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Abstract

Thyroid hormones are biologically active small molecules responsible for growth and development regulation, basal metabolic rate, and lipid and carbohydrate metabolism. Liquid chromatography mass spectrometry (LC–MS) can be used to quantify thyroid hormones blood level with high speed and selectivity, aiming to improve the diagnosis and treatment of the severe pathological conditions in which they are implicated, i.e., hypo- and hyperthyroidism. In this work, the gas-phase behavior of the isomeric thyroid hormones triiodothyronine (T3) and reverse triiodothyronine (rT3) in their deprotonated form was studied at a molecular level using MS-based techniques. Previously reported collision-induced dissociation experiments yielded distinct spectra despite the high structural similarity of the two compounds, suggesting different charge sites to be responsible. Infrared multiple photon dissociation spectroscopy on [T3-H]− and [rT3-H]− was performed, and the results were interpreted using DFT and MP2 calculations, assessing the prevalence of T3 in the carboxylate form and rT3 as a phenolate isomer. The different deprotonation sites of the two isomers were also found to drive their ion-mobility behavior. In fact, [T3-H]− and [rT3-H]− were successfully separated. Drift times were correlated with collisional cross section values of 209 and 215 Å2 for [T3-H]− and [rT3-H]−, respectively. Calculations suggested the charge site to be the main parameter involved in the different mobilities of the two anions. Finally, bare [T3-H]− and [rT3-H]− were made to react with neutral acetylacetone and trifluoroacetic acid, confirming rT3 to be more acidic than T3 in agreement with the calculated gas-phase acidities of T3 and rT3 equal to 1345 and 1326 kJ mol–1, respectively.

Sample Preparation

Triiodothyronine (3,5,3′-triiodothyronine, T3) and reverse triiodothyronine (3,3′,5′-triiodothyronine, rT3) were bought from Sigma-Aldrich, Milan, IT and used without further purification. T3 was dissolved in methanol of MS-grade to a final concentration of 10–5 M with the addition of ammonia (0.1% v/v). rT3 is provided as a methanolic solution already basified with ammonia, which was diluted in methanol to reach the same concentration as T3, i.e., 10–5 M. Acetylacetone, trifluoroacetic acid (TFA), and diethylmethoxyborane (DEMB) were commercial products obtained from Sigma-Aldrich to be used as neutral reagents for ion–molecule reactions.

IRMPD Analysis

IRMPD spectra in the 800–1800 cm–1 frequency range were recorded at the free-electron laser (FEL) beamline of the Centre Laser Infrarouge d’Orsay (CLIO). The FEL beamline (operated at 42.3 MeV for the present experiments) is coupled with a hybrid FT-ICR tandem mass spectrometer (APEX-Qe Bruker Daltonics) equipped with a 7.0 T actively shielded magnet and a quadrupole–hexapole interface. The ions of interest were generated by direct injection of the T3 and rT3 solutions into the ESI source of the mass spectrometer, mass isolated in the quadrupole sector, and accumulated in the hexapole for 1 ms before being irradiated in the FT-ICR cell with the IR FEL light operating at a repetition rate of 25 Hz. Irradiation times were 220 and 300 ms for [T3-H]− and [rT3-H]−, respectively. The photofragmentation products were mass analyzed, and IRMPD spectra were obtained by plotting the photofragmentation yield R (corresponding to −log[Ip/(Ip + ΣIf)], where Ip and ΣIf are the integrated abundances of the precursor ion and the sum of the fragments, respectively) as a function of the photon energy.

Abstract

The homocysteine pathway in the preconception period should be evaluated to highlight micronutrient deficiencies and warrants optimal multivitamin supplementation, before Assisted Reproduction, as preconception care. We conducted a retrospective study aimed at investigating the role of vitamin B complex (5-methyltetrahydrofolate, vitamin B12, vitamin B6) supplement use compared with the role of only folic acid supplement use, in relation to clinical pregnancy and live birth in infertile women undergoing homologous ART. We investigated 269 Caucasian women referred to the Centre for Assisted Reproductive Technology for homologous ART. In these women, 111 (Group A) were daily supplemented with vitamin B complex and 158 (Group B) with only folic acid. In group A the mean number of Metaphase II oocytes and the 2PN Fertilization Rate were higher in comparison to group A (p = 0.04; p = 0.05, respectively). A higher percentage of women in group A had a clinical pregnancy and live birth in comparison to group B (p = 0.01; p = 0.02, respectively). Vitamin B complex supplementation remained independently associated, after multivariable adjustment, with clinical pregnancy (OR 2.03, p = 0.008) and live birth (OR 1.83, p = 0.03). Women supplemented with 5-MTHF and vitamin B12, have a higher chance of clinical pregnancy and live birth in comparison to those supplemented with only folic acid.

