PVA solutions were prepared by heating a proprietary blend of PVA in water (previously optimized for electrospinning on the NS-1WS500U) at 80°C until the polymer was completely dissolved. TFV (a gift from CONRAD) was added to polymer solutions at 0, 5, 10, 20, 40, 60, and 80 wt% TFV theoretical drug loading (defined as mass drug/mass fiber). Drug precipitate was observed in all pH-unadjusted (pH 3) TFV solutions. Given that TFV has a pKa of 4.1[20 (link)], we hypothesized that by increasing the pH, we could increase drug solubility in the polymer solutions. The pH of each of the TFV-containing solutions was adjusted from pH ~3.3 to a final pH of 7.0 using 10 M sodium hydroxide. A bench top conductivity meter (Thermo Scientific Orion Star A212), pH meter (Thermo Scientific Orion Star A111), and surface tensiometer (Kibron AquaPi ) were used to measure solution parameters in duplicate for both pH 3 and pH 7 solutions. Solution viscosity was measured using an ARG2 rheometer (TA Instruments) fitted with cone and plate geometry (1°58’48” cone angle, 40 mm diameter). A frequency sweep test was performed with a constant strain of 0.04 and ramping up angular frequency from 1–628.3 rad/s at 10 points/decade.
Orion star a212
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia
The Orion Star A212 is a benchtop pH meter designed for laboratory use. It provides accurate pH measurements and features a large, easy-to-read display. The device is capable of measuring pH, mV, and temperature.
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10 protocols using orion star a212
1
Optimizing Drug-Loaded PVA Nanofibers
2
Bac-FeOx Purification and Hydrothermal Synthesis
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Bac-FeOxs were purified by washing the samples multiple times with DI water with a centrifuge (Sigma 3–18, John Morris, Australia) at 4200 rpm for 20 min until the conductivity (Thermo Scientific, Orion Star A212, Australia) of the samples reached that of DI water. This indicated that the salt impurities were successfully removed from the samples. X-ray diffraction (XRD, Rigaku MiniFlex 600, Japan) was also used to confirm the purity of the samples when no salt (i.e. sodium chloride, NaCl) peaks were measured. The samples were then dried at 60 °C in an oven overnight. For the hydrothermal process, 2.5 g of the purified Bac-FeOxs (P-Bac-FeOxs) were homogeneously mixed in 40 ml DI water for 30 min and the pH of the solution was adjusted from to 1–6 (HCl) or 8–10 (NH4OH) before transferring into a 50 ml Teflon-lined autoclave. The temperature of the reaction was fixed at 200 °C and was kept for 20 h. After the reaction the samples were washed with DI water (2×) for 10 min in a centrifuge (4200 rpm) then dried overnight at 60 °C in an oven and stored at 25 °C.
3
Quantifying Cold Stress Responses in Rice
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For cold stress treatment, 5-week-old wild-type and transgenic rice plants were transferred to cold room at 4°C for 6 days, after which plants were recovered at 28°C and their growth patterns were monitored as described previously (Byun et al., 2015 (link)). Total chlorophyll (chlorophyll a + chlorophyll b) was extracted from wild-type and transgenic leaves before and after cold treatment according to Lichtenthaler (1987) (link) with modifications as described by Min et al. (2016) (link). The amounts of chlorophyll a and chlorophyll b were measured at 664.2 and 648.6 nm, respectively, by ELISA microplate reader (VERSAmax, Molecular Devices, USA).
Electrolyte leakage analysis was conducted using 8-day-old rice seedlings before and after cold stress treatments in different time points (0, 6, and 11 days) at 4°C. Seedlings of wild-type and transgenic plants were soaked in a test tube containing 35 mL of distilled water on an orbital shaker (200 rpm) at 28°C for overnight. The electrolyte conductivity of each sample was determined before and after autoclaving by using conductivity meter (Orion Star A212, Thermo Scientific, USA) by the method of Min et al. (2016) (link).
Electrolyte leakage analysis was conducted using 8-day-old rice seedlings before and after cold stress treatments in different time points (0, 6, and 11 days) at 4°C. Seedlings of wild-type and transgenic plants were soaked in a test tube containing 35 mL of distilled water on an orbital shaker (200 rpm) at 28°C for overnight. The electrolyte conductivity of each sample was determined before and after autoclaving by using conductivity meter (Orion Star A212, Thermo Scientific, USA) by the method of Min et al. (2016) (link).
