Total fungal DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions with slight modifications. Fungal cells of tested Trichoderma strains were harvested by centrifugation of liquid cultures and dried on filter paper at room temperature (21 ± 2 °C), prior to grinding in liquid nitrogen with quartz sand (Sigma-Aldrich). Proteinase K and RNase A were added to the lysis buffer. The purity and quantity of isolated DNA were evaluated by agarose gel electrophoresis followed by ethidium bromide staining, and by determination of A260/A280 using a NanoDrop 8000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Finally, the DNA concentration was adjusted to 1 ng/μL by diluting the samples with 10 mmol/L Tris-HCl, pH 8.0.
Quartz sand
Manufactured by Merck Group
Sourced in United States
Quartz sand is a naturally occurring mineral composed primarily of silicon dioxide (SiO2). It is a common raw material used in various industrial and scientific applications, including the production of glass, ceramics, and as a filtration medium. Quartz sand exhibits high mechanical strength, chemical stability, and thermal resistance, making it a versatile material for laboratory equipment and other specialized uses.
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Lab products found in correlation
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Ag nitrate, by Merck Group (1 mentions)
Potassium permanganate, by Merck Group (1 mentions)
Coulter counter, by Beckman Coulter (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Absolute ethanol, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Polystyrene round bottom tube, by BD (1 mentions)
Kaolin, by Fujifilm (1 mentions)
Acetone, by Fujifilm (1 mentions)
Acetonitrile, by Fujifilm (1 mentions)
Benzo a pyrene, by Fujifilm (1 mentions)
Flash ea 1112, by Thermo Fisher Scientific (1 mentions)
13 protocols using quartz sand
1
Trichoderma DNA Isolation Protocol
2
Quantitative Glucokinase Activity Assay
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Glucokinase activity was measured by a modified enzyme-linked assay3 (link). Synechococcus cells cultivated in BG11 medium were centrifuged and resuspended in 1 mL of breakage buffer (50 mM Tris-HCl, pH 7.4) pre-cooled to 4 °C. Quartz sand (Sigma) was added to the cell suspension, and the cells were disrupted by vortex mixing at 4 °C for 30 min. After centrifugation, 60 µL of the supernatant was transferred to a 96-well plate, to which 200 µL of a reaction buffer (100 mM Tris-HCl, pH 7.8, 2.5 mM ATP, 4 mM MgCl2, 20 mM KCl, 0.2 mM NADP+, 10 mM glucose, and 5 U/mL glucose-6-phosphate dehydrogenase) was added, and the absorbance at 340 nm was measured every 5 min in the dark. The slope of A510, which increased with time, was calculated, and the specific enzyme activity of glucokinase in the reaction mixture was calculated according to the Lambert-Beer law.
3
Protein Aggregation Detection Protocol
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Protein aggregate detection was done as described before (49 (link)). Briefly, total protein extracts were prepared by homogenization of plant material in 1.0 ml per 0.2 g fresh weight of protein isolation buffer [25 mM HEPES, pH 7.5, 200 mM NaCl, 0.5 mM Na2EDTA, 0.1% (v/v) Triton X-100, 5 mM ϵ-amino-N-caproic acid, 1 mM benzamidine], using a mortar and pestle and then a Cole-Parmer PTFE glass tissue grinder. The soluble and insoluble fractions were separated from 1.0 ml of total extract by centrifugation at 16 000 × g for 15 min at 4°C. The soluble fraction was prepared by adding 0.25 volume of 5× sample buffer and heating for 2 min at 95°C. The insoluble pelleted fraction was washed five times by repeated resuspension in protein extraction buffer containing 0.1 g of quartz sand (Sigma-Aldrich). The pellet (insoluble fraction) was resuspended in 400 ml hot 2× SDS-PAGE sample buffer and clarified by centrifugation at 1500 × g for 1 min. Samples were separated by SDS-PAGE and stained; the whole lanes have been quantified by Image Lab 5.1 (Bio-Rad) and ratios calculated.
4
Characterization of Quartz Sand Properties
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Quartz sand (Sigma-Aldrich, St. Louis, MO, USA) with sizes ranging from 300 to 355 μm was adopted to pack the columns. Metal oxides and other impurities were extensively removed from the sand before packing the columns using the method of Elimelech and O’Melia [15 (link)]. Briefly, the sand was soaked with 1 M HNO3 solution for 12 h, and then rinsed with DI water and dried in an oven at 105 °C. The zeta potentials of sand were measured following the method in previous studies [16 (link),17 (link),18 (link)]. Briefly, 7 g cleaned sand was sonicated for 20 min in 12 mL NaCl solution with the ISs of 0.001, 0.01, 0.1, or 0.2 M. Samples of the supernatant were diluted 10-fold in a background NaCl solution and zeta potentials were measured using Zetasizer Nano ZS (Malvern Instruments Ltd., Southborough, MA, USA).
