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2.1. Materials

Antimony with a purity of 99% (obtained from Stanchem Sp. z o.o. (Niemce, Poland)) and potassium manganate(VII) with a purity of 99% (obtained from STANDARD P.P.H. (Lublin, Poland)) were used to produce pyrotechnic compositions. The grain analysis of both components was performed using a FRITSCH ANALYSETTE 22 MicroTec plus laser particle size analyzer. The samples were dosed into the measuring cells by means of a vacuum dosing unit with the application of ultrasound in order to break up the agglomerates of particles. Volume-based particle size distributions were determined on the basis of laser beam diffraction. Measurements were made in the range of 0.08–2000 µm. The detected grains were divided into 102 classes in terms of their diameter (n = 102). The beam obscuration value was 8.0%.

1. Introduction

Two dominant peaks are visible on the DTA curve for the Sb/KMnO4 composition (with 60 wt% antimony content) heated in the air atmosphere []. The first one is the exothermic decomposition of KMnO4, which takes place at a temperature of about 300 °C. Another sharp exotherm (ignition of the composition), occurring at a temperature of about 500 °C, comes from the oxidation of Sb with oxygen from the air (as evidenced by the rapid increase in the mass of the sample in the TG curve). An important observation of the authors of Ref. [] is the finding of a wide, exothermic peak on the DTA curve without changing the mass of the sample above the temperature of 500 °C during the test with inert gas (nitrogen). This fact is the basis for a hypothesis that, in such conditions, secondary reactions can take place in the system. On the basis of the area under the DTA curve, the total heat of the reaction for the tested composition in the air atmosphere was determined at ∆H = 2.11 kJ g−1. The total thermal effect of the reaction in a nitrogen atmosphere was not determined in Ref. [].

1. Introduction

Based on the results of the thermal analysis presented in the literature, the combustion products should be expected to contain compounds derived from the thermal decomposition of potassium manganate(VII) and the products of antimony oxidation by the released oxygen. The occurrence of secondary reactions between the products of KMnO4 decomposition and the oxidation products of Sb is not confirmed. However, this possibility should not be rejected. In Ref. [], on the basis of the DTA and TG curves, the two-stage decomposition of KMnO4 was confirmed and the reactions reported in the literature for the first stage of the thermal decomposition of KMnO4 at approx. 290 °C and, for the second stage, at 620 °C were quoted. The solid products of KMnO4 decomposition can be: K2MnO4, K3MnO4 and solid solutions of K2O and MnO2. According to the authors of Ref. [], Sb2O3 and Sb2O4 are the most likely products of antimony oxidation during thermal analysis conducted in the air atmosphere. The first one occurs between 300 and 500 °C, the second one at 600 °C.

1. Introduction

Two dominant peaks are visible on the DTA curve for the Sb/KMnO4 composition (with 60 wt% antimony content) heated in the air atmosphere []. The first one is the exothermic decomposition of KMnO4, which takes place at a temperature of about 300 °C. Another sharp exotherm (ignition of the composition), occurring at a temperature of about 500 °C, comes from the oxidation of Sb with oxygen from the air (as evidenced by the rapid increase in the mass of the sample in the TG curve). An important observation of the authors of Ref. [] is the finding of a wide, exothermic peak on the DTA curve without changing the mass of the sample above the temperature of 500 °C during the test with inert gas (nitrogen). This fact is the basis for a hypothesis that, in such conditions, secondary reactions can take place in the system. On the basis of the area under the DTA curve, the total heat of the reaction for the tested composition in the air atmosphere was determined at ∆H = 2.11 kJ g−1. The total thermal effect of the reaction in a nitrogen atmosphere was not determined in Ref. [].

