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Supplementary Materials and Methods
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Subjects and sampling
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Fifty four obese Chinese adults (body mass index [BMI] > 26 kg/m 2, 31 females and 23 males)
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and 54 sex- and age-matched lean control (18≤ BMI ≤23 kg/m2) subjects in this study were selected
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from a case-control study in which over 500 pairs of overweight/obese and normal-weight control
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people were recruited from November 2007 to January 2008 (Sun et al. 2010). In brief, the 108
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volunteers were 35- to 54-year-old Chinese Han people who had lived in the urban area of Shanghai,
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China, for at least 10 years. Individuals who were alcoholic, diabetic, with organic/infectious diseases,
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with severe psychological disorders or physical disabilities, pregnant/lactating, or had diarrhea or
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antibiotic treatment within 3 months prior to the recruitment were excluded. For each volunteer in this
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study, the waist and hip circumference, waist-hip ratio (WHR), fasting glucose level, systolic and
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diastolic blood pressure (SBP and DBP) were adopted from previously published study on over 500
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pairs of overweight/obese and normal-weight control people (Sun et al. 2010), and re-calculated in the
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context of this study. The volunteers were asked to keep their normal diets before and at the time of
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sampling. Freshly passed feces were collected from each volunteer and stored at -80°C before analysis,
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and fecal DNA was extracted using InviMag® Stool DNA Kit (Invitek GmbH, Berlin, Germany) in
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accordance with the manufacturer’s instruction. The protocol of the study was approved by the
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Institutional Review Board of Institute for Nutritional Sciences, Shanghai Institutes for Biological
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Sciences, Chinese Academy of Sciences, and was performed in accordance with the Declaration of
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Helsinki and the later amendments. Written informed consent was obtained from each volunteer before
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the participation in the study.
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Quantitative Real-time PCR
Real-time PCR was performed on a DNA Engine Opticon 3 system (MJ Research, Waltham, MA,
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USA). Primer pairs, FPR-2(5’-GGAGGAAGAAGGTCTTCGG-3’) and Fprau645R
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(5’-AATTCCGCCTACCTCTGCACT-3’) (Ramirez-Farias et al. 2009), and Uni-1 (331F)
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5’-TCCTACGGGAGGCAGCAGT-3’ and Uni-2 (797R)
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5’-GGACTACCAGGGTATCTAATCCTGTT-3’ (Nadkarni et al. 2002) were used to quantify fecal F.
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prausnitzii and total bacteria, respectively.
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Each 25-μl reaction mixture contained 0.75U TaKaRa rTaq polymerase (Takara, Dalian, China),
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12.5μl of 2×Sybmix (BioEasy SYBR Green I Real-time PCR Kit, Hangzhou, China), 25 pmol of each
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primer, and extracted fecal DNA (40 ng and 20 ng for quantification of F. prausnitzii and total bacteria,
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respectively). F. prausnitzii were quantified with the following program: 3 min at 95°C, 40 cycles of 30
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s at 95°C, 30 s at 60°C, 30 s at 72°C, and 10 s at 83°C for fluorescence detection. Total bacterial were
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quantified with the following program: 4 min at 95°C, 40 cycles of 15 s at 95°C, 1 min at 60°C, 5s at
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80°C for fluorescence detection. To confirm the specificity of the PCR reaction, melting curve analysis
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was performed after amplification by increasing the temperature at a rate of 0.5°C per 10 s from 60°C
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to 95°C with continuous fluorescence monitoring.
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The gene copy number of bacteria in feces was quantified using standard curves constructed from
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known concentrations of plasmid DNA ranging from 1×108 to 1×102 copies/μl for F. prausnitzii and
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1×109 to 1×103 copies/μl for total bacteria.
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Each PCR reaction was performed in triplicate. The abundance of F. prausnitzii was calculated by
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dividing the gene copy number of F. prausnitzii by that of total bacteria, and expressed as the
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percentage of F. prausnitzii accounting to the total bacteria.
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Statistical analysis
Comparison between volunteer groups was performed with Student’s t test for data with normal
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distribution and with Mann-Whitney U test for data without normal distribution. Spearman’s rank
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correlation was performed to study the association between the abundance of F. prausnitzii and clinical
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parameters. All statistical analysis was carried out with PAST software (Hammer et al. 2001), and P
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values less than 0.05 were taken as statistically significant.
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Supplementary References
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Hammer O, Harper DAT, Ryan PD (2001) PAST: Paleontological Statistics Software Package for
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Education and Data Analysis. Palaeontologia Electronica 4 (1):9
Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time
PCR using a broad-range (universal) probe and primers set. Microbiology 148 (Pt 1):257-266
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Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, Louis P (2009) Effect of inulin on the
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human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium
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prausnitzii. Br J Nutr 101 (4):541-550
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Sun L, Yu Z, Ye X, Zou S, Li H, Yu D, Wu H, Chen Y, Dore J, Clement K, Hu FB, Lin X (2010) A
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marker of endotoxemia is associated with obesity and related metabolic disorders in
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apparently healthy Chinese. Diabetes Care 33 (9):1925-1932
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