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The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.

Jinmudan (JM) tea, produced using Camellia sinensis, cultivar JM, is popular among consumers because of its strong floral and fruity aroma. Aroma profiles of white, green, black, and oolong teas, which were obtained from JM and Tanyangcaicha (TY, control and indigenous variety), were investigated. Sensory evaluation was used to describe the quality characteristics of the tea samples. A combination of headspace solid-phase microextraction, gas chromatography–mass spectrometry (HS-SPME/GC–MS), and chemometrics was used to extract and analyze the volatiles in the samples; 162 volatiles were identified and categorized on the basis of their chemical structures. The proportions of chemical categories differed among the teas. The same type of tea obtained from JM and TY differed significantly in concentrations and categories of volatiles. This study will improve our understanding of the profile of volatile compounds in JM teas and create a basis for investigating the biological characteristics of JM teas.

Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger (P-Rex) proteins control many fundamental cellular functions including cell migration, actin cytoskeletal rearrangement and adhesion in many cancers. However, P-Rex1 expression and its prognostic effect and possible clinical value are not clearly elucidated in human oral squamous cell carcinoma (OSCC). Here, OSCC tissue microarrays were used to verify the expression levels of P-Rex1, coinhibitory immune checkpoints and tumor associated macrophage (TAM) markers, and to analyze the relationship between PREX1 expression levels and clinicopathological characteristics in OSCC. The study found that P-Rex1 expression was elevated in OSCC compared to dysplasia and normal mucosa (P

Objective Adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC) are the most frequent malignancies in the salivary glands. The purpose of this study was to explore the roles of sineoculis homeobox homolog 1 (SIX1), malic enzyme 2 (ME2) and AP-2 complex subunit mu (AP2M1) in AdCC and MEC, as well as the potential relationship between SIX1, ME2, AP2M1 and proliferation marker cyclin D1. Material and methods Immunohistochemistry was performed on human salivary gland tissue microarrays that contained 76 normal salivary glands (NSGs), 14 pleomorphic adenomas (PMAs), 81 AdCCs and 52 MECs. Results We observed that the expression levels of SIX1 and ME2 were significantly elevated in AdCC and MEC when compared with NSG and PMA. In addition, we detected that AP2M1 was overexpressed in AdCC and MEC when compared with NSG. We also found that SIX1 and AP2M1 were positively associated with cyclin D1. Conclusions These results may prove that SIX1 and AP2M1 are involved in the proliferation of AdCC and MEC to cause tumor growth.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future., Design Type(s)sequence assembly objective • sequence annotation objectiveMeasurement Type(s)whole genome sequencing assay • BAC • transcription profiling assayTechnology Type(s)DNA sequencing • RNA sequencingFactor Type(s)developmental stageSample Characteristic(s)Camellia sinensis var. sinensis • leaf • apical bud • stem • flower • fruit • root Machine-accessible metadata file describing the reported data (ISA-Tab format)

Expression of the family with sequence similarity 3 member C (FAM3C) is necessary for the epithelial-mesenchymal transition (EMT). However, the expression level and clinicopathological significance of FAM3C in oral squamous cell carcinoma (OSCC) has not been thoroughly elucidated to date. We performed immunohistochemical staining on human OSCC specimens with FAM3C, co-inhibitory immune checkpoints, EMT markers, and cancer stem cells (CSCs) markers to analyze the expression levels and clinicopathological features of FAM3C in OSCC. There were 210 primary OSCC specimens, 69 oral epithelial dysplasia and 42 normal oral mucosae in our human OSCC tissue microarrays cohort. We observed that FAM3C expression was upregulated in OSCC compared with normal mucosa and epithelial dysplasia (P 0.001). Moreover, patients with higher FAM3C expression levels had a worse prognosis than those with lower expression levels (P0.05). Also, FAM3C expression was positively correlated with the immune checkpoints PD-L1, VISTA, and B7-H4, the EMT marker Slug and the CSC markers SOX2 and ALDH1. In conclusion, these findings suggested that overexpression of FAM3C in human OSCC may predict a poor prognosis for OSCC patients.

While caffeine is one of the most important bioactive metabolites for tea as the most consumed non-alcohol beverage, its biosynthesis and catabolism in tea plants are still not fully understood. Here, we integrated purine alkaloid profiling and transcriptome analysis on shoot tips and roots fed with caffeine, theophylline, or theobromine to gain further understanding of caffeine biosynthesis and degradation. Shoot tips and roots easily took up and accumulated high concentrations of alkaloids, but roots showed much faster caffeine and theophylline degradation rates than shoot tips, which only degraded theophylline significantly but almost did not degrade caffeine. Clearly feedback inhibition on caffeine synthesis or inter-conversion between caffeine, theophylline, and theobromine, and 3-methylxanthine had been observed in alkaloids-fed shoot tips and roots, and these were also evidenced by significant repression of TCS and MXMT genes critical for caffeine biosynthesis. Among these responsively repressed genes, two highly expressed genes TCS-4 and TCS-8 were characterized for their enzyme activity. While we failed to detect TCS-4 activity, TCS-8 displayed N-methyltransferase activities towards multiple substrates, supporting the complex metabolic network in caffeine biosynthesis in tea plants since at least 13 TCS-like N-methyltransferase genes may function redundantly. This study provides new insight into complex metabolic networks of purine alkaloids in tea plants.

