Identification and Functional Analysis of Two Alcohol Dehydrogenase Genes Involved in Catalyzing the Reduction of (Z)-3-Hexenal into (Z)-3-Hexenol in Tea Plants (Camellia sinensis).

Alcohol dehydrogenase (ADH) is a vital enzyme in the biosynthesis pathway of six-carbon volatiles in plants. However, little is known about its functions in tea plants. Here, we identified two ADH genes (CsADH1 and CsADH2). An in vitro protein expression assay showed that both CsADH1 and CsADH2 proteins can catalyze the reduction of (Z)-3-hexenal into (Z)-3-hexenol. Subcellular localization revealed that both CsADH1 and CsADH2 proteins were predominantly localized in the nucleus and cytosol. CsADH1 had high transcripts in young stems in autumn, while CsADH2 showed extremely high expression levels in stems and roots. The expression of CsADH2 was mainly downregulated under ABA treatment, while CsADH1 and CsADH2 transcripts were significantly lower under MeJA treatment at 12 and 24 h. Under cold treatment, CsADH1 transcripts first decreased and then increased, while CsADH2 demonstrated an almost opposite expression pattern. Notably, CsADH2 was significantly upregulated under simulated Ectropis obliqua invasion. Gene suppression by antisense oligonucleotides (AsODNs) demonstrated that AsODN_ADH2 treatment significantly reduced CsADH2 transcripts and the abundance of (Z)-3-hexenol products. The results indicate that the two CsADH genes may play an important role in response to (a)biotic stresses and in the process of (Z)-3-hexenol biosynthesis.

Weakly supervised learning analysis of Aß plaque distribution in the whole rat brain.

Alzheimer's disease (AD) is a great challenge for the world and hardly to be cured, partly because of the lack of animal models that fully mimic pathological progress. Recently, a rat model exhibiting the most pathological symptoms of AD has been reported. However, high-resolution imaging and accurate quantification of beta-amyloid (Aß) plaques in the whole rat brain have not been fulfilled due to substantial technical challenges. In this paper, a high-efficiency data analysis pipeline is proposed to quantify Aß plaques in whole rat brain through several terabytes of image data acquired by a high-speed volumetric imaging approach we have developed previously. A novel segmentation framework applying a high-performance weakly supervised learning method which can dramatically reduce the human labeling consumption is described in this study. The effectiveness of our segmentation framework is validated with different metrics. The segmented Aß plaques were mapped to a standard rat brain atlas for quantitative analysis of the Aß distribution in each brain area. This pipeline may also be applied to the segmentation and accurate quantification of other non-specific morphology objects.

High Internal Phase Emulsions Synergistically Stabilized by Sodium Carboxymethyl Cellulose and Palm Kernel Oil Ethoxylates as an Essential Oil Delivery System.

High internal phase emulsions (HIPEs) with an internal phase fraction of 84 vol % were prepared using carboxymethyl cellulose (CMC) and palm kernel oil ethoxylates (SOE-N-60) as a dual emulsifier. Effects of the oil-phase volume fraction, CMC concentration, and SOE-N-60 concentration on oil-in-water HIPEs stability were systematically studied by a Mastersizer 2000 instrument, Lx POL polarizing microscope, rheometer, etc. The bioavailability of tea tree oil can be effectively protected using HIPEs as a delivery system. The experimental results showed that, with the increase of the concentrations of CMC and SOE-N-60, the droplet size of HIPEs gradually decreases and the HIPEs showed good static stability. In addition, it was observed by scanning electron microscopy that the polyHIPEs materials using HIPEs stabilized by different SOE-N-60 and CMC concentrations as templates had different structures. Moreover, the synergism between CMC and SOE-N-60 surfactants plays a significant role in the preparation and stability of HIPEs.

The Biosynthesis of Main Taste Compounds Is Coordinately Regulated by miRNAs and Phytohormones in Tea Plant (Camellia sinensis).

Based on the abundance of taste compounds in leaves at different leaf positions on the same shoot, green tea made from one bud and one leaf, or even just one bud, has the best quality. To elucidate the mechanism underlying the regulation of the biosynthesis of these compounds, we profiled the metabolome, transcriptome, sRNA, degradome, and WGCNA using leaves from five leaf positions of shoots. Through this analysis, we found 139 miRNA-target pairs related to taste compound biosynthesis and 96 miRNA-target pairs involved in phytohormone synthesis or signal transduction. Moreover, miR166-HD-ZIP, miR169-NF-YA, IAA, ZA, ABA, and JA were positively related to the accumulation of gallated catechin, caffeine, and theanine. However, miR396-GRF, miR393-bHLH, miR156-SBP, and SA were negatively correlated with these compounds. Among these important pairs, the miR396-GRF and miR156-SBP pairs were further validated by using qRT-PCR, Northern blots, and cotransformation. This is the first report describing that miRNA-TF pairs and phytohormones might synergistically regulate the biosynthesis of taste compounds in the leaves of tea plants.

