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1.
Life Sci ; 248: 117445, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32081664

RESUMO

AIMS: Atherosclerosis (AS) is a common cardiovascular disease with complicated pathogenesis. Long non-coding RNAs (lncRNAs) have been reported to be associated with AS progression. We aimed to explore the role and underlying mechanism of HOXA transcript at the distal tip (HOTTIP) in AS. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HOTTIP, miR-490-3p and high mobility group B 1 (HMGB1) in AS patients' sera and oxidized low-density lipoprotein (ox-LDL) induced human aortic vascular smooth muscle cells (HA-VSMCs). Cell Counting Kit-8 (CCK-8) assay and transwell assay were conducted to evaluate the proliferation and migration of HA-VSMCs, respectively. Western blot assay was carried out to determine the levels of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), MMP9 and HMGB1. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the targeting association between HOTTIP and miR-490-3p, as well as miR-490-3p and HMGB1. KEY FINDINGS: HOTTIP and HMGB1 were upregulated and miR-490-3p was downregulated in the sera of AS patients and ox-LDL-stimulated HA-VSMCs. HOTTIP knockdown suppressed ox-LDL induced proliferation and migration in HA-VSMCs. MiR-490-3p was identified as a target of HOTTIP and HOTTIP overexpression abolished the inhibition on cell proliferation and migration mediated by miR-490-3p in ox-LDL-induced HA-VSMCs. Moreover, miR-490-3p inhibition promoted cell proliferation and migration by directly targeting HMGB1 in ox-LDL-induced HA-VSMCs. Besides, HOTTIP knockdown repressed the activation of PI3K-AKT signaling pathway. SIGNIFICANCE: HOTTIP knockdown suppressed cell proliferation and migration by regulating miR-490-3p/HMGB1 axis and PI3K-AKT pathway in ox-LDL-induced HA-VSMCs.


Assuntos
Aterosclerose/genética , Proteína HMGB1/genética , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Longo não Codificante/genética , Aorta/metabolismo , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Proteína HMGB1/metabolismo , Humanos , Lipoproteínas LDL/farmacologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
2.
J Agric Food Chem ; 67(49): 13758-13766, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31789514

RESUMO

Probiotics, such as Lactobacillus, have been proven to be effective in maintaining intestinal homeostasis. The modulatory effect of Lactobacillus on intestinal epithelial development in early life is still unclear. In this study, Lactobacillus isolates with good probiotic abilities were screened and orally administered to detect their regulatory effect on intestinal development in chickens. L. reuteri 22 was isolated from chickens and chosen for subsequent chicken experiments due to its strong acid and bile salt resistance and ability to adhere to epithelial cells. The 3-day-old chickens were orally administrated with 108 CFU L. reuteri 22 for consecutive 7 days. L. reuteri 22 increased Lgr5 mRNA expression (3.23 ± 0.40, P = 0.001) and activated the Wnt/ß-catenin signaling pathway, with increasing expression of proliferating cell nuclear antigen (PCNA) (49.27 ± 9.81, P = 0.021) to support the proliferation of chicken intestinal epithelial cells. Moreover, L. reuteri 22 also inhibited the Notch signaling pathway to induce intestinal stem cell differentiation into goblet cells with increased mucin 2 (Muc-2) expression (1.72 ± 0.34, P = 0.047). L. reuteri 22 significantly enhanced lysozyme mRNA expression (2.32 ± 0.55, P = 0.019) to improve intestinal innate mucosal immunity. This study demonstrated that L. reuteri administration could regulate chicken intestinal epithelium development to ensure the function of the intestinal mucosal barrier, which is beneficial for newborn animals.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Caliciformes/efeitos dos fármacos , Lactobacillus reuteri/fisiologia , Probióticos/farmacologia , Animais , Galinhas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Mucina-2/genética , Mucina-2/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismo
3.
DNA Cell Biol ; 38(12): 1564-1576, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31633379