Global warming has multifarious effects on crop growth and productivity. Nonetheless, the effects of moderate-high temperatures and melatonin on tea yield and quality remain unclear. In this study, we found that melatonin, a universal growth stimulatory molecule, not only promotes photosynthesis and biomass accumulation in tea plants (Camellia sinensis L.) but also improves tea quality under sub high temperature (SHT). SHT increased the dry biomass and photosynthesis by 40.8% and 28.1%, respectively, and exogenous melatonin caused a further improvement. Moreover, SHT increased the total polyphenol concentrations and decreased the free amino acid concentrations, leading to a significant increase (68.2%) in polyphenol to free amino acid ratio. However, melatonin decreased the polyphenol to free amino acid ratio by delicately improving the concentrations of polyphenols and amino acids. Consistent with the total polyphenol, melatonin increased the concentrations of (-)-catechin, (-)-gallocatechin (GC), and (-)-epigallocatechin-3-gallate (EGCG) in tea leaves. The qRT-PCR analysis revealed that melatonin increased the transcript levels of catechins biosynthesis genes, such as CsCHS, CsCH1, CsF3H, CsDFR, CsANS, CsLAR, and CsANR under SHT. Meanwhile, the theanine concentration was decreased by SHT, which was attributed to the attenuated expression of CsGS, CsGOGAT, CsGDH, and CsTS1. Nonetheless, melatonin significantly increased those transcripts and the content of theanine under SHT. Melatonin also increased the caffeine content by inducing the expression of CsTIDH, CssAMS, and CsTCS1. These results suggest that melatonin could positively alter tea growth and quality by modulating the photosynthesis and biosynthesis of polyphenols, amino acids, and caffeine in tea leaves under SHT.

Tea plant is an important economic crop, which is used to produce the world's oldest and most widely consumed tea beverages. Here, we present a high-quality reference genome assembly of the tea plant (Camellia sinensis var. sinensis) consisting of 15 pseudo-chromosomes. LTR retrotransposons (LTR-RTs) account for 70.38% of the genome, and we present evidence that LTR-RTs play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns. Genes, particularly those coding for terpene biosynthesis proteins, associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome. Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations, supporting the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction. Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome, particularly those associated with tea quality and stress resistance. The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cultivars of tea plants.

Aim: Immunohistochemistry was used to detect the expression of Stathmin 1 and Serine 38 phospho-Stathmin 1 (p-Stathmin 1S38) in head and neck squamous cell carcinoma (HNSCC) and research its correlation with clinical parameters, survival and expression of immune checkpoint molecules. Results: Stathmin 1 and p-Stathmin 1S38 overexpression in primary HNSCC is associated with poor overall survival. Stathmin 1 expression is related to tumor size, category and lymph node status. Stathmin 1 expression correlates with PD-L1, TIM3, VISTA, B7-H3, B7-H4, LAG-3 and p-STAT3 expression in HNSCC. P-Stathmin 1S38 expression correlates with PD-L1, VISTA, B7-H4, LAG-3 and p-STAT3 in HNSCC. Conclusion: We found expression of Stathmin 1 and p-Stathmin 1S38 indicates poor survival in HNSCC and may be associated with immune suppression.

The immune system plays a critical role in the establishment, development, and progression of head and neck squamous cell carcinoma (HNSCC). As treatment with single-immune checkpoint agent results in a lower response rate in patients, it is important to investigate new strategies to maintain favorable anti-tumor immune response. Herein, the combination immunotherapeutic value of CTLA4 blockade and SFKs inhibition was assessed in transgenic HNSCC mouse model. Our present work showed that tumor growth was not entirely controlled when HNSCC model mice were administered anti-CTLA4 chemotherapeutic treatment. Moreover, it was observed that Src family kinases (SFKs) were hyper-activated and lack of anti-tumor immune responses following anti-CTLA4 chemotherapeutic treatment. We hypothesized that activation of SFKs is a mechanism of anti-CTLA4 immunotherapy resistance. We, therefore, carried out combined drug therapy using anti-CTLA4 mAbs and an SFKs' inhibitor, dasatinib. As expected, dasatinib and anti-CTLA4 synergistically inhibited tumor growth in Tgfbr1/Pten 2cKO mice. Furthermore, dasatinib and anti-CTLA4 combined to reduce the number of myeloid-derived suppressor cells and Tregs, increasing the CD8+ T cell-to-Tregs ratio. We also found that combining dasatinib with anti-CTLA4 therapy significantly attenuated the expression of p-STAT3Y705 and Ki67 in tumoral environment. These results suggest that combination therapy with SFKs inhibitors may be a useful therapeutic approach to increase the efficacy of anti-CTLA4 immunotherapy in HNSCC.

IQ-domain GTPase-activating protein 1 (IQGAP1) is associated with the development and progression of many human cancers. We aimed to investigate the expression and clinicopathological significances of IQGAP1 in head and neck squamous cell carcinoma (HNSCC). In this study, immunohistochemical staining of IQGAP1, co-inhibitory immune checkpoint molecules and macrophage markers were performed in human HNSCC samples to analyze the expression and correlation with clinicopathological characteristics. Immunoreactivity of IQGAP1 was also detected in immunocompetent mouse HNSCC tissue. We found that IQGAP1 expression level was significantly increased in human HNSCC compared with dysplasia and normal mucosa, and the expression of IQGAP1 in HNSCC was positively associated with advanced lymph node status. Besides, our data indicated that patients with higher IQGAP1 expression exhibited poor overall survival compared with patients with lower IQGAP1 expression. Furthermore, this study demonstrated that IQGAP1 expression was positively associated with TIM3, Galectin-9 (TIM3 ligand), B7H4, macrophage markers CD68 and CD163. In conclusion, these findings suggest that over-expression of IQGAP1 in human HNSCC may indicate poor prognosis.

Background T-cell immunoglobulin mucin 3 (TIM3) is a negative immune checkpoint and plays a crucial part in tumor-induced immune suppression. However, the mechanism of TIM3 in regulating immunosuppression in head and neck squamous cell carcinoma (HNSCC) was still not quite clear. Methods We carried out the immunohistochemistry staining of HNSCC tissue microarrays. Through quantification of the histoscore, we performed the correlation analysis among the TIM3, Galectin-9, Foxp3, CD68 and CD163. The effects of TIM3 on regulatory T cells (Tregs) and macrophages were detected by utilizing the Tgfbr1/Pten 2cKO HNSCC mouse model. Flow cytometry were used to analysis the percent of Tregs, macrophages and IFN-γ. Results We demonstrated the close association among TIM3/Galectin-9 pathway, regulatory T cell marker (Foxp3) and macrophage marker (CD68, CD163) in human HNSCC. In the transgenic HNSCC mouse model, blockade of TIM3 by the anti-TIM3 monoclonal antibody induced a reduction of CD4+CD25+Foxp3+ Tregs. Meanwhile, the population of TIM3+ Tregs was also decreased. However, the population of CD206+ macrophages was not significantly declined. The increased IFN-γ production on CD8+ T cells in anti-TIM3 treatment mice showed that the antitumor immune response was enhanced through suppression of these negative immune factors. Conclusions The present study demonstrated that TIM3 was associated with the immunosuppression in HNSCC. And targeting TIM3 can enhance anti-tumor immune response by decreasing Tregs in HNSCC.

The upregulation of co-inhibitory immune checkpoints hampers the immune response toward tumor cells and facilitates the tumor cells ability to evade immunosurveillance. Specific inhibitory immune checkpoint delivers inhibitory signals to T cells using multiple mechanisms. More in-depth understanding of the co-inhibitory immune checkpoints could be exploited for head and neck squamous cell carcinoma (HNSCC) treatment. In this review, we summarize the expression and the mechanism of partial co-inhibitory immune checkpoint signals and discuss targeting co-inhibitory immune checkpoints as an immunotherapeutic target for cancer therapy. This review may provide a better understanding of the co-inhibitory immune checkpoints and could promote applications of immunotherapy.

Amino acids are constituents of proteins, precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. However, the amino acid transport in tea plant is rarely reported. In this study, six cationic amino acid transporter (CAT) family genes were cloned. Phylogenetic analysis categorized these CsCATs into four subgroups. These CsCATs all contain the 12–14 transmembrane domains and the conserved CAT motifs. Their expression was tissue-specific, with higher expression levels in root and stem and correlated to the abundances of key free amino acids such as Theanine. Some CsCATs expression responded to some abiotic stress conditions and to the exogenous application of theanine (Thea), glutamine or ethylamine hydrochloride, an ethylamine precursor for Thea biosynthesis. Our results indicated that the CsCATs expression is regulated by amino acid contents and is sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants.

Phenolic compounds, such as flavanols, flavones, flavonols, anthocyanins, and phenolic acids, contribute to a plant’s adaptation to their ecological niches. These compounds serve as attractants of organisms beneficial to the plants or as defence against other biotic and abiotic factors during the plant evolution. In this paper, the accumulation patterns of phenolic compounds in the eight evergreen woody core eudicots indicated divergent and convergent evolution of plant secondary metabolites. The results indicated some phenolic acids and flavonols were distributed in all the studied eight evergreen woody plants, within Oleaceae, Theaceae, Caprifoliaceae and Celastraceae, with different accumulation profiles. The flavan-3-ols, monomeric catechins, and polymeric proanthocyanidins, were undetectable in Osmanthus fragrans, Ligustrum lucidum, and Fraxinus mariesii of Oleaceae, but highly accumulated in the other five species, especially in Camellia sinensis of Theaceae. It indicated Camellia sinensis evolved great ecological adaptive capacity to prevent herbivores, insect pests, and microbial pathogens.

Immature myeloid cells including myeloid‐derived suppressor cells (MDSCs) and tumour‐associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7‐H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7‐H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7‐H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7‐H3 blockade as a future therapeutic strategy to treat patients with HNSCC.

T‐cell immunoglobulin mucin 3 (TIM3) contributes to immune suppression during progression of many cancers, but the precise role of TIM3 in head and neck squamous cell carcinoma (HNSCC) is not clearly understood. In this study, we report that TIM3 expression was significantly up‐regulated in patients with HNSCC and associated with lymph node metastasis. Additionally, TIM3 expression was increased in patients with recurrent HNSCC and patients with preradiotherapy or prechemotherapy. We also characterized CD8+ T cells and CD11b+ CD33+ myeloid‐derived suppressor cells (MDSCs) in human HNSCC, and found that their expression was positively correlated with TIM3 expression. To determine the underlying mechanism of TIM3 in immune response during HNSCC progression, we utilized the Tgfbr1/Pten 2cKO HNSCC mouse model with TIM3 overexpression. Treatment with anti‐TIM3 monoclonal antibody effectively suppressed tumor growth through restoring effector T‐cell function by targeting CD4+ TIM3+ cells and CD8+ TIM3+ cells and decreasing MDSCs. Our findings demonstrate TIM3 expression in patients with HNSCC and suggest anti‐TIM3 immunotherapy as a novel therapeutic approach for effective treatment of HNSCC.

Caffeine (Cf, 1,3,7-trimethylxanthine), a major secondary metabolite of many higher plants, is widely used in popular non-alcoholic beverages, and in the pharmaceutical and health industries. Currently, this valuable chemical is mainly manufactured by chemical synthesis. In this study, we developed a novel approach for de novo caffeine production in metabolically engineered Escherichia coli. Xanthine-to-caffeine conversion was first achieved by the expression of a plant-derived gene encoding tea caffeine synthase (TCS1). Caffeine accumulation was then increased using two metabolic strategies: higher-level expression of the target enzymes, and enhancement of xanthine and S-adenosyl-L-methionine biosynthesis. The final strain (BL21/pRSF-eCS1-SAM2-vgb-eGUD1) produced up to 21.46 ± 1.03 mg L−1 caffeine from 20 g L−1 of glucose in shake flask culture, yielding caffeine up to 2.96 mg g−1 glucose, which represents the highest titer of caffeine produced by fermentation reported to date. This novel microbial conversion also represents an innovative approach to produce value-added methylxanthine chemicals from cheap carbon sources.

In addressing caffeine toxicity to the producing cells, engineering a transporter that can move caffeine from cytoplasm across the cell membrane to the extracellular space, thus enhancing caffeine resistance and potentially increasing the yield in yeast, is important. An ABC-transporter bfr1 from Schizosaccharomyces pombe was cloned and transformed into S. cerevisiae, resulting in enhancing caffeine resistance. Afterward, a library of randomly mutagenized bfr1 mutants through error-prone PCR was generated. One mutant was identified with drastically increased caffeine resistance (15 mg/mL). Sequencing and structural analysis illustrated that many of the mutations occurred at the cytosolic domain. Site-directed mutagenesis of these mutations confirmed at least one amino acid that conferred enhancing caffeine resistance in the mutated bfr1. These data demonstrated engineering ABC-transporters can be an efficient way to reduce product toxicity in heterologous systems.

