HIF, Hypoxia Research & Stem Cell News

PKI-179: An orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor.

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PKI-179: An orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor.

Bioorg Med Chem Lett. 2010 Jul 30;

Authors: Venkatesan AM, Chen Z, Santos OD, Dehnhardt C, Santos ED, Ayral-Kaloustian S, Mallon R, Hollander I, Feldberg L, Lucas J, Yu K, Chaudhary I, Mansour TS

A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed.

PMID: 20797855 [PubMed - as supplied by publisher]

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Autosomal dominant polycystic kidney disease and mTOR inhibitors: the narrow road between hope and disappointment.

Autosomal dominant polycystic kidney disease and mTOR inhibitors: the narrow road between hope and disappointment.

Nephrol Dial Transplant. 2010 Aug 26;

Authors: Ponticelli C, Locatelli F

PMID: 20798121 [PubMed - as supplied by publisher]

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Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes.

Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes.

Genes Dev. 2010 Aug 27;

Authors: Takai H, Xie Y, de Lange T, Pavletich NP

We reported previously that the stability of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs) depends on their interaction with Tel2, the ortholog of yeast Tel2 and Caenorhabditis elegans Clk-2. Here we provide evidence that Tel2 acts with Hsp90 in the maturation of PIKK complexes. Quantitative immunoblotting showed that the abundance of Tel2 is low compared with the PIKKs, and Tel2 preferentially bound newly synthesized ATM, ATR, mTOR, and DNA-PKcs. Tel2 complexes contained, in addition to Tti1-Tti2, the Hsp90 chaperone, and inhibition of Hsp90 interfered with the interaction of Tel2 with the PIKKs. Analysis of in vivo labeled nascent protein complexes showed that Tel2 and Hsp90 mediate the formation of the mTOR TORC1 and TORC2 complexes and the association of ATR with ATRIP. The structure of yeast Tel2, reported here, shows that Tel2 consists of HEAT-like helical repeats that assemble into two separate alpha-solenoids. Through mutagenesis, we identify a surface patch of conserved residues involved in binding to the Tti1-Tti2 complex in vitro. In vivo, mutation of this conserved patch affects cell growth, levels of PIKKs, and ATM/ATR-mediated checkpoint signaling, highlighting the importance of Tti1-Tti2 binding to the function of Tel2. Taken together, our data suggest that the Tel2-Tti1-Tti2 complex is a PIKK-specific cochaperone for Hsp90.

PMID: 20801936 [PubMed - as supplied by publisher]

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mTOR Inhibition Abrogates Insulin-Mediated Mammary Tumor Progression in Type 2 Diabetes.

mTOR Inhibition Abrogates Insulin-Mediated Mammary Tumor Progression in Type 2 Diabetes.

Endocr Relat Cancer. 2010 Aug 27;

Authors: Fierz Y, Novosyadlyy R, Vijayakumar A, Yakar S, Leroith D

Type 2 diabetes increases breast cancer risk and mortality, and hyperinsulinemia is a major mediator of this effect. The mammalian target of rapamycin (mTOR) is activated by insulin and is a key regulator of mammary tumor progression. Pharmacological and genetic mTOR ablation suppresses tumor growth in numerous mammary tumor models in the non-diabetic setting. However, the role of the mTOR pathway in type 2 diabetes-induced tumor growth remains elusive. Herein, we investigated whether the mTOR pathway is implicated in insulin-induced mammary tumor progression in a transgenic mouse model of type 2 diabetes (MKR mice) and evaluated the impact of mTOR inhibition on the diabetic state. Mammary tumor progression was studied in the double transgenic MMTV-Polyoma virus middle T antigen (PyVmT)/MKR mice and by orthotopic inoculation of PyVmT- and Neu/ErbB2- driven mammary tumor cells (Met-1 and MCNeuA cells, respectively). mTOR inhibition by rapamycin markedly suppressed tumor growth in both wild type and MKR mice. In diabetic animals, however, the promoting action of insulin on tumor growth was completely blunted by rapamycin despite a worsening of the carbohydrate and lipid metabolism. Taken together, pharmacological mTOR blockade is sufficient to abrogate mammary tumor progression in the setting of hyperinsulinemia and thus, mTOR inhibitors may be an attractive therapeutic modality for breast cancer patients with type 2 diabetes. Careful monitoring of the metabolic state however, is important as dose adaptations of glucose- and/or lipid-lowering therapy might be necessary.

PMID: 20801951 [PubMed - as supplied by publisher]

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mTOR Signaling is Involved in Indomethacin and Nimesulide Suppression of Colorectal Cancer Cell Growth via a COX-2 Independent Pathway.

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mTOR Signaling is Involved in Indomethacin and Nimesulide Suppression of Colorectal Cancer Cell Growth via a COX-2 Independent Pathway.

