Durapore filter

Seawater samples were collected manually in autoclaved glass bottles from the control and oil treated tanks daily for 7 days. One sample of 1000 mL of water was collected using a pipe placed in the middle of each tank at the start of the experiment (T0, after 24 h of acclimation to laboratory conditions and prior to oil addition) and then 700 mL was collected each following day for 7 days. Seawater samples were immediately brought to two ESPs placed in a separate room, one dedicated to processing exposed and one dedicated to processing samples from non-exposed tanks. The glass bottles were connected to the ESP instruments which processed further the samples automatically. The ESPs filtered the samples onto 25 mm diameter, 0.22 µm pore size hydrophilic Durapore filters (Millipore, Burlington, MA, USA) and archived the filters after addition of RNAlater™ stabilization solution (Ambion, Austin, TX, USA) for RNA preservation [15 (link),42 (link)]. After each operation, the filters were retrieved from the ESP and stored at −80 °C until use. In total, 15 filters were collected during the experiment (1 filter/day/condition and the initial seawater), of which 5 were selected for the metatranscriptomics analysis.

The biological and environmental samples were collected at local noon at 11-time points between 14 December 2011 and 10 January 2013 from the Isfjorden Adventfjorden time series station (IsA); located on the west coast of Spitsbergen, Svalbard (N 78°15.6, E 15°31.8, Fig. 1). At each of the 11 sampling dates, 30 l of seawater was sequentially sampled from 25 m depth using a 10 l Niskin bottle (KC Denmark), and immediately processed on board. Samples were kept in dark and cold conditions while prefiltered by gravity through 10 μm nylon mesh (KC Denmark) and then onto 8–12 47 mm 0.45 μm Durapore filters (Millipore) using vacuum pumps. Each filter was fixed in 600 μl LB buffer (RNAqueous Total RNA Isolation Kit, Invitrogen, Thermo Fisher Scientific) 5–20 min after sampling, flash-frozen in liquid nitrogen and stored at −80 °C.
At each sampling date, a vertical profile of environmental variables was obtained using a handheld SAIV 204 STD/CTD probe. However, in this paper we present only the data for 25 m depth where the samples were taken from (see^{10 (link)} for complete profile). Photosynthetically active radiation (PAR), size-fractionated chlorophyll a and nutrient concentrations (nitrate/nitrite, phosphate, silicate), were obtained as described in^{10 (link)}.

The ability of MNT₁ to provide liposome leakage was demonstrated on unilamellar phosphatidylcholine (Sigma-Aldrich, Burlington, MA, USA) liposomes loaded with the fluorescent dye calcein (Fluka, Seelze, Germany). Unilamellar liposomes were loaded with fluorescent dye calcein to the concentration of 100 mM which is self-quenching. To do this fresh lipid suspension in liposome buffer containing HEPES (20 mM), MES (20 mM), citrate (20 mM), and sodium chloride (150 mM), pH 7.4 was sonicated until clear, using a W-181-T sonicator (Ulta Sonic Finland LTD, Lanti, Finland; 40 kHz, 90 W, 0 °C, 30 min), Then the resulting liposomes were passed 10 times through 0.22 µm Durapore filters (Millipore, Burlington, MA, USA) for size standardization. The liposomes were stored at 4 °C under an argon atmosphere. Liposomes were purified on PD-10 prior to experiment, then the same day incubated for 30 min with 100 nM MNT₁ in liposome buffer at the different pHs at room temperature in triplicate. After that, samples were diluted tenfold in liposome buffer pH 7.5, and the fluorescence of free calcein (leaked from liposomes) was measured at 520 nm (excitation at 490 nm). As a positive control (100% calcein leakage), a 0.5% Triton X-100-containing sample was used. Parallel probes where MNT₁ was omitted served to assess background calcein leakage.

The ability of the new MNT to provide liposome leakage was demonstrated on unilamellar liposomes from egg lecithin (Khimpharmzavod, Kharkov, Ukraine) loaded with the fluorescent dye calcein (Fluka, Munich, Germany) in accordance with a previously published protocol (Khramtsov et al., 2008 (link)). In brief, the unilamellar liposomes loaded with fluorescent calcein up to the concentration of fluorescence quenching (100 mM) were prepared by sonicating fresh lipid suspension in 20 mM HEPES, 20 mM MES, 20 mM citrate, 150 mM NaCl, pH 7.4 (liposome buffer) until clear, using a W-181-T sonicator (Finnsonik, Lahti, Finland; 40 kHz, 90 W, 0°C, 30 min), and passed 10 times through Durapore filters with 0.22 μm pore diameter (Millipore, Burlington, MA, USA) to standardize liposomes sizes. The liposomes were stored under an argon atmosphere at 4°C for several months. PD-10-purified liposomes were incubated with 100 nM MNT for 30 min in liposome buffer at indicated pH (3–7.5) in triplicates after that samples were diluted tenfold in liposome buffer, pH 7.5 and fluorescence of leaked calcein was measured at 520 nm at excitation wavelength 490 nm. As a positive control (100% calcein leakage) we used addition Triton X-100 up to 0.5%. The samples without MNT were used as background leakage.

Utilizing unilamellar phosphatidylcholine liposomes (Fluka, Seelze, Germany) loaded with the fluorescent dye calcein (Sigma-Aldrich, Burlington, MA, USA), MNT₁′s capacity to induce liposome leakage was established. Unilamellar liposomes were loaded with fluorescent dye calcein to the fluorescence self-quenching concentration of 100 mM. Using a W-181-T sonicator (Ulta Sonic Finland Ltd., Lanti, Finland; 40 kHz, 90 W, 0 °C, 30 min), fresh lipid suspension was sonicated until clear in liposome buffer containing HEPES (20 mM), MES (20 mM), citrate (20 mM), and sodium chloride (150 mM), pH 7.4. For size standardization, the resultant liposomes were run through Durapore filters with a pore size of 0.22 µm (Millipore, Burlington, MA, USA) for size standardization ten times. The liposomes were kept in an argon environment at 4 °C. Before the experiment, liposomes were purified on PD-10. They were then treated for 30 min in triplicate at room temperature with 100 nM MNT₁ in liposome buffer at various pH levels. After that, samples were diluted tenfold in liposome buffer, pH 7.5, and the fluorescence of free calcein (leaked from liposomes) was measured at 520 nm (excitation at 490 nm). Samples with 0.5% Triton X-100 were utilized as a positive control (100% calcein leakage). Parallel samples with MNT1 deleted were used to measure background calcein leakage.