BACKGROUND: Non‐volatile compounds play a key role in the quality and price of Keemun black tea (KBT). The non‐volatile compounds in KBT samples from different producing areas normally vary greatly. The development of rapid methods for tracing the geographical origin of KBT is useful. In this study, we develop models for the discrimination of KBT's geographical origin based on non‐volatile compounds. RESULTS: Seventy‐two KBT samples were collected from five towns in Anhui province to determine 13 KBT compounds by high‐performance liquid chromatography (HPLC). Analysis of variance showed that the content of 13 compounds in KBT indicated significant differences (P

Tea is a natural nonalcoholic beverage consumed worldwide. And grafting technique has been widely used to improve tea quality and quantity. Camellia sinensis cv. Xianghongdian 1 is an excellent variety of Lu’an guapian tea in China, because of its clones, early sprouting, high yielding, superior quality, and strong stress resistance characters. In this study, we grafted shoots from Xianghongdian 1 onto the rootstocks of low-yielding indigenous tea plants cv. Qimenzhong to improve their varietal characteristics. In order to investigate the variations of metabolites and transcription patterns in the grafted tea plants, the leaves were collected from 1-year-old grafted tea plants, whereas the leaves from scions and rootstocks were served as controls. The metabolic profiles and the gene expression patterns in the target pathways were determined and correlated. The contents of caffeine and total catechins were both higher in the grafting leaf compared with those in scion leaf and rootstock leaf, while the total free amino acid content was lower. All these metabolic changes were correlated to the variations in the transcripts expression patterns. It indicated a feasibility of enhancing the agronomic and economic traits in the short-term grafting tea plants through asexual propagation. And the long-term effects should undergo further investigation.

Objectives Dysregulation of immune cells in the tumor microenvironment is a hallmark of head and neck squamous cell carcinoma (HNSCC). Increased infiltration of pDCs has been reported in the microenvironment of HNSCC. However, the precise immunological role of pDC and the therapeutic effects of pDC depletion in HNSCC need to be further investigated. Materials and methods CD317 antibodies were applied for depleting pDCs in an immunocompetent transgenic HNSCC mouse model. Tumor volume was monitored. Flow cytometric analysis was conducted for studying the immune profile changes after pDC depletion. In addition, immunohistochemical staining was carried out in a human HNSCC tissue microarray for detecting the infiltration of pDCs. We also analyzed the survival implication of pDCs and its correlation with other immune related markers in human HNSCC. Results pDC depletion in the transgenic HNSCC mouse model significantly delayed tumor growth. After pDCs were depleted, T cells were markedly revitalized, and the proportions of regulatory T cells (Tregs) and monocytic myeloid-derived suppressor cells (MDSCs) were decreased. In human HNSCC microenvironment, pDC infiltration was upregulated and its high infiltration conferred a poor prognosis. Moreover, pDC infiltration was closely correlated with the expression of Foxp-3, PD-1, TIM-3, and LAG-3. Conclusion Our findings demonstrated that pDCs play a negative immunomodulatory role in HNSCC and may present as a target for effective immunotherapy.

Background Keemun black tea (KBT) is one of the most popular tea beverages in China as a result of its unique flavor and potential health benefits. The geographical origin of KBT influences its quality and price. The present study aimed to apply a head-space solid phase microextraction approach and gas chromatography-mass spectrometry combined with chemometric analysis to profile the volatile compounds of KBT collected from five production areas. Results Thirty-one peaks were detected in 61 KBT samples. Hierarchical cluster analysis, principal component analysis (PCA), k-nearest neighbor (k-NN) and stepwise linear discriminant analysis (SLDA) were employed to visualize the volatile fractions. The results of unsupervised statistical tools were compared using a test for similarities and distinctions, which showed that different sources may be associated. A satisfying combination of average recognition (91.7%) and cross-validation prediction abilities (84.6%) was obtained for the PCA-k-NN. Among all of the statistical tools, SLDA provided promising results, with 100% recognition and 96.4% prediction ability. Conclusion The results obtained in the present study indicate that the volatile compounds can be used as indicators to identify the geographical origin of KBT. © 2019 Society of Chemical Industry.

Objective The specific purposes of this study were (1) to undertake a thorough systematic review and meta-analysis based only on randomized clinical trials (RCTs) to compare the rates of survival and complications of short implants to those of long implants; (2) to compare the surgical time and cost of short implants to those of long implants. Methods RCTs were identified from the major electronic databases (MEDLINE, Embase and Cochrane Library) using the keywords “dental implant,” “short implant” and “atrophic maxilla,” and a quantitative meta-analysis was conducted. The survival rate of implants and complications were the primary outcome measures, and other parameters assessed included costs and surgical time. Results Seven RCTs that met the inclusion criteria included 554 implants (265 implants in the short implant group). There was no significant difference in survival rate between two groups (RR: 1.00; 95% CI: [0.97, 1.03]; p = .96; seven trials, 554 participants). Compared with long implant group, the short implant group had a lower complications and the effect measure was significant (RR: 0.58; 95% CI: [0.37, 0.90]; p = .02; seven trials, 554 participants). Conclusion This systematic review showed that no difference between the survival rates of short implants (5–8 mm) and long implants (>8 mm); complications in short implants are lower than that in long implants. However, further studies are required to substantiate our findings.

Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST searches showed high homology to HPL sequences from other plants. The His-tag expression vector pET-32a(+)/CsHPL was constructed and transferred into Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E. coli was about 60 kDa. The enzyme activity of CsHPL was 0.20 μmol·min(-1)·mg(-1). Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea plants after Ectropis obliqua attack, suggesting that it may be an important candidate for defense against insects in tea plants.

