scholarly journals Effect of MT3 on Retinal and Choroidal TGF-β2 and HAS2 Expressions in Form Deprivation Myopia of Guinea Pig

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Tao Li ◽  
Xiaodong Zhou ◽  
Bing Li ◽  
Bo Jiang
Keyword(s):  
Protein Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Protein Expression Level ◽  
Tissue Concentrations ◽  
Treatment Groups ◽  
Protein Expressions ◽  
Mrna And Protein Expression ◽  
Dose Dependent ◽  
Dose Dependent Effect

Purpose. To confirm its dose-dependent effect on form deprivation myopia and evaluate the effect of MT3 at different tissue concentrations on changes in mRNA and protein expression for TGF-β2 and HAS2. Methods. MT3 was intravitreally injected into deprived eyes at two-day intervals. Refraction was measured by streak retinoscopy after cycloplegia. The axial dimensions were measured by A-scan ultrasound. The quantitative RT-PCR and Western blot were used to detect the changes of TGF-β2 and HAS2 expressions in the retina and choroid of guinea pigs. Results. MT3 treatment produced a significant dose-dependent reduction in relative myopia compared to FD group (both p<0.001). There were statistically significant increases in retinal and choroidal mRNA levels for both TGF-β2 and HAS2 after injections of 10 μM of MT3, when compared to the FD group. There were no significant differences in retinal and choroidal TGF-β2 protein expression levels between the MT3 treatment groups and FD group (all p>0.05). The injections of 10 μM of MT3 caused a marked decrease in retinal HAS2 protein expression level, when compared to the FD group (p=0.001). Conclusion. MT3 can inhibit form deprivation myopia, and MT3 treatment can result in changes of retinal and choroidal TGF-β2 and HAS2 mRNA and protein expressions.

scholarly journals FGF21 Ameliorates Hepatic Fibrosis by Multiple Mechanisms

2021 ◽  
Author(s):  
Fanrui Meng ◽  
Mir Hassan Khoso ◽  
Kai Kang ◽  
Qi He ◽  
Yukai Cao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Hepatic Fibrosis ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Model ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Mrna And Protein Expression ◽  
Dose Dependent

Abstract Previous study reports that FGF21 could ameliorate hepatic fibrosis, but its mechanisms have not been fully investigated. In this study, three models were used to investigate the mechanism by which FGF21 alleviates liver fibrosis. CCL4 and DMN were respectively used to induce hepatic fibrosis animal models. Our results demonstrated that liver index and liver function were deteriorated in both models. HE and Masson’s staining showed that the damaged tissue architectonics were observed in the mice of both models. Treatment with FGF21 significantly ameliorated these changes. ELISA analysis showed that the serum levels of IL-1β, IL-6 and TNF-α were significantly elevated in both models. However, administration of FGF21 significantly reduced these inflammatory cytokines. RT-PCR and Western blot analysis showed that mRNA and protein expression of collagenI, α-SMA and TGF-β were significantly decreased by treatment with FGF21. PDGF-BB stimulant was used to establish the experimental cell model in HSCs. RT-PCR and Western blot analysis demonstrated that the expression of collagenI and α-SMA were significantly upregulated by this stimulant in model group. Interestingly, our results showed that mRNA and protein expression of leptin were also significantly induced in PDGF-BB treated HSCs. Administration of FGF21 could significantly reduce leptin expression in a dose dependent manner and these effects were reversed in siRNA (against β-klotho) transfected HSCs. Furthermore, the leptin signaling pathways related protein p-ERK/t-ERK, p-STAT3/STAT3 and TGF-β were significantly downregulated by FGF21 treatment in a dose dependent manner. The expression of SOCS3 and Nrf-2 were enhanced by treatment with FGF21. The underlying mechanism may be that FGF21 regulates leptin-STAT3 axis via Nrf-2 and SOCS3 pathway in activated HSCs.

scholarly journals Degradation of connexin 50 protein causes waterclefts in human lens