2.5. Heat of Combustion

Combustion heat measurements were made using the KL-12 calorimeter produced by Precyzja Bit from Poland. A cylindrical steel calorimetric bomb with a capacity of 350 mL was placed in a steel vessel containing distilled water. The calorimetric system has been described in detail in []. Pellets of Sb/KMnO4 compositions weighing from 4 to 9 g were pressed. The diameter of the pellets was 15 mm. A resistance wire made of the Kanthal D alloy (69% Fe, 22% Cr, 6% Al, 3% Zn and Si) was pressed into each pellet. The wire was 20 cm long, 2 mm in diameter and had a mass of 0.045 g. The resistance of the wire was 42.88 Ω m−1. The part of the wire that was pressed into the pellet was coiled into a spiral to increase its contact surface with the composition. The wire did not burn down during the tests but was broken due to the rapid increase of temperature. The heat released as a result of glow-up of the wire was not taken into account in determining the heat of combustion of the samples. The samples were placed in 4 mL quartz crucibles. Combustion heat measurements were made in an atmosphere of inert gas (argon) at various initial pressures.

2.5. Changes in Soluble Sugar Concentration and PgSWEETs Expression Pattern of Pomegranate Fruit and Leaf after Tripotassium Phosphate Treatment

When the pomegranate fruit enters the color turning period (4–5 weeks before harvest), the growth of the fruit and the accumulation of sugar require nutrients, and the demand for fertilizer increases at this stage, which is the most demanding stage in the whole growth process. At this time, in addition to basic and reasonable fertilization, proper foliar application can supplement sufficient nutrients to improve fruit growth, increase sweetness, and enable successful coloring. Therefore, we started foliar spraying of “Big Seed” pomegranate with tripotassium phosphate solution from September 20 (pomegranate fruit color change phase) to 10 October. Compared with the control group, the results showed that fruit TSS/TA, fruit and leaf total soluble sugar contents were significantly enhanced after foliar spraying with tripotassium phosphate (A–C).

2.5. Changes in Soluble Sugar Concentration and PgSWEETs Expression Pattern of Pomegranate Fruit and Leaf after Tripotassium Phosphate Treatment

To investigate the effect of foliar application of tripotassium phosphate on the fruit sugar transporter, the transcript levels of 16 PgSWEET genes were determined (D). The expression of SWEETs in pomegranate fruits was generally low from the color turning stage to the ripening stage, and the expression of the same gene varied greatly at different times. Overall, the expression of SWEETs in fruits was relatively high on 20 September, and the expression of nine genes (PgSWEET1c, 3, 5, 7a, 7b, 9, 11, 15 and 17a) was the highest compared with other periods. Foliar application of tripotassium phosphate also altered the expression pattern of SWEETs in fruits, with the highest expression of PgSWEET2 and 16b in the treatment group on 30 September and the highest expression of PgSWEET1e and 16a in the treatment group on 10 October. In addition, foliar fertilization decreased the expression of PgSWEET10 compared to the control group.

Abstract

The kinetics and mechanism of ternesite formation (calcium sulfosilicate, Ca5(SiO4)2SO4, C5S2$) were investigated by studying the reaction between beta-dicalcium silicate (β-C2S) and calcium sulfate dihydrate (CaSO4∙2H2O). Mineralogical composition development was monitored using X-ray diffraction (XRD) and backscattered scanning electron microscopy (BSEM) coupled to energy-dispersive X-ray spectroscopy (EDS). Ternesite can form in the 1100 to 1200 °C range by the solid-phase reaction of β-C2S and CaSO4. The formation of ternesite is favored by increasing the sintering temperature or extending the sintering time. The solid-phase reaction is carried out by diffusion of CaSO4 to β-C2S. The kinetics equation of ternesite is consistent with three-dimensional diffusion models (3-D model, D3 model or Jander model). The equation of the D3 model is 1 − 2α/3 − (1 − α)2/3 = kt. On the basis of the Arrhenius equation, the activation energy of ternesite is 239.8 kJ/mol.

1. Introduction

However, the sintering mechanism and formation kinetics of ternesite have not been systematically studied. In this paper, the formation of ternesite from β-C2S and CaSO4∙2H2O was investigated in detail through isothermal treatment by mixing synthetic β-C2S and CaSO4∙2H2O from 1100 to 1200 °C.

