Antipyrine

In order to investigate placental transport without adversely affecting the integrity of the cell layers, the toxic potential of the NPs was investigated using the MTS assay (see Supplementary Fig. S7). Concentrations of up to 100 µg/ml of 70 nm PS NPs did not decrease the viability of BeWo cell or HPECs after 24 h of treatment. Previous publications showed that also 49 nm PS NPs, antipyrine, indomethacin, Na-F and FITC-dextran (40 kDa) did not affect the viability of BeWo cells^{47 (link),64 (link)}. Therefore reported concentrations were chosen for translocation studies of the model compounds and NPs to allow a comparison to previous results. These were 5 µM Na-F, 5 µM FITC-dextran (40 kDa), 100 µM antipyrine, 100 µM indomethacin, 0.5 mg/ml 49 nm PS NPs and 50 µg/ml 70 nm PS NPs.
Translocation studies were performed with co-cultured membranes as well as separate monolayers (BeWo on the apical or HPEC on the basolateral side of the insert) cultivated for 3 d as described above. Transfer studies with Na-F, FITC-dextran (40 kDa; Sigma-Aldrich, Buchs, Switzerland) and PS NPs were conducted using phenol red-free EM to avoid interference with fluorescence spectroscopic measurements. Na-F, FITC-dextran, 70 nm PS NPs and 49 nm PS NPs were added to the apical chamber. At each time point (0, 0.25, 2, 4, 6, 8, 24 h) samples (50 µl) were taken from the basolateral chamber and renewed with fresh medium. Fluorescent signals of the samples were measured at an excitation of 485 nm and an emission of 528 nm using a microplate reader (Mithras² LB 943, Berthold Technologies GmbH, Zug, Switzerland). Translocation of antipyrine (Sigma-Aldrich, Buchs, Switzerland) and indomethacin (Sigma-Aldrich, Buchs, Switzerland) was investigated applying the same experimental conditions. Only differences were the use of EM with phenol red and sample volumes of 200 µl (basolateral) at each time point (0, 0.25, 1, 2, 6 h). Analysis of these samples was done by high performance liquid chromatography (HPLC). These studies were conducted in static and shaken (horizontal shaking at 50 rpm) conditions to determine the potential influence of simple shaking on transfer rates.
The mass transported (ΔQ_n) was calculated for each time point and corrected for the mass taken before: $$\mathrm{\Delta }Qn=C_n\ast V_w+\sum _{j=1}^{n-1}{V}_s\ast C_j$$ with the concentration measured at time t_n (C_n), the volume of the well (V_w, 1.5 ml) and the sample volume (V_s). The sum of the amount removed during previous sampling is added respectively (Σ). Results were then expressed as basolateral amount of the initial dose (ID) in %. Equilibrium between the apical and basolateral chamber would be reached if 75% of the ID would pass the barrier and would be found in the basolateral chamber (V_apical 0.5 ml, V_basolateral 1.5 ml). The permeability factor was calculated with the following two equations: $$P=\frac{\mathrm{\Delta }Q/\mathrm{\Delta }t}{A\ast C_0}$$ $$P_e=\frac{1}{\left(\frac{1}{P_c}-\frac{1}{P_m}\right)}$$ where the permeability factor (P; cm s⁻¹) is first calculated as the quotient of the amount transported (ΔQ; mg) at a specific time point (Δt; sec) divided by the product of the membrane surface area (cm²) and the initial concentration of the substance (C_0; mg cm⁻³). P_e describes the apparent permeability factor corrected for the influence of the membrane. Therefore, the permeability value across the membrane P_m was subtracted from the permeability across the cells (P_c; monolayer or co-culture).

The commercial trypanocidal drug used was diminazine aceturate (Veriben® containing 1.05 g diminazine aceturate + 2.36 g antipyrine, (Ceva Santé Animale, France; batch number- 719A1).

