Isothiocyanate

The long-FFQ consisted of 172 food and beverage items and nine frequency categories, ranging from almost never to seven or more times per day (or to 10 or more glasses per day, for beverages). It asked about the usual consumption of listed foods during the previous year. The food list was initially developed according to percentage contributions based on absolute values of energy and intake of 14 target nutrients from weighed food records in 1989–1991^{8 (link)} and used for the Japan Public Health Center-based prospective Study,^{8 (link)–12 (link)} for which it was modified for middle-aged and elderly residents in a wide variety of areas of Japan. With regard to this modification, the following criteria were considered: calculation for an additional 17 nutrient items, such as fiber and folate, change of foods contributing to the absolute nutrient intake according to the updated Standard Tables of Food Composition in Japan,^{13 ,14} and dietary regionality and change in generation for the present cohort (data not shown). As a result, 33 foods were added, and 5 foods and beverages were excluded.^{15 (link)} Moreover, six foods were also added to account for potential inter-individual variation in specific nutrients, such as isothiocyanate and isoflavone. With regard to alcoholic beverages, choices of intake amount were changed from the initial JPHC-FFQ.
To develop the food list for the short-FFQ, we selected and combined items and supporting questions from the original long-FFQ. We selected the three major foods and beverages that contributed to inter-individual variation for each of 40 nutrients according to a cumulative R² for the 40 nutrients,¹⁶ based on the multiple regression coefficient with total intake of a specific nutrient as the dependent variable and its intake from each food as the explanatory variable. Inter-individual variation was calculated by gender among 45 869 men and 52 989 women who responded to the JPHC Study 10-year follow-up survey. Consequently, cumulative R² for the nutrients ranged from 0.4 to 1.0. For potential inter-individual variation in intake of specific food groups, some foods, such as coffee, were added. Ultimately, 66 food and beverage items were selected for the short-FFQ. In this validation study, information on alcoholic beverages was substituted with those from the long-FFQ (united with overall information of lifestyle), because these questions were not included in the short-FFQ. This was because information on alcoholic beverage intake was structured in pages for lifestyle other than diet, such as smoking status and physical activity, and the reproducibility of alcoholic beverage intake was relatively high even if questionnaires were administered at a 1-year interval.^{17 (link),18 (link)}Intakes of energy, 53 nutrients, and 29 food groups were calculated using the Standard Tables of Food Composition in Japan 2010,¹⁹ Standard Tables of Food Composition in Japan Fifth Revised and Enlarged Edition 2005 For Fatty Acids,²⁰ and a specifically developed food composition table for isoflavones in Japanese foods.^{21 (link)}

Two flowers per day from anthesis, two and three days after pollination were fixed in 4% formaldehyde freshly prepared from paraformaldehyde in 1x phosphate saline buffer (PBS) pH7.3, left overnight at 4ºC, and conserved then at 0.1% formaldehyde solution [83 (link)]. Then the pistils were dehydrated in an acetone series (30%, 50%, 70%, 90%, 100%), and embedded in Technovit 8100 (Kulzer and Co, Germany) for two days. The resin was polymerized at 4ºC, and sectioned at 4 μm thickness. Sections were placed in a drop of water on a slide covered with 2% (3-Aminopropyl) triethoxysilane - APTEX (Sigma-Aldrich), and dried at room temperature. Callose was identified with the anticallose antibody (AntiCal) that recognises linear β-(1,3)-glucan segments (anti-β-(1,3)-glucan; immunoglobulin G1), Biosupplies, Australia [49 (link)]. As a secondary antibody, Alexa 488 fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG was used (F-1763; Sigma). Additionally, a monoclonal antibody (mAbs) JIM13 [84 (link)] against AGPs glycosyl epitopes, and one mAb JIM11 [85 (link)] against extensin epitopes were obtained from Carbosource Services (University of Georgia, USA). Secondary antibodies were anti-rat IgG conjugated with the same Alexa 488 used above. Sections were incubated for 5 min in PBS pH7.3 followed by 5% bovine serum albumin (BSA) in PBS for 5 min. Then, sections were incubated at room temperature for 1h with AntiCal primary mAb, JIM13, and JIM11. After that, three washes in PBS of 5 minutes each preceded the incubation for 45 min in the dark with a 1/25 diluted secondary fluorescein isothiocyanate (FITC) conjugated with the antibody in 1% BSA in PBS, followed by three washes in PBS [83 (link)]. Sections were counterstained with calcofluor white for cellulose [86 (link)], mounted in PBS or Mowiol, and examined under a LEICA DM2500 epifluorescence microscope connected to a LEICA DFC320 camera. Filters were 355/455 nm for calcofluor white and 470/525 nm for the Alexa 488 fluorescein label of the antibodies (White Level?=?255; Black Level = 0; ϒ?=?1). Exposur (Exp) times were adapted to the best compromise in overlapping photographs for each antibody: AntiCal, Exp.?=?15.30ms (Calcofluor Exp. = 1.20ms); JIM13 Exp.?=?2.52ms (Calcofluor?=?0.41ms); JIM11, Exp. = 31.59 ms (Calcofluor Exp. = 1.40ms). Brightness and contrasts were adjusted to obtain the sharpest images with the Leica Application Suite software.

The extraction of PEITC was performed as previously described [3 (link)]. Briefly, 5.0 g of lyophilized watercress flowers were suspended in phosphate-buffered saline (PBS; pH 7) (150 mL) containing a catalytic amount of ascorbic acid. The formed suspension was incubated at 37 °C under continuous stirring for 1 h in order to stimulate the hydrolysis of glucosinolates. The resulting solution (150 mL) was filtered over Whatman filter paper (pore size: 4.0–12 μm) and the filtrates were subsequently extracted by convectional liquid–liquid extraction using n-hexanes (3 washes × 150 mL each). Isolation and drying of the organic phase over magnesium sulphate followed by concentration to dryness under reduced pressure yielded a phenethyl isothiocyanate-enriched fraction as a viscous oil (2.1 g, 1.91 mL, yield: 42%). The yield was calculated based on the mass of dry extract.

On the 22^nd day of age, 3 birds per pen (12/group) were weighted and submitted to feed restriction for 12 h to induce a gut permeability challenge. Fluorescein isothiocyanate dextran (FITC-d, 100 mg, MW 4000; Sigma-Aldrich, St. Louis, MO, USA) was utilized to assess intestinal permeability, following the method described by Baxter et al. [4 (link)]. In particular, birds were orally administered FITC-d dissolved in phosphate-buffered saline (PBS). Three additional birds per group were gavaged by PBS only and were used as serum blank controls for each group, as previously outlined [4 (link)]. In each group, subsequently, after a 2 h duration, blood samples were collected from the jugular vein and left at room temperature for 3 h before undergoing centrifugation (500× g for 15 min) to obtain the serum. The serum was then diluted with PBS (1:1 PBS), and the presence of FITC-d in the serum was quantified using a multi-mode microplate fluorescence reader (Perkin-Elmer, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The standard curve was plotted according to absorption of standards prepared by spiking FITC-d at a range of concentrations (0–0.5 μg/mL). The amount of FITC-d in the serum for each bird was reported as µg of FITC-d per milliliter of serum.