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Supplementary methods:Daily macronutrient intakeDaily macronutrient intake was calculated utilising a validated food frequency questionnaire, designed to capture dietary intake for the preceding 12 months (Dietary Questionnaire for Epidemiological studies v2). ADDIN EN.CITE <EndNote><Cite><Author>Office</Author><Year>2015</Year><RecNum>365</RecNum><DisplayText><style face="superscript">1</style></DisplayText><record><rec-number>365</rec-number><foreign-keys><key app="EN" db-id="x9ef0xf20r99x4etwz5x50av5s59adadedvx" timestamp="1446540653">365</key></foreign-keys><ref-type name="Report">27</ref-type><contributors><authors><author>Nutriton Assessement Office</author></authors><tertiary-authors><author>Cancer Council Victoria</author></tertiary-authors><subsidiary-authors><author>Cancer Epidemiology Centre</author></subsidiary-authors></contributors><titles><title>Dietary Questionnaire for Epidemiological Studies Version 2 (DQES v2). User Guide</title></titles><dates><year>2015</year><pub-dates><date>January 2015</date></pub-dates></dates><publisher>Cancer Council Victoria</publisher><work-type>Document</work-type><urls></urls><access-date>02/04/2015</access-date></record></Cite></EndNote>1 Macronutrient composition was expressed as a percentage of total energy intake. ADDIN EN.CITE <EndNote><Cite><Author>(MoH)</Author><Year>2015</Year><RecNum>366</RecNum><DisplayText><style face="superscript">2</style></DisplayText><record><rec-number>366</rec-number><foreign-keys><key app="EN" db-id="x9ef0xf20r99x4etwz5x50av5s59adadedvx" timestamp="1446883909">366</key></foreign-keys><ref-type name="Report">27</ref-type><contributors><authors><author>Australian National Health and Medical Research Council (NHMRC) and the New Zealand Ministry of Health (MoH)</author></authors><tertiary-authors><author>Australian National Health and Medical Research Council (NHMRC) and the New Zealand Ministry of Health (MoH)</author></tertiary-authors></contributors><titles><title>Nutrient Reference Values for Australia and New Zealand</title></titles><dates><year>2015</year></dates><urls><related-urls><url> Anthropometric measurements. Height, weight, waist circumference, and blood pressure were obtained, and BMI was calculated. Peripheral venous blood samples were obtained after an overnight fast and serum alanine aminotransferase, bilirubin, albumin, creatinine, fasting triglyceride and high density lipoprotein (UniCel? DxC 800 Biochemical Analyser, Beckman Coulter), prothrombin time (ACL TOP 700 Haemostasis Testing System, Instrumentation Laboratory) and platelet count (XE-5000 Automated Haematology System, Sysmex) were measured. HCV RNA levels (COBAS TaqMan HCV quantitative assay, Version 2, Roche) were measured from peripheral blood samples collected within 3 months. The presence of the MetS was evaluated as per IDF/AHA/NHLBI 2009 consensus criteria.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5BbGJlcnRpPC9BdXRob3I+PFllYXI+MjAwOTwvWWVhcj48
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ADDIN EN.CITE.DATA 3 Hepatic stiffness Liver stiffness measurement (LSM) was determined by transient elastography (Fibroscan? 502 Touch, Echosens, Paris, France) performed after a three hour fast and in the recumbent position, by trained operators (A.S.R. and M.B.). The measurement reported was the median of at least 10 readings. Criteria for acceptability were 10 valid readings, at least 60% success rate, with an interquartile/median LSM<30.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5GcmllZHJpY2gtUnVzdDwvQXV0aG9yPjxZZWFyPjIwMDg8
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ADDIN EN.CITE.DATA 5Small intestinal paracellular permeabilitySmall intestinal paracellular permeability was measured within six weeks of transient elastography by a dual sugar enteral challenge. Subjects avoided alcohol for 48 hours prior to testing, and after an overnight fast drank a solution of 5g (14607?mol) lactulose (AlphaPharm, Millers Point, Australia) and 1g (5488?mol) rhamnose (Sigma Aldrich, Castle Hill, Australia) in 100mL water. Ninety minutes post-ingestion, peripheral venous blood was drawn and the concentration of lactulose and rhamnose were measured in plasma by HPLC with pulsed amperometric detection (Dionex, Sunnyvale, CA) and Chromeleon? Chromatography Management System (Version 6.80, Dionex), as per the method of Haase et al. ADDIN EN.CITE <EndNote><Cite><Author>Haase</Author><Year>2000</Year><RecNum>27</RecNum><DisplayText><style face="superscript">6</style></DisplayText><record><rec-number>27</rec-number><foreign-keys><key app="EN" db-id="x9ef0xf20r99x4etwz5x50av5s59adadedvx" timestamp="0">27</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Haase, A. M.</author><author>Kukuruzovic, R. H.</author><author>Dunn, K.</author><author>Bright, A.