Anandamide-Induced Cell Death: Dual Effects in Primary Rat Decidual Cell Cultures

Placenta 30 (2009) 686–692

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Anandamide-Induced Cell Death: Dual Effects in Primary Rat Decidual Cell Cultures B.M. Fonseca a, b, G. Correia-da-Silva a, b, N.A. Teixeira a, b, * a b

´cia, Universidade do Porto, Porto, Portugal Serviço de Bioquı´mica, Faculdade de Farma Instituto de Biologia Molecular e Celular – IBMC, Universidade do Porto, Porto, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 29 May 2009

Anandamide (AEA) belongs to an emerging class of lipid mediators collectively termed ‘‘endocannabinoids’’. This endogenously synthesized compound has been implicated in multiple processes, mainly related to the regulation of cell growth/death. During pregnancy endometrial fibroblast-like stromal cells proliferate and differentiate into decidual cells, forming the decidua. After reaching its maximum development, the decidua undergoes regression, which appears to be associated with apoptosis. In order to study the role of this endocannabinoid in this process, the effects of AEA upon cell viability and cell death in primary rat decidual cell cultures was investigated. The results obtained demonstrated that AEA induces cell death, in a dose-dependent manner which is associated with morphological alterations, such as nuclear condensation, DNA fragmentation and upregulation of caspase-3/7 activities. Moreover, these effects were attenuated by AM251, a selective antagonist for the cannabinoid receptor CB1. High concentrations induced a dramatic effect in cell viability and morphology, though methyl-b-cyclodextrin (MCD), a membrane cholesterol depletor completely reversed the cytotoxic effect. These findings suggest that AEA in the uterine environment may play an important role in regulating apoptosis through CB1 and thereby modulate decidual stability and regression during pregnancy. However, it cannot be discarded the hypothesis that AEA, in high concentrations, represent a deleterious factor during this complex process. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Endocannabinoids Anandamide Decidual cells Apoptosis

1. Introduction The uterus is constituted by different cell types and in response to the implanting blastocyst undergoes a synchronized process by which the endometrial stroma transforms into a dense cellular matrix, denominated decidua [1,2]. Decidualization initiates on the antimesometrial region, where implantation occurs and extends thereafter to the mesometrial pole, which will be invaded by the trophoblast cells giving rise to the placenta. This extensive tissue reorganization is a well-coordinated process and is followed by an active process of cell death involving apoptosis and secondary necrosis [3–6]. The discovery of tetra-hydro-cannabinol (THC), the active component of Cannabis sativa, led to the finding of two specific cannabinoid receptors, CB1 and CB2 [7–9]. Although CB1 receptor is predominantly localized in the brain and CB2 in the immune cells,

* Correspondence to: Nate´rcia A. Teixeira, Faculdade de Farma´cia da Universidade do Porto, Serviço de Bioquimica, R. Anı´bal Cunha no. 164, 4050-047 Porto, Portugal. Tel.: þ351 222 078 900; fax: þ351 222 003 977. E-mail address: [email protected] (N.A. Teixeira). 0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.05.012

they are also present in other tissues, like in the reproductive tract [10]. Both receptors are coupled to G-proteins and act via adenylate cyclase and calcium channels [7,11]. The first endogenous ligand for these receptors was found in 1992, and was designated as anandamide (AEA) [12]. Since then many other related compounds have been found, like 2-arachidonylglycerol (2-AG) [13]. Anandamide has also been shown to act as an agonist on the transient receptor potential vanilloid 1 (TRPV1 or VR1), which is a non-selective cation channel [14,15]. There is increasing evidence that endocannabinoids are involved in a broad range of central and peripheral actions, including adverse effects on pregnancy and embryonic development. It is now known that AEA has an important role in the synchronization between the embryo and the uterus, acting as a regulator of the ‘‘implantation window’’ [16–18]. It has also been shown that AEA induces cell death in a wide variety of cells through the activation of different receptors [19–21], though there are also some evidences that endocannaboids protect primary neurons from apoptosis [22], stimulate proliferation of cancer cells [23] or enhance the proliferative effect of erythropoietin [24]. Most of the work performed in pregnancy is related to the implantation period and there is a lack of information during

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decidualization and early placental development, which is an important response strategy to allow remodeling of the implantation chamber. As there is a relationship between AEA and cell death in various cell types and it is known that AEA has dramatic effects on embryo development and implantation, the aim of this study was to investigate whether AEA could be relevant to the programmed cell death that occurs during decidual regression.

