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Microbial action and moist-heat action are crucial factors that influence the piling fermentation (PF) of Pu-erh tea. However, their effects on the quality of Pu-erh tea remain unclear. In this study, the effects of spontaneous PF (SPPF) and sterile PF (STPF) on the chemical profile of Pu-erh tea were investigated for the first time, and sun-dried green tea was used as a raw material to determine the factors contributing to the unique quality of Pu-erh tea. The results indicated that the SPPF-processed samples had a stale and mellow taste, whereas the STPF-processed samples had a sweet and mellow taste. Through metabolomics-based analysis, 21 potential markers of microbial action (including kaempferol, quercetin, and dulcitol) and 10 potential markers of moist-heat action (including ellagic acid, β-glucogallin, and ascorbic acid) were screened among 186 differential metabolites. Correlation analysis with taste revealed that metabolites upregulated by moist-heat and microbial action were the main factors contributing to the staler mellow taste of the SPPF-processed samples and the sweeter mellow taste of the STPF-processed samples. Kaempferol, quercetin, and ellagic acid were the main active substances formed under microbial action. This study provides new knowledge regarding the quality formation mechanism of Pu-erh tea.

Integrin β4 (ITGB4) is a member of the integrin family, which plays a crucial role in mediating cell adhesion to the extracellular matrix. Recent studies have demonstrated that ITGB4 is involved in tumorigenesis and metastasis during the development of cancer. However, the role of ITGB4 in oral squamous cell carcinoma (OSCC) remains unclear. A Multiplex immunohistochemistry (OPAL™, mIHC) assay was employed to stain ITGB4, ALDH1, PD-L1, cytokeratin (CK), CD8 and PD-1 in a human OSCC tissue microarray, containing 26 normal oral epithelium samples, 21 oral epithelium dysplasia samples and 76 OSCC samples. The expression pattern and clinicopathological characteristics of ITGB4 were analyzed and compared with those of PD-1, PD-L1, ALDH1 and CD8. The correlation between subgroups of tumor cells, including ITGB4+PD-L1+ and ITGB4+ALDH1+, and subgroups of T cells, including CD8+ and CD8+PD-1+, was evaluated using two-tailed Pearson’s statistics. A Kaplan–Meier curve was built, and a log-rank test was performed to analyze the survival rate of different subgroups. The mIHC staining results show that ITGB4 was mostly expressed in the tumor cells, with a significant increase in the OSCC specimens compared with normal oral epithelium and oral epithelium dysplasia. The paired analysis, conducted between the OSCC tumor tissue and normal paracancer mucosa, confirmed the results. The study further revealed that ITGB4+PD-L1+ cancer cells, but not ITGB4+ALDH1+ cancer cells, were significantly associated with the infiltration of CD8+ T cells (positivity p = 0.005, positive number p = 0.03). Additionally, ITGB4+PD-L1+ tumor cells were positively correlated with CD8+PD-1+ T cells (positivity p = 0.02, positive number p = 0.03). Most intriguingly, the subgroup of ITGB4/PD-L1high with CD8/PD-1high displayed the best prognosis compared with the other considered subgroups. The results show that the expression of ITGB4 was increased in OSCC compared with normal oral mucosa. Furthermore, a specific subgroup with high levels of expression of ITGB4/PD-L1 and CD8/PD-1 was found to have a relatively better prognosis compared with the other subgroups. Ultimately, this study sheds light on the potential role of ITGB4 in OSCC and provides a basis for further investigation.

