Agonist Stimulation, Talin-1, and Kindlin-3 Are Crucial for α¶bβ3 activation in a Human Megakaryoblastic Cell Line, CMK

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1137-1137
Author(s):  
Tsuyoshi Nakazawa ◽  
Seiji Tadokoro ◽  
Kazunobu Kiyomizu ◽  
Hirokazu Kashiwagi ◽  
Shigenori Honda ◽  
...  
Keyword(s):  
Cell Line ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Wild Type ◽  
Over Expression ◽  
Agonist Stimulation ◽  
Activated State ◽  
Critical Residue ◽  
Critical Residues

Abstract Abstract 1137 Integrin αIIbβ3 plays a central role in platelet aggregation, by changing its conformation from low-affinity to high-affinity state for ligand via inside-out signaling (integrin activation). However, detailed mechanism of αIIbβ3 activation remains to be determined. We have developed a powerful system to analyze the mechanism of physiological agonist-induced αIIbβ3 activation, by employing a human megakaryoblastic leukemia cell line, CMK. In contrast to exogenously expressed αIIbβ3 in CHO cells, endogenously expressed αIIbβ3 in GPIb-positive CMK cells can be activated by physiological agonists such as thrombin and protease-activated receptor-actibvating peptide (PAR-AP). An activated state of αIIbβ3 induced by agonists was only transient and the activated αIIbβ3 gradually returned inactive state. A robust αIIbβ3 activation was detected by an initial velocity analysis for FITC-PAC-1 binding. In this study, we performed direct genetic manipulation to examine an effect of talin-1 and kindlin-3 on αIIbβ3 activation in CMK. We measured the initial PAC-1 binding velocity by incubating FITC-PAC-1 and PAR1-TRAP with CMK cells only for 2 minutes. Over-expression of full length talin-1 (FL-TLN), talin head domain (THD) or kindlin-3 significantly augmented PAC-1 binding after 50 μM PAR1-TRAP stimulation. In sharp contrast to the CHO system, αIIbβ3 activation was not induced without any agonist stimulation in CMK system even if THD was over-expressed. Overexpression of FL-TLN mutant in critical residues for its head-rod interaction (FL-TLN-K318A or FL-TLN-M319A) more strongly augmented αIIbβ3 activation, as compared with wild-type FL-TLN. In contrast, overexpression of THD with the same mutation did not augment it, as compared with wild-type THD. These findings confirmed an autoinhibition mechanism for talin-1. FL-TLN mutants or THD mutants in critical residues for the interaction with β3 cytoplasmic tail (W359A, L325R, S365D, S379R, Q381V or K324D) or in the critical residue for the interaction with membrane (K322D) failed to augnent αIIbβ3 activation. In order to gain stable talin-1 or kindlin-3 knocked-down cell line, we used lentivirus mediated RNAi technology. In brief, lentiviral particles containing short-hairpin RNAs targeting talin-1 mRNA were integrated in CMK, and DsRed2 which is expressed with shRNA was used as transfection marker. Stable clones were obtained by limitting dilution. In these clones the expression of endogenous talin-1 or kindlin-3 was reduced to approximately half of non-silencing cells. Knock-down each molecule resulted in a robust decrease in PAC-1 binding induced by PAR1-TRAP. The impaired αIIbβ3 activation in kindlin-3 knocked down cells was rescued by exogenously expressed kindlin-3. Interestingly, the impaired αIIbβ3 activation in kindlin-3 knocked down CMK cells was also rescued by overexpression of THD. However, the increase in αIIbβ3 activation by PAR1-TRAP after over-expression of THD in kindlin-3 knocked-down cells was still smaller, as compared with that in THD overexpressed wild-type cells. Those results suggest that talin-1 and kindlin-3 play a crucial role in αIIbβ3 activation co-operatively. Taken together, this experimental system with CMK using RNAi and overexpression technology provides a newly alternative way to investigate molecular mechanism for the αIIbβ3 activation induced by physiological agonists. Disclosures: No relevant conflicts of interest to declare.