4
Cell Death Induction in N. benthamiana
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Four-week-old N. benthamiana plants were infiltrated with A. tumefaciens carrying the constructs of interest. One day after A. tumefaciens infiltration, the same leaves were infiltrated with 30 μg/mL Brefeldin A (BFA; B7651, Sigma; ER stress/cell death inducer), 20 μg/mL tunicamycin (TM; ab120296, Abcam; ER stress/cell death inducer), or 0.2% DMSO (mock treatment).
The extent of cell death was measured quantitatively by monitoring electrolyte leakage. Four leaf discs (diameter, 1 cm) of infiltrated leaves were punched out for each sample immediately after BFA/TM/DMSO infiltration (at 1 dpi of A. tumefaciens infiltration). Then, the leaf discs were placed in a 12-well plate containing 5 mL of distilled water per well for 1 h to remove ions released by sampling-related injury. After washing, the leaf discs were carefully transferred to a fresh 12-well plate containing 5 mL of distilled water per well, which was incubated in a growth chamber for 24 h. Conductivity measurements were taken with ORION STAR A212 conductivity meter (Thermo Scientific) 24 h after treatment.
The extent of cell death was measured quantitatively by monitoring electrolyte leakage. Four leaf discs (diameter, 1 cm) of infiltrated leaves were punched out for each sample immediately after BFA/TM/DMSO infiltration (at 1 dpi of A. tumefaciens infiltration). Then, the leaf discs were placed in a 12-well plate containing 5 mL of distilled water per well for 1 h to remove ions released by sampling-related injury. After washing, the leaf discs were carefully transferred to a fresh 12-well plate containing 5 mL of distilled water per well, which was incubated in a growth chamber for 24 h. Conductivity measurements were taken with ORION STAR A212 conductivity meter (Thermo Scientific) 24 h after treatment.
5
Water Loss, Chlorophyll, and Electrolyte Leakage Assays for Stress-Tolerant Plant Lines
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For water loss rate measurement of wild-type and Ubi:CaPUB1 transgenic lines, the cut aerial parts of 5-week-old plants were incubated at 25°C under dim light and their weights were estimated at various time points. The water loss rates were expressed as a percentage of initial fresh weights as described previously (Cho et al., 2011 (link)).
Total chlorophyll (chlorophyll a + chlorophyll b) was extracted from drought and cold stress-treated wild-type and Ubi:CaPUB1 transgenic rice leaves as previously described (Lichtenthaler, 1987 ) with slight modifications. Chlorophyll content was estimated with a spectrophotometer (model DU800; Beckman Coulter). Chlorophyll a and chlorophyll b were measured at 664.2 nm and 648.6 nm, respectively. Total chlorophyll content was calculated using the following formula: chlorophyll a + chlorophyll b (mg/g DW) = 8.1 × (5.24 A648.6 + 22.24 A664.2)/DW.
An electrolyte leakage assay was conducted with the leaves of 5-week-old wild-type and Ubi:CaPUB1 rice plants after 7 days of 4°C treatment. The conductivity of each sample was estimated before and after autoclaving with a conductivity meter (Orion Star A212, Thermo Scientific, USA).
Total chlorophyll (chlorophyll a + chlorophyll b) was extracted from drought and cold stress-treated wild-type and Ubi:CaPUB1 transgenic rice leaves as previously described (Lichtenthaler, 1987 ) with slight modifications. Chlorophyll content was estimated with a spectrophotometer (model DU800; Beckman Coulter). Chlorophyll a and chlorophyll b were measured at 664.2 nm and 648.6 nm, respectively. Total chlorophyll content was calculated using the following formula: chlorophyll a + chlorophyll b (mg/g DW) = 8.1 × (5.24 A648.6 + 22.24 A664.2)/DW.
An electrolyte leakage assay was conducted with the leaves of 5-week-old wild-type and Ubi:CaPUB1 rice plants after 7 days of 4°C treatment. The conductivity of each sample was estimated before and after autoclaving with a conductivity meter (Orion Star A212, Thermo Scientific, USA).
