Extran

Mice were evaluated in the open field arena to analyze anxiety and general motor performance one day before CFC. The open field consisted of a white acrylic arena, 40 cm width × 40 cm length × 25 cm height. To facilitate the analysis of locomotion and the time spent at the center and periphery zones, the floor was divided into 16 drawn squares (10 cm × 10 cm each square) where 4 were central and 12 were peripheral. Each mouse was individually placed in a corner of the arena and was allowed to explore freely. Behavior was videotaped for 5 minutes. The total number of line crossings (from one square to another), the time spent at the center or periphery zones, and the displacement track from each mouse were manually assessed by the experimenter through repeated analysis of videos recorded during the test sessions. Caution was taken to counterbalance the introduction of the mice to each of the four corners of the arena. Before every trial, the arena was wiped with a cleaning solution consisting of 10% EtOH and 10% Extran (Merck, Darmstadt, Germany) diluted in distilled water.

Personal and written instructions were given to each child and their parents on how to collect the 24 h urine sample at home using preservative free, Extran-cleaned (Extran, MA03; Merck, Darmstadt, Germany) 1-L plastic containers. The urine samples were stored at −18 to −20 °C until they were thawed for analysis. Creatinine excretion was quantified with a creatinine analyzer (Beckman-2; Beckman Instruments, Fullerton, CA, USA) based on the kinetic Jaffe’ procedure. To minimize possible errors in urine collection, samples with a daily creatinine excretion < 0.1 mmol/kg were not considered in the analysis [39 (link)]. Urinary urea was measured using the urease Berthelot method (Randox Laboratories, Crumlin, UK). Acid–base analytes, i.e., 24 h pH, titratable acidity (mEq/L), ammonium (mmol/L), and bicarbonate (mmol/L) were quantified by the three phase acid–base titration method [40 ], using a Mettler Toledo endpoint titrator (Mettler Toledo, Giessen, Germany), and NAE was then calculated by summing titratable acid and ammonium minus bicarbonate. Urinary phosphate excretion was determined with a Dionex 2000i/SP ion chromatograph that contained an ion Pac AS4A column (Dionex GmbH, Idstein, Germany).

Before cell seeding, the scaffolds were cleaned by consecutive soaking in Sodium Dodecyl Sulfate (SDS, Carl Roth GmbH, Ludwigshafen, Germany) and Extran (Merck, Germany) solutions (2% w/v and 5% w/v, respectively) for 5 min. After drying, BG scaffolds were sterilized at 160 °C for 2 h in a furnace, meanwhile, polymer-coated scaffolds were put under UV light for 3 h. BG, KHC2000/BG, KHC2000E2000/BG, and PCL/BG scaffolds were then preconditioned in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher, Karlsruhe, Germany) without phenol red at 37 °C, 2% O_2, and 10% CO₂ (Galaxy 48 R, Fredericton, NB, Canada) for six days before cell seeding.

Seawater samples were collected in Otsuchi Bay, located on the Pacific coast of northeastern Japan (39°20′36″N, 141°56′47″E; water depth: 46 m) on January 24, March 11, May 30, and July 24 of 2013. Dynamics of bacteria and viruses in Otsuchi Bay have been described elsewhere (Nagata et al., In press ). Samples were collected at a depth of 5 m using a 12-L Niskin bottle (Sea-Bird Scientific, United States), gravity filtered through a 20-μm mesh-size nylon, and then used for subsequent analysis. Prescreening was used to remove larger particles, including occasionally abundant diatom cells (Tachibana et al., 2017 (link)) that can clog filters. Bacteria associated with particles larger than 20 μm were not considered in this study. All sampling tanks, cylinders, and incubation bottles used for the experiments were thoroughly cleaned through sonicating in alkaline detergent (5% Extran, Merck Ltd., Germany) and then soaked overnight. Then, the containers were rinsed with hot water to remove the detergent, and then rinsed with Milli-Q water. After that, the containers were soaked in 1 N HCl overnight and thoroughly rinsed with Milli-Q water. Before each incubation experiment, the incubation bottles and cylinders were rinsed with virus-free seawater (see below) three times to wet their inner surfaces.

Each year, participants are requested to collect a 24-h urine sample. All micturitions from the 24-h sampling period are collected in provided Extran-cleaned (Extran, MA03, Merck, Darmstadt, Germany) preservative-free 1-L plastic containers and stored immediately at ≤−12 °C. After transport to the study centre, the samples are stored at ≤−20 °C until analysed.
uHA was measured photometrically, in triplicate, with the following modifications to the method from Tomokuni and Ogata [18 (link)]: (a) placement of the reagents in an ice bath to moderate the exothermic reaction; (b) prolongation of the reaction time of the urine with benzenesulfonyl chloride and pyridine to 60 min; (c) replacement of ethanol with methanol as the diluent; and (d) photometrical measurement at 436 nm. Photometric determination of uHA was performed on two spectrometers (Lambda 11UV/VIS Spectrometer, Perkin Elmer, Überlingen, Germany and Campspec UV/VIS Spectrophotometer M107, Spectronic Campspec Ltd., Leeds, UK) with conformity of measurements on both spectrometers ensured by Bland–Altman plots. The inter- and intra-assay precision for both spectrometers, expressed as coefficient of variation (CV), was 6.3% and 3.8%, respectively.
uHA excretions from all 24-h urine samples collected during adolescence (2 to 5 samples per person, mean = 4.5) were averaged to reflect habitually ingested flavonoids.