Characterization of the key odorants in floral aroma green tea based on GC-E-Nose, GC-IMS, GC-MS and aroma recombination and investigation of the dynamic changes and aroma formation during processing.

To characterize the key odorants of floral aroma green tea (FAGT) and reveal its dynamic evolution during processing, the volatile metabolites in FAGT during the whole processing were analyzed by integrated volatolomics techniques, relative odor activity value (rOAV), aroma recombination, and multivariate statistical analysis. The volatile profiles undergone significant changes during processing, especially in the withering and fixation stages. A total of 184 volatile compounds were identified (∼53.26% by GC-MS). Among them, 7 volatiles with rOAV > 1 were identified as characteristic odorants of FAGT, and most of these compounds reached the highest in withering stage. According to the formation pathways, these key odorants could be divided into four categories: fatty acid-derived volatiles, glycoside-derived volatiles, amino acid-derived volatiles, and carotenoid-derived volatiles. Our study provides a comprehensive strategy to elucidate changes in volatile profiles during processing and lays a theoretical foundation for the targeted processing of high-quality green tea.

Insight into aroma dynamic changes during the whole manufacturing process of chestnut-like aroma green tea by combining GC-E-Nose, GC-IMS, and GC × GC-TOFMS.

Processing is the crucial factor for green tea aroma quality. In this study, the aroma dynamic changes throughout the manufacturing process of chestnut-like aroma green tea were investigated with gas chromatography electronic nose (GC-E-Nose), gas chromatography-ion mobility spectrometry (GC-IMS), and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS). GC-IMS identified 33 volatile compounds while GC × GC-TOFMS identified 211 volatile components. Drying exerted the greatest influence on the volatile components of chestnut-like aroma green tea, and promoted the generation of heterocyclic compounds and sulfur compounds which were commonly generated via the Maillard reaction during the roasting stage. A large number of heterocyclic compounds such as 1-methyl-1H-pyrrole, pyrrole, methylpyrazine, furfural, 2-ethyl-5-methylpyrazine, 1-ethyl-1H-pyrrole-2-carboxaldehyde, and 3-acetylpyrrole were newly formed during the drying process. This study also validated the suitability of GC-E-Nose combined with GC-IMS and GC × GC-TOFMS for tracking the changes in volatile components of green tea throughout the manufacturing process.

Resveratrol inhibits MUC5AC expression by regulating SPDEF in lung cancer cells.

BACKGROUND: MUC5AC was recently identified to play important roles in the proliferation and metastasis of malignant mucinous lung tumor cells. Resveratrol (Res), a natural compound with anticancer effects in lung cancer cells, has been reported to inhibit mucin production in airway epithelial cells. This study aimed to investigate the inhibitory effect of Res on MUC5AC expression in lung mucinous adenocarcinoma cells and the potential mechanisms. METHODS: Mucus-producing A549 human lung carcinoma cells were used to test the effects of Res on SPDEF and MUC5AC expression. Gene and protein expression was assessed by real-time quantitative PCR (qPCR), immunofluorescence and western blotting assays. SPDEF lentivirus was used to upregulate SPDEF expression levels in mucus-producing A549 human lung carcinoma cells. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. RESULTS: Res decreased MUC5AC expression in an SPDEF-dependent manner in mucus-producing A549 human lung carcinoma cells, and this change was accompanied by decreased ERK expression and AKT pathway activation. Moreover, SPDEF was found to be overexpressed in lung adenocarcinoma (LUAD), especially in mucinous adenocarcinoma. In-vitro functional assays showed that overexpression of SPDEF reduced the chemosensitivity of A549 cells to cisplatin (DDP). In addition, Res treatment increased A549 cell chemosensitivity to DDP by inhibiting the SPDEF-MUC5AC axis. CONCLUSION: Our results indicate that the SPDEF-MUC5AC axis is associated with DDP sensitivity, and that Res decreases SPDEF and MUC5AC expression by inhibiting ERK and AKT signaling in A549 cells, which provides a potential pharmacotherapy for the prevention and therapeutic management of mucinous adenocarcinoma.

Rapid characterization of the volatile profiles in Pu-erh tea by gas phase electronic nose and microchamber/thermal extractor combined with TD-GC-MS.