Hematological factors associated with immunity, inflammation, and metabolism in patients with systemic lupus erythematosus: Data from a Zhuang cohort in Southwest China.

INTRODUCTION: Hematological parameters play important role in multiple diseases. This study was to investigate the possible association of the routine hematological parameters involved in immunity, inflammation, and metabolism with systemic lupus erythematosus (SLE) in patients of Zhuang ethnicity in Guangxi, southwest China. METHODS: The medical records of 195 Zhuang SLE patients between January 2013 and November 2018 were retrospectively reviewed. Random forest algorithm and multivariate logistic regression were used to identify the feature hematological parameters in patients with SLE. Association rules were explored between each parameter and immunity- (IgG, IgA, IgM, C3, and C4), inflammation- (ESR, hs-CRP, and CAR), and metabolism- (TG, TC, HDL-C, LDL-C, TP, PA, ALB, and UA) related indexes. RESULTS: Random forest algorithm and logistic regression analysis showed that neutrophil-to-lymphocyte ratio (NLR), red blood cell distribution width (RDW), and platelet-to-lymphocyte ratio (PLR) were the feature parameters for distinguishing SLE patients from healthy controls. According to the ROC curves, the optimal cutoff values to predict SLE were 1.98 for NLR, 13.35 for RDW, and 145.64 for PLR. Association rule analysis showed that NLR was strongly associated with C3, hs-CRP, TG, ALB, and UA; RDW was strongly associated with C3, C4, hs-CRP, TG, and ALB; PLR was strongly associated with IgG, hs-CRP, HDL-C, and UA. CONCLUSIONS: Neutrophil-to-lymphocyte ratio, RDW, and PLR may serve as effective predictors of dysregulation in immunity, inflammation, and metabolism. These three indicators may be potential for cardiovascular risk assessment in Zhuang SLE patients in southwest China.

Comprehensive co-expression analysis provides novel insights into temporal variation of flavonoids in fresh leaves of the tea plant (Camellia sinensis).

Flavonoids are the major class of characteristic secondary compounds in Camellia sinensis that affect quality of tea. However, the temporal variation and the underlying regulatory mechanism of flavonoid biosynthesis during different growth months require a further investigation. Here, we combined analyses of the metabolomics and transcriptomics to tea leaves freshly collected during five different months for a comprehensive understanding of flavonoid metabolism regulation in tea plants. Through loading plot analysis, significant changes in the contents of metabolites during growing months were discovered, and further co-expression and association analysis indicated that one flavone glycoside (naringenin-7-O-glucoside) and two flavonol glycosides (quercetin-3-O-galactoside and kaemferol-3-O-(6″-O-p-courmaroyl)-glucoside) were evaluated as growth markers, which may explain the high bitterness and astringency of August teas; additionally, the high levels of two flavan-3-ols (gallocatechin and catechin gallate) may contribute to the flavor formation of April tea. Meanwhile, multiple flavonoid-related structural genes, MYB and bHLH transcription factors exhibit specific expression patterns to modulate the biosynthesis of these key flavonoids. A co-expression regulatory sub-network was constructed based on profiles of differentially expressed genes; one CsbHLH and six transcription factors (three CsbHLHs and three CsMYBs) exhibited negative and positive roles in the regulation of flavonoid biosynthetic genes, respectively. Taken together, our results provide new insights into the regulation of principle flavonoids for unique flavor of tea regulated by many flavonoid-related structural genes and transcription factors during different growth months.

Revealing of MicroRNA Involved Regulatory Gene Networks on Terpenoid Biosynthesis in Camellia sinensis in Different Growing Time Points.

Tea, made from leaves of Camellia sinensis, has long been consumed worldwide for its unique taste and aroma. Terpenoids play important roles not only in tea beverage aroma formation, but also in the productivity and quality of tea plantation due to their significant contribution to light harvesting pigments and phytohormones. To date, however, the regulation of terpenoid synthase genes remains unclear. Herein, the analyses of metabolomics, sRNAs, degradome, and transcriptomics were performed and integrated for identifying key regulatory miRNA-target circuits on terpenoid biosynthesis in leaf tissues over five different months in which the amount of terpenoids in tea leaves varies greatly. Four classes of miRNA-TF pairs that might play a central role in the regulation of terpenoid biosynthesis were also uncovered. Ultimately, a hypothetical model was proposed that mature miRNAs maintained by light regulator at both the transcriptional and posttranscriptional levels negatively regulate the targets to control terpenoid biosynthesis.

