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magnesium stearate and silicon dioxide. The probiotic formulation con- sisted of B. longum APC1472, whereas the placebo contained maltodex- trin. The product was stored at − 20 °C until distributed to the study par- ticipant and the participant was instructed to keep the product refriger- ated. Participants returned any leftover product at their next visit, and the excess product was counted to check for compliance. 3.7. Collection and analysis of blood samples Fasting blood samples were taken into EDTA tubes, fasting defined as refraining from food overnight (at least 10 h), however drinking water was allowed throughout the duration of the fast. Samples for the analy- sis of active ghrelin were immediately treated with AEBSF (final concen- tration 1 mg/mL, Sigma, A8456), centrifuged and the resulting plasma was treated with HCl (final concentration 0.05 N). Blood samples for the analysis using the U-PLEX assays were treated with DPP-IV inhibitor (fi- nal concentration 1%, Sigma, DPP4) and centrifuged. Blood plasma sam- ples for other analyses did not undergo any additional processing, except for centrifugation. Centrifugation was performed at 1000 g for 10 min at 4 °C, after which samples were aliquoted and either processed or stored at − 80 °C for future analysis. Blood plasma from visit 1 was used to measure urea, creatinine, bilirubin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyl transferase, total protein, albumin, globulin, calcium, magnesium, phosphate, uric acid, cholesterol, HDL cholesterol, LDL cholesterol, total triglycerides, glucose, full blood count + 5-part diff. Safety blood, haematology and biochemistry para- meters were analysed by Biomnis-Eurofins Ireland. Blood from visits 2, 3 and 4 was used to measure total cholesterol, LDL, HDL, triglycerides HbA1c, glucose and insulin by Biomnis-Eurofins Ireland. Furthermore, blood plasma was assessed for active ghrelin lev- els using an ELISA (EMD Millipore, EZGRA-88BK) which was performed according to the manufacturer's instructions. Plates were read at 405 nm with a correction at 590 nm using the synergy HT plate reader (Biotek instruments). Blood plasma was also assessed for metabolic and inflam- matory biomarkers using custom U-PLEX assays (MSD, K151ACM-2), which were also performed according to the manufacturer's instructions. Blood plasma samples were diluted 1:3 for the U-PLEX assays. U-PLEX markers were linked as following; Plate 1: 1) Leptin, 2) PYY, 3) GLP-1 – total, 4) IFN γ , 5) Il-4, 7) TNF- α , 8) Il-10, 9) C-peptide, 10) Ghrelin – total; Plate 2: 1) GLP-1 – active. The working solution was supple- mented with DPP-IV inhibitor (final concentration 1%, Sigma, DPP4). Plates were read using the MESO QuickPlex SQ 120. Duplicates with ≥ 20% coefficient of variability were re-analysed. Samples did not undergo any additional freeze-thaw cycles. 3.8. Collection and analysis of cortisol awakening response samples To monitor the cortisol awakening response, saliva from visits 2 and 4 was collected in Salivette devices (Sarstedt, 51.1534.500) im- mediately upon awakening, and after 30, 45 and 60 min. Participants were instructed to keep samples in the fridge until delivery at the visit time, after which they were centrifuged at 1500 g for 5 min, the saliva was harvested and immediately stored at − 80 °C. Salivary cor- tisol concentrations were quantified using ELISA kits (Enzo life sci- ences, ADI-901 – 071), which were performed according to the manufac- turer's instructions. Saliva samples were diluted 1:2. Plates were read at 405 nm with a correction at 580 nm using the synergy HT plate reader (Biotek instruments). Duplicates with ≥ 20% coefficient of variability were re-analysed. Samples did not undergo any additional freeze-thaw cycles. Cortisol awakening response was calculated using the area under the curve increase (AUCi). Briefly, data from the 30-, 45- and 60-minute time-points were normalized (delta) to the samples taken immediately
upon awakening, after which the sum was taken of the 30-, 45- and 60-minute time-points [52].
