Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells

Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells Itallia V. Pacentine, Peter G. Barr-Gillespie This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-913374/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. Co-application with vanadate (VO43-) enhanced the tip labeling, which is consistent with myosin isoforms being responsible; by contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+ stereocilia of the same length range. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ. Scientific Communication Health Economics & Outcomes Research Cy3-ATP Labeling Permeabilized Hair Cells ATP-utilizing enzymes stereocilia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 07 Nov, 2021 Reviews received at journal 21 Oct, 2021 Reviewers agreed at journal 06 Oct, 2021 Reviewers agreed at journal 03 Oct, 2021 Reviewers invited by journal 30 Sep, 2021 Editor assigned by journal 24 Sep, 2021 Editor invited by journal 24 Sep, 2021 Submission checks completed at journal 24 Sep, 2021 First submitted to journal 16 Sep, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-913374","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":53977733,"identity":"0eb6e007-88b3-4ea5-a29c-b95afcab9de7","order_by":0,"name":"Itallia V. Pacentine","email":"","orcid":"","institution":"Oregon Health \u0026 Science University","correspondingAuthor":false,"prefix":"","firstName":"Itallia","middleName":"V.","lastName":"Pacentine","suffix":""},{"id":53977734,"identity":"784e02ed-f598-46d5-9c07-26da3f020238","order_by":1,"name":"Peter G. Barr-Gillespie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDACZubGAyCan4EHzGdsIKyFsQGsRbKBaC0MUC0GB4jVIu8O1PLhz5084+O9hz/zMNjIbjhAQIvhYcaGgzPbnhWbnTmXJs3DkGZMWEszY8Nh3obDidtu5Jgx8zAcTiROy58/hxM3z8gxBjrsP2Et8sAQO8zABjRcIscA6LADhLUYALUc7AX6ReLMGTPJOQbJxjMJ2tJ/+OCDH8AQ42/vMf7wpsJOto+gLRAFBxKgXALKwbY0oGgZBaNgFIyCUYAFAAB9wkxAD5qS+QAAAABJRU5ErkJggg==","orcid":"","institution":"Oregon Health \u0026 Science University","correspondingAuthor":true,"prefix":"","firstName":"Peter","middleName":"G.","lastName":"Barr-Gillespie","suffix":""}],"badges":[],"createdAt":"2021-09-16 19:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-913374/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-913374/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14024267,"identity":"617d3df4-15a7-4acf-87ed-7ea847892e4f","added_by":"auto","created_at":"2021-09-27 15:35:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209794,"visible":true,"origin":"","legend":"Experimental materials and apparatus. (a) We expect that the hydrolysis of Cy3-ATP\nproceeds according to this scheme, which is thought to hold for most myosin isoforms. In this scheme, \nactin is represented by A and myosin by M. Nucleoside triphosphates such as Cy3-ATP bind to [[1]] and \ndissociate [2] the actomyosin complex. After hydrolysis of the nucleotide [4], actin and myosin reassociate \n[5]. A fraction of the nucleotide is hydrolyzed without dissociating the actomyosin complex [3]. The \nhydrolysis products, inorganic phosphate (Pi) and Cy3-ADP, are released sequentially [6] and [9]. \nBetween the release of Pi and that of Cy3-ADP, the myosin proceeds through a power stroke and a \nkinetically irreversible step [8]. Vanadate can bind before Cy3-ADP release; the slow dissociation of \nvanadate will trap Cy3-ADP on myosin for a prolonged time [7]. Although specific rate constants may \ndiffer, this mechanism is thought to hold for all myosin isoforms [32]. (b) Perfusion chamber. (c) Perfusion \nchamber mounted on microscope, with a tissue paper wicking liquid out of the chamber on one side and \ndrawing liquid (blue) into the chamber following the direction of the arrow","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1/1fb0f1ce74be012c9f8d2a79.jpg"},{"id":14024755,"identity":"fa0eb548-b4e9-4381-b7b3-c12e18336a05","added_by":"auto","created_at":"2021-09-27 15:38:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1380678,"visible":true,"origin":"","legend":"Cy3-ATP labels the tips of cochlear stereocilia. (a) IHCs labeled with Cy3-ATP at P3.5. Z-projections using maximum intensity. Increasing the image gain (right) shows that shorter stereocilia also \nhave signal at their tips (yellow arrowheads). (b) IHCs labeled at P6.5. The top two examples are single \nsection images; the bottom two examples are z-projections using maximum intensity. (c) IHCs labeled at \nP8.5. The top left example is a single section image; the other three examples are z-projections using \nmaximum intensity. (d) IHCs at P14.5; z-projections at maximum intensity. (e) IHCs at P9.5; z-projections \nusing maximum intensity. (f) Cochlear tissue at P9.5 labeled with phalloidin alone; z-projections using \nmaximum intensity. The single row of inner hair cells (white arrowhead) shifted away from the three rows \nof outer hair cells (yellow arrowhead) in one region. (g-h) IHCs at P6 or P9.5; z-projections using \nmaximum intensity. (i) OHCs labeled at P9.5; z-projections using maximum intensity. (j) IHCs labeled at \nP7.5; z-projections using maximum intensity. Left images are before exposure to ADP, middle images \nare during exposure to 500 μM ADP, and right-images are after ADP has been washed out. Panel full \nheights: a, 9 μm (left) and 3.5 μm (right); b-c, 6.7 μm; d, 9.3 μm; e, 40 μm; f, 399 μm; g-i, 9.3 μm; j, \n7.0 μm. Panel a is from a lattice-SIM processed images; all others are Airyscan. Actin was labeled with \nphalloidin 488. All images except f were taken during exposure to 5 μM Cy3-ATP in C57B/6 mice.