Journal: Investigative Ophthalmology & Visual Science

Article Title: NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage

doi: 10.1167/iovs.65.12.37

Figure Lengend Snippet: Induction of retinal organoids from NMNAT1-KO hiPSC. ( A ) Bright field photographs of the retinal organoids made from control or NMNAT1-KO hiPSCs were taken at the cultured days 7, 11, 14, and 21, and week 8. Representative photographs from 10 to 20 colonies of few independent cultures are shown. ( B, C ) Colonies at day 15 ( B ) and 21 ( C ) were immunostained with anti-TUBB3 antibody, and nuclei were visualized by DAPI staining. In B , the red square regions of upper panels are enlarged in the lower panels . Representative photographs of six samples from two independent cultures are shown. ( D, E ) Colonies at day 63 were frozen sectioned, and the sections were immunostained with anti-Ki67 and -BRN3B antibodies ( D ), or anti-CRX or -RXRG antibodies ( E ). Representative photographs of three sections from three independent organoids of two independent cultures are shown. Nuclei were visualized by staining with DAPI ( B – E ). Scale bars are 500 µm ( A, B, C ) and 100 µm ( D, E ).

Article Snippet: Immunohistochemistry of frozen sections was done as described previously., Primary antibodies used were mouse monoclonal antibodies against Ki67 (BD Biosciences, #55060924), TUBB3 (R&D Systems, MAB1195), goat polyclonal antibody against human BRN3B (Santa Cruz, sc-60267), CRX (Abnova Corp., 4G11, H00001406-M02), RXRG (Santa Cruz, A-2, sc-365252), and chick polyclonal antibody against GFP (Abcam, ab13970).

Techniques: Control, Cell Culture, Staining

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

Journal: Pharmaceutics

Article Title: Stepwise Structural Simplification of the Dihydroxyanthraquinone Moiety of a Multitarget Rhein-Based Anti-Alzheimer Lead to Improve Drug Metabolism and Pharmacokinetic Properties

doi: 10.3390/pharmaceutics16080982

Figure Lengend Snippet: In vitro inhibitory activity of the new compounds 4 – 7 , the lead rhein–huprine hybrid 3 , and reference compounds on hAChE, hBChE, hBACE-1, and Aβ42 and tau aggregation.

Article Snippet: The human recombinant AChE (hAChE) and human serum BChE (hBChE) inhibitory activities of the new compounds 4 – 7 were tested by the widely used method of Ellman [ ], using enzymes supplied by Sigma (Milan, Italy).

Techniques: In Vitro, Activity Assay

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