Phosphorylated tau in cerebrospinal fluid-derived extracellular vesicles in Alzheimer's disease: a pilot study.


Journal

Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288

Informations de publication

Date de publication:
25 Oct 2024
Historique:
received: 05 07 2024
accepted: 04 10 2024
medline: 26 10 2024
pubmed: 26 10 2024
entrez: 25 10 2024
Statut: epublish

Résumé

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder characterized by brain aggregation of β-amyloid (Aβ) peptides and phosphorylated tau (P-tau) proteins. Extracellular vesicles (EVs) can be isolated and studied for potential roles in disease. While several studies have tested plasma-derived EVs in AD, few have analyzed EVs from cerebrospinal fluid (CSF), which are potentially more closely related to brain changes. This study included 20 AD patients and 20 cognitively unimpaired (CU) participants. Using a novel EV isolation method based on acoustic trapping, we isolated and purified EVs from minimal CSF volumes. EVs were lysed and analyzed by immunoassays for P-tau217 and P-tau181. Isolation was confirmed through transmission electron microscopy and the presence of EV-specific markers (CD9, CD63, CD81, ATP1A3). Nanoparticle tracking analysis revealed a high variance in EV distribution. AD patients exhibited increased P-tau181 and decreased P-tau217 in EVs, leading to a higher EV P-tau181/P-tau217 ratio compared to CU. No significant differences in EV counts or sizes were observed between AD and CU groups. This study is the first to use acoustic trapping to isolate EVs from CSF and demonstrates differential P-tau content in AD-derived EVs, warranting further research to understand the relationship between these EV changes and brain pathology.

Identifiants

pubmed: 39455624
doi: 10.1038/s41598-024-75406-0
pii: 10.1038/s41598-024-75406-0
doi:

Substances chimiques

tau Proteins 0
Biomarkers 0

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

25419

Informations de copyright

© 2024. The Author(s).

