2 methyl 2 butanol

Flavor compounds in SFB samples were extracted as described previously (48 (link)) and quantified using a Clarus 500 gas chromatograph (PerkinElmer, Waltham, MA) equipped with flame ionization detectors (FIDs) and a 50-m by 0.25-mm by 0.2-μm CP-Wax 57 CB column (Agilent, Santa Clara, CA) as described previously (28 (link)). The compounds were quantitated by comparison with the internal standards 2-methyl-2-butanol, pentyl acetate, and 2-ethylbutyric acid (Sigma-Aldrich, Shanghai, China).

SCFAs were purchased from Sigma-Aldrich Inc. (St. Louis, MO, United States). SCFAs were dissolved in endotoxin-free distilled water (Dai Han Pharm Co. Ltd., Seoul, South Korea) and filtered with a syringe filter (0.2 μm) purchased from Corning (Corning, NY, United States) prior to use. Luria-Bertani (LB) broth was purchased from LPS solution (Daejeon, South Korea). Trypticase soy broth (TSB) and Bacto agar were purchased from BD Biosciences (Franklin Lakes, NJ, United States). 2,2,2-Tribromoethanol and 2-methyl-2-butanol were purchased from Sigma-Aldrich Inc. Amsacrine (AMSA) was purchased from Abcam (Cambridge, United Kingdom). Hematoxylin and eosin were purchased from Sigma-Aldrich Inc. and BBC Biochemical (Mount Vernon, WA, United States), respectively. Crystal violet and safranin were purchased from Sigma-Aldrich Inc. Iodide solution was purchased from Samchun Chemicals (Seoul, South Korea).

All procedures on mice were conducted at CSSI (Centro Servizi Stabulario Interdipartimentale), approved by the Ethical Committee of University of Modena and Reggio Emilia and by the Italian Ministero della Salute (346/2015-PR) and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C3H/HeN (rd1) mice were purchased from Envigo Italy (Udine, IT). Mice were maintained in 12-h light/dark cycles and had free access to food and water. For intravitreal administration, mice were anesthetized with an intraperitoneal injection of 250 mg/kg body weight of avertin (1.25% (w/v) 2,2,2-tribromoethanol and 2.5% (v/v) 2-methyl-2-butanol; Sigma, Milan, IT). Subsequently, eyes were intravitreously injected via a trans-scleral trans-choroidal approach with 0.5 μl of PEDF recombinant protein (6 pmol) or of PEDF recombinant protein (6 pmol) with 100 μM CLX 3A1 (final intravitreal concentration; AnaSpec) or of 17-mer (6 pmol) or of 17-mer[R99A] (6 pmol) or of 17-mer[H105A] (2 pmol) peptides or of PEDF together with 10-fold molar excess of P1 peptide (a peptide from PEDF-R encompassing the PEDF binding domain)^{11 (link)}. Control eyes received vehicle only (mock injected). Mice were sacrificed 16 h post-injection.

2,2,2-Tribromoethanol (avertin) and 2-Methyl-2-butanol were obtained from Sigma-Aldrich (Missouri, United States). Saline was purchased from JW Pharmaceutical (Seoul, Korea). The animal marker was purchased from Muromachi Kikai Co., Ltd (Tokyo, Japan).

Monobasic and dibasic potassium phosphate, magnesium sulfate, p-nitrophenyl butyrate, 4-(1,1,3,3-tetramethylbutyl) phenyl-polyethylene glycol (Triton X-100), p-nitrophenol, 2-methyl-2-butanol, yeast extract, bacteriological peptone, glucose, agar, olive oil and Tween 80 were purchased from Sigma Aldrich-Fluka (Toluca, Mexico) and local suppliers. The commercial Candida antarctica B lipase (CaLB) from Novozyme was used to validate the first measurements in the SIA system.

C57BL/6J female mice were anesthetized by intraperitoneal administration of a freshly prepared mixture of tribromoethanol solution (25mg/ml, Thermofisher, Belgium). Tribromoethanol was dissolved in 2-methyl-2-butanol (1g/mL, Sigma, MA) and was subsequently diluted to working concentrations in nano-pure H₂O. Mice were randomly assigned to experimental groups of mock infection, PA14 infection or PA14ΔexoU infection. With mice held at an upright position, intranasal inoculation of either sterile HBSS+, PA14 or PA14ΔexoU (45 μl, 1.3e⁷ CFU/mL) was administered by placing 15μl over the nostrils 3X. Subjects were placed in the recovery cage for either 6 or 18 hours. After incubation, mice were re-anesthetized.