2.1. Raw Materials and Samples Preparation

Then, the sieved C2S and CaSO4∙2H2O were proportioned in a molar ratio of 2:1 and mixed well. The mixture was pressed into disks. The disks were heated to 1100, 1125, 1150, 1175, and 1200 °C. At every temperature, the disks were sintered for 0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 10, or 12 h. Then, the sintered disks were removed from the resistance furnace and cooled immediately.

Abstract

The success and longevity of a resin composite restoration is determined by its good bonding to the tooth structure, with the adhesion being a challenge to dentin due to its complexity and structural heterogeneity. The present study aimed to compare the adhesive strength of dentin conditioned with 18% ethylenediaminetetraacetic acid (EDTA) versus 35% phosphoric acid (H3PO4) in human premolars. Materials and Methods: This in vitro experimental study was performed on 40 human premolars. The occlusal thirds were sectioned and randomly placed into four groups according to the type of dentin conditioning: Group 1 (control), Group 2 (18% EDTA), Group 3 (35% H3PO4) and Group 4 (18% EDTA plus 35% H3PO4). Then, 10,000 thermocycles between 5 +/− 2 °C and 55 +/− 2 °C were applied. Adhesive strength was tested by shearing with a digital universal testing machine at a crosshead speed of 0.75 mm/min. The values obtained were analyzed in megapascals (MPa). The mean and standard deviation were used as measures of central tendency and dispersion. In addition, a one-factor intergroup ANOVA test was applied with Tukey’s post hoc test considering a significance level of p < 0.05. Results: The 18% EDTA and 18% EDTA plus 35% H3PO4 showed significantly higher adhesive strength compared to the control group that did not receive dentin conditioning (p = 0.047 and p < 0.001, respectively). However, the group conditioned with 18% EDTA did not present significant differences compared to the group conditioned with 35% H3PO4 (p = 0.997). In addition, the group conditioned with 18% EDTA plus 35% H3PO4 showed significantly higher adhesive strength compared to the groups conditioned with 18% EDTA (p = 0.002) and 35% H3PO4 (p = 0.001). Conclusion: The adhesion of bulk fill resin composite to dentin was favorable when preconditioning was performed using 18% EDTA followed by 35% H3PO4. In contrast, when both etchants were used separately, the bulk fill resin composite showed similar bond strength values in both cases, but significantly lower compared to their sequential application.

2.4. Dentin Conditioning and Resin Composite Bonding

The procedure for each group was as follows ():Group 1: No dentin etching. Only rinsed in water for 5 s, and excess moisture was dried with sterile gauze. Then, a layer of Tetric® N-Bond adhesive was placed with a microbrush, and air was gently applied for 5 s. Finally, a block of Tetric® N-Ceram Bulk-Fill resin composite color A2 was placed and light cured perpendicularly for 10 s at a maximum distance of 1 mm to the upper surface with a Bluephase N LED (Ivoclar Vivadent AG, Schaan, Liechtenstein) at an intensity of 1200 mW/cm2 for 10 s. (). The light intensity of curing unit was previously verified using a radiometer (Bluephase Meter II, Ivoclar Vivadent AG, Schaan, Liechtenstein).Group 2: Dentin etching was performed with 18% EDTA gel for 90 s. Then it was washed with water for 10 s, and the excess moisture was dried with sterile gauze. Then, the adhesive was applied, and the resin composite was placed using the same procedure as group 1 [].Group 3: Dentin etching was performed with 35% H3PO4 gel for 15 s. Then, it was washed with water for 10 s, and the excess moisture was dried with sterile gauze. Then, the adhesive was applied, and the resin composite was placed using the same procedure as groups 1 and 2.Group 4: Dentin etching was performed with 18% EDTA gel for 90 s. Then, it was washed with water for 10 s, and the excess moisture was dried with sterile gauze. Then, 35% H3PO4 gel was applied for 15 s. Then, it was again washed with water, and excess moisture was dried with sterile gauze. Then, the adhesive was applied, and the resin composite was placed using the same procedure as in groups 1, 2 and 3.

