Journal: Microbiome

Article Title: Elucidating the role of the gut microbiota in the physiological effects of dietary fiber

doi: 10.1186/s40168-022-01248-5

Figure Lengend Snippet: Schematic representation of the ex vivo detection assay based on BONCAT. Stool samples stored frozen were thawed, filtered, and washed in PBS and then incubated in the presence of AX and the cellular activity marker L -azidohomoalanine (AHA) to detect AX-stimulated bacterial cells. A no-amendment control, containing only AHA, was incubated to detect possible basal activity in the absence of AX. Microscopic inspection showed no BONCAT signal for all controls; thus, no basal activity was detected. AX-incubated samples were then fixed in ethanol and active cells were stained using a Cu(I)-catalyzed click reaction using a Cy5 dye solution. A and B A representative picture of fecal microbiota incubated for 6 h A with AX and B without AX (BONCAT control). Stimulated cells, shown in pink as a Cy5-positive BONCAT signal, were sorted by FACS, with all microbial cells shown in blue (DAPI stained). DNA was extracted from both sorted cells and samples at 0-h and 6-h anaerobic incubations. The 16S rRNA gene was amplified by PCR and amplicons were sequenced using the Illumina Miseq platform. AX, arabinoxylan; BONCAT, bioorthogonal non-canonical amino acid tagging; FACS, fluorescence-activated cell sorting

Article Snippet: To prepare the Cy5 dye solution, 1.25 μl of 20 mM CuSO 4 , 2.50 μl of 50 mM THPTA (Baseclick, Germany), and 0.30 μl of Cy5 alkyne dye (Jena Bioscience, Germany) were left in the dark for 3 min to react and then added to 221 μl of 1× PBS, 12.5 μl of 100 mM sodium ascorbate (Sigma-Aldrich, Austria), and 12.5 μl of 100mM aminoguanidine hydrochloride (Sigma-Aldrich, Austria).

Techniques: Ex Vivo, Detection Assay, Incubation, Activity Assay, Marker, Staining, Amplification, Fluorescence, FACS

Journal: Redox Biology

Article Title: Noncanonical inhibition of topoisomerase II alpha by oxidative stress metabolites

doi: 10.1016/j.redox.2025.103504

Figure Lengend Snippet: Lipid-derived electrophiles are inhibitors of TOP2A activity. (A) Chemical structures of HNE, HPNE, and ONE, which are electrophiles derived from lipids under conditions of cellular oxidative stress. The structures differ predominantly by the group at the 4-carbon position, which is altered as redox reactions occur. HPNE is a precursor of HNE, and ONE is an end-product. (B) Catenated kDNA decatenation assay to evaluate effects of LDEs on human TOP2A catalytic activity. LDEs (0.5 mM) were pre-incubated with purified human TOP2A (5U = 98.5 ng/rxn) for 10 min on ice prior to addition of kDNA (0.4 μg/rxn) for 10 min at 37 °C. Merbarone, a control catalytic inhibitor, was used at the same conditions. Vehicle-only controls are shown alongside each agent. Fully decatenated (Decat) kDNA migrates through the gel, while in lanes where hTOP2A catalytic activity is inhibited, a portion of kDNA remains catenated (Cat) and cannot move into the gel. (C) Supercoiled plasmid DNA (pBR322) assay to evaluate effects of LDEs on human TOP2A DNA relaxation activity. LDEs were used at 0.5 mM for a 10 min pre-incubation with TOP2A on ice prior to addition of plasmid DNA (0.3 μg/rxn). Etoposide, a control inhibitor of DNA relaxation by TOP2A, was used at the same conditions. Agent-matched vehicle-only controls are also shown. Presence of relaxed (Rel) DNA indicates expected activity by hTOP2A, while residual supercoiled (SC) plasmid DNA demonstrates inhibition of TOP2A DNA processing.

Article Snippet: Agents were pre-incubated with purified human TOP2A for 10 min on ice, and then pBR322 plasmid DNA (Promega, 4361 bp; 0.3 μg/rxn at 650 g/bp = 106 fmol/rxn in 20 μL volume = 5.3 nM) was added for 15 min at 37 °C.

Techniques: Derivative Assay, Activity Assay, Incubation, Purification, Control, Plasmid Preparation, Inhibition