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BACKGROUND Debate ensues regarding the stronger indicator of obesity, body mass index (BMI), or waist circumference (WC), in association with hypertension. Furthermore, little research has been done to compare BMI and WC in their associations with prehypertension. METHODS A cross-sectional survey was undertaken in 33 communities randomly selected from 3 cities in Northeastern China during 2009–2010, using a total of 25,196 adults aged 18–74 years with an overall response rate of 87.4%. Diagnoses of prehypertension and hypertension were based on the criteria set by the Seventh Report of the Joint National Committee. Generalized overweight and obesity were defined as BMI 25–29.9 and ≥30kg/m2, respectively. Abdominal overweight and obesity were defined as WC 94–102 and >102cm in men and WC 80–88 and >88cm in women, respectively. RESULTS Both BMI and WC were positively related to the prevalence rate of elevated blood pressure. Among younger individuals, aged 18–44 years, WC seemed to be associated with a higher odds ratio (OR) than BMI for hypertension (e.g., for men, 17.18 (BMI) vs. 23.28 (WC) for obesity), in contrast, BMI seemed to be associated with a higher OR than WC for prehypertension (e.g., for men, 5.99 (BMI) vs. 1.51 (WC) for obesity). Among older individuals, aged ≥45 years, the adjusted OR for hypertension was modestly higher for WC than for BMI in men. In women, the adjusted OR was modestly higher for BMI than for WC. CONCLUSIONS In this Chinese population, the association of body fat and abdominal fat with hypertension and prehypertension depends on age and sex. [ABSTRACT FROM PUBLISHER]

Abstract: Trigonelline is a major component in coffee seeds and may contribute to the bitter taste of the resultant beverage. To determine the trigonelline biosynthetic pathway in coffee fruits, we investigated the metabolic fate of [carboxyl-14C]nicotinic acid riboside and in situ activity of related enzymes. Exogenously supplied [carboxyl-14C]nicotinic acid riboside was rapidly converted to nicotinic acid mononucleotide and was utilized for NAD synthesis. Nicotinic acid riboside was also used for trigonelline synthesis, but this process took longer than NAD synthesis. These results indicate that an efficient nicotinic acid riboside salvage system functions in coffee fruits, and that trigonelline is synthesized mainly from nicotinic acid produced by the degradation of NAD. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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.

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Intelligent identification of black tea fermentation quality is becoming a bottleneck to industrial automation. This study presents at-line rapid detection of black tea fermentation quality at industrial scale based on low-cost micro-near-infrared spectroscopy (NIRS) and laboratory-made computer vision system (CVS). High-performance liquid chromatography and a spectrophotometer were used for determining the content of catechins and theaflavins, and the color of tea samples, respectively. Hierarchical cluster analysis combined with sensory evaluation was used to group samples through different fermentation degrees. A principal component analysis-support vector machine (SVM) model was developed to discriminate the black tea fermentation degree using color, spectral, and data fusion information; high accuracy (calibration = 95.89%, prediction = 89.19%) was achieved using mid-level data fusion. In addition, SVM model for theaflavins content prediction was established. The results indicated that the micro-NIRS combined with CVS proved a portable and low-cost tool for evaluating the black tea fermentation quality., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 Institute of Food Technologists®.)

