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Research
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Partners and Sponsors

Felipe opazo

Publications of Felipe Opazo

2024

2023

2022

2021

2020

2019

2018

2017

2016

2015

2014

2012

2010

2009

2008

2004

2024

Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.

Markusson S, Raasakka A, Schröder M, Sograte-Idrissi S, Rahimi AM, Asadpour O, Körner H, Lodygin D, Eichel-Vogel MA, Chowdhury R, Sutinen A, Muruganandam G, Iyer M, Cooper MH, Weigel MK, Ambiel N, Werner HB, Zuchero JB, Opazo F, Kursula P.

bioRxiv [Preprint]. 2024 Jun 1:2024.05.25.595513. doi: 10.1101/2024.05.25.595513.

Spatial proteomics in neurons at single-protein resolution

Unterauer EM, Shetab Boushehri S, Jevdokimenko K, Masullo LA, Ganji M, Sograte-Idrissi S, Kowalewski R, Strauss S, Reinhardt SCM, Perovic A, Marr C, Opazo F, Fornasiero EF, Jungmann R.

Cell. 2024 Mar 28;187(7):1785-1800.e16. doi: 10.1016/j.cell.2024.02.045.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

Alsouri S, Ambrose A, Mougios N, Paglilla N, Mayr F, Choi K, Loeber J, Chapuy B, Haeupl B, Opazo F, Oellerich T, Gold M, Engelke M.

Eur J Immunol. 2024 Mar;54(3):e2350774. doi: 10.1002/eji.202350774. Epub 2024 Feb 1.

Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations.

DʼEste E, Lukinavičius G, Lincoln R, Opazo F, Fornasiero EF.

Trends Cell Biol. 2024 Jan 5:S0962-8924(23)00239-8. doi: 10.1016/j.tcb.2023.12.001. Online ahead of print.

2023

Protein target highlights in CASP15: Analysis of models by structure providers.

Alexander LT, Durairaj J, Kryshtafovych A, Abriata LA, Bayo Y, Bhabha G, Breyton C, Caulton SG, Chen J, Degroux S, Ekiert DC, Erlandsen BS, Freddolino PL, Gilzer D, Greening C, Grimes JM, Grinter R, Gurusaran M, Hartmann MD, Hitchman CJ, Keown JR, Kropp A, Kursula P, Lovering AL, Lemaitre B, Lia A, Liu S, Logotheti M, Lu S, Markússon S, Miller MD, Minasov G, Niemann HH, Opazo F, Phillips GN Jr, Davies OR, Rommelaere S, Rosas-Lemus M, Roversi P, Satchell K, Smith N, Wilson MA, Wu KL, Xia X, Xiao H, Zhang W, Zhou ZH, Fidelis K, Topf M, Moult J, Schwede T.

Proteins. 2023 Dec;91(12):1571-1599. doi: 10.1002/prot.26545. Epub 2023 Jul 26.

Protein nanobarcodes enable single-step multiplexed fluorescence imaging.

de Jong-Bolm D, Sadeghi M, Bogaciu CA, Bao G, Klaehn G, Hoff M, Mittelmeier L, Basmanav FB, Opazo F, Noé F, Rizzoli SO.

PLoS Biol. 2023 Dec 11;21(12):e3002427. doi: 10.1371/journal.pbio.3002427. eCollection 2023 Dec.

A Versatile Synaptotagmin-1 Nanobody Provides Perturbation-Free Live Synaptic Imaging And Low Linkage-Error in Super-Resolution Microscopy.

Queiroz Zetune Villa Real K, Mougios N, Rehm R, Sograte-Idrissi S, Albert L, Rahimi AM, Maidorn M, Hentze J, Martínez-Carranza M, Hosseini H, Saal KA, Oleksiievets N, Prigge M, Tsukanov R, Stenmark P, Fornasiero EF, Opazo F.

Small Methods. 2023 Jul 8:e2300218. doi: 10.1002/smtd.202300218. Online ahead of print.

Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives.

Kilisch M, Gere-Becker M, Wüstefeld L, Bonnas C, Crauel A, Mechmershausen M, Martens H, Götzke H, Opazo F, Frey S.

Int J Mol Sci. 2023 Apr 14;24(8):7294. doi: 10.3390/ijms24087294.

Heat denaturation enables multicolor X10-STED microscopy.

Saal KA, Shaib AH, Mougios N, Crzan D, Opazo F, Rizzoli SO.

Sci Rep. 2023 Apr 1;13(1):5366. doi: 10.1038/s41598-023-32524-5.

Visualizing proteins by expansion microscopy.

Shaib AH, Chouaib AA, Chowdhury R, Mihaylov D, Zhang C, Imani V, Georgiev SV, Mougios N, Monga M, Reshetniak S, Mimoso T, Chen H, Fatehbasharzad P, Crzan D, Saal KA, Alawar N, Eilts J, Kang J, Alvarez L, Trenkwalder C, Mollenhauer B, Outeiro TF, Köster S, Preobraschenski J, Becherer U, Moser T, Boyden ES, Aricescu AR, Sauer M, Opazo F, Rizzoli SO.

