Initial [14C]methylamine uptake rates (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) for amino acids were measured as described previously (Marini et al., 1997 (link)). Single colonies were grown in liquid YNB-N supplemented with 6% glucose and 500 μg/mL L -proline to logarithmic phase and were centrifuged at an OD600 of 0.5 to 0.8. Cells were washed twice in sterile water and resuspended in 50 mM KH2PO4 buffer pH 5, to a final OD600 of 5. Before the uptake measurements an aliquot of yeast cells was supplemented with 20 mM glucose, incubated at 30°C for 5 min at 1,000 rpm. To start the reaction an equal amount of pre-warmed KH2PO4 buffer containing 15 kBq of [14C]methylamine and unlabelled methylamine (0–15 mM) was added. Cells were incubated at 30°C, 1,000 rpm, and 45 μl subsamples were taken after 1, 2, 3, and 4 min, diluted in 5 ml KH2PO4/sorbitol buffer, separated from the incubation buffer on glass fibre filters (Whatman), and washed twice with the same buffer. Radioactivity retained on the filter was assayed by liquid scintillation spectrometry (Packard).
Glass fibre filter
Manufactured by Cytiva
Sourced in United Kingdom
Glass fiber filters are porous mats made from randomly oriented, extremely fine glass fibers. They are designed to capture and retain a wide range of particulates and contaminants from liquids or gases passing through them. The filters offer high mechanical strength, good chemical resistance, and consistent performance across a variety of applications.
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8 protocols using glass fibre filter
1
Measuring Methylamine Uptake in Yeast
2
Aerobic Oxidative Functionalization Catalyst
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The starting material was dissolved in H2O/MeCN (4 : 1). Subsequently, the catalyst was added as a solid (in the reported amounts, see ESI† ). An oxygen atmosphere was applied by attaching an oxygen cylinder to the system. After a reaction time of 20–48 h at 45 °C, active carbon was added to the reaction mixture, which was then filtered over Celite and 5 layers of Whatman glass fibre filters (420 μm thick, 0.7 μm pore size). The filtrate was lyophilised.
3
Measuring Urchin Assimilation Efficiency
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To measure assimilation efficiency, sea urchins were placed individually in 2.5-l beakers filled with filtered seawater (0.45 μm) for 24 h and during this period they were not fed. Faeces from each beaker were drawn off, filtered on pre-ashed and weighed glass fibre filters (Whatman GFC), and rinsed with distilled water to remove the salt. Filters were then dried in an oven at 60 °C, weighed after 24 h, ashed in a muffle furnace at 450 °C for 4 h and re-weighed (Conover 1966 ; Reid et al. 2010 ). Weight determinations were performed using a Mettler Toledo, XS105 Dual Range analytical balance (0.01 mg readability). The same procedure was applied in triplicate on diet samples. In faeces and algae, organic content (OC) was calculated as ash-free dry weight obtained as the difference between dry and ash weight. Absorption efficiency (AE) was determined according to Conover (1966 ): AE = [(DietOC − FaecesOC) / (1 − FaecesOC) × DietOC] × 100, where DietOC and FaecesOC are the organic fractions in algae and in faeces, respectively.
4
Isolation and Preservation of Alexandrium Cells
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Bottles were sealed and carefully turned over 7 times to ensure equal mixing of the culture. The direct grazing experiment samples were pre-filtered through a submerged 64 µm nylon mesh to remove copepods and copepods eggs. 60–70 mL were sub-sampled for enumeration and sizing of cells and chains using a coulter counter (Micromeritics, Norcross, USA) mounted with a 100 µm orifice tube and continuous stirring. A known volume of the subsamples was then suction filtered onto glass-fibre filters (Whatman) and stored at −20 °C until toxin extraction and analysis.
The remaining culture was concentrated on a 10 µm nylon mesh to collect the A. fundyense cells. The 10 µm mesh was rinsed into a 50 mL centrifuge tube with sterile filtered seawater and immediately centrifuged at 4 °C for 5 min for RNA samples. The supernatant was discharged and the pellet immediately mixed with 1 mL 60 °C hot TriReagent (Sigma-Aldrich, Steinheim, Germany) and transferred to a cryovial with acid washed glass beads. The cryovial was vortexed for 10 s and submerged into liquid nitrogen. The samples were stored at −80 °C until RNA isolation.
