Analar

Tumour and foetal brain tissue were disrupted using an immersion processor and cells were washed in PBS and pelleted before resuspension in TRIzol (Invitrogen, Carlsbad, CA, USA). After 5min of incubation at RT, 0.2 ml of chloroform (AnalaR, Merck, Whitehouse Station, NJ, USA) was added to each 1 ml TRIzol. The phases were mixed and centrifuged (13000 rpm) for 15 min at 4°C. The upper phase was collected and adjusted to 35% ethanol by adding 100% pure ethanol (AnalaR, Merck, Whitehouse Station, NJ, USA). The solution was applied to an RNeasy column (Qiagen, Germantown, MD, USA) and RNA was purified according to the manufacturer's instructions. The RNA was eluted using 50 µl diethyl pyrocarbonate (DEPC)-treated water and precipitated by the addition of 0.1 volume NaOAc (Sigma, St Louis, MO, USA), 2.5 volume of 100% ethanol and 1 µl glycogen (Ambion, Foster City, CA, USA). After 1 hr at −20°C, the samples were centrifuged (13000 rpm) for 20 min at 4°C. The RNA pellet was washed twice with 80% ethanol and air-dried prior to resuspension in 10 µl DEPC-treated water. RNA integrity was measured with an Agilent 2100 Bioanalyser (Agilent, Santa Clara, CA, USA).

The growth with different polysaccharide feeds (substrates) was investigated. The ciliate growth was examined with no particulate substrate (NoPOS), with Xylan (Xylan from beech wood, Sigma-Aldrich), carboxymethyl cellulose (CMC, Sigma-Aldrich), crystalline cellulose (CC, Sigmacel 20, Sigma-Aldrich), rice starch (RS, ANALAR, Hopkins and Williams GB), and Inulin (Inulin from Dahlia tubers, Sigma-Aldrich).

Melting points were recorded on an advanced melting point apparatus, SMP30, Stuart (Bibby Scientific, London, UK). Microanalytical data were gathered with a Vario Elementar apparatus (Shimadzu-Japan). Elemental analyses of all compounds were within ± 0.4% of the theoretical values. The IR spectra (KBr) were recorded on a Perkin Elmer 1650 spectrometer (USA). ¹H and ¹³C NMR spectra (500 MHz for ¹H and 125 MHz for ¹³C NMR) were recorded on JEOL ECA-500 (Shimadzu) instruments.
Chemical shifts were expressed in ppm relative to SiMe₄ as an internal standard in DMSO-d₆ or (CDCl₃) as a solvent. Mass spectra were recorded on a 70 eV Finnigan SSQ 7000 spectrometer (Thermo-Instrument System Incorporation, USA). Follow up of the reaction and the purity of the compounds was checked on aluminum plates coated with silica gel (Merck, Germany) on Microwave Advanced Flexible Synthesis Platform 1900 W (flexiWAVE—Milestone, Italy), producing continuous irradiation and equipped with a simultaneous external air-cooling system. Chemicals and solvents (Analar ≥ 99%) were purchased from Sigma-Aldrich (USA).