Abstract

Methods. Eighty-nine nonvital premolar and molar teeth with periapical lesions (PAI: 3‒4) without any clinical symptoms were included. The patients were randomly assigned to three groups according to the final irrigation solutions used: G1: 2 mL of Qmix (n=29), G2: 2 mL of 17% ethylenediaminetetraacetic acid (EDTA) (n=30), and G3: 2 mL of 2% chlorhexidine gluconate (CHX) (n=30). All the patients were prescribed 100 mg of flurbiprofen to use as needed for pain. The patients were asked to rate their pain status according to the verbal rating scale at 12, 24, 48, and 72 hours, and one week. The data were analyzed using Pearson’s chi-squared test, Fisher’s exact test, and chi-squared analysis with Monte Carlo simulation. The significance level was set at P≤0.05.

Abstract

Results. No significant differences were observed in postoperative pain rates at 12, 48, and 72 hours and one week (P>0.05). However, in the Qmix group, a significantly lower pain level was observed at 24 hours with EDTA and CHX (P=0.019). The rate of mild pain in the EDTA group at 72 hours (18.8%) was significantly higher in premolar teeth than in molar teeth (P=0.012). The moderate pain level in the EDTA group at 12 hours was significantly higher in those>60 years of age (P=0.008).

Patient selection

The method and design of this prospective, randomized, double-blind clinical trial were approved by the Ethics Board of the University (No: 929). The study protocol was published and registered at www.clinicaltrials.gov (identifier: ). Before inclusion, all the patients were informed of the study protocol, aims, complications, and possible risks, and written informed consent was obtained from each patient. Patients who met the inclusion criteria were treated by a single endodontist (SIY) from the Faculty of Dentistry, Department of Endodontics, between October 2019 and February 2020.

Root canal treatment procedures

Group 2 (EDTA, n = 30): After applying 2 mL of saline solution to each root canal for 30 sec, 2 mL of 17% EDTA was applied for 60 seconds.

Abstract

This study aimed to determine the effects of dietary lanthanum chloride on the growth and health performance of juvenile Clarias gariepinus when fed diets experimentally contaminated with mixtures of aflatoxin B1 and fumonisin B1. A control diet, (mycotoxin free, diet A), mycotoxin contaminated (diet B), and two mycotoxin-contaminated diets amended with lanthanum chloride (200 mg/kg, diet C; and 400 mg/kg, diet D), were fed to 450 fish divided equally into five groups (each with three replicates) for 56 days. The fish were randomly sampled at the time points: day 7, 28 and day 56 for the zootechnical, hematological and serum biochemical evaluations. The fish fed the diets amended with lanthanum chloride exhibited significantly (p < 0.05) better performance indices compared with the fish fed only the mycotoxin-contaminated diet. Lanthanum chloride elicited significant (p < 0.05) increases in erythrocytes and leucocytes count and significant (p < 0.05) reduction in serum transaminase, alkaline phosphatase, lactate dehydrogenase activities, urea and uric acid concentrations in the fish fed the diets contaminated with mixtures of aflatoxin B1 and fumonisin B1. The study indicates that juvenile Clarias gariepinus may be beneficially cultured with mycotoxin-contaminated grains amended with 200 to 400 mg/kg lanthanum chloride.

2.1. Fiber Extraction and Treatment

Butea parviflora (BP) is a woody climber belonging to the Fabaceae family. It is found in the regions of Kanniyakumari, Tamil Nadu. The whole plant is fibrous, but the strength and texture vary from the outer to inner space. Due to its strength, bark fibers of Butea plant are used by locals for tying, knotting, and binding requirements. Bark contains 1% rotenone, which is used as an insecticide. In order to separate mature fibers from the plant, a metal teeth tool is used. Collected fibers are treated in distilled water and potassium permanganate; treatment involves soaking of fibers in 0.1 M NaOH solution for 20 min, followed by 15 min in the same molarity of potassium permanganate at 27 °C. Treated fibers are rinsed in distilled water. Fibers are first drained in shade, then dried completely using a vacuum desiccator. Variation in soaking time and concentration of KMnO4 will bring varying results. Different treatments at 0.025% and 0.05% of KMnO4 on the Musa plant eliminated the non-cellulosic debris and manifested the crystalline and tensile property of fiber. The delignification process that takes place during permanganate action facilitates the rearrangement of microfibrils, resulting in an improvement in crystallinity. Moreover, the wetting of fibers in 0.1% KMnO4 induces capillary forces that increase their resistance to water []. . presents water and KMnO4 treated BP fibers.

