Biosan

For quantification of the free zinc concentration with the low molecular weight fluorescent probe Zinpyr-1 the formula by Grynkiewicz et al. is applied [39 (link)]: $\left[Z{n}^{2+}\right]=K_D·\frac{F-F_{min}}{F_{max}-F}$
Herein, K_D indicates the dissociation constant of the zinc:Zinpyr-1 complex of 0.7 nM [18 (link)], F_min the autofluorescence of the probe in the absence of zinc, F the fluorescence of the probe in the sample, and F_max represents the maximum fluorescence of the zinc-saturated probe. Notably, the dilution of the serum in the assay is not relevant for determining the free zinc concentration by this method, as outlined in detail in supplemental information SI 1.
In order to minimize the required sample volume, all fluorescence parameters were measured sequentially in the same well, starting with F, followed by the addition of a chelator to yield F_min, and finally addition of excess zinc sulfate to reach F_max. Incubations were carried out using a thermo-shaker (PST-60-HL4, Biosan, Riga, Latvia) at 37 °C and 250 rpm. Fluorescence measurements were performed at 37 °C at an excitation wavelength of 507 nm, emission wavelength of 526 nm, and bandwidths of 5 nm using a SPARK fluorescence plate reader (Tecan, Männedorf, Switzerland).
Assay-buffer (50 mM HEPES in bidistilled water adjusted to pH 6.5 with sodium hydroxide solution), was depleted of multivalent cations with Chelex^® 100 Resin according to the manufacturer’s protocol. This process increases the pH of the assay-buffer to a final value of 7.5. Measurements were performed in a volume of 100 µL of a 1:50 dilution in assay buffer in the wells of a 96-well-plate (Sarstedt, Nümbrecht, Germany). Samples were analyzed in triplicates, leading to a net sample requirement of 6 µL. Outer wells were not used for measurements, but filled with buffer, leaving a capacity of 60 wells, or 20 samples, per 96 well-plate.
Based on the results of the optimization outlined below the assay was finally performed with Zinpyr-1 (final concentration 0.05 µM) dissolved in 98 µL assay-buffer, equilibrated for 20 min before incubation was started by addition of 2 µL HS. Incubation times for F, F_min, and F_max were 90, 20, and 90 min, respectively. For determination of F_min a final concentration of 100 µM of EDTA, and for F_max 500 µM zinc sulfate was applied. Serum samples were routinely stored at –21 °C. Results remained unchanged by five freeze/thaw cycles, indicating that this is without effect on the determined free zinc concentration (data not shown).

The DNA substrates were prepared by annealing the oligonucleotides (Fig. 1b; Supplementary Table S10) purchased from Integrated DNA Technologies in 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA and 100 mM NaCl as described37 (link). In the electrophoretic mobility shift assay (EMSA) the proteins were incubated with 1 μM oligonucleotides in a binding buffer (50 mM Hepes, 0.1 mM EDTA, 300 mM NaCl, 10% glycerol (V/V), 0.1 mM TCEP) for 30 minutes in an ice bath. The mixtures were resolved on 0.5% agarose gels in 0.5X Tris/Borate/EDTA buffer at +4 °C degrees, 80 volts for 80 minutes. The gels were stained using GelRed (Biotium) and visualized using a gel imaging system (Bio-Rad).
The fluorescence polarization assay (FP) was done in black 96-well U-bottom propylene plates (Greiner BioOne). Notably, the concentration of the protein samples were quantified prior to the measurements using calculated extinction coefficients and absorbance at 280 nm. The DNA amount was determined by the supplier (Integrated DNA Technologies) and the measured absorbance at 260 nm was found to agree with the calculated concentration. The reaction was carried out in 10 mM Hepes pH 8.0, 0.1 mM TCEP, 0.1 mM EDTA, 150 mM NaCl and 10% glycerol. DNA oligonucleotides 36–42 (Fig. 1b) were used. Specifically, 5 nM DNA was used and the protein concentration was titrated from 0–4 μM for ARTD2_FL, and from 0–8 μM for the other constructs (Fig. 1a). The plates were incubated at 25 °C with shaking (300 rpm) in a PST-60 HL Plus shaker (Biosan) for 60 minutes. The fluorescence polarization was measured using a Tecan Infinite M1000 at the excitation and emission wavelengths of 475 and 520 nM, respectively. The experiment was done in triplicate and the measurements were fitted using Graphpad Prism version 5.04 for windows (GraphPad Software). Stoichiometry of ARTD2_FL DNA binding was measured with FP as described by Updegrove et al.38 (link). We used DNA concentrations of 250 nM, which is significantly higher than the measured K_D. The protein was titrated from 0–4 μM.