Transformation by Mutant IDH and (R)-2HG Is Reversible.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2413-2413
Author(s):  
Julie-Aurore Losman ◽  
Ryan E. Looper ◽  
William G. Kaelin
Keyword(s):  
Cancer Cells ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Wild Type ◽  
Monocytic Differentiation ◽  
Idh Mutations ◽  
Estrogen Withdrawal ◽  
Mutant Idh1

Abstract Abstract 2413 Somatic mutations in IDH1 and IDH2 are common in normal karyotype AML as well as in other clonal myeloid disorders and several solid tumors. Mutant IDH overproduces the R-enantiomer of 2-hydroxyglutarate, (R)-2HG (Dang, et al Nature 462: 739, 2009), which is hypothesized to alter the epigenetic landscape of cancer cells by inhibiting the activity of α-ketoglutarate-dependent enzymes, including the TET family of 5-methylcytosine hydroxylases and the jumonji-domain-containing family of histone demethylases. There is considerable interest in developing inhibitors of mutant IDH in the hope that, by decreasing (R)-2HG production in cancer cells, their epigenetic regulation can be restored. However, there is, to date, no evidence that transformation by mutant IDH is reversible or that inhibiting production of (R)-2HG has any effect on cancers harboring IDH mutations. Herein we report that in two different myeloid transformation assays, transformation by (R)-2HG and mutant IDH1 is reversible by removal of (R)-2HG. We previously reported that stable expression of a tumor-derived mutant IDH1 (IDH1R132H) induces growth factor independence and blocks EPO-induced differentiation in the human TF-1 erythroleukemia cell line, and that treatment of TF-1 cells with a cell-permeable form of (R)-2HG, TFMB-(R)-2HG, is sufficient to recapitulate this phenotype (Late Breaking Abstract #LBA-4, ASH 2011). We have extended these studies and found that transformation by TFMB-(R)-2HG is reversible and that this reversibility is influenced by the duration and intensity (dose) of (R)-2HG exposure. We developed a second myeloid transformation assay using a murine myeloid leukemia cell line that is transformed by expression of a HoxB8-ER fusion protein when cultured in the presence of estrogen. Upon estrogen withdrawal, the cells undergo monocytic differentiation and apoptosis. We expressed wild-type IDH1 or IDH1R132H in the cells and found that cells expressing wild-type IDH1 differentiate normally, but cells expressing IDH1R132H do not upregulate monocytic markers CD11b/Mac1 and Gr1 upon estrogen withdrawal. Furthermore, treatment of the IDH1R132H-expressing cells with an inhibitor of mutant IDH1 restores their ability to undergo monocytic differentiation upon estrogen withdrawal. Our findings suggest that continued exposure to (R)-2HG is required to maintain the cellular changes induced by mutant IDH, and further suggest that targeting (R)-2HG production may have therapeutic efficacy in the treatment of cancers harboring IDH mutations. Disclosures: No relevant conflicts of interest to declare.

Detection of the V617F Mutation of JAK2 Using a Novel Fully Automated SNP Super-Rapid Detector.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4640-4640
Author(s):  
Ruriko Tanaka ◽  
Junya Kuroda ◽  
Eishi Ashihara ◽  
Takayuki Ishikawa ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Cell Line ◽  
Residual Disease ◽  
Detection System ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Wild Type ◽  
Snp Detection

Abstract The JAK2 V617F substitution mutation (JAK2V617F) is one of the genetic hallmarks of chronic myeloproliferative diseases (CMPDs), such as polycythemia vera (PV), essential thrombocythemia (ET), or chronic idiopathic myelofibrosis (CIMF). Accurate and rapid detection of this mutation is essential for diagnosing and treating CMPDs today. We have developed a novel, rapid, sensitive and fully-automated single nucleotide polymorphism (SNP) detection system, termed ARKRAY SNP Detection System (ASDS), and used it to detect JAK2V617F in patients with CMPDs. With ASDS, diagnosis requires only 100ml of whole blood (or DNA), and the system automatically performs DNA extraction and PCR. The detection of both wild type and mutant jak2 alleles from PCR amplicons was measured by the increase in fluorescence produced by the dissociation of a JAK2V617F-specific guanine-quenching probe, and was completed within 75 minutes. In dilution assays of HEL cells (a JAK2V617F-positive leukemia cell line) using MYL cells (a chronic myelogenous leukemia (CML)-derived cell line with wild type JAK2), the system reliably quantified the mutation in a cell population containing as few as 1.0% mutant cells (Figure). We tested 44 samples from CMPDs patients using ASDS and direct sequencing (DS) (13 PV, 23 ET, 3 CIMF, 1 chronic myelomonocytic leukemia (CMMoL), 1 chronic neutrophilic leukemia (CNL), 3 unclassifiable CMPD (uCMPD)), which included samples from 3 post-allogenic bone marrow transplantation (BMT) patients with CIMF or uCMPD. Using ASDS, we detected JAK2V617F in 12/13 PV, 13/23 ET, 0/1 CIMF without BMT, 1/1 CMMoL, 0/1 CNL and 1/3 uCMPD. Overall, these results were comparable to previous results using relatively sensitive detection strategies, such as allele-specific PCR (AS-PCR). One of the 3 post-BMT CIMF samples was positive for JAK2V617F, which indicated that there were residual disease clones after BMT. ASDS detected JAK2V617F in one PV and eight ET patients, while DS failed to detect the mutation in these same samples, which clearly indicated that ASDS has a higher sensitivity than DS. JAK2V617F was absent in all samples from secondary erythrocytemia and healthy volunteers. Collectively, these results demonstrate that ASDS is a powerful and convenient tool for detecting JAK2V617F. With its associated high sensitivity, convenience and rapidity, this system will enable “Point-of-care” testing in clinical laboratories and “Patient-oriented” therapy for CMPDs. ASDS could also be applied to the detection of other point mutations relevant to cancer treatment, such as mutations in the BCR-ABL kinase domain that are associated with CML. Figure Figure