</author><author>Brewster, D. R.</author></authors></contributors><auth-address>NT Clinical School (Flinders University), Royal Darwin Hospital, Casuarina, Northern Territory, Australia.</auth-address><titles><title>Dual sugar permeability testing in diarrheal disease</title><secondary-title>J Pediatr</secondary-title><alt-title>The Journal of pediatrics</alt-title></titles><pages>232-7</pages><volume>136</volume><number>2</number><edition>2000/02/05</edition><keywords><keyword>Blood Specimen Collection</keyword><keyword>Case-Control Studies</keyword><keyword>Chromatography, High Pressure Liquid</keyword><keyword>Diarrhea, Infantile/ physiopathology</keyword><keyword>Female</keyword><keyword>Gastroenteritis/ physiopathology</keyword><keyword>Humans</keyword><keyword>Infant</keyword><keyword>Intestinal Absorption/ physiology</keyword><keyword>Intestinal Mucosa/physiology</keyword><keyword>Lactulose/ diagnostic use</keyword><keyword>Male</keyword><keyword>Permeability</keyword><keyword>Reproducibility of Results</keyword><keyword>Rhamnose/ diagnostic use</keyword><keyword>Urinalysis</keyword></keywords><dates><year>2000</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>0022-3476 (Print)
0022-3476 (Linking)</isbn><accession-num>10657831</accession-num><urls></urls><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>6 Results were considered acceptable if the coefficient of variation (CV) of the calibration standards was <15% for each HPLC run, and the sample values fell within the range obtained for the standards.?Small intestinal permeability was measured as the ratio of Lactulose: Rhamnose (L: R) x 100.Peripheral blood endotoxemiaPlasma endotoxin concentrations were measured as a marker for bacterial translocation, using a kinetic chromogenic limulus amebocyte lysate (LAL) assay (Endosafe? Endochrome-K?, Charles River, SC, USA). Plasma was diluted 1:20 with endotoxin free water (LAL reagent water, Charles River) and heated for a 20 mins at 75°C, and the assay performed as per the manufacturer. Optical Density measurements to derive the endotoxin concentration were performed using the Tecan Sunrise? plate reader and Endoscan? Version V software (Charles River, SC, USA), using the kinetic chromogenic function. A reference standard curve was generated by polynomial regression. Results were accepted based on the following criteria as per the manufacturer: correlation coefficient of standard curve ≥0.98; CV of standards <15%; CV of duplicates <20%; endotoxin recovery in samples of 50–200%. The lowest limit of endotoxin detection in the sample was 0.02 EU/mL.Mucosa-associated Microbiota assessmentDuodenal tissue collected from endoscopy was snap frozen on dry ice. Genomic DNA (gDNA) was isolated from tissue specimens using a modification of the “repeated bead beating plus column purification” method described by Yu and Morrison. ADDIN EN.CITE <EndNote><Cite><Author>Yu</Author><Year>2004</Year><RecNum>443</RecNum><DisplayText><style face="superscript">7</style></DisplayText><record><rec-number>443</rec-number><foreign-keys><key app="EN" db-id="x9ef0xf20r99x4etwz5x50av5s59adadedvx" timestamp="1457395453">443</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Yu, Z.</author><author>Morrison, M.</author></authors></contributors><auth-address>Dept. of Animal Sciences, Ohio State University, 2027 Coffey Road, OH 43210, USA. yu.226@osu.edu</auth-address><titles><title>Improved extraction of PCR-quality community DNA from digesta and fecal samples</title><secondary-title>Biotechniques</secondary-title><alt-title>BioTechniques</alt-title></titles><periodical><full-title>Biotechniques</full-title><abbr-1>BioTechniques</abbr-1></periodical><alt-periodical><full-title>Biotechniques</full-title><abbr-1>BioTechniques</abbr-1></alt-periodical><pages>808-12</pages><volume>36</volume><number>5</number><edition>2004/05/22</edition><keywords><keyword>Animals</keyword><keyword>Cattle</keyword><keyword>Chemical Precipitation</keyword><keyword>DNA, Bacterial/*isolation & purification</keyword><keyword>Feces/chemistry/*microbiology</keyword><keyword>Gastrointestinal Contents/chemistry/*microbiology</keyword><keyword>Polymerase Chain Reaction/*methods</keyword><keyword>Specimen Handling/*methods</keyword><keyword>Ultracentrifugation/*methods</keyword></keywords><dates><year>2004</year><pub-dates><date>May</date></pub-dates></dates><isbn>0736-6205 (Print)
