Discovery of Novel N-Phenyltriazinone Derivatives Containing Oxime Ether or Oxime Ester Moieties as Promising Protoporphyrinogen IX Oxidase Inhibitors.

Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) is one of the most important targets for the discovery of green herbicides. In order to find novel PPO inhibitors with a higher herbicidal activity, a series of novel N-phenyltriazinone derivatives containing oxime ether and oxime ester groups were designed and synthesized based on the strategy of pharmacophore and scaffold hopping. Bioassay results revealed that some compounds showed herbicidal activities; especially, compound B16 exhibited broad-spectrum and excellent 100% herbicidal effects to Echinochloa crusgalli, Digitaria sanguinalis, Setaria faberii, Abutilon juncea, Amaranthus retroflexus, and Portulaca oleracea at a concentration of 37.5 g a.i./ha, which were comparable to trifludimoxazin. Nicotiana tabacum PPO (NtPPO) enzyme inhibitory assay indicated that B16 showed an excellent enzyme inhibitory activity with a value of 32.14 nM, which was similar to that of trifludimoxazin (31.33 nM). Meanwhile, compound B16 revealed more safety for crops (rice, maize, wheat, peanut, soybean, and cotton) than trifludimoxazin at a dose of 150 g a.i./ha. Moreover, molecular docking and molecular dynamics simulation further showed that B16 has a very strong and stable binding to NtPPO. It indicated that B16 can be used as a potential PPO inhibitor and herbicide candidate for application in the field.

VqMAPK3/VqMAPK6, VqWRKY33, and VqNSTS3 constitute a regulatory node in enhancing resistance to powdery mildew in grapevine.

Grapevine powdery mildew is caused by Erysiphe necator, which seriously harms grape production in the world. Stilbene synthase makes phytoalexins that contribute to the resistance of grapevine against powdery mildew. A novel VqNSTS3 was identified and cloned from Chinese wild Vitis quinquangularis accession Danfeng-2. The novel VqNSTS3 was transferred into susceptible 'Thompson Seedless' by Agrobacterium-mediated transformation. The transgenic plants showed resistance to the disease and activated other resistance-related genes. VqNSTS3 expression in grapevine is regulated by VqWRKY33, and which binds to TTGACC in the VqNSTS3 promoter. Furthermore, VqWRKY33 was phosphorylated by VqMAPK3/VqMAPK6 and thus led to enhanced signal transduction and increased VqNSTS3 expression. ProVqNSTS3::VqNSTS3-GFP of transgenic VqNSTS3 in Arabidopsis thaliana was observed to move to and wrap the pathogen's haustoria and block invasion by Golovinomyces cichoracearum. These results demonstrate that stilbene accumulation of novel VqNSTS3 of the Chinese wild Vitis quinquangularis accession Danfeng-2 prevented pathogen invasion and enhanced resistance to powdery mildew. Therefore, VqNSTS3 can be used in generating powdery mildew-resistant grapevines.

Alfin-like transcription factor VqAL4 regulates a stilbene synthase to enhance powdery mildew resistance in grapevine.

Resveratrol is a phytoalexin that is synthesized by stilbene synthase (STS). Resveratrol in the human diet is known to have beneficial effects on health. We previously identified six novel STS (VqNSTS) transcripts from the transcriptome data of Vitis quinquangularis accession Danfeng-2. However, the functions of and defensive mechanisms triggered by these VqNSTS transcripts remain unknown. In the present study, we demonstrate that the expression of five of these six novel members, VqNSTS2-VqNSTS6, can be induced by the powdery mildew-causing fungus Uncinula necator. Additionally, overexpression of VqNSTS4 in the V. vinifera susceptible cultivar Thompson Seedless promoted accumulation of stilbenes and enhanced resistance to U. necator by activating salicylic acid (SA) signalling. Furthermore, our results indicate that the Alfin-like (AL) transcription factor VqAL4 can directly bind to the G-rich element (CACCTC) in the VqNSTS4 promoter and activate gene expression. Moreover, overexpression of VqAL4 in Thompson Seedless enhanced resistance to U. necator by promoting stilbene accumulation and activating SA signalling. Conversely, RNA interference-mediated silencing of VqNSTS4 and VqAL4 resulted in increased susceptibility to U. necator. Collectively, our results reveal that VqNSTS4, regulated by VqAL4, enhances grapevine resistance to powdery mildew by activating SA signalling. Our findings may be useful to improve disease resistance in perennial fruit trees.

VqBGH40a isolated from Chinese wild Vitis quinquangularis degrades trans-piceid and enhances trans-resveratrol.

