5 μg/ml). Molecular sizes of the amplified DNA fragments were estimated by comparison with 1-kb DNA molecular size markers (Invitrogen Life Technologies). RAPD-PCR profiles were acquired by Gel Doc EQ System (Bio-Rad Laboratories) and compared using Fingerprinting II Informatix™ Software (Bio-Rad). The similarity of the electrophoretic profiles was evaluated by determining the Dice coefficients of similarity and using the UPGMA method. Gas-chromatography mass spectrometry/solid-phase microextraction (GC-MS/SPME) analysis

After preconditioning according to the manufacturer’s instructions, the carboxen-polydimethylsiloxane coated fiber (85 μm) and the manual SPME holder (Supelco Inc., Bellefonte, PA, USA) were used. Before head space sampling, the fiber was exposed to CFTRinh-172 supplier GC inlet for 5 min for Idasanutlin concentration thermal desorption at 250°C. Three grams of faecal sample were placed into 10 ml glass vials and added of 10 μl of 4-methyl-2-pentanol BAY 63-2521 cost (final concentration of 4 mg/l), as the internal standard.

Samples were then equilibrated for 10 min at 45°C. SPME fiber was exposed to each sample for 40 min. Both phases of equilibration and absorption were carried out under stirring condition. The fiber was then inserted into the injection port of the GC for 5 min of sample desorption. GC-MS analyses were carried out on an Agilent 7890A gas-chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent 5975C mass selective detector operating in electron impact mode (ionization voltage 70 eV). A Supelcowax 10 capillary column (60 m length, 0.32 mm ID) was used (Supelco, Bellefonte, PA, USA). The temperature program was: 50°C for 1 min, 4.5°C/min to 65°C and 10°C/min to 230°C, which was held for 25 min. Injector, interface and ion source temperatures were 250, 250 and 230°C, respectively. The mass-to-charge ratio interval was 30-350 a.m.u. at 2.9 scans per second. Injections were carried out in splitless mode and helium (1 ml/min) was used as the carrier gas. Sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) was used as the internal standard. Identification of molecules was

carried out based on comparison of their retention times with those of pure compounds (Sigma-Aldrich, Milan, Italy). Identification was confirmed by searching mass spectra Dichloromethane dehalogenase in the available databases (NIST version 2005 and Wiley Vers. 1996) and literature [57]. Quantitative data of the identified compounds were obtained by interpolation of the relative areas versus the internal standard area [33]. 1H Nuclear Magnetic Resonance (NMR) spectroscopy analysis To study the water soluble fraction of the faeces by means of 1H NMR spectroscopy, 40 mg of thawed faecal or urine mass were thoroughly homogenized by vortex-mixing with 400 μl of cold deuterium oxide (D2O) at pH 7.4 ± 0.02, containing 1 mM TSP as the internal standard. Mixtures were centrifuged at 14,000 rpm for 5 min and the supernatant was collected.