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2.5. Determination of caspase-3/7 activity The caspase-GloTM-3/7 assay (Promega Corporation, Madison, WI, USA) was used for the evaluation of caspase-3 and -7 activities. The cells were seeded (2  104/well) in 96-well microplates, for 12 h and the caspase-GloTM-3/7 reagents were added directly to the cells and luminometer readings were taken after 3 h of incubation at room temperature. The resultant luminescence was measured in relative light units (RLU) using a 96-well Microplate Luminometer (BioTek Instruments, Vermont, USA). 2.6. TdT-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)

2. Material and methods 2.1. Animals Female Wistar rats (200–250 g) were mated with fertile males and the day spermatozoa were found in the vaginal smear was designated day 1 of pregnancy. Animals were killed on day 10 of pregnancy by cervical dislocation and the uterine horns dissected. The homogenates for the positive controls were obtained from brain for CB1 and TRPV1 and from spleen for CB2. All procedures involving animals were conducted in accordance with the guidelines of the Ethics Committee of the Institute of Molecular and Cellular Biology, Oporto University.

2.2. Primary decidual cell culture Mesometrial decidua was separated from the antimesometrial decidua, minced and digested with dispase II (2.4 U/ml) (Roche Diagnostics GmbH, Mannhein, Germany), colagenase (50 U/ml) (Sigma Chemical Co, St. Louis, MO, USA) and Dnase I (200 U/ml) (Roche Diagnostics GmbH, Mannhein, Germany) in HBSS (Gibco/Invitrogen Corporation, Carlsbad, CA, USA) for 1 h at 37  C. Non-digested tissue was removed by filtration through a 60-mm mesh and decidual cells were collected by centrifugation at 200g for 10 min as described previously [25]. Erythrocytes were removed by addition of NH4Cl solution (0.83%) followed by incubation on ice for 10 min. Cells were cultured at 37  C in Dulbecco’s minimum essential medium (DMEM) (Gibco/Invitrogen Corporation, Carlsbad, CA, USA) supplemented with antibiotic– antimycotic solution (200 U/ml penicillin G, 0.5 mg/ml amphotericine B and 200 mg/ml streptomycin) (Gibco/Invitrogen Corporation, Carlsbad, CA, USA) and 10% FBS (Gibco/Invitrogen Corporation, Carlsbad, CA, USA) under a 95% air-5% CO2 humidified atmosphere for 4 h. After adhesion, cells were cultured for 12–48 h in DMEM without FBS in the presence or absence of varying concentrations of anandamide (0.1–25 mM) (Tocris Bioscience, Bristol, UK). Medium was replaced every 24 h. Purity of cultures was evaluated by cellular morphology and a2-macroglobulin expression was evaluated as a decidual marker. In order to study the involvement of anandamide-binding receptors, selective antagonists, SR141716A (0.1–1 mM), SR144528 (0.1–1 mM) and Capsazepine (1–10 mM) for CB1, CB2 and TRPV1, respectively (Tocris Bioscience, Bristol, UK), were pre-incubated 15 min before AEA treatment. In methyl-cyclodextrin (MCD) (Sigma Chemical Co, St. Louis, MO, USA) experiments, the cells were pre-incubated for 1 h. Control experiments with equimolar concentrations of ethanol alone (data not shown) or with each antagonist (1–10 mM) and MCD (1–5 mM) did not show any significant effects on the parameters investigated in this study. Stock solutions were prepared in ethanol and stored in aliquots at 80  C.