RESUMO

The molecular mechanism of tumorigenesis of the prevalent cancer hepatocellular carcinoma (HCC) is unclear. In this study, through weighted gene coexpression network analysis, a coexpression network was constructed by selecting the top 25% most variant genes in the dataset GSE62232. The average linkage hierarchical clustering identified 24 modules, and among them, the pink module associated with prognosis of HCC was screened. Five gene candidates (PCNA, RFC4, PTTG1, H2AFZ, and RRM1) with a common network in the module were screened after the protein-protein interaction network complex was combined with the coexpression network. After progression and survival analysis, all candidates were identified as real core genes. According to the Human Protein Atlas and the Oncomine database, these genes were dysregulated in HCC samples. The receiver operating characteristic curve proved that the expression levels of the core genes had high diagnostic efficacy. The results of gene set enrichment analysis and functional enrichment analysis demonstrated the importance of the cell cycle-related pathways in HCC progression and prognosis. In conclusion, the five real core genes and cell cycle-related pathways identified in this study could greatly improve the knowledge about HCC progression and contribute to HCC treatment.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/patologia , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Hepáticas/patologia , Transcriptoma , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Progressão da Doença , Perfilação da Expressão Gênica , Histonas/genética , Humanos , Neoplasias Hepáticas/genética , Prognóstico , Antígeno Nuclear de Célula em Proliferação/genética , Mapas de Interação de Proteínas , Curva ROC , Proteína de Replicação C/genética , Securina/genética , Proteínas Supressoras de Tumor/genética
4.
Food Funct ; 10(10): 6351-6361, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31503268

RESUMO

Nonalcoholic steatohepatitis (NASH) increases hepatocellular carcinoma (HCC) risk. We hypothesized that the hepatoprotective anti-inflammatory benefits of catechin-rich green tea extract (GTE) would protect against HCC progression by inhibiting NASH-associated liver injury and pro-oncogenic responses. We used an HCC model in high-fat (HF)-fed mice that mimics early oncogenic events during NASH without inducing tumorigenesis and premature mortality. Male C57BL/6J mice (4-weeks old) were fed a HF diet containing GTE at 0% or 2%. Mice were administered saline or diethylnitrosamine (DEN; 60 mg kg-1, i.p.) at 5-weeks and 7-weeks of age. NASH, inflammation, fibrosis, and oncogenic responses were assessed at 25-weeks of age. Saline-treated mice showed prominent histopathological signs of steatosis and hepatocellular ballooning. Although DEN did not impact adiposity, steatosis, ballooning and hepatic lipid accumulation, these parameters were attenuated by GTE regardless of DEN. Hepatic lipid peroxidation and fibrosis that were increased by DEN were attenuated by GTE. Hepatic TLR4, MCP1 and TNFα mRNA levels were unaffected by DEN, whereas iNOS was increased by DEN. These transcripts were lowered by GTE. GTE attenuated the frequency of PCNA+ hepatocytes and mRNA expression of cyclin D1, MIB1 and Ki-67 that were otherwise increased by DEN. GTE increase APAF1 mRNA that was otherwise lowered by DEN. Relative to saline-treated mice, DEN increased mRNA levels of oncostatin M, gp130, c-Fos, c-Myc and survivin; each was lowered by GTE in DEN-treated mice. These findings indicate that GTE may protect against hepatic oncogenesis by limiting early steps in the carcinogenic cascade related to NASH-associated HCC.


Assuntos
Camellia sinensis/química , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Extratos Vegetais/administração & dosagem , Substâncias Protetoras/administração & dosagem , Animais , Anti-Inflamatórios/administração & dosagem , Carcinogênese , Dieta Hiperlipídica/efeitos adversos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/patologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
5.
J Biochem Mol Toxicol ; 33(11): e22398, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31557371

RESUMO

Cyclophosphamide (CTX) has been broadly used in the clinic for the treatment of autoimmune disorders and ovarian cancer. The process of chemotherapy has significant toxicity in the reproductive system as it has detrimental effects on folliculogenesis, which leads to an irreversible premature ovarian failure (POF). Coenzyme Q10 (CoQ10) has positive impacts on the reproductive system due to its antioxidant properties, protecting the cells from free-radical oxidative damage and apoptosis. However, little is known about the possible synergistic effect of CTX and CoQ10 on the expression of genes involved in folliculogenesis, such as proliferation cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSHR). A total of 32 NMRI mice were applied and divided into four groups, including healthy control, CTX, CTX + CoQ10, and CoQ10 groups. The effects of CoQ10 on CTX-induced ovarian injury and folliculogenesis were examined by histopathological and real-time quantitative reverse transcription-polymerase chain reaction analyses. The rates of fertilization (in vitro fertilization), embryo development, as well as the level of reactive oxygen species (ROS) in metaphase II (MII) mouse oocytes after PMSG/HCC treatment were also assessed. Results showed that the treatment with CTX decreased the mRNA expression of PCNA and FSHR, IVF rate, and embryo development whereas the application of CoQ10 successfully reversed those factors. CoQ10 administration significantly enhanced histological morphology and decreased ROS levels and the number of atretic follicles in the ovary of CTX-treated mice. In conclusion, it seems that the protective effect of CoQ10 is exerted via the antioxidant and proliferative properties of this substance on CTX-induced ovarian damage.