6
Electrolyte Leakage Method for Cell Membrane Injury
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Electrolyte leakage was the conductometrical method used for assessing the injury percentage of cell membrane (Bajji et al. 2002 (link)). Five fresh, complete leaves of each sample were washed in deionized water for a few seconds, embedded in test-tubes containing 20 mL of deionized water and took the first conductometric measurement (ECi); then incubated on a shaking platform at ambient temperature for 3 h and measured again (ECf) by a conductivity meter (Orion Star A212, Thermo Scientific, USA). After autoclaving the solution at 100 °C for 20 min to rupture completely the membranes allowing ion leakage of the entire cell contents and cooling to ambient temperature, the third measurement was done (ECt). The membrane damage of soybean leaves’ cells was evaluated as the injury percentage compared with control and was calculated as where Rs and Rc represent (ECf − ECi)/(ECt − ECi) for aphid and/or cyanobacteria treated tissues and control, respectively.
7
Soil Electrical Conductivity and pH Measurement
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Soil electrical conductivity and pH were measured in 1:1 extract (soil: water) following the method described by Rhoades28 and Mclean27 , respectively. 30g of each soil was placed in a centrifuge tube, 30 ml of distilled water was added, placed on a shaker, shaken for two hours, and centrifuged to extract the solution from the soil. The solution was then filtrated using filter paper. After that, the EC and pH for the extracted solution were measured using an EC meter (Orion Star A212, Thermo Scientific) and a pH meter (Orion Star A211, Thermo Scientific), respectively.
8
Biochar Production from Olive Pomace
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Olive pomace (locally known as Jift) was obtained from a local Olive mill and used as feedstock for biochar production. Jift samples were subjected to different pyrolysis temperatures (300, 400, 500, and 600 °C) using a muffle furnace for 1 h without an oxygen supply. After pyrolysis, biochar was ground and passed through a 0.25 mm sieve.
Electrical conductivity (EC) was measured using an EC meter (Orion Star A212, Thermo Scientific) in a 1:20 (biochar: water) solution9 (link). pH was measured using a pH meter (Orion Star A211, Thermo Scientific) using boiling water at a ratio of 1:10 (biochar: water) solution22 . The ash content of biochar was measured following the ASTM D2866-11 method23 . Briefly, 1 g biochar was combusted in a muffle furnace for 6 h at 650 °C, cooled down in a desiccator, and then ash content was measured by sample mass differences.
Electrical conductivity (EC) was measured using an EC meter (Orion Star A212, Thermo Scientific) in a 1:20 (biochar: water) solution9 (link). pH was measured using a pH meter (Orion Star A211, Thermo Scientific) using boiling water at a ratio of 1:10 (biochar: water) solution22 . The ash content of biochar was measured following the ASTM D2866-11 method23 . Briefly, 1 g biochar was combusted in a muffle furnace for 6 h at 650 °C, cooled down in a desiccator, and then ash content was measured by sample mass differences.
9
Electrical Conductivity Measurement Protocol
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The electrical conductivity (EC) of samples was measured at 20°C with the aid of a conductivity meter (Thermo Scientific, Orion Star A212) (Svečnjak et al., 2019) .
10
Purification and Annealing of Bacterial Biofilm
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The collected biofilm samples were suspended in mili-Q water at 1:40 w/v ratio (i.e. 1 g biofilm in 40 mL of water) and vortexed for 10 minutes to obtain a good suspension. This suspension was subjected to repeated centrifugation at 4200 rpm for 15 min and re-suspension until the conductivity (Thermo Scientific, Orion Star A212, Australia) of the sample reached that of mili-Q water itself, indicating effective removal of salt impurities. This purified biofilm sample was dried in air at 70 °C. A specific amount of the purified biofilm sample, referred to as Bac-FeOxNWs, (100 mg in this case) was subsequently loaded in a silica boat and annealed at three different temperatures including 600, 800, and 1000 ˚C in a tube furnace (Brother Furnace, China) fitted with a quartz tube. Table 1 summarizes the preparation conditions of all the Bac-FeOxNWs (i.e. name, annealing temperature, and color) and a schematic of the shape and morphology of samples after annealing at different temperatures is included in Scheme 2. The annealed samples of biofilm (i.e. Bac-FeOxNWs-600, Bac-FeOxNWs-800, and Bac-FeOxNWs-1000) were washed once more with mili-Q water after thermal annealing, air dried at 70 °C in oven and stored under inert atmosphere until further use.
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