The aroma profile of raw pu'er tea (RPT) depends on its storage duration (2-10 years) and storage conditions (wet-hot or dry-cold environment). We analyzed the major odorants of RPT samples by performing metabolomic analysis and by using the molecular sensory science approach. Under dry-cold storage conditions, tea leaves had more carotenoid derivatives, glycoside-derived volatiles, and phenolic volatiles, resulting in "fresh," "floral," and "sweet" aroma. Under wet-hot storage conditions, tea leaves had more methoxybenzenes, which contributed considerably to their "stale" and "woody" aroma. We identified 11 and 4 compounds as the odor markers of RPTs when stored in dry-cold and wet-hot environments, respectively. Our findings provide a scientific basis for optimal storage that yields the desired aroma profile., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 eff value)of each myocardial segment(except for apical segment)were measured and normalized to those of the aorta.All the data were quantitatively evaluated using ANOVA or Friedman test. Results Iodine density and Z eff value of myocardial segments in middle plane were significantly different(all P<0.001).The iodine density and Z eff value showed no significant difference between segments in basal and apical plane(all P > 0.05). Conclusions Iodine density and Z eff value of myocardial segments can be quantitatively evaluated using dual-layer detector energy spectral CT.Resting myocardial perfusion of segments in middle plane are significantly different in patients with normal coronary artery.

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 < 0.0001). In addition, patients who expressed high PREX1 had a poorer prognosis than those who expressed low PREX1 (P = 0.0070). Furthermore, positive correlations were found between P-Rex1 expression and the immune checkpoints PD-L1, Galectin-9 and B7-H4, and the TAM markers CD68, CD206 and CD163. In short, these findings implicated that overexpression of P-Rex1 may predict a poor prognosis in human OSCC.

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., (This journal is © The Royal Society of Chemistry 2020.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 (P < 0.05). 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., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Main Conclusion: 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.

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-STAT3 Y705 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.

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.

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.

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., (© 2017 Wiley Periodicals, Inc.)

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 signaling was activated in a Tgfbr1/Pten-knockout HNSCC mouse model. Notch signaling 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 signaling 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 signaling pathway may decrease MDSCs, TAMs, Tregs and immune checkpoint molecules in HNSCC., (© 2017 UICC.)

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., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

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.

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.

B7 Homolog 3 (B7-H3), a newly identified member of the B7 family, is over-expressed in various human cancers and plays a vital role in tumor progression. To identify the expression pattern of B7-H3 in human salivary adenoid cystic carcinoma (AdCC) and its underlying mechanisms, we characterized B7-H3 expression in AdCC tissue microarrays using immunohistochemical staining, and analyzed potentially associated molecules. The results showed that B7-H3 was highly expressed in salivary AdCC, compared with normal salivary glands. Statistical analyses of immunohistochemical staining showed that B7-H3 was closely correlated with Slug and p-STAT3. Functional studies showed that knockdown of B7-H3 in AdCC cell lines using RNA interference did not influence cell growth and apoptosis, but decreased migration and invasion in vitro . Further mechanism studies suggested that B7-H3 influenced the migration and invasion of AdCC cells by regulating the epithelial-mesenchymal transition via JAK2/STAT3 pathway components. Collectively, these findings suggested that B7-H3 may be a potential therapeutic target for AdCC.

Salivary adenoid cystic carcinoma (AdCC) is a common head and neck cancer with the propensity for local spread and distant metastasis. In our previous study, elevated expression of Anterior gradient 2 (AGR2) was detected in head and neck squamous cell carcinoma (HNSCC), associated with epithelial-mesenchymal transition (EMT) and cancer stemness. However, to date, the expression and function of AGR2 in AdCC has yet to be elucidated. In the present study, human AdCC tissue microarrays including 18 cases of normal salivary gland (NSG), 12 cases of pleomorphic adenoma (PMA) and 72 cases of AdCC were employed for immunohistochemical staining analysis. Results indicated that AGR2, which was remarkably correlated with Ki-67, transforming growth factor beta-1 (TGF-β1) and CD147, was significantly elevated in human salivary AdCC tissues. Knockdown of AGR2 significantly repressed the proliferation and migration of human SACC-83 and SACC-LM cell lines. Additionally, AGR2 silencing obviously reversed the EMT phenomena induced by TGF-β1. Taken together, our present study revealed the potential pro-metastasis role of AGR2 in AdCC, indicating that AGR2 might be a novel therapeutic target of AdCC with distant metastasis.

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.

