Hymecromone

The NA activity of influenza A and B viruses was measured by a fluorescence-based assay using the fluorogenic substrate MUNANA (Sigma-Aldrich, St Louis, MO), based on the method of Potier et al. [13] (link). Substrate cleavage by the NA enzyme releases the fluorescent product 4-methylumbelliferone (4-MU) (Sigma-Aldrich, St Louis, MO). Fluorescence was measured every 60 s for 60 min at 37°C in a Synergy 2 multimode microplate reader (BioTek Instruments, Winooski, VT), using excitation and emission wavelengths of 360 nm and 460 nm, respectively, and a sensitivity setting of 60%. Under these conditions, blank samples of enzyme buffer generated a background signal of approximately 10 relative fluorescence units (RFU) and a dynamic range to detect increasing concentrations of 4-MU fluorescence over 5 orders of magnitude. Two-fold virus dilutions were prepared in enzyme buffer [32.5 mM of 2-(N-morpholino) ethanesulfonic acid (MES), 4 mM of calcium chloride, pH 6.5] and added (100 µL/well) in duplicate to a flat-bottom 96-well opaque black plate (Corning, Tewksbury, MA). After preincubation for 20–30 min at 37°C, the MUNANA substrate (separately pre-incubated for 20–30 min at 37°C) was added to all wells (50 µL/well) to achieve a final concentration of 100 µM. Immediately after adding the MUNANA substrate, the plate was transferred to a 37°C prewarmed Synergy 2 multimode microplate reader. The fluorescence signal from the enzyme buffer and MUNANA substrate alone in the absence of enzyme was subtracted as background from the signals obtained in the other wells. A standard curve was generated for each experiment using 4-MU diluted in enzyme buffer at final concentrations of 0.05 µM to 26.67 µM. Background-corrected RFU was converted to 4-MU concentration (µM) and used to determine the percentage of substrate consumed during the reaction. Enzymatic reactions were performed under conditions of ≤15% MUNANA substrate conversion to product during the reaction time.

Premature senescence was tested in fibroblasts treated with different concentrations of H₂O₂ for 1.5 h using senescence-associated (beta)-galactosidase (SA-β-gal) as a biomarker of senescence. After H₂O₂ treatment, the medium was replaced with fresh complete medium, and cells were cultured for 72 h before staining for SA-β-Gal activity as previously described [26 (link)]. Quantification of premature senescence was determined by calculating the rate of conversion of 4-methylumbellliferyl-β-D-galactopyranoside (MUG) to the fluorescent product, 4-methylumbelliferone (4-MU), at pH 6.0 as previously described [30 (link)]. The relative increase in 4-MU fluorescence per mg protein was determined by subtracting the untreated control values from the H₂O₂-treated values.

GLA activities were determined in Fabry mouse tissues using previously described methods (Desnick et al., 1973 (link)). In brief, tissue samples were homogenized in chilled reporter lysis buffer (Promega) and protease inhibitor (Pierce) was added to the lysates. Protein concentrations were determined using the Bio-Rad Colorimetric Protein Assay Kit. 10 μL of tissue lysate was added to an equal volume of 10 mM 4-methylumbelliferyl-α-D-galactopyranoside (Sigma-Aldrich), dissolved in assay buffer (0.2 M citrate, 0.4 M phosphate buffer, pH 4.4), and 0.1 M N-acetylgalactosamine (Sigma Aldrich), the latter to inhibit α-galactosidase B activity (Mayes et al., 1981 (link)). Following a 30 min incubation at 37°C, reactions were terminated by the addition of 480 μL of 0.1 M ethylenediamine, pH 10.3. The amount of 4-methylumbelliferone (4-MU) produced was determined by measuring fluorescence using a Synergy H1 fluorometer (BioTek). Tissue α-Gal A activities were expressed as nmol of 4-MU produced per h per mg of total protein (nmol/h/mg). Measurement of plasma GLA activities in wildtype mice for PK studies was performed as described above with the following modifications: lysates were incubated with 5 mM 4-methylumbelliferyl α-D-galactopyranoside in assay buffer [20 mM citrate, 30 mM sodium phosphate (pH 4.4), 0.1 M N-acetylgalactosamine, and 4 mg/mL BSA], and the reaction was stopped by addition of stop buffer (0.1 M Glycine, 0.1 N NaOH], as previously described (Shen et al., 2016 (link)).
AGA activity was measured with 1 mM L-aspartic acid β-(7- amido-4-methylcoumarin) in 10% SuperBlock and 90% 50 mM Tris-HC (pH 7.5) for 60 min at 37°C, and then adding 100 µL of stop buffer [0.2 M glycine, 0.175 M NaOH (pH 10.6)], as previously described (Mononen et al., 1993 (link)). GUSB enzyme assay was performed using 10 mM 4-methylumbelliferyl-β-D-glucuronide (Merck) in 0.1 M sodium acetate (pH 4.6) at 37°C for 30 min, and reactions were stopped by 0.1 M sodium carbonate (Grubb et al., 2008 (link)). GAA activity assay was performed with 3 mM 4-methylumbelliferyl-a-D-glucopyranoside (Merck) in assay buffer (30 mM sodium citrate, 40 mM sodium phosphate dibasic, pH 4.0) at 37°C for 3 h (Flanagan et al., 2009 (link)). Reactions were stopped by the addition of an equal volume of 0.4 M glycine, pH 10.8. IDS activity assay was performed with 2.5 mM 4-Methylumbelliferyl sulfate potassium salt (Merck) in 50 mM sodium acetate, at 37°C for 4 h (Dean et al., 2006 (link)). Reactions were stopped with glycine carbonate buffer (pH 10.7). Fluorescence was measured by microplate reader with 360/40 nm excitation and 440/30 nm emission filters.

For GUS staining, 2-week-old seedlings were immersed in staining solution [containing 1 mM X-Gluc, 100 mM sodium phosphate, pH 7.0; 1 mM potassium ferricyanide (K₃Fe(CN)₆), 1 mM potassium ferrocyanide (K₄Fe(CN)₆), 10 mM EDTA, pH 8.0; 0.1% Triton X-100] (prepared just before use and stored in the dark). Samples were treated in the dark in a shaker at 100 rpm at 37ºC overnight. After washed in ddH₂O for 2–3 times, seedlings were boiled in de-staining solution (containing glacial acetic acid: anhydrous ethanol = 3:1) for 10 min till became completely transparent, then photographed. Fluorometric GUS activity was detected using 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) as the substrate. Samples (0.1 g) were harvested and homogenized in extraction buffer (Cat. SL7161, Coolaber, Beijing), after being centrifuged at 12,000 rpm for 10 min, aliquots of supernatant were incubated for 10, 20 min at 37ºC in extraction buffer containing 1 mM 4-MUG. The reaction was terminated by addition of 0.2 M Na₂CO₃. Fluorescence was then measured on a fluorescent spectrophotometer (F97pro, Shanghai) with 4-methylumbelliferone (4-MU) as standard. Protein concentration was measured using Bradford Kit (Cat. SK1060, Coolaber, Beijing) with bovine serum albumin (BSA) as standard.