Establishment and Characterization of a Novel Human Myeloid Leukemia Cell Line, AMU-AML1, Carrying t(12;22)(p13;q11) with No Fusion of MN1-TEL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4375-4375
Author(s):  
Mayuko Goto ◽  
Ichiro Hanamura ◽  
Motohiro Wakabayashi ◽  
Hisao Nagoshi ◽  
Tomohiko Taki ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Chromosome Band ◽  
Structural Abnormality ◽  
Myeloid Leukemia Cell

Abstract Abstract 4375 Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis. In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1. Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of DMSA Modified Fe3O4 and Multidrug Resistance Modulator HZ08 on the Reversal of Multidrug Resistance in K562/A02 Cell Line

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5235-5235
Author(s):  
Fei Shen ◽  
Mingfei Zhou ◽  
Xuzhang Lu ◽  
Yanping Chen ◽  
Baoan Chen
Keyword(s):  
Multidrug Resistance ◽  
Cell Line ◽  
Real Time ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Morphological Changes ◽  
Leukemia Cell ◽  
Cell Counting ◽  
Leukemia Cell Line ◽  
Gene And Protein Expression

Abstract The objective of the present study was to investigate the reversible effect of HZ08 and daunorubicin(DNR) combined with dimercaptosuccinic acid modified iron oxide (DMSA-Fe3O4) magnetic nanoparticles (MNPs) in human chronic leukemia cell line K562/A02, and the mechanism potentially involved. The growth inhibition rate of K562/A02 cells was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis and intracellular concentration of DNR were detected by flow cytometry (FCM). DAPI staining was used to view apoptotic cellular morphology. Subsequently, transcription levels of MDR1 mRNA and expression levels of P-glycoprotein (P-gp) and caspase-3 were determined by real time polymerase chain reaction (real-time PCR) and Western blotting analysis, respectively. group clearly exhibited more morphological changes (severe structural alterations) than other groups. In addition, transcription of MDR1 gene and protein expression of P-gp and caspase-3 of K562/A02 cells were regulated at the most remarkable extent in DNR-HZ08-MNPs group when compared with other groups. These findings suggest that the remarkable effects of the novel DNR-HZ08-MNPs on multidrug resistant K562/A02 leukemia cells would be a promising strategy for overcoming multidrug resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1

2021 ◽  
Vol 9 ◽  
Author(s):  
Iveta Gažová ◽  
Lucas Lefevre ◽  
Stephen J. Bush ◽  
Rocio Rojo ◽  
David A. Hume ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemia Cell Line ◽  
Acute Monocytic Leukemia ◽  
Wild Type ◽  
Monocytic Leukemia ◽  
Damage Repair ◽  
Apparent Loss

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.

Leukemia Cell-Derived Exosomes Targeted Dendritic Cells Can Induce More Strong Anti-Leukemia Response Than Exosomes Alone

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4278-4278
Author(s):  
Chang Sheng ◽  
Jing Sun ◽  
Chun Wang ◽  
Siguo Hao
Keyword(s):  
Cell Line ◽  
K562 Cell ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Disease Free Survival ◽  
Membrane Vesicles ◽  
Biological Properties ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Hematopoietic Stem