Aroma plays an important role in the quality of Pu-erh tea. However, the quality evaluation of Pu-erh tea aroma is heavily relied on the experience of sensory evaluation, and the theoretical research is relatively scarce. In the present work, the volatile compounds in Pu-erh tea were characterized by using gas phase electronic nose (e-nose) and microchamber/thermal extractor (µ-CTE) combined with thermal desorption coupled to gas chromatography-mass spectrometry (TD-GC-MS). A satisfactory discrimination model (R2 Y = 0.95, Q2  = 0.807) was obtained by using orthogonal partial least squares discriminant analysis (OPLS-DA) based on the odor fingerprint of different brands of Pu-erh tea. In addition, based on the double criterion of multivariate analysis with VIP >1.0 and univariate analysis with p ≤ 0.001, 39 volatile components were identified to contribute greatly to the discrimination of five brands of Pu-erh tea. The results suggested that gas phase e-nose and µ-CTE combined with TD-GC/MS were simple, rapid techniques to characterize the volatile compounds in Pu-erh tea and were allowed to effectively distinguish different brands of Pu-erh tea, which would provide an important reference on the quality assessment of Pu-erh tea. PRACTICAL APPLICATION: This work demonstrates that the volatile compounds in Pu-erh tea are simply and rapidly characterized by using µ-CTE/TD-GC/MS and gas phase e-nose, allowing to effectively distinguish different brands of Pu-erh tea, which can provide an important reference for the quality assessment and authentication of Pu-erh tea.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Investigation on green tea lipids and their metabolic variations during manufacturing by nontargeted lipidomics.

Lipids are hydrophobic metabolites implicated in tea flavor quality. Understanding their transformations during tea manufacture is of particular interest. To date, the detailed lipid composition and variations during green tea manufacture are largely unknown. Herein, we performed a comprehensive characterization of the dynamic changes of lipids during green tea manufacture, by applying nontargeted lipidomics using ultrahigh performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Exactive/MS) combined with chemometric tools. Totally, 283 lipid species were detected, covering 20 subclasses. Significant lipidomic variations were observed during green tea manufacture, especially in the fixation stage, mainly associated with chlorophyll decomposition, phosphatidic acids (PAs) reduction and glycolipids degradation, which potentially contribute to tea color and aroma quality. Specifically, the most prominent decrease of PAs content during green tea manufacture was identified for the first time. This study provides insights into the lipid metabolic fates upon green tea manufacture, and their roles in green tea sensory quality.

Aroma dynamic characteristics during the process of variable-temperature final firing of Congou black tea by electronic nose and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry.

The drying technology is crucial to the quality of Congou black tea. In this study, the aroma dynamic characteristics during the variable-temperature final firing of Congou black tea was investigated by electronic nose (e-nose) and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS). Varying drying temperatures and time obtained distinctly different types of aroma characteristics such as faint scent, floral aroma, and sweet fragrance. GC × GC-TOFMS identified a total of 243 volatile compounds. Clear discrimination among different variable-temperature final firing samples was achieved by using partial least squares discriminant analysis (R2Y = 0.95, Q2 = 0.727). Based on a dual criterion of variable importance in the projection value (VIP > 1.0) and one-way ANOVA (p < 0.05), ninety-one specific volatile biomarkers were identified, including 2,6-dimethyl-2,6-octadiene and 2,5-diethylpyrazine with VIP > 1.5. In addition, for the overall odor perception, e-nose was able to distinguish the subtle difference during the variable-temperature final firing process.

Influence of fixation methods on the chestnut-like aroma of green tea and dynamics of key aroma substances.

