scholarly journals Modulation of O-GlcNAcylation Regulates Autophagy in Cortical Astrocytes

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Md. Ataur Rahman ◽  
Hongik Hwang ◽  
Yoonjeong Cho ◽  
Hyewhon Rhim
Keyword(s):  
Posttranslational Modification ◽  
Autophagic Flux ◽  
Single Pair ◽  
Cortical Astrocytes ◽  
Intracellular Proteins ◽  
Protein Functions ◽  
Nutrient Signaling ◽  
Autophagic Degradation ◽  
Recycling Pathway ◽  
Lc3 Puncta

The addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) to serine and threonine residues is a common posttranslational modification of intracellular proteins which modulates protein functions and neurodegenerative diseases, controlled by a single pair of enzymes, O-GlcNAcase (OGA), and O-GlcNAcylation transferase (OGT). Autophagy is a cellular recycling pathway activated by stress and nutrient signaling; however, the mechanism by which O-GlcNAcylation modification regulates autophagy in cortical astrocytes is poorly understood. Here, we report that increased O-GlcNAcylation by the suppression of OGA activity using thiamet-G and OGA siRNA did not affect autophagy, whereas decreased O-GlcNAcylation caused by OGT inhibition by alloxan and OGT siRNA increased autophagy. OGT inhibitor and siRNA accumulated LC3 puncta, and cotreatment with chloroquine (CQ), an autophagy inhibitor, significantly increased LC3 puncta and LC3-II protein, confirming that decreased O-GlcNAcylation promotes autophagic flux. In particular, we found that OGT knockdown increases the fusion between autophagosomes as well as lysosomes and stimulates autophagy to promote lysosomal-associated membrane protein 1 (LAMP-1). Additionally, decreasing O-GlcNAcylation by treatment with alloxan, OGT siRNA, and OGA overexpression significantly decreased the level of autophagy substrate SQSTM1/p62, indicating that autophagic degradation was activated. Together, our study reveals a mechanism by which the modulation of O-GlcNAcylation modification regulates autophagy in mouse cortical astrocytes.

scholarly journals Antioxidant Compound, Oxyresveratrol, Inhibits APP Production through the AMPK/ULK1/mTOR-Mediated Autophagy Pathway in Mouse Cortical Astrocytes

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 408
Author(s):  
Md. Ataur Rahman ◽  
Yoonjeong Cho ◽  
Ghilsoo Nam ◽  
Hyewhon Rhim
Keyword(s):  
Radical Scavenging ◽  
Production Model ◽  
Autophagic Flux ◽  
Small Interfering Rnas ◽  
Compound C ◽  
Cortical Astrocytes ◽  
Autophagy Inhibitor ◽  
Wide Range ◽  
Autophagy Pathway ◽  
Lc3 Puncta

Oxyresveratrol (OxyR), a well-known polyphenolic phytoalexin, possesses a wide range of pharmacological and biological properties, comprising antioxidant, anti-inflammatory, free radical scavenging, anti-cancer, and neuroprotective activities. Autophagy is a cellular self-degradation system that removes aggregated or misfolded intracellular components via the autophagosome-lysosomal pathway. Astrocyte accumulation is one of the earliest neuropathological changes in Alzheimer’s disease (AD), and amyloid precursor protein (APP) is the hallmark of AD. OxyR could affect APP modulation via the autophagy pathway. Here, we have reported that OxyR promotes autophagy signaling and attenuates APP production in primary cortical astrocytes based on immunofluorescence and immunoblotting assay results. Co-treatment with the late-stage autophagy inhibitor chloroquine (CQ) and OxyR caused significantly higher microtubule-associated protein light chain 3 (LC3)-II protein levels and LC3 puncta counts, demonstrating that OxyR stimulated autophagic flux. We also found that OxyR significantly reduced the levels of the autophagy substrate p62/SQSTM1, and p62 levels were significantly augmented by co-treatment with OxyR and CQ, because of the impaired deficiency of p62 in autolysosome. Likewise, pretreatment with the autophagy inhibitor, 3-methyladenine (3-MA), resulted in significantly fewer OxyR-induced LC3 puncta and lower LC3-II expression, suggesting that OxyR-mediated autophagy was dependent on the class III PI3-kinase pathway. In contrast, OxyR caused significantly lower LC3-II protein expression when pretreated with compound C, an AMP-activated protein kinase (AMPK) inhibitor, indicating that AMPK signaling regulated the OxyR-induced autophagic pathway. Additionally, co-treatment with OxyR with rapamycin intended to inhibit the mammalian target of rapamycin (mTOR) caused significantly lower levels of phospho-S6 ribosomal protein (pS6) and higher LC3-II expression, implying that OxyR-mediated autophagy was dependent on the mTOR pathway. Conversely, OxyR treatment significantly upregulated unc-51-like autophagy activating kinase 1 (ULK1) expression, and ULK1 small interfering RNAs (siRNA) caused significantly lower OxyR-induced LC3 puncta counts and LC3-II expression, indicating that ULK1 was essential for initiating OxyR-induced autophagy. However, we found that OxyR treatment astrocytes significantly increased the expression of lysosome-associated membrane protein 1 (LAMP1). Finally, we established a stress-induced APP production model using corticosterone (CORT) in cortical astrocytes, which produced significantly more APP than the equivalent using dexamethasone (DEX). In our experiment we found that CORT-induced APP production was significantly attenuated by OxyR through the autophagy pathway. Therefore, our study reveals that OxyR regulates AMPK/ULK1/mTOR-dependent autophagy induction and APP reduction in mouse cortical astrocytes.

