A pathologist

blinded to clinical characteristics assessed liver histology for presence of steatosis, inflammation, and fibrosis. The presence of NASH was determined using the Brunt scoring system, which is a validated and reproducible tool for the evaluation of NASH.31 The stool collection kit included a plastic collection/storage container with a tightly closing lid, an insulated bag, and cooling elements. Patients were asked to collect one sample within 24 hours of their next clinic appointment. The samples were immediately frozen in the patients’ home freezer and transported to the hospital using the cooling elements and the insulated bag, similar to previously published methods.5 Stools were then stored at −80°C until analysis.

The stool was thawed, immediately homogenized with a masticator blender, and 0.1 g was used for DNA extraction using the Alpelisib datasheet E.Z.N.A. stool DNA Isolation Kit (Omega, Norcross, GA), as per the manufacturer’s protocol. The extraction protocol was modified to include a lysozyme digestion step (incubation at 37°C for 30 minutes). DNA concentration and purity were measured using ThermoScientific Nanodrop 1000 Spectrophotometer (ThermoScientific, Rockford, IL). DNA samples were subsequently stored at −20°C. Fifty nanograms of the extracted DNA were used for the quantification of fecal bifidobacteria, Bacteroides/Prevotella, Clostridium leptum, C. coccoides, Escherichia coli, as well as total bacteria and Archaea, www.selleckchem.com/products/Rapamycin.html selleck chemical by quantitative polymerase chain reaction (qPCR) using a 7900HT

thermocycler from Applied Biosystems (Foster City, CA) under default thermocycling conditions. Custom-made TaqMan primers for total bacteria,32 C. coccoides,32 C. leptum,32 Bacteroides/Prevotella,32, 33 bifidobacteria,32 and Archaea34 were used. Real-time PCR for E. coli was done using SYBR Green Gene Expression master mix (Applied Biosystems) and the specific forward and reverse primer.32 Number of cells of each microorganism in fecal samples was calculated by interpolation from standard curves and expressed as log cell counts/g feces. Bacteroides/Prevotella counts were considered representative of the Bacteroidetes phylum (as previously33) and will herein be referred to as Bacteroidetes. Results are expressed as median (range) as the data were not normally distributed. Kruskal-Wallis test was used to compare the three groups for demographic, dietary, and laboratory data (Stata v. 12, College Station, TX). Nonparametric tests were used for statistical comparisons of the results of the fecal analyses as well (Kruskal-Wallis; Stata v. 12 and GraphPad Prism v. 4.0, GraphPad Software, La Jolla, CA). For the microbiota, high and low outliers were defined as numbers higher than the third quartile plus 1.5 times the interquartile range (IQR) and lower than the first quartile minus 1.5 times the IQR, respectively.