2.3. Morphological studies The morphological changes during the time of culture and the characteristics of apoptotic morphology (cell shrinkage, nuclear condensation and appearance of apoptotic bodies) were evaluated by Giemsa staining and by phase contrast microscopy. To evaluate nuclear morphology, Hoechst 33342, a cell permeant blue fluorescent DNA stain, was used. Cells were fixed with 4% paraformaldehyde and exposed to 0.5 mg/ml Hoechst 33342 in PBS for 20 min, at room temperature, and examined under a fluorescence microscope equipped with an excitation filter with maximum transmission at 360/40 nm.

DNA fragmentation was assessed using an Apoptosis Detection System, Fluorescein (Roche Molecular Biochemicals, Mannheim, Germany). Briefly, cells were cultured onto chamber slides (2.5  105 cells/well) and fragmented DNA of apoptotic cells was measured by the catalytic incorporation of fluorescein-1,2-dUTP at the 30 -OH ends using the enzyme terminal deoxynucleotidyl transferase (TdT). The fluorescein-1,2-dUTP-labeled DNA was visualized using a fluorescent microscope. Specimens were counterstained with propidium iodide (PI) (Sigma Chemical Co, St. Louis, MO, USA), a red fluorescent nucleic acid stain. For the positive control, cells were incubated with staurosporine or DNase I (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at room temperature, and for the negative control cells were incubated with fluorescein-tagged dNTP without TdT. Examination and cell counting were performed using a microscope equipped with appropriate filter combinations. In each experiment 7–10 independent fields, in a total of 500 cells, were counted per condition. 2.7. Immunocytochemistry Cells (2.5  105 cells/well) were seeded on eight-well glass chamber slides and incubated for 12–48 h in the presence or absence of anandamide. After incubation, cells were fixed for 15 min at room temperature in methanol. After fixation, cells were permeabilized with PBS, 5% BSA, 0.1% Triton X-100 and 0.2% Tween-20 solution for 15 min at room temperature. Expression of anandamide-binding receptors and a2macroglobulin, a decidual marker, was analysed using an avidin–biotin alkaline phosphatase complex immunocytochemical technique (Vectastain ABC kit, Vector Laboratories, CA, USA). The slides were incubated overnight at 4  C with the primary rabbit anti-a2-macroglobulin (1:500) (Abcam, Cambridge, UK), anti-CB1 (1:100), antiCB2 (1:100) or goat anti-TRPV1 (1:100) (Santa Cruz, CA, USA). After washing with PBS they were incubated with diluted biotinylated secondary antibody for 30 min, followed by incubation with Vectastain ABC-AP reagent as recommended in the kit instructions. The reaction was developed by incubation with Sigma Fast RedÔ tablets (SigmaÒ FastTM Fast Red-TR/Naphtol). Negative controls were performed with the inclusion of rabbit or goat IgG instead of the primary antibody. The slides were counterstained with Mayer’s Hematoxylin solution (Sigma Chemical Co, St. Louis, USA) and mounted in Aquamont improved medium (BDH Laboratory Supplies, Pool, England). 2.8. Protein extraction and Western blot analysis Cells were scraped from the plates, sonicated on ice, and the protein fraction was obtained after centrifugation at 14,000g for 10 min. Samples (50 mg) were subjected to SDS-polyacrylamide gel electrophoresis and proteins were transferred onto nitrocellulose membranes. Western blots were performed with the anti-a2macroglobulin (1:500), anti-CB1 (1:100), anti-CB2 (1:100) or anti-TRPV1 (1:100), and then with peroxidase-conjugated secondary antibody. Finally blots were subjected to a chemiluminescence detection kit (Super Signal West Pico; Pierce, Rockford, USA). 2.9. Statistical analysis Statistical analysis was carried out by ANOVA, followed by the Bonferroni post hoc test to make pairwise comparisons of individual means (GraphPad PRISM v. 4.0, GraphPad Software, Inc., San Diego, CA, USA) when significance was indicated. The results are the mean of three independent experiments performed in triplicate. Data were expressed as mean  SEM and differences were considered to be statistically significant at P < 0.05.

