Production regions discrimination of Huangguanyin oolong tea by using the content of chemical components and rare earth elements.

Oolong tea is one of the most popular tea beverages in China. Tea cultivars, processing technology and origin of production affect the quality and price of oolong teas. To investigate the differences in Huangguanyin oolong tea from different production regions, the chemical components, mineral elements and rare earth elements of Huangguanyin oolong tea produced in Yunxiao (YX) and Wuyishan (WY) were analyzed by using spectrophotometry methods, targeted metabolomics and inductive plasma coupled mass spectrometry (ICP-MS). The results of spectrophotometry methods revealed that there were significant differences in thearubigin, tea polyphenols and water extract between Huangguanyin oolong teas from different production regions. Targeted metabolomics identified a total of 31 chemical components in Huangguanyin oolong teas from the two production regions, of which 14 chemical components were significantly different and contributed to the regional differentiation of Huangguanyin oolong tea. Yunxiao Huangguanyin had relatively higher contents of (-)-Epigallocatechin-3-O-(3-O-methylgallate) (EGCG3″Me), ornithine (Orn) and histidine (His), while Wuyishan Huangguanyin had relatively higher contents of glutamic acid (Glu), γ-aminobutyric acid (GABA), ß-aminobutyric acid (ß-ABA) and other components. Moreover, ICP-MS identified a total of 15 mineral elements and 15 rare earth elements in Huangguanyin oolong tea from the two production regions, of which 15 elements were significantly different between YX and WY, and contributed to the regional differentiation of Huangguanyin oolong tea. K had a relatively higher content in Yunxiao Huangguanyin, while rare earth elements had relatively higher contents in Wuyishan Huangguanyin. The classification results by the production region showed that the discrimination rate of the support vector machine (SVM) model based on the 14 different chemical components reached 88.89%, while the SVM model based on the 15 elements reached 100%. Therefore, we used targeted metabolomics and ICP-MS techniques to screen and explore the chemical components, mineral elements and rare earth elements differences among two production regions, which indicated the feasibility of Huangguanyin oolong tea classification by production regions in the study. The results will provide some reference for the distinction between the two production regions of Huangguanyin oolong tea.

Allele-specific expression and chromatin accessibility contribute to heterosis in tea plants (Camellia sinensis).

Heterosis is extensively used to improve crop productivity, yet its allelic and chromatin regulation remains unclear. Based on our resolved genomes of the maternal TGY and paternal HD, we analyzed the contribution of allele-specific expression (ASE) and chromatin accessibility of JGY and HGY, the artificial hybrids of oolong tea with the largest cultivated area in China. The ASE genes (ASEGs) of tea hybrids with maternal-biased were mainly related to the energy and terpenoid metabolism pathways, whereas the ASEGs with paternal-biased tend to be enriched in glutathione metabolism, and these parental bias of hybrids may coordinate and lead to the acquisition of heterosis in more biological pathways. ATAC-seq results showed that hybrids have significantly higher accessible chromatin regions (ACRs) compared with their parents, which may confer broader and stronger transcriptional activity of genes in hybrids. The number of ACRs with significantly increased accessibility in hybrids was much greater than decreased, and the associated alleles were also affected by differential ACRs across different parents, suggesting enhanced positive chromatin regulation and potential genetic effects in hybrids. Core ASEGs of terpene and purine alkaloid metabolism pathways with significant positive heterosis have greater chromatin accessibility in hybrids, and were potentially regulated by several members of the MYB, DOF and TRB families. The binding motif of CsMYB85 in the promoter ACR of the rate-limiting enzyme CsDXS was verified by DAP-seq. These results suggest that higher numbers and more accessible ACRs in hybrids contribute to the regulation of ASEGs, thereby affecting the formation of heterotic metabolites.

[The albino mechanism of a new theanine-rich tea cultivar 'Fuhuang 2'].

