Researchers identify genes that maintain healthy neurons thecrick
) objectives were acquired as single images, whereas the rest of the images were acquired with the 40× or 63× objective as Z-stacks and three slices were maximally projected to obtain the images used in the figure panels.
For histological analysis, fixed brains were dehydrated and embedded in paraffin, after completion of 4% PFA fixation. 4-μm sections were prepared and stained with H&E. Images were acquired using Olympus VS120 Slide Scanner.Rat cortical and hippocampal neurons were cultured from E16.5 embryos from a WT Long Evans mother. All the hippocampi and all the cortices from one litter were dissected, and all tissues from the same brain area were pooled together.
For the kinase assay, 500 ng Raph1 was incubated with 50 ng NDR1 in buffer containing 20 mM Tris–HCl, pH 7.5, 10 mM MgCl, l μM okadaic acid, 1 mM DTT, 1× protease inhibitor cocktail , 100 μM ATP and 0.5 mM 6-benzyl-ATPγS at 30°C, with rotation, for 30 min. The reactions were quenched with 20 mM EDTA and alkylated with 5 mM p-nitrobenzyl mesylate for 30 min at room temperature. The proteins were solubilised in sample buffer containing 0.
. At DIV12, the neurons were washed with cold PBS and then incubated with 1 mg/ml biotin , with shaking, at 4°C for 12 min. The following steps, before cell lysis, were all carried out on ice, using cold solutions. After biotin incubation, the neurons were washed with PBS twice and the biotinylation reaction was quenched with a buffer containing 1 mg/ml BSA in PBS.
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The effect of high-polyphenol Mediterranean diet on visceral adiposity: the DIRECT PLUS randomized controlled trial - BMC MedicineBackground Mediterranean (MED) diet is a rich source of polyphenols, which benefit adiposity by several mechanisms. We explored the effect of the green-MED diet, twice fortified in dietary polyphenols and lower in red/processed meat, on visceral adipose tissue (VAT). Methods In the 18-month Dietary Intervention Randomized Controlled Trial PoLyphenols UnproceSsed (DIRECT-PLUS) weight-loss trial, 294 participants were randomized to (A) healthy dietary guidelines (HDG), (B) MED, or (C) green-MED diets, all combined with physical activity. Both isocaloric MED groups consumed 28 g/day of walnuts (+ 440 mg/day polyphenols). The green-MED group further consumed green tea (3–4 cups/day) and Wolffia globosa (duckweed strain) plant green shake (100 g frozen cubes/day) (+ 800mg/day polyphenols) and reduced red meat intake. We used magnetic resonance imaging (MRI) to quantify the abdominal adipose tissues. Results Participants (age=51 years; 88% men; body mass index=31.2 kg/m2; 29% VAT) had an 89.8% retention rate and 79.3% completed eligible MRIs. While both MED diets reached similar moderate weight (MED: − 2.7%, green-MED: − 3.9%) and waist circumference (MED: − 4.7%, green-MED: − 5.7%) loss, the green-MED dieters doubled the VAT loss (HDG: − 4.2%, MED: − 6.0%, green-MED: − 14.1%; p | 0.05, independent of age, sex, waist circumference, or weight loss). Higher dietary consumption of green tea, walnuts, and Wolffia globosa; lower red meat intake; higher total plasma polyphenols (mainly hippuric acid), and elevated urine urolithin A polyphenol were significantly related to greater VAT loss (p | 0.05, multivariate models). Conclusions A green-MED diet, enriched with plant-based polyphenols and lower in red/processed meat, may be a potent intervention to promote visceral adiposity regression. Trial registration ClinicalTrials.gov , NCT03020186
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