Références

Möller, H. J., Graeber, M. The case described by Alois Alzheimer in 1911. Eur. Arch. Psychiatry Clin. Neurosci. 248, 111–122. https://doi.org/10.1007/s004060050027 (1998).
Gustavsson, A. et al. Global estimates on the number of persons across the Alzheimer's disease continuum. Alzheimer's Dement. 19, 658–670. https://doi.org/10.1002/alz.12694 (2023).
Mantzavinos, V. & Alexiou, A. Biomarkers for Alzheimer’s Disease diagnosis. Curr. Alzheimer Res. 14(11), 1149–1154 (2017).
Selkoe, D. J. Alzheimer’s disease is a synaptic failure. Sci. (1979). 298, 789–791 (2002).
Kalluri, R. & LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Sci. 367(6478), eaau6977 (2020).
Yates, A. G. et al. In sickness and in health: the functional role of extracellular vesicles in physiology and pathology in vivo. J. Extracell. Vesicles 11(1), e12151 (2022).
Bongiovanni, L., Andriessen, A., Wauben, M. H. M. & Hoen, E. N. M. N.-’t & de Bruin, A. Extracellular vesicles: Novel opportunities to understand and detect neoplastic diseases. Vet. Pathol. 58, 453–471 (2021).
pubmed: 33813952 pmcid: 8064535 doi: 10.1177/0300985821999328
Pitt, J. M., Kroemer, G. & Zitvogel, L. Extracellular vesicles: masters of intercellular communication and potential clinical interventions. J. Clin. Invest. 126, 1139–1143 (2016).
pubmed: 27035805 pmcid: 4811136 doi: 10.1172/JCI87316
Nieland, L., Mahjoum, S., Grandell, E., Breyne, K. & Breakefield, X. O. Engineered EVs designed to target diseases of the CNS. J. Controlled Release. 356, 493–506 (2023).
doi: 10.1016/j.jconrel.2023.03.009
Liang, T. et al. The emerging double-edged sword role of exosomes in Alzheimer’s disease. Front. Aging Neurosci. 15, 1209115 (2023).
Gabrielli, M., Tozzi, F., Verderio, C. & Origlia, N. Emerging roles of Extracellular vesicles in Alzheimer’s Disease: focus on synaptic dysfunction and vesicle–Neuron Interaction. Cells. 12, 63 (2022).
pubmed: 36611856 pmcid: 9818402 doi: 10.3390/cells12010063
Barthélemy, N. R. et al. Highly accurate blood test for Alzheimer’s disease is similar or superior to clinical cerebrospinal fluid tests. Nat. Med. 30, 1085–1095 (2024).
pubmed: 38382645 pmcid: 11031399 doi: 10.1038/s41591-024-02869-z
Asai, H. et al. Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat. Neurosci. 18, 1584–1593 (2015).
pubmed: 26436904 pmcid: 4694577 doi: 10.1038/nn.4132
Guix, F. et al. Detection of aggregation-competent tau in Neuron-Derived Extracellular vesicles. Int. J. Mol. Sci. 19, 663 (2018).
pubmed: 29495441 pmcid: 5877524 doi: 10.3390/ijms19030663
Lyu, C. et al. The Disease Associated Tau35 Fragment has an increased propensity to Aggregate compared to full-length tau. Front. Mol. Biosci. 8, 779240 (2021).
Gibbons, G. S., Lee, V. M. Y. & Trojanowski, J. Q. Mechanisms of cell-to-cell transmission of pathological tau. JAMA Neurol. 76, 101 (2019).
pubmed: 30193298 pmcid: 6382549 doi: 10.1001/jamaneurol.2018.2505
Saman, S. et al. Exosome-associated tau is secreted in Tauopathy models and is selectively phosphorylated in Cerebrospinal Fluid in Early Alzheimer Disease. J. Biol. Chem. 287, 3842–3849 (2012).
pubmed: 22057275 doi: 10.1074/jbc.M111.277061
Wang, Y. et al. The release and trans-synaptic transmission of tau via exosomes. Mol. Neurodegener. 12, 5 (2017).
pubmed: 28086931 pmcid: 5237256 doi: 10.1186/s13024-016-0143-y
Lee, S., Mankhong, S. & Kang, J. H. Extracellular vesicle as a source of Alzheimer’s biomarkers: opportunities and challenges. Int. J. Mol. Sci. 20, 1728 (2019).
pubmed: 30965555 pmcid: 6479979 doi: 10.3390/ijms20071728