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1163-1171
Author(s):  
Yosuke Nakazawa ◽  
Teppei Shibata ◽  
Noriaki Nagai ◽  
Eri Kubo ◽  
Hiroomi Tamura ◽  
...  
Keyword(s):  
Light Scattering ◽  
Adhesion Molecules ◽  
Adhesion Molecule ◽  
Human Lens ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Protein Expressions ◽  
Mrna And Protein Expression ◽  
Connexin 50 ◽  
Cataract Patients

AbstractCataracts are mainly classified into three types: cortical cataracts, nuclear cataracts, and posterior subcapsular cataracts. In addition, retrodots and waterclefts are cataract subtypes that cause decreased visual function. To maintain an orderly and tightly packed arrangement to minimize light scattering, adhesion molecules such as connexins and aquaporin 0 (AQP0) are highly expressed in the lens. We hypothesized that some main and/or subcataract type(s) are correlated with adhesion molecule degradation. Lens samples were collected from cataract patients during cataract surgery, and mRNA and protein expression levels were measured by real-time RT-PCR and western blotting, respectively. The mRNA levels of adhesion molecules were not significantly different among any cataract types. Moreover, AQP0 and connexin 46 protein expressions were unchanged among patients. However, connexin 50 protein level was significantly decreased in the lens of patients with WC cataract subtype. P62 and LC3B proteins were detected in the WC patients’ lenses, but not in other patients’ lenses. These results suggest that more research is needed on the subtypes of cataracts besides the three major types of cataract for tailor-made cataract therapy.

scholarly journals Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice

2023 ◽  
Vol 83 ◽  
Author(s):  
F. J. Siyal ◽  
R. A. Siddiqui ◽  
Z. Memon ◽  
Z. Aslam ◽  
U. Nisar ◽  
...  
Keyword(s):  
Protein Expression ◽  
Viral Vectors ◽  
Anterior Cingulate ◽  
Dihydroxyacetone Phosphate ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Over Expression ◽  
Protein Expressions ◽  
Anxiety Behavior ◽  
Dose Dependent

Abstract The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

scholarly journals Cloning and Expression Analysis of Two Kdm Lysine Demethylases in the Testes of Mature Yaks and Their Sterile Hybrids

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 521
Author(s):  
Zhenhua Shen ◽  
Lin Huang ◽  
Suyu Jin ◽  
Yucai Zheng
Keyword(s):  
Amino Acids ◽  
Protein Expression ◽  
Male Sterility ◽  
Histone Methylation ◽  
Real Time Pcr ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Coding Sequences ◽  
Mrna And Protein Expression ◽  
Sterile Hybrids

The objective of this study was to explore the molecular mechanism for male sterility of yak hybrids based on two demethylases. Total RNA was extracted from the testes of adult yaks (n = 10) and yak hybrids (cattle–yaks, n = 10). The coding sequences (CDS) of two lysine demethylases (KDMs), KDM1A and KDM4B, were cloned by RT-PCR. The levels of KDM1A and KDM4B in yaks and cattle–yaks testes were detected using Real-time PCR and Western blotting for mRNA and protein, respectively. In addition, the histone methylation modifications of H3K36me3 and H3K27me3 were compared between testes of yaks and cattle–yaks using ELISA. The CDS of KDM1A and KDM4B were obtained from yak testes. The results showed that the CDS of KDM1A exhibited two variants: variant 1 has a CDS of 2622 bp, encoding 873 amino acids, while variant 2 has a CDS of 2562 bp, encoding 853 amino acids. The CDS of the KDM4B gene was 3351 bp in length, encoding 1116 amino acids. The mRNA and protein expression of KDM1A and KDM4B, as well as the level of H3K36me3, were dramatically decreased in the testes of cattle–yaks compared with yaks. The present results suggest that the male sterility of cattle–yaks might be associated with reduced histone methylation modifications.

scholarly journals Hypoxia increases KIAA1199/CEMIP expression and enhances cell migration in pancreatic cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takuya Oba ◽  
Norihiro Sato ◽  
Yasuhiro Adachi ◽  
Takao Amaike ◽  
Yuzan Kudo ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Expression ◽  
Hypoxia Inducible Factor ◽  
Ductal Adenocarcinoma ◽  
Rt Pcr ◽  
Transwell Migration Assay ◽  
Migration Assay ◽  
Hypoxic Conditions ◽  
Mrna And Protein Expression ◽  
Interfering Rna