Characterization and Alternative Splicing Profiles of the Lipoxygenase Gene Family in Tea Plant (Camellia sinensis).

Oxylipins, including jasmonic acid (JA) and volatiles, are important for signaling in plants, and these are formed by the lipoxygenase (LOX) enzyme family. There is a large gap in understanding of the underlying molecular basis of their roles in tea plants. Here, we identified 11 CsLOX genes from the tea plant (Camellia sinensis), and characterized their phylogeny, gene structure and protein features into three subclasses. We then examined their enzymatic activities, LOX expression and alternative splicing of transcripts during development and in response to abiotic or biotic stresses in tea plants. In vitro expressed protein assays showed that the CsLOX2, 3 and 9 enzymatically function to produce 9/13-HPOT, 13-HPOT and 9-HPOT, respectively. CsLOX2 and CsLOX9 green fluorescent protein (GFP) fusion proteins localized to chloroplasts and the cytoplasm, respectively. RNA sequencing, quantitative reverse transcription-PCR and Northern blot analysis suggested that CsLOX5, 6 and 9 were predominantly expressed in seeds, flowers and roots, respectively. CsLOX2, 3, 4, 6 and 7 were up-regulated after attack by the insect Ectropis oblique, while CsLOX1 was induced after infection with the pathogen Glomerella cingulata. CsLOX3, 7 and 10 were up-regulated by JA but not ABA or salicylic acid. Long-term cold stress down-regulated CsLOX expression while a short duration of cold induced the expression of CsLOX1, 6 and 7. Alternatively spliced transcripts of six CsLOX genes were dynamically regulated through time and varied in relative abundances under the investigated stresses; we propose a mechanism of competing or compensating regulation between isoforms. This study improves our understanding of evolution of LOXs and regulation of their diverse functions in plants.

GSTM1 and GSTT1 Gene Polymorphisms, Gene-Gene Interaction, and Esophageal Carcinoma Risk: Evidence from an Updated Meta-Analysis.

BACKGROUND: Published data regarding the association between GSTM1 and/or GSTT1 gene polymorphisms and esophageal cancer (EC) susceptibility remain inconclusive. To clarify these associations, a meta-analysis was conducted. METHODS: We conducted a comprehensive search in PubMed, Embase, and the China National Knowledge Infrastructure (CNKI) for all such manuscripts published as of May 1, 2017. The pooled odds ratio (ORs) with confidence intervals (95% CI) were estimated for each study to assess the strength of the association. A subgroup analysis, a sensitivity analysis, and a publication bias analysis were also performed. RESULTS: Data from 41 studies comprising 5291 EC cases and 8191 controls were available for analysis of the GSTM1 polymorphism, and data from 31 studies comprising 4330 EC cases and 6558 controls were available for analysis of the GSTT1 polymorphism. Analyses of the GSTM1 polymorphisms demonstrated that there was a significantly increased EC risk in GSTM1 null genotype carriers (OR = 1.319, 95% CI = 1.125-1.546, p for heterogeneity <0.001). In subgroup analyses by ethnicity, and categories of EC, a significantly increased EC risk was found in Asians (OR = 1.457, 95% CI = 1.212-1.751, p for heterogeneity <0.001) and patients whose histological type was unknown. Analyses of the GSTT1 polymorphisms indicated a positive correlation between the GSTT1 null genotype and the EC risk (OR = 1.233, 95% CI = 1.044-1.455, p for heterogeneity <0.001). In subgroup analyses stratified by ethnicity and categories of EC, similar statistical associations were observed in Asians, esophageal squamous cell carcinoma (ESCC) patients, and ESCC on Asian population. In the GSTM1-GSTT1 interaction analysis, we discovered remarkably enhanced EC risk for patients with the GSTM1 and GSTT1 dual null genotypes (OR = 1.962, 95% CI = 1.178-3.268, p for heterogeneity <0.001) compared with the reference GSTM1 and GSTT1 dual positive genotype. CONCLUSIONS: We conclude that the GSTM1 and GSTT1 null genotypes are associated with increased genetic susceptibility to EC in the overall human population, particularly among Asians. In addition, our findings suggest that persons with a null genotype for both the GSTM1 and GSTT1 genes are at higher risk of developing EC. Further well-designed studies are needed to confirm these associations.

[The relationship between p120ctn translocation and malignant features of hepatocellular carcinoma].