4.Methods – murine and human microbiota 4.1. Murine and human microbiota sequencing
Murine caecal DNA was isolated using the QIAamp Fast DNA Stool Mini kit (Qiagen) as previously described and kept at − 20 °C until further analysis [53]. Isolated DNA was quantified on a NanoDrop ND2000 spectrophotometer (Thermo Scientific, DE) and used for 16S ribosomal RNA sequencing by Illumina MiSeq System (Illumina Inc., USA) according to the manufacturer's instructions. Briefly, PCR ampli- cons (primers for V3-V4 hypervariable region of the 16S rRNA gene: F (5 ′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGAC AGCCTACGGGNG- GCWGCAG-3 ′ ) and R (5 ′ -GTCTCGTGGGCTCGGAGATGTGTATA AGA- GACAGGACTACH VGGGTATCTAATCC-3 ′ ) were purified and libraries prepared as previously described [53]. Briefly, the 16S V3-V4 ampli- cons were generated using Kapa HiFi HS ready mix and purified using the Agencourt AMPure XP system (Beckman Coulter Genomics, Take- ley, UK). The Nextera XT Index Kit (Illumina Inc., USA) was used to barcode each sample. PCR products were cleaned using AMPure XP beads and a magnetic 96-well plate. Final barcoded amplicons were measured using the Qubit dsDNA High Sensitivity assay kit on the Qubit 3.0 fluorometer, diluted to 5 ng/µL and pooled. The PCR products from both PCR steps (Amplicon & Indexing) were visualised in agarose gels stained with SYBR Safe DNA gel stain (Invitrogen). Samples were se- quenced at Clinical-Microbiomics, Denmark on the Illumina MiSeq plat- form using a 2 × 300 bp kit. After sequencing, reads were assembled, processed and analysed as previously described [53]. In the microbiota composition analysis, LDA Effect Size (LEfSe: Linear Discriminant Analy- sis Effect Size) was used as an algorithm with default settings on the interface Galaxy (http://huttenhower.sph.harvard.edu/lefse/) [54] to identify taxa with differentiating abundances. The differentially abun- dant features are ranked by effect size after undergoing linear discrim- inant analysis (LDA), using an effect size threshold of 2 (log10 scale). In non-technical terms, LEfSe pre-selects features that are different be- tween groups and then tries to fit a model to see how well these features explain the groups. The score is an average between the effect size and how well the model fits, after which they are transformed to a value be- tween − 6 and 6. Principal coordinates Analysis (PCoA) was performed based on Bray-Curtis beta diversity distances using the Adonis function in the “ vegan ” (2.4 – 3) package for R (version 3.3.1). For the human intervention study, faecal sample collection and DNA extraction was performed as previously described (see supplementary material for details) [55]. The DNA samples were processed according to the Illumina 16S Metagenomic Sequencing Library Preparation in- structions as described above for the murine DNA samples. Final bar- coded amplicons were measured using the Qubit dsDNA High Sensitiv- ity assay kit on the Qubit 3.0 fluorometer, diluted to 8.3 ng/µL, pooled and sent for sequencing. Microbiome analysis was carried out in R (ver- sion 3.6.1) with Rstudio (version 1.2.1335). DADA2 was used to denoise and call amplicon sequence variants (ASVs). Taxonomy was assigned using the SILVA SSUREf database version 132. ASVs unknown on a genus level were excluded, as well as ASVs present in two or fewer sam- ples. The ALDEx2 library used to compute the centred log-ratio trans- formed values of the remaining taxa [56]. For principal components analysis (PCA), a pairwise implementation of the adonis() PERMANOVA function in the vegan library followed by the Bonferroni-Holm correc- tion was used to test for difference in β -diversity in terms of Aitchi- son distance (source: Oksanen, Jari, et al. "Package ‘ vegan ’ ." Commu- nity ecology package, version 2.9 (2013): 1 – 295). Differential abun- dance was assessed using a pairwise implementation of the aldex.test() function, followed by Benjamini-Hochberg correction. In all
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