3. Results 2.4. Cell viability Cells were cultured in ninety-six well plates at a density of 2.5  105/ml for 12–48 h in the presence or absence of AEA (0.1–25 mM). After incubation, MTT (0.5 mg/ml final concentration) (Sigma Chemical Co, St. Louis, MO, USA) was added to each well and the plate was incubated for 3 h at 37  C. The formazan was quantified spectrophotometrically by addition of DMSO:isopropanol mixture (3:1). LDH release was measured using CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The results of cell viability are the mean of four independent experiments performed in triplicate and are expressed as a percentage of the untreated control cells.

3.1. Anandamide effects in rat primary decidual cells The exposure of rat primary decidual cells to AEA in concentrations between (5–25 mM) resulted in a decrease in cell viability in a time and dose-dependent manner (Fig. 1A). The loss of viability for 10 mM AEA increased significantly between 12 and 24 h of treatment, reducing the cell viability to 78% at 24 h (P < 0.05) (Fig. 1A). In contrast, after 12 h or 24 h of treatment with

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Fig. 1. Effects of AEA on cultured primary decidual cell viability estimated by MTT assay and LDH release. (A) Cell viability of AEA-treated cells with different concentrations (0.1–25 mM) and at different times (12, 24, 48 h). (B) LDH release increased after 25 mM of AEA treatment. (***P < 0.001, **P < 0.01, *P < 0.05 vs. Control).

a concentration of 25 mM a dramatic decrease was observed (P < 0.05) (Fig. 1A). Cell viability was reduced markedly after 48 h of treatment reaching only 58% and 25% of viable cells, respectively for 10 mM and 25 mM (Fig. 1A). To understand the process associated with the reduction in cell viability, LDH release in the culture medium was evaluated. In lower

concentrations till 10 mM, no alteration was observed, however, at 25 mM of AEA, LDH levels were approximately 70% and 82% higher than in control cells, at 12 and 24 h, respectively (Fig. 1B). To get further insight into this effect cell morphology was studied by phase contrast microscopy and Giemsa staining (Fig. 2). With AEA 10 mM (Fig. 2C, D) general morphology was preserved

Fig. 2. Morphological study by phase contrast microscopy and Giemsa staining of decidual cells in culture in the presence or absence of anandamide. (A, B) Untreated decidual cells with normal morphology in phase contrast microscopy and Giemsa staining. (C, D) Treatment with 10 mM of AEA-induced changes in cell morphology; some cells present a distended cytoplasm. In Giemsa it was also possible to observe chromatin condensation. (E, F) Treatment with 25 mM revealed abnormal morphology of decidual cells and a reduction in cell density. (Original magnification, 400).

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though, it was possible to observe some cells presenting chromatin condensation in Giemsa staining (Fig. 2D). After 24 h of incubation, 25 mM of AEA caused an intense reduction in cell density and a dramatic change in cell morphology (Fig. 2E, F) compared to the untreated cells (Fig. 2A, B). The decidual cells from day 10 of pregnancy cultured for 12, 24 and 48 h express anandamide-binding receptors and a2-macroglobulin, the major product synthesized by the decidual cells, which is considered a marker of rat decidual cells (Fig. 3).

3.2. Anandamide-induced apoptosis To further get insight into the process associated with the decrease in cell viability TUNEL and Hoechst 33342 staining were used (Fig. 4). Treated cells with AEA (10 mM) showed chromatin condensation, indicative of apoptosis, compared to the untreated cells. Although sporadic apoptotic nuclei could be seen in untreated cells, they became more frequent after 24 h of AEA incubation (10 mM) (Fig. 4A, C). AEA-treated cells (10 mM) revealed an increase of 10% TUNEL-positive cells, after 24 h of treatment (P < 0.05) (Fig. 4B,D,E).