Preprint: BioRxiv. 30 March 2023. doi: 10.1101/2022.08.03.502284.

View at BioRXIV

Trafficking proteins show limited differences in mobility across different postsynaptic spines.

Mougios N, Opazo F, Rizzoli SO, Reshetniak S.

iScience. 2023 Jan 13;26(2):105971. doi: 10.1016/j.isci.2023.105971. eCollection 2023 Feb 17.

2022

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses.

Chakrabarti R, Jaime Tobón LM, Slitin L, Redondo Canales M, Hoch G, Slashcheva M, Fritsch E, Bodensiek K, Özçete ÖD, Gültas M, Michanski S, Opazo F, Neef J, Pangrsic T, Moser T, Wichmann C.

Elife. 2022 Dec 23;11:e79494. doi: 10.7554/eLife.79494.

Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications.

Iannielli A, Luoni M, Giannelli SG, Ferese R, Ordazzo G, Fossati M, Raimondi A, Opazo F, Corti O, Prehn JHM, Gambardella S, Melki R, Broccoli V.

Cell Death Dis. 2022 Oct 19;13(10):881. doi: 10.1038/s41419-022-05330-6.

An iodine-containing probe as a tool for molecular detection in secondary ion mass spectrometry.

Kabatas Glowacki S, Agüi-Gonzalez P, Sograte-Idrissi S, Jähne S, Opazo F, Phan NTN, Rizzoli SO.

Chem Commun (Camb). 2022 Jul 5;58(54):7558-7561. doi: 10.1039/d2cc02290g.

Rapidly Signal-enhanced Metabolites for Atomic Scale Monitoring of Living Cells with Magnetic Resonance.

Ding Y, Korchak S, Mamone S, Jagtap AP, Stevanato G, Sternkopf S, Moll D, Schroeder H, Becker S, Fischer A, Gerhardt E, Outeiro TF, Opazo F, Griesinger C, Glöggler S.

Chem Methods 2022 Apr 07. doi: 10.1002/cmtd.202200023.

View at Chemistry Europe

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells.

Oleksiievets N, Sargsyan Y, Thiele JC, Mougios N, Sograte-Idrissi S, Nevskyi O, Gregor I, Opazo F, Thoms S, Enderlein J, Tsukanov R.

Commun Biol. 2022 Jan 11;5(1):38. doi: 10.1038/s42003-021-02976-4.

2021

A fluorescent probe for STED microscopy to study NIP-specific B cells.

Glowacki SK, Gomes de Castro MA, Yip KM, Asadpour O, Münchhalfen M, Engels N, Opazo F.

Analyst. 2021 Jul 6. doi: 10.1039/d1an00601k. Online ahead of print.

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer.

Kilisch M, Götzke H, Gere-Becker M, Crauel A, Opazo F, Frey S.

Biomolecules. 2021 Feb 12;11(2):269. doi: 10.3390/biom11020269.

2020

Correction: Circumvention of common labelling artefacts using secondary nanobodies.

Sograte-Idrissi S , Schlichthaerle T , Duque-Afonso CJ , Alevra M , Strauss S , Moser T , Jungmann R , Rizzoli SO , Opazo F .

Nanoscale. 2020 Dec 23;12(48):24543. doi: 10.1039/d0nr90279a.

Quantum Defects as a Toolbox for the Covalent Functionalization of Carbon Nanotubes with Peptides and Proteins.

Mann FA, Herrmann N, Opazo F, Kruss S.

Angew Chem Int Ed Engl. 2020 Sep 28;59(40):17732-17738. doi: 10.1002/anie.202003825. Epub 2020 Jul 13.

Quantum Defects as a Toolbox for the Covalent Functionalization of Carbon Nanotubes with Peptides and Proteins.

Mann FA, Herrmann N, Opazo F, Kruss S.

Angew Chem Int Ed Engl. 2020 Jun 8. doi: 10.1002/anie.202003825. Online ahead of print.

A Nanobody-Based Fluorescent Reporter Reveals Human α-Synuclein in the Cell Cytosol.

Gerdes C, Waal N, Offner T, Fornasiero EF, Wender N, Verbarg H, Manzini I, Trenkwalder C, Mollenhauer B, Strohäker T, Zweckstetter M, Becker S, Rizzoli SO, Basmanav FB, Opazo F.

Nat Commun. 2020 Jun 1;11(1):2729. doi: 10.1038/s41467-020-16575-0.

Circumvention of common labelling artefacts using secondary nanobodies.

Sograte-Idrissi S, Schlichthaerle T, Duque-Afonso CJ, Alevra M, Strauss S, Moser T, