± SD from three or four technical replicates. Statistical analysis was performed using an unpaired Student’s t -test. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.

Article Snippet: Recombinant human BChE (Sinobiological, Beijing, China) was used as a control.

Techniques: Clone Assay, Western Blot, Control, Molecular Weight, Activity Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

doi: 10.3390/ijms252011320

Figure Lengend Snippet: Detection of HEK-293T cell clones with double-copy integration of rhBCHE. ( A ) PCR genotyping of the rhBCHE-forward clones targeting the AAVS1 and GRIK1 loci in HEK-293T cells. The lanes within red rectangles indicate forward knock-in patterns, while the red asterisks indicate reverse knock-in patterns at the GRIK1 locus. ( B ) Western blot analysis. AF44: clone with single-copy forward integration of rhBCHE at the AAVS1 locus; AF44-GF5 and AF44-GF10: clone with single-copy forward integration of rhBCHE at both the AAVS1 and GRIK1 loci; AF44-GR6: clone with single-copy forward integration of rhBCHE at the AAVS1 locus and single-copy reverse integration of rhBCHE at the GRIK1 locus. Tubulin served as the loading control. ( C ) Deglycosylation of the intracellular protein was analyzed by Western blot with GAPDH as the loading control. ( D ) Deglycosylation of the supernatant protein was analyzed by Western blot. ( E ) Multimerization of the supernatant protein was detected by Western blot. The blue arrow indicates the dimer protein band, and the green arrow indicates the monomer protein band. ( F ) Ellman activity assay of the supernatant protein. The rhBChE activity of AF44-GR6 was significantly higher than that of AF44. Data are presented as the mean ± SD from three technical replicates. An unpaired Student’s t -test was used for statistical analysis. (*) p < 0.05.

Article Snippet: Recombinant human BChE (Sinobiological, Beijing, China) was used as a control.

Techniques: Clone Assay, Knock-In, Western Blot, Control, Activity Assay