The tea tree is a perennial woody plant, and pruning is one of the most crucial cultivation measurements for tea plantation management. To date, the relationship between long-term pruning and metabolic flux enhancement in tea trees has not been studied. In this research, 11-year-old pruned tea trees from four different cultivars were randomly selected for transcriptome analysis and characteristic secondary metabolite analysis together with controls. The findings revealed that epigallocatechin gallate (EGCG) accumulation in pruned tea trees was significantly higher than that in unpruned tea trees. SCPL1A expression (encoding a class of serine carboxypeptidase), which has been reported to have a catalytic ability during EGCG biosynthesis, together with LAR, encoding leucoanthocyanidin reductase, was upregulated in the pruned tea trees. Moreover, metabolic flux enhancement and transcriptome analysis revealed low EGCG accumulation in the leaves of unpruned tea trees. Because of the bitter and astringent taste of EGCG, these results provide a certain understanding to the lower bitterness and astringency in teas from "ancient tea trees", growing in the wild with no trimming, than teas produced from pruned plantation trees.

Objectives The aims of this study were to investigate the relationship between Golgi phosphoprotein 2 (GOLPH2) and oral squamous cell carcinoma (OSCC) and explore the clinical significance of GOLPH2 in OSCC. Methods Tissue microarrays from human OSCC samples were stained for GOLPH2 expression and clinicopathologic features. Kaplan-Meier analysis was used to compare the survival of patients with high GOLPH2 expression and patients with low GOLPH2 expression. Results We found GOLPH2 is highly expressed in OSCC tissue, and the GOLPH2 expression in metastatic lymph nodes is higher than in tumor tissue. Our data indicate that patients with higher GOLPH2 expression have poor overall survival compared with those with lower GOLPH2 expression. This study demonstrated that GOLPH2 was associated with CD44, SOX2, Slug, B7-H3, B7-H4, TIM3, and VISTA. Conclusions These findings suggest GOLPH2 is a potential marker for estimating the patient's prognosis and may be a target for molecular-targeted therapy against OSCC.

A normal tea plant with one albino branch was discovered. RNA sequencing, albinism phenotype and ultrastructural observations provided a valuable understanding of the albino mechanism in tea plants. Tea plants with a specific color (white or yellow) have been studied extensively. A normal tea plant (Camellia sinensis cv. quntizhong) with one albino branch was discovered in a local tea plantation in Huangshan, Anhui, China. The pure albino leaves on this special branch had accumulated a fairly high content of amino acids, especially theanine (45.31 mg/g DW), and had a low concentration of polyphenols and an extremely low chlorophyll (Chl) content compared with control leaves. Ultrastructural observation of an albino leaf revealed no chloroplasts, whereas it was viable in the control leaf. RNA sequencing and differentially expressed gene (DEG) analysis were performed on the albino leaves and on control leaves from a normal green branch. The related genes involved in theanine and polyphenol biosynthesis were also investigated in this study. DEG expression patterns in Chl biosynthesis or degradation, carotenoid biosynthesis or degradation, chloroplast development, and biosynthesis were influenced in the albino leaves. Chloroplast deletion in albino leaves had probably destroyed the balance of carbon and nitrogen metabolism, leading to a high accumulation of free amino acids and a low concentration of polyphenols in the albino leaves. The obtained results can provide insight into the mechanism underlying this special albino branch phenotype, and are a valuable contribution toward understanding the albino mechanism in tea plants.

Aim: p21-activated kinase 2 (PAK2) is overexpressed in several tumors but the expression of PAK2 in oral squamous cell carcinomas (OSCCs) remains unclear. Materials & methods: Immunohistochemistry was performed on human tissue microarrays containing 165 primary OSCC, 48 oral epithelial dysplasia and 43 normal oral mucosa. Results: PAK2 expression was increased in primary OSCC compared with normal mucosa and significantly increased in primary OSCC grade III compared with grade I, but independent of overall survival rate. Moreover, the expression of PAK2 was statistically correlated with Lck/Yes novel tyrosine kinase (LYN), zinc finger transcription factor Slug, tumor-associated macrophage marker CD163 and LAG3. Conclusion: Overexpression of PAK2 in OSCC may be associated with an advanced pathology grade.

Significance A high-quality genome assembly of Camellia sinensis var. sinensis facilitates genomic, transcriptomic, and metabolomic analyses of the quality traits that make tea one of the world’s most-consumed beverages. The specific gene family members critical for biosynthesis of key tea metabolites, monomeric galloylated catechins and theanine, are indicated and found to have evolved specifically for these functions in the tea plant lineage. Two whole-genome duplications, critical to gene family evolution for these two metabolites, are identified and dated, but are shown to account for less amplification than subsequent paralogous duplications. These studies lay the foundation for future research to understand and utilize the genes that determine tea quality and its diversity within tea germplasm., Tea, one of the world’s most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Head and neck squamous cell carcinoma (HNSCC) is considered as an immunosuppressive disease, with impaired tumor-infiltrating T lymphocytes and increased suppressive immune cells. The efficacy of CD47 antibodies in immune checkpoint therapy is not clearly understood in HNSCC. In this study, human tissue microarrays and immunocompetent transgenic mouse models were used to explore the expression of CD47 and the use of CD47 antibodies in HNSCC. We identified overexpression of CD47 in HNSCC as compared with the control normal human tissue and also in HNSCC mouse models. The expression of CD47 also correlated with clinicopathological parameters as well as outcome. Furthermore, inhibition of CD47 delayed tumor growth and improved tumor microenvironment by stimulating effector T cells and decreasing suppressive immune cells and regulating the function of CD11b(+) Ly6G(+) MDSC. Our data suggest that CD47 blockade may be a potential immunotherapeutic target in human HNSCC.

