Amine Switchable Hydrophilic Solvent Vortex-Assisted Homogeneous Liquid-Liquid Microextraction and GC-MS for the Enrichment and Determination of 2, 6-DIPA Additive in Biodegradable Film.

2, 6-diisopropylaniline (2, 6-DIPA) is a crucial non-intentionally organic additive that allows the assessment of the production processes, formulation qualities, and performance variations in biodegradable mulching film. Moreover, its release into the environment may have certain effects on human health. Hence, this study developed simultaneous heating hydrolysis-extraction and amine switchable hydrophilic solvent vortex-assisted homogeneous liquid-liquid microextraction for the gas chromatography-mass spectrometry analysis of the 2, 6-DIPA additive and its corresponding isocyanates in poly(butylene adipate-co-terephthalate) (PBAT) biodegradable agricultural mulching films. The heating hydrolysis-extraction conditions and factors influencing the efficiency of homogeneous liquid-liquid microextraction, such as the type and volume of amine, homogeneous-phase and phase separation transition pH, and extraction time were investigated and optimized. The optimum heating hydrolysis-extraction conditions were found to be a H2SO4 concentration of 2.5 M, heating temperature of 87.8 °C, and hydrolysis-extraction time of 3.0 h. As a switchable hydrophilic solvent, dipropylamine does not require a dispersant. Vortex assistance is helpful to speed up the extraction. Under the optimum experimental conditions, this method exhibits a better linearity (0.0144~7.200 µg mL-1 with R = 0.9986), low limit of detection and quantification (0.0033 µg g-1 and 0.0103 µg g-1), high extraction recovery (92.5~105.4%), desirable intra- and inter-day precision (relative standard deviation less than 4.1% and 4.7%), and high enrichment factor (90.9). Finally, this method was successfully applied to detect the content of the additive 2, 6-DIPA in PBAT biodegradable agricultural mulching films, thus facilitating production process monitoring or safety assessments.

Rapid and Precise Differentiation and Authentication of Agricultural Products via Deep Learning-Assisted Multiplex SERS Fingerprinting.

Accurate and rapid differentiation and authentication of agricultural products based on their origin and quality are crucial to ensuring food safety and quality control. However, similar chemical compositions and complex matrices often hinder precise identification, particularly for adulterated samples. Herein, we propose a novel method combining multiplex surface-enhanced Raman scattering (SERS) fingerprinting with a one-dimensional convolutional neural network (1D-CNN), which enables the effective differentiation of the category, origin, and grade of agricultural products. This strategy leverages three different SERS-active nanoparticles as multiplex sensors, each tailored to selectively amplify the signals of preferentially adsorbed chemicals within the sample. By strategically combining SERS spectra from different NPs, a 'SERS super-fingerprint' is constructed, offering a more comprehensive representation of the characteristic information on agricultural products. Subsequently, utilizing a custom-designed 1D-CNN model for feature extraction from the 'super-fingerprint' significantly enhances the predictive accuracy for agricultural products. This strategy successfully identified various agricultural products and simulated adulterated samples with exceptional accuracy, reaching 97.7% and 94.8%, respectively. Notably, the entire identification process, encompassing sample preparation, SERS measurement, and deep learning analysis, takes only 35 min. This development of deep learning-assisted multiplex SERS fingerprinting establishes a rapid and reliable method for the identification and authentication of agricultural products.

Development and Application of Portable Reflectometric Spectroscopy Combined with Solid-Phase Extraction for Determination of Potassium in Flue-Cured Tobacco Leaves.