Assuntos
Antioxidantes/farmacologia , Ciclofosfamida/farmacologia , Insuficiência Ovariana Primária/induzido quimicamente , Antígeno Nuclear de Célula em Proliferação/genética , Receptores do FSH/genética , Ubiquinona/análogos & derivados , Regulação para Cima/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Ciclofosfamida/administração & dosagem , Sinergismo Farmacológico , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Modelos Animais , Oócitos/metabolismo , Ovário/efeitos dos fármacos , Ovário/patologia , Indução da Ovulação , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquinona/administração & dosagem , Ubiquinona/farmacologia
6.
Genetics ; 213(2): 449-463, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451562

RESUMO

In Saccharomyces cerevisiae, transcriptional silencing at HML and HMR maintains mating-type identity. The repressive chromatin structure at these loci is replicated every cell cycle and must be re-established quickly to prevent transcription of the genes at these loci. Mutations in a component of the replisome, the proliferating cell nuclear antigen (PCNA), encoded by POL30, cause a loss of transcriptional silencing at HMR We used an assay that captures transient losses of silencing at HML and HMR to perform extended genetic analyses of the pol30-6, pol30-8, and pol30-79 alleles. All three alleles destabilized silencing only transiently and only in cycling cells. Whereas pol30-8 caused loss of silencing by disrupting the function of Chromatin Assembly Factor 1, pol30-6 and pol30-79 acted through a separate genetic pathway, but one still dependent on histone chaperones. Surprisingly, the silencing-loss phenotypes of pol30-6 and pol30-79 depended on ploidy, but not on POL30 dosage or mating-type identity. Separately from silencing loss, the pol30-6 and pol30-79 alleles also displayed high levels of mitotic recombination in diploids. These results established that histone trafficking involving PCNA at replication forks is crucial to the maintenance of chromatin state and genome stability during DNA replication. They also raised the possibility that increased ploidy may protect chromatin states when the replisome is perturbed.


Assuntos
Ciclo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Fator 1 de Modelagem da Cromatina/genética , Replicação do DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Heterocromatina/genética , Histonas/genética , Mutação , Ploidias , Fase S/genética , Saccharomyces cerevisiae/genética
7.
Mol Med Rep ; 20(2): 1977-1985, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257482

RESUMO

Basaloid squamous cell carcinomas (BSCCs) in oral lesions are extremely rare, and the histology is not well understood. Histologically, they are often similar to conventional squamous cell carcinoma (SCC). The present study was designed with an aim to distinguish BSCC from SCC using claudin­4, occludin, SRY­box 2 (SOX2) and proliferating cell nuclear antigen (PCNA) immunoreactivities and staining patterns. Three BSCCs (with abundant, with moderate, and without squamous components) specimens and 20 SCC specimens were selected for comparison of their immunoreactivity. These specimens were stained with claudin­4, occludin, SOX2 and PCNA. In addition to histological analysis, the expression of claudin­4, occludin and PCNA was determined in oral cancer HSC2 and HSC3 cells with or without SOX2 overexpression, and cell proliferation was determined by XTT assay. Claudin­4 had strong and occludin had weak immunoreactivity as detected in the membrane of squamous components of BSCC but not in cancer cells. No obvious detection of squamous components and cancer cells were observed in SCC. SOX2 and PCNA immunoreactivities in SCC had dot­like staining patterns in the nuclei of partial and marginal cancer cells. In contrast, in BSCCs, SOX2 and PCNA had diffuse staining patterns in almost all cancer cells. SOX2 overexpression had little effect on the expression levels of claudin­4, occludin and PCNA. It also had little effect on the cell proliferation of HSC2 and HSC3 cells. Differences in immunoreactivity and staining pattern may be valuable to distinguish between BSCC and SCC in diagnosis.