P21 activated kinase 2 (PAK2) is a member of Group I PAKs family and highly expressed in various cancers. Current studies have demonstrated that PAK2 played a pivotal role in tumor progression. However, the role of PAK2 in salivary adenoid cystic carcinoma is still unclear. This study aims to explore the expression and the function of PAK2 in AdCC. Human salivary gland tissue microarray, including 18 normal salivary glands (NSG), 12 pleomorphic adenoma (PMA) and 72 AdCC, and immunohistochemistry were used to evaluate the expression of PAK2. The result showed that PAK2 was significantly increased in AdCC compared with NSG and PMA. Then the Pearson correlation analysis using serial tissue sections showed a close correlation of PAK2 with Cyclin D1, Phospho-STAT3 at Tyrosine 705 (p-STAT3) and Ki-67. Further in vitro study utilizing PAK2 knockdown via siRNA transfection revealed significantly reduced migration and proliferation of AdCC cell lines compared with control group. Knockdown of PAK2 decreased the expression of Cyclin D1 in AdCC cell lines. In addition, the inhibition of STAT3 reduced the expression of PAK2 in AdCC cell lines. These findings suggested that PAK2 promotes AdCC cell migration and proliferation and may be a potential therapeutic target.

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 < 0.05). Moreover, the high-expression of KEAP1 had significant correlations with LC3 (P < 0.001, R = 0.3195), and LAMP2 (P < 0.001, R = 0.3346) and NRF2 (P < 0.05, R = 0.2246) by using the Pearson correlation coefficient test. Our findings demonstrated that up-regulation of LC3, LAMP2, KEAP1 and NRF2 were associated with carcinogenesis and progression of SACC patients, suggesting that they may be useful molecular targets in salivary adenoid cystic carcinoma.

In this study, we accessed the expression and correlation of p-STAT3 with Survivin, Cyclin D1, CD147, Slug and Ki67 by immunohistochemical staining of human tissue microarray which contains 72 adenoid cystic carcinoma (AdCC), 12 pleomorphic adenoma (PMA) and 18 normal salivary gland (NSG) using digital pathological scanner and scoring system. We found that the expression of p-STAT3, Survivin, Slug, Cyclin D1 and CD147 was significantly increased in AdCC as compared with PMA and (or) NSG (p<0.05). While, the level of p-STAT3 and expression of Cyclin D1 and CD147 was not associated with pathological type of human AdCC (p>0.05). Correlation analysis of these proteins revealed that p-STAT3 up-regulates the expression of Survivin, Slug, Cyclin D1 and CD147 (p<0.05). Moreover, the activation of STAT3 was associated with proliferation marker Ki-67 (p<0.05). Selective inhibition of STAT3 by a small molecule S3I-201 significantly reduced human SACC-83 and SACC-LM cells proliferation, migration and invasion with the corresponding decrease in expression of Survivin, Slug, Cyclin D1 and CD147. These findings indicate that high phosphorylation level of STAT3 in AdCC is related to Survivin, Slug, Cyclin D1 and CD147. We suggest that the inhibition of STAT3 may be a novel strategy for neoadjuvant chemotherapeutic treatment of AdCC.

Pyridine compounds, including nicotinic acid and nicotinamide, are key metabolites of both the salvage pathway for NAD and the biosynthesis of related secondary compounds. We examined the in situ metabolic fate of [carbonyl-(14)C]nicotinamide, [2-(14)C]nicotinic acid and [carboxyl-(14)C]nicotinic acid riboside in tissue segments of tea (Camellia sinensis) plants, and determined the activity of enzymes involved in pyridine metabolism in protein extracts from young tea leaves. Exogenously supplied (14)C-labelled nicotinamide was readily converted to nicotinic acid, and some nicotinic acid was salvaged to nicotinic acid mononucleotide and then utilized for the synthesis of NAD and NADP. The nicotinic acid riboside salvage pathway discovered recently in mungbean cotyledons is also operative in tea leaves. Nicotinic acid was converted to nicotinic acid N-glucoside, but not to trigonelline (N-methylnicotinic acid), in any part of tea seedlings. Active catabolism of nicotinic acid was observed in tea leaves. The fate of [2-(14)C]nicotinic acid indicates that glutaric acid is a major catabolite of nicotinic acid; it was further metabolised, and carbon atoms were finally released as CO(2). The catabolic pathway observed in tea leaves appears to start with the nicotinic acid N-glucoside formation; this pathway differs from catabolic pathways observed in microorganisms. Profiles of pyridine metabolism in tea plants are discussed.