To explore the mechanism of tea albino variation and high theanine formation, 'Fuyun 6' and a new theanine-rich tea cultivar 'Fuhuang 2' were as materials in this study, pigment content, metabolome and transcriptome of the two cultivars were analyzed by ultramicroelectron microscopy, widely targeted metabolomics, targeted metabolomics and transcriptomics. The results showed that five catechins, theobromine, caffeine, and 20 free amino acids, including theanine, glutamine, arginine, etc., were identified by targeted metabolomics. The amino acid content of 'Fuhuang 2' was significantly higher than that of 'Fuyun 6', and the theanine content was as high as 57.37 mg/g in 'Fuhuang 2'. The ultrastructure of leaves showed that the chloroplast cell structure of 'Fuhuang 2' was fuzzy, most of the grana lamellae were arranged in disorder, with large gaps, and the thylakoids were filiform. The determination of pigments showed that compared with 'Fuyun 6', the contents of chlorophyll A and B, carotenoids, flavonoids and other pigments of 'Fuhuang 2' decreased significantly, some important pigment-related-genes, such as chlorophyllase (CLH), 9-cis-epoxycarotenoid dioxygenase (NCED), flavonoid 3ß-hydroxylase (F3H) and flavonoid 3', 5'-hydroxylase (F3'5'H) were significantly changed. Compared with 'Fuyun 6', 'Fuhuang 2' identified 138 significantly changed metabolites (SCMs) and 658 differentially expressed genes (DEGs). KEGG enrichment analysis showed that SCMs and DEGs were significantly enriched in amino acid biosynthesis, glutathione metabolism and TCA cycle. In general, the albino phenotype of 'Fuhuang 2' may be caused by a deficiency in photosynthetic proteins, chlorophyll metabolism genes and chlorophyll content. The accumulation of high theanine in 'Fuhuang 2' may be due to the low nitrogen consumption in yellowed leaves and the lack of carbon skeleton, amino and nitrogen resources are stored more effectively, resulting in the up regulation of metabolites and related gene expression in the amino acid synthesis pathway, theanine has become a significant accumulation of nitrogen-containing compounds in yellowed leaves.

Autologous Bioactive Compound Concentrated Growth Factor Ameliorates Fistula Healing of Anal Fistula in a Pig Model and Promotes Proliferation and Migration of Human Skin Fibroblasts via Regulating the MEK/ERK Pathway.

Recent evidence suggested that autologous concentrated growth factor (CGF), a new bioactive compound from autologous blood is used widely as an ingenious biomaterial in tissue regeneration with anti-inflammatory properties. This study investigated whether CGF could be involved in the treatment of fistula healing in the anal fistula. For this purpose, the porcine anal fistula model was conducted using the rubber band ligation method and collected pig autogenic CGF to treat the fistulas. CGF treatment promoted fistula healing, which was reflected in the downregulation of inflammatory factors, upregulation of growth factors, and promoted epithelial-mesenchymal transition with increased collagen synthesis. Besides, 16S rRNA gene sequencing analysis of fistula tissues between the control and CGF groups showed that the microbial populations exhibiting significant differences were VadinCA02, Blastomonas, Deinococcus, Devosia, Sphingomonas, Rubrobacteria, and GW_34. CGF of volunteers were collected to process small interfering RNA- (siRNA-) ERK or siRNA-negative control transfected human skin fibroblasts (HSF). The results showed that CGF also promoted the proliferation and extracellular matrix-related functions in HSF, as well as activated the MEK/ERK pathway in vitro and in vivo. Finally, knockdown ERK reversed the effects of CGF in promoting wound healing in HSF. Collectively, our results suggest that the CGF as the bioactive compound from autologous blood exhibited great potential for repairing fistulas as well as promoting the proliferation and migration of human skin fibroblasts by triggering MEK/ERK signaling. These findings provided a fresh perspective for understanding the role of CGF in the management of fistulas.

Changes in Non-Volatile and Volatile Metabolites Associated with Heterosis in Tea Plants (Camellia sinensis).

