Chromates

Plasma Alarin and Adipsin levels were examined using the Human Alarin, Adipsin ELISA Kit (Sunred Biological Technology, Shanghai, China) in a plate-washing -incubation CombiWash device (Human Diagnostics, Wiesbaden, Germany) in accordance with the study procedures determined in the kit catalogue, and the absorbance measurement was taken with a Chromate 4300 Microplate Reader (Awareness Technology, Palm City, USA).
Aqueous analyzes were performed according to a previously published methods [15 (link)]. Two Aqueous liquids and blood samples were enriched with increasing amounts of Adipsin or Alarin. The percentage recovery was calculated as follows: recovered value/expected value × 100.
The measurement range human alarin kit was 5 to 1500 pg/mL and the sensitivity was determined by the manufacturer at 4.638 pg/mL. The intra-assay and inter-assay coefficients of variation for alarin were < 10% and < 12%, respectively. The measurement range of the human Adipsin kit 0.5 to 100 ng/mL and the sensitivity was the determined by the manufacturer at 0.472 ng/mL. The intra-assay and inter-assay coefficients of variation for Alarin were < 10% and < 12%, respectively.

A putative promoter region was searched upstream of the start codon of chrA_SO using the BPROM Softberry online software (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb). A DNA fragment corresponding to the 400 bp upstream from the ATG was amplified by PCR using S. oneidensis MR1-R chromosomal DNA, digested with XbaI and SpeI, and ligated upstream of the lacZ gene of vector pACYC184-lacZ, which was previously constructed by cloning the β-galactosidase-encoding gene lacZ from pGE593 into the vector pACYC184 (S1 Table) [30 (link),31 (link)]. The ligation product was transformed into E. coli strain MC1061. The resulting plasmid called pchrA_SO::lacZ was introduced into S. oneidensis MR1-R by conjugation. Plasmid construction was checked by DNA sequencing. As a control, another fusion, named pmxd₄₅₀::lacZ, was similarly constructed using a DNA fragment corresponding to the 450 bp upstream from the ATG of mxdA [32 (link)]. S. oneidensis MR1-R strain containing pchrA_SO::lacZ or pmxd₄₅₀::lacZ was grown overnight on LB-agar plate. The overnight culture was diluted to an OD_600nm = 0.1 in fresh liquid LB medium containing various concentrations of chromate (0, 0.05, 0.1 and 0.2 mM) and incubated in semi-aerobiosis at 28°C for 16 hours prior to β-galactosidase activity quantification. The β-galactosidase activity was determined by a Miller assay adapted for use with plate reader [33 ]. Briefly, cells were transferred into a microtiter plate and OD_600nm was measured using a Tecan Spark 10M microplate reader. Cells were then lysed using lysozyme and PopCulture reagent (Sigma-Aldrich) prior to incubation with Z buffer (62 mM Na₂HPO₄, 45 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM β-mercaptoethanol). O-nitrophenyl-ß-D-galactopyranoside (ONPG) was added to the mix before kinetic quantifications of OD_420nm. Slope of the obtained curves were calculated and β-galactosidase activity (arbitrary units) was determined by the following equation:
$\left(1000\times slope\right)/\left({OD}_{600nm}\times volumeofreaction\left(\mu L\right)\right).$

As for the collection of blood samples, antecubital venous blood samples (approximately 10 mL) were obtained from all patients included in this study on the morning of the operation day. The blood samples collected from each participant were put into ethylenediamine tetraacetic (EDTA) tubes with added aprotinin and taken to the laboratory within 30 min. The blood samples taken for the measurement of afamin level were centrifuged in Nüve (Nüve Sanayi Malzemeleri İmalat ve Ticaret A.Ş, Ankara, Turkey) NF800 centrifuge devices at 4000 rpm for 10 min, and following the separation of the serum part, the serum samples were transferred to Eppendorf tubes and stored at −80 °C until the study day. Insulin (fasting), HgbA1c, TSH, T4, total cholesterol, triglyceride, LDL, HDL, and VLDL serum values within the scope of this study were taken during the preoperative evaluation one week before the surgery. Antecubital venous samples (approximately 25 mL) were collected at 6 months postoperatively while the patients had an empty stomach. The serum part of the blood samples collected for the measurement of the afamin level was separated and stored at −80 °C. Insulin (fasting), HgbA1c, TSH, T4, total cholesterol, triglyceride, LDL, HDL, and VLDL values were evaluated routinely in the hospital laboratory. Afamin levels were measured by the enzyme-linked immunosorbent assay (ELISA) method. Afamin was stored at room temperature for about half an hour with Human Afamin ELISA kits manufactured by AFG Scientific (catalog number EK 714650, AFG Bioscience LLC, USA). After washing with an Awareness brand Stat Fax 2600 device, afamin levels were evaluated with an Awareness brand ChroMate 4300 device and immunoassay 450 wavelength method. The values were measured according to the manufacturer’s instructions.

Supernatants collected from platelets treated with LL-37 or the control condition (Section 4.2) and the secretion of BPI, MPO, HNP1 (R & D System), and Azu (Sino Biological, Beijing, China) were quantified by ELISA (enzyme-linked immunoadsorbent assay) using an optical density. A capture antibody was used for each antimicrobial molecule and incubated overnight. Subsequently, blocking was performed using bovine albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) for one hour. Next, 100 µL of the sample was placed in each well per condition and left to incubate for two hours. Afterwards, the wells were washed with PBS buffer (Phosphate buffered saline) three times, and a detection antibody was added, followed by a secondary antibody coupled to HRP (rabbit peroxidase), according to the instructions from each supplier of the ELISA Kits. Readings were performed at 450 nm using a microplate reader (Model 4300, Awareness Technology Inc. Chromate, Palm City, FL, USA) with the Leica Suite 345 X software application (LAS-X, Leica-Microsystems, Wetzlar, Germany).