Bitterness quantification and simulated taste mechanism of theasinensin A from tea.

Theasinensin A is an important quality chemical component in tea, but its taste characteristics and the related mechanism are still unclear. The bitterness quantification and simulated taste mechanism of theasinensin A were researched. The results showed that theasinensin A was significantly correlated with the bitterness of tea. The bitterness threshold of theasinensin A was identified as 65 µmol/L for the first time. The dose-over-threshold (DOT) value of theasinensin A was significantly higher than that of caffeine in black tea soup. The concentration-bitterness curve and time-intensity curve of theasinensin A were constructed. The bitterness contribution of theasinensin A in black tea was higher than in oolong and green tea. Theasinensin A had the highest affinity with bitterness receptor protein TAS2R16, which was compared to TAS2R13 and TAS2R14. Theasinensin A was mainly bound to a half-open cavity at the N-terminal of TAS2R13, TAS2R14, and TAS2R16. The different binding capacity, hydrogen bond, and hydrophobic accumulation effect of theasinensin A and bitterness receptor proteins might be the reason why theasinensin A presented different bitterness senses in human oral cavity.

DLC1 inhibits colon adenocarcinoma cell migration by promoting secretion of the neurotrophic factor MANF.

DLC1 (deleted in liver cancer-1) is downregulated or deleted in colorectal cancer (CRC) tissues and functions as a potent tumor suppressor, but the underlying molecular mechanism remains elusive. We found that the conditioned medium (CM) collected from DLC1-overexpressed SW1116 cells inhibited the migration of colon adenocarcinoma cells HCT116 and SW1116, but had no effect on proliferation, which suggested DLC1-mediated secretory components containing a specific inhibitor for colon adenocarcinoma cell migration. Analysis by mass spectrometry identified mesencephalic astrocyte-derived neurotrophic factor (MANF) as a candidate. More importantly, exogenous MANF significantly inhibited the migration of colon adenocarcinoma cells HCT116 and SW1116, but did not affect proliferation. Mechanistically, DLC1 reduced the retention of MANF in ER by competing the interaction between MANF and GRP78. Taken together, these data provided new insights into the suppressive effects of DLC1 on CRC, and revealed the potential of MANF in the treatment of CRC.

[Identification and single-cell sequencing analysis of rare tumor cells in malignant pleural effusion of lung cancer patients].

Tumor heterogeneity refers to distinct genomic or phenotypic characteristics of tumor cells. Under the environmental or drug stress, tumor cells exhibit different responses, corresponding to different properties of cell proliferation, invasion, metastasis and drug resistance. In particular, a small fraction of tumor cells are capable of detaching from primary tumor sites and initiating distant metastases. Thus, tumor heterogeneity sets the basis for tumor resistance and metastasis. Traditional methods in studying tumor heterogeneity are mainly based on bulk cells from different locations in primary tumors, lacking analysis at the single-cell level and of metastatic tumor cells. This study establishes a single-cell method to study metastatic tumor cells in malignant pleural effusions of lung cancer patients. Metabolically active tumor cells in malignant pleural effusions are firstly identified with a metabolic marker 2-NBDG, a fluorescent glucose analog. These metabolically active tumor cells are confirmed to harbor the same driver oncogenic mutations by Sanger sequencing, followed by high-throughput sequencing to analyze copy number variation profiles. Our results show metastatic tumor cells in pleural effusion have the same driver mutations but different features in copy number variation patterns. The study provides new insights to understand the mechanism of tumor metastasis.

Far-Infrared Optimization of the Fragrance-Improving Process with Temperature and Humidity Control for Green Tea.

In this study, a Box-Behnken design was used to explore the effect of a new technology on green tea fragrance improvement and to optimize fragrance-improving with a bilayer far-infrared fragrance-improving machine with temperature and humidity control. Based on the results of previous single-factor experiments, the main biochemical composition and sensory evaluation scores of the fragrance-improved samples were used as investigation indices. The new fragrance-improving technology was compared with the traditional far-infrared fragrance-improving process, roller pot fragrance improvement, and hot air rotary fragrance improvement. The results show that the optimal parameter combination of the new technology consists of a temperature of 128.00 °C, relative humidity of 70.00 g/h, and transmission speed of 435.00 r/min. With these process parameters, the amino acids, tea polyphenols, flavonoids, soluble sugar, catechins, and caffeine in the fragrance-improved samples reached 3.86%, 32.29%, 5.59%, 4.45%, 8.97%, and 2.75%, respectively. The quality material weight value was 11.72%. The shape, color, taste, and aroma of the fragrance-improved samples made using these parameters were found to be best, with a sensory quality score of 87.40, which is significantly higher than that of other fragrance-improving methods. The energy consumption was 0.19 RMB/kg, which was reduced by more than 50% compared with the other methods, and the production efficiency was more than 30% higher than the traditional methods. This new far-infrared fragrance-improving technology overcomes the yellowish and grayish color of fragrance-improved tea samples that is caused by the traditional fragrance-improving approach, and will provide technical guidance for actual green tea production. PRACTICAL APPLICATION: Our proposed approach innovatively integrates humidity and rotational speed as factors for fragrance improvement in Chinese tea process. The findings of this work provide new technical for fragrance improvement processes.

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.

Single-cell gene variation analysis method for single gland.

