Allegra 25r

CNFs (Sigma Aldrich) had a diameter ≈100 nm and length ≈20–200 µm (manufacturer's specifications). Varying ratios of CNFs (0, 1:10, 1:2, and 1:1) were suspended in GO solution and were sonicated using a bath sonicator (Branson Ultrasonic, Danbury, CT) for 1 h at 60 W followed by centrifugation at 800 rpm for 5 min (Allegra 25R, Beckman Coulter, Indianapolis, IN) to sediment CNF bundles. The concentrated GO:CNF suspensions were degassed to remove any bubbles. Reducing agents including sodium iodide and ascorbic acid, at a concentration of 10 wt% to induce self‐assembly of CGS, were added into suspensions; and it was poured into cylindrical molds. The suspensions formed the CGS at 80 °C within 24 h. The CGS had dimensions of 6 × 2 mm (diameter × height). To form a 2D CGS, the same steps were used to form 3D CGS. Subsequently, the CGS was placed between glass slides and then weight of 1 kg was applied for 1 h. The CGSs were neutralized by washing with deionized water until pH of supernatant equilibrated to 7. Collected CGSs were autoclaved and stored at 4 °C.

The right lobe of the lung was immediately frozen at the end of the study and the tissue stored at 70 °C until analysis. In previous studies we used the right lobe of the lung for biochemical analysis and the left lobe for pathological evaluations [3 (link)]. The lung tissue nuclei were extracted as follows. Approximately, 0.15 g (1/3 of the right lobe) was placed in ice cold buffer A (20 mM Tris/HCl pH 7.8, 1 mM EDTA, 0.1 M NaCl, 1 mM phenylmethylsufonyl fluoride, and 1 μg/mL of each of the following: aprotinin, leupeptin and pepstain) and homogenized with a glass-teflon homogenizer (Con-Torque Eberbach Company Ann Arbor, MI). The homogenate was then centrifuged at 2000 × g for 15 min (Allegra 25R, Beckman Coulter). The supernatant was then removed, and the pellet was re-extracted using buffer B (buffer A with NaCl increased to 0.6 M) by tearing the pellet and vortexing in order to maximize the nuclear amount in the sample. The solution was then covered with mineral oil and spun at 190,000 × g using an ultracentrifuge (Optima L-90 K, Bechman Coulter). The supernatant was then removed by syringe and stored at −70 °C until analysis. Protein concentration was quantified using a spectrophotometer (UV-1601PC, Shimadzu).

To determine activities of MMP2 and MMP9, 3 × 10⁶ cells were plated into 15-cm dishes in OFCS. After 16 h, cells were incubated with 1 μM RGD peptide (Santa Cruz Biotechnology) or vehicle (DMSO). After 72 h, supernatants were collected and cleared of cells and debris at 500 g for 10 min and 5,500 g for 20 min, respectively, using an Allegra 25R centrifuge (Beckman Coulter). Subsequently, supernatants were concentrated at 5,500 g on VivaSpin 20 columns (GE Healthcare Life Sciences) for 30 min. Concentrated samples were diluted 1:1 in Tris-glycine SDS gel loading buffer (Novex, Invitrogen) and separated by electrophoresis on 10% Tris-glycine gels containing 0.1% gelatine (Novex, Invitrogen) at 125 Volts for 90 min. Gels were incubated in 1× Zymogram renaturation and developing buffer (Novex, Invitrogen) according to the manufacturer's specification. Subsequently, gels were stained with SimplyBlue SafeStain (Novex, Invitrogen) for 6–12 h with 3–5 changes until protein bands were clearly visible.

Polyphenolic substances of approximately 50 g of healthy, frozen whole grapes were extracted with 50 mL methanol/water/formic acid (50:48.5:1.5, v/v/v/v) according to the method described by Portu et al. [76 (link)] with slight modifications. The methanol acid mixture was added to the frozen gapes and homogenized (Moulinex 180 W grinder, Alençon, France), sonicated for 10 min at 50 Hz (Grant XUB5, Cambridge, England), and centrifuged at 5000 rpm for 10 min (Allegra 25R, Beckman Coulter, DE, USA). The supernatant was separated, and the resulting pellet was extracted up to three times. Supernatants obtained were combined in a flask and the volume was brought to 200 mL with the extraction mixture and stored at −20 °C until analysis. Three extractions were performed for each sample of a given cultivar.
For the analysis of ANs, the extract was injected directly into the HPLC.
Isolation of non-anthocyanin compounds was carried out based on previous works [30 (link)]. First, grape phenolic extracts (3 mL) were diluted with 9 mL of 0.1 N HCl and passed through PCX SPE cartridges, (Chromafil PET 20/25, Machery-Nagel, Düren, Germany). The non-anthocyanin phenolic substances were eluted with 3 × 5 mL of methanol.
The eluate containing FLAVO, FLAVA, HCA, and HBA was separately dried in a rotary evaporator (40 °C) and redissolved in 1.5 mL of 20% (v/v) methanol aqueous solution.

Neuron viability was determined using a previously described method of ethidium bromide and acridine orange staining (54 (link)). SK-N-BE(2) cells were seeded in a 96-well plate and, upon reaching 80 to 90% confluence, were inoculated with either wild-type SINV or one of the capping mutants at an MOI of 30 IU/cell. After a 1-h adsorption period, the inoculum was removed, the cells were washed once with 1× PBS, and DMEM/F12 medium was added. At 24 hpi, cell viability was assessed using ethidium bromide/acridine orange staining as described in Ribble et al. (54 (link)). Briefly, the 96-well plate was centrifuged at 1,000 rpm for 5 min using an Allegra 25R model centrifuge (Beckman Coulter) with inserts for 96-well plates. Following centrifugation, 8 μl of EB/AO dye solution (100 μg/ml ethidium bromide and 100 μg/ml acridine orange in 1× PBS) was added to each well. Cells were viewed using an epifluorescence microscope. Tests were done in triplicate, and a minimum of 100 total cells per well were counted using ImageJ.