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1/4d225d39250b9e586bc4cbc7.png"},{"id":14023877,"identity":"574b87d2-2b86-44f9-bffb-6eb0248a2273","added_by":"auto","created_at":"2021-09-27 15:32:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":518446,"visible":true,"origin":"","legend":"Cy3-ATP tip signal is sensitive to vanadate trapping and is not binding to actin. Actin was \nstained with phalloidin-. All images are Airyscan in C57B/6 mice. (a) A single inner hair cell during a \ntime-lapse experiment, z-projections with averaged intensity. (b) Images of the same hair cell during a \nsecond time-lapse experiment performed 5 minutes after the experiment in (a), with 1 mM vanadate in \nthe initial solution. (c) Graphs of fluorescence signal at the tips of stereocilia from the cell in b and c, n=7 \nstereocilia at 12 time points for two conditions (with or without vanadate). The full experiment described \nin b and c was repeated with similar results (N=2 mice). Statistical significance was determined by \nmultiple t-tests with a two-stage step-up method [46]; Q=1%. *p\u003c0.001 **p\u003c0.0001. (d) Single section \nimages of two inner hair cells during an exposure and wash regiment. The orange bar represents 12 \nminutes of exposure to a co-application of 20 μM latrunculin A and 20 μM cytochalasin D. The black bar \nrepresents 8 minutes of wash with intracellular solution (1 min to perform wash, 5 min wait for cells to \nclear, 2 min with concurrent exposure to Cy3-ATP). The blue bar represents 2 min of exposure to 5 μM \nCy3-ATP. N=6 mice. Scale: All panel heights are 8.9 μm.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1/374cfad3e70ac9c496513aef.jpg"},{"id":14023882,"identity":"359bbc7c-7b69-4351-86d8-34c75112f4b5","added_by":"auto","created_at":"2021-09-27 15:32:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":627442,"visible":true,"origin":"","legend":"Cy3-ATP labeling in isolated utricle stereocilia. (a-j) Examples of isolated stereocilia from \nutricles of P9.5-P11.5 C57B/6 mice using Airyscan imaging. a and b show low- and high-gain images of \na single field from P9.5 utricle. Yellow arrowheads indicate weak Cy3-ATP signal at stereocilia taper \nregions. Yellow arrowheads in j indicate probable kinocilia. Panel full heights: a-b and f-g, 14 μm; c, \n12 μm; d-e, 5.8 μm; h, 3.4 μm; i, 8.3 μm; j, 13.6 μm.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1/57f8de4d86ad1c6a406b3f0b.jpg"},{"id":14023878,"identity":"9c7cbfb4-763a-40ef-b032-109b2c7089c0","added_by":"auto","created_at":"2021-09-27 15:32:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":838620,"visible":true,"origin":"","legend":"Cy3-ATP signal is significantly reduced in Myo15ash2/sh2 mice, but not Myo7a 8J/8J mice. (a-d) Maximum intensity z-projections of cochlear hair cells. (a) IHCs from Myo15ash2 /+ mice. (b) IHCs from \nMyo15ash2/sh2 mice. (c) IHCs from Myo7a8J/+ mice. (d) IHCs from Myo7a 8J/8J mice. (e) Stereocilia isolated \nfrom Myo15ash2/+ mouse utricles. (f) Stereocilia isolated from Myo15a sh2/sh2 mouse utricles. (g)-(i) \nQuantitation of utricle stereocilia from 5 Myo15ash2/+ animals and 4 Myo15ash2/sh2 animals. (g) Number \nof stereocilia binned by length of stereocilium (1 μm bins); single Gaussian fits. Myo15ash2/+: R2 = 0.8694, \nMyo15ash2/sh2: R2 = 0.9956. (h) Tip signal dependence on length for individual stereocilia. Y-axis is \nlogarithmic; all negative values were converted to 0.1 (35 converted to 0.1 of n=300 for Myo15ash2 /+; 99 \nconverted of n=450 for Myo15ash2/sh2). Data were fit with linear regression: Myo15ash2/+, 21x + 27 (R2 = \n0.1754); Myo15ash2/sh2, 12x + 11 (R2 = 0.0284). (i) Tip signal divided by length (mean ± SEM). Statistical \nsignificance determined by multiple t-tests with a two-stage step-up method [46]; Q=1%. **p\u003c0.0001. \nNegative values were converted to zeros (Overall: 35 converted of n=298 for Myo15ash2 /+, 99 converted \nof n=450 for Myo15ash2/sh2; Overall \u003c4 μm: 25 converted of n=143 total Myo15ash2 /+, 99 converted of \nn=450 for Myo15ash2/sh2; Overall \u003c4 μm n\u003e450: 98 converted of n=530 for Myo15ash2 /+, 99 converted of \nn=450 for Myo15ash2/sh2). (j) Stereocilia isolated from Myo7a8J /+ mouse