Resveratrol (3,5,4'-trihydroxy-stilbene) is a phytoalexin that can prevent plants from pathogen attacks. Piceid is the glycosylation product of resveratrol and the main storage form of stilbenes in grapevines. Here, we reported the function of a ß-glycoside hydrolase gene, VqBGH40a, from the Chinese wild grapevine Vitis quinquangularis accession Danfeng-2 in the regulation of plant resistance to powdery mildew (Uncinula necator). VqBGH40a belonging to ß-glycoside hydrolase family 1 encoded 506 amino acids and was located on the cytomembrane. Its optimal induction condition was 28 or 30℃, for 4 h, with 0.1 mM IPTG in a prokaryotic expression system. Enzyme activity detection showed that purified VqBGH40a could hydrolyze trans-piceid to form trans-resveratrol in vitro. VqBGH40a was transiently overexpressed in Danfeng-2 leaves and then artificially inoculated with powdery mildew showed that VqBGH40a protein could hydrolyze trans-piceid in vivo. Additionally, a comparative family analysis between VqBGH40a and 38 VviBGHs was performed. Overall, these results demonstrate that VqBGH40a can hydrolyze trans-piceid, enhance trans-resveratrol content, and participate in the defense mechanism of grapevine against powdery mildew.

Circ_0058124 Aggravates the Progression of Papillary Thyroid Carcinoma by Activating LMO4 Expression via Targeting miR-370-3p.

BACKGROUND: Thyroid cancer is the most common malignant tumor in the endocrine system. Papillary thyroid carcinoma (PTC) accounts for the vast majority of cases in this cancer. Recently, the vital role of circular RNA (circRNA) has been acknowledged in various cancers, and this study aimed to investigate the role of circ_0058124 and related mechanism of its action in PTC. MATERIALS AND METHODS: The expression of circ_0058124, miR-370-3p and LIM domain only (LMO4) was detected by qRT-PCR in tissue samples (PTC tissues or normal tissues, n=20) and cell lines (non-cancer cell line, Nthy-ori 3-1, and PTC cell lines, IHH-4 and TPC-1). For functional analysis, cell proliferation was investigated using CCK-8 assay and colony formation assay. Cell migration and invasion were determined using transwell assay, and cell migration was also assessed by wound healing assay. Cell apoptosis was monitored by flow cytometry assay. For mechanism analysis, the interaction between miR-370-3p and circ_0058124 or LMO4 predicted by the bioinformatics analysis was validated by dual-luciferase reporter assay or RIP assay. The effect of circ_0058124 on tumor growth in vivo was identified by establishing the Xenograft model. RESULTS: The expression of circ_0058124 was enhanced in PTC tissues and cells. Circ_0058124 knockdown inhibited viability, colony formation, migration and invasion and promoted apoptosis of PTC cells. Besides, circ_0058124 knockdown also blocked tumor growth in vivo. miR-370-3p was a target of circ_0058124, and circ_0058124 regulated the expression of LMO4, a target of miR-370-3p, by targeting miR-370-3p. Rescue experiments presented that miR-370-3p inhibition reversed the inhibitory effects of circ_0058124 knockdown on PTC development, and LMO4 overexpression reversed the effect of miR-370-3p restoration on PTC development. CONCLUSION: Circ_0058124 promoted the development of PTC by mediating the miR-370-3p/LMO4 axis, and circ_0058124, functioned as an oncogene in PTC, might be used as a promising biomarker for PTC diagnosis and treatment.

An Integrated Analysis of mRNAs and miRNAs Microarray Profiles to Screen miRNA Signatures Involved in Nasopharyngeal Carcinoma.

OBJECTIVE: We aim to identify several microRNAs (miRNAs/miRs)-messenger RNAs (mRNAs) biomarkers correlated to nasopharyngeal carcinoma (NPC) based on an integrated analysis of miRNA and mRNAs microarray expression profiles. METHODS: The available mRNA and miRNA microarray datasets were retrieved from Gene Expression Omnibus (GEO) database according to pre-determined screening criteria. Differentially expressed miRNA and mRNAs (DEmiRNAs and DEmRNAs) were extracted between NPC and noncancerous nasopharyngeal tissues. The target genes of DEmiRNAs were predicted with miRTarBase followed by the construction of DEmiRNAs-target DEmRNAs network, and functional analyses were performed. The DEmiRNAs expressions were validated and the performance of these DEmiRNAs was assessed by the area under the curve (AUC) values. Finally, the correlations between DEmiRNAs and specific clinical factors were analyzed. RESULTS: There were 1140 interaction pairs (including let-7d/f-MYC/HMGA2 and miR-452-ITGA9) in DEmiRNAs-target DEmRNAs network. The GO annotation analysis showed that several genes such as MYC, HMGA2 and ITGA9 primarily participated in cellular process. KEGG analysis showed that these targets were associated with cell cycle and cancer-related pathways. Down-regulated let-7(-d and -f) and up-regulated miR-452 were verified in datasets. The AUC values of these 3 DEmiRNAs (let-7d, let-7-f and miR-452) was 0.803, 0.835 and 0.735, respectively. Besides, miR-452 was significantly related to survival rate of NPC patients. CONCLUSION: The findings implied let-7d/f-MYC/HMGA2 and miR-452-ITGA9 might be promising targets for the detection and treatment of NPC.

Association of MUC2, MUC5AC and MUC5B genes with the recurrence of nasal polyps.

