Secondary metabolites with fungicide potentials from the deep-sea seamount-derived fungus Talaromyces scorteus AS-242.

Marine natural products play an important role in biopesticides. Seven new secondary metabolites with different structural classes, including two cycloheptapeptides, scortide A (1) and scortide B (2), two 19-nor-diterpenoids, talascortene H (3) and talascortene I (4), two diterpenoid acids, talascortene J (5) and talascortene K (6), and one triterpenoid, talascortene L (7) were isolated and identified from the sea-anemone-derived endozoic fungus Talaromyces scorteus AS-242. Their structures were comprehensively assigned by spectroscopic data analysis, single-crystal X-ray diffraction, tandem mass spectrometry, and electronic circular dichroism (ECD) calculations. The result of the antimicrobial assay demonstrated that compounds 1 - 6 have inhibitory activity against several human, aquatic, and plant pathogens with minimum inhibitory concentration (MIC) values ranging from 1 to 64 µg/mL. Specially, compounds 2 and 4 showed significant activities against the pathogenic fungus Curvularia spicifera with the MIC value of 1 µg/mL, providing an experimental basis of 2 and 4 with the potential as lead compounds to be developed into biopesticides.

Access to Reverse Glycosyl Azides and Rare Sugar-Based Glycosyl Azides via Radical Decarboxylative Azidation: Divergent Synthesis of 4'-C-Azidonucleosides as Potential Antiviral Agents.

The radical decarboxylative azidation of structurally diverse uronic acids has been established as an efficient approach to reverse glycosyl azides and rare sugar-derived glycosyl azides under the action of Ag2CO3, 3-pyridinesulfonyl azide, and K2S2O8. The power of this method has been highlighted by the divergent synthesis of 4'-C-azidonucleosides using Vorbrüggen glycosylation of nucleobases with 4-C-azidofuranosyl acetates. The antiviral assessment of the resulting nucleosides revealed one compound as a potential inhibitor of covalently closed circular DNA.

A Versatile Thioesterase Involved in Dimerization during Cinnamoyl Lipid Biosynthesis.

The cinnamoyl lipid compound youssoufene A1 (1), featuring a unique dearomatic carbon-bridged dimeric skeleton, exhibits increased inhibition against multidrug resistant Enterococcus faecalis as compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatization/dimerization remains unknown. In this study, an unusual "gene-within-gene" thioesterase (TE) gene ysfF was functionally characterized. The gene was found to naturally encodes two proteins, an entire YsfF with α/ß-hydrolase and 4-hydroxybenzoyl-CoA thioesterase (4-HBT)-like enzyme domains, and a nested YsfFHBT (4-HBT-like enzyme). Using an intracellular tagged carrier-protein tracking (ITCT) strategy, in vitro reconstitution and in vivo experiments, we found that: i) both domains of YsfF displayed thioesterase activities; ii) YsfF/YsfFHBT could accomplish the 6π-electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP-tethered dearomatization/dimerization process, possibly through an unprecedented Michael-type addition reaction. Moreover, site-directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6π-electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.

Fucoidan from the cell wall of Silvetia siliquosa with immunomodulatory effect on RAW 264.7 cells.

Silvetia siliquosa, the only species of the family Fucaceae in China, is used as a medicine food homology. Fucoidan from S. siliquosa was extracted by hot water twice thoroughly (13 % of total yield), and a purified fucoidan SSF with a molecular weight of 93 kD was obtained. Chemical composition analysis demonstrated that SSF was primarily composed of sulfate (21.68 wt%) and fucose (84 % of all neutral monosaccharides). IR, methylation analysis, NMR and ESI-MS results indicated SSF had the backbone of mainly (1 â†’ 3)-α-L-fucopyranose and minor (1 â†’ 4)-α-L-fucopyranose, with little 1,3 and 1,4 branched ß-D-Xylp and ß-D-Galp. The in vitro immunomodulatory test on RAW 264.7 cells showed that SSF could up-regulate the expression of immune related factors and proteins in a concentration-dependent manner, but the immunomodulatory effect disappeared from desulfated SSF. This research indicated that highly sulfated fucan possessed immunomodulatory effect and the importance of sulfate groups in the activity of SSF.

