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27 protocols using chromafil

1

Comprehensive Characterization of Nanoparticles

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The solution was utilized for UV-Vis, TEM, single particle ICP-MS (sp-ICP-MS), dynamic light scattering (DLS), and zeta potential. The suspension was centrifuged at 5000 rpm for 15 min, filtered (0.22 m, Chromafil®, Macherey-Nagel, Düren, Germany), and the precipitate was used for Fourier transform infrared spectra (FTIR) and X-ray diffraction (XRD) analyses.
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2

Micropollutant Transformation in Wastewater Treatment

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Batch experiments to assess the primary transformation of the micropollutants were performed in 1 L reactors with two carriers and 500 mL biologically treated wastewater at pH 7±0.2. A pH-adjustment to pH 7 with 10 mM NaH 2 PO 4 and NaOH was made in the experiment with carriers from Klippan WWTP. The micropollutants were spiked as a mixture to a concentration of 4 µg/L in the Klippan experiment and to 4 µg/L and 100 µg/L in the Bad Ragaz experiment. To test for abiotic transformation, a control reactor with tap water without carriers and a micropollutant spike of 4 µg/L was run in parallel. Samples (5 mL) for micropollutant analysis were taken after 10 min, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h and 120 h. All samples were immediately filtered (0.45 µm -Chromafil, Macherey-Nagel) and stored at -20 °C until analysis.
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3

Quantification of Bioactive Compounds in Lamium album

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The concentrations of bioactive compounds in the methanolic extracts were determined using a UPLC-PDA-TQD system, which consisted of the Acquity UPLC chromatograph (Waters, Manchester, MA, USA) coupled with a photodiode detector (PDA eλ Detector) (Waters, Manchester, MA, USA) and an electrospray ionization (ESI) triple quadrupole mass spectrometer (TQD) (Waters, Manchester, MA, USA). Mobile phase consisted of two solutions: line A-water containing 0.1% HCOOH and line B methanol containing 0.1% HCOOH. The phenolics were separated at 30 • C on the analytical columna Waters ACQUITY UPLC BEH C18 (150 × 2.1 mm/ID, with 1.7 µm particle size) (Waters, Manchester, MA, USA) with flow rate 0.35 mL/min and using gradient elution: from 10 to 60% of B line during 15 min followed by the return to the initial conditions. Temperature of samples in the autosampler was 10 • C. UV spectral data for all peaks were recorded in the range of 190-450 nm. Data processing was done using Empower 3 (Waters, Manchester, MA, USA). All samples of Lamium album extracts were filtered through a 0.20 µm syringe filter (Chromafil, Macherey-Nagel, Duren, Germany) before analyses and were injected to the chromagraphic system in triplicate. Identification of bioactive compounds was done using matching retention times, UV and mass spectra (MRM) data of standards.
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4

Soil Pore Water Extraction Protocol

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Because centrifugal extraction did not yield enough soil pore water, soil pore water was extracted by saturating of 20 g of wet soil sampled from the different depths, from columns treated for 28 days. After 24 h of equilibration, water was centrifuged through glass wool at 2000 g for 35 min (Hermle Z400K, Germany). The collected water was filtered through a 0.45 mm cellulose acetate syringe filter (Chromafil, Macherey-Nagel, Germany). Glass wool and filters were conditioned by soaking them in a solution 0.1 M of CuNO 3 (99.9%, Sigma Aldrich) overnight before use, in order to avoid adsorption of Ag on the surface of the glass fibres and filters (Cornelis et al., 2010) . Water leachate of the columns was accumulated in a Petri dish at the bottom of the columns after 12, 19 and 21 days of exposure and stored in a À20 C freezer until chemical analysis.
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5

Nanoparticle Production Yield Calculation

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The production yields of the obtained nanoparticles from different batches were calculated according to Equation (1): Production yield (%)=Spray dried nanoparticles (mg)Drug (mg)+Polymer (mg)×100
Drug loading (DL) and entrapment efficiency (EE) were determined by HPLC analysis. Ten milligrams of each batch of nanoparticles were dissolved in 5 mL mobile phase and sonicated for 5 min into an ultrasonic bath (Sonorex Bandelin electronic, Berlin, Germany) until dissolution of the particles and extraction of DRB was achieved. The blend was then centrifuged at 5000 rpm for 15 min and filtered (0.22 µm, Chromafil®, Macherey-Nagel, Düren, Germany). The supernatant was recovered and the concentration of DRB was determined via HPLC (UltiMate 3000, Thermo Scientific, Waltham, MA, USA) under the following conditions: a 254 nm wavelength; a 5 µL injection volume; an Inertsil® ODS-3 HPLC column of 5 µm, 150 × 4.6 mm (GL Sciences, Tokyo, Japan); a 1 mL/min flow rate; a 25 °C oven temperature; a 20 °C sampler temperature; and mobile phase: 0.29% sodium dodecyl sulfate in 0.2% aqueous solution of phosphoric acid:acetonitril (1:1). The drug loading and entrapment efficiency were calculated using Equations (2) and (3), respectively.
DL (%)=Drug amount in the formulationAmount of nanoparticles×100
EE (%)=Actual drug contentTheoretical drug content×100
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6