rhEPO produced in Chinese hamster ovary (CHO) cell lines was provided
by the European Pharmacopoeia as a chemical reference substance (CRS-batch
1). Each sample vial contained 100 μg of rhEPO (EPO-CRS; a mixture
of epoetin alpha and beta), 0.1 mg of Tween-20, 30 mg of trehalose,
3 mg of arginine, 4.5 mg of NaCl, and 3.5 mg of Na₂HPO₄. The content of each vial was dissolved in water to obtain
a 1000 mg·L^–1 solution of rhEPO. Two rhEPOs
produced in CHO cell lines were provided by the Center of Molecular
Immunology (Havana, Cuba): EPOCIM (batch 1) and NeuroEPO plus (batch
1). EPOCIM vials contained 963 mg·L^–1 rhEPO
and 0.02% (m/v) Tween-20 in citrate buffer at a pH of 6.9. NeuroEPO
plus vials contained 1090 mg·L^–1 rhEPO and
0.02% (m/v) Tween-20 in phosphate buffer at a pH of 6.3. Excipients
of low molecular mass were removed from rhEPO samples by centrifugal
filtration using Microcon-10 kDa centrifugal filters (Millipore, Molsheim,
France) as described in a previous work.^{7 (link)} Samples were centrifuged at room temperature in a Mikro 20 centrifuge
(Hettich, Tuttligen, Germany). The filter membrane was initially washed
with water at 13,000 g for 10 min. Then, the sample was centrifuged,
and the residue was washed three times with an appropriate volume
of water under the same centrifugal conditions. Finally, the residue
was recovered from the upper reservoir by centrifugation upside down
into a new vial (3 min at 1000 g), and sufficient water was added
to adjust rhEPO concentration to 1000 mg·L^–1. Aliquots were evaporated to dryness in a Savant SPD-111V SpeedVac
concentrator (Thermo-Fisher Scientific, Waltham, MA, USA) and stored
at −20 °C until enzymatic digestion.
rhEPO samples
were first reduced and alkylated to facilitate digestion. Briefly,
an aliquot of 50 μg of dried glycoprotein was dissolved in 50
μL of digestion buffer (50 mM NH₄HCO₃,
pH 7.9), and 2.5 μL of 0.5 M DTT in digestion buffer was added.
The mixture was incubated in a thermoshaker at 56 °C for 30 min.
Then, alkylation was carried out by adding 7 μL of 50 mM IAA
in digestion buffer and shaking for 30 min at room temperature in
the dark. Low molecular mass reagents were removed using Microcon
YM-10 centrifugal filters (Millipore) as described above. The final
glycoprotein residue was dissolved in digestion buffer to obtain a
final concentration of 1000 mg·L^–1. Aliquots
of 50 μL of reduced and alkylated rhEPO solution were digested
in an enzyme to a protein ratio of 1:40 (m/m) and incubated at 37
°C for 18 h (trypsin digestion) and then to a protein ratio of
1:20 (m/m) and incubated at 25 °C for 18 h (Glu-C digestion).
Digestions were stopped by heating at 100 °C for 10 min, and
samples were dried in a SpeedVac before storage at −20 °C
until analysis.^{7 (link)} Incubations were performed
in a TS-100 thermoshaker (Biosan, Riga, Latvian Republic). pH measurements
were carried out using a Crison 2002 potentiometer and a Crison electrode
52-03 (Crison instruments, Barcelona, Spain).

The quantitative evaluation was performed by means of the minimum inhibitory concentration (MIC) method for the same eight standard microbial strands. The method was performed in accordance with the EUCAST protocols [36 (link)], with slight modifications. 96-wells titer plates, containing the extracts diluted in liquid MH medium and inoculated with 20 µL microbial suspension, were used. Extract stock solutions were diluted using a two-fold serial dilution system in ten consecutive wells, from the initial concentration (1/1) to the highest (1/512). The total broth volume was brought to 200 µL. Microbial inoculum in MH broth as positive control and microbial inoculum in 30% ethanol as negative control were prepared and placed in wells 11 and 12, respectively. For bacteria, the plates were incubated at 37 °C for 24 h, and at 28 °C for 48 h for Candida. MIC values were determined as the lowest concentration of the extracts’ dilution that inhibited the growth of the microbial cultures (having the same OD as the negative control), compared to the positive control, as established by a decreased value of absorbance at 450 nm (HiPo MPP-96, Biosan, Latvia). MIC50 was determined as well, representing the MIC value at which ≥50% of the bacterial/yeast cells were inhibited in their growth, considered as the well with the OD value similar to the average between the positive and negative control.

In order to determine the cytotoxic activity of olive bud essential oils and extracts, a cell viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) was performed on three cell lines: human breast adenocarcinoma (MDA-MB-231), human breast metastatic adenocarcinoma (MCF-7), and human ovarian carcinoma (OVCAR-3) cell line (LGC Standards) [38 (link),39 (link)]. MDA-MB-231, MCF-7, and OVCAR-3 cell lines were incubated overnight in 96-well plates at a density of 9000 cells/well for MDA-MB-231 and MCF-7 and 6000 cells/well for OVCAR-3 followed by incubation with test extracts at concentrations in the range of 1–200 µg/mL for 24 h, 48 h, and 72 h (in triplicate). Afterward, cells were incubated with 0.5 g MTT/L at 37 °C for 2 h; the medium was removed, and 10% dimethylsulfoxide (DMSO) was added for another 10 min at 37 °C. The indicator of metabolically active cells, formazan, was formed and measured at 570 nm using a microplate reader (BioSan, Riga, Latvia). The half maximal inhibitory concentration (IC₅₀) value is a quantitative measure that indicates how much of a particular inhibitory substance is needed to inhibit, in vitro, a given biological process or component by 50%. We performed its calculation with Microsoft Excel 2016 with data normalization by the measurements of untreated controls. To determine the differences between tested concentrations, analysis of variance (one-way ANOVA) was performed using Past 3.X software (version 3.14, University of Oslo, Oslo, Norway), with the significance level at p < 0.05.