Ann Surg Oncol. 2010 Aug 28;

Authors: Zhang YJ, Bao YJ, Dai Q, Yang WY, Cheng P, Zhu LM, Wang BJ, Jiang FH

BACKGROUND: Inhibition of mammalian target of rapamycin (mTOR) represents an attractive target for anticancer therapy, but its role in suppression of colorectal cancer (CRC) cell growth by cyclooxygenase-2 (COX-2) inhibitors is unclear. Here, we analyzed the effect of indomethacin (Indo, a nonselective COX-2 inhibitor) and nimesulide (Nim, a selective COX-2 inhibitor) on mTOR signaling in CRC cells in vitro and in vivo to determine the dependence of this effect on COX-2. METHODS: Human CRC cell lines with varying COX-2 expression levels were treated with Indo and Nim. Western blot test was performed to detect mTOR-related components (mTOR, p70s6 K, and 4EBP1), and cell viability, cell cycle, and apoptosis were assessed. HCT116 and SW1116 cells were injected into athymic nude mice to establish a CRC xenograft model. After treatment with Nim, tumor volume, mTOR signaling, and apoptosis were evaluated in this model. HT29 and SW1116 cells were also treated with Nim after transfection with COX-2-specific small interfering RNA (siRNA) to assess dependence of COX-2 on mTOR signaling under drug treatment. RESULTS: Both Indo and Nim reduced mTOR signaling activity in CRC cells that differ in their COX-2 expression in vitro and in vivo. Additionally, Indo and Nim could reduce the mTOR signaling activity after COX-2 silencing in CRC cells. CONCLUSIONS: mTOR signaling is involved in Indo- and Nim-mediated suppression of CRC growth via a COX-2 independent pathway. This study unveils a novel mechanism through which COX-2 inhibitors exerts their anticancer effects and further emphasizes targeting mTOR signaling in anticancer therapy.

PMID: 20803081 [PubMed - as supplied by publisher]

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Steady-State Kinetic and Inhibition Studies of the mTOR Kinase Domain and mTOR Complexes.

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Steady-State Kinetic and Inhibition Studies of the mTOR Kinase Domain and mTOR Complexes.

Biochemistry. 2010 Aug 30;

Authors: Tao Z, Barker J, Shi SD, Gehring M, Sun S

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase and a major controller of cell growth. In cells, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. The mTORC1 complex can phosphorylate 4EBP1 and S6K1, two key regulators of translation initiation, whereas mTORC2 phosphorylates AKT1, an event required for AKT1 activation. Here, we expressed and purified human mTORC1 and mTORC2 from HEK-293 cells using FLAG-M2 affinity chromatography. Western blotting analysis using phospho-specific antibodies indicated that recombinant mTORC1 and mTORC2 exhibit distinct substrate preferences in vitro, consistent with their roles in cells. To better understand the enzymatic properties of mTOR alone and mTOR in its complex form, steady-state kinetic profiles of truncated mTOR containing kinase domain (residues 1360-2549) and mTORC1 were determined. The results revealed that mTORC1 is catalytically less active than truncated mTOR, as evidenced by 4.7- and 3.1-fold decrease in catalytic efficiency, kcat/Km, for ATP and 4EBP1, respectively. We also found that truncated mTOR undergoes autophosphorylation through an intramolecular mechanism. Mass spectrometric analysis identified two novel mTOR autophosphorylation sites, Ser2454 and one of Thr2473/Thr2474, in addition to the previously reported Ser2481 site. Truncated mTOR and mTORC1 were completely inhibited by ATP competitive inhibitors PI103 and BEZ235, while partially inhibited by rapamycin/FKBP12 in a noncompetitive fashion toward ATP. All inhibitors tested exhibited similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain. Our studies presented here provide the first detailed kinetic studies of a recombinant mTOR complex.

PMID: 20804212 [PubMed - as supplied by publisher]

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Phospholipase D-mTOR requirement for the Warburg effect in human cancer cells.

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Phospholipase D-mTOR requirement for the Warburg effect in human cancer cells.

Cancer Lett. 2010 Aug 28;

Authors: Toschi A, Lee E, Thompson S, Gadir N, Yellen P, Drain CM, Ohh M, Foster DA

A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property – known as the “Warburg effect” or aerobic glycolysis – contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxia-inducible factor (HIF) – a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the alpha subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.

PMID: 20805015 [PubMed - as supplied by publisher]

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Effect of rapamycin, an mTOR inhibitor, on radiation sensitivity of lung cancer cells having different p53 gene status.

Effect of rapamycin, an mTOR inhibitor, on radiation sensitivity of lung cancer cells having different p53 gene status.