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca2+ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca2+ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca2+ solution followed by a 2 mM Ca2+ solution. Under 2 mM Ca2+ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca2+ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca2+ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (Vrev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca2+ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca2+ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca2+ solution followed by a 2 mM Ca2+ solution. Under 2 mM Ca2+ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca2+ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca2+ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (Vrev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

In control experiments, we ensured that an intact plasma membrane expression of the mutants could only be achieved in the presence of the UAA and the expression of the respective tRNA/aaRS pair. In the absence of the latter, only weak background fluorescence was detectable in the cytosol, but not in the plasma membrane (Supplementary Fig. ) in line with abolished UV-mediated Ca2+ influx comparable to untransfected cells (Supplementary Fig. ). Furthermore, examination of the UV-evoked Ca2+ currents of cells co-expressing Orai1 A137Bpa-YFP and Orai1-CFP wild-type as a function of the Orai1 A137Bpa:Orai1 fluorescence intensity ratio showed a tendency for higher maximum currents with increasing ratio and highest UV-induced activation when only Orai1 A137Bpa was expressed (Supplementary Fig. ), indicating efficient UAA incorporation. Moreover, rapid UV-mediated activation occured independent of endogenous STIM and Orai isoforms, as shown in CRISPR/Cas9 STIM1/STIM2 double-knockout (DKO), CRISPR/Cas9 STIM1/Orai1 DKO and CRISPR/Cas9 Orai1/Orai2/Orai3 triple KO (TKO) cells (Supplementary Fig. ), although to slightly lower maximum current levels, likely owing to lower expression levels in the KO compared to wild-type HEK293 cells (Supplementary Fig. ). Moreover, we showed that ER Ca2+ levels detected by LAR-GECO1 in HEK293 cells expressing either Orai1 or Orai1 A137Bpa compared to untransfected cells remained comparable before and after UV light irradiation and subsequent thapsigargin (TG)-induced store-depletion led to a similar decrease in Ca2+ levels (Supplementary Fig. ), thus excluding that UV light-triggered Ca2+ entry leads to Ca2+ overload in the ER.

Abstract

Nitrosative stress promotes protein glycoxidation, and both processes can occur during an infection with the SARS-CoV-2 virus. Therefore, the aim of this study was to assess selected nitrosative stress parameters and protein glycoxidation products in COVID-19 patients and convalescents relative to healthy subjects, including in reference to the severity of COVID-19 symptoms. The diagnostic utility of nitrosative stress and protein glycoxidation biomarkers was also evaluated in COVID-19 patients. The study involved 218 patients with COVID-19, 69 convalescents, and 48 healthy subjects. Nitrosative stress parameters (NO, S-nitrosothiols, nitrotyrosine) and protein glycoxidation products (tryptophan, kynurenine, N-formylkynurenine, dityrosine, AGEs) were measured in the blood plasma or serum with the use of colorimetric/fluorometric methods. The levels of NO (p = 0.0480), S-nitrosothiols (p = 0.0004), nitrotyrosine (p = 0.0175), kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan fluorescence was significantly (p < 0.0001) lower in COVID-19 patients than in the control group. Significant differences in the analyzed parameters were observed in different stages of COVID-19. In turn, the concentrations of kynurenine (p < 0.0001), N-formylkynurenine (p < 0.0001), dityrosine (p < 0.0001), and AGEs (p < 0.0001) were significantly higher, whereas tryptophan levels were significantly (p < 0.0001) lower in convalescents than in healthy controls. The ROC analysis revealed that protein glycoxidation products can be useful for diagnosing infections with the SARS-CoV-2 virus because they differentiate COVID-19 patients (KN: sensitivity—91.20%, specificity—92.00%; NFK: sensitivity—92.37%, specificity—92.00%; AGEs: sensitivity—99,02%, specificity—100%) and convalescents (KN: sensitivity—82.22%, specificity—84.00%; NFK: sensitivity—82,86%, specificity—86,00%; DT: sensitivity—100%, specificity—100%; AGE: sensitivity—100%, specificity—100%) from healthy subjects with high sensitivity and specificity. Nitrosative stress and protein glycoxidation are intensified both during and after an infection with the SARS-CoV-2 virus. The levels of redox biomarkers fluctuate in different stages of the disease. Circulating biomarkers of nitrosative stress/protein glycoxidation have potential diagnostic utility in both COVID-19 patients and convalescents.

