{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T22:31:41Z","timestamp":1760481101495,"version":"build-2065373602"},"reference-count":54,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2020,6,30]],"date-time":"2020-06-30T00:00:00Z","timestamp":1593475200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Health Director's New Innovator Award","award":["DP2GM119131"],"award-info":[{"award-number":["DP2GM119131"]}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["CA031845"],"award-info":[{"award-number":["CA031845"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["DBI-1553577"],"award-info":[{"award-number":["DBI-1553577"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001082","name":"Camille and Henry Dreyfus Foundation","doi-asserted-by":"publisher","award":["Teacher-Scholar Award"],"award-info":[{"award-number":["Teacher-Scholar Award"]}],"id":[{"id":"10.13039\/100001082","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP\u2212 cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.<\/jats:p>","DOI":"10.3390\/ijms21134649","type":"journal-article","created":{"date-parts":[[2020,6,30]],"date-time":"2020-06-30T09:36:04Z","timestamp":1593509764000},"page":"4649","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9906-2791","authenticated-orcid":false,"given":"Sarah A.","family":"Overall","sequence":"first","affiliation":[{"name":"Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lauren E.","family":"Price","sequence":"additional","affiliation":[{"name":"Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brice J.","family":"Albert","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chukun","family":"Gao","sequence":"additional","affiliation":[{"name":"Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicholas","family":"Alaniva","sequence":"additional","affiliation":[{"name":"Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland"},{"name":"Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick T.","family":"Judge","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA"},{"name":"Department of Biochemistry, Biophysics and Structural Biology, Washington University in St. Louis, St. Louis, MO 63110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erika L.","family":"Sesti","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul A.","family":"Wender","sequence":"additional","affiliation":[{"name":"Departments of Chemistry and Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8678-4656","authenticated-orcid":false,"given":"George B.","family":"Kyei","sequence":"additional","affiliation":[{"name":"Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA"},{"name":"Noguchi Memorial Institute for Medical Research, University of Ghana, Accra 00233, Ghana"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander B.","family":"Barnes","sequence":"additional","affiliation":[{"name":"Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1021\/acscentsci.7b00475","article-title":"REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment","volume":"4","author":"Yang","year":"2018","journal-title":"ACS Central Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1038\/nsmb.3194","article-title":"Solid-state NMR structure of a pathogenic fibril of full-length human \u03b1-synuclein","volume":"23","author":"Tuttle","year":"2016","journal-title":"Nat. Struct. Mol. Boil."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"16742","DOI":"10.1073\/pnas.262663499","article-title":"A structural model for Alzheimer\u2019s \u03b2-amyloid fibrils based on experimental constraints from solid state NMR","volume":"99","author":"Petkova","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1546","DOI":"10.1073\/pnas.1521933113","article-title":"Huntingtin exon 1 fibrils feature an interdigitated \u03b2-hairpin\u2013based polyglutamine core","volume":"113","author":"Hoop","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2747","DOI":"10.1038\/s41467-018-05199-0","article-title":"Molecular architecture of fungal cell walls revealed by solid-state NMR","volume":"9","author":"Kang","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.ssnmr.2016.08.001","article-title":"Multidimensional solid-state NMR spectroscopy of plant cell walls","volume":"78","author":"Wang","year":"2016","journal-title":"Solid State Nucl. Magn. Reson."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1976","DOI":"10.1021\/ja908687k","article-title":"Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies","volume":"132","author":"Han","year":"2010","journal-title":"J. Am. Chem. Soc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"941","DOI":"10.1016\/j.bpj.2014.05.048","article-title":"Insights into the Structure and Dynamics of Measles Virus Nucleocapsids by 1H-detected Solid-state NMR","volume":"107","author":"Felletti","year":"2014","journal-title":"Biophys. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1016\/j.jmb.2013.11.010","article-title":"Solid-state NMR spectroscopy of the HIV gp41 membrane fusion protein supports intermolecular antiparallel \u03b2 sheet fusion peptide structure in the final six-helix bundle state","volume":"426","author":"Sackett","year":"2013","journal-title":"J. Mol. Boil."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"10926","DOI":"10.1073\/pnas.1501430112","article-title":"Viral fusion protein transmembrane domain adopts \u03b2-strand structure to facilitate membrane topological changes for virus\u2013cell fusion","volume":"112","author":"Yao","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.jsb.2018.05.007","article-title":"Spectral comparisons of mammalian cells and intact organelles by solid-state NMR","volume":"206","author":"Werby","year":"2018","journal-title":"J. Struct. Boil."