AbstractPancreatic ductal adenocarcinoma (PDAC) is characterised by dense desmoplasia and hypoxic microenvironment. Our previous reports demonstrated that hyaluronan (HA), especially low-molecular-weight HA, provides a favourable microenvironment for PDAC progression. However, the effect of hypoxia on HA metabolism remains unknown. Using quantitative real-time RT-PCR and western blot analysis, we analysed the changes in the expression of HA-synthesizing enzymes (HAS2 and HAS3) and HA-degrading enzymes (HYAL1, KIAA1199/CEMIP) in PDAC cell lines under hypoxic conditions. Hypoxia increased the mRNA and protein expression of KIAA1199, whereas it decreased HYAL1 expression. The expression of HAS3 was increased and HAS2 remained unchanged in response to hypoxia. The effect of KIAA1199 on hypoxia-induced cell migration was determined using a transwell migration assay and small-interfering RNA (siRNA). Hypoxia enhanced the migratory ability of PDAC cells, which was inhibited by KIAA1199 knockdown. We also used immunohistochemistry to analyse the protein expression of hypoxia inducible factor (HIF) 1α and KIAA1199 in PDAC tissues. There was a significant immunohistochemically positive correlation between KIAA1199 and HIF1α. These findings suggest that hypoxia-induced KIAA1199 expression may contribute to enhanced motility in PDAC.

Abstract 239: MicroRNAs hsa-miR-584 and hsa-miR-31 Regulate Expression of Human Angiotensnogen Gene

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Brahmaraju Mopidevi ◽  
Shreekrishna Maharjan ◽  
Sudhir Jain ◽  
Varunkumar G. Pandey ◽  
Ashok Kumar
Keyword(s):  
Luciferase Activity ◽  
Negative Control ◽  
Hek293 Cells ◽  
Multiple Cloning Site ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Hep3b Cells ◽  
Agt Gene ◽  
Dose Dependent ◽  
Mirna Expression Vector

Background: Hypertension is a risk factor for stroke, myocardial infarction, and congestive heart failure. A single nucleotide polymorphism (SNP) (rs7079) in 3’UTR of human angiotensinogen (hAGT) gene is associated with elevated blood pressure. We have used TargetScan V6.0, miRanda miRNA prediction algorithms and found that two microRNAs hsa-miR-584 and hsa-miR-31 may bind differentially to rs7079 and alter the expression of hAGT gene. METHODS: The effect of hsa-miR-584 and hsa-miR-31 miRNAs on endogenous AGT expression levels in Hep3B cells were studied by transfection of individual miRNA mimics followed by quantitative real time PCR (Q-RT-PCR) of the hAGT gene. In addition, the 600 bp 3’UTR of hAGT gene containing either rs7079C or rs7079A was PCR amplified and cloned in to the multiple cloning site of pMIR-REPORT™ miRNA Expression Vector containing luciferase gene. These reporter constructs were then co-transfected with either miRNA mimics or inhibitors to study the effect of miRNAs on luciferase activity in Hep3B cells since these cells express hAGT gene. These experiments were also performed in HEK293 cells which do not express the hAGT gene. Results: Q-RT-PCR showed that hsa-miR-584 and hsa-miR-31 mimics at 50nM concentration reduced endogenous hAGT mRNA levels by 38% and 30% respectively compared to the mock (without any microRNA) or negative control (which does not have any binding site for eukaryotic 3’UTRs) in Hep3B cells. Furthermore hsa-miR-584 and hsa-miR-31 showed 40% and 25% reduced luciferase activity of the construct containing rs7079 C allele. On the other hand these miRNAs did not affect the luciferase activity in the presence of rs7079 A. When dose dependent anti miRNA inhibitors were transfected along with miRNA mimics, these mimics abolished the microRNA induced down-regulation of luciferase activity. Conclusion: hsa-miR-584 and hsa-miR-31 miRNAs bind strongly to the hAGT 3’UTR containing rs7079 C allele as compared to rs7079 A allele and may down regulate the expression of AGT gene containing rs7079 C allele. This may be one of the possible mechanism involved in association of rs7079 A allele with human hypertension.