Unlike that of other crops, the growth of tea plants can be promoted by aluminum, but its regulation mechanism remains unclear. Some endophytes can also promote growth of plant hosts. In this paper, tea roots treated with aluminum were used to study the growth-promoting traits and aluminum tolerance of endophytes. Meta-16S rDNA analysis revealed that Burkholderia was enriched in tea roots after aluminum treatment, and it was the dominant strain for hydroponic tea roots and field tea roots. Actinomycetes constituted the dominant strains in hydroponic tea seedlings treated with aluminum. Sixteen endophytic bacteria, including 12 strains of Firmicutes, 2 strains of Proteobacteria and 2 strains of Actinomycetes, were isolated and identified from hydroponic tea roots treated with different aluminum concentrations. Growth-promoting activity analysis showed that the isolated endophytic bacteria all had more than one plant growth-promoting trait. Among them, B4 (Bacillus nealsonii), B8 (Brevibacterium frigoritolerans) and A2 (Nocardia nova) bacteria each had three growth-promoting traits. Aluminum tolerance ability analysis indicated that endophyte A1 (Leifsonia shinshuensis) had the strongest aluminum tolerance ability, up to 200 mg l−1 aluminum. Plant–bacteria interactions showed that endophytes A1, A2 and B4 and their synthetic community all had a growth-promoting effect on the growth of wheat lateral roots. Moreover, endophytes A1 and B4 alleviated aluminum stress in wheat. Endophyte A1 also promoted the growth of tea cuttings, especially lateral roots, with/without aluminum. Taken together, aluminum enhanced the distribution of aluminum-tolerant and growth-promoting bacteria, thereby promoting the growth of tea roots. This study provides a new aspect for research on the mechanism by which aluminum promotes tea plant growth.

Bacteria survived and thrived inside any part of plant without damaging its host are called endophytic bacteria. Many studies have focused on plant–endophytic bacterium interactions and their Plant-growth-promoting (PGP) abilities. Albino tea plants are widely investigated because of their high amino acid concentrations. Previous studies mainly elucidated specific metabolite mechanisms in albino tea plants, while the present study identified the variation in diversity and endophytic bacterial abundance in albino and non-albino tea leaves using culture-dependent and culture-independent methods. With different culture media, we identified a total of 110 isolates, of which 58 endophytic bacteria belonged to three phyla (Proteobacteria, Firmicutes, and Actinobacteria). Moreover, we identified a total of 1856 operational taxonomic units as 513 endophytic bacteria belonging to 27 phyla including Proteobacteria, Firmicutes, and Actinobacteria. In addition, comparing to non-albino tea leaves, endophytic bacteria obtained from albino tea leaves showed higher diversity and abundance. Endophytic bacteria isolated from albino leaves also exhibited higher PGP properties than those from non-albino ones, and their PGP effects on rice and tea cuttings were examined. The results indicated differences in endophytic bacteria between albino and non-albino leaves. Specific endophytic bacteria isolated from albino leaves may have a relationship with the albino phenotype. Our study indicated that PGP bacteria isolated from albino leaves are expected to become a plant growth promoter in the future.

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.

Methyl jasmonate (MeJA), a volatile organic compound, is a principal flowery aromatic compound in tea. During the processing of black tea, MeJA is produced by jasmonic acid carboxyl methyltransferase (JMT) of the jasmonic acid (JA) substrate, forming a specific floral fragrance. CsJMT was cloned from tea leaves; the three-dimensional structure of CsJMT was predicted. Enzyme activity was identified, and protein purification was investigated. Site-directed deletions revealed that N-10, S-22, and Q-25 residues in the beginning amino acids played a key functional role in enzyme activity. The expression patterns of CsJMT in tea organs differed; the highest expression of CsJMT was observed in the fermentation process of black tea. These results aid in further understanding the synthesis of MeJA during black tea processing, which is crucial for improving black tea quality using specific fragrances and could be applied to the aromatic compound regulation and tea breeding improvement in further studies.

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.

Herein, a new indicator-displacement array (IDA) sensor was developed for the quality evaluation of black tea fermentation. On the principle of the reversible covalent binding of phenylboronic acid and catechol, phenylboronic acids were selected as acceptors for targeted binding to polyphenols. Pyrocatechol violet and alizarin red S were used as indicators of the reaction. The IDA sensors have sensitive differential responses to fermented tea samples, achieving an assessment of the fermentation degree with accuracies of 80.39-88.00% by support vector machine (SVM). In addition, the key polyphenol components of the fermentation process were accurately predicted by the IDA and SVM regression with ratio of prediction to deviation values of 1.55-1.72, 2.03-2.21, and 2.03-2.08 for total polyphenols, total catechins, and epigallocatechin-3-O-gallate, respectively. In conclusion, the developed IDA sensor is capable of the in-situ quality monitoring of black tea fermentation, with the advantages being cost-effectiveness, sensitivity, and rapidity.