Mitra, P., Gupta, S. & Sharma, P. Extracellular vesicles (EVs) as a window to the brain: potential, challenges and Future perspectives. Indian J. Clin. Biochem. 38, 1–3 (2023).
pubmed: 36684493 pmcid: 9852378 doi: 10.1007/s12291-023-01111-w
Muraoka, S. et al. Enrichment of Neurodegenerative Microglia Signature in brain-derived extracellular vesicles isolated from Alzheimer’s Disease Mouse models. J. Proteome Res. 20, 1733–1743 (2021).
pubmed: 33534581 pmcid: 7944570 doi: 10.1021/acs.jproteome.0c00934
Goetzl, E. J., Abner, E. L., Jicha, G. A., Kapogiannis, D. & Schwartz, J. B. Declining levels of functionally specialized synaptic proteins in plasma neuronal exosomes with progression of Alzheimer’s disease. FASEB J. 32, 888–893 (2018).
pubmed: 29025866 doi: 10.1096/fj.201700731R
Goetzl, E. J. et al. Low neural exosomal levels of cellular survival factors in Alzheimer’s disease. Ann. Clin. Transl Neurol. 2, 769–773 (2015).
pubmed: 26273689 pmcid: 4531059 doi: 10.1002/acn3.211
Jones, M. E. et al. A genetic variant of the wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer’s disease. Sci. Adv. 9(2), eabo7421 (2023).
Tang, Z. & Dai, C. Heat shock factor 1 is a direct anti-amyloid factor: connecting neurodegeneration and uncontrolled growth. Neural Regen Res. 17, 559 (2022).
pubmed: 34380889 doi: 10.4103/1673-5374.320983
Lu, T. et al. REST and stress resistance in ageing and Alzheimer’s disease. Nature. 507, 448–454 (2014).
pubmed: 24670762 pmcid: 4110979 doi: 10.1038/nature13163
Ruan, Z. Extracellular vesicles drive tau spreading in Alzheimer’s disease. Neural Regen Res. 17, 328 (2022).
pubmed: 34269203 doi: 10.4103/1673-5374.317975
Imanbekova, M. et al. Identification of amyloid beta in small extracellular vesicles via Raman spectroscopy. Nanoscale Adv. 3, 4119–4132 (2021).
pubmed: 34355118 pmcid: 8276787 doi: 10.1039/D1NA00330E
Ruan, Z. et al. Alzheimer’s disease brain-derived extracellular vesicles spread tau pathology in interneurons. Brain. 144, 288–309 (2021).
pubmed: 33246331 doi: 10.1093/brain/awaa376
Su, H. et al. Characterization of brain-derived extracellular vesicle lipids in Alzheimer’s disease. J. Extracell. Vesicles 10(7), e12089 (2021).
Hansson, O. Biomarkers for neurodegenerative diseases. Nat. Med. 27, 954–963 (2021).
pubmed: 34083813 doi: 10.1038/s41591-021-01382-x
Simon, M. J. & Iliff, J. J. Regulation of cerebrospinal fluid (CSF) flow in neurodegenerative, neurovascular and neuroinflammatory disease. Biochim. et Biophys. Acta (BBA) - Mol. Basis Disease. 1862, 442–451 (2016).
doi: 10.1016/j.bbadis.2015.10.014
Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles. 7, 1535750 (2018).
pubmed: 30637094 pmcid: 6322352 doi: 10.1080/20013078.2018.1535750
Ter-Ovanesyan, D. et al. Framework for rapid comparison of extracellular vesicle isolation methods. Elife 10, e70725 (2021).
Vacchi, E. et al. Profiling inflammatory extracellular vesicles in plasma and cerebrospinal fluid: an optimized diagnostic model for Parkinson’s Disease. Biomedicines. 9, 230 (2021).
pubmed: 33669043 pmcid: 7996605 doi: 10.3390/biomedicines9030230
Skalnikova et al. Isolation and characterization of small extracellular vesicles from Porcine Blood plasma, Cerebrospinal Fluid, and seminal plasma. Proteomes. 7, 17 (2019).
pubmed: 31027284 doi: 10.3390/proteomes7020017
Ku, A. et al. Acoustic Enrichment of Extracellular vesicles from Biological fluids. Anal. Chem. 90, 8011–8019 (2018).
pubmed: 29806448 pmcid: 7556308 doi: 10.1021/acs.analchem.8b00914