2.1. Study Population

In this retrospective study, we investigated Caucasian women referred to the Internal Medicine Clinic (which consists of a multidisciplinary team focused on the prevention of cardiovascular risk) at the Centre for Assisted Reproductive Technology, Careggi University Hospital (Florence, Italy) from May 2018 to April 2020, for homologous ART. Included in the study were 269 women supplemented with vitamin B complex (400 µg 5-methyltetrahydrofolate, 5 µg vitamin B12, 3 mg vitamin B6) or only folic acid (400 µg) (according to gynecologist’s prescription).

3. Results

A total of 269 infertile women participated in this study, mean age 36.9 (±3.7) yrs. We observed that 111 women (Group A) were daily supplemented with vitamin B complex (400 µg 5-MTHF, 5 µg vitamin B12, 3 mg vitamin B6) and 158 women (Group B) supplemented with only folic acid (400 µg folic acid). In terms of markers of ovarian reserve, mean AMH was 2.9 (±3.2) ng/mL, mean FSH was 8.6 (±4) IU/mL and mean AFC was 10.1 (± 6.9).

Abstract

In this study, the effects of calcium chloride (CaCl2) addition on the physical and oxidative stabilities of filled hydrogel were investigated. The results revealed that CaCl2 significantly enhanced the particle size, interfacial layer thickness, apparent viscosity, and viscoelastic behavior of filled hydrogels and decreased their light and whiteness values (p < 0.05). This phenomenon was mainly attributed to the strong binding ability between Ca2+ and protein/pectin mixtures, which were present in the interfacial area or aqueous phase, as verified by cryo-scanning electron microscopy results. Moreover, lower levels of CaCl2 (2 or 4 mM) significantly enhanced the oxidative stability of filled hydrogels (p < 0.05), particularly at a concentration of 4 mM. However, a higher level of CaCl2 (6 or 8 mM) resulted in an electrostatic shielding effect, which resulted in the aggregation of multiple droplets and the flocculation of the filled hydrogels, which negatively affected the oxidative stability of filled hydrogels. The findings of this study indicated that appropriate Ca2+ levels (4 mM) improved the physical and oxidative stability of filled hydrogel, and this finding may provide useful insights for the development of effective delivery systems for specific applications.

2.4. Fabrication of the Filled Hydrogel

Filled hydrogels were prepared based on the method by Matalanis and McClements [] with some modifications, which are shown in . Briefly, the as-prepared O/W emulsion, the lower phase, and upper phase were mixed at a mass ratio of 1:1:18, after which the mixture was stirred at a speed of 10,000 rpm for 45 min, while maintaining a pH of 5.0. CaCl2 solution (50 mM, pH 5.0) was added to as-prepared sample to achieve final Ca2+ levels of 0, 2, 4, 6, and 8 mM in the final systems. Lastly, sodium azide (0.02%, w/w) was added to the filled hydrogels to inhibit microbial growth.

2.5. Particle Size and Zeta-Potential

The particle size and surface charge of freshly prepared and 10-days-stored filled hydrogels were analyzed by using a Nano ZS dynamic light scattering instrument (Malvern instruments Ltd., Worcestershire, UK) based on the method proposed by Hinderink, Schröder, Sagis, and Berton-Carabin [].

2.1. Materials

Zinc nitrate hexahydrate (Zn(NO3)2·6H2O, Loba Chemie, Mumbai, India 98%) and zinc acetate dihydrate (Zn(CH3COO)2·2H2O, Daejung Chemicals, Gyeonggi-do, Korea 98%) were chosen as zinc precursors of ZnO powders. Hexamethylenetetramine ((CH2)6N4, Ajax Finechem, New South Wales, Australia), monoethanolamine (HOC2H4NH2, Sigma Aldrich, St. Louis, MO, USA, 99%), and 2-methoxyethanol (HOC2H4OCH3, Alfa Aesar, Ward Hill, MA, USA 99%) were used as received without further purification. Microscope slides were used as substrates representing window glass.

2.2. Synthesis of ZnO with Different Ratios of ZA/ZN

The synthesis of ZnO particles with different molar ratios of zinc acetate (ZA) and zinc nitrate (ZN) was performed using a simple precipitation method []. The synthesis flow chart in summarizes the method used to produce ZnO powders. Typically, the synthesis of ZnO particles began with the preparation of ZA and ZN precursor solutions. The ZA solution was prepared by dissolving 0.6 M ZA and 0.4 M monoethanolamine in 2-methoxyethanol at room temperature with continuous stirring for 30 min before heating at 90 °C for 1 h. The ZN solution was produced by dissolving 0.015 M ZN and 0.015 M hexamethylenetetramine in deionized (DI) water. The ZN solution was subsequently stirred until the solution became clear. Then, ZA and ZN solutions were mixed with ZA/ZN at mole ratios of 80/1, 40/1, and 20/1. During the mixing procedure, the precipitates formed instantly in the mixed solutions. The mixed solutions were subsequently incubated in a temperature-controlled oven at 90 °C for 2 h. The precipitates were washed through several rounds of rinsing with ethanol and then DI water and with centrifugation, then dried in the oven for 12 h. Finally, the samples were calcined at 550 °C for 6 h in a muffle furnace (CWF 1200, Carbolite Gero, Neuhausen, Germany). Calcination is required for the oxidative transformation of Zn(OH)42- into ZnO []. The ZnO particles prepared with the various mole ratios of ZA/ZN at 80/1, 40/1, and 20/1 are referred to as ZnO-80/1, ZnO-40/1, and ZnO-20/1, respectively.