Assuntos
Carcinoma de Células Escamosas/genética , Claudina-4/genética , Ocludina/genética , Antígeno Nuclear de Célula em Proliferação/genética , Fatores de Transcrição SOXB1/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasia de Células Basais/genética , Neoplasia de Células Basais/patologia
8.
Molecules ; 24(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340453

RESUMO

Dendrobium officinale is a herb in traditional Chinese medicine where D. officinale polysaccharides (DOP) are the main active ingredient. This study aimed at evaluating DOP efficiency at inhibiting 1-Methyl-2-nitro-1-nitrosoguanidine (MNNG) induced precancerous lesions of gastric cancer (PLGC) in rats through the Wnt/b-catenin pathway and analyzing the variations of serum endogenous metabolites. PLGC was established in male Sprague-Dawley (SD) rats by administering 150 µg/mL MNNG in drinking water for 7 months and giving 0.1 mL of 10% NaCl once weekly during the initial 20 weeks. Treatment with DOP inhibited the progress of PLGC through decreasing the expression of ß-catenin by immunohistochemical analysis. The futher study indicated DOP downregulated gene expression of Wnt2ß, Gsk3ß, PCNA, CyclinD1, and ß-catenin, as well as protein expression of Wnt2ß, PCNA, and ß-catenin. On the other hand, there were nine endogenous metabolites identified after the DOP treatment. Among these, the most significant one is betaine because of its strong antioxidant activity, leading to an anti-tumor effect. DOP can inhibit MNNG-induced PLGC models via regulating Wnt/ß-catenin pathway and by changing endogenous metabolites.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Dendrobium/química , Regulação Neoplásica da Expressão Gênica , Polissacarídeos/farmacologia , Lesões Pré-Cancerosas/prevenção & controle , Neoplasias Gástricas/prevenção & controle , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Betaína/sangue , Ciclina D1/genética , Ciclina D1/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Metaboloma/genética , Metilnitronitrosoguanidina/toxicidade , Extratos Vegetais/química , Polissacarídeos/isolamento & purificação , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Neoplasias Gástricas/induzido quimicamente , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
9.
Eur J Pharmacol ; 860: 172560, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31344364

RESUMO

Plants, fruits, and vegetables containing the bioflavonoid quercetin are widely used in food, beverages, and medicines; however, the effects of quercetin on reproductive processes and the possible mechanisms of quercetin action require extensive investigation. The aim of our study was to examine the direct effects of quercetin on basic ovarian cell functions and their response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I), known hormonal stimulators of reproduction. We analyzed the effects of quercetin alone (0, 1, 10, and 100 ng/ml) on cultured porcine ovarian granulosa cells or isolated ovarian follicles; or of quercetin (10 ng/ml) in combination with FSH (0, 0.01, 0.1, or 1 IU/ml) or IGF-I (0, 1, 10, or 100 ng/ml) on cultured porcine granulosa cells. The expression of proliferative (PCNA, cyclin B1) and apoptotic (BAX) markers, as well as markers for release of progesterone (P4), testosterone (T), and leptin (L), were measured by quantitative immunocytochemistry, Western immunoblotting, RT-qPCR, and EIA/RIA. Addition of quercetin reduced the accumulation of PCNA and cyclin B1, as well as their transcript levels, promoted the accumulation of BAX, decreased the release of P4 and L, and increased the release of T in cultured granulosa cells. In ovarian follicles, quercetin reduced the levels of both P4 and T. Exposure to FSH stimulated PCNA and decreased BAX accumulation, and increased the release of P4, T, and L. Quercetin inhibited and even reversed the effects of FSH. Like FSH, IGF-I also promoted granulosa cell proliferation and suppressed apoptosis. Quercetin did not modify IGF-I effects. These data suggest that the plant molecule quercetin can directly down-regulate basal ovarian cell functions (proliferation, apoptosis, and release of ovarian steroid and peptide hormones) and their response to the stimulatory activity of the upstream hormonal stimulator FSH.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Quercetina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Leptina/metabolismo , Progesterona/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , RNA Mensageiro/genética , Suínos , Testosterona/metabolismo
10.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31278166

RESUMO

Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron-sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data hence suggest that the FeS cluster in human Pol δ is an important co-factor that despite its C-terminal location has an impact on both DNA polymerase and exonuclease activities, and can influence the fidelity of DNA synthesis.