Gardiner, C. et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J. Extracell. Vesicles 5, 32945 (2016).
Bryl-Górecka, P. et al. Effect of exercise on the plasma vesicular proteome: a methodological study comparing acoustic trapping and centrifugation. Lab. Chip. 18, 3101–3111 (2018).
pubmed: 30178811 doi: 10.1039/C8LC00686E
Wang, Y. M. et al. Analysis of circulating non-coding RNAs in a non-invasive and cost-effective manner. TRAC Trends Anal. Chem. 117, 242–262 (2019).
doi: 10.1016/j.trac.2019.07.001
Havers, M., Broman, A., Lenshof, A. & Laurell, T. Advancement and obstacles in microfluidics-based isolation of extracellular vesicles. Anal. Bioanal Chem. 415, 1265–1285 (2023).
pubmed: 36284018 doi: 10.1007/s00216-022-04362-3
Macías, M. et al. Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis. Clin. Chem. Lab. Med. (CCLM). 57, 1539–1545 (2019).
pubmed: 30990781 doi: 10.1515/cclm-2018-1297
Broman, A. et al. Multinodal Acoustic Trapping enables high Capacity and High Throughput Enrichment of Extracellular vesicles and Microparticles in miRNA and MS Proteomics studies. Anal. Chem. 93, 3929–3937 (2021).
pubmed: 33592145 pmcid: 8023533 doi: 10.1021/acs.analchem.0c04772
Palm, F. et al. Phenotypic characterization of Acoustically Enriched Extracellular vesicles from Pathogen-activated platelets. J. Innate Immun. 15, 599–613 (2023).
pubmed: 37245510 pmcid: 10620552 doi: 10.1159/000531266
Gonzalez-Ortiz, F. et al. Plasma phospho-tau in Alzheimer’s disease: towards diagnostic and therapeutic trial applications. Mol. Neurodegener. 18, 18 (2023).
pubmed: 36927491 pmcid: 10022272 doi: 10.1186/s13024-023-00605-8
Thijssen, E. H. et al. Plasma phosphorylated tau 217 and phosphorylated tau 181 as biomarkers in Alzheimer’s disease and frontotemporal lobar degeneration: a retrospective diagnostic performance study. Lancet Neurol. 20, 739–752 (2021).
pubmed: 34418401 pmcid: 8711249 doi: 10.1016/S1474-4422(21)00214-3
Barthélemy, N. R. et al. A soluble phosphorylated tau signature links tau, amyloid and the evolution of stages of dominantly inherited Alzheimer’s disease. Nat. Med. 26, 398–407 (2020).
pubmed: 32161412 pmcid: 7309367 doi: 10.1038/s41591-020-0781-z
Mattsson-Carlgren, N. et al. Aβ deposition is associated with increases in soluble and phosphorylated tau that precede a positive tau PET in Alzheimer’s disease. Sci. Adv. 6(16), eaaz2387 (2020).
Havers, M., Baasch, T., Lenshof, A., Evander, M. & Laurell, T. Silica seed particles improve the efficiency and throughput of nanoparticle acoustic trapping. Phys. Rev. Appl. 21, 034016 (2024).
doi: 10.1103/PhysRevApplied.21.034016
Andreu, Z. & Yanez-Mo, M. Tetraspanins in extracellular vesicle formation and function. Front. Immunol. 5, 442 (2014).
Zhang, Z. et al. Comprehensive characterization of human brain-derived extracellular vesicles using multiple isolation methods: implications for diagnostic and therapeutic applications. J. Extracell. Vesicles 12(8), e12358 (2023).
Janelidze, S. et al. Detecting amyloid positivity in early Alzheimer’s disease using combinations of plasma Aβ42/Aβ40 and p-tau. Alzheimer’s Dement. 18, 283–293 (2022).
doi: 10.1002/alz.12395
Gobom, J. et al. Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid. Clin. Chem. Lab. Med. (CCLM). 60, 207–219 (2022).
pubmed: 34773730 doi: 10.1515/cclm-2021-0651
You, Y. et al. ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids. Sci. Adv. 9(37), eadi3647 (2023).
Ogaki, A., Ikegaya, Y. & Koyama, R. Extracellular vesicles taken up by astrocytes. Int. J. Mol. Sci. 22, 10553 (2021).