Fixation is the key process to ensure green tea quality; however, the effect of various fixation methods on the formation of green tea with a chestnut-like aroma and the evolution of key volatile compounds has not been assessed to date. In this study, we compared four types of fixation methods for green tea: roller-hot air-steam, roller-hot air, roller-steam, and single roller. Infrared-assisted headspace solid-phase microextraction and gas chromatography-tandem dual mass spectrometry technology were used to detect the volatile compounds of green tea samples during processing. Partial least-squares discriminant analysis (PLS-DA), multiple experiment viewer (MEV), odor activity value (OAV), and least-significant difference analyzes were then applied to clarify the best fixation method for forming a chestnut-like aroma and associated compounds, and to explore the change law of key volatile compounds using different green tea fixation processes. One hundred and eighty-four volatile compounds were detected in the processed samples, with roller-hot air fixation found as the optimal method for generating an intense and long-lasting chestnut-like aroma and floral taste, based on sensory evaluation. The PLS-DA model clearly distinguished the four kinds of fixation samples and obtained 32 differential volatile compounds. Combining OAVs with screening by MEV analysis, 2,6,10,10-tetramethyl-1-oxaspiro [4.5] dec-6-ene, linalool, cedrol, 3-methyl-butanal, trans-ß-ionone, and τ-cadinol emerged as key differential volatile compounds between green teas with and without a chestnut-like aroma. The evolution of these six differential volatile compounds throughout the tea-making process confirmed that rolling-hot air coupling treatment is most conducive to produce a chestnut-like aroma, which is beneficial to form and transform 2,6,10,10-tetramethyl-1-oxaspiro[4.5] dec-6-ene, 3-methyl-butanal, and τ-cadinol with baking aromas and fruity substances. These results provide a theoretical basis and technical guidance for the precise and directional processing of high-quality green tea with a chestnut-like aroma.

Quantitation of pyrazines in roasted green tea by infrared-assisted extraction coupled to headspace solid-phase microextraction in combination with GC-QqQ-MS/MS.

Pyrazines play an important role in the characteristic flavor of roasted green tea due to powerful strong odours and low sensory thresholds. It is important to analyze these compounds reliably and rapidly in roasted green tea. In this study, infrared-assisted extraction coupled to headspace solid-phase microextraction (IRAE-HS-SPME) and gas chromatography-triple quadrupole-tandem mass spectrometry (GC-QqQ-MS/MS) were developed and validated to determine the pyrazines in roasted green tea. Good linear correlation coefficients (0.9955-0.9996) were obtained over the concentration ranges of 10-5000 ng/mL. The limits of detection (LODs) and limits of quantification (LOQs) for the pyrazines were in the range of 1.46-3.27 ng/mL and 4.89-10.90 ng/mL, respectively. The average recoveries varied from 84% to 119%. The method was used to analyze the pyrazines in roasted green tea manufactured by different final firing methods, the results revealed that microwave final firing method had maximum contents of pyrazines, and significantly improved the aroma quality. In addition, there were great disparities of pyrazines in flatten-shaped green tea and strip-shaped green tea according to the appearance. The result is expected to better understand the role of pyrazines related to aroma quality of roasted green tea and improve processing technology.

Quality Evaluation of Green and Dark Tea Grade Using Electronic Nose and Multivariate Statistical Analysis.

Aroma assessment remains difficult and uncertain in the present sensory assessment system. It is highly desirable to develop a new assessment method to discriminate the quality of various teas in the tea market. In the present work, based on linear discriminant analysis and principal component analysis, the aroma of dry and wet samples of different Xi-hu Longjing and Pu-erh teas were tested and differentiated by electronic noses (e-nose). The results confirm that e-nose can discriminate different priced Xi-hu Longjing tea samples in the range of 80-800 RMB/500 g and varying storage years of Pu-erh tea samples. Furthermore, for the detection of both dry and wet samples of Longjing and Pu-erh teas, the results reveal that all samples have specific aroma characteristics that e-nose can recognize. More importantly, contribution analysis in sensors indicates that nitrogen oxides, methane and alcohols are the characteristic components that contribute to the fragrances of different priced Xi-hu Longjing teas, while nitrogen oxides, aromatic benzene and amines make the fragrances of Pu-erh teas with different storage years disparate. PRACTICAL APPLICATION: This work demonstrates that e-nose can rapidly distinguish tea products with different price levels and varying storage years. With the advantages of ease of use, high portability and flexibility, e-nose will be widely expanded and applied in refined processing and the development of flavored foods.

Rutin, γ-Aminobutyric Acid, Gallic Acid, and Caffeine Negatively Affect the Sweet-Mellow Taste of Congou Black Tea Infusions.