Potassium (K) plays important roles in the energy and substance conversion of tobacco metabolism and is also regarded as one of the important indicators of tobacco quality evaluation. However, the K quantitative analytical method shows poor performance in terms of being easy-to-use, cost-effective, and portable. Here, we developed a rapid and simple method for the determination of K content in flue-cured tobacco leaves, including water extraction with 100 °C heating, purification with solid-phase extraction (SPE), and analysis with portable reflectometric spectroscopy based on K test strips. The method development consisted of optimization of the extraction and test strip reaction conditions, screening of SPE sorbent materials, and evaluation of the matrix effect. Under the optimum conditions, good linearity was observed in 0.20-0.90 mg/mL with a correlation coefficient >0.999. The extraction recoveries were found to be in the range of 98.0-99.5% with a repeatability and reproducibility of 1.15-1.98% and 2.04-3.26%, respectively. The sample measured range was calculated to be 0.76-3.68% K. Excellent agreement was found in accuracy between the developed reflectometric spectroscopy method and the standard method. The developed method was applied to analyze the K content in different cultivars, and the content varied greatly among the samples with lowest and highest contents for Y28 and Guiyan 5 cultivars, respectively. This study can provide a reliable approach for K analysis, which may become available on-site in a quick on-farm test.

Comparative study on the structural and in vitro digestion properties of starch within potato parenchyma cells under different cooking methods.

To study the effects of cooking methods on the structure and digestion changes of starch encapsulated by cellular structure, intact potato parenchyma cells were successfully isolated and then subjected to different domestic cooking methods, including baking, frying, boiling, and autoclaving. The morphology, crystalline structure, thermal properties, and in vitro starch digestibility of cooked cell samples were investigated. Our results indicated that potato cell walls remained intact and performed as physical barriers preventing the diffusion/absorption of α-amylase to intracellular starch substrates after baking or frying treatment. However, boiling or autoclaving treatment destroyed cell wall structure, and the disrupted cellular structure reduced the digestion rate, likely by inhibiting diffusion of amylase through a weakened cell wall barrier, but could not lower the final digestion extent when compared to the pure starch. These findings suggested that potato products with lower glycemic index can be obtained by baking or frying treatment.

Determination of Residual Diisocyanates and Related Diamines in Biodegradable Mulch Films Using N-Ethoxycarbonylation Derivatization and GC-MS.

Diisocyanates are highly reactive compounds with two functional isocyanate groups. The exposure of diisocyanates is associated with severely adverse health effects, such as asthma, inflammation in the respiratory tract, and cancer. The hydrolysis product from diisocyanates to related diamines is also a potential carcinogen. Here, we developed an effective, accurate, and precise method for simultaneous determination of residual diisocyanates and related diamines in biodegradable mulch films, based on N-ethoxycarbonylation derivatization and gas chromatography-mass spectrometry. The method development included the optimization of ultrasonic hydrolysis and extraction, screening of N-ethoxycarbonylation conditions with ethyl chloroformate, evaluation of the diamines degradation, and analysis of the fragmentation mechanisms. Under the optimum experimental conditions, good linearity was observed with R2 > 0.999. The extraction recoveries were found in the range of 93.9−101.2% with repeatabilities and reproducibilities in 0.89−8.12% and 2.12−10.56%, respectively. The limits of detection ranged from 0.0025 to 0.057 µg/mL. The developed method was applied to commercial polybutylene adipate co-terephthalate (PBAT) biodegradable mulch film samples for analysis of the diverse residual diisocyanates and related diamine additives. The components varied greatly among the sample from different origin. Overall, this study provides a reliable method for assessing safety in biodegradable mulch films.

Chiral determination of nornicotine, anatabine and anabasine in tobacco by achiral gas chromatography with (1S)-(-)-camphanic chloride derivatization: Application to enantiomeric profiling of cultivars and curing processes.