pubmed: 34638890 pmcid: 8508591 doi: 10.3390/ijms221910553
Janelidze, S. et al. Cerebrospinal fluid p-tau217 performs better than p-tau181 as a biomarker of Alzheimer’s disease. Nat. Commun. 11, 1683 (2020).
pubmed: 32246036 pmcid: 7125218 doi: 10.1038/s41467-020-15436-0
Janelidze, S. et al. Head-to-head comparison of 10 plasma phospho-tau assays in prodromal Alzheimer’s disease. Brain. 146, 1592–1601 (2023).
pubmed: 36087307 doi: 10.1093/brain/awac333
Mendes, A. J. et al. Head-to-head study of diagnostic accuracy of plasma and cerebrospinal fluid p-tau217 versus p-tau181 and p-tau231 in a memory clinic cohort. J. Neurol. 271, 2053–2066 (2024).
pubmed: 38195896 pmcid: 10972950 doi: 10.1007/s00415-023-12148-5
Yamada, K. et al. Neuronal activity regulates extracellular tau in vivo. J. Exp. Med. 211, 387–393 (2014).
pubmed: 24534188 pmcid: 3949564 doi: 10.1084/jem.20131685
Mudher, A. et al. What is the evidence that tau pathology spreads through prion-like propagation? Acta Neuropathol. Commun. 5, 99 (2017).
pubmed: 29258615 pmcid: 5735872 doi: 10.1186/s40478-017-0488-7
Dujardin, S. et al. Ectosomes: a new mechanism for non-exosomal secretion of Tau Protein. PLoS One. 9, e100760 (2014).
pubmed: 24971751 pmcid: 4074092 doi: 10.1371/journal.pone.0100760
Frost, B., Jacks, R. L. & Diamond, M. I. Propagation of tau misfolding from the outside to the Inside of a cell. J. Biol. Chem. 284, 12845–12852 (2009).
pubmed: 19282288 pmcid: 2676015 doi: 10.1074/jbc.M808759200
Guo, J. L. & Lee, V. M. Y. Seeding of normal tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles. J. Biol. Chem. 286, 15317–15331 (2011).
pubmed: 21372138 pmcid: 3083182 doi: 10.1074/jbc.M110.209296
Yamada, K. et al. In vivo Microdialysis reveals Age-Dependent decrease of Brain interstitial fluid tau levels in P301S Human Tau Transgenic mice. J. Neurosci. 31, 13110–13117 (2011).
pubmed: 21917794 pmcid: 4299126 doi: 10.1523/JNEUROSCI.2569-11.2011
Mulcahy, L. A., Pink, R. C. & Carter, D. R. F. Routes and mechanisms of extracellular vesicle uptake. J. Extracell. Vesicles 3, 10.3402/jev.v3.24641 (2014).
Montecalvo, A. et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood. 119, 756–766 (2012).
pubmed: 22031862 pmcid: 3265200 doi: 10.1182/blood-2011-02-338004
Prada, I. & Meldolesi, J. Binding and Fusion of Extracellular vesicles to the plasma membrane of their cell targets. Int. J. Mol. Sci. 17, 1296 (2016).
pubmed: 27517914 pmcid: 5000693 doi: 10.3390/ijms17081296
Esmaeili, A., Alini, M., Baghaban Eslaminejad, M. & Hosseini, S. Engineering strategies for customizing extracellular vesicle uptake in a therapeutic context. Stem Cell Res Ther 13, 129 Saman, S. et al. Exosome-associated Tau Is Secreted in Tauopathy Models and Is Selectively Phosphorylated in Cerebrospinal Fluid in Early Alzheimer Disease. Journal of Biological Chemistry 287, 3842–3849 (2012). (2022).
Longobardi, A. et al. Cerebrospinal Fluid EV concentration and size are altered in Alzheimer’s Disease and Dementia with Lewy Bodies. Cells. 11, 462 (2022).
pubmed: 35159272 pmcid: 8834088 doi: 10.3390/cells11030462
Gerritzen, M. J. H., Martens, D. E., Wijffels, R. H. & Stork, M. High throughput nanoparticle tracking analysis for monitoring outer membrane vesicle production. J. Extracell. Vesicles 6(1), 1333883(2017).
Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell. Biol. 20, 332–343 (2018).
pubmed: 29459780 pmcid: 5931706 doi: 10.1038/s41556-018-0040-4
Comfort, N. et al. Isolation and characterization of extracellular vesicles in saliva of children with asthma. Extracell. Vesicles Circ. Nucl. Acids. https://doi.org/10.20517/evcna.2020.09 (2021).
