UHPLC-HRMS-Based Untargeted Lipidomics Reveal Mechanism of Antifungal Activity of Carvacrol against Aspergillus flavus

Aspergillus flavus is a common contaminant in grain, oil and their products. Its metabolite aflatoxin B1 (AFB1) has been proved to be highly carcinogenic. Therefore, it is of great importance to find possible antifungal substances to inhibit the growth and toxin production of Aspergillus flavus. Carvacrol (CV) was reported as a potent antifungal monoterpene derived from plants. In this paper, the antifungal effects and mechanism of CV on Aspergillus flavus were investigated. CV was shown good inhibition on the growth of Aspergillus flavus and the production of AFB1. CV used in concentrations ranging from 0, 50, 100 and 200 μg/mL inhibited the germination of spores, mycelia growth and AFB1 production dose-dependently. To explore the antifungal mechanism of CV on Aspergillus flavus, we also detected the ergosterol content of Aspergillus flavus mycelia, employed Scanning Electron Microscopy (SEM) to observe mycelia morphology and utilized Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS) to explore the lipidome profiles of Aspergillus flavus. The results showed that the production of ergosterol of mycelia was reduced as the CV treatment concentration increased. SEM photographs demonstrated a rough surface and a reduction in the thickness of hyphae in Aspergillus flavus treated with CV (200 µg/mL). In positive ion mode, 21 lipids of Aspergillus flavus mycelium were downregulated, and 11 lipids were upregulated after treatment with 200-µg/mL CV. In negative ion mode, nine lipids of Aspergillus flavus mycelium were downregulated, and seven lipids upregulated after treatment with 200-µg/mL CV. In addition, the analysis of different lipid metabolic pathways between the control and 200-µg/mL CV-treated groups demonstrated that glycerophospholipid metabolism was the most enriched pathway related to CV treatment.

Simultaneous determination of rivaroxaban and sitagliptin in rat plasma by LC–MS/MS and its application to pharmacokinetic drug-drug interaction study

A sensitive and accurate LC-MS/MS method was developed and validated for the simultaneous quantification of rivaroxaban (RIV) and sitagliptin (SIT) in rat plasma using apixaban as internal standard (IS). An Agilent Eclipse plus C18 column (2.1 × 100 mm, 3.5 µm, Agilent) was used for chromatographic separation with isocratic elution. Multiple reaction monitoring (MRM) using positive-ion ESI mode to monitor ion transitions of m/z 436.8→144.9 for RIV, m/z 407.7→173.8 for SIT, m/z 459.8→442.8 for IS. The procedure of method validation included selectivity, linearity, precision, accuracy, matrix effect, extraction recovery and stability were conducted according to the guidelines of EMA and FDA. The results indicated that no obvious drug-drug interactions occurred might be owing to their differences in metabolic pathways.

Intestinal Metabolomics of Juvenile Lenok (Brachymystax Lenok) in Response to Heat Stress

Changes in the metabolic profile within the intestine of lenok (Brachymystax lenok) when challenged to acute and lethal heat stress (HS) are studied using no-target HPLC-MS/MS metabonomic analysis. Of 51 differentially expressed metabolites identified in response to HS, 34 occurred in the positive ion mode and 17 in negative ion mode (VIP > 1, P < 0.05). Changes in metabolites (i.e. alpha-D-glucose, stachyose and L-lactate) related to carbohydrate and glycolysis are identified in HS-treated lenok. Fatty acid β-oxidation in HS-treated lenok was inhibited by accumulation of acetyl carnitine, palmitoylcarnitine, carnitine, and erucic acid. Many amino acids (L-tryptophan, D-proline, L-leucine, L-phenylalanine, L-aspartate, L-tyrosine, L-methionine, L-histidine and L-glutamine) decreased to support energy demands in HS-treated lenok. Oxidative damage in HS-treated lenok was indicated by decreased glycerophospholipid metabolites (i.e. glycerophosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine), and increased oxylipin production (12-HETE and 9R, 10S-EpOME). Oxidative stress increased formation of eicosanoids and dicarboxylic acids, overwhelming the mitochondrial β-oxidation pathway, while minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation) were likely to be activated in HS-treated lenok.