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In order to confirm the occurrence of apoptosis, it was also evaluated the activity of caspase-3/7 by luminescence (Fig. 4F). An increase of 11.3% in caspase-3/7 activity was observed after 12 h of culture with AEA (10 mM) in comparison to the control cells (P < 0.05) in conditions used (Fig. 4F). 3.3. The involvement of anandamide-binding receptors inducing cell death It is known that most of the biological effects of AEA are mediated by its receptors CB1, CB2, or VR1. As referred before, all receptors were expressed in rat primary decidual cells and to verify their involvement in decidual cell apoptosis, CB1, CB2, and VR1 were blocked with selective antagonists, SR141716A (AM251), SR144528 (AM630) and Capsazepine (CAPZ), respectively. The results showed that treatment with AM251 (1 mM), but not with AM630 (1 mM) or CAPZ, even at 10 mM, prevented cell death induced by AEA (10 mM) (Fig. 5A). In addition, pre-treatment with the selective CB1 antagonist, AM251 (1 mM), reverted the number of TUNEL-positive cells obtained by AEA treatment to identical levels observed in control (Fig. 4E). It also prevented caspase-3/7 activation (Fig. 4F),

Fig. 3. Immunolocalization of a2-macroglobulin and anandamide-binding receptors in primary decidual cells. Decidual cells obtained from day 10 of pregnancy were cultured for 24 h in DMEM. a2-macroglobulin (A), CB1 (B), CB2 (C) and TRPV1 (D) expression was found in decidual cells by immunocytochemistry and Western blot analysis (F). (E) Negative control obtained by omitting the primary antibody. (PC – positive control).

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confirming the results observed previously and suggesting that CB1 is required for the induction of apoptosis.

3.4. The role of membrane cholesterol in anandamide-induced cell death

In order to confirm that MCD effects in cytotoxicity reduction were not due to a direct interaction between AEA and MCD, cells were washed before addition of AEA to the cell cultures. The inhibitory activity of MCD observed previously was maintained (Fig. 5E). 4. Discussion

Previous studies have shown in other cell types that AEA may induce cell death independently of CB1, CB2 or VR1 through interaction with membrane cholesterol [26,27]. In order to clarify the mechanisms associated with the dramatic reduction of cell viability and LDH release after treatment with 25 mM of AEA, methyl-cyclodextrin (MCD), a membrane cholesterol-depleting agent, was used. To analyze the effect of MCD on lipid raft integrity, cells were pre-treated for 1 h with MCD (0.25–1 mM) followed by AEA at 10 and 25 mM. Interestingly, in the latter concentration, pre-treated cells with MCD (0.25–1 mM) showed an increase in cell viability from 50% to 94% (Fig. 5B), indicating that a specific lipid raft-disrupting effect of MCD under our experimental conditions, was important for AEA cytotoxicity in rat primary decidual cells. It was also possible to observe a significant decrease in LDH release with AEA (25 mM) in MCD-pre-treated cells (Fig. 5C). However, we have not found any effect in MCD pre-treatment in cell viability at 10 mM of AEA (Fig. 5D).

The uterus undergoes morphological and physiological changes during gestation to accommodate the developing conceptus, being apoptosis and secondary necrosis important mechanisms involved in the regression of decidual tissue [3,4]. Anandamide is the main endogenous cannabinoid ligand and its levels are spatiotemporally regulated in the uterus during early pregnancy, showing a dual function dependent on the concentration. It has been shown that low levels allow a successful implantation, while high levels are detrimental [16,28,29]. It has been referred that this AEA-biphasic action is coupled with CB1 receptor, which works as a ‘‘sensor’’ to AEA-levels. The underlying mechanisms at higher concentrations are related to the inhibition of calcium mobilization and at lower concentrations to the activation of ERK signalling pathway [16]. Additionally, it has been described important effects of AEA in the regulation of cell survival/death [21] and as decidual

Fig. 4. Decidual cells stained with Hoescht and TUNEL assay. Graphic representation of TUNEL-positive cells and Caspase-3/7 activity of control and AEA-treated cells. (A) Hoescht staining of untreated cells showed few cells with condensed chromatin (arrows) compared to AEA-treated cells (10 mM) (C). TUNEL and PI staining (TUNELþ, yellow; PI, red) also revealed differences between treatment (D) and control (B). Arrows indicate the TUNELþ cells. (E) Number of TUNELþ cells of controls compared to AEA-treated cells. (F) Evaluation of caspase-3/7 activity in primary decidual cells after 12 h of culture with AEA (10 mM) reveals an enhancement of 11% of caspase activity. (Original magnification, 200). (*P < 0.05 vs. Control).