doi: 10.20517/evcna.2020.09 pubmed: 34368811 pmcid: 8340923
Kugeratski, F. G. et al. Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker. Nat. Cell. Biol. 23, 631–641 (2021).
pubmed: 34108659 pmcid: 9290189 doi: 10.1038/s41556-021-00693-y
Okada-Tsuchioka, M. et al. Tetraspanin heterogeneity of small extracellular vesicles in human biofluids and brain tissue. Biochem. Biophys. Res. Commun. 627, 146–151 (2022).
pubmed: 36037746 doi: 10.1016/j.bbrc.2022.08.025
Wang, P. et al. α-Synuclein-carrying astrocytic extracellular vesicles in Parkinson pathogenesis and diagnosis. Transl Neurodegener. 12, 40 (2023).
pubmed: 37620916 pmcid: 10463943 doi: 10.1186/s40035-023-00372-y
Ahmad, S., Srivastava, R. K., Singh, P., Naik, U. P. & Srivastava, A. K. Role of Extracellular vesicles in Glia-Neuron Intercellular Communication. Front. Mol. Neurosci. 15, 844194 (2022).
Smith, A. M. et al. Diverse human astrocyte and microglial transcriptional responses to Alzheimer’s pathology. Acta Neuropathol. 143, 75–91 (2022).
pubmed: 34767070 doi: 10.1007/s00401-021-02372-6
Li, T., Tan, X., Li, S., Al-Nusaif, M. & Le, W. Role of glia-derived extracellular vesicles in neurodegenerative diseases. Front. Aging Neurosci. 13, 765395 (2021).
Agosta, F. et al. Myeloid microvesicles in cerebrospinal fluid are associated with myelin damage and neuronal loss in mild cognitive impairment and < scp > A lzheimer disease. Ann. Neurol. 76, 813–825 (2014).
pubmed: 25087695 doi: 10.1002/ana.24235
Clayton, K. et al. Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model. Mol. Neurodegener. 16, 18 (2021).
pubmed: 33752701 pmcid: 7986521 doi: 10.1186/s13024-021-00440-9
Gabrielli, M., Raffaele, S., Fumagalli, M. & Verderio, C. The multiple faces of extracellular vesicles released by microglia: where are we 10 years after? Front. Cell. Neurosci. 16, 984690 (2022).
van Hezel, M. E., Nieuwland, R., van Bruggen, R. & Juffermans, N. P. The ability of Extracellular vesicles to induce a pro-inflammatory host response. Int. J. Mol. Sci. 18, 1285 (2017).
pubmed: 28621719 pmcid: 5486107 doi: 10.3390/ijms18061285
Osteikoetxea, X. et al. Differential detergent sensitivity of extracellular vesicle subpopulations. Org. Biomol. Chem. 13, 9775–9782 (2015).
pubmed: 26264754 doi: 10.1039/C5OB01451D

Auteurs

Roman Sattarov (R)

Clinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden. Roman.Sattarov@med.lu.se.

Megan Havers (M)

Department of Biomedical Engineering, Lund University, Lund, Sweden. Megan.Havers@bme.lth.se.

Camilla Orbjörn (C)

Clinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden.

Erik Stomrud (E)

Clinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden.
Memory Clinic, Skåne University Hospital, Malmö, Sweden.
Wallenberg Center for Molecular Medicine, Lund University, Lund, Sweden.

Shorena Janelidze (S)

Clinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden.
Wallenberg Center for Molecular Medicine, Lund University, Lund, Sweden.

Thomas Laurell (T)

Department of Biomedical Engineering, Lund University, Lund, Sweden.

Niklas Mattsson-Carlgren (N)

Clinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Lund, Sweden. Niklas.Mattsson-Carlgren@med.lu.se.
Wallenberg Center for Molecular Medicine, Lund University, Lund, Sweden. Niklas.Mattsson-Carlgren@med.lu.se.
Department of Neurology, Skåne University Hospital, Lund, Sweden. Niklas.Mattsson-Carlgren@med.lu.se.

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