Multiple covariates were involved in this study including gender, age, education level, geographical location, income level, smoking history, alcohol drinking status, physical activity (PA), urbanization levels, body mass index (BMI), total energy intake, dietary protein, dietary fat, dietary carbohydrate, and dietary sodium. Age was divided into three groups (18–44 years, 50–59 years, and 60 years and above). Education level was divided into two groups (junior high school or below and senior high school or above). Residence was separated into two groups (urban and rural areas). In view of the differences between the north and the south, regions were divided into the north (Beijing, Liaoning, Heilongjiang, Shandong, Henan, and Shaanxi) and the south (Shanghai, Jiangsu, Zhejiang, Hubei, Hunan, Guangxi, Chongqing, Guizhou, and Yunnan). Annual per capita household income was divided into three groups (low, medium, and high by the tertiles). Smoking history and drinking past year were divided into two groups (yes and no), respectively. Physical activities included occupational, household chores, leisure time, and transportation, and calculated into a metabolic equivalent of task (METs h/week) based on the American College of Sports Medicine Association’s recommended standard, and were then divided into three groups (low, medium, and high by the tertiles) [36 (link)]. Urbanization levels were calculated based on the economic environment of the community and the cultural and social environment and divided into three groups (low, medium, and high by the tertiles). BMI was calculated as body weight (kg) divided by the square of height (m²) and divided into three groups (<18.5 kg/m², 18.5–23.9 kg/m², and ≥24.0 kg/m²).
WC was measured using an inelastic flexible ruler, and weight and height were measured using an electronic weight scale and portable SECA206 stadiometer. Cholesterol oxidase-phenol and amino phenazone methods were used to measure TG and HDL-C. Blood pressure was measured using a standard mercury sphygmomanometer (Korotkoff sound). The participants were in a seated position in a quiet room for at least five minutes of rest and with the bladder emptied. The average value of three consecutive standard measurements was taken as the result for each participant. Fasting plasma glucose was measured using the hexokinase method with a Roche 702 instrument. All measurements were performed by trained professional technicians with strict quality control.

Referring to the one variable at a time (OVAT) technique, all factors were held constant in these experiments, with only the targeted variable being changed to optimize process variables. Briefly, the optical density of prepared cultures of each isolate, S3, S10, and S18, were adjusted at (1.0) at a wavelength of 600 nm. The inocula (2%) were inoculated into the phenol (1 g/L)-MSM medium as the only carbon source. The bacterial growth was monitored at 0, 1, 2, 3, and 4 days. Additionally, phenol degradation was recorded after four days of incubation in a shaking incubator (120 rpm) at different conditions of temperatures (25, 30, 35, and 40 °C), pH values (6, 7, and 8), and different nitrogen sources in growth medium MSM that contain peptone, yeast extract, urea extract, NaNO₃, KNO₃, and NH₄Cl at a concentration of 2 g/L. Buffer solutions with different pH ranges, i.e., acetate buffer for pH 6.0, phosphate buffer for pH 6.5, 7.0, and 7.5, and Tris-HCl buffer for pH 9 were used to achieve different pH values [31 (link)].
The phenol concentrations were measured at 520 nm using 4-amino antipyrine following American Public Health Association-recommended procedures [6 ]. In summary, 0.3 mL of 2% aqueous 4-amino antipyrine solution and 1 mL of 2 N NH₄OH were added to a 5 mL sample. After adequately mixing the contents, 1 mL of 2% K₃FeCN₆ is added. At pH 10, the OH group reacts with 4-amino antipyrine and K₄Fe(CN)₆ to produce a purple-red color. Quantitative analysis of phenol degradation was conducted by measuring the phenol content in the supernatant of each sample and calculating degradation using the following equation

The phenol content was determined by comparing the absorbance at 520 nm to the phenol standard curve. Where A is the initial concentration of phenol (Zero h. sample), and B is the phenol concentration in the sample taken at various time intervals.