scholarly journals Silencing of PARP2 Blocks Autophagic Degradation

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 380 ◽  
Author(s):  
Laura Jankó ◽  
Zsanett Sári ◽  
Tünde Kovács ◽  
Gréta Kis ◽  
Magdolna Szántó ◽  
...  
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Serum Starvation ◽  
Autophagic Flux ◽  
Embryonic Fibroblasts ◽  
Nicotinamide Riboside ◽  
Chloroquine Treatment ◽  
Autophagic Degradation ◽  
Sirt1 Inhibitor

Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD+. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD+ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.

scholarly journals Assessing methods to quantitatively validate TGFβ-dependent autophagy

Biology Open ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. bio055103
Author(s):  
Charles B. Trelford ◽  
Gianni M. Di Guglielmo
Keyword(s):  
Cell Lines ◽  
Transforming Growth Factor Beta ◽  
Epithelial To Mesenchymal Transition ◽  
Transforming Growth Factor ◽  
Nsclc Cell ◽  
Serum Starvation ◽  
Autophagic Flux ◽  
Mesenchymal Transition ◽  
Protein Levels ◽  
Lc3 Puncta

ABSTRACTTransforming growth factor beta (TGFβ) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGFβ may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGFβ1 modulates autophagy related (ATG) gene expression and ATG protein levels. We also assessed microtubule-associated protein light chain 3 (LC3) lipidation, LC3 puncta formation and autophagosome-lysosome co-localization in non-small cell lung cancer (NSCLC) cell lines. These experimental approaches were validated using pharmacological autophagy inhibitors (chloroquine and spautin-1) and an autophagy activator (MG132). We found that TGFβ1, chloroquine and MG132 had little effect on ATG protein levels but increased LC3 lipidation, LC3 puncta formation and autophagosome-lysosome co-localization. Since similar outcomes were observed using chloroquine and MG132, we concluded that several techniques employed to assess TGFβ-dependent autophagy may not differentiate between the activation of autophagy versus lysosomal inhibition. Thus, NSCLC cell lines stably expressing a GFP-LC3-RFP-LC3ΔG autophagic flux probe were used to assess TGFβ-mediated autophagy. Using this approach, we observed that TGFβ, MG132 and serum starvation increased autophagic flux, whereas chloroquine and spautin-1 decreased autophagic flux. Finally, we demonstrated that ATG5 and ATG7 are critical for TGFβ-dependent autophagy in NSCLC cells. The application of this model will fuel future experiments to characterize TGFβ-dependent autophagy, which is necessary to understand the molecular processes that link, TGFβ, autophagy and tumorigenesis.