The Electrodynamic Mechanism of Collisionless Multicomponent Plasma Expansion in Vacuum Discharges: From Estimates to Kinetic Theory

This paper is devoted to the study of collisionless multicomponent plasma expansion in vacuum discharges. Based on the fundamental principles of physical kinetics formulated for vacuum discharge plasma, an answer is given to the following question: What is the main mechanism of cathode plasma transport from cathode to anode, which ensures non-thermal metallic positive ion movement? Theoretical modeling is provided based on the Vlasov–Poisson system of equations for a current flow in a planar vacuum discharge gap. It was shown that the non-thermal plasma expansion is of a purely electrodynamic nature, caused by the formation of a “potential hump” in the interelectrode space and its subsequent movement under certain conditions consistent with plasma electrodynamic transportation. The presented results reveal two cases of the described phenomenon: (1) the dynamics of single-component cathode plasma and (2) multicomponent plasma (consisting of multiple charged ions) expansion.

Determination of oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin in mouse blood by UPLC-MS/MS and its application to pharmacokinetics

In this study, a UPLC-MS/MS method was developed to measure the concentrations of the flavonoids oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin in the blank mouse blood, and the method was then used in the measurement of the pharmacokinetics of the compounds in mice. Oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin were administered intravenously at a dose of 5 mg kg−1, and the mouse blood (20 μL) was withdrawn from the caudal vein 0.08333, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 h after administration. The mobile phase used for chromatographic separation by gradient elution was composed of acetonitrile and water (0.1% formic acid). The analytes were detected by operating in electrospray ionization (ESI) positive-ion mode using multiple reactions monitoring (MRM). The intra-day and inter-day accuracy ranged from 86.2 to 109.3%, the intra-day precision was less than 14%, and the inter-day precision was less than 15%. The matrix effect ranged from 85.3 to 111.3%, and the recovery of the analytes after protein precipitation were all above 78.2%. This method had the advantages of high sensitivity, accuracy, and recovery, and it had excellent selectivity, which enabled it to be applied to measuring the pharmacokinetics of the analytes in mice.

Evaluation of 6 MALDI-Matrices for 10 μm lipid imaging and on-tissue MSn with AP-MALDI-Orbitrap

Mass spectrometry imaging (MSI) is a technique uniquely suited to localize and identify lipids in a tissue sample. Using an AP-MALDI UHR source coupled to an Orbitrap Elite, numerous lipid locations and structures can be determined in high mass resolution spectra and at cellular spatial resolution, but careful sample preparation is necessary. We tested 11 protocols on serial brain sections for the commonly used MALDI matrices, CHCA, Norharmane, DHB, DHAP, THAP, and DAN, in combination with tissue washing and matrix additives, to determine the lipid coverage, signal intensity, and spatial resolution achievable with AP-MALDI. In positive ion mode, the most lipids could be detected with CHCA and THAP, while THAP and DAN without additional treatment offered the best signal intensities. In negative ion mode, DAN showed the best lipid coverage and DHAP performed superior for Gangliosides. DHB produced intense cholesterol signals in the white matter. 155 lipids were assigned in positive (THAP), 137 in negative ion mode (DAN) and 76 lipids were identified using on tissue tandem-MS. The spatial resolution achievable with DAN was 10 μm, confirmed with on tissue line-scans. This enabled the association of lipid species to single neurons in AP-MALDI images. The results show that the performance of AP-MALDI is comparable to vacuum MALDI techniques for lipid imaging.

Flagellin FLiC Enhances Resistance of Upland Cotton to Verticillium dahliae

The mechanism by which flagellin induces an immune response in plants is still unclear. The purpose of this study is to reveal the effect and mechanism of flagellin in inducing plants to produce an immune response to increase the resistance to Verticillium dahliae (VD). The full-length flagellin gene C (FliC) was cloned from an endophytic bacteria (Pseudomonas) in the root of upland cotton cultivar Zhongmiansuo 41. The FliC protein purified in vitro has 47.50% and 32.42% biocontrol effects on resistant and susceptible cotton cultivars, respectively. FLiC can induce allergic reactions in tobacco leaf cells and immune responses in cotton plants. Smearing FLiC to cotton and performing RNA-seq analysis, it is significantly enriched in the activity of positive ion transporters such as potassium ions and calcium ions (Ca2+), diterpenoid biosynthesis, phenylpropane biosynthesis and other disease-resistant metabolic pathways. FLiC inhibits the expression of calcium antiporter activity gene (GhCAA) to accelerate intracellular Ca2+ influx and stimulate the increase of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) content. The coordinated regulation of Ca2+, H2O2 and NO enhances disease resistance. The resistance of transgenic FLiC gene Arabidopsis to VD was significantly improved. The FLiC gene can be used as an anti-VD gene and as a regulator to improve resistance to VD.