A comparison of the levels protein glycoxidation products in patients infected with the SAR-CoV-2 virus, convalescents, and healthy controls, including in reference to the severity of COVID-19

Tryptophan concentration was significantly lower in COVID-19 patients than in the control group (p < 0.0001) and convalescents (p = 0.0498), and significantly lower in convalescents than in the control group (p < 0.0001). In turn, KN, NFK, DT, and AGE levels were significantly higher in COVID-19 patients than in healthy controls (p < 0.0001), and significantly higher in convalescents than in the control group (p < 0.0001). In addition, KN and NFK levels were higher in patients infected with the SARS-CoV-2 virus than in the control group (p = 0.0009, p = 0.0394) (Fig. A–E). An analysis of protein glycoxidation products revealed no significant differences in COVID-19 patients with different severity of symptoms evaluated based on the MEWS (Table ). However, tryptophan concentration decreased, whereas KN, NFK, and DT concentrations increased with the aggravation of disease symptoms (Table ). In turn, a comparison of patients with mild/moderate symptoms and moderately severe/severe symptoms revealed that the concentrations of all protein glycoxidation products were higher in MEWS 3 + 4 subgroups than in MEWS 1 + 2 subgroups (tryptophan p = 0.0021, KN p = 0.0051, NFK = 0.0187, dityrosine p = 0.0213, AGEs p = 0.0065) (Fig. E,G–J).Figure 2Comparison of the levels protein glycoxidation products: in COVID-19 patients, convalescents, and the control group (A, B, C, D, E); in COVID-19 patients with mild/moderate (1 + 2) and moderately severe/severe (3 + 4) symptoms (F, G, H, I, J). Key: AGEs advanced glycation end products.

Discussion

According to the literature, oxidative and nitrosative stress influences the pathogenesis and severity of infections with the SARS-CoV-2 virus,,, and the present study was undertaken to evaluate nitrosative stress parameters (NO, S-nitrosothiols, and NT) and protein glycoxidation products (tryptophan, KN, NFK, DT, and AGEs) in COVID-19 patients and convalescents, and to examine the above parameters in COVID-19 patients with different severity of disease symptoms evaluated based on the MEWS. The concentrations of NO, NT, S-nitrosothiols, KN, NFK, DT, and AGEs were higher, whereas tryptophan concentration was lower in COVID-19 patients than in the control group and convalescents. The study demonstrated that in patients with a history of COVID-19, the levels of KN, NFK, DT, and AGEs were significantly higher, whereas tryptophan concentration was significantly lower than in persons who had never been infected with the SARS-CoV-2 virus. Significant differences in nitrosative stress parameters and protein glycoxidation products were observed among COVID-19 patients with different disease severity. In addition, protein glycoxidation products were useful diagnostic biomarkers for differentiating COVID-19 patients and convalescents from healthy subjects. This is the first study to analyze changes in the levels of nitrosative stress and protein glycoxidation products in convalescents, and it provides valuable information for predicting long-term consequences of COVID-19.