Fermentation is a key black tea processing step and makes an important contribution to quality formation. Current approaches to fermentation monitoring are costly or laboratory-based. Here, we first evaluated the potential of at-line computer vision for detecting fermentation quality in a tea factory. A self-built industrial camera was used to collect tea samples at various fermentation durations. The correlations of color variables that were extracted from the images with key quality indicators in the tea samples were verified. Subsequently, partial least-squares regression models based on the color variables showed high prediction accuracy with residual prediction deviation values of 4.13, 3.53, and 3.39 for catechins, theaflavins and chlorophylls, respectively. Finally, the spatial and temporal distributions of indicators during fermentation were mapped to visualize the fermentation quality. This study realized low-cost, at-line and real-time detection for black tea fermentation, which provides technical support for the industrial and intelligent production of black tea.

Camellia sinensis (L.) O. Kuntze is used to produce tea, a beverage consumed worldwide. Catechins are major medically active components of C. sinensis and can be used clinically to treat hyperglycaemia, hypertension, and cancer. In this study, we aimed to identify the genes involved in catechins biosynthesis. To this end, we analysed transcriptome data from two different cultivars of C. sinensis using DNBSEQ technology. In total,47,717 unigenes were obtained from two cultivars of C. sinensis, of which 9429 were predicted as new unigenes. In our analyses of the Kyoto Encyclopedia of Genes and Genomes database, 212 unigenes encoding 13 key enzymes involved in catechins biosynthesis were identified; the structures of leucoanthocyanidin reductase and anthocyanidin reductase were spatially modelled. Some of these key enzymes were verified by real-time quantitative polymerase chain reaction, and multiple genes encoding plant resistance proteins or transcription factors were identified and analysed. Furthermore, two microRNAs involved in the regulation of catechins biosynthesis were explored. Differentially expressed genes involved in the flavonoid biosynthesis pathway were identified from pairwise comparisons of genes from different cultivars of tea plants. Overall, our findings expanded the number of publicly available transcript datasets for this valuable plant species and identified candidate genes related to the biosynthesis of C. sinensis catechins, thereby establishing a foundation for further in-depth studies of catechins biosynthesis in varieties or cultivars of C. sinensis.

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.

Our study investigated the expression of malic enzyme 2 (ME2) in human oral squamous cell carcinoma (OSCC) and associated pathological and clinical pattern. We demonstrated that human OSCC tissues expressed a high level of ME2, and the overexpression of ME2 is closely connected to a high pathological grade, lymphatic metastasis, large tumor size and human papillomavirus (HPV) (P < 0.001). Similarly, high levels of ME2 expression in OSCC tissue were shown to be correlated with poor prognosis (P < 0.05). The expression of ME2 was correlated with Slug, SOX2, and aldehyde dehydrogenase-1 (ALDH1) immunoreactivity.ME2 was shown to be overexpressed in OSCC tissue and indicated a poor prognosis for OSCC. ME2 may be correlated with several immune markers.

Accumulating evidence indicates that colonized microbes play a crucial role in regulating health and disease in the human body. Detecting microbes should be essential for understanding the relationship between microbes and diseases, as well as increasing our ability to detect diseases. Here, a combined metabolic labeling strategy was developed to identify different bacterial species and microbiota by the use of three different fluorescent metabolite derivatives emitting red, green, and blue (RGB) fluorescence. Upon co-incubation with microbes, these fluorescent metabolite derivatives are incorporated into bacteria, generating unique true-color fingerprints for different bacterial species and different microbiota. A portable spectrometer was also fabricated to automate the colorimetric analysis in combination with a smartphone to conveniently identify different bacterial species and microbiota. Herein, the effectiveness of this system was demonstrated by the identification of certain bacterial species and microbiota in mice with different diseases, such as skin infections and bacteremia. By analyzing the microbiota fingerprints of saliva samples from clinical patients and healthy people, this system was proved to precisely distinguish oral squamous cell carcinoma (OSCC, n = 29) samples from precancerous (n = 10) and healthy (n = 5) samples.