P6273Inhibitory phosphorylation of RIP1 at Ser320 induces nuclear translocation of TFEB, leading to suppression of necroptosis in cardiomyocytes

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
K Abe ◽  
T Yano ◽  
T Sato ◽  
H Kouzu ◽  
A Kuno ◽  
...  
Keyword(s):  
Cell Death ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Phosphorylation Site ◽  
Autophagic Flux ◽  
Downstream Target ◽  
Lysosome Biogenesis ◽  
Ldh Release ◽  
Lc3 Puncta

Abstract Background Necroptosis, a form of programmed necrosis, has been shown to contribute to the pathogenesis of various diseases including ischemia/reperfusion injury and heart failure. We recently reported that necroptotic signals suppresses autophagy in cardiomyocytes and that rapamycin, an mTORC1 inhibitor, not only promotes autophagy but also protect the cells from necroptosis. Purpose We examined the mechanism by which rapamycin suppresses necroptosis of cardiomyocytes, focusing on regulation of RIP1 activity and autophagic flux. Methods and results In H9c2 cardiomyoblasts, necroptosis was induced by treatment with TNF and z-VAD-fmk (zVAD) for 24 h, and cell death was determined by LDH release (as % of total). The treatment with TNF/zVAD increased LDH release from 3.4±1.3% to 46.1±2.3%, and LDH release was suppressed by necrostatin-1 (5.9±0.9%), a RIP1 inhibitor, and by rapamycin (23.5±1.4%). The protective effect of rapamycin was mimicked by Ku-0063794, an mTORC1/2 inhibitor. TNF/zVAD induced RIP1-RIP3 complex formation, together with suppression of TNF-induced RIP1 cleavage, which was mitigated by rapamycin. In addition, rapamycin not only suppressed TNF/zVAD-induced phosphorylation of RIP1-Ser166, an index of RIP1 activation, but also increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site. In cells transfected with RIP1-S320A, which lack Ser320 for inhibitory phosphorylation, rapamycin failed to suppress TNF/zVAD-induced RIP1-RIP3 binding and cell death. Immunoblot analyses showed that TNF/zVAD significantly increased level of LC3-II. The accumulation of LC3-II protein was not further increased by bafilomycin A1 (100 nM), an inhibitor of lysosomal protein degradation, indicating that accumulation of LC3-II by TNF/zVAD reflected suppression of autophagic flux. Inhibition of RIP1 by necrostatin-1 attenuated TNF/zVAD-induced accumulation of LC3 II. The restoration of autophagic flux in TNF/zVAD-treated cells by necrostatin-1 was confirmed by monitoring tandem RFP-GFP-LC3 transfected cells; necrostatin-1 increased a ratio of RFP-LC3-puncta (autolysosomes) to RFP-GFP-LC3-puncta (autophagosomes) in TNF/zVAD-treated cells. In addition, necrostatin-1 and rapamycin induced nuclear translocation of TFEB, a regulator of lysosome biogenesis, which was associated with upregulation of MCOLN1 mRNA, a downstream target of TFEB. Restoration of autophagic flux in TNF/zVAD-treated cells by necrostatin-1 was inhibited by siRNA-mediated knockdown of TFEB. Conclusion Activation of TFEB by inhibitiory phosphorylation of RIP1-Ser320 is a primary mechanism of cytoprotection afforded by mTORC1 inhibition against necroptosis.

scholarly journals Gintonin stimulates autophagic flux in primary cortical astrocytes

2020 ◽  
Vol 44 (1) ◽  
pp. 67-78 ◽  
Author(s):  
Md. Ataur Rahman ◽  
Hongik Hwang ◽  
Seung-Yeol Nah ◽  
Hyewhon Rhim
Keyword(s):  
Autophagic Flux ◽  
Cortical Astrocytes

scholarly journals Saponin Formosanin C-Induced Ferritinophagy and Ferroptosis in Human Hepatocellular Carcinoma Cells

Antioxidants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 682 ◽  
Author(s):  
Pin-Lun Lin ◽  
Han-Hsuan Tang ◽  
Shan-Ying Wu ◽  
Ning-Sing Shaw ◽  
Chun-Li Su
Keyword(s):  
Cell Death ◽  
Significant Negative Correlation ◽  
Degradation Pathway ◽  
Autophagic Flux ◽  
Ferritin Heavy Chain ◽  
Gfp Reporter ◽  
Dependent Cell ◽  
Ros Formation ◽  
Lc3 Puncta ◽  
Human Hepatocellular Carcinoma Cells

Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.

scholarly journals Phagocytosis of Candida albicans Inhibits Autophagic Flux in Macrophages

2018 ◽  
Vol 2018 ◽  
pp. 1-14
Author(s):  
Zhimin Duan ◽  
Qing Chen ◽  
Leilei Du ◽  
Jianbo Tong ◽  
Song Xu ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Acridine Orange ◽  
Brdu Incorporation ◽  
Autophagic Flux ◽  
Marker Protein ◽  
Independent Manner ◽  
Single Membrane ◽  
Molecular Pattern ◽  
Lc3 Puncta

Autophagy machinery has roles in the defense against microorganisms such as Candida albicans. Lipidated LC3, the marker protein of autophagy, participates in the elimination of C. albicans by forming a single-membrane phagosome; this process is called LC3-associated phagocytosis (LAP). However, the influence of C. albicans on autophagic flux is not clear. In this study, we found that C. albicans inhibited LC3 turnover in macrophages. After the phagocytosis of C. albicans in macrophages, we observed fewer acridine orange-positive vacuoles and RFP-GFP-LC3 puncta without colocalization with phagocytized C. albicans. However, phagocytosis of C. albicans led to LC3 recruitment, but p62 and ATG9A did not colocalize with LC3 or C. albicans. These effects are due to an MTOR-independent pathway. Nevertheless, we found that the C. albicans pattern-associated molecular pattern β-glucan increased LC3 turnover. In addition, phagocytosis of C. albicans caused a decrease in BrdU incorporation. Blocking autophagic flux aggravated this effect. Our findings suggest that phagocytosis of C. albicans decreases autophagic flux but induces LAP in an MTOR-independent manner in macrophages. Occupation of LC3 by recruiting engulfed C. albicans might contribute to the inhibition of autophagic flux. Our study highlights the coordinated machinery between canonical autophagy and LAP that defends against C. albicans challenge.

Treatment with the Ketone Body D-β-hydroxybutyrate Attenuates Autophagy Activated by NMDA and Reduces Excitotoxic Neuronal Damage in the Rat Striatum In Vivo

2020 ◽  
Vol 26 (12) ◽  
pp. 1377-1387
Author(s):  
Teresa Montiel ◽  
Luis A. Montes-Ortega ◽  
Susana Flores-Yáñez ◽  
Lourdes Massieu
Keyword(s):  
Molecular Mechanisms ◽  
Neuronal Damage ◽  
Building Blocks ◽  
Lysosomal Membrane ◽  
Rat Striatum ◽  
Autophagic Flux ◽  
Lesion Volume ◽  
Continuous Epidural Infusion ◽  
Autophagic Degradation

Background: The ketone bodies (KB), β-hydroxybutyrate (BHB) and acetoacetate, have been proposed for the treatment of acute and chronic neurological disorders, however, the molecular mechanisms involved in KB protection are not well understood. KB can substitute for glucose and support mitochondrial metabolism increasing cell survival. We have reported that the D-isomer of BHB (D-BHB) stimulates autophagic degradation during glucose deprivation in cultured neurons increasing cell viability. Autophagy is a lysosomal degradation process of damaged proteins and organelles activated during nutrient deprivation to obtain building blocks and energy. However, impaired or excessive autophagy can contribute to neuronal death. Objective: The aim of the present study was to test whether D-BHB can preserve autophagic function in an in vivo model of excitotoxic damage induced by the administration of the glutamate receptor agonist, N-methyl-Daspartate (NMDA), in the rat striatum. Methods: D-BHB was administered through an intravenous injection followed by either an intraperitoneal injection (i.v+i.p) or a continuous epidural infusion (i.v+pump), or through a continuous infusion of D-BHB alone. Changes in the autophagy proteins ATG7, ATG5, BECLIN 1 (BECN1), LC3, Sequestrosome1/p62 (SQSTM1/ p62) and the lysosomal membrane protein LAMP2, were evaluated by immunoblot. The lesion volume was measured in cresyl violet-stained brain sections. Results: Autophagy is activated early after NMDA injection but autophagic degradation is impaired due to the cleavage of LAMP2. Twenty-four h after NMDA intrastriatal injection, the autophagic flux is re-established, but LAMP2 cleavage is still observed. The administration of D-BHB through the i.v+pump protocol reduced the content of autophagic proteins and the cleavage of LAMP2, suggesting decreased autophagosome formation and lysosomal membrane preservation, improving autophagic degradation. D-BHB also reduced brain injury. The i.v+i.p administration protocol and the infusion of D-BHB alone showed no effect on autophagy activation or degradation.