Assuntos
DNA Polimerase III/química , DNA/biossíntese , Proteínas com Ferro-Enxofre/química , Motivos de Aminoácidos , Domínio Catalítico/genética , Cisteína/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Ativação Enzimática/genética , Humanos , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo , Modelos Moleculares , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo
11.
Int J Med Sci ; 16(5): 751-756, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217743

RESUMO

Background: Increasing research has recently been focused on the supplementary use of drugs such as bisphosphonates that are known to influence bone turnover to prevent and treat periprosthetic bone loss and subsequent implant loosening following total joint replacements. However, there are still concerns about the conflicting effects of bisphosphonate treatment on osteoblastic bone formation in the literature. Methods: In this study, we investigate the role of zoledronate (ZOL) in regulating cell cycle distribution and differentiation in mouse MC3T3-E1 preosteoblasts and also explore the mechanism underlying this effect of ZOL. We examined the expression levels of osteocalcin (OCN) by quantitative polymerase chain reaction (qPCR), the total amount of CDK6, p21 and p27 proteins by Western blot analysis, and the cell cycle distribution by flow cytometric analysis in mouse MC3T3-E1 preosteoblasts to evaluate the effect of ZOL. Small interfering RNAs (siRNAs) were used to assess the individual contributions of genes to specific osteoblast phenotypes. Results: In addition to increased OCN expression, we found that ZOL treatment induces the G0/G1 arrest and results in the increase of p21 and p27 expressions and decrease of CDK6 expression in mouse MC3T3-E1 preosteoblasts. Both p21 and p27 mediates ZOL-induced cell cycle exit; however, p21, but not p27, is responsible for the increase of ZOL-induced OCN expression in these cells. Conclusions: These results endorse that ZOL might have an anabolic effect on osteoblasts. The CDK inhibitor p21 plays a key role in regulating osteoblast differentiation by controlling proliferation-related events in mouse MC3T3-E1 preosteoblasts.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteogênese/genética , Ácido Zoledrônico/farmacologia , Quinases Ativadas por p21/genética , Células 3T3 , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 6 Dependente de Ciclina/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Osteogênese/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/genética
12.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180053

RESUMO

Sepsis is the systemic inflammatory response caused by infection. Cardiac dysfunction is an acknowledged result of sepsis. Shengjiang Powder, a prescribed traditional Chinese medicine, showed anti-infection and antipyretic functions in our previous study. In this study, we established a septic rat model via cecal ligation puncture (CLP) to evaluate the effects of Shengjiang Powder on sepsis and the involvement of P38 mitogen activated protein kinase (P38-MAPK) signaling. The 10 main ingredients of Shengjiang Powder were identified by LC-MS. The results of this study indicated that Shengjiang Powder at a concentration of 3.0 g/kg with SB203580 (an inhibitor of P38-MAPK) could improve myocardial injury, ameliorate the histopathological abnormalities, decrease apoptosis and upregulate proliferating cell nuclear antigen (PCNA) levels in myocardial tissues. Further, cytokine mRNA expression levels (tumor necrosis factor - alpha, TNF-α and interleukin 6, IL-6) were decreased by Shengjiang Powder and SB203580 in the myocardial tissues. Furthermore, the p-P38 protein level in myocardial tissues was upregulated in septic rats but decreased upon treatment with Shengjiang Powder and SB203580; however, the relative protein level of P38 showed no significant changes. Collectively, Shengjiang Powder showed a myocardial protective effect on rats with CLP-induced sepsis.


Assuntos
Anti-Inflamatórios/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Traumatismos Cardíacos/prevenção & controle , Miocárdio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Anti-Inflamatórios/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Regulação da Expressão Gênica , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Imidazóis/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Piridinas/farmacologia , Ratos , Ratos Wistar , Sepse , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Mol Med Rep ; 20(1): 604-612, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180541

RESUMO

Emerging evidence suggests the critical function of microRNAs in regulating the growth of cancer cells. In the present study, it was demonstrated that miR­221­3p was overexpressed in non­small cell lung cancer (NSCLC) tissues and cell lines compared with that noted in the normal controls. Downregulation of miR­221­3p suppressed the proliferation, colony formation and invasion of NSCLC cells. To further understand the molecular mechanisms underlying the potential oncogenic function of miR­221­3p in NSCLC, the downstream targets of miR­221­3p were predicted using bioinformatic databases. The prediction suggested the cell cycle regulator p27 as one of the targets of miR­221­3p. Molecular experiments showed that miR­221­3p was able to bind with the 3'­untranslated region (UTR) of p27 and decreased the expression of p27 in NSCLC cells. Consistent with the suppressive role of p27 in controlling cell cycle progression, overexpression of miR­221­3p decreased the expression of p27 and promoted cell cycle progression from G1 to S phase. Collectively, our findings identified miR­221­3p as a novel regulator of NSCLC cell growth via modulating the expression of p27.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Antígeno Nuclear de Célula em Proliferação/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Humanos , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Regulação para Cima
14.
mBio ; 10(3)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186330