Single-cell analysis of heterogeneity has become the cutting-edge technology for profound understandings of relationships between cell populations. At present, common methods used in single cellular genomic research are mainly microfluidic technologies (Fluidigm) or based on microwells, both requiring a uniform size of cells at the entrance. However, the size of cells in specific tissues can vary from type to type. To address this issue, we need to establish a method to identify genomic features of individual cells of different sizes. In this paper, we developed a robust method in the analysis of single cellular genomic mutations among gastric tissues. Briefly, the single gastric gland was isolated from the whole tissue, and further enzymatically digested into single cells of various sizes by trypsin. These single cells were then spread on the polyethylene naphthalene slides and selected by the laser microdissection method. Whole genome amplification (WGA) and capillary electrophoresis were performed subsequently to detect single cell microsatellite. This method enabled us to detect the existence of microsatellite instability (MSI) of each single cell within the intestinal metaplasia, and to carry out a flexible and fine analysis of single cells with different sizes in tissues and glands. This reliable and practical method is well performed in both low and high-throughput genome analysis when combined with cell labeling methods, thus providing a novel and highly flexible way to study tissue heterogeneity on the single cell scale.

Fast isolation and ex vivo culture of circulating tumor cells from the peripheral blood of lung cancer patients.

Circulating tumor cells (CTCs) are free tumor cells shed from tumor site and enter into blood circulation. CTCs represent a reliable source of tumor cells for the molecular characteristics of the original tumor. However, the extraordinary rarity of CTCs makes the subsequent molecular and functional analysis technically challenging. Here, we describe a one-step microfludics-based immunomagnetic isolation method to isolate CTCs directly from the whole blood of lung adenocarcinoma patients. This method avoids harsh sample preparation and enrichment steps, and therefore preserves the viability (>90%) of CTCs during the in vitro isolation. The isolated CTCs are enriched in small volume (80 µL) and cultured ex vivo that leads to successful ex vivo expansion. The expanded CTCs can be frozen and thawed, which shows cell line property. Genetic sequencing on EGFR、KRAS、PIK3CA、TP53 and BRAF and metabolic assay (2-NBDG) are utilized to characterize the expanded CTCs. Our results demostrated that this method is suitable for ex vivo expansion of CTCs facilitates. The genomic, proteomic and metabolic analyses of CTCs have guiding significance in tumor precise treatment.

Genetic diversity and population structure in Bactrocera correcta (Diptera: Tephritidae) inferred from mtDNA cox1 and microsatellite markers.

Bactrocera correcta is one of the most destructive pests of horticultural crops in tropical and subtropical regions. Despite the economic risk, the population genetics of this pest have remained relatively unexplored. This study explores population genetic structure and contemporary gene flow in B. correcta in Chinese Yunnan Province and attempts to place observed patterns within the broader geographical context of the species' total range. Based on combined data from mtDNA cox1 sequences and 12 microsatellite loci obtained from 793 individuals located in 7 countries, overall genetic structuring was low. The expansion history of this species, including likely human-mediated dispersal, may have played a role in shaping the observed weak structure. The study suggested a close relationship between Yunnan Province and adjacent countries, with evidence for Western and/or Southern Yunnan as the invasive origin of B. correcta within Yunnan Province. The information gleaned from this analysis of gene flow and population structure has broad implications for quarantine, trade and management of this pest, especially in China where it is expanding northward. Future studies should concentrate effort on sampling South Asian populations, which would enable better inferences of the ancestral location of B. correcta and its invasion history into and throughout Asia.

[Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer].

Circulating tumor cells (CTCs) are cells that shed from a primary tumor and enter the peripheral blood circulation. The CTCs are closely associated with tumor development and metastasis because of its high heterogeneity. However, there are still no effective methods to detect single-cell heterogeneity of the CTCs. To this end, we developed a method to detect gene mutation in CTCs at the single-cell level and applied it to the detection of EGFR gene mutation in single lung cancer CTC. Specifically, the rare CTCs were captured from blood using an integrated microfluidic system, and then were released into a microchip with thousands of nanoliter wells to isolate single CTC. The single CTC was then transferred into a PCR tube under the microscope for single-cell genome amplification and detection of EGFR gene mutation. We firstly modified chip and capillary and optimized PCR conditions (annealing temperature, number of cycles) using non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H1650 and NCI-H1975 as samples, which showed maximal amplification after 30 cycles with an annealing temperature at 59℃. We then successfully detected blood samples from NSCLC patients using this method. 5 CTCs were obtained from 2 mL patient's blood and the sequencing of EGFR exons 18, 19, 20 and 21 showed no mutations. Our results demonstrated that this method is sensitive enough to detect gene mutation in single CTC and has guiding significance in clinic research.

Description of a new species and record of Bactrocera Macquart (Diptera,Tephritidae) from China.

One new species, Bactrocera (Zeugodacus) anala Chen et Zhou, sp.nov, and one newly recorded species, B. (Z.) armillata (Hering, 1938), from China are described and illustrated. The male of B. (Z.) armillata (Hering) was discovered for the first time and as a result the species is moved from subgenus Bactrocera to subgenus Zeugodacus. In addition, the morphological differences and comparing illustrations of B. (Z.) adusta (Wang et Zhao) and B. (Z.) biguttata (Bezzi), are provided.