Trifluroacetic acid

Nisin A (95%, isolated from Lactococcus lactis in sauerkraut) was obtained from Handary, Belgium. Dextran (from Leuconostoc mesenteroides, 100–200 kDa), Dextran 60 (from L. mesenteroides, ~ 60 kDa), alginic acid (from brown algae, low viscosity (4–12 cps), M/G ratio 1.43 as determined by ¹³C SS-NMR below), sodium (meta) periodate (> 99%), ethylene glycol (99%), N-hydroxysulfosuccinimide sodium salt (S-NHS, > 98%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, crystalline), adipic acid dihydrazide (ADH, > 98%), hydroxylamine hydrochloride, glycol chitosan (degree of deacetylation: > 60%), trifluroacetic acid (TFA, > 99%), acetonitrile (ACN, > 99.9%), phosphate-buffered saline (tablets, PBS), and sodium dodecyl sulphate were all purchased from Sigma-Aldrich, Ireland. Dimethyl sulfoxide (DMSO, for molecular biology) was purchased from VWR International. Liquid nitrogen was supplied by BOC Gases (Ireland). Glycine (lab grade), 2,4,6-trinitrobenzene sulfonic acid (5% w/v, MeOH), potassium chloride (99%) and hydrochloric acid (37%, fuming) were purchased from Fisher Scientific, Ireland. Pullulan/Dextran standards for light scattering/tetra detection (TDS3030) were provided by Particular Sciences, Dublin, Ireland. Staphylococcus aureus (DSM 20231) was purchased from the Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures.

Hyaluronan polyaldehyde was provided by Contipro a.s. 3-(4-hydroxyphenyl)propionic acid (HPA), 2-(N-morpholino)ethanesulfonic acid hydrate (MES), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), gelatine from porcine skin gel (Bloom strength of 300; Type A), dimethylformamide, tetrahydrofuran, ethylacetate, methanol, trifluroacetic acid, and horseradish peroxidase – type I (HRP) were purchased from Sigma Aldrich. Hydrogen peroxide 30% was purchased from Penta s.r.o.

MeOH – methanol, CHCl₃ – chloroform, CH₂Cl₂ – dichloromethane, Na₂SO₄ – sodium sulfate, formic acid, TFA – trifluroacetic acid (Sigma-Aldrich, St. Louis, MO) were used as solvents for the extraction of crude extracts preparation and successive partition of the aqueous phase. DMSO – dimethyl sulfoxide, isopropanol, PBS – phosphate-buffered saline and PI – propidium iodide were used for screening biological activity of ascidians.

For sequencing, protein bands on the gel stained with Simply Blue safe stain (Invitrogen) were sliced and dehydrated with 2:1 mixture of acetonitrile (ACN): 50 mM ammonium bicarbonate (Sigma-Aldrich) and washed with 25 mM ammonium bicarbonate. Reduction and alkylation were done with 10 mM dithiothreitol (Sigma-Aldrich) and 100 mM idoacetamide (Sigma-Aldrich) respectively. Digestion was completed with trypsin (sequencing grade, Promega). Finally, extractions were carried out by increasing grade of extraction buffer having 50, 60 and 80% ACN containing 0.1% trifluroacetic acid (Sigma-Aldrich). After purification using ZIP-Tip, the solutions were loaded into the column (injection volume- 10.0 μL, cap pump flow rate- 2.00 μL/min, nano pump flow rate- 0.30 μL/min) on HR-LCMS (1290 infinity UHPLC and Nano HPLC with 6550 iFunnel Q-TOF, Agilent). Sophisticated Analytical Instrument Facility operated by Indian Institute of Technology, Mumbai, India conducted the protein sequencing analyses.

Phospholipid extraction was performed with modifications to a previously published protocol^{47 (link)}. All extractions were performed under dim red light. Retina homogenates from 8-week-old strain 129/Sv mice or bovine OS were extracted by the addition of 3 ml of 1:2 (vol/vol) mixture of chloroform (Sigma) and acidified methanol (Fisher) (one L methanol + 8 µl trifluroacetic acid (Sigma)) followed by brief vortexing and incubation on ice for 10 min. Next, 1.3 ml of 0.3 M NaCl were added and the samples extracted twice into one ml chloroform with centrifugation at 1750 × g for 10 min at 10 °C to separate phases. The pooled chloroform layers were transferred to 16 × 100 mm borosilicate glass test tubes and evaporated to dryness under a stream of nitrogen. Samples were dissolved in 100 µl of acidified methanol and analyzed by reverse-phase LC^{48 (link)} on an Agilent 1100 series chromatograph equipped with a photodiode-array detector using an Phenomenex Primeshere C18-HC 110 A column (250 × 4.6 mm) and a 15–0% water gradient in acidified methanol (8 µl TFA/L methanol) at a flow rate of 1.0 (gradient) to 2.4 (isocratic) ml per min. Spectra (190–550 nm) were acquired for all eluted peaks. The identity of each eluted peak was established by comparing the spectra and elution times with those of authentic retinoid and N-ret-PE standards. Sample peaks were quantitated by peak area.

Genistein (98%), kaempferol (99%), trifluroacetic acid (99%), and formic acid (98%) were purchased from Sigma, St. Louis, MO. Daidzein (98%) was purchased from Organic Technologies, Coshocton, OH. Dimethyl sulfoxide (DMSO; 99.9%), methanol (99.8%), trifluroacetic acid was purchased from ThermoFisher, Waltman, MA. Deionized (DI) water was used for the preparation of all diets.