5
Sand Attachment Assay for E. coli Biofilm
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Sand attachment assay was conducted according to the method described by Hinsa et al. (2003) (link). E. coli cells were grown for 24 h on LB agar at 28°C. Colonies were scraped off with a loop and resuspended in 5 ml PBS. OD600nm was measured and normalized in LB without salt (LBns) to give a starting population of 106 CFU ml-1. quartz sand was pre-weighed into 1.5 ml tubes and sterilized by autoclaving. Then, 0.5 g of sterilized quartz sand (Sigma) was added to wells of a 96-well plate and 1 ml of the inoculated LBns was added to each well. Plates were incubated static in the dark at 28°C for 48 h. After incubation, the LBns in each well was removed, serially diluted, and plated out to determine planktonic cell count. For biofilm cell count, sand in the 96-well plate was pipetted into pre-weighed 1.5 ml tubes and 500 μl of PBS was used to wash the sand five times to remove unattached cells. All liquid was removed and tubes were re-weighed. Then 500 μl of PBS was added and the tubes were vortexed for 30 s, sonicated (4 min, 100% power) and vortexed again for 30 s. The liquid fraction was then serially diluted and plated onto LB agar to determine the biofilm count. The bacterial cell counts were normalized to the weight of the sand.
6
Imaging EcoFAB Plant-Fungus Interaction Protocol
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Imaging EcoFABs were filled with quartz sand (Sigma-Aldrich, 50–70 mesh particle size) prior to autoclaving. Prior to planting, sand Imaging EcoFABs were filled to capacity with 10% MS. For gel-filled Imaging EcoFABs, 1.5 g/L phytagel was prepared in 10% MS, autoclaved, and filled into Imaging EcoFABs to capacity while still hot. As described above, B. distachyon seedlings were prepared and allowed to grow for 1 week prior to inoculation with fungal strains. The Neurospora crassa strain was kindly provided by Dr. N. Louise Glass (UC Berkeley, Berkeley, CA, USA) [14 (link)]. Plants were inoculated at 105N. crassa spores per mL of Imaging EcoFAB volume (three plants per treatment or media). Plants were grown in the growth chamber (setting described above) for 2 days prior to imaging.
7
Enzyme Activity Analysis of Fruit Peel
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The activities of PAL (EC 4.3.1.5), CHS (EC 3.2.1.74), CHI (EC3.2.1.14), and FHT (EC 1.14.11.9) were analyzed for the peel from the fruit following the control and MeSA and SA treatments. The sample preparation for all of these enzymes was according to Halbwirth et al., [40 (link)] with minor changes.
Briefly, for each sample, ~0.4 g fruit peel frozen in liquid nitrogen was ground and homogenized in a ceramic mortar with 0.2 g quartz sand (Sigma-Aldrich, St. Louis, MI, USA), 0.2 g Polyclar AT, and 3 mL Dellus buffer, which contained 0.1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 40 mM sucrose, 0.75 mM polyethylene glycol 20,000, 0.1 M sodium ascorbate, 1 mM dithioerythritol, and 0.025 mM CaCl2 (pH 7.3). Before use, the oxygen was removed from the buffer. Here, the mixture HEPES and sucrose were prepared to 150 mL with bi-distilled water and boiled for 20 min to a final volume of 100 mL. This was cooled in an ice bath to 30 °C under a nitrogen flow, and then the polyethylene glycol, sodium ascorbate, and dithioerythritol were added [41 (link)]. The preparation of the fruit peel was completed by centrifugation at 13,000× g for 10 min at 4 °C.
Briefly, for each sample, ~0.4 g fruit peel frozen in liquid nitrogen was ground and homogenized in a ceramic mortar with 0.2 g quartz sand (Sigma-Aldrich, St. Louis, MI, USA), 0.2 g Polyclar AT, and 3 mL Dellus buffer, which contained 0.1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 40 mM sucrose, 0.75 mM polyethylene glycol 20,000, 0.1 M sodium ascorbate, 1 mM dithioerythritol, and 0.025 mM CaCl2 (pH 7.3). Before use, the oxygen was removed from the buffer. Here, the mixture HEPES and sucrose were prepared to 150 mL with bi-distilled water and boiled for 20 min to a final volume of 100 mL. This was cooled in an ice bath to 30 °C under a nitrogen flow, and then the polyethylene glycol, sodium ascorbate, and dithioerythritol were added [41 (link)]. The preparation of the fruit peel was completed by centrifugation at 13,000× g for 10 min at 4 °C.
8
Eggshell-Derived Catalyst Preparation
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Potassium permanganate (KMnO4; ≥ 99%), manganese(II) acetate (MnAc (C4H6MnO4); 98%), Ag nitrate (AgNO3; ≥ 99%), quartz sand (SiO2), and paraformaldehyde (pFA (HO(CH2O)nH); 95%) were procured from Sigma-Aldrich (St. Louis, Missouri, United States of America (USA)).