A Modelling Study on Visitor Usage Intentions with AR-Based Content for Sports Venues / Un estudio de modelado sobre las intenciones de uso de los visitantes con contenido basado en AR para instalaciones deportivas

This study examined the development of users' behavioural intentions to visit sports venues when using AR-based content using the stimulus-organism-response model. The study centred on individuals who had experienced the AR-based content at Gocheuk Sky Dome. This study presents a structural model that illustrates the connections between three attributes of Augmented Reality (AR)—interactivity, vividness, and novelty (stimulus)—and their influence on the flow experience. This, in turn, affects the intention to depend on AR-based content for visiting a sports venue. A survey was conducted to evaluate the model, targeting young visitors in their 20s and 30s who are knowledgeable about AR technology. The study's findings indicate that novelty had the greatest influence on flow, followed by vividness, while interactivity did not show any significant impact. Flow had a significant impact on all four experiences: education, entertainment, aesthetics, and escapism. The positive impact on the intention to visit AR-based sports venues was observed only in relation to entertainment and education experiences. The role of consumer cultural innovativeness was examined as a moderator in the proposed structure model for visiting AR-based sports venues. These insights offer valuable information for the development of AR-based content in countries with culturally significant tourist destinations for sports enthusiasts. They also guide destination marketers in promoting AR in the tourism industry.(AU)

ß-Glycosylations with 2-Deoxy-2-(2,4-dinitrobenzenesulfonyl)-amino-glucosyl/galactosyl Selenoglycosides: Assembly of Partially N-Acetylated ß-(1 → 6)-Oligoglucosaminosides.

An efficient protocol has been established for ß-glycosylations with 2-deoxy-2-(2,4-dinitrobenzenesulfonyl)amino (2dDNsNH)-glucopyranosyl/galactopyranosyl selenoglycosides using PhSeCl/AgOTf as an activating system. The reaction features highly ß-selective glycosylation with a wide range of alcohol acceptors that are either sterically hindered or poorly nucleophilic. Thioglycoside- and selenoglycoside-based alcohols prove to be viable nucleophiles, opening up new opportunities for one-pot construction of oligosaccharides. The power of this approach is highlighted by the efficient assembly of tri-, hexa-, and nonasaccharides composed of ß-(1 → 6)-glucosaminosyl residues based on one-pot preparation of a triglucosaminosyl thioglycoside with DNs, phthaloyl, and 2,2,2-trichloroethoxycarbonyl as the protecting groups of amino groups. These glycans are potential antigens for developing glycoconjugate vaccines against microbial infections.

DUSP10 alleviates ischemic stroke-induced neuronal damage by restricting p38/JNK pathway.

Neuronal apoptosis is considered one of the hallmarks of ischemic stroke. Dual specificity phosphatase 10 (DUSP10), a member of the dual-specificity phosphatase family, which is involved in the regulation of apoptosis process. This study aimed to investigate the effect of on apoptosis in primary cortical neurons exposed to oxygen-glucose deprivation and reoxygenation (OGD/R) and mice suffered from transient middle cerebral artery occlusion and reperfusion (MCAO/R). The results showed that DUSP10 overexpression improved survival and reduced apoptosis in neurons subjected to OGD/R, which was manifested by decreased apoptotic proteins (cleaved caspase 3 and bax) and TUNEL+ cells, as well as increased the anti-apoptotic protein (bcl-2). DUSP10 overexpression inhibited the p38/JNK signaling pathway after OGD/R treatment, whilst DUSP10 knockdown had opposite effects. In addition, the p38 inhibitor SB203580 or JNK inhibitor SP600125 attenuated the increased apoptosis of OGD/R-stimulated neurons treated with DUSP10 silencing. Consistently, DUSP10 knockdown exacerbated infarct volume in MCAO/R injury. The data of Nissl staining and TUNEL-NeuN double staining revealed that DUSP10 interference aggravated neuronal damage in the ischemic penumbra of mice. Furthermore, DUSP10 inhibition activated the p38/JNK axis accompanied by enhanced phosphorylation of p38 and JNK in vivo. In summary, DUSP10 is a neuroprotective agent against ischemic stroke-induced neuronal damage via suppressing the p38/JNK signaling pathway.

