UHPLC-HRMS-Based Untargeted Lipidomics Reveal Mechanism of Antifungal Activity of Carvacrol against <i>Aspergillus flavus</i>

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

Associations of CYP24A1 Copy Number Variation with Vitamin D Deficiency and Insulin Secretion

Vitamin D plays an important role in insulin secretion. As the enzyme that initiates degradation of the active metabolite of vitamin D (1,25-(OH)2 vitamin D), 24-hydroxylase encoded by CYP24A1 may be associated with insulin secretion. In this study, we aimed at investigating the association between copy number of CYP24A1 and the concentration of insulin. Included in the study were 1528 rural people from Henan Province of China. The copy number of CYP24A1 and the concentrations of serum 25(OH) vitamin D3 and insulin were determined. Association between copy number of CYP24A1 and vitamin D deficiency was investigated with logistic regression model. Correlation between copy number of CYP24A1 and serum insulin was observed by Spearman correlation. The results suggested that copy number variation of CYP24A1 was associated with vitamin D deficiency. Higher copy number of CYP24A1 was a risk factor for vitamin D deficiency (adjusted odds ratio: 1.199; 95% confidence interval: 1.028–1.397; P = 0.021). Furthermore, copy number of CYP24A1 was positive correlated with the concentration of serum insulin (r = 0.115; P < 0.001), regardless of vitamin D status, age, and body mass index (BMI). Increased copy number of CYP24A1 is associated with not only vitamin D deficiency but also increased serum insulin. Vitamin D supplement may be beneficial to individuals with high copy number of CYP24A1. Novelty Increased copy number of CYP24A1 was a risk factor of vitamin D deficiency. Increased copy number of CYP24A1 was associated with increased serum concentration of insulin independent of age, BMI, and vitamin D status.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies

A Heat and Mass Transfer Model of Peanut Convective Drying Based on a Two-Component Structure

In order to optimize the convective drying process parameters of peanuts and to provide a theoretical basis for the scientific use of energy in the drying process, this study took single-particle peanuts as the research object and analyzed the heat and mass transfer process during convective drying. In addition, a 3D two-component moisture heat transfer model for peanuts was constructed based on the mass balance and heat balance theorem. Moreover, the changes in the internal temperature and concentration fields of peanut pods during the whole drying process were investigated by simulations using COMSOL Multiphysics. The model was validated by thin-layer drying experiments, compared with the one-component model, and combined with low-field NMR technology to further analyze the internal moisture distribution state of peanut kernel drying process. The results show that both models can effectively simulate the peanut thin-layer drying process, and consistency is found between the experimental and simulated values, with the maximum errors of 10.25%, 9.10%, and 7.60% between the simulated moisture content and the experimental values for the two-component model, peanut shell, and peanut kernel models, respectively. Free water and part of the weakly bound water was the main water lost by peanuts during the drying process, the change in oil content was small, and the bound water content was basically unchanged. The results of the study provide a theoretical basis to accurately predict the moisture content within different components of peanuts and reveal the mechanism of moisture and heat migration during the drying process of peanut pods