0736-6205</isbn><accession-num>15152600</accession-num><urls></urls><remote-database-provider>Nlm</remote-database-provider><language>eng</language></record></Cite></EndNote>7 Following bead-based cell lysis, the gDNA was purified using the Maxwell MDx AS1000 automated DNA purification system (Promega, Madison, WI) and Maxwell Tissue DNA Purification Kit (Promega), according to the manufacturer’s protocol. All steps were performed in a biosafety laminar flow hood, and tissue was handled with sterile techniques. Bacterial DNA was selectively amplified from total gDNA by PCR with primers 917F (GAATTGRCGGGGRCC; bacterial domain specific) and 1392R (ACGGGCGGTGWGTRC; universal) (Integrated DNA Technologies, Singapore), that target the V6-V8 hypervariable region of the bacterial 16S rRNA gene and also contained Illumina adapter sequences. Subsequent barcoding of the PCR amplicons and downstream manipulations and preparation for amplicon sequencing using the Illumina MiSeq platform were performed as previously described.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5aaG9uZzwvQXV0aG9yPjxZZWFyPjIwMTY8L1llYXI+PFJl
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ADDIN EN.CITE.DATA 9 Operational Taxonomic Units (OTU) were assigned using the Greengenes database (version 13.8) with a sequence similarity of 97% applied. ADDIN EN.CITE <EndNote><Cite><Author>McDonald</Author><Year>2012</Year><RecNum>446</RecNum><DisplayText><style face="superscript">10</style></DisplayText><record><rec-number>446</rec-number><foreign-keys><key app="EN" db-id="x9ef0xf20r99x4etwz5x50av5s59adadedvx" timestamp="1457491758">446</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>McDonald, D.</author><author>Price, M. N.</author><author>Goodrich, J.</author><author>Nawrocki, E. P.</author><author>DeSantis, T. Z.</author><author>Probst, A.</author><author>Andersen, G. L.</author><author>Knight, R.</author><author>Hugenholtz, P.</author></authors></contributors><auth-address>Department of Chemistry & Biochemistry and Biofrontiers Institute, University of Colorado, Boulder, CO, USA.</auth-address><titles><title>An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea</title><secondary-title>Isme j</secondary-title><alt-title>The ISME journal</alt-title></titles><periodical><full-title>Isme j</full-title><abbr-1>The ISME journal</abbr-1></periodical><alt-periodical><full-title>Isme j</full-title><abbr-1>The ISME journal</abbr-1></alt-periodical><pages>610-8</pages><volume>6</volume><number>3</number><edition>2011/12/03</edition><keywords><keyword>Archaea/*classification/genetics</keyword><keyword>Bacteria/*classification/genetics</keyword><keyword>Classification/*methods</keyword><keyword>*Databases, Genetic</keyword><keyword>Metagenomics</keyword><keyword>*Phylogeny</keyword><keyword>RNA, Ribosomal, 16S/genetics</keyword><keyword>Software</keyword></keywords><dates><year>2012</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>1751-7362</isbn><accession-num>22134646</accession-num><urls></urls><custom2>Pmc3280142</custom2><electronic-resource-num>10.1038/ismej.2011.139</electronic-resource-num><remote-database-provider>Nlm</remote-database-provider><language>eng</language></record></Cite></EndNote>10 OTUs with an abundance of less than 0.1%, and those present in the negative controls were removed from the overall analysis to generate a list of OTUs considered representative of gut MAM. The absolute number of OTUs present in each sample was normalised using cumulative sum scaling (CSS) to account for variations in sampling depth.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5QYXVsc29uPC9BdXRob3I+PFllYXI+MjAxMzwvWWVhcj48
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ADDIN EN.CITE.DATA 11 Total sum scaling was used for relative abundance measurements. Statistical analysis of microbiota dataAlpha diversity (derived from CSS data) was assessed using the Shannon index (to account for both the number of species, as well as their relative abundance). ADDIN EN.CITE <EndNote><Cite><Author>Spellerberg</Author><Year>2003</Year><RecNum>639</RecNum><DisplayText><style face="superscript">12</style></DisplayText><record><rec-number>639</rec-number><foreign-keys><key app="EN" db-id="x9ef0xf20r99x4etwz5x50av5s59adadedvx" timestamp="1467181933">639</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Spellerberg, I.F.</author><author>Fedor, P.J.</author></authors></contributors><titles><title>A tribute to Claude Shannon (1916–2001) and a plea for more rigorous use of species richness, species diversity and the ‘Shannon–Wiener’ Index</title><secondary-title>Global Ecology & Biogeography</secondary-title></titles><periodical><full-title>Global Ecology & Biogeography</full-title></periodical><pages>177-179</pages><volume>2003</volume><number>12</number><dates><year>2003</year></dates><publisher>Blackwell Publishing</publisher><urls></urls></record></Cite></EndNote>12 Differential abundance of microbial taxa (derived from raw data, to reduce potential biases of transformed data) was analysed by both differential expression analysis based on the negative binomial distribution (DESeq2) and analysis of composition of microbiomes (ANCOM)PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NYW5kYWw8L0F1dGhvcj48WWVhcj4yMDE1PC9ZZWFyPjxS
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