Heterosis or hybrid vigor is extensively used in plant breeding. However, the contribution of metabolites to heterosis is still elusive. Here, we systematically identified the non-volatile and volatile metabolites of two hybrids and their parents in Camellia sinensis. The metabolomics analysis showed prevalent non-additive accumulation in hybrids, among which the non-additive nucleotides, alkaloids, organic acids, and tannins contribute to the positive heterosis of hybrids, including typical inosine, guanosine, adenosine, caffeine, succinic acid, adipic acid, xylonic acid, and gallic acid. The catechins and free amino acids in hybrids showed negative heterosis compared to its maternal cultivar TGY. Furthermore, the significant accumulation of non-additive terpenes combined with the mild heterosis of other types of volatiles contributes to the aroma of tea plant hybrids. The genetics of volatiles from different parents affect the aroma of hybrids processed into oolong tea. The comprehensive heterosis of these non-additive metabolites may play an important role in the formation of desirable breeding traits for hybrids. Our results provide insights into the utilization of heterosis breeding and the regulation of heterosis metabolites in tea plants.

Identification of Co-Expressed Genes Related to Theacrine Synthesis in Tea Flowers at Different Developmental Stages.

Jiaocheng kucha is the first reported tea germplasm resource which contains theacrine founded in Fujian Province. Currently, the anabolic mechanism of theacrine within tea leaves is clear, but there are few studies focused on its flowers. In order to further explore the mechanism of theacrine synthesis and related genes in flowers, current study applied Jiaocheng kucha flowers (JC) as test materials and Fuding Dabaicha flowers (FD) as control materials to make transcriptome sequencing, and determination of purine alkaloid content in three different developmental periods (flower bud stage, whitening stage and full opening stage). The results showed that the flower in all stages of JC contained theacrine. The theacrine in the flower bud stage was significantly higher than in the other stages. The differentially expressed genes (DEGs) at three different developmental stages were screened from the transcriptome data, and were in a total of 5642, 8640 and 8465. These DEGs related to the synthesis of theacrine were primarily annotated to the pathways of purine alkaloids. Among them, the number of DEGs in xanthine synthesis pathway was the largest and upregulated in JC, while it was the smallest in caffeine synthesis pathway and downregulated in JC. Further weighted gene co-expression network (WGCNA) indicated that ADSL (CsTGY03G0002327), ADSL (CsTGY09G0001824) and UAZ (CsTGY06G0002694) may be a hub gene for the regulation of theacrine metabolism in JC. Our results will contribute to the identification of candidate genes related to the synthesis of theacrine in tea flowers, and explore the molecular mechanism of theacrine synthesis in JC at different developmental stages.

The Comparison of Laparoscopic Colorectal Resection with Natural Orifice Specimen Extraction versus Mini-Laparotomy Specimen Extraction for Colorectal Tumours: A Systematic Review and Meta-Analysis of Short-Term Outcomes.

AIM: The aims of this study were to compare the short-term outcomes of natural orifice specimen extraction surgery (NOSES) and conventional laparoscopic surgery (CLAPS) for colorectal tumours and to evaluate the safety and feasibility of NOSES in colorectal resection. METHODS: A literature review was performed on the PubMed, Cochrane Library, and Embase databases up to March 2019. Papers conforming to the inclusion criteria were used for further analysis. The short-term outcomes included intraoperative outcomes and postoperative recovery results. The weighted mean difference (WMD) was calculated for continuous outcomes and odds ratio (OR) for dichotomous results. Study quality was evaluated using the Newcastle-Ottawa Quality Assessment Scale (NOS) or the 6-item Jadad scale. RESULTS: Eight studies comprising 686 patients met the inclusion criteria. Compared with CLAPS, NOSES had more advantages in terms of postoperative complications, postoperative pain, recovery of gastrointestinal function, duration of hospital stay, and cosmetic results. The lymph nodes harvested and intraoperative blood loss in NOSES were comparable with CLAPS; however, a prolonged operative time was observed in NOSES. CONCLUSIONS: NOSES was shown to be a safe and viable alternative to CLAPS in colorectal oncology in terms of short-term results. Further long-term and randomized trials are required.

