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Abstract

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells.

2.5. Structure Elucidation

The structures of 1 and 2 () were elucidated using 1D (1H and 13C, ) and 2D (HSQC, HMBC, HSQC-TOCSY and COSY, COSY only recorded for 2). NMR experiments in methanol-d3 and UHPLC-HR-MS analysis. The compounds were determined to consist of a polar ornithine head group linked to a mono-hydroxylated 15:0 (1)/16:0 (2) iso-fatty acid through an amide bond. The structures of the individual variants of 1 and 2 could not be determined individually, but the presence of two stereoisomers in the 5-position could be observed as two near isochronous C-5 resonances and an unresolvable H-5 multiplet pattern.

Experimental design

Thirty male rats (8 weeks old, 172 ± 16 g) were randomly divided into 3 groups: A) sham (SHO) group; B) unilateral ureteral obstruction (UUO) group; C) ATRA treat (ATRA) group, 10 rats per group. Before surgery the rats were performed under general anesthesia (40 mg/kg ip sodium pentobarbital). The left ureter was either completely ligated with two separate silk ties without cutting off the ureter (group B/C) or manipulated similarly but not ligated (group A). ATRA dissolved in corn oil at a dose of 15 mg/kg (group C) or vehicle corn oil (group A/B) was orally administered from 3 days before surgery and on each day until rats were sacrificed after 14 and 28 days (n = 5 for each time point for all groups). Kidneys were removed for histological analysis or Western blot assay.

ARTA improves histopathologic changes in UUO rats

After ureteral ligation, the major signs of renal damage were hydronephrosis and thin renal cortex. From d 14 to d 28, the renal tubular dilation, loss of integrality for tubular brush border, and severity for inflammatory infiltration was significantly enhanced in UUO group when compared with SHO group (). In parallel, the renal interstitial area gradually widened and fibrosis gradually developed in UUO group, and the ATRA group was weaker than UUO group, as showed by the staining of Masson trichrome (). Meanwhile, RIF score was notably up-regulated in UUO rats when compared with that of sham rats. Compared with UUO rats, the RIF score was markedly down-regulated in rats of ATRA group at d 14 and d 28. The values were shown in .

ATRA affects the expressions of angpt-1, TGF-β1, Col-IV and FN in UUO rats

An imbalance of ECM synthesis and degradation was an important event in RIF, including expression of cytokines (e.g., TGF-β1) [,] and mesenchymal markers (e.g., Col-IV and FN) [,]. To assess the effect of ATRA on ECM in the renal interstitial, Angpt-1, TGF-β1, Col-IV and FN protein expression levels were determined by immunohistochemistry in UUO rats treated with or without ATRA, respectively. As shown in , in the group of UUO, TGF-β1, Col-IV and FN expressions were significantly up-regulated, whereas Angpt-1 expression was notably down-regulated than the group of SHO from d 14 to d 28 (p < 0.05). ATRA treatment can reduce TGF-β1, Col-IV and FN expressions and improved Angpt-1 expression at d 14 and d 28 than the UUO group (p < 0.05; ).

ATRA restores angpt-1 expression in UUO rats

To investigate the effect of ATRA on ECM accumulation in the RIF rats, Angpt-1 protein expression was determined using Western-blot method. As shown in , when compared with the group of sham, the expression of Angpt-1 in UUO group was markedly down-regulated from day of d 14 to d 28 (p < 0.05; ). Interestingly, ATRA treatment in group of UUO resulted in increased the expression of Angpt-1 on days 14 and 28 compared to the group of UUO (p < 0.05; ).

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.