Although mucins were suggested to contribute to the pathogenesis of nasal polyposis, the correlation between the expression levels of MUC5AC, MUC5B and MUC2, and the recurrence of nasal polyps, has not been extensively investigated. The present study aimed to investigate the association of the levels of mucin (MUC) 2, MUC5AC and MUC5B with the recurrence of nasal polyps. A total of 56 patients with nasal polyps who underwent functional endoscopic sinus surgery at the Tianjin Third Central Hospital (Tianjin, China) between June 2007 and June 2010 were included and baseline characteristics were recorded. Reverse transcription-quantitative PCR analysis was used to determine the expression levels of the MUC2, MUC5AC and MUC5B genes at six months following the operation. The recurrence rate was calculated at one year following the operation. Spearman's rank correlation was used to determine the association between the reduction in the expression levels of MUC2, MUC5AC and MUC5B, and the recurrence rate of nasal polyps. There were no significant differences observed in the baseline characteristics between patients with and without the recurrence of nasal polyps. Prior to treatment, the expression levels of MUC5AC, MUC5B and MUC2 in patients with nasal polyps were significantly increased compared with those in the paranasal tissues and normal nasal mucosa. The expression levels of MUC5AC, MUC5B and MUC2 were similar between patients with and without recurrent nasal polyps. In addition, significantly decreased MUC5AC, MUC5B and MUC2 gene expression levels were observed in patients without recurrence of nasal polyps compared with those with recurrence of nasal polyps at six months following the operation. The decreased values of MUC5AC, MUC5B and MUC2 in patients with recurrence and without recurrence of nasal polyps compared with baselines were significantly negatively correlated with the recurrence rate of nasal polyps. In conclusion, the present results provided novel data for the diagnosis and treatment of patients with recurrent nasal polyps.

DRL1, Encoding A NAC Transcription Factor, Is Involved in Leaf Senescence in Grapevine.

The NAC (for NAM, ATAF1,2, and CUC2) proteins family are plant-specific transcription factors, which play important roles in leaf development and response to environmental stresses. In this study, an NAC gene, DRL1, isolated from grapevine Vitis vinifera L. "Yatomi Rose", was shown to be involved in leaf senescence. The quantity of DRL1 transcripts decreased with advancing leaf senescence in grapevine. Overexpressing the DRL1 gene in tobacco plants significantly delayed leaf senescence with respect to chlorophyll concentration, potential quantum efficiency of photosystem II (Fv/Fm), and ion leakage. Moreover, exogenous abscisic acid (ABA) markedly reduced the expression of DRL1, and the ABA and salicylic acid (SA) concentration was lower in the DRL1-overexpressing transgenic plants than in the wild-type plants. The DRL1 transgenic plants exhibited reduced sensitivity to ABA-induced senescence but no significant change in the sensitivity to jasmonic acid-, SA- or ethylene-induced senescence. Transcriptomic analysis and RNA expression studies also indicated that the transcript abundance of genes associated with ABA biosynthesis and regulation, including 9-cis-epoxycarotenoid dioxygenase (NCED1), NCED5, zeaxanthin epoxidase1 (ZEP1), ABA DEFICIENT2 (ABA2), ABA4, and ABA INSENSITIVE 2 (ABI2), was markedly reduced in the DRL1-overexpressing plants. These results suggested that DRL1 plays a role as a negative regulator of leaf senescence by regulating ABA synthesis.

Effect of combined treatment with clozapine and metformin on fasting blood glucose, insulin level, and expression of the glucose transporter-2 (GLUT2) in Sprague-Dawley rats.

BACKGROUND: Antipsychotic medications can cause an increase in blood glucose and the development of type II diabetes. Metformin may ameliorate these side effects. OBJECTIVE: Assess whether or not metformin reduces the abnormalities in glucose metabolism that occur with use of the antipsychotic clozapine. METHODS: Eighteen adult Sprague-Dawley (SD) rats were divided into three groups that were intragastrically administered saline, clozapine (20 mg/kg), or a combination of clozapine (20 mg/kg) and metformin (78 mg/kg) for 28 days. Fasting blood glucose was assessed at baseline and every seven days thereafter. The animals were euthanized on the 28(th) day at which time aortic blood was obtained to assess blood insulin and C-peptide by radioimmunoassay, and pancreatic tissue samples were collected and used to determine the expression of the glucose transporter-2 (GLUT2) by real-time polymerase chain reaction (RT-PCR) and Western Blot. RESULTS: Fasting blood glucose in the clozapine group was significantly higher at the 14(th), 21(st), and 28(th) day compared to baseline, but rats receiving clozapine and metformin only had significantly elevated levels on the 14(th) day of treatment. However, repeated measures ANOVA found no statistically significant differences in blood glucose levels over time between the three groups (p=0.136). Multiple comparison tests found that the mean insulin level in the clozapine+metformin group was significantly lower than the levels in the clozapine and saline groups. There were statistically significant differences in the expression of GLUT2 mRNA (clozapine+metformin group < clozapine group < saline group) and in the expression of GLUT2 protein (clozapine+metformin group, clozapine group < saline group). CONCLUSION: This study found a non-significant increase in fasting blood glucose in SD rats treated with clozapine that was partially counteracted by concurrent administration of metformin. Rats administered clozapine showed the expected decrease in the expression of GLUT2, but concurrent administration of metformin and clozapine for 28 days did not show the expected normalization of the expression levels of GLUT2.