Camellia sinensis (L.) Kuntze and Camellia oleifera C. Abel (Theaceae) are closely related perennial woody shrubs, but the accumulation of metabolites and gene expression patterns are quite different between these two species. In order to understand the mechanisms behind the accumulation and biosynthesis of tea-specific secondary metabolites and the key genes that regulate their target pathways, 1-year-old clone cuttings of C. sinensis and C. oleifera were grafted in both directions, and self-grafted C. sinensis were used as controls. The transcriptomes and metabolomes of leaves and roots from the grafts were analyzed. We found that 1375 unigenes were up-regulated in the leaves of the CS-CO grafts (C. sinensis scion, C. oleifera stock), while 2437 unigenes were down-regulated. OPLS-DA models established for 7230 and 3223 mass spectra peaks were obtained in the positive and negative modes by LC-MS detection. Association analysis of the secondary metabolism pathways was performed, and the relative gene expressions of 14 genes from the transcriptome screening were verified by qRT-PCR. Among the differential metabolites screened and identified, we found that the relative levels of theanine and caffeine decreased significantly, and that many of the genes in these metabolic pathways were also down-regulated. In contrast, the levels of flavonoids apparently increased, and the expression of related genes in the flavonoid biosynthetic pathway were mostly up-regulated.

Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that releases floral scent and plays an important role in the sweet flowery aroma of tea. During the withering process for white tea producing, MeSA was generated by salicylic acid carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral scent was formed. In this study, we first cloned a CsSAMT from tea leaves (GenBank accession no. MG459470) and used Escherichia coli and Saccharomyces cerevisiae to express the recombinant CsSAMT. The enzyme activity in prokaryotic and eukaryotic expression systems was identified, and the protein purification, substrate specificity, pH, and temperature optima were investigated. It was shown that CsSAMT located in the chloroplast, and the gene expression profiles were quite different in tea organs. The obtained results might give a new understanding for tea aroma formation, optimization, and regulation and have great significance for improving the specific quality of white tea.

The adenosine-induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto-5-nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4+ and CD8+ T cells was associated with an "exhausted" phenotype. Further, treatment with anti-CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the "exhausted" phenotype of CD4+ and CD8+ T cells through downregulation of total expression of PD-1 and CTLA-4 on T cells. Whereas the population of CD4+ CD73hi /CD8+ CD73hi T cells expressed higher CTLA-4 and PD-1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.

Although conventional chemoradiotherapy is effective for most anal squamous cell carcinoma (ASCC) patients, HPV-negative ASCC patients respond poorly to this treatment and new therapeutic approach is required. Our group has previously established an HPV-negative ASCC mouse model and demonstrated that signal transducer and activation of transcription 3 (STAT3) is hyper-activated in the model. Here, we show that in vivo inhibition of STAT3 by S3I-201 effectively delays tumor growth in ASCC mouse model indicated by significantly smaller tumor size and burden in the treatment group compared with control group at the same point. Further analysis shows that survivin and Ki67, important biomarkers for tumor cell survival and proliferation, are significantly reduced after S3I-201 treatment. Additionally, flow cytometry and immunohistofluorescent assays reveal decreased Myeloid-derived suppressor cell (MDSC) and tumor-associated macrophage (TAM) populations in the S3I-201 treatment group, which indicates a reversion of the immunosuppressive environment, unraveling the potential role for S3I-201 in immunosuppression in ASCC. Together these results for the first time demonstrated the anti-tumor effects of STAT3 inhibitor S3I-201 in HPV-negative ASCC mouse model and its multiple effects on cancer cells and immune system. Thus we conclude that S3I-201 may be a novel therapeutic approach for HPV-negative ASCC patients.

Angiogenesis is an essential event in tumor growth and metastasis, and immune system also contributes to the tumor evasion. Emerging evidences have suggested the bidirectional link between angiogenesis and immunosuppression. Myeloid-derived suppressor cell (MDSC) is a kind of immunosuppressive cells and plays an important role in this process. However, the actual regulatory mechanisms of angiogenesis and MDSCs in head and neck squamous cell carcinoma (HNSCC) were unclear. In this study, through analyzing the immunohistochemistry staining of human HNSCC tissue microarray, we found that the microvascular density (MVD) was significantly increased in HNSCC patients. We also characterized angiogenic factors p-STAT3, VEGFA, CK2, and MDSCs marker CD11b in HNSCC tissue array, and found the close expression correlation among these markers. To determine the role of JAK2/STAT3 pathway in tumor microenvironment of HNSCC, we utilized AG490 (an inhibitor of JAK2/STAT3) for further research. Results showed that inhibition of JAK2/STAT3 suppressed angiogenesis by decreasing VEGFA and HIF1-α both in vitro and vivo. Moreover, in HNSCC transgenic mouse model, inhibiting JAK2/STAT3 not only suppressed angiogenesis but also reduced MDSCs in the tumor microenvironment through suppressing VEGFA and CK2. Our findings demonstrated the close relationship between angiogenesis and MDSCs in HNSCC, and inhibition of JAK2/STAT3 could reduce tumor-induced angiogenesis and decrease MDSCs.

Background Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC. Methods The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model. Results Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4+Foxp3+ regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4+ Foxp3+ Tregs and enhanced the anti-tumor response of CD8+ T cells. Conclusions These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0665-0) contains supplementary material, which is available to authorized users.

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1β, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1β concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1β levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1β production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4+ and CD8+ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1+ and Tim3+ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1β pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1β pathway may provide a novel approach for HNSCC therapy.

Immune evasion is a hallmark feature of cancer, and it plays an important role in tumour initiation and progression. In addition, tumour immune evasion severely hampers the desired antitumour effect in multiple cancers. In this study, we aimed to investigate the role of the Notch pathway in immune evasion in the head and neck squamous cell carcinoma (HNSCC) microenvironment. We first demonstrated that Notch1 signalling was activated in a Tgfbr1/Pten-knockout HNSCC mouse model. Notch signalling inhibition using a γ-secretase inhibitor (GSI-IX, DAPT) decreased tumour burden in the mouse model after prophylactic treatment. In addition, flow cytometry analysis indicated that Notch signalling inhibition reduced the sub-population of myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs), and regulatory T cells (Tregs), as well as immune checkpoint molecules (PD1, CTLA4, TIM3 and LAG3), in the circulation and in the tumour. Immunohistochemistry (IHC) of human HNSCC tissues demonstrated that elevation of the Notch1 downstream target HES1 was correlated with MDSC, TAM and Treg markers and with immune checkpoint molecules. These results suggest that modulating the Notch signalling pathway may decrease MDSCs, TAMs, Tregs and immune checkpoint molecules in HNSCC. This article is protected by copyright. All rights reserved.