scholarly journals Midazolam Enhances Mutant Huntingtin Protein Accumulation via Impairment of Autophagic Degradation In Vitro

2018 ◽  
Vol 48 (2) ◽  
pp. 683-691 ◽  
Author(s):  
Jiqian Zhang ◽  
Wei Dai ◽  
Pengcheng Geng ◽  
Li Zhang ◽  
Qilian Tan ◽  
...  
Keyword(s):  
Cell Viability ◽  
Pc12 Cell ◽  
Cathepsin D ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Mutant Huntingtin ◽  
Polyglutamine Diseases ◽  
Huntingtin Protein ◽  
Mutant Huntingtin Protein ◽  
Autophagic Degradation

Background/Aims: Autophagy is a well-known pathway to “clean” the misfolded mutant huntingtin protein (mHtt), which plays a considerable role in polyglutamine diseases. To date, there have been few studies of the choice of anesthetic during surgery in patients with polyglutamine diseases and evaluation of the effects and underlying mechanisms of anesthetics in these patients. Methods: GFP-Htt (Q74)-PC12 cells, which stably express green fluorescent protein-tagged Htt protein containing 74 glutamine repeating units, were used throughout this study. Cells were treated with 15 μM midazolam and 100 mM trehalose (positive control), and the induction of autophagy and autophagic degradation were assessed by detecting changes in autophagy-related proteins and substrates, and cell viability was assessed using the MTT assay. Overexpression of cathepsin D by plasmid transfection was used to restore midazolam-impaired autophagic degradation. Results: Midazolam increased intracellular mHtt levels in a time- and dose-dependent manner. Additionally, enhancing or blocking autophagic flux by trehalose or chloroquine could decrease or increase midazolam-induced mHtt elevation, respectively. Midazolam induced autophagy in the mTOR-dependent signaling pathway, but autophagic degradation was impaired, with a continuous rise in p62 and LC3 II levels and decrease in cathepsin D. However, overexpression of cathepsin D reversed the effects of midazolam. Midazolam led to a 20% decrease in GFP-Htt (Q74)-PC12 cell viability, which could be abrogated by overexpression of cathepsin D. Conclusions: Midazolam increased mHtt levels and decreased Htt (Q74)-PC12 cell viability via impairment of autophagic degradation, which could be restored by overexpression of cathepsin D.

scholarly journals TRPM2 promotes autophagic degradation in vascular smooth muscle cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiannan Zhao ◽  
Jingxuan Li ◽  
Wing-Hung Ko ◽  
Yiu-Wa Kwan ◽  
Liwen Jiang ◽  
...  
Keyword(s):  
Cell Death ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Muscle Cells ◽  
Transient Receptor Potential Channel ◽  
Cell Types ◽  
Autophagic Flux ◽  
Autophagic Degradation

AbstractTransient receptor potential channel M2 (TRPM2) is a Ca2+-permeable channel that is activated by reactive oxygen species (ROS). In many cell types, ROS activate TRPM2 to induce excessive Ca2+ influx, resulting in Ca2+ overload and consequent cell death. Recent studies suggest that TRPM2 may also regulate autophagy in pericytes and cancer cells by acting on the early step of autophagy, i.e. autophagic induction. However, there is no report on the role of TRPM2 in autophagic degradation, which is the late stage of autophagy. In the present study, we found abundant TRPM2 expression in lysosomes/autolysosomes in the primary cultured mouse aortic smooth muscle cells (mASMCs). Nutrient starvation stimulated autophagic flux in mASMCs mainly by promoting autophagic degradation. This starvation-induced autophagic degradation was reduced by TRPM2 knockout. Importantly, starvation-induced lysosomal/autolysosomal acidification and cell death were also substantially reduced by TRPM2 knockout. Taken together, the present study uncovered a novel mechanism that lysosomal TRPM2 facilitates lysosomal acidification to stimulate excessive autolysosome degradation and consequent cell death.

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