Abstract

Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.

Introduction

In this work, we demonstrate SE of up to 35.5 ± 0.8% obtained in Vmax. Plasmids designed for and commonly used in E. coli were transferred to the engineered V. natriegens strain Vmax™ Express for successful expression of recombinant proteins containing p-azido-L-phenylalanine (AzF) (Scheme A). Additionally, the newly introduced azido-group was used as coupling site for 19F-containing tags, applicable in NMR studies.Scheme 1A Structure of p-azido-L-phenylalanine. B Work flow for EYFP expression: Co-transformation of plasmids into E. coli BL21(DE3) and V. natriegens Vmax™ Express cells, selection of variants by a 96-well-plate screening, shaking flask expressions with best variants

Abstract

Jojoba oil (JO) extracted from seeds has outstanding properties, including anti-inflammatory, antioxidant, and antibacterial activities, and can be stored forlong periodsof time. The unique properties of jojoba oil depend on its chemical composition; therefore, the effect of the jojoba genotype on the chemical properties and active components of the seed oil was evaluated in this study. Oil samples were collected from 15 elite Egyptian jojoba lines. The chemical composition, such as moisture, crude fiber, crude oil, ash, and crude protein of elite lines’ seeds was determined to investigate the variation among them based on the jojoba genotype. In addition, the iodine value was obtained to measure the degree of jojoba oil unsaturation, whereas the peroxide number was determined as an indicator of the damage level in jojoba oil. Fatty acid composition was studied to compare elite jojoba lines. Fatty acid profiles varied significantly depending on the jojoba genotype. Gadoleic acid exhibited the highest percentage value (67.85–75.50%) in the extracted jojoba oil, followed by erucic acid (12.60–14.81%) and oleic acid (7.86–10.99%). The iodine value, peroxide number, and fatty acid composition of the tested elite jojoba lines were compared withthose reported by the International Jojoba Export Council (IJEC). The results showed that the chemical properties of jojoba oils varied significantly, depending on the jojoba genotype.

2.1. Chemical Composition of the JO

The chemical composition of the seeds of elite jojoba lines was determined to study the effect of jojoba genotype on the chemical constituents of the seeds. Notably, the crude oil, moisture, ash, crude protein, and crude fiber contents varied depending on the jojoba genotype properties, as listed in . The results obtained from the chemical composition analysis revealed that S3, S6, and S14 showed higher oil percentage values of 48.11, 48.05, and 48.15, respectively, compared to the other tested lines. In contrast, S2, S4, and S9 had lower oil contents of approximately 42%. Gad et al. reported similar results and found that oil weight represented approximately 52% of the total weight of jojoba seeds. JO, the only unsaturated liquid wax extracted from plant sources in large quantities, has a chemical composition similar to sperm whale oil []. This similarity enhances the applicability of JO as an inexpensive replacement for sperm whale oil. The total protein content of jojoba seeds was determined by a colorimetric method using a Biuret solution. S7 had the highest protein content (12.55%), whereas S1 seeds had the lowest protein content (11.93%) compared to the other lines. Cappillino et al. and Mohamed [,] determined the total protein content of jojoba seeds and found similar protein contents of 15.2 and 14.72%, respectively. Additionally, the moisture percentage in the seeds of the different elite jojoba lines was determined to study the potential variation among these lines based on their chemical constituents. In this respect, there was a notable change in the moisture content rangingfrom 3.4 to 5.2%. The crude fiber content of jojoba seeds differed significantly as the change in jojoba lines was in the range of 9.94–17.74%.

2.3. Fatty Acid Profile

Gadoleic acid (C20:1), also known as gondoic acid, is an unsaturated omega-9 fatty acid. It representsthe main fatty acid in the oil of jojoba seeds and exhibitsthe highest percentage value, ranging from 67.85% to 75.50%, compared to the other fatty acids, as shown in . It is similar to human sebum, which acquires high absorption in the human skin and can thereforemoisturize the skin without a greasy effect []. The high content of gadoleic acid in the oils of elite jojoba lines enhances their applicability in the pharmaceutical and cosmetic industries. In addition, the presence of gadoleic acid, an unsaturated fatty acid, at high concentrations confirmed the high iodine values determined for different elite jojoba lines.

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.