The remaining culture was concentrated on a 10 µm nylon mesh to collect the A. fundyense cells. The 10 µm mesh was rinsed into a 50 mL centrifuge tube with sterile filtered seawater and immediately centrifuged at 4 °C for 5 min for RNA samples. The supernatant was discharged and the pellet immediately mixed with 1 mL 60 °C hot TriReagent (Sigma-Aldrich, Steinheim, Germany) and transferred to a cryovial with acid washed glass beads. The cryovial was vortexed for 10 s and submerged into liquid nitrogen. The samples were stored at −80 °C until RNA isolation.
5
Analytical Protocols for Persistent Organic Pollutants
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All labelled and unlabelled reference standards of PAHs, PAEs, AP 1-2 EOs and APs were purchased by Sigma-Aldrich (St. Louis, MO, USA) and LGC (Teddington, UK). All solvents and reagents were supplied by Sigma-Aldrich. Solid-phase extraction cartridges were purchased from Agilent (Santa Clara, CA, USA). Glass fibre filters were obtained from Whatman (Maidstone, UK). For full details, see section S1.1 of Supplementary Material.
Chemical name of target analytes, abbreviations, logarithms of the octanol-water partition coefficients (log K OW ) and attributes of priority or priority hazardous substance (European Parliament and Council of the European Union, 2013), are reported in Table 1.
Chemical name of target analytes, abbreviations, logarithms of the octanol-water partition coefficients (log K OW ) and attributes of priority or priority hazardous substance (European Parliament and Council of the European Union, 2013), are reported in Table 1.
6
Environmental Water DNA Extraction
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A 10 L sample of collected environmental water was passed through a 47 mm GF/D (2.7 μm pore size) glass-fibre filter (Whatman). After filtering the samples, the filters were fixed by adding 15 mL of ethanol46 (link). The filter was divided into two equal parts with scissors before DNA extraction; one of the two pieces was used for DNA extraction in order to properly fit the extraction kit. DNA extraction from the filter was performed using a DNeasy Blood & Tissue Kit47 (link). The extracted eDNA was eluted in 400 µL (twice with 200 µL) of buffer AE supplied with the kit. The extracted eDNA was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and subjected to PCR under the same conditions as used for Sanger sequencing to confirm PCR amplification.
7
Silicon Anode Fabrication and Electrochemical Characterization
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The battery investigated here is a half-cell consisting of a 200 μm-thick silicon powder electrode with a diameter of 1.5 mm and an active mass of 175 μg, a 250 μm glass fibre separator (Whatman glass fibre filter), and a metallic lithium electrode (Alpha Aesar, lithium foil, 99.9%). The sample is wet with 30 μl standard LP50 electrolyte (1 M LiPF6 in 1 : 1 ethylene carbonate : diethyl carbonate, BASF) and assembled in the custom-made cell in an argon-filled glove box (O2 < 0.2 ppm). The silicon electrode is fabricated by combining amorphous/polycrystalline silicon powder (50 wt%, Alpha Aesar, 1–20 μm) and carbon black (25 wt%, Imerys, C65) with a pre-mixed solution containing 5 wt% PVDF (25 wt%, Kynar Flex®, HSV900) in NMP and agitated in a high shear mixer for 10 min. After ultra-sonication for 5 min and air bubble removal for 2 h, 200 μm thick films are coated on copper foil using a doctor blade. The resulting electrode sheets are dried in a vacuum oven at 120 °C for 8 h.
8
Measurement of Photosynthetic Oxygen Fluxes
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BS samples (1 ml) were supplied with the metabolites required to support CO2 assimilation and filtered via gentle vacuum onto a glass fibre filter (Whatman, Buckinghamshire, UK) through a 10.2‐mm aperture. A disc of 10.0 mm was cut from the centre of the filter, saturated with 50 µl of the activity buffer with the metabolites and loaded onto a steel mesh within a 1‐ml stainless steel cuvette. The cuvette was equilibrated to 25°C via a circulating water bath (Julabo, Seelbach, Germany). The bottom was sealed with a gas‐permeable Teflon membrane (Hansatech, Norfolk, UK) while the top lid had a quartz window through which halogen light was supplied through a fibre optic and a septum. The cuvette was purged with compressed air and then injected with 2% 18O2 (99%, Sigma‐Aldrich) and 2% CO2 (BOC, Sydney, Australia). After 5 min in darkness for equilibration, gross rates of O2 production and O2 consumption were measured for 5 min in the dark and for 5 min at 1000 µmol m−2 sec−1 via Delta V MIMS (Thermo Electron Corp, Bremen, Germany) resolving the evolution of 16O2 (the product of splitting H216O at natural abundance) from consumption of artificially enriched 18O2. Rates were calculated according to Beckmann et al. (2009 (link)).
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