Du 7400 spectrophotometer

Spectrophotometric assays were made at 22°C with a Beckman DU 7400 spectrophotometer, monitoring absorbance at 340 nm every 15 s. Assays (100 µl) contained 25 mM Tris-HCl, pH 8.0, 5 mM KCl, 2 mM MgCl₂, 10 µg bovine serum albumin, 40 µM of a purified mixture of NADHX epimers, and 1 mM ADP. Reactions using purified proteins were started by adding 2 µg of either native or K192A YjeF protein. Two micrograms of Arabidopsis NAD(P)HX epimerase domain protein [6 (link)] was added at 4 min. NADHX dehydratase assays of cell lysates were started by adding 25 μl of desalted lysate.

Absorption spectra were measured with a Beckmann DU-7400 spectrophotometer. For spectral analysis, purified oligonucleotides were dialyzed against deionized water Milli-Q. Purification of oligonucleotides with the aid of HPLC was carried out on a Waters HPLC system consisting of aWaters 262 Pump, Waters 2487 UV detector, and Waters 600S Controller with MonoQ HR 5/5 column. Gels were visualized using the Typhoon FLA 7000 bioimaging analyzer, and the radioactivity associated with bands was quantitated with AIDA image analyzer software (Raytest, Germany).

The human vaginal cell line A431 (ATCC, Manassas, VA, USA) was used as an in vitro model of infection with CVEC strains to determine the transcription of virulence genes. This model is frequently used to study host-pathogen interactions [17 (link),18 (link)]. Each E. coli strain was seeded in BHI broth and incubated at 37 °C for 12 h under constant stirring. Following the instructions of the RNAprotect Bacteria Reagent Handbook (Qiagen), each E. coli strain was seeded in BHI broth and incubated at 37 °C for 12 h under constant stirring. The bacterial culture was diluted 1:4 using phosphate-buffered saline and the bacterial concentration was estimated by optical density at 600 nm in a Beckman DU-7400 spectrophotometer (optical density of 600 nm = 1.0 corresponded to 1x10⁹ cells/mL). From this preparations, dilutions were made to obtain a concentration of 2 x 10⁶ cells/mL. In 24-well plates, fifty microliters of the dilution were inoculated on the surface of a 180,000 A431 cell monolayer and incubated with 1 mL of F12K plus 10% bovine foetal serum at 37 °C for 72 h in a 5% CO₂ atmosphere and saturated humidity. After incubation A431 cells reach full confluency with ~250,000 cells. This represents a multiplicity of infection of 8 E. coli per A431 cell. The maintenance medium (F12K plus 10% bovine foetal serum) was changed every 24 h. Each experiment was done in triplicate.

Plasma metabolite data are from Liu and Swanson (2014) (link), who measured plasma concentrations of triglycerides (TRIG), glycerol and β-hydroxybutyrate (BUTY) from the same individual birds for which CORT was measured in the present study. We measured plasma metabolites with commercially available spectrophotometric assay kits (Sigma-Aldrich Corp., St Louis, MO, USA) using a Beckman DU-7400 spectrophotometer (Liu and Swanson, 2014 (link)). We did not obtain sufficient volumes of blood from all individuals for all plasma metabolite and CORT measurements, so we determined the relationships between plasma CORT and plasma metabolites from a subset of individuals, for which we had measurements of both CORT and plasma metabolites.

Ultraviolet (UV) spectra were measured on a DU^® 7400 spectrophotometer (Beckman, München, Germany). ¹H and ¹³C Nuclear magnetic resonance (NMR) spectra were recorded on Ultrashield 400 MHz and 100 MHz spectrometer respectively (Bruker DPX, Faellanden, Germany). Mass spectra were recorded on a mass spectrometer (Thermo Quest Finnigan, San Jose, CA, USA). For in vitro experiments, ultracentrifuge (Sigma, St. Louis, MO, USA), CO₂ incubator (WTC Binder, Tuttlingen, Germany), Biosafety cabinet (Clean air, Chennai, India), autopipettes, ELISA plate reader (Labsystems, Helsinki, Finland) and Neubauer chamber (HBG, Gießen, Germany) were used. Extracts were dried in vacuo using a vacuum rotary evaporator (Buchi R-210, Flawil, Switzerland).

Colloidal stability was spectrophotometrically studied monitoring the turbidity of the nanocarriers (Beckman DU 7400 spectrophotometer) working at 570 nm in simple saline media (pH 7.4). The salts used were NaCl and CaCl₂. From the analysis of aggregation kinetics, we calculated two important parameters in colloidal-stability studies, the critical coagulation concentration (CCC), defined as the minimum salt concentration needed for the most rapid aggregation, and the critical stabilization concentration (CSC) defined as the minimum salt concentration at which the system begins to re-stabilize when salinity is progressively increased and related to the surface hydrophilicity34 .