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regression is accompanied by extensive cell death we tested anandamide effects in rat primary decidual cell cultures in order to investigate the role of this endocannabinoid during this period of pregnancy. Our results showed that anandamide-induced cell death in a time-dependent manner, and between concentrations of 5–25 mM this effect was also dose-dependent. Moreover, for higher levels, AEA was able to induce LDH release, probably due to a cytotoxic effect. The reduction in cell viability was associated with morphologic and molecular alterations, characteristic of an apoptotic cell death, such as chromatin condensation as shown by Giemsa and Hoescht staining and an increase in TUNEL-positive cells for lower doses. It has been referred in literature that AEA induces apoptosis via CB1 or TRPV1 depending on cell type [30–32]. Specifically in rat primary decidual cells, we found that AEA at 10 mM can induce apoptosis mediated by CB1. Once again, our results showed a dual response for AEA depending on the concentration. While lower concentrations caused apoptosis, which was reversed by blockade of CB1, higher concentrations provoked dramatic effects in cell viability and morphology. In order to get insight into this mechanism methyl-b-cyclodextrin was used. Numerous studies have referred the use of this compound and its reversible effects on membrane cholesterol content and caveolae structure are well documented [33]. It has been reported the involvement of lipid rafts in AEA-induced cell

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death in different cell types and that MCD blocks in vitro apoptosis induced by AEA in glioma cells [27] and hepatocytes [26]. However, we have not found any effect of MCD in AEA-mediated cell death at low concentrations. In contrast, it was demonstrated that pre-treatment with MCD increased cell viability and caused a considerable reduction in LDH release only in the case of high concentrations of AEA. Thus, it is reasonable to suggest that high levels of AEA exert direct effects in rat decidual cells through greater access due to cholesterol-rich lipid rafts and that depletion of membrane cholesterol inhibits this process, while at lower levels it induces apoptosis mediated by CB1 with the involvement of a caspase-3/7dependent pathway. However, the local regulation of apoptosis in the decidua is complex and requires a careful examination of different pathways. Further studies are in progress to address this issue. Taken together, these results suggest that AEA may be involved in the natural remodeling process occurring during decidual regression through cannabinoid receptor 1. As already highlighted, the anandamide signalling mediated by CB1 is crucial to various female reproductive events that include development of embryos, oviductal transport and implantation. Our results also suggest, for the first time, the involvement of AEA and CB1 in decidual remodeling. However, it cannot be discarded the hypothesis that AEA, in higher levels, represent a deleterious factor during this complex process and that a similar mechanism for exocannabinoids may occur during drug consumption in pregnancy.

Fig. 5. The effect of cannabinoid and vanniloid receptors and methyl-b-cyclodextrin (MCD) in AEA-induced death of rat primary decidual cells. (A) Cell death induced by low doses of AEA (10 mM) is only reduced when CB1 receptor is blocked (AM251). (B) Membrane cholesterol depletion by methyl-b-cyclodextrin (MCD) prevents AEA-induced cell death at higher concentrations (25 mM). (C) MCD pre-treatment reduces LDH release. (D) MCD pre-treatment does not prevent cell death at low concentrations of AEA. (E) Cells were pre-incubated with MCD followed or not by washing (W-0.5). AEA addition demonstrates similar effects in both cases suggesting that there are no direct interaction between AEA and MCD. (*Indicates P < 0.05 vs. AEA (10 mM) and **indicates P < 0.05 vs. AEA (25 mM)).

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Acknowledgments This work was supported by Fundaça˜o para a Cieˆncia e Tecnologia (FCT), Portugal. B.M.R.F. is a recipient of a Ph.D. grant of FCT (SFRH/BD/29856/2006).

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