utricles. (k) Stereocilia isolated \nfrom Myo7a8J/8J mouse utricles. (l)-(n) Quantitation of utricle stereocilia from 6 Myo7a8J /+ animals and \n6 Myo7a8J/8J animals. (l) Number of stereocilia binned by length of stereocilium (1 μm bins); single \nGaussian fits. Myo7a8J/+ data were fit with a single Gaussian, R2 = 0.9274; data for Myo7a8J/8J were fit \nwith a sum of two Gaussians. (m) Tip signal dependence on length for individual stereocilia. Y-axis is \nlogarithmic, negative values were converted to 0.1 (n=22 converted of n=400 total Myo7a8J /+, n=36 \nconverted of n=400 total Myo7a8J/8J). Data were fit with linear regression: Myo7a8J /+: 41x – 62 \n(R2 = 0.3549); Myo7a8J/8J: 34x - 26 (R2 = 0.2981). (n) Tip signal divided by length (mean ± SEM). \nStatistical significance determined as in (H); ns, not significant. Negative values were converted to zeros \n(Overall: 22 converted of n=400 for Myo7a8J/+, 36 converted of n=400 for Myo7a8J/8J ; Overall \u003e1 μm: 20 \nconverted of n=386 for Myo7a8J/+, 33 converted of n=387 for Myo7a8J/8J ). Panel full heights: e-f and j-k, \n10.1 μm","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1/35eb7c89595466fd8fc51421.png"},{"id":14024932,"identity":"bc4b8b7c-e5f6-4e10-b614-bf443aba0f4c","added_by":"auto","created_at":"2021-09-27 15:41:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":528373,"visible":true,"origin":"","legend":"Both MYO3A and MYO7A are normally localized in Myo15a sh2/sh2 mice. All images are \nAiryscan and are single sections unless stated. (a-d) IHCs from Myo15ash2/+ (a) and Myo15ash2/sh2 (b) \ncochleas labeled with anti-MYO3A. Similar results were seen in 1 Myo15ash2 /+ animals and 3 \nMyo15ash2/sh2 animals (applies to c-d too). (c-d) Vestibular hair cells from Myo15ash2 /+ (c) and \nMyo15ash2/sh2 (d) utricles labeled with anti-MYO3A. Viewed in profile using x-z reslice, except for top \npanels in d. (e-f) IHCs from Myo15ash2/+ (e) and Myo15ash2/sh2 (f) cochleas labeled with anti-MYO7A. \nMYO3A. Similar results were seen in 4 Myo15ash2/+ animals and 4 Myo15ash2/sh2 animals (applies to g-h \ntoo). (g-h) Vestibular hair cells from Myo15ash2/+ (g) and Myo15ash2/sh2 (h) utricles labeled with anti-MYO7A. Viewed in profile using x-z reslice. Panel full heights: a-b and e-f, 23 μm; c-d and g-h, 7.8 μm.","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1/63592b374d35579d573f96bf.jpg"},{"id":14024946,"identity":"11ab015a-7266-4f45-b252-19a6852ec5c3","added_by":"auto","created_at":"2021-09-27 15:41:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":933070,"visible":true,"origin":"","legend":"","description":"","filename":"Cy3ATP20210921a.pdf","url":"https://assets-eu.researchsquare.com/files/rs-913374/v1_covered.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCy3-ATP Labeling of Unfixed, Permeabilized Hair Cells\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"This preprint is available for \u003ca href='/article/rs-913374/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cy3-ATP Labeling , Permeabilized Hair Cells, ATP-utilizing enzymes, stereocilia","lastPublishedDoi":"10.21203/rs.3.rs-913374/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-913374/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. Co-application with vanadate (VO43-) enhanced the tip labeling, which is consistent with myosin isoforms being responsible; by contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+ stereocilia of the same length range. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.","manuscriptTitle":"Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-27 15:32:34","doi":"10.21203/rs.3.rs-913374/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-11-08T04:03:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-10-21T17:01:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"015d2b99-7409-44e0-b70e-17f91eb8384b","date":"2021-10-06T04:47:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c5052af9-72cb-48f7-969a-ebf85a537409","date":"2021-10-04T00:05:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-09-30T10:36:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-09-24T09:48:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-09-24T08:22:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-09-24T08:18:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-09-16T19:57:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"553da926-59cb-434c-87de-7282707677d7","owner":[],"postedDate":"September 27th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":7463370,"name":"Scientific Communication"},{"id":7463371,"name":"Health Economics \u0026 Outcomes Research"}],"tags":[],"updatedAt":"2021-12-01T11:44:16+00:00","versionOfRecord":[],"versionCreatedAt":"2021-09-27 15:32:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-913374","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-913374","identity":"rs-913374","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}