Abstract Abstract 4278 Exosomes are the newest family number of ‘bioactive vesicles’ that play important roles in antigen presentation. Recent clinical trials about exosomes have shown minor clinical benefit. However, exosomes derived from leukemia cell lines were less studied, In this study, we investigated the biological properties and anti-leukemia immunity of leukemia cell-derived exosomes (LEX). The results showed that like other tumor cells, similarly, leukemia cells can release exosomes too. Transmission electron microscopy showed that exosomes derived from NB4 cell, a promyloblastic leukemia cell line and K562 cell, a chronic myeloid leukemia cell line were membrane vesicles with diameter about 50–100μ m, and loading their specific moleculars such as RAR-α and ABL and adherent moleculars such as ICAM-1. Similarly, our data also demonstrated the presence of Hsp70 in these two leukemia cell-derived exosomes. The data from two-dimensional electrophoresis of K562 cell-derived exosomes showed that most of molecules expressing on leukemia cells were detected on their exosomes, indicating that LEX load most of proteins expressed on their original leukemia cells. Out data also demonstrated that LEX can induce anti-leukemia CTL response and LEX-targeted DC can induce more efficient anti-leukemia immunity compared with LEX alone. In conclusion, exosome-based therapies could be considered as a promising strategy to prolong disease-free survival in patients with leukemia after consolidation or hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

The Effect of Over-Expression of miR-20a on Cell Proliferation of Human T Cell Leukemia Cell Line

2018 ◽  
Vol 64 (10/2018) ◽  
Author(s):  
Razavi M ◽  
Vahabpour R ◽  
Ranji N ◽  
Sanati MH ◽  
Mapar M ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Cell Line ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Over Expression ◽  
Human T Cell

scholarly journals RNA Cytosine Methyltransferases NSUN1 and NSUN2 Mediate the Lineage-Associated Resistance to Venetoclax in Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-14
Author(s):  
Shaun Wood ◽  
Amber Willbanks ◽  
Jason Xiaojun Cheng
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Synergistic Effects ◽  
Leukemia Cell ◽  
Resistant Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Leukemia Cell Lines

Background: Combinations of venetoclax/ABT-199, a small molecule that selectively inhibits anti-apoptotic protein BCL2, with hypomethylating agents (HMAs), such as 5-azacytidine (5-AZA, azacidtine) and decitabine have demonstrated remarkable synergistic effects and resulted in high response rates and significant overall survivals in patients with refractory MDS/AML (Ram, et al. Annual Hematology 2019; DiNardo et al. Blood 2019). However, resistance to venetoclax-based therapies has emerged as a major therapeutic barrier and been linked to monocytic clones in leukemia (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). Our recent study demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, mediate the lineage-associated resistance to 5-AZA through formation of a drug-resistant elongating RNA-Polymerase-II (eRNAPII) complex at nascent RNA (Cheng et al. Nature Communications 2018). This study aims to address the role of NSUN1 and NSUN2 in mediating venetoclax resistance in leukemia. Experimental Design and Methods: Experiments, including drug-induced cell growth inhibition, western blot, and co-immunoprecipitation, were performed on leukemia cell lines with various lineages to assess lineage-associated venetoclax resistance and identify the key factors/proteins involved in such resistance. Venetoclax-resistant cell lines were established from drug sensitive lines in order to elucidate mechanisms underlying resistance and cell lineage plasticity. Knockdown of NSUN1 and NSUN2 expression was performed to determine their roles in venetoclax-resistant cell lines. Results: Our experimental results have demonstrated monocyte differentiation-associated resistance to venetoclax in leukemia cell lines of different lineages (Figure 1A), which is consistent with previous published studies (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). The degree of lineage-associated venetoclax resistance is closely correlated with the expression of eRNAPII, NSUN2 and NSUN1. Importantly, venetoclax strongly induces expression of eRNAPII and NSUN1 (Figure 1B). We established venetoclax-resistant leukemia cell line (K1VR) from original venetoclax-sensitive granulocytic leukemia cell line Kasumi-1 and confirmed the importance of NSUN1 and NSUN2 in mediating venetoclax resistance in those leukemia cells. siRNA knockdown of NSUN1 or NSUN2 expression inhibits growth of leukemia cells and re-sensitizes the venetoclax-resistant K1VR leukemia cells to a low dose of venetoclax (Figure 1C). Conclusion: Our study has demonstrated that RNA cytosine methyltransferases NSUN1 and NSUN2 mediate monocyte-associated resistance to venetoclax in leukemia cells. We are currently extending our study to clinical specimens. Our study may lead to development of novel RNA epigenetics-driven strategies to predict and overcome the resistance to venetoclax-based therapies. Disclosures No relevant conflicts of interest to declare.

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