[Association of serum miRNA6086 expression with postoperative anal fistula recurrence].

OBJECTIVE: To investigate the association between serum miRNA-6086 expression level and anal fistula recurrence. METHODS: Clinical data and serum samples of 60 patients with anal fistula and mix hemorrhoid identified by pathology undergoing resection in our department from August 2015 to August 2016 were collected. In addition, serum samples of 20 patients matching with fistula group in age, gender and body weight and receiving only hemorroidectomy were collected as control during the same period. Serum miRNA6086 expression level was detected by real-time quantitative RT-PCR method, and the association of serum miRNA6086 expression level with clinicopathologic features was analyzed. Univariate ANOVA test and multivariate logistic regression analysis were performed to analyze the association between serum miRNA6086 expression level and anal fistula recurrence. RESULTS: The relative expression of serum miRNA6086 in fistula group was 65.85±15.57, which was significantly up-regulated for 4.87 folds of 13.52±7.32 in control group(P<0.05). In fistula group, 24 cases(40%) developed anal fistula recurrence, whose serum miRNA6086 expression was significantly higher compared to 36 cases without recurrence (74.06±12.92 vs. 60.38±14.90, P<0.05). No associations of serum miRNA6086 expression with age, gender, BMI, drug history, acute phase were observed (P>0.05), while association of serum miRNA6086 expression level with the type, number and position of anal fistula was significant (all P<0.05). Univariated analysis indicated that anal fistula type (χ2=6.890, P=0.009), anal fistula number (χ2=0.554, P<0.001) and serum miRNA6086 expression (χ2=11.390, P=0.001) were significantly associated with anal fistula recurrence. Multivariate logistic regression analysis showed that complex anal fistula (OR=4.75, 95%CI: 1.84 to 12.01, P=0.001) and high expression of serum miRNA6086 (OR=3.22, 95%CI:1.31 to 8.22, P=0.011) were independent risk factors of anal fistula recurrence. CONCLUSION: Up-regulated expression of serum miRNA6086 is associated to the anal fistula type and may be valuable in predicting the prognosis.

Comparative evaluation of colonoscopy-assisted transanal minimally invasive surgery via glove port and endoscopic submucosal dissection for early rectal tumor.

BACKGROUND: Early rectal tumor is usually managed by local excision. A novel method-colonoscopy-assisted transanal minimally invasive surgery via glove port (CA-TAMIS-GP)-for resecting early rectal tumor was developed and compared with endoscopic submucosal dissection (ESD). MATERIALS AND METHODS: We performed CA-TAMIS-GP surgery on 26 patients from January 2014 to February 2016. For better analysis, we retrospectively collected data from 31 patients who underwent ESD between October 2012 and December 2013; overall, 57 patients diagnosed with early rectal tumor were included in this study. Perioperative conditions and long-term outcomes of both groups were compared. RESULTS: All lesions were dissected completely and successfully without conversion to open surgery or major complications. On histopathologic examination, all specimens in this study had negative margins. All patients had uneventful postoperative recoveries, except 3 patients of CA-TAMIS-GP with minor hematochezia, which resolved spontaneously; 7 ESD patients had late-onset bleeding and 3 needed colonoscopic hemostasis; 2 patients in each group had mild fever. The CA-TAMIS-GP group had a shorter operation time, less hemorrhage, and a lower average consumable cost than the ESD group (P < 0.05); moreover, the CA-TAMIS-GP group had no recurrence or long-term complications during a follow-up of 10-32 months, whereas3 patients in the ESD group developed local recurrence during a follow-up of 24-36 months. CONCLUSIONS: The CA-TAMIS-GP is a new method that is safe and effective in patients with early rectal tumor and appears to have a shorter operation time and less blood loss as compared with ESD.