Abstract 492: Arachidonic Acid Induces Activation of Platelet PF4 and Par-1 mRNA, Which Is Attenuated by Aspirin

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Liang Hu ◽  
Michael A Nardi ◽  
Michael Merolla ◽  
Yajaira Suarez ◽  
Jeffrey Berger
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activation ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Thiazole Orange ◽  
Platelet Activity ◽  
Protein Levels ◽  
Reticulated Platelets ◽  
Mrna And Protein Expression ◽  
Dose Dependent

Arachidonic acid (AA) is converted to thromboxane A2 via the cyclooxygenase pathway; however its exact mechanism of platelet activation is uncertain. Inhibition of this pathway via aspirin highlights the importance of this pathway in decreasing thrombotic events. In the present study, we investigate the effect of AA on platelet activity indicators (leukocyte- and monocyte-platelet aggregation [LPA, MPA] and reticulated platelets [RP]), as well as the expression (mRNA and protein) of platelet markers PF4 and Par-1, previously well established platelet transcripts with quantitative determinations. To this end, whole blood was incubated with AA (150mM) for 30 min at room temperature in the absence or presence of aspirin (1mM) prior to addition of antibodies for platelet activity indicators, and isolating platelets for mRNA and protein expression. LPA and MPA were significantly increased after AA stimulation in a dose dependent manner, and were inhibited by aspirin treatment. AA significantly increased PF4 and Par-1 protein level as determined by flow cytometry and western blot assays. Pretreatment with aspirin also attenuated this increase in protein levels. Surprisingly, AA stimulation significantly increased thiazole orange staining (a measure of nucleic acids), another marker of increased platelet activity. Importantly, these results suggest that AA-mediated platelet activation produced an overall increase in platelet total RNA content. To confirm these findings, we analyzed the mRNA expression of PF4 and Par-1 by quantitative real time PCR from platelets treated with AA. Interestingly, AA significantly up-regulated the platelet mRNA transcripts of PF4 and Par-1 by 40% to 60%, and pretreatment with aspirin completely attenuated this effect supporting the specificity of the AA effect on platelet RNA. Altogether, these data suggest that platelet mRNA is affected by AA stimulation, which is attenuated by pretreatment with aspirin. However, the mechanisms responsible for the increased mRNA levels and expression of PF4 and Par-1 (processing of pre-RNA to mRNA) require further investigation. Importantly, our findings provide novel insight regarding platelet activation and a better understanding of mediators in the processes of thrombosis and hemostasis.

Kaempferol Induces DNA Damage and Inhibits DNA Repair Associated Protein Expressions in Human Promyelocytic Leukemia HL-60 Cells

2015 ◽  
Vol 43 (02) ◽  
pp. 365-382 ◽  
Author(s):  
Lung-Yuan Wu ◽  
Hsu-Feng Lu ◽  
Yu-Cheng Chou ◽  
Yung-Luen Shih ◽  
Da-Tian Bau ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Expression ◽  
Ataxia Telangiectasia ◽  
Repair System ◽  
Dapi Staining ◽  
Viable Cells ◽  
Protein Expressions ◽  
Dose Dependent

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O6-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60 cells, which may be the factors for kaempferol induced cell death in vitro.

scholarly journals Different Sources of Copper Effect on Intestinal Epithelial Cell: Toxicity, Oxidative Stress, and Metabolism

Metabolites ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 11
Author(s):  
Runxian Li ◽  
Yang Wen ◽  
Gang Lin ◽  
Chengzhen Meng ◽  
Pingli He ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Epithelial Cell ◽  
Protein Expression ◽  
Intestinal Epithelial Cell ◽  
Cell Damage ◽  
Intestinal Epithelial ◽  
Protein Expression Level ◽  
Treatment Groups ◽  
All Treatment

