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standard holding cages with free access to food and water in the ani- mal care facility of University College Cork. The holding room temper- ature (21 ± 1 °C) and humidity (55 ± 10%) were controlled under a 12 h light/dark cycle (lights on 7.00 AM, lights off 7.00 PM). The mice were fed a low-fat diet (LFD) (10% fat (kcal/100 g), D12450B, Research Diet, USA) or a high-fat diet (HFD) (45% fat (kcal/100 g), D12451, Re- search Diet, USA) for 16 weeks. Food intake was recorded once per week and calculated on the basis of two mice per cage and five cages per group. The data were reported as cumulative food intake per mouse. Bodyweight was monitored weekly for 15 weeks. Experiments were con- ducted in accordance with the European Directive 86/609/EEC and the Recommendation 2007/526/65/EC and were approved by the Animal Experimentation Ethics Committee of University College Cork. 2.2. In vivo probiotic administration Bifidobacterium longum APC1472 was grown anaerobically in De Man, Rogosa and Sharpe (MRS) medium as previously described [37]. The bacterial cell pellet was washed and concentrated in sterile phos- phate buffered saline (PBS) containing 25% Glycerol (v/v) to an end concentration of ~7.5 × 10 9 CFU/mL, aliquoted and stored at − 80 °C. Aliquots were defrosted daily just prior to the start of the dark phase and diluted to ~2 × 10 8 CFU/mL in drinking water for administration to LFD-fed and HFD-fed mice for 16 weeks. Water intake was moni- tored throughout the experiment. Drinking water containing an equiv- alent end concentration of sterile PBS (2% v/v) and glycerol (0.5% v/ v) was administered to control mice. Water was replaced for probiotic/ vehicle-free water every morning. B. longum APC1472 survival in drink- ing water (distilled water) in ambient temperature and oxygen content was tested over 24 h prior to the start of the experiment. Bacteria counts (CFU/mL) did not decrease over 1 log unit for the first 12 h suggesting adequate viability of the strain upon the time of consumption ( Figure S1A) . No significant changes in water intake were observed within the Glucose tolerance was assessed after 15 weeks of treatment as pre- viously described [49], with minor modifications. Briefly, mice were fasted for 7 h during the light phase, with free access to water. Glucose levels were measured in tail vein blood using a glucometer (Bayer, UK) immediately before and 15, 30, 60, 90 and 120 min after intraperitoneal injection of glucose (1 g/kg of body weight in sterile saline). 2.4. Murine tissue sampling Mice were euthanized by decapitation. Trunk blood was collected in tubes containing 25 μ M dipeptidyl peptidase IV (DPP-IV) inhibitor, 2x protease inhibitor cocktail (Roche) (diluted in PBS) and 0.1% Na 2 EDTA for an expected blood volume of 400 µL, centrifuged at 3500 g for 15 min at 4 °C and placed on dry ice until storage at − 80 °C for further analysis. Adipose depots (epididymal, subcutaneous, mesenteric and retroperitoneal) were dissected and weighed. Whole-brains were collected and placed for 8 – 10 s into ice-cold isopentane. All tissues were frozen on dry ice and subsequently stored at − 80 °C for further analysis. 2.5. Murine biochemical analysis same diet groups ( Figure S1B ). 2.3. In vivo glucose tolerance test Plasma insulin and leptin levels were analysed by ELISA using the MILLIPLEX® MAP Mouse Metabolic Hormone Magnetic Bead Panel (Millipore, MMHMAG-44 K) accordingly to the manufacturer's instruc- tions. Plasma ghrelin levels were analysed using the Rat/Mouse Ghrelin
(Total) ELISA Kits (Millipore, EZGRA-88 K). Triglycerides levels were analysed with a Triglyceride Quantification Kit (Abcam Ltd, ab65336) following the to manufacturer's instructions. Corticosterone levels were assayed using ELISA kits (Enzo Life Sciences, ADI-900 – 097) according to the manufacturer's instructions. 2.6. Murine RNA isolation and quantitative real-time PCR Hypothalamus was dissected with a forceps (macropunch) from the frozen brain on dry ice and immediately processed for RNA extraction. Hypothalamus and epididymal adipose tissue total RNA were extracted using the mirVana ™ miRNA Isolation kit (Ambion/Life Technologies, AM1560) and RNeasy® Lipid Tissue Mini Kit (Qiagen, 74,804), re- spectively with DNase treatment using Turbo DNA-free (Ambion/life Technologies, AM1907) according to the manufacturer's recommenda- tions. Equal amounts of RNA were first reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosys- tems, 4,368,814). Real-time PCR was performed using TaqMan Univer- sal Master Mix II, no Uracil-N glycoslyase (UNG) on a LightCycler®480 System (Roche). Mouse β -actin control mix Probe dye: VIC-MGB (Ap- plied Biosystems, 4352341E) was used as an endogenous control. Tar- get genes were amplified with probes designed by Integrated DNA Tech- nologies (Table S1). Cycle threshold (Ct) values were recorded, normal- ized to its endogenous control and transformed to relative gene expres- sion value using the 2 −ΔΔ Ct method [50]. Each sample was analysed in triplicate for both target gene and endogenous control. The gene expres- sion levels for each animal was calculated considering the mean from each of these triplicates. 3.Methods – human intervention study 3.1. Human intervention study outline This study has a parallel-controlled design. In total, 150 individu- als were screened, after which 124 were randomized into the treatment groups (Placebo: n = 50; Treatment: n = 74). The aim of the first visit of the participant was to assess participants for their eligibility to par- ticipate in the study and explain which procedures would be under- taken. Subjects were given an appointment for the next visit within a 3-week period. At the second visit, all baseline data and biologics were recorded, which was also done after 6 (visit 3) and 12 weeks (visit 4) of placebo or B. longum APC1472 treatment. Vital signs, anthropometric measurements and medical history were recorded. For women of child- bearing age, a urine sample was collected for a pregnancy test. Fasting blood samples (20 mL) were collected to assess glucose, insulin, HbA1c, lipid profiles, satiety/appetite hormone profiles, and inflammatory pro- files. Saliva samples were collected for the assessment of the cortisol awakening response, as well as a stool sample for the microbiota analy- sis and short-chain fatty acid (SCFA) quantification. Questionnaires were administered to assess self-reported perceived stress, anxiety, hunger/ satiety, exercise and diet. Participants were asked to take one capsule per day, providing a daily dose of 1 × 10 10 CFU. Subjects, study facilitators, nurses and re- search analysts were kept blind as to in which group they belonged. The randomisation of treatment schedules was carried out by a com- puter-generated program. The remaining study product was collected to check for compliance following visits 3 and 4 [51]. 3.2. Inclusion and exclusion criteria The inclusion criteria were as follows: subjects had to give written informed consent; had to be between 18 and 65 years of age; had a BMI between 28 and 34.9; had a W/H ratio ≥ 0.88 for males and ≥ 0.83 for females; had to be willing to consume the investigational product daily for the duration of the study. Subjects were excluded if they were
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