RESUMO

The PCNA (proliferating cell nuclear antigen) ring plays central roles during DNA replication and repair. The yeast Elg1 RFC-like complex (RLC) is the principal unloader of chromatin-bound PCNA and thus plays a central role in maintaining genome stability. Here we identify a role for Elg1 in the unloading of PCNA during DNA damage. Using DNA damage checkpoint (DC)-inducible and replication checkpoint (RC)-inducible strains, we show that Elg1 is essential for eliciting the signal in the DC branch. In the absence of Elg1 activity, the Rad9 (53BP1) and Dpb11 (TopBP1) adaptor proteins are recruited but fail to be phosphorylated by Mec1 (ATR), resulting in a lack of checkpoint activation. The chromatin immunoprecipitation of PCNA at the Lac operator sites reveals that accumulated local PCNA influences the checkpoint activation process in elg1 mutants. Our data suggest that Elg1 participates in a mechanism that may coordinate PCNA unloading during DNA repair with DNA damage checkpoint induction.IMPORTANCE The Elg1protein forms an RFC-like complex in charge of unloading PCNA from chromatin during DNA replication and repair. Mutations in the ELG1 gene caused genomic instability in all organisms tested and cancer in mammals. Here we show that Elg1 plays a role in the induction of the DNA damage checkpoint, a cellular response to DNA damage. We show that this defect is due to a defect in the signal amplification process during induction. Thus, cells coordinate the cell's response and the PCNA unloading through the activity of Elg1.


Assuntos
Proteínas de Transporte/genética , Dano ao DNA , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Mutação , Ligação Proteica
15.
Mol Med Rep ; 20(2): 903-914, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173190

RESUMO

Metastasis is the most lethal stage of cancer progression. The present study aimed to investigate the underlying molecular mechanisms of melanoma metastasis using bioinformatics. Using the microarray dataset GSE8401 from the Gene Expression Omnibus database, which included 52 biopsy specimens from patients with melanoma metastasis and 31 biopsy specimens from patients with primary melanoma, differentially expressed genes (DEGs) were identified, subsequent to data preprocessing with the affy package, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A protein­protein interaction (PPI) network was constructed. Mutated genes were analyzed with 80 mutated cases with melanoma from The Cancer Genome Atlas. The overall survival of key candidate DEGs, which were within a filtering of degree >30 criteria in the PPI network and involved three or more KEGG signaling pathways, and genes with a high mutation frequency were delineated. The expression analysis of key candidate DEGs, mutant genes and their associated genes were performed on UALCAN. Of the 1,187 DEGs obtained, 505 were upregulated and 682 were downregulated. 'Extracellular exosome' processes, the 'amoebiasis' pathway, the 'ECM­receptor interaction' pathway and the 'focal adhesion' signaling pathway were significantly enriched and identified as important processes or signaling pathways. The overall survival analysis of phosphoinositide­3­kinase regulator subunit 3 (PIK3R3), centromere protein M (CENPM), aurora kinase A (AURKA), laminin subunit α 1 (LAMA1), proliferating cell nuclear antigen (PCNA), adenylate cyclase 1 (ADCY1), BUB1 mitotic checkpoint serine/threonine kinase (BUB1), NDC80 kinetochore complex component (NDC80) and protein kinase C α (PRKCA) in DEGs was statistically significant. Mutation gene analysis identified that BRCA1­associated protein 1 (BAP1) had a higher mutation frequency and survival analysis, and its associated genes in the BAP1­associated PPI network, including ASXL transcriptional regulator 1 (ASXL1), proteasome 26S subunit, non­ATPase 3 (PSMD3), proteasome 26S subunit, non ATPase 11 (PSMD11) and ubiquitin C (UBC), were statistically significantly associated with the overall survival of patients with melanoma. The expression levels of PRKCA, BUB1, BAP1 and ASXL1 were significantly different between primary melanoma and metastatic melanoma. Based on the present study, 'extracellular exosome' processes, 'amoebiasis' pathways, 'ECM­receptor interaction' pathways and 'focal adhesion' signaling pathways may be important in the formation of metastases from melanoma. The involved genes, including PIK3R3, CENPM, AURKA, LAMA1, PCNA, ADCY1, BUB1, NDC80 and PRKCA, and mutation associated genes, including BAP1, ASXL1, PSMD3, PSMD11 and UBC, may serve important roles in metastases of melanoma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/genética , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Laminina/genética , Laminina/metabolismo , Melanoma/metabolismo , Melanoma/mortalidade , Melanoma/patologia , Anotação de Sequência Molecular , Mutação , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Análise de Sobrevida
16.
BMC Plant Biol ; 19(1): 257, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31200645

