Journal:

Article Title:

doi:

Figure Lengend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication. In order to determine whether 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-dC) induce similar changes in HIV-1 mutation frequencies and spectra, U373-MAGI cells were treated with DMSO (no-drug control), 5-aza-C, or 5-aza-dC. 5-Aza-C and 5-aza-dC were added 2 h before infection at the EC75 (∼260 or 3.8 μM, respectively). Cells were infected at an MOI of 1.0 with NL4-3 MIG-VSVG and were collected 72 h postinfection for the purification of genomic DNA. PCR was performed to prepare multiple amplicons (Gag, Pol, Vif, Env, Nef) from proviral DNA; these were then pooled, used to prepare libraries, and analyzed by 2 × 250 paired-end sequencing on the Illumina MiSeq system. Plasmid control amplifications were performed to determine the levels of background errors resulting from PCR and sequencing. Mutation frequencies for each amplicon, expressed as the number of mutations per base pair, were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases). Data represent means ± standard deviations for three independent biological replicates; N.S., not significant (P > 0.05).

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Mutagenesis, Infection, Purification, Sequencing, Plasmid Preparation, Amplification

Journal:

Article Title:

doi:

Figure Lengend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar patterns of mutation during HIV-1 replication. Using the Illumina sequencing data, G-to-C and C-to-G transversion frequencies were determined at every individual guanine (124 in total) or cytosine (116 in total) position within the sequences of the five amplicons. Mutation frequencies for each amplicon were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases) and are represented as mutations per base pair (m/bp). 5-Aza-C- and 5-aza-dC-induced mutation frequencies were then plotted against each other for each sequence position, and the resulting data were subjected to linear regression and correlation analyses. Data represent averages for three independent biological replicates. R2 denotes the extent to which the best-fit regression line explains the observed variability in the data; P indicates the significance of the correlation; and m indicates the slope of the best-fit regression line.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Mutagenesis, Sequencing, Amplification

Journal:

Article Title:

doi:

Figure Lengend Snippet: 5-Aza-dCTP levels are comparable in cells treated with 5-aza-C or 5-aza-dC. In order to determine the extent to which 5-aza-C is reduced to 5-aza-dC intracellularly, U373-MAGI cells were incubated with varying concentrations (EC25, EC50, or EC75) of 5-aza-C or 5-aza-dC. Cells were collected for analysis 4 h after drug addition, a time corresponding to the expected time of early reverse transcription. LC–MS-MS was then used to determine the relative levels of 5-aza-dCTP and 2′-deoxyriboguanylurea 5′-triphosphate (dRGU-TP). dRGU-TP is the final hydrolysis product of 5-aza-dCTP and is potentially relevant to antiviral activity. Data represent means ± standard deviations for three independent experiments, normalized to the EC75 of 5-aza-dC. N.S., not significant; *, P < 0.05.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: HIV-1 RT incorporates 5-aza-CTP much less efficiently than 5-aza-dCTP in vitro. The relative abilities of HIV-1 RT to incorporate 5-aza-CTP, 5-aza-dCTP, CTP, and dCTP were determined using an in vitro single nucleotide extension assay. The HIV-1 RT was incubated with a radiolabeled primer (18 nt) annealed to a DNA template (19 nt) in the presence of each compound at 5 nM to 500 μM (a 10-fold series, from left to right). Additional control reactions were performed by incubating with all four standard dNTPs (50 μM) (+) or by omitting RT (−). All of the reactions were analyzed on the same gel.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: In Vitro, Incubation

Journal:

Article Title:

doi:

Figure Lengend Snippet: Model of 5-azacytidine- and 5-aza-2′-deoxycytidine-mediated HIV-1 mutagenesis. 5-Aza-C and 5-aza-dC are first transported into the cell by facilitated diffusion through human equilibrative nucleoside transporter 1 (hENT1). 5-Aza-C and 5-aza-dC are then phosphorylated by uridine-cytidine kinase (UCK) and deoxycytidine kinase (dCK), respectively, to the monophosphate forms. Nucleoside monophosphate and diphosphate kinases (NMPK and NDPK) phosphorylate the monophosphate and diphosphate forms, respectively, resulting in the formation of 5-aza-CTP and 5-aza-dCTP. 5-Aza-CTP can be incorporated during the transcription of viral genomic RNA, resulting in C-to-G transversions. 5-Aza-CDP is also reduced to 5-aza-dCDP, ultimately forming 5-aza-dCTP. 5-Aza-dCTP is incorporated during reverse transcription, resulting primarily in G-to-C transversions (reflecting minus-strand incorporation) but also in low levels of C-to-G transversions. The mechanism by which C-to-G transversions are formed is still unclear, but they may be due to plus-strand incorporation of 5-aza-dCTP or minus-strand incorporation of 5-aza-dCTP hydrolysis products.

Article Snippet: 5-Aza-CTP and 5-aza-dCTP were purchased from American Advanced Scientific (College Station, TX) and Jena Bioscience (Jena, Germany), respectively.