Ultrahigh-performance liquid chromatography-high-resolution mass spectrometry-based plasma metabolomics study of thymoma and thymic hyperplasia.

RATIONALE: Thymoma is a rare malignant tumor but it is the most common primary tumor of the anterior mediastinum. The current imaging methods for thymoma screening suffer from false positive rate problems, and thymoma pathogenesis remains elusive. Study of thymoma metabolic characteristics could provide clues for improving the diagnosis and understanding the pathogenesis of thymoma. METHODS: Metabolic profiling of plasma from thymoma and thymic hyperplasia patients was performed using ultrahigh-performance liquid chromatography combined with high-resolution mass spectrometry in both positive and negative ionization modes. After pre- and post-processing, the dataset was divided into three age groups and statistical analysis was performed to select differential metabolites of thymoma. For feature identification, experimental tandem mass spectra were matched to those of databases and available chemical standards, and also manually annotated with plausible chemical structures to ensure high identification confidence. RESULTS: A total of 47 differential metabolites were identified in thymoma. Significantly higher levels of histidine, sphinganine 1-phosphate, lactic acid dimer, phenylacetylglutamine, LPC (18:3) and LPC (16:1), and significantly lower levels of phenylalanine, indole-3-propionic acid (IPA), hippuric acid and mesobilirubinogen were associated with thymoma. Tryptophan level in thymoma-associated myasthenia gravis (TAMG) was significantly lower than that of the MG(-) group. IPA and hippuric acid abundances exhibited increasing trends from indolent to aggressive thymoma. CONCLUSIONS: Our study revealed aberrant aromatic amino acid metabolism and fatty acid oxidation might be associated with thymoma. The identified unique metabolic characteristics of thymoma may provide valuable information for study of the molecular mechanism of thymoma pathogenesis, and improvement of diagnosis and discovery of new therapeutic strategies for thymoma.

Protein Kinase CK2 Modulates the Calcium Sensitivity of Type 3 Small-conductance Calcium-activated Potassium Channels in Colonic Platelet-derived Growth Factor Receptor Alpha-positive Cells From Streptozotocin-induced Diabetic Mice.

Background/Aims: The gastrointestinal symptom of diabetes mellitus, chronic constipation, seriously affects patients' life. Whereas, the mechanism of chronic constipation is still ambiguous, resulting in a lack of effective therapies for this symptom. As a part of the smooth muscle cells, interstitial cells of Cajal, and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells syncytium (SIP syncytium), PDGFRα+ cells play an important role in regulating colonic motility. According to our previous study, in PDGFRα+ cells in colons of diabetic mice, the function of the P2Y1 purinergic receptor/type 3 small-conductance calcium-activated potassium (SK3) channel signaling pathway is strengthened, which may lead to colonic dysmotility. The purpose of this study is to investigate the changes in SK3 channel properties of PDGFRα+ cells in diabetic mice. Methods: Whole-cell patch clamp, Western blotting, superoxide dismutase activity measurement, and malondialdehyde measurement were main methods in the present study. Results: The present study revealed that when dialysed with low calcium ion (Ca2+) solution, the SK3 current density was significantly decreased in PDGFRα+ cells from diabetic mice. However, the SK3 current density in PDGFRα+ cells was enhanced from diabetic mice when dialysed with high Ca2+ solution. Moreover, hydrogen peroxide-treatment mimicked this phenomenon in SK3 transgenic HEK293 cells. The subunit of SK3 channels, protein kinase CK2, was up-regulated in colonic muscle layers and hydrogen peroxide-treated HEK293 cells. Additionally, protein phosphatase 2A, the subunit of SK3 channels, was not changed in streptozotocin-treated mouse colons or hydrogen peroxide-treated HEK293 cells. Conclusion: The diabetic oxidative stress-induced upregulation of CK2 contributed to modulating SK3 channel sensitivity to Ca2+ in colonic PDGFRα+ cells, which may result in colonic dysmotility in diabetic mice.