RESUMO

BACKGROUND: Proliferating cell nuclear antigen (PCNA), a conserved trimeric ring complex, is loaded onto replication fork through a hetero-pentameric AAA+ ATPase complex termed replication factor C (RFC) to maintain genome stability. Although architectures of PCNA-RFC complex in yeast have been revealed, the functions of PCNA and protein-protein interactions of PCNA-RFC complex in higher plants are not very clear. Here, essential regions mediating interactions between PCNA and RFC subunits in Arabidopsis and rice were investigated via yeast-two-hybrid method and bimolecular fluorescence complementation techniques. RESULTS: We observed that OsPCNA could interact with all OsRFC subunits, while protein-protein interactions only exist between Arabidopsis RFC2/3/4/5 and AtPCNA1/2. The truncated analyses indicated that the C-terminal of Arabidopsis RFC2/3/4/5 and rice RFC1/2 is essential for binding PCNA while the region of rice RFC3/4/5 mediating interaction with PCNA distributed both at the N- and C-terminal. On the other hand, we found that the C- and N-terminal of Arabidopsis and rice PCNA contribute equally to PCNA-PCNA interaction, and the interdomain connecting loop (IDCL) domain and C-terminal of PCNAs are indispensable for interacting RFC subunits. CONCLUSIONS: These results indicated that Arabidopsis and rice PCNAs are highly conserved in sequence, structure and pattern of interacting with other PCNA monomer. Nevertheless, there are also significant differences between the Arabidopsis and rice RFC subunits in binding PCNA. Taken together, our results could be helpful for revealing the biological functions of plant RFC-PCNA complex.


Assuntos
Arabidopsis/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , Arabidopsis/genética , Sequência Conservada , Oryza/genética , Proteínas de Plantas/genética , Antígeno Nuclear de Célula em Proliferação/genética
17.
Nucleic Acids Res ; 47(13): 6826-6841, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31114918

RESUMO

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair (MMR) as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. Here we show that timely removal of PCNA from DNA by the Elg1 complex is important to prevent mutations. Although premature unloading of PCNA generally increases mutation rate, the mutator phenotype of elg1Δ is attenuated by PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA. In contrast, the elg1Δ mutator phenotype is exacerbated by PCNA mutants that accumulate on DNA due to enhanced electrostatic PCNA-DNA interactions. Epistasis analysis suggests that PCNA over-accumulation on DNA interferes with both MMR and MMR-independent process(es). In elg1Δ, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of MMR intermediates. Our results suggest that PCNA retention controlled by the Elg1 complex is critical for efficient MMR: PCNA needs to be on DNA long enough to enable MMR, but if it is retained too long it interferes with downstream repair steps.


Assuntos
Proteínas de Transporte/fisiologia , Reparo de Erro de Pareamento de DNA , DNA Fúngico/metabolismo , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Cristalografia por Raios X , Replicação do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Genes Fúngicos , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/metabolismo , Conformação de Ácido Nucleico , Mutação Puntual , Antígeno Nuclear de Célula em Proliferação/fisiologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação
18.
Immunopharmacol Immunotoxicol ; 41(3): 446-454, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31124391