Int J Oncol. 2010 Oct;37(4):1001-10

Authors: Nagata Y, Takahashi A, Ohnishi K, Ota I, Ohnishi T, Tojo T, Taniguchi S

Activation to a large extent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and mutations in the p53 gene are involved in lung cancer therapeutic resistance. The mammalian target of rapamycin (mTOR) acts as a downstream effector for Akt. Activation of the Akt/mTOR signal is a contributing factor to decreased radiation sensitivity. The purpose of this study was to examine whether the effect of rapamycin on radiation sensitivity is affected by cellular p53 gene status. Cellular radiation sensitivity was evaluated by using two human non-small cell lung cancer (NSCLC) cell lines with the same genetic background except for their p53 gene status (H1299/wtp53 and H1299/mp53). The cells were treated with rapamycin and/or radiation. Cell viability, cell proliferation, apoptosis, cell cycle and Akt/mTOR signaling activity were explored. Rapamycin synergistically enhanced the cytotoxicity of radiation, promoting the induction of apoptosis. Moreover, the combined treatment augmented the cytostatic effects of radiation regardless of cellular p53 gene status. Rapamycin in combination with radiation increased G1 arrest and suppressed progression to S phase in both cell lines. Furthermore, the combined treatment conduced to a prominent p53-independent down-regulation of the mTOR signal and pro-survival molecule, cyclin D1. Rapamycin can enhance the effect of radiation through the repression of pro-survival signals and the reduction in the apoptotic threshold. Taken together, inhibition of the mTOR signal may be a promising strategy for radiosensitization with no relevance to p53 gene status from the aspects of cell lethality and cell growth depression.

PMID: 20811722 [PubMed - in process]

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2-Arylthiazolidine-4-carboxylic acid amides (ATCAA) target dual pathways in cancer cells: 5′-AMP-activated protein kinase (AMPK)/mTOR and PI3K/Akt/mTOR pathways.

2-Arylthiazolidine-4-carboxylic acid amides (ATCAA) target dual pathways in cancer cells: 5′-AMP-activated protein kinase (AMPK)/mTOR and PI3K/Akt/mTOR pathways.

Int J Oncol. 2010 Oct;37(4):1023-30

Authors: Li CM, Narayanan R, Lu Y, Hurh E, Coss CC, Barrett CM, Miller DD, Dalton JT

Phosphatidylinositol-3-kinase (PI3K)/Akt and 5′-AMP-activated protein kinase (AMPK) are attractive targets for anti-cancer drug development. Inhibition of Akt or activation of AMPK is cytotoxic to human cancer cells in vitro and in vivo. We previously demonstrated that 2-arylthiazolidine-4-carboxylic acid amides (ATCAA) are effective cytotoxic agents in prostate and melanoma cancer cell lines, with IC50 values in the low/sub micromolar range. Using in vitro and in vivo studies, we further characterized the anti-cancer efficacy and mechanism of action of ATCAA-10, a potent lead. ATCAA-10 exhibited equal potency on both MES/SA and P-glycoprotein over-expressing multidrug resistant MES/SA/Dx5 cells, suggesting that ATCAA-10 may overcome multiple drug resistance. Cell-free kinase binding assays excluded the direct binding of ATCAA-10 to several kinases, including IGF-1R, EGFR, FGFR and PDGFR. However, in A549 and HeLa cells, ATCAA-10 effectively dephosphorylated Akt, with concomitant phosphorylation of AMPK. Determination of intracellular ATP and AMP concentrations revealed that ATCAA-10 activated AMPK by altering the intracellular AMP/ATP ratio. ATCAA-10 exhibited favorable pharmacokinetic properties in both mice and rats, including low clearance, low hepatic extraction rate, moderate volume of distribution and long half-life. In addition, ATCAA-10 inhibited A549 tumor xenograft growth with 46% tumor growth inhibition (TGI) at 20 mg/kg dose. Taken together; these results suggest that ATCAA-10 modulates the activity of two signaling pathways, PI3K/AKT/mTOR and AMPK/mTOR, resulting in the inhibition of cancer cell growth.

PMID: 20811725 [PubMed - in process]

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The role of the mTOR pathway in regulating food intake.

The role of the mTOR pathway in regulating food intake.

Curr Opin Drug Discov Devel. 2010 Sep;13(5):604-12

Authors: Wiczer BM, Thomas G

mTOR is a principal effector of nutrient action, integrating nutritional inputs from glucose, amino acids and fatty acids, as well as growth factor and hormonal signals. The mTOR signaling pathway plays a vital role in regulating cell growth and proliferation, and has been studied extensively in a variety of metabolic and cancer models. However, only recently has the mTOR signaling pathway become implicated in the regulation of food intake. This review focuses on recent studies describing the role of hypothalamic and gastric mTOR signaling in suppressing food intake, and discusses the potential mechanisms through which this regulation occurs.

PMID: 20812152 [PubMed - in process]

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Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage.

Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage.