Copper (Cu) is widely used in the swine industry to improve the growth performance of pigs. However, high doses of copper will induce cell damage and toxicity. The aim of this study was to evaluate toxicity, bioavailability, and effects on metabolic processes of varying copper sources using porcine intestinal epithelial cells (IPEC-J2) as a model. The IPEC-J2 were treated with two doses (30 and 120 μM) of CuSO4, Cu Glycine (Cu-Gly), and Cu proteinate (Cu-Pro) for 10 h, respectively. Cell damage and cellular copper metabolism were measured by the changes in cell viability, copper uptake, oxidative stress biomarkers, and gene/protein expression levels. The results showed that cell viability and ratio of reduced and oxidized glutathione (GSH/GSSG) decreased significantly in all treatment groups; intracellular copper content increased significantly in all treatment groups; total superoxide dismutase (SOD) activity increased significantly in the 120 μM exposed groups; SOD1 protein expression levels were significantly upregulated in 30 μM Cu-Pro, 120 μM Cu-Gly, and 120 μM Cu-Pro treatment groups; intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) content increased significantly in 30 μM treatment groups and 120 μM CuSO4 treatment group. CTR1 and ATP7A gene expression were significantly downregulated in the 120 μM exposed groups. While upregulation of ATOX1 expression was observed in the presence of 120 μM Cu-Gly and Cu-Pro. ASCT2 gene expression was significantly upregulated after 120 μM Cu-Glycine and CuSO4 exposure, and PepT1 gene expression was significantly upregulated after Cu-Pro exposure. In addition, CTR1 protein expression level decreased after 120 μM CuSO4 and Cu-Gly exposure. PepT1 protein expression level was only upregulated after 120 μM Cu-Pro exposure. These findings indicated that extra copper supplementation can induce intestinal epithelial cell injury, and different forms of copper may have differing effects on cell metabolism.

scholarly journals Comparison of CXC Chemokines ENA-78 and Interleukin-8 Expression in Helicobacter pylori-Associated Gastritis

2001 ◽  
Vol 69 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Gabriele Rieder ◽  
Wolfgang Einsiedl ◽  
Rudolf A. Hatz ◽  
Manfred Stolte ◽  
Georg A. Enders ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Expression ◽  
Outer Membrane Proteins ◽  
Water Soluble ◽  
Interleukin 8 ◽  
Gastric Epithelium ◽  
Mrna Levels ◽  
Rt Pcr ◽  
H Pylori

ABSTRACT Colonization of the gastric mucosa with Helicobacter pylori is associated with a dense infiltration of granulocytes into the lamina propria in the active phase of gastritis. In this study, we investigated the involvement of epithelial cell-derived neutrophil-activating protein 78 (ENA-78) in development of H. pylori-associated gastritis. Antral biopsies from 27 patients with H. pylori-associated gastritis and 25 from H. pylori-negative individuals were first analyzed for ENA-78 and interleukin-8 (IL-8) mRNA by semiquantitative reverse transcription (RT)-PCR. In H. pylori-positive patients, significantly elevated levels were found for both chemokines (P < 0.05). Only IL-8 mRNA levels differed significantly (P< 0.05) in H. pylori-infected individuals who had serum antibodies for cytotoxin-associated protein CagA versus H. pylori-infected CagA-negative persons. Quantification of ENA-78 transcript levels by competitive RT-PCR yielded a significant 45-fold upregulation for ENA-78 transcripts in biopsies of H. pylori-positive versus H. pylori-negative patients (P < 0.05). In contrast to earlier findings with IL-8, the degree of ENA-78 mRNA upregulation was independent of the grade of activity of gastritis. Immunofluorescence studies on tissues of antral biopsies localized ENA-78 protein expression mainly to the gastric epithelium of H. pylori-positive patients, while control tissues were negative. Upregulation of ENA-78 and IL-8 mRNA and protein expression was also observed in an in vitro system using a gastric adenocarcinoma cell line. Only viable H. pyloriyielded a strong ENA-78 and IL-8 induction, while H. pyloriouter membrane proteins or water-soluble proteins had no significant effect. These data provide evidence for the importance of both IL-8 and ENA-78 in the development and perpetuation of H. pylori-associated gastritis.

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