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1038\/nature07839","article-title":"High-resolution multi-dimensional NMR spectroscopy of proteins in human cells","volume":"458","author":"Inomata","year":"2009","journal-title":"Nature"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/nature07814","article-title":"Protein structure determination in living cells by in-cell NMR spectroscopy","volume":"458","author":"Sakakibara","year":"2009","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"11904","DOI":"10.1073\/pnas.0604667103","article-title":"Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes","volume":"103","author":"Selenko","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nature16531","article-title":"Structural disorder of monomeric \u03b1-synuclein persists in mammalian cells","volume":"530","author":"Theillet","year":"2016","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Poulhazan, A., Arnold, A.A., Warschawski, D., and Marcotte, I. (2018). Unambiguous Ex Situ and in Cell 2D 13C Solid-State NMR Characterization of Starch and Its Constituents. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19123817"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15711","DOI":"10.1021\/ja5088453","article-title":"Amplifying Dynamic Nuclear Polarization of Frozen Solutions by Incorporating Dielectric Particles","volume":"136","author":"Kubicki","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ssnmr.2018.12.003","article-title":"Hyperpolarized MAS NMR of unfolded and misfolded proteins","volume":"98","author":"Uluca","year":"2019","journal-title":"Solid State Nucl. Magn. Reson."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1021\/acs.jpcb.5b12134","article-title":"Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies","volume":"120","author":"Gupta","year":"2016","journal-title":"J. Phys. Chem. B"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1038\/nchembio.2551","article-title":"The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors","volume":"14","author":"Joedicke","year":"2018","journal-title":"Nat. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1542","DOI":"10.1002\/anie.201410249","article-title":"A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins","volume":"54","author":"Smith","year":"2014","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1007\/s10858-015-9919-6","article-title":"Dynamic nuclear polarization of membrane proteins: Covalently bound spin-labels at protein-protein interfaces","volume":"61","author":"Wylie","year":"2015","journal-title":"J. Biomol. NMR"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4508","DOI":"10.1002\/anie.201008244","article-title":"Proton-Detected Solid-State NMR Spectroscopy of Fibrillar and Membrane Proteins","volume":"50","author":"Linser","year":"2011","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4741","DOI":"10.1021\/acs.biochem.8b00257","article-title":"Dynamic Nuclear Polarization NMR in Human Cells Using Fluorescent Polarizing Agents","volume":"57","author":"Albert","year":"2019","journal-title":"Biochemistry"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"10858","DOI":"10.1002\/anie.201304657","article-title":"Highly Efficient, Water-Soluble Polarizing Agents for Dynamic Nuclear Polarization at High Frequency","volume":"52","author":"Sauvee","year":"2013","journal-title":"Angew. Chemie Int. Ed."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"11021","DOI":"10.1096\/fj.201900931R","article-title":"Nitroxide spin\u2013labeled peptides for DNP-NMR in-cell studies","volume":"33","author":"Sani","year":"2019","journal-title":"FASEB J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2474","DOI":"10.1002\/cbic.201900297","article-title":"Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy","volume":"20","author":"Schlagnitweit","year":"2019","journal-title":"ChemBioChem"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"12969","DOI":"10.1002\/anie.201903246","article-title":"DNP-Supported Solid-State NMR Spectroscopy of Proteins Inside Mammalian Cells","volume":"58","author":"Narasimhan","year":"2019","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1016\/j.bbamem.2014.07.008","article-title":"Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization","volume":"1848","author":"Yamamoto","year":"2014","journal-title":"Biochim. et Biophys. Acta (BBA) - Bioenerg."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1126\/science.278.5341.1295","article-title":"Identification of a Reservoir for HIV-1 in Patients on Highly Active Antiretroviral Therapy","volume":"278","author":"Finzi","year":"1997","journal-title":"Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1126\/science.278.5341.1291","article-title":"Recovery of Replication-Competent HIV Despite Prolonged Suppression of Plasma Viremia","volume":"278","author":"Wong","year":"1997","journal-title":"Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1038\/387183a0","article-title":"Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection","volume":"387","author":"Chun","year":"1997","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1056\/NEJM199709113371102","article-title":"Treatment with Indinavir, Zidovudine, and Lamivudine in Adults with Human Immunodeficiency Virus Infection and Prior Antiretroviral Therapy","volume":"337","author":"Gulick","year":"1997","journal-title":"New Engl. J. Med."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1056\/NEJM199709113371101","article-title":"A Controlled Trial of Two Nucleoside Analogues plus Indinavir in Persons with Human Immunodeficiency Virus Infection and CD4 Cell Counts of 200 per Cubic Millimeter or Less","volume":"337","author":"Hammer","year":"1997","journal-title":"New Engl. J. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1016\/j.drudis.2012.12.008","article-title":"Targeting HIV latency: Pharmacologic strategies toward eradication","volume":"18","author":"Xing","year":"2012","journal-title":"Drug Discov. Today"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.virol.2018.05.006","article-title":"Characterization of designed, synthetically accessible bryostatin analog HIV latency reversing agents","volume":"520","author":"Marsden","year":"2018","journal-title":"Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1038\/nchem.1395","article-title":"Designed, synthetically accessible bryostatin analogues potently induce activation of latent HIV reservoirs in vitro","volume":"4","author":"DeChristopher","year":"2012","journal-title":"Nat. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"7456","DOI":"10.1038\/s41598-017-07814-4","article-title":"Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation","volume":"7","author":"Albert","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"466","DOI":"10.2119\/molmed.2011.00076","article-title":"Histone Deacetylase Inhibitors for Purging HIV-1 from the Latent Reservoir","volume":"17","author":"Matalon","year":"2011","journal-title":"Mol. Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1038\/nature11286","article-title":"Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy","volume":"487","author":"Archin","year":"2012","journal-title":"Nature"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3678","DOI":"10.1021\/jacs.5b00886","article-title":"Toward a Biorelevant Structure of Protein Kinase C Bound Modulators: Design, Synthesis, and Evaluation of Labeled Bryostatin Analogues for Analysis with Rotational Echo Double Resonance NMR Spectroscopy","volume":"137","author":"Loy","year":"2015","journal-title":"J. Am. Chem. Soc."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1868","DOI":"10.1093\/emboj\/cdg188","article-title":"HIV reproducibly establishes a latent infection after acute infection of T cells in vitro","volume":"22","author":"Jordan","year":"2003","journal-title":"EMBO J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1726","DOI":"10.1093\/emboj\/20.7.1726","article-title":"The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation","volume":"20","author":"Jordan","year":"2001","journal-title":"EMBO J."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"42008","DOI":"10.1074\/jbc.M402124200","article-title":"Prostratin antagonizes HIV latency by activating NF-kB","volume":"279","author":"William","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"5974","DOI":"10.1073\/pnas.86.15.5974","article-title":"Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat","volume":"86","author":"Duh","year":"1989","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.jmr.2019.04.013","article-title":"Stability of nitroxide biradical TOTAPOL in biological samples","volume":"303","author":"McCoy","year":"2019","journal-title":"J. Magn. Reson."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5070","DOI":"10.1002\/anie.201100886","article-title":"Long-Range Distance Measurements on Nucleic Acids in Cells by Pulsed EPR Spectroscopy","volume":"50","author":"Romainczyk","year":"2011","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"8228","DOI":"10.1021\/ja906104e","article-title":"Distance Determination in Proteins inside Xenopus laevis Oocytes by Double Electron\u2212Electron Resonance Experiments","volume":"132","author":"Igarashi","year":"2010","journal-title":"J. Am. Chem. Soc."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1089\/aid.1997.13.1077","article-title":"Use of a Green Fluorescent Protein as a Marker for Human Immunodeficiency Virus Type 1 Infection","volume":"13","author":"Kathleen","year":"1997","journal-title":"AIDS Res. Hum. Retrovir."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"280","DOI":"10.3389\/fmicb.2011.00280","article-title":"Fluorescent Reporter Signals, EGFP, and DsRed, Encoded in HIV-1 Facilitate the Detection of Productively Infected Cells and Cell-Associated Viral Replication Levels","volume":"2","author":"Terahara","year":"2012","journal-title":"Front. Microbiol."},{"key":"ref_51","first-page":"591","article-title":"The role of TNF-alpha in T-cell-mediated inflammation depends on the Th1\/Th2 cytokine balance","volume":"82","author":"Rook","year":"1994","journal-title":"Immunology"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/j.tim.2015.01.013","article-title":"Towards an HIV-1 cure: Measuring the latent reservoir","volume":"23","author":"Bruner","year":"2015","journal-title":"Trends Microbiol."},{"key":"ref_53","unstructured":"(2020, June 29). RPMI 1640. Available online: https:\/\/www.thermofisher.com\/ch\/en\/home\/technical-resources\/media-formulation.114.html."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1002\/mrc.984","article-title":"Modelling one- and two-dimensional solid-state NMR spectra","volume":"40","author":"Massiot","year":"2001","journal-title":"Magn. Reson. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/13\/4649\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:45:03Z","timestamp":1760175903000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/13\/4649"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,30]]},"references-count":54,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2020,7]]}},"alternative-id":["ijms21134649"],"URL":"https:\/\/doi.org\/10.3390\/ijms21134649","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2020,6,30]]}}}