The alkaloid enantiomers are well-known to have different physiological and pharmacological effects, and to play an important role in enantioselectivity metabolism with enzymes catalysis in tobacco plants. Here, we developed an improved method for simultaneous and high-precision determination of the individual enantiomers of nornicotine, anatabine and anabasine in four tobacco matrices, based on an achiral gas chromatography-nitrogen phosphorus detector (GCNPD) with commonly available Rtx-200 column using (1S)-(-)-camphanic chloride derivatization. The method development consists of the optimization of extraction and derivatization, screening of achiral column, analysis of the fragmentation mechanisms and evaluation of matrix effect (ME). Under the optimized experimental conditions, the current method exhibited excellent detection capability for the alkaloid enantiomers, with coefficients of determination (R2) > 0.9989 and normality test of residuals P > 0.05 in linear regression parameters. The ME can be neglected for the camphanic derivatives. The limit of detection (LOD) and limit of quantitation (LOQ) ranged from 0.087 to 0.24 µg g - 1 and 0.29 to 0.81 µg g - 1, respectively. The recoveries and within-laboratory relative standard deviations (RSDR) were 94.3%~104.2% and 0.51%~3.89%, respectively. The developed method was successfully applied to determine the enantiomeric profiling of cultivars and curing processes. Tobacco cultivars had a significant impact on the nornicotine, anatabine, anabasine concentration and enantiomeric fraction (EF) of (R)-nornicotine, whereas the only significant change induced by the curing processes was an increase in the EF of (R)-anabasine.

Overexpression of Family with Sequence Similarity 83, Member A (FAM83A) Predicts Poor Clinical Outcomes in Lung Adenocarcinoma.

BACKGROUND The aim of this study was to explore the expression levels of family with sequence similarity 83, member A (FAM83A) in lung adenocarcinoma (LUAD) and investigate its clinical prognostic value. MATERIAL AND METHODS Bioinformatics mining methods were used to predict the differential expression levels of FAM83A mRNA in LUAD and normal lung tissues based on the TCGA and Oncomine databases. Immunohistochemical staining was performed to demonstrate the FAM83A protein expression levels in 83 cases of LUAD combined with paired normal lung tissues. The correlation between clinicopathologic factors and FAM83A differential expression levels in LUAD was explored by the chi-square test. Kaplan-Meier univariate and Cox multivariate survival analyses were performed to investigate the clinical prognostic value of FAM83A expression in LUAD patients. RESULTS Results from TCGA and Oncomine databases revealed that FAM83A mRNA expression level was significantly higher in LUAD than that in normal lung tissues (both P<0.05). Immunohistochemical findings demonstrated that the high positive rate of FAM83A in LUAD was 73.49% (61/83), while that of matched normal lung tissues was only 22.89% (19/83). Moreover, LUAD patients with FAM83A mRNA or high protein levels had dramatically lower OS times than those with FAM83A mRNA or low protein levels (All P<0.05). Lastly, Cox multivariate survival analysis showed that FAM83A differential expression level (low vs. high) was the only independent factor predicting the prognosis of LUAD patients (P=0.001). CONCLUSIONS FAM83A was overexpressed in LUAD, and FAM83A overexpression could be used as an independent factor of poor prognosis in LUAD patients.

An improved in situ acetylation with dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry for the sensitive determination of phenols in mainstream tobacco smoke.

Phenols in tobacco smoke can adversely affect health with serious consequences that include cardiovascular toxicity, tumor promotion and genotoxic activity. Hence, an improved method involving in situ acetylation and dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS) was developed for the determination of 39 phenols in mainstream tobacco smoke (MTS). The in situ acetylation was optimized using four protocols, after which the effects of experimental variables on acetylation efficiency were studied using a multifactorial experimental design. The optimum conditions were found to involve an initial 75 µL volume of acetic anhydride, 140 mg of NaHCO3 and 72 mg of K2CO3. The phenolic acetates were then subjected to DLLME, after which they were identified and quantified by GC-MS. A total of 32 additional phenols were tentatively identified. Good linearity was observed with R > 0.999 and each lack-of-fit P > 0.05. The relative recoveries were in the range of 94.8-104.3% with repeatabilities and reproducibilities less than 5.5% and 6.8%, respectively. The limits of detection ranged from 1.12 to 1.74 ng cig-1, with high enrichment factors between 87 and 144. This method was applied to the MTS from three commercial cigarettes with different tar levels. The results provide valuable information for assessing the risks of phenols.

Free amino acids, biogenic amines, and ammonium profiling in tobacco from different geographical origins using microwave-assisted extraction followed by ultra high performance liquid chromatography.