Withering is a key process that affects the aroma of Keemun black tea (KBT). In this study, the aroma composition of KBT through natural withering, sun withering, and warm-air withering was analysed using gas chromatography-mass spectrometry. The results revealed significant differences in the three samples. Gas chromatography-olfactometry and aroma extract dilution analysis were performed with screening through a relative odour activity value (rOAV) 1. In total, 11 aroma-active compounds (geraniol, (Z)-4-heptenal, 1-octen-3-ol, (E)-β-ionone, 3-methylbutanal, linalool, β-damascenone, (E, E)-2,4-decadienal, methional, (E, E)-2,4-nonadienal, and (E)-2-nonenal) were found to be responsible for the differences in aroma caused by different withering methods. Linalool (rOAV, 161) and geraniol (rOAV, 785) were responsible for the higher flowery and fruity aromas when sun withering was applied, whereas methional (rOAV, 124) contributed to the intense roasty aroma when warm-air withering was employed. Moreover, our results were verified by quantitative descriptive analysis and addition experiments.

Epigenetic alterations, including DNA methylation, play crucial roles in the tumor. Epigenetic drugs like DNA methyltransferase-1 (DNMT1) inhibitors have been exhibited positive effects in cancer treatment. However, the role of DNMT1 in oral squamous cell carcinoma (OSCC) is less clearly described. What is more, the effects on the immune microenvironment of DNMT1 have not become appreciated. In this research, we determine the expression levels of DNMT1 and the association of prognosis by analyzing human OSCC tissue microarrays. Two different types of immunocompetent mouse OSCC models were established to explore the effects of DNMT1 inhibitor on the tumor microenvironment(TME). We identified DNMT1 was highly expressed both in human and mouse OSCC tissues. The expression levels of DNMT1 was also correlated with the immunosuppressive molecules and tumor-promoter such as VISTA, PD-L1, B7-H4, and PAK2, indicating a worse prognosis. Of particular concern is that DNMT1 inhibition improved TME and delayed tumor growth by decreasing myeloid-derived suppressor cells (MDSCs) and increasing tumor-infiltrating T cells. Our data suggests that DNMT1 play a key role in OSCC and has a possible immunotherapeutic marker treatment.

Crush-tear-curl (CTC) black tea is a popular beverage, owing to its unique taste characteristics and health benefits. However, differences in the taste quality and chemical profiles of CTC black tea from different geographical regions remain unclear. In this study, 28 CTC black tea samples were collected from six geographical regions and analyzed using electronic tongue and ultrahigh performance liquid chromatography-Orbitrap-mass spectroscopy. The e-tongue analysis indicated that each region's CTC black tea has its own relatively prominent taste characteristics: Sri Lanka (more umami and astringent), North India (more umami), China (more sweetness and astringent), South India (moderate umami and sweetness), and Kenya (moderate umami and astringent). Based on multivariate statistical analysis, 78 metabolites were tentatively identified and used as potential markers for CTC black tea of different origins, mainly including amino acids, flavone/flavonol glycosides, and pigments. Different metabolites, which contributed to the taste characteristics of CTC black tea, were clarified by partial least squares regression correlation analysis. Our findings may serve as useful references for future studies on origin traceability and quality characteristic determination of CTC black teas. PRACTICAL APPLICATION: This study provides useful references for future studies on the origin traceability and taste characteristic determination of CTC black teas from different geographical regions.

Objective To investigate the role of dual-layer detector energy spectral CT in resting myocardial perfusion imaging for patients with normal coronary artery. Methods One hundred and fifty-six patients with suspected coronary heart disease underwent dual-layer detector energy spectral CT coronary angiography,and resting myocardial perfusion imaging was performed for 28 patients with normal coronary artery.According to American Heart Association's 17-segmentmodel,the iodine density and effective atomic number(Z

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.

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.

Although anti-PD-1 immunotherapy is widely used to treat melanoma, its efficacy still has to be improved. In this work, we present a therapeutic method that combines immunotherapy and starvation therapy to achieve better antitumor efficacy. We designed the CMSN-GOx method, in which mesoporous silica nanoparticles (MSN) are loaded with glucose oxidase (GOx) and then encapsulate the surfaces of cancer cell membranes to realize starvation therapy. By functionalizing the MSN's biomimetic surfaces, we can synthesize nanoparticles that can escape the host immune system and homologous target. These attributes enable the nanoparticles to have improved cancer targeting ability and enrichment in tumor tissues. Our synthetic CMSN-GOx complex can ablate tumors and induce dendritic cell maturity to stimulate an antitumor immune response. We performed an in vivo analysis of these nanoparticles and determined that our combined therapy CMSN-GOx plus PD-1 exhibits a better antitumor therapeutic effect than therapies using CMSN-GOx or PD-1 alone. Additionally, we used the positron emission tomography imaging to measuring the level of glucose metabolism in tumor tissues, for which we investigate the effect with the cancer therapy in vivo.