MicroRNA-24 inhibits serotonin reuptake transporter expression and aggravates irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common chronic functional gastrointestinal disorder. MicroRNAs (miRNAs) have been widely demonstrated to take part in various physiological and pathological processes. In the present study, the role of miR-24 in the pathogenesis of IBS and the potential mechanism in this process were evaluated. Human intestinal mucosa epithelial cells of colon from IBS patients and healthy subjects were collected. An IBS mouse model was established with the induction of trinitro-benzene-sulfonic acid (TNBS). The expression levels of miR-24 and serotonin reuptake transporter (SERT) were analyzed using Real-time PCR and western blot in both human specimen and mice. miR-24 was upregulated in IBS patients and mice intestinal mucosa epithelial cells. Luciferase reporter assay showed that SERT was a potential target gene of miR-24. The treatment of miR-24 inhibitor increased pain threshold and nociceptive threshold levels and reduced MPO activity in proximal colon of IBS mice, and up-regulated the mRNA and protein expression levels of SERT in intestinal mucosa epithelial cells. miR-24 played a role in the pathogenesis of IBS probably through regulating SERT expression.

[Expression of prostaglandin transporter in colorectal cancer tissues and its relationship with clinicopathological features].

OBJECTIVE: To investigate the expression of prostaglandin transporter (PGT) in colorectal cancer (CRC) tissues and its relationship with clinicopathological features. METHODS: The mRNA and protein levels of PGT were determined by real-time PCR, Western blot and immunohistochemical methods in cancer tissues and adjacent normal tissue from 80 patients with colorectal cancer and their relationship with clinicopathological features was analyzed. RESULTS: Compared with the adjacent normal tissue of colorectal cancer, the PGT mRNA relative expression (0.57 ± 0.33 vs. 2.33 ± 1.20) and the PGT protein expression in cancer tissues decreased significantly [PGT/GAPDH 0.45 ± 0.16 vs. 0.78 ± 0.23, integral A 718.7 ± 359.4 vs. 10412.0 ± 6423.3, average A 0.03 ± 0.01 vs. 0.12 ± 0.09, all P<0.01]. Lower mRNA and protein expressions of PGT in colorectal cancer were associated with depth of invasion T3 to T4 and TNM stage III( to IIII( (P<0.01), while not associated with gender, age, tumor location and differentiation degree (all P>0.05). CONCLUSION: Expression levels of PGT mRNA and protein in colorectal cancer tissue are significantly down-regulation. PGT expression is associated with invasion depth and late stages.

[Clinical significance of the detection of serum neutrophil gelatinase-associated lipocalin in human colorectal cancer].

OBJECTIVE: To explore the feasibility and clinical significance of the detection of serum neutrophil gelatinase-associated lipocalin (NGAL) in human colorectal cancer. METHODS: Levels of NGAL in serum samples from 133 healthy people, 125 colorectal polyps patients and 100 colorectal cancer patients respectively were determined by sandwich ELISA assay. Relationship of NGAL level with clinicopathological features of colorectal cancer patients was analyzed. The optimal cut-off value of serum NGAL for diagnosing colorectal cancer was determined by ROC curve and compared with CEA and CA19-9. Univariate and multivariate analyses were performed to examine the relationship of NGAL level with the prognosis of patients with colorectal cancer. RESULTS: The median serum NGAL protein level in 100 colorectal cancer cases was 67.96 (53.30-79.86) µg/L, significantly higher than that in healthy people and colorectal polyps patients. The differences were statistically significant (all P<0.01). Serum NGAL protein level was significantly associated with tumor diameter, TNM stage, lymph node metastasis and vascular involvement (P<0.05). The optimal cut-off point of serum NGAL protein level for diagnosing colorectal cancer was 49.78 µg/L, and the sensitivity and specificity were 88% and 81% respectively. As for colorectal cancer patients with stage I, the sensitivity of serum NGAL (78.9%) was significantly higher as compared to CA19-9 (31.6%) and CEA (36.8%); as for those with stage II, the sensitivity of serum NGAL(88.0%) was also significantly higher compared to CA19-9 (48.0%) and CEA (52.0%). Kaplan-Meier analysis showed that patients with positive NGAL (≥49.78 µg/L) had worse survival than those with negative NGAL (P=0.002). Multivariate analysis showed that NGAL was an independent prognostic factor (HR=2.060, 95%CI:1.023-4.150, P=0.043). CONCLUSIONS: NGAL can be served as the novel malignant biological phenotype marker for human colorectal cancer and can be used for the risk stratification. NGAL may be an independent prognostic factor in colorectal cancer.