Aging (Albany NY). 2010 Aug 24;

Authors: Panieri E, Toietta G, Mele M, Labate V, Ranieri SC, Fusco S, Tesori V, Antonini A, Maulucci G, De Spirito M, Galeotti T, Pani G

Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies.

PMID: 20739737 [PubMed - as supplied by publisher]

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ReproCELL Announces New Line Of Services In Neuronal Research Using Human IPS Cells

ReproCELL, Inc. (CEO: Chikafumi Yokoyama PhD) is pleased to announce a new line of products to be launched in the autumn of 2010, offering neuronal cells derived from human iPS (induced pluripotent stem) cells…

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Justice Dept. Asks Federal Judge To Allow Stem-Cell Research While Case Is Appealed

The Wall Street Journal: “The Obama administration asked a federal judge Tuesday to allow the government to continue funding embryonic stem-cell research while a case challenging the program makes its way through the courts. The judge, Royce Lamberth in Washington, D.C…

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For the first time, researchers identify and isolate adult mammary stem cells in mice

Source: Fred Hutchinson Cancer Research Center
Date: August 31, 2010

Summary:

SEATTLE – For the first time, researchers at Fred Hutchinson Cancer Research Center have identified and isolated adult mammary stem cells in mice. Long-term implications of this research may include the use of such cells to regenerate breast tissue, provide a better understanding of the role of adult stem cells in breast cancer development, and develop potential new targets for anti-cancer drugs. The findings, by Larry Rohrschneider, Ph.D., a member of the Basic Sciences Division at the Hutchinson Center, and Lixia Bai, M.D., Ph.D., a research associate in his lab, are published in the Sept. 1 issue of Genes & Development.

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Scientific breakthrough to pave the way for human stem cell factories

Source: University of Nottingham
Date: 31 August 2010

Summary:

Large scale, cost-effective stem cell factories able to keep up with demand for new therapies to treat a range of human illnesses are a step closer to reality, thanks to a scientific breakthrough involving researchers at The University of Nottingham. In a paper published in the September edition of Nature Materials, a team of Nottingham scientists led by Professor Morgan Alexander in the University’s School of Pharmacy, reveal they have discovered some man-made acrylate polymers which allow stem cells to reproduce while maintaining their pluripotency. Stem cells tagged with a fluorescent agent which allow them to be seen were placed onto the polymer spots. The scientists were then able to watch the stem cells and observe which polymers were most successful at promoting the most growth while also maintaining the pluripotency of the stem cells.

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Functional motor neuron subtypes generated from embryonic stem cells

Scientists have devised a method for coaxing mouse embryonic stem cells into forming a highly specific motor neuron subtype. The research provides new insight into motor neuron differentiation and may prove useful for devising and testing future therapies for motor neuron diseases.

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‘Basal-like’ breast cancer does not originate from basal stem cells

New research uncovers a case of mistaken identity that may have a significant impact on future breast cancer prevention and treatment strategies. The study suggests that despite their “stem cell-like” characteristics, most aggressive breast tumors are not derived from normal mammary gland stem cells.

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Live imaging puts new light on stem cell division

A long-held assumption about asymmetrical division of stem cells has cracked. Researchers report that the mitotic spindle does not act alone — that cortical proteins help to position a cleavage furrow in the right location.

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Cancer-causing gene crucial in stem cell development, study finds

A research team has shown for the first time that a gene called Myc, which is traditionally thought of as a cancer-causing gene, may be far more important in the development and persistence of stem cells than was known before.

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The effect of progressive hypoxia on spontaneous activity in single and shoaling golden grey mullet Liza aurata.

The effect of progressive hypoxia on spontaneous activity in single and shoaling golden grey mullet Liza aurata.

J Fish Biol. 2009 Nov;75(7):1615-25

Authors: Lefrançois C, Ferrari RS, Moreira da Silva J, Domenici P

The hypothesis of a differential effect of hypoxia on activity in shoaling v. solitary fish was tested in golden grey mullet Liza aurata. In both solitary and shoaling fish, (1) swimming activity increased significantly at </= 60% air saturation compared with normoxia, (2) aquatic surface respiration increased significantly below 15% air saturation and (3) a significant increase in ventilation frequency was observed between 40 and 10% air saturation. Swimming activity was higher in shoaling than in solitary fish, possibly due to social interactions in shoaling fish. Despite showing a higher swimming activity, shoaling individuals showed similar ventilation frequency to the less active solitary fish. This suggests that shoaling exerts a calming effect on L. aurata, probably related to increased safety while in numbers. In addition, shoaling fish spent a higher proportion of time performing aquatic surface respiration at 15% air saturation (i.e. the highest oxygen level at which aquatic surface respiration was observed) than solitary fish, possibly because of (1) synchronization of aquatic surface respiration in shoaling fish and (2) lower risk of predation perceived by shoaling fish.

PMID: 20738637 [PubMed - in process]

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