RESUMO

Context: Atherosclerosis is a chronic inflammatory disease in which the plaques were built up inside of the artery. Interleukin-8 (IL-8, CXCL8) is an inflammatory factor, known to play an important role in the development of atherosclerosis. G31P is an antagonist of the IL-8 receptor, which plays roles in vascular smooth muscle cell (VSMC) proliferation and migration. Objective: This study is to investigate the therapeutic effect of G31P on atherosclerosis through a mouse model. Materials and methods: A mouse model of atherosclerosis was generated through feeding the ApoE-/- mice with high fat diet for 12 weeks. G31P was injected subcutaneously into the mice. The levels of keratinocyte chemoattractant (KC), CXCR2, TNF-α, and IFN-γ were analyzed through ELISA. The expressions of MMP-2, MMP-9, PCNA, and Mef2a in aortic tissues were detected through RT-qPCR. In A7r5 cells, the levels of p-ERK, ROCK1, and ROCK2 were analyzed by western blot. Intracellular calcium levels were measured through Fluo-3 AM assay. Results and disccussion: G31P suppressed the abnormal lipid profile and decreased the levels of KC, MMP-2, MMP-9, PCNA, and Mef2a in a mouse model of atherosclerosis. In addition, G31P also inhibited the expressions of p-ERK, ROCK1, ROCK2, and decreased the calcium concentrations in A7r5 cells. Conclusions: These findings indicate the potential therapeutic effects of G31P in suppressing the development of atherosclerosis by antagonizing the IL-8 receptor. G31P inhibits the proliferation and migration of VSMCs through regulating the Rho-kinase, ERK, and calcium-dependent pathways.


Assuntos
Aorta/imunologia , Aterosclerose/tratamento farmacológico , Interleucina-8/farmacologia , Músculo Liso Vascular/imunologia , Miócitos de Músculo Liso/imunologia , Fragmentos de Peptídeos/farmacologia , Placa Aterosclerótica/tratamento farmacológico , Animais , Aorta/patologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Knockout para ApoE , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/imunologia , Quinases Associadas a rho/genética , Quinases Associadas a rho/imunologia
19.
Cell Mol Life Sci ; 76(24): 4923-4943, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31134302

RESUMO

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.


Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação a DNA/química , DNA/química , Antígeno Nuclear de Célula em Proliferação/química , DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Espectroscopia de Ressonância Magnética , Antígeno Nuclear de Célula em Proliferação/genética , Conformação Proteica
20.
J BUON ; 24(2): 715-719, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128028

RESUMO

PURPOSE: To analyze the correlations of cluster of differentiation 24 (CD24), B7-H4 and proliferating cell nuclear antigen (PCNA) with clinicopathological indexes of ovarian cancer by detecting the expressions of the three indexes, and further explore the clinical value of CD24, B7-H4 and PCNA detection in early diagnosis of ovarian cancer and screening of high-risk patients. METHODS: Gynecological paraffin-embedded blocks archived in the Department of Pathology of Suzhou Municipal Hospital from January 2015 to June 2017 were selected. There were 30 patients with benign epithelial ovarian tumor and 50 patients with malignant tumor according to the medical history and pathological data. In addition, 20 patients with normal ovarian tissues were enrolled as controls. Immunohistochemical streptavidin-peroxidase (SP) method was applied to detect CD24, B7-H4 and PCNA in different ovarian tissues, which were ultimately analyzed in combination with clinicopathological factors such as grade of differentiation and with or without lymph node metastasis. RESULTS: The expression levels of CD24, B7-H4 and PCNA in ovarian cancer tissues were remarkably higher than those in benign ovarian tumor tissues and normal ovarian tissues, and the expression levels of the three indexes in poorly- and moderately-differentiated ovarian cancer were notably higher than those in well-differentiated ovarian cancer (p<0.05). The expressions of CD24, B7-H4 and PCNA in ovarian cancer tissues with lymph node metastasis were significantly increased compared with those in ovarian cancer tissues without lymph node metastasis (p<0.05). The expressions of CD24, B7-H4 and PCNA in ovarian cancer tissues were positively correlated with each other (p<0.05). CONCLUSIONS: The expressions of CD24, B7-H4 and PCNA have correlations with the occurrence, development, invasion and metastasis of ovarian cancer, and the combined detection may have clinical guiding significance for early diagnosis of ovarian cancer and screening of high-risk patients.


Assuntos
Antígeno CD24/genética , Neoplasias Ovarianas/genética , Antígeno Nuclear de Célula em Proliferação/genética , Inibidor 1 da Ativação de Células T com Domínio V-Set/genética , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/patologia , Neoplasias Ovarianas/patologia
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