The sweet-mellow taste sensation is a unique and typical feature of premium congou black tea infusions. To explore the key taste-active compounds that influence the sweet-mellow taste, a sensory and molecular characterization was performed on thirty-three congou black tea infusions presenting different taste qualities, including the sweet-mellow, mellow-pure, or less-mellow taste. An integrated application of quantitative analysis of 48 taste-active compounds, taste contribution analysis, and further validation by taste supplementation experiments, combined with human sensory evaluation revealed that caffeine, γ-aminobutyric acid, rutin, succinic acid, citric acid, and gallic acid negatively affect the sweet-mellow taste, whereas glucose, sucrose, and ornithine positively contribute to the sweet-mellow taste of congou black tea infusions. Particularly, rutin, γ-aminobutyric acid, gallic acid, and caffeine, which impart the major inhibitory effect to the manifestation of the sweet-mellow taste, were identified as the key influencing components through stepwise screening and validation experiments. A modest level of these compounds was found to be favorable for the development and manifestation of the sweet-mellow taste. These compounds might potentially serve as the regulatory targets for oriented-manufacturing of high-quality sweet-mellow congou black tea.

Rapid determination by near infrared spectroscopy of theaflavins-to-thearubigins ratio during Congou black tea fermentation process.

The theaflavin-to-thearubigin ratio (TF/TR) is an important parameter for evaluating the degree of fermentation and quality characteristics of Congou black tea. Near infrared (NIR) spectroscopy, one of the most promising techniques for evaluating large-scale tea processing quality, in association with chemometrics, can be used as a selection tool when a fast determination of the requested parameters is required. The aim of this work is to develop a unique model for the determination of TF/TR. First, 11 key wavelength variables were screened by synergy interval partial least-squares regression (SI-PLS) and competitive adaptive reweighted sampling (CARS). Based on these characteristic variables, a new extreme learning machine (ELM) combined with an adaptive boosting (ADABOOST) algorithm (ELM-ADABOOST) was applied to construct the nonlinear prediction model for TF/TR, and an independent external set was used for the validation. A determinate coefficient (Rp2) of 0.893, root mean square error of prediction (RMSEP) of 0.0044, RSD below 10%, and RPD above 3 were acquired in the prediction model. These results demonstrate that NIR can be used to rapidly determine the TF/TR value during fermentation, and it effectively simplify the model and improve the prediction accuracy when combined with the SI-CARS variable.

Characterization of the volatile components in green tea by IRAE-HS-SPME/GC-MS combined with multivariate analysis.

In the present work, a novel infrared-assisted extraction coupled to headspace solid-phase microextraction (IRAE-HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of the volatile components in green tea. The extraction parameters such as fiber type, sample amount, infrared power, extraction time, and infrared lamp distance were optimized by orthogonal experimental design. Under optimum conditions, a total of 82 volatile compounds in 21 green tea samples from different geographical origins were identified. Compared with classical water-bath heating, the proposed technique has remarkable advantages of considerably reducing the analytical time and high efficiency. In addition, an effective classification of green teas based on their volatile profiles was achieved by partial least square-discriminant analysis (PLS-DA) and hierarchical clustering analysis (HCA). Furthermore, the application of a dual criterion based on the variable importance in the projection (VIP) values of the PLS-DA models and on the category from one-way univariate analysis (ANOVA) allowed the identification of 12 potential volatile markers, which were considered to make the most important contribution to the discrimination of the samples. The results suggest that IRAE-HS-SPME/GC-MS technique combined with multivariate analysis offers a valuable tool to assess geographical traceability of different tea varieties.

Comparison of headspace solid-phase microextraction with conventional extraction for the analysis of the volatile components in Melia azedarach.

The volatile compositions of Melia azedarach were studied by headspace solid-phase microextraction (HS-SPME). The result was compared with that obtained by soxhlet extraction (SE) and ultrasonic extraction (UAE). 79 compounds were identified in this study, among which 64 compounds were first reported. The experimental parameters including fiber type (PDMS, PDMS-DVB and CAR-PDMS), desorption time, extraction temperature and time were investigated. 37 compounds were obtained by HS-SPME, including curcumene (33.25%), α-cadinol (11.16%), α-muurolene (8.72%), copaene (5.04%), ß-bisabolene (3.41%), and α-selinene (2.97%). The result suggested that the HS-SPME method is a powerful analytic tool and complementary to traditional methods for the determination of the volatile compounds in Chinese herbs.

A genomic atlas of mouse hypothalamic development.

The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.