Commercial CaCO3 (> 98%) was supplied by Showa Co., Ltd. (Tokyo, Japan). Air (99.999% (21% oxygen (O2) in nitrogen (N2) balance)), N2 (99.999%), and hydrogen (H2; 99.999%) cylinders were purchased from Union Gas Co., Ltd. (Yongin, Republic of Korea). All the chemicals were used as received without any additional purification. Chicken eggs, supplied by the Korea egg distribution association (Seoul, Republic of Korea), were purchased from a local market. Tap water was used to hard-boil the eggs. The eggshells were then peeled off and thoroughly washed with deionized (DI) H2O to remove surface impurities. Subsequently, the eggshells were dried at 50°C for 12 h in a convection oven (ED-CO150, Han Yang Scientific Equipment Co., Ltd., Seoul, Republic of Korea).
The dried eggshells were then ground (< 1.18 mm) for preparing the catalysts.
Commercial CaCO3 (> 98%) was supplied by Showa Co., Ltd. (Tokyo, Japan). Air (99.999% (21% oxygen (O2) in nitrogen (N2) balance)), N2 (99.999%), and hydrogen (H2; 99.999%) cylinders were purchased from Union Gas Co., Ltd. (Yongin, Republic of Korea). All the chemicals were used as received without any additional purification. Chicken eggs, supplied by the Korea egg distribution association (Seoul, Republic of Korea), were purchased from a local market. Tap water was used to hard-boil the eggs. The eggshells were then peeled off and thoroughly washed with deionized (DI) H2O to remove surface impurities. Subsequently, the eggshells were dried at 50°C for 12 h in a convection oven (ED-CO150, Han Yang Scientific Equipment Co., Ltd., Seoul, Republic of Korea).
The dried eggshells were then ground (< 1.18 mm) for preparing the catalysts.
9
Nylon Microplastics Impacts on Chironomid Larvae
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Nylon 6 powder (particles < 50 µm with a mean size of 13-19 measured using a Coulter Counter (Multisizer 3, Beckman, USA), density 1.13 g cm -3 ) was purchased from Goodfellow, UK. The powder was soaked in Nile Red solution (8 µg mL -1 in 80:20 methanol: water solution) to provide a fluorescent label that would allow the detection of particles within the chironomid gut. After labelling, the carrier solvent was evaporated at room temperature for approx. 24 h with occasional mixing until the powder was completely dry. Particles were then rinsed in deionised water to remove any unbound dye, filtered onto 1.2 Whatman GF/C glass microfiber filter papers (GE Healthcare Life Sciences, UK) and redried at 60°C. Experimental treatments consisted of exposure in sediment either with or without microplastics (1% nylon powder by mass). Microplasticspiked sediments were prepared by mixing 0.8 g of the labelled nylon powder with quartz sand (Sigma Aldrich) and making up to 80 g, Masses of eggs freshly laid from the colony were transferred to trays containing reconstituted water with 1.2 mg L -1 hydrated CaSO 4 , 0.08 mg L -1 KCl, 2.44 mg L -1 MgSO 4 7H 2 O, and 1.92 mg L -1 Na 2 CO 3 , conductivity of 160 cm -1 , pH 7.2 and hardness 16 mg L -1 (US EPA 2000). Larvae were maintained with constant aeration and fed TetraMin ® fish food three times per week until they reached the fourth instar.
10
Western Blot Analysis for HeLa and C. elegans Protein Extracts
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For HeLa cells, protein extracts were recovered in sample buffer (0.25 mM Tris-Cl, pH 6.8; 10% SDS; 50% glycerol; and 5% β-mercaptoethanol), resolved by SDS-PAGE (10% acrylamide) and transferred onto nitrocellulose membranes using a TransBlot Turbo™ system (BioRad), at 0.3 A for 1 h. For C. elegans, 60 adult worms/condition were harvested, washed 3 times in M9 containing 0.1% Triton X-100, centrifuged at 750 g, resuspended in sample buffer supplemented with quartz sand (Sigma) corresponding to 1/3 of the final volume. 3 cycles of 5 min boiling at 95ºC followed by 5 min vortexing were performed. Samples were resolved by SDS-PAGE (7,5% acrylamide) and transferred onto nitrocellulose membranes (Hybond ECL; GE Healthcare) at 0.22 mA for 2 h at 4ºC. Primary and secondary HRP-conjugated antibodies were diluted in TBS-Tween 0.1% (150 mM NaCl; 50 mM Tris-HCl, pH 7.4; and 0.1% Tween) with 5% (m/v) milk. Washes were performed with TBS-Tween 0.2%. Signal was detected using Western Blotting Substrate (Thermo Fisher Scientific) and collected in a ChemiDoc™ XRS+ System with Image Lab™ Software (BioRad).
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