An experimental research into endostatin microbubble combined with focused ultrasound for anti-tumor angiogenesis in colon cancer.

OBJECTIVE: to evaluate the therapeutic effect of targeted endostatin-loaded microbubbles, combined with improved, focused, directional ultrasound radiation for inhibition of subcutaneous translocation in situ colon tumor angiogenesis in colon cancer. METHODS: 65 BALB/c nude mice with subcutaneous translocation in situ colon tumors were randomly divided into five groups. Group A was the control group, without any treatments. In Group B, the mouse was treated with ultrasonic radiation. In Group C, the mouse was treated with ultrasonic radiation combined with empty SonoVue microbubbles. In Group D, the mouse was treated with ultrasonic radiation combined with empty Targestar-SA microbubbles. In Group E, the mouse was treated with ultrasonic radiation combined with endostatin microbubbles. The tumor size was measured before and 1, 14, and 28 days after irradiation. The peak intensity (PI), regional blood volume (RBV) and regional blood flow (RBF) were recorded using contrast-enhanced ultrasound. The tumor tissue was removed for pathological examination; the tumor necrosis area and microvascular density (MVD) were evaluated by immunohistochemistry. RESULTS: Tumors in Groups C, D and E were significantly smaller than in Groups A and B at 28 days after irradiation, with Group E the smallest. PI, RBF and RBV of Groups C, D, and E were significantly decreased 28 days after radiation with Group E the lowest, and significantly lower than Groups A and B (all P < 0.05). The tumor tissue necrosis area of Group E was clearly greater while MVD was obviously lower than the other groups (all P < 0.01) at 28 days after treatment. CONCLUSION: The targeted endostatin microbubbles, combined with focused, directional ultrasound radiation can damage tumor microvasculature of subcutaneous colon translocation in situ colon cancer, as well as inhibit the tumor angiogenesis.

[Multi-gene methylation detection increases positive methylation rate in colorectal cancer].

OBJECTIVE: To study whether combined detection of the methylation status of vimentin, sFRP1, and HPP1 gene can increase the positive methylation rate in colorectal cancer. METHODS: Tissue samples were collected from 90 patients with colorectal cancer, 60 patients with adenomatous polyp, and 20 healthy controls. DNA was extracted and the methylation status of vimentin, sFRP1, and HPP1 gene was detected by Methylation-specific PCR (MSP). The relationship between clinicopathologic features of colorectal cancer and gene methylation was analyzed. RESULTS: The methylation rates of vimentin, sFRP1, and HPP1 were 66.7%, 68.9%, and 72.2% in colorectal cancer, 53.3%, 55.0%, and 50.0% in colorectal adenomas, and 0, 0, and 5.0% in healthy controls, respectively. The methylation of each of the three genes in colorectal cancer tissues was higher than colorectal adenomas and healthy controls(P<0.05). The diagnostic sensitivity by combining three methylation markers was 93.3% in colorectal cancer, 76.7% in colorectal adenomas, which was higher than the sensitivity using single gene testing(P<0.05). No significant associations existed between the methylation status of the three genes and clinical characteristics including sex, age, tumor location, lymph node metastases, distant metastasis, and TNM stage(P>0.05). CONCLUSIONS: DNA methylation levels of vimentin, sFRP1 and HPP1 are significantly higher in colorectal cancer tissue. Combined detection significantly improves the positive rate of methylation, and may be used as early diagnosis method for colorectal cancer.