Techniques: Mutagenesis, Diffusion-based Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: GSDME Increases Chemotherapeutic Drug Sensitivity by Inducing Pyroptosis in Retinoblastoma Cells

doi: 10.1155/2022/2371807

Figure Lengend Snippet: After GSDME expression was upregulated by decitabine, pyroptosis was increased. (a and b) The toxicity of decitabine to Y79 and WERI-RB-1 cells was detected by CCK-8 assay at different concentrations of decitabine. (c and d) The mRNA expression of GSDME was detected using RT-PCR after treatment with different concentrations of decitabine for 3 days (the cells were centrifuged, and decitabine was replaced every day). (e and f) The protein level of GSDME in cells was quantitatively analysed by western blot. (g and h) The protein level of GSDME in cells was detected using immunofluorescence. (i and j) CCK-8 assay of Y79 and WERI-RB-1 cell viability after treatment with carboplatin or decitabine plus carboplatin. (k) The death morphologies of the three cell lines were observed using an Olympus microscope. Y79 and WERI-RB-1 cells were treated with decitabine plus carboplatin; ARPE-19 cells were treated with decitabine; black arrows, apoptotic cells; white arrows, pyroptotic cells. (l–p) GSDME-N and its pathway proteins were detected by WB. (q and r) Flow cytometry showed the ratio of FITC monostaining to FITC and PI double-positive staining after treatment with decitabine plus carboplatin, and quantitative maps were drawn for carboplatin-treated cells after 24 h. (s) LDH in the cell supernatant was detected by ELISA. Each experiment was repeated at least three times. Scale bar, 100 μ m.

Article Snippet: When the tumor was approximately 100 mm 3 , the second group was injected with 0.5 mg/kg decitabine (MedChemExpress, USA) 1 time/day for 7 days in total.

Techniques: Expressing, CCK-8 Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Microscopy, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: GSDME Increases Chemotherapeutic Drug Sensitivity by Inducing Pyroptosis in Retinoblastoma Cells

doi: 10.1155/2022/2371807

Figure Lengend Snippet: IC 50 values and statistical analyses of decitabine and carboplatin treatments in RB cell lines.

Article Snippet: When the tumor was approximately 100 mm 3 , the second group was injected with 0.5 mg/kg decitabine (MedChemExpress, USA) 1 time/day for 7 days in total.

Techniques:

Journal: Cell Reports Methods

Article Title: RECOVER identifies synergistic drug combinations in vitro through sequential model optimization

doi: 10.1016/j.crmeth.2023.100599

Figure Lengend Snippet:

Article Snippet: Ixabepilone , MedChem Express , HY-10222.

Techniques: Software, Recombinant

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on TH striatal levels. ( A ) Representative example of striatal TH immunofluorescence. Scale bar = 1000 μm and ( B ) TH striatal intensity in SHAM and GDX males and SHAM and GDX female mice. Values shown are the means pixel intensity ± SEM of 6-16 mice per group. Two-way ANOVA, Tukey post-hoc test: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques: Immunofluorescence

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on striatal microglial density. ( A ) Representative example of striatal Iba1 immunofluorescence. Scale bar = 20 μm; ( B ) Iba1 positive cells count in the striatum in SHAM and GDX males and SHAM and GDX female mice. Values shown are the mean number of Iba1 positive cells per mm 3 ± SEM of 4 images of 4-6 mice per group and 7 to 25 microglia per image. Two-way ANOVA, Tukey post-hoc test: * p < 0.05 and ** p < 0.01 and ( C ) Correlations between striatal DA contents with striatal microglial density (Iba1 positive cell counts).

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques: Immunofluorescence

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on striatal microglial doublet. ( A ) Representative example of striatal microglial doublet indicated with white arrowheads. Scale bars = 5 μm; ( B ) Microglial doublet counts in the striatum in SHAM and GDX males and SHAM and GDX female mice. Values shown are the mean number of doublets per image ± SEM 4 images of 4-6 mice per group and from 0 to 4 microglial doublet per image. Two-way ANOVA, Tukey post-hoc test: * p < 0.05 and **** p < 0.0001 and ( C ) Correlations between striatal DA contents with striatal microglial doublet counts.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques:

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Three-dimension morphological analysis of striatal microglia schematic and effect of mice gonadal status (SHAM, GDX), MPTP, and dutasteride (DUT) treatment on striatal microglial morphology in 3D. ( A ) Number of arborization terminal points representing the number of ramifications terminal points in the whole microglia. Values shown are the sum of terminal points of microglia ± SEM ( B ) Number of arborization branch points representing the number of ramifications branching points in the whole microglia. Values shown are the sum of branch points of microglia ± SEM; ( C ) Number of arborization segments representing the number of ramification segments in the whole microglia. Values shown are the sum of the segment of microglia ± SEM; ( D ) Arborization length defined as the sum of the lengths of all branches within the entire microglia. Values shown are the sum of the length of each branch of each microglia ± SEM; ( E ) Sholl intersections defined as the sum of branches intersections with concentric circles (spheres in 3D) spaced 1 μm each. Values shown are the sum of Sholl intersection of each microglia ± SEM; ( F ) Radius expressed as maximum reach from the nucleus where the concentric circle (spheres in 3D) presenting the intersection of the branches. Values shown are concentric circle numbers having the last branch intersection of microglia. The mean and SEM of 4-6 mice per group and from 1 to 14 microglia per mice is shown. Each point represents one microglia. Two-way ANOVA, Tukey post-hoc test: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques:

Journal: Pharmaceuticals

Article Title: Three-Dimensional Analysis of Sex- and Gonadal Status- Dependent Microglial Activation in a Mouse Model of Parkinson’s Disease

doi: 10.3390/ph16020152

Figure Lengend Snippet: Experimental design. Male and female mice were gonadectomized (GDX) or sham operated (SHAM) and received intraperitoneal injection of dutasteride (DUT) or vehicle and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) or saline.

Article Snippet: Mice that received dutasteride (Toronto Research Chemicals, Toronto, ON, Canada) (5 mg/kg, intraperitoneal) or vehicle only (0.9% saline with 1% tween 80, i.p.) were injected once daily for 10 days.

Techniques: Injection, Saline