Because a host's immune system is affected by host-microbiota interactions, means of modulating the microbiota could be leveraged to augment the effectiveness of cancer therapies. Here we report that patients with oral squamous cell carcinoma (OSCC) whose tumours contained higher levels of bacteria of the genus Peptostreptococcus had higher probability of long-term survival. We then show that in mice with murine OSCC tumours injected with oral microbiota from patients with OSCCs, antitumour responses were enhanced by the subcutaneous delivery of an adhesive hydrogel incorporating silver nanoparticles (which inhibited the growth of bacteria competing with Peptostreptococcus) alongside the intratumoural delivery of the bacterium P. anaerobius (which upregulated the levels of Peptostreptococcus). We also show that in mice with subcutaneous or orthotopic murine OSCC tumours, combination therapy with the two components (nanoparticle-incorporating hydrogel and exogenous P. anaerobius) synergized with checkpoint inhibition with programmed death-1. Our findings suggest that biomaterials can be designed to modulate human microbiota to augment antitumour immune responses.

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.

Background Caffeine acts as an antifungal agent; however, its underlying inhibition mechanism remains unclear. To investigate variations in caffeine antifungal activity, this study was evaluate the inhibition mechanisms of caffeine on phytopathogens Botryosphaeria dothidea and Colletotrichum gloeosporioides through transcriptomic analysis, which causes leaf necrosis and anthracnose on tea leaves, respectively.Results Our in vitro results shows that the antifungal activity of caffeine quite different between B. dothidea and C. gloeosporioides. Furthermore, microscopic observation and bioactivity assay results proved that caffeine destroyed the fungal protective layers (cell wall and cell membrane) and subcellular organelles. The transcriptomes of these two fungi exposed to different caffeine concentrations were analyzed by high-throughput sequencing and the results showed that caffeine repressed gene expression involved in the pathways of mRNA surveillance, ribosome biogenesis in eukaryotes, aminoacyl-tRNA biosynthesis, etc. Particularly, caffeine affected gene expression in melanin biosynthesis in C. gloeosporioides, resulting in the disturbances of melanin production and fungal pathogenicity. Manually infected tea leaves showed melanin pigment was important to appressorium development and fungal infection by histopathological observation and fungal biomass quantification. Besides, C. gloeosporioides successfully enters into the host tissue even plant could build multilayer defenses against the fungus, this might be due to the variation in pathogen targets and quantities of secondary metabolites by the host cells.Conclusions Overall, our findings indicate that caffeine in the environment can reduce the fungal growth and physiological activities. However, the genome-wide expression showed different transcriptional profiles between these fungal isolates. Thus, we believe that target of the caffeine varied among isolates and melanin may play a major role in fungus caffeine tolerance. Further in-depth research required to conclude this hypothesis.

Caffeine and catechin are two main components of instant green tea, and are essential components of tea quality. This paper mainly focuses on the feasibility of rapidly determining instant green tea components by using a portable near infrared (NIR) spectrometer. The two main components (caffeine and catechin) were studied. In addition, the instrument performance levels of portable and benchtop NIR spectrometers were studied and compared. Quantitative models developed using portable and benchtop spectrometers for measuring caffeine, total catechins, and four individual catechins were established and compared. After preprocessing using standard normal variate (SNV), the Rp values of the caffeine, total catechins, (-)-epigallocatechin, (-)-epigallocatechin 3-gallate, (-)-epicatechin, and (-)-epicatechin gallate in the partial least squares models for a portable NIR spectrometer were 0.974, 0.962, 0.669, 0.945, 0.942 and 0.905, respectively. For a benchtop NIR spectrometer, Rp values were 0.993, 0.958, 0.883, 0.955, 0.966 and 0.936, respectively. Passing-Bablok regression method results indicated no significant differences between the two instruments. A genetic algorithm (GA) and the successive projections algorithm (SPA) were used to screen the wavelength of the NIR spectrum and establish the model. The GA obtained more robust modeling results. This study concludes that the developed portable spectroscopy system combined with appropriate variable selection methods can be effectively used for rapid determination of caffeine, total catechins, and four individual catechins in instant green tea.

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.