This work describes a rapid, stable, and accurate method for determining the free amino acids, biogenic amines, and ammonium in tobacco. The target analytes were extracted with microwave-assisted extraction and then derivatized with diethyl ethoxymethylenemalonate, followed by ultra high performance liquid chromatography analysis. The experimental design used to optimize the microwave-assisted extraction conditions showed that the optimal extraction time was 10 min with a temperature of 60°C. The stability of aminoenone derivatives was improved by keeping the pH near 9.0, and there was no obvious degradation during the 80°C heating and room temperature storage. Under optimal conditions, this method showed good linearity (R2 > 0.999) and sensitivity (limits of detection 0.010-0.081 µg/mL). The extraction recoveries were between 88.4 and 106.5%, while the repeatability and reproducibility ranged from 0.48 to 5.12% and from 1.56 to 6.52%, respectively. The newly developed method was employed to analyze the tobacco from different geographical origins. Principal component analysis showed that four geographical origins of tobacco could be clearly distinguished and that each had their characteristic components. The proposed method also showed great potential for further investigations on nitrogen metabolism in plants.

Two C3H Type Zinc Finger Protein Genes, CpCZF1 and CpCZF2, from Chimonanthus praecox Affect Stamen Development in Arabidopsis.

Wintersweet (Chimonanthus praecox) is a popular garden plant because of its flowering time, sweet fragrance, and ornamental value. However, research into the molecular mechanism that regulates flower development in wintersweet is still limited. In this study, we sought to investigate the molecular characteristics, expression patterns, and potential functions of two C3H-type zinc finger (CZF) protein genes, CpCZF1 and CpCZF2, which were isolated from the wintersweet flowers based on the flower developmental transcriptome database. CpCZF1 and CpCZF2 were more highly expressed in flower organs than in vegetative tissues, and during the flower development, their expression profiles were associated with flower primordial differentiation, especially that of petal and stamen primordial differentiation. Overexpression of either CpCZF1 or CpCZF2 caused alterations on stamens in transgenic Arabidopsis. The expression levels of the stamen identity-related genes, such as AGAMOUS (AG), PISTILLATA (PI), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), SEPALLATA3 (SEP3), APETALA1 (AP1), APETALA2 (AP2), and boundary gene RABBIT EAR (RBE) were significantly up-regulated in CpCZF1 overexpression lines. Additionally, the transcripts of AG, PI, APETALA3SEP1-3, AP1, and RBE were markedly increased in CpCZF2 overexpressed plant inflorescences. Moreover, CpCZF1 and CpCZF2 could interact with each other by using yeast two-hybrid and bimolecular fluorescence complementation assays. Our results suggest that CpCZF1 and CpCZF2 may be involved in the regulation of stamen development and cause the formation of abnormal flowers in transgenic Arabidopsis plants.

[Effects of biochar on the micro-ecology of tobacco-planting soil and physiology of flue-cured tobacco].

Biochar is one of the research hotspots in the field of the agroforestry waste utilization. A field experiment was carried out to investigate the effects of different amounts of tobacco stem biochar (0, 1, 10, 50 t · hm⁻²) on soil micro-ecology and physiological properties of flue-cured tobacco. The results showed that soil water content (SWC) increased at all tobacco growth stages as the amounts of biochar applications increased. There were significant differences of SWC between the treatment of 50 t · hm⁻² and other treatments at the period of tobacco vigorous growth. As the application of biochar increased, the total soil porosity and capillary porosity increased, while soil bacteria, actinomyces, fungi amount increased firstly and then decreased. The amount of soil bacteria, actinomyces, fungi reached the maximum at the treatment of 10 t · hm⁻². Soil respiration rate (SRR) at earlier stage increased with the increase of biochar application. Compared with the control, SSR under biochar treatments increased by 7.9%-36.9%, and there were significant differences of SRR between high biochar application treatments (50 t · hm⁻² and 10 t · hm⁻²) and the control. Biochar improved leaf water potential, carotenoid and chlorophyll contents. Meanwhile, the dry mass of root, shoot and total dry mass under biochar application were higher than that of the control. These results indicated that the biochar played active roles in improving tobacco-planting soil micro-ecology and regulating physiological properties of flue-cured tobacco.