In Vitro Drug Release from Casein Nanoparticles

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In vitro release of DRB from the casein nanoparticles was performed using the dialysis bag method. A dialysis membrane (Sigma, MWCO 12 kDa) was hydrated in distilled water for 24 h. An accurately weighed amount of nanoparticles (equivalent to 1.5 mg DRB) was dispersed in 5 mL from dissolution media and then transferred to the dialysis bag, which was closed using a plastic clamp. Each bag was placed into a beaker containing 20 mL of each fresh dissolution medium and kept under constant mild agitation on an electromagnetic stirrer at (37.0 ± 0.5) °C. Aliquots (2 mL) were taken and subsequently replaced at predetermined time intervals with fresh medium. Each sample was filtered through a 0.45 μm membrane filter (Chromafil®, Macherey-Nagel, Düren, Germany) and the pH was adjusted to pH 2 using phosphoric acid. The analysis for drug content was performed by HPLC as described in Section 2.2.1. The drug release study was performed in two different dissolution media (PBS pH 5 and PBS pH 7.4) for 30 h. The mean results of triplicate measurements and standard deviations were reported.
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7

Enzymatic Synthesis of 3-Ketodihydrosphingosine

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A step by step protocol can be found in the supplemental material.
Initially, samples containing 0.1 M HEPES pH 7.5, 2.5 mM MgCl2, 0.5 mM TCEP, 0.5 mM ATP, 20 μM CoA, 0.5 mM palmitic acid (50 mM stock solution in DMSO, final DMSO concentration 1% [vol/vol]), 5 mM serine, 20 μg/mL FadD, and 60 μg/mL SPT were incubated at 37°C for different time points. For extraction of 3-ketodihydrosphingosine, to confirm its formation, an equal volume of methanol was added after 2 h. The sample was filtered through a 0.2 μm PTFE filter (Chromafil, Macherey-Nagel) and analyzed by HPLC-HRMS. In all other cases, the reaction was stopped by adding ammonium molybdate (see PPi assay). Later, samples with a final DMSO concentration of 10% (vol/vol) were set up. For samples containing inhibitors (dissolved in DMSO), the final DMSO concentration was also kept at 10% (vol/vol). For blank samples, CoA was replaced with water and for samples measuring FadD activity alone SPT was replaced with buffer C. For measurements with different serine concentrations, appropriate stock solutions were used to set up the reactions. The PPi assay was performed as described before (23 (link)). All measurements were done including technical and biological triplicates.
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8

Screening Fungal Cultures for Depsipeptide Analogues

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For screening the fungal rice cultures for the presence of different depsipeptide analogues using LC-ion-trap MS, 0.15 g aliquots weighed (after lyophilized and ground into powder) of each culture were extracted using 1.5 mL of methanol/water (9:1, v/v) by shaking on an orbital shaker (225 min−1, 90 min) and by sonication for 20 min. After centrifugation using a Beckman J2-MC centrifuge (Beckman Coulter Inc., Fullerton, CA) at 15,000× g, for 10 min, extracts were filtered through a 0.22 µm nylon membrane (Costar, Corning Inc., Corning, NY, USA) and transferred to chromatography vials. For quantification of mycotoxins (BEA, ENNs) by LC-MS/MS, individual culture aliquots (1 g) were mixed with 2 mL of acetonitrile-water (9:1, v/v) and after homogenization (homogenizer H500, Pol-Ekoaparatura, Poland), centrifugation (at 4500× g for 5 min), and filtration (0.20 µm syringe filter—Chromafil, Macherey-Nagel, Duren, Germany) were prepared for chromatographic analysis.
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9

Mycotoxin Extraction and Quantification

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Mycotoxin extraction was performed by adding 5 mL of the extraction solvents (acetonitrile: water, 86:16, v/v) to 0.5 g of dried roots or leaves of the infected and non-infected wheat seedlings, vortexing (for about 30 s) and mixing using a horizontal shaker for 24 h. After extraction, the samples were centrifuged at 7,500 rpm for 10 min. Then, approximately 2 mL of supernatant was filtered through a 0.2 μm syringe filter (Chromafil, Macherey-Nagel, Duren, Germany) and poured into vials for chromatographic analysis. For the analysis, the method reported by Uwineza et al. (2022 (link)) was followed with some modifications (Perczak et al., 2020 (link)). The compounds were quantitatively analyzed using multiple reaction monitoring. The mycotoxin concentrations (μg/g) were calculated using a calibration curve based on commercial single-component preparations of DON, 3- and 15-AcDON, ZEN, and ZEN-14S. All samples were analyzed in triplicate.
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10

Characterization of Synthesized Nanoparticles

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After NPs preparation, the solution was used for UV-Vis, transmission electron microscopy (TEM), single-particle ICP-MS (sp-ICP-MS), dynamic light scattering (DLS), and zeta potential. For Fourier transform infrared spectra (FTIR) and X-ray diffraction (XRD) analysis, the suspension was centrifuged at 5000 rpm for 15 min and filtered (0.22 µm, Chromafil®, Macherey-Nagel, Düren, Germany), and the precipitate was used.
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