Immunotherapy with immune checkpoint molecule-specific monoclonal antibody have obtained encouraging results from preclinical studies and clinical trials, which promoted us to explore whether this kind of immunotherapy could be applicable to head and neck squamous cell carcinoma (HNSCC). Lymphocyte activation gene-3 (LAG-3) is an immune checkpoint control protein that negatively regulates T cells and immune response. Here, using the human tissue samples, we report these findings that LAG-3 is overexpressed on tumor-infiltrating lymphocytes (TILs; p < 0.001) and its overexpression correlates with the high pathological grades, lager tumor size and positive lymph node status in human primary HNSCC. Survival analysis identifies LAG-3 as a prognostic factor independent of tumor size and pathological grades for primary HNSCC patients with negative lymph node status (p = 0.014). Study in immunocompetent genetically defined HNSCC mouse model reports that LAG-3 is upregulated on CD4+ T cells, CD8+ T cells and CD4+Foxp3+ regulatory T cells (Tregs). In vivo study, administration of LAG-3-specific antibody retards tumor growth in a way associated with enhanced systemic antitumor response by potentiating the antitumor response of CD8+ T cells and decreasing the population of immunosuppressive cells. Taken together, our results offer a preclinical proof supporting the immunomodulatory effects of LAG-3 and suggest a potential therapeutic target of immunotherapy for HNSCC.

The tea plant, Camellia sinensis (L.) O. Kuntze, is a crucial commercial crop in China. In September 2016, a new spot disease was observed on the leaves of a mature (age: 10 years) tea plant in Chizhou, Anhui Province, China. The symptoms began with brown lesions with irregular margins that expanded to 10 mm. Brown to gray color changes in the centers of the mature lesions with dark brown edges were noticed. The disease incidence reached 20% of tea plants in 100 ha of tea fields. The infected parts (junction of diseased and healthy region) were cut into small pieces (5 to 7 mm²) and subsequently underwent surface sterilization (Chen et al. 2016). Thereafter, the pieces were blotted dry on sterilized filter paper and placed on water agar plates, which were incubated in the dark at 28°C until fungal hyphae started to grow from the pieces. The single-hyphal tip was then transferred onto potato dextrose agar plates for further analysis. After 7 to 10 days, the purified colony produced yellowish green colonies with sparse aerial hyphae, and yellow with scattered black dots appeared on the back. Pycnidia were black, globose, nonostiole, 27.5 to 112.1 μm (mean, 62.0 µm; n = 50) in diameter, and they contained smooth ovoid conidia with round ends 3.9 to 8.1 µm in length (mean, 5.6 µm; n = 50) by 2.2 to 3.9 µm in width (mean, 3.1 µm; n = 50). Chlamydospores and swollen hyphal bodies were present. On oatmeal agar, the colonies were gray with sparse aerial hyphae; yellowish green occurred on the reverse side. The colonies turned purplish pink with the addition of 1% NaOH. The same colonial characters were noticed on malt extract agar, and yellow with dark brown was noticed on the dorsal side. From the morphological data, the isolate was identified as Phoma sp. (Boerema et al. 2004). To identify the fungal isolate, DNA was extracted (Fungal DNA extraction kit, Biomiga, China), amplified, and sequenced using universal primers (ITS1/ITS4) (White et al. 1990) and the D1/D2 region of 28S rDNA (LR0R/TW13) (Hamayun et al. 2009). BLAST analysis of GenBank data (National Center for Biotechnology Information) showed 100 and 99% similarity with Phoma herbarum for KU204761 and LT966056, respectively. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and upper and middle leaves were injured on the upper surface through acupuncture (Gilbert and Webb 2007) with slight modifications (two needle pricks per leaf, using a sterile needle). A conidial suspension (10⁶/ml) was spread over the wounds, and sterile water was applied to the wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 12 to 15 days after inoculation, lesions appeared on the wounded leaves, with symptoms similar to those previously described, whereas the wounded control leaves and leaves without puncture remained healthy. The experiment was performed twice, and the same fungus was isolated both times from the infected leaves. P. herbarum causes leaf spot disease in other plants, such as Elaeis guineensis and Vetiveria zizanioides in China, Bituminaria bituminosa in Australia, and Salvia nemorosa in Italy. To our knowledge, this is the first report of P. herbarum causing leaf spot disease on tea plant leaves in China, which has a strong influence on tea plant cultivation.

Objective Our aim is to evaluate the expression of SATB1 in human oral squamous cell carcinomas (OSCC) and its role in the invasiveness and metastasis of OSCC. Subjects and methods A human OSCC tissue microarray was used to evaluate the expression pattern of SATB1. SATB1 mRNA knockdown was performed in human OSCC cell lines SCC25 and Cal27 to assess the function of SATB1 in the invasiveness and metastasis of OSCC. Results SATB1 is highly expressed in human OSCC determined by immunohistochemistry, and its nuclear/cytoplasmic ratio of histoscore is significantly correlated with patients’ prognosis. Reduced cell motility, invasiveness, expression of epithelial to mesenchymal transition (EMT) markers (N-cadherin and β-catenin), and elevated expression of epithelial markers were observed in SATB1-knockdown cells in in vitro studies. Depletion of SATB1 also restored a cobblestone-like morphology in TGF-β1-treated cells. Conclusions These findings suggest SATB1 may play an important role in OSCC invasiveness and metastasis.