Aim: To investigate the effect of high glucose and mycophenolate (MMF) on the expression of MCP-1 in human mesangial cells (HMCs) and fibronectin (FN)., Methods: The HMCs were divided randomly into five groups: control group (5 mmol/L glucose), high glucose group (30 mmol/L glucose), mannitol group (5 mmol/L glucose and 25 mmol/L mannitol), high glucose+MMF-10 group (30 mmol/L glucose plus 10 μg/mL mycophenolate) and high glucose+MMF-100 group (30 mmol/L glucose plus 100 μg/mL mycophenolate). We detected the levels of MCP-1 and fibronectin in each group at 24 h, 48 h and 72 h, respectively. The expression levels of the MCP-1 mRNA were detected by RT-PCR, and the protein expression of MCP-1 and fibronectin was measured by ELISA., Results: Compared with the control group, the levels of the MCP-1 and FN in high glucose group were significantly increased with the expression peak at 48 h (P<0.01). The MMF with different concentration could inhibit the expression of MCP-1 and FN in time- and dose-dependent manner (P<0.05)., Conclusion: Mycophenolate could inhibit the expressions of MCP-1 and FN in human mesangial cells and it might be expected to delay the development and progression of glomerular sclerosis and interstitial fibrosis.

Objective: To isolate the long coding sequence of human novel gene C17orf62 which is named as C17orf62-L by us, and analyze its effects on cell viability, subcellular localization, and expression profile in multiple cell lines., Methods: RT-PCR (reverse transcription polymerase chain reaction ) was used to clone C17orf62-L which encoded 187 amino acids from human multi-tissue cDNA library. We used bioinformatics analysis to identify structural characteristics of C17orf62-L, and RT-PCR to detect its expression. By Laser Scanning Confocal Microscopy we identified its subcellular localization of C17orf62-L. Furthermore, flow cytometry experiment was used to validate whether overexpression of C17orf62-L could influence cell phenotypes and Western blot was used to study related mechanisms., Results: C17orf62-L was cloned and constructed into the pcDNA-and pEGFP-expression plasmids. C17orf62-L had signal peptide and transmembrane domain.C17orf62-L was widely expressed in multiple cell lines and was validated partial co-localization with Golgi apparatus. Functional studies showed C17orf62-L could induce cell death accompanied with rising of cleaved PARP(poly ADP-ribose polymerase)., Conclusion: Human C17orf62 is a novel cell death inducing gene.

The metabolic fate of [carbonyl-14C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [14C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-14C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3h after administration. Radioactivity from [carbonyl-14C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves., (2009 Elsevier Ltd. All rights reserved.)

Objective: To find novel isoform of PIK3IP1 and analyze their effects on cell viability, subcellular localization, and expression profile in cell lines., Methods: RT-PCR was used to clone PIK3IP1 and its novel splicing isoform PIK3IP1-v1 from multi-tissue cDNA pool. By cell-based assays, we studied how PIK3IP1 and PIK3IP1-v1 affected the activity of Renila luciferase and morphological change in the HEK 293T cells. Furthermore, flow cytometry experiment was used to validate that overexpression of both splicing isoforms could induce cell apoptosis. Bioinformatics analysis was used to identify structural characteristics of these two splicing isoforms. By fluorescence microscopy assay, we identified their subcellular localization. RT-PCR was used to detect the expression of PIK3IP1 in the cell lines., Results: PIK3IP1 and its novel splicing isoform PIK3IP1-v1 were cloned and constructed into the pcDNA-and pEGFP-expression plasmids. They both had signal peptide and transmembrane domain. Nevertheless, PIK3IP1-v1 was in absence of an extracellular Kringle domain. They could inhibit the activity of Renila luciferase and induce cell apoptosis. Simultaneously, both splicing isoforms are validated with subcellular localization on cell membrane and lowly expressed in many cell lines., Conclusion: PIK3IP1-v1 is a novel splicing isoform of PIK3IP1. Both of them are located on cell membrane and can induce cell apoptosis.