ICOS/ICOSLG and PD-1 Co-Expression is Associated with the Progression of Colorectal Precancerous- Carcinoma Immune Microenvironment.

Purpose: This study aimed to investigate the expression of inducible T-cell co-stimulator (ICOS) and its ligand (ICOSLG), along with their association with clinicopathological features and influence on the immune profile in colorectal cancer (CRC). Patients and Methods: The Cancer Genome Atlas Colorectal Adenocarcinoma cohorts were used. We also analyzed 131 clinical samples of colon lesions, including precancerous lesions (hyperplastic polyps, low-grade dysplasia, and high-grade dysplasia) and CRC tissues. We conducted immunohistochemical (IHC) assays and multiple IHC (mIHC) of CD4+, Foxp3+ tumor-infiltrating lymphocytes (TILs), and PD-1/PD-L1 immune checkpoints in precancerous lesions and CRC samples from our patient subsets to determine changes and correlations in ICOS and ICOSLG expression during progression through the adenoma-carcinoma pathway. Results: High expression of ICOS and ICOSLG was a significant factor in CRC in multiple analyses and was positively correlated with CD4+/Foxp3+ TIL density and PD-1/PD-L1 expression, which increased with the sequential progression of lesions from precancerous tissues to carcinoma. Multivariable logistic regression analysis suggested that the location and expression level of ICOS/ICOSLG may be involved in precancerous-carcinoma progression. The co-expression status of PD-1 and ICOS/ ICOSLG could stratify patients with colorectal lesions into three groups of low, moderate, and high risk of progression. According to this classification and mIHC assays, we found a strong correlation between increased PD-1+ICOS+ or PD-1+ICOSLG+ co-expression and CRC, which might be deemed an independent factor in carcinogenesis. Conclusion: Increased ICOS/ICOSLG expression may be associated with the progressive formation of Foxp3+ TILs in the immune microenvironment and may further promote the development of the abnormal cytology of colorectal lesions from precancerous neoplasia to CRC. Our findings support the interpretation that enhanced co-expression of PD-1+ICOS+ or PD-1+ICOSLG+ contributes to the immune-active microenvironment of the colorectal adenoma-carcinoma sequence.

Agricultural water allocation with climate change based on gray wolf optimization in a semi-arid region of China.

Background: We quantified and evaluated the allocation of soil and water resources in the Aksu River Basin to measure the consequences of climate change on an agricultural irrigation system. Methods: We first simulated future climate scenarios in the Aksu River Basin by using a statistical downscaling model (SDSM). We then formulated the optimal allocation scheme of agricultural water as a multiobjective optimization problem and obtained the Pareto optimal solution using the multi-objective grey wolf optimizer (MOGWO). Finally, optimal allocations of water and land resources in the basin at different times were obtained using an analytic hierarchy process (AHP). Results: (1) The SDSM is able to simulate future climate change scenarios in the Aksu River Basin. Evapotranspiration (ET0) will increase significantly with variation as will the amount of available water albeit slightly. (2) To alleviate water pressure, the area of cropland should be reduced by 127.5 km2 under RCP4.5 and 377.2 km2 under RCP8.5 scenarios. (3) To be sustainable, the allocation ratio of forest land and water body should increase to 39% of the total water resource in the Aksu River Basin by 2050.