V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint regulatory molecule, suppresses T cell mediated immune responses. The aim of the present study was to profile the immunological expression, clinical significance and correlation of VISTA in human oral squamous cell carcinoma (OSCC). Human tissue microarrays, containing 165 primary OSCCs, 48 oral epithelial dysplasias and 43 normal oral mucosae, were applied to investigate the expression levels of VISTA, CD8, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death ligand 1 (PD-L1), PI3Kα p110, IL13Rα2, phospho-STAT3 at tyrosine 705 (p-STAT3) and myeloid-derived suppressor cell (MDSC) markers (CD11b and CD33) by immunohistochemistry and digital pathology analysis. The results demonstrated that the protein level of VISTA was significantly higher in human OSCC specimens, and that VISTA expression in primary OSCCs was correlated with lymph node status. VISTA expression did not serve as an independent predictor for poor prognosis, while patient subgroup with VISTA high and CD8 low expression (22/165) had significantly poorer overall survival compared with other subgroups based on the multivariate and Cox hazard analyses among the primary OSCC patients in the present cohort. Additionally, the expression of VISTA was significantly correlated with PD-L1, CTLA-4, IL13Rα2, PI3K, p-STAT3, CD11b and CD33 according to Pearson’s correlation coefficient test. Taken together, the results indicated that the VISTA high and CD8 low group, as an immunosuppressive subgroup, might be associated with a poor prognosis in primary OSCC. These findings indicated that VISTA might be a potential immunotherapeutic target in OSCC treatment.

Checkpoint blockade therapy utilizing monoclonal antibodies to reactivate T cells and recover their antitumor activity makes an epoch in cancer immunotherapy. The role of B7-H4, a novel negative immune checkpoint, in oral squamous cell carcinoma (OSCC) has still not been elucidated. In this study, tissue samples from human OSCC, which contains 165 primary OSCC, 48 oral epithelial dysplasia and 43 normal oral mucosa specimens, and Tgfbr1/Pten 2cKO mice OSCC model were stained with B7-H4 antibody to analyze the correlations between B7-H4 expression and clinicopathological characteristics. Kaplan–Meier analysis was used to compare the survival of patients with high B7-H4 expression and patients with low B7-H4 expression. We found B7-H4 is highly expressed in human OSCC tissue, and the B7-H4 expression level was associated with the clinicopathological parameters containing pathological grade and lymph node status. Moreover, we confirmed that B7-H4 was overexpressed in Tgfbr1/Pten 2cKO mice OSCC model. Our data also indicated that patients with high B7-H4 expression had poor overall survival compared with those with low B7-H4 expression. Furthermore, this study demonstrated that B7-H4 was positively associated with PD-L1, CD11b, CD33, PI3Kα p110, and p-S6 (S235/236). Taken together, these findings suggest B7-H4 is a potential target in the treatment of OSCC.

Immature myeloid cells such as myeloid-derived suppressor cells (MDSCs) and M2 macrophages play a vital role in the tumor immune escape and tumor progression. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4), as a negative immune checkpoint, is highly expressed in numerous solid tumors. However, precise functions of CTLA4 in head and neck squamous cell carcinoma (HNSCC) have not yet been elucidated. In this study, we demonstrated that the ratio of CD8(+)/CTLA4 can be used as a potential index with a clinical prognostic value for HNSCC. Using immunocompetent transgenic mouse model with spontaneous HNSCC, we directly observed that targeting CTLA4 decreases MDSCs and M2 macrophages and promotes T cell activation in both tumor microenvironment and macro-environment. In all, our study provides direct evidence in vivo and proposes a rationale for CTLA4 inhibition as a future therapeutic strategy in patients with HNSCC.

Salivary Adenoid Cystic Carcinoma (SACC) is a tumor characterized by inevitable local progression and terminal hematogenous metastasis. This study aimed to investigate the expression of LC3, LAMP2, KEAP1 and NRF2 in SACC. Human salivary gland tissue microarray which contains 74 SACC, 12 pleomorphic adenoma and 18 normal salivary gland specimens. High expression of LC3, LAMP2, KEAP1 and NRF2 were found in SACC patients, and LC3, LAMP2, KEAP1 and NRF2 expression were significantly higher in SACC than as compared with pleomorphic adenoma and (or) normal salivary gland. The expression of NRF2 was correlated with pathological type of human SACC (P

Caffeine, a purine alkaloid, was not detected in leaves of two Mascarocoffea species, Coffea millotii and Coffea perrier. Trigonelline, a pyridine alkaloid, occurred in these species, but the levels (3−4 μmol/g fresh weight) were much lower than that of Robusta coffee (Coffea canephora) (36 μmol/g fresh weight). Feeding experiments with [8-14C]adenine indicated that purine alkaloid biosynthesis was terminated at 7-methylxanthine formation and as a consequence theobromine and caffeine were not produced in Coffea millotii and Coffea perrier. The adenine salvage activity was lower but its degradation activity was higher in leaves of these Mascarocoffea species than those in Coffea canephora. The metabolic fate of the purine nucleosides, [8-14C]inosine, [8-14C]guanosine and [8-14C]xanthosine was investigated in leaves of Coffea millotii. The biosynthesis of 7-methylxanthine, but not theobromine or caffeine, from these precursors was detected. Large amounts of these purine nucleosides were catabolized via allantoin. Limited amounts of [8-14C]inosine and [8-14C]guanosine were salvaged and utilized for RNA synthesis, however, no [8-14C]xanthosine salvage was observed. Little or no 14C-metabolites were observed when [8-14C]theobromine and [8-14C]caffeine were applied to leaf disks of Coffea millotii. From the results obtained in this study, possible metabolic pathways of purines in Mascarocoffea species are discussed.

Cumulative evidence suggests that constitutively activated signal transducer and activator of transcription (STAT3) may contribute to sustaining immunosuppressive status, and that inhibiting STAT3 signaling represents a potential strategy to improve antitumor immunity. In the present study, we observed that high levels phosphorylated of STAT3 are significantly associated with the markers for both myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in human head and neck squamous cell carcinoma (HNSCC). Additionally, we showed that targeting STAT3 signaling with a tolerable selective inhibitor S3I-201 significantly decreased immature myeloid cells such as MDSCs, TAMs and iDCs in genetically defined mice HNSCC model. These findings highlight that targeting STAT3 signaling may be effective to enhance antitumor immunity via myeloid suppressor cells in HNSCC.