OBJECTIVE: To investigate the effect of catenin p120 (p120ctn) translocation on the malignant features of hepatocellular carcinoma and its interrelation with beta-catenin in E-cadherin-mediated cell signaling. METHODS: Expression and translocation of p120ctn, tyrosine phosphorylation, and its binding capacity to E-cadherin were detected by DNA transfection, immunoblotting and immunoprecipitation. Cellular localization of p120ctn and beta-catenin was detected by immunofluorescent microscopy. Cell adhesion, cell migration and cell proliferation were also studied. RESULTS: Expression of p120ctn increased after cells transfected with p120ctn isoform 3A, and it was located mainly at cell-cell contact region. Its binding to E-cadherin was enhanced. After EGF stimulation, tyrosine phosphorylation of p120ctn was increased, membrane expression of p120ctn and beta-catenin was decreased while cytosol expression was increased. It was translocated into the nucleus, cell adhesiveness was increased but mobility decreased. With over-expression of p120ctn, beta-catenin was recruited by nucleus export. Cell proliferation was reduced but it was increased after EGF treatment. CONCLUSION: p120tn plays an important role in cell adhesion, migration and proliferation of hepatocellular carcinoma, and its tyrosine phosphorylation might contribute to this mechanism. There might be a competitive relationship between p120ctn and beta-catenin.

[p120(ctn) tyrosine phosphorylation are involved in the biologic behavior changes of hepatocellular carcinoma cells].

OBJECTIVE: In hepatocellular carcinoma cells, the tyrosine phosphorylation of p120(ctn) was stimulated by epidermal growth factor (EGF) to investigate the relationship between the tyrosine phosphorylation of p120(ctn) and the translocation of p120(ctn), also the relationship between the tyrosine phosphorylation of p120(ctn) and the biological behaviour of hepatocellular carcinoma cells. The role of p120(ctn) in the cell adhesion and signaling of hepatocellular carcinoma is to be investigated. METHODS: In BEL-7404 human hepatocellular carcinoma cells, the tyrosine phosphotyrosine of p120(ctn) stimulated by EGF were detected by immunoprecipitation (IP) and Immunoblotting (IB). The cellular distribution and translocation of p120(ctn) and beta-catenin were detected and examined by indirect intracellular immunofluorescence. Cell morphology and cell adhesion potential were also detected using correspondent methods. Antisense nucleotide of p120(ctn) was transfected into BEL-7404 cells. RESULTS: The tyrosine phosphorylation of p120(ctn) was enhanced after EGF treatment than control, especially at 20min after EGF treatment; When BEL-7404 cells were transfected with antiseuse nucleotide of p120(ctn) before EGF treatment, the tyrosine phosphorylation of p120(ctn) stimulated by EGF was obviously lowered. We also observed that the tyrosine phosphorylation of p120(ctn) stimulated by EGF was accompanied by the nuclear translocation of p120(ctn); the similar translocation was also observed in beta-catenin after EGF stimulation. At the meantime, cell adhesion potential was reduced after EGF treatment and cell morphology became thin, elongated and irregular, speudopods increased. CONCLUSIONS: In BEL-7404 cells,the tyrosine phosphorylation of p120(ctn) could be stimulated by EGF, which was accompanied by the nuclear accumulation of p120(ctn). The tyrosine phosphorylation of p120(ctn) stimulated by EGF was also in correlation with the changes of cell adhesion and cell morphology. The results indicated that the tyrosine phosphorylation of p120(ctn) cell correlated with the translocation of p120(ctn) and the biological behavior of cells.

[Expression of P120 catenin in the cell lines and tissues of normal liver and hepatocellular carcinoma].

OBJECTIVE: To explore P120 catenin expression in normal liver cell line, hepatocellular carcinoma cell line, normal liver tissue and hepatocellular carcinoma tissues, further exploring the relationship-between catenin and hepatocellular carcinoma. METHODS: Reverse transcript polymerase chain reaction (RT-PCR), immunofluorescence microscopy and immunohistochemistry methods were used to detect P120 catenin expression in the cells and tissues mentioned above. RESULTS: RT-PCR results showed that P120 catenin 1A and 3A isoform mRNA can be detected in all the cells and tissues. Immunofluorescence microscopy and immunohistochemistry results showed that P120 catenin expressed at cell to cell contact region and at cell membrane in normal liver cells and in normal liver tissues, while in hepatocellular carcinoma cell lines and in hepatocellular carcinoma tissues, the molecule expression increased in cytosol, membrane expression reduced, and there was nuclear translocation. CONCLUSION: Hepatocellular carcinoma was found to have relation with abnormal P120 catenin expression in this study, which suggested the process of abnormal cell signaling and adhesion functions during the onset and development of hepatocellular carcinoma.