Objective: To investigate the relationship of anti-tuberculosis immunity with perforin (PFN), granzyme B (GzmB), interferon-gamma (IFN-gamma), and interleukin-2 (IL-2) expression by T lymphocyte subsets., Methods: Sixty mice were randomly allocated into a tuberculosis group and a control group (n = 30 each). Surface markers of T lymphocytes were stained with CD(3)PerCP, CD(4)FTTC, CD(8)APC, and intracellular cytotoxic molecules with PE-PFN, PE-GzmB, PE-IFN-gamma, and PE-IL-2 multi-color-labeled monoclonal antibodies, and analyzed at the single cell level. The relation between T lymphocyte subsets expression PFN, GzmB, IFN-gamma, IL-2 and antituberculosis immunity by flow cytometer., Results: (1) CD(4)(+), CD(8)(+), and CD(4)(+)CD(8)(+) (DP) T lymphocytes all expressed PFN, GzmB, IFN-gamma and IL-2 to some degrees. The expressions of PFN and GzmB were much higher in CD(8)(+) T lymphocytes than those in CD(4)(+) T lymphocytes, while the expressions of IFN-gamma and IL-2 were higher in CD(4)(+) T lymphocytes. (2) The counts of T lymphocyte subsets and the percentages of T lymphocyte subsets to total lymphocytes may or may not reflect the cellular immunity consistently. (3) There was no significant difference in T lymphocytes expressing PFN between the tuberculosis group and the control group. But the counts of CD(3)(+), CD(4)(+), DP and CD(8)(+) T lymphocytes and the percentages of CD(3)(+), DP and CD(8)(+) cells expressing GzmB were significantly increased in the tuberculosis group (t value from -3.72 to 4.13, all P < 0.05). (4) IFN-gamma expressing CD(3)(+) and CD(4)(+) lymphocytes were increased significantly in the tuberculosis group. The counts of CD(8)(+) and DP T lymphocytes and the percentages of CD(3)(+), CD(4)(+), CD(8)(+), and DP cells that expressed IL-2 were decreased significantly in the tuberculosis group (t value from 2.62 to 3.46, all P < 0.05)., Conclusion: CD(4)(+), CD(8)(+) and DP lymphocytes all can express PFN, GzmB, IFN-gamma and IL-2 at different degree levels.

Activator protein-1(AP-1) is an important transcription factor, and dysregulation of its activity has been associated with many human diseases, including various cancers. A novel human gene AC3-33 (GenBank name: c30rf33, Accession No. FLJ31139), which can suppress PMA and Ionomycin induced activation of AP-1, was identified from 650 human func-tion-known genes by using the high throughput-high content cell-based screening technology. The gene whose full cDNA length is 1391 bp containing 6 exons and 5 introns is located in the human chromosome 3q25.31. The predicted protein encoded by this gene contains 251 amino acids with a theoretical molecular weight of 29 kDa. Expression of the AC3-33 gene is widly found in adrenal glands and cervix. The amino acid sequences of AC3-33 is highly conserved, and has no homology to other known proteins. Subcellular localization studies show that the AC3-33 protein was localized in the cytoplasm. Our preliminary results showed that AC3-33 is an important novel gene related to supress AP-1 activity.