Clinicopathological analysis of superior mediastinal lymph node metastases in medullary thyroid carcinoma / 中华肿瘤杂志

Objective: To investigate the clinicopathological characteristics of superior mediastinal lymph node metastases (sMLNM) in medullary thyroid carcinoma (MTC). Methods: This retrospective analysis enrolled the patients who were treated for sMLNM of MTC in our hospital from May 2012 to January 2021. All patients were suspected of sMLNM due to preoperative imaging. According to the pathological results, the patients were divided into two groups named sMLNM group and the negative superior-mediastinal-lymph-node group. We collected and analyzed the clinical features, pathological features, pre- and post-operative calcitonin (Ctn), and carcinoembryonic antigen (CEA) levels of the two groups. Logistic regression analysis was used to analyze risk factors, and receiver operation characteristic (ROC) curves were drawn to determine the optimal cut-off values of preoperative Ctn and preoperative CEA for predicting sMLNM. Results: Among the 94 patients, 69 cases were in the sMLNM group and 25 cases were in the non-SMLNM group. Preoperative Ctn level (P=0.003), preoperative CEA level (P=0.010), distant metastasis (P=0.022), extracapsular lymph node invasion (P=0.013), the number of central lymph node metastases (P=0.002) were related to sMLNM, but the multivariate analysis did not find any independent risk factors. The optimal threshold for predicting sMLNM by pre-operative Ctn is 1500 pg/ml and AUC is 0.759 (95% CI: 0.646, 0.872). The sensitivity, specificity, positive predictive value, and negative predictive value of diagnosis are 61.2%, 77.3%, 89.1%, 39.5%, respectively. In patients who underwent mediastinal lymph node dissection through transsternal approach, the metastatic possibility of different levels from high to low were level 2R (82.3%, 28/34), level 2L (58.8%, 20/34), level 4R (58.8%, 20/34), level 3 (23.5%, 8/34), level 4L (11.8%, 4/34). Postoperative complications occurred in 41 cases (43.6%), and there was no perioperative death in all cases. 14.8% (12/81) of the patients achieved biochemical complete response (Ctn≤12 pg/ml) one month after surgery, 5 of these patients were in sMLNM group. Conclusions: For patients who have highly suspicious sMLNM through imaging, combining with preoperative Ctn diagnosis can improve the accuracy of diagnosis, especially for patients with preoperative Ctn over 1 500 pg/ml. The superior mediastinal lymph node dissection for the primary sternotomy should include at least the superior mediastinal levels 2-4 to avoid residual lesions. The strategy of surgery needs to be cautiously performed. Although the probability of biochemical cure in sMLNM cases is low, nearly 40% of patients can still benefit from the operation at the biochemical level.

Access to 6-Deoxy-heptose Constructs by One Carbon Homologation of Hexoses with Malononitrile: Divergent Synthesis of Campylobacter jejuni Strain 81-176 Capsular Trisaccharide Repeating Unit Derivatives.

An efficient approach to 6-deoxy-heptose constructs has been established by one-carbon homologation of the sugar chain of hexoses. The reaction features the formation of sugar-based α-substituted propanedinitriles and ensuing diverse oxidative transformations under mild reaction conditions that are compatible with a wide range of sugars bearing various functional/protecting groups. The applications of this method are demonstrated by a divergent assembly of Campylobacter jejuni strain 81-176 capsular trisaccharide repeating unit derivatives.

Direct ß-Mannosylation of Primary Alcohol Acceptors: Trisaccharide Iteration Assembly of ß-1,6-Oligomannosides Corresponding to Kakelokelose.

Gold(I)-catalyzed stereoselective ß-glycosylation of primary alcohols is achieved using the orthogonally protected mannosyl α-ortho-hexynylbenzoate (OABz) donors devoid of 4,6-O-tethering groups used in conventionally constructing ß-mannosidic bonds. The potential of this methodology is showcased by the first assembly of ß-1,6-tri/hexa-/nonamannosides and related sulfated congeners through a convergent strategy. The synthesis features the stereocontrolled ß-glycosylation of α-trimannosyl OABz donors and the late-stage sulfonation. This work is expected to expedite the preparation of ß-1,6-mannans and functionalized derivatives.

Involvement of PAR2 in platelet-derived growth factor receptor-α-positive cell proliferation in the colon of diabetic mice.

Our previous study indicated that streptozotocin (STZ)-induced diabetes leads to colonic platelet-derived growth factor receptor-α-positive (PDGFRα+ ) cell proliferation accompanied by slow colonic transit in mice; however, the mechanism of this effect is unclear. The present study used western blotting, immunohistochemistry, and quantitative PCR to investigate whether proteinase-activated receptor 2 (PAR2) mediates PDGFRα+ cell proliferation. Our results showed that PDGFRα, PAR2, and Ki-67 coexpression was increased in the diabetic colonic muscle layer. PDGFRα and PAR2 mRNA and protein expression levels were also markedly enhanced in the diabetic colonic muscle layer. Mice treated with 2-furoyl-LIGRLO-amide (2-F-L-a), a PAR2 agonist, exhibited significant colon elongation and increased smooth muscle weight. In the 2-F-L-a-treated mice, PDGFRα, PAR2, and Ki-67 coexpression was increased and PDGFRα and PAR2 mRNA and protein expression was significantly enhanced in the colonic smooth muscle layer. 2-F-L-a also increased proliferation and PDGFRα expression in NIH/3T3 cells cultured in high glucose, while LY294002, a PI3K antagonist, decreased cell proliferation and PDGFRα expression. PI3K and Akt protein and mRNA expression and p-Akt protein expression in diabetic and 2-F-L-a-treated mice were markedly reduced in colonic smooth muscle. 2-F-L-a also reduced PI3K, Akt, and p-Akt protein expression in NIH/3T3 cells, while the PI3K antagonist LY294002 increased this expression. The results indicate that PAR2 is involved in the proliferation of PDGFRα+ cells through the PI3K/Akt signaling pathway in the colon of STZ-induced diabetic mice, which may contribute to the slow transit and constipation that are associated with diabetes.

Comparison of Different Labeling Techniques for the LC-MS Profiling of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) exhibit various biological activities for infants, such as serving as prebiotics, blocking pathogens, and aiding in brain development. HMOs are a complex mixture of hetero-oligosaccharides that are generally highly branched, containing multiple structural isomers and no intrinsic chromophores, presenting a challenge to both their resolution and quantitative detection. While liquid chromatography-mass spectrometry (LC-MS) has become the primary strategy for analysis of various compounds, the very polar and chromophore-free properties of native glycans hinder their separation in LC and ionization in MS. Various labeling approaches have been developed to achieve separation of glycans with higher resolution and greater sensitivity of detection. Here, we compared five commonly used labeling techniques [by 2-aminobenzamide, 2-aminopyridine, 2-aminobenzoic acid (2-AA), 2,6-diaminopyridine, and 1-phenyl-3-methyl-5-pyrazolone] for analyzing HMOs specifically under hydrophilic-interaction chromatography-mass spectrometry (HILIC-MS) conditions. The 2-AA labeling showed the most consistent deprotonated molecular ions, the enhanced sensitivity with the least structural selectivity, and the sequencing-informative tandem MS fragmentation spectra for the widest range of HMOs; therefore, this labeling technique was selected for further optimization under the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) conditions. The combination strategy of 2-AA labeling and PGC-MS techniques provided online decontamination (removal of excess 2-AA, salts, and lactose) and resolute detection of many HMOs, enabling us to characterize the profiles of complicated HMO mixtures comprehensively in a simple protocol.

Induction of long-term potentiation at Schaffer collateral-CA1 synapses in mice hippocampus after IMPX977 administration.

Objective: To investigate the effects of IMPX977 on long term potentiation (LTP) at Schaffer collateral-CA1 synapses in vitro and on methyl CpG binding protein 2 (Mecp2) expression in mice cortex and hippocampus. Methods: Thirty-two C57BL/6 mice were randomly divided into four groups: control, olive oil (vehicle), IMPX977 low (5 mg/kg) and high (15 mg/kg) groups. Mice were administrated every other day orally for two weeks. Extracellular recording technique in vitro was used to record the effects of IMPX977 on Schaffer collateral-CA1 LTP pathway in acute mice hippocampal slices. The Mecp2 protein expression level was detected by Western blotting. Results: Compared to the control group, vehicle did not alter the synaptic transmission in Schaffer collateral-CA1 synapses, however, IMPX977 at concentrations of 5 mg/kg and 15 mg/kg significantly enhanced fEPSP (field excitatory postsynaptic potential) slope in Schaffer collateral-CA1 pathway to (179.6 ± 17.8)% and (191.4 ± 21.4)%, individually 60 min after HFS, IMPX977 improved LTP induction significantly at Schaffer collateral-CA1 pathway at least. Also, IMPX977 significantly elevated MeCP2 protein level in cortex. Conclusion: The effects of IMPX977 on synaptic transmission and Mecp2 protein expression provided convincing evidence that IMPX977 could be promising new drug candidates for Rett syndrome treatment.

Diabetes-induced damage of gastric nitric oxide neurons mediated by P2X7R in diabetic mice.

It is generally considered that enteric neuropathy is one of the causative factors in diabetic gastroparesis. Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice. However, the underlying mechanism remains unclear. The present study was designed to clarify the relationship between neuronal P2X7R and NOS neuron damage. The effect of P2X7R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-PCR，immunofluorescence, western blot, isometric force recording, intracellular calcium ([Ca2+]i) measurement and whole-cell patch clamp techniques. The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice, meanwhile, electric field stimulation-induced NOS sensitive relaxation was significantly suppressed. Myenteric neurons expressed P2X7R and pannexin1, and the mRNA and protein level of P2X7R and pannexin1 were up-regulated in diabetic mice. BzATP, a P2X7R activator, evoked [Ca2+]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca2+]i was more obvious in Hek293-P2X7R cells than in Hek293 cells. Application of BzATP activated an inward current of Hek293-P2X7R in a dose dependent manner. Hek293-P2X7R but not untransfected Hek293 cells could take up of YO-PRO-1. In addition, the uptake of YO-PRO-1 by Hek293-P2X7R was blocked by oxATP, a P2X7 antagonist and CBX, a pannexin1 inhibitor. The results suggest that the P2X7R of enteric neurons may be involved in diabetes-induced NOS neuron damage via combining with pannexin-1 to form transmembrane pores which induce macromolecular substances and calcium into the cells.

Rs6265 polymorphism in brain-derived neurotrophic factor (Val/Val and Val/Met) promotes proliferation of bladder cancer cells by suppressing microRNA-205 and enhancing expression of cyclin J.

BACKGROUND: In this study, we evaluated the effect of rs6265 polymorphism on the expression of brain-derived neurotrophic factor (BDNF) and relevant downstream targets, as well as the involvement of this polymorphism in bladder cancer. METHOD: A computational analysis and luciferase assays were used to explore the interaction among BDNF, miR-205, and cyclin J (CCNJ). Real-time polymerase chain reaction (RT-PCR) and Western blot analysis were carried out to determine the effect of rs6265 polymorphism on the expression of BDNF and relevant downstream genes. RESULT: BDNF directly inhibited miR-205 expression but enhanced the expression of CCNJ, which was identified as a virtual target gene of miR-205. Furthermore, the inhibitory effect of BDNF carrying the Val genotype, defined as BDNF (Val), on miR-205 expression was much stronger than that of BDNF (Met), while the inductive effect of BDNF (Val) on CCNJ expression was much weaker than that of BDNF (Met). miR-205 and CCNJ small interfering RNA (siRNA) were found to reduce cell proliferation and arrest the cells in G0/G1 phase. In addition, miR-205 expression in patients carrying BDNF genotyped as Met/Met (defined as Met/Met group) was much higher than patients carrying BDNF genotyped as Val/Val and Val/Met (defined as Val/Val group and Val/Met group). As an inhibitor of CCNJ expression, the inhibitory effect of miR-205 was much higher in the Met/Met group than that in the Val/Val and Val/Met groups. CONCLUSION: In summary, we suggested that the rs6265 polymorphism in BDNF upregulates the expression of CCNJ in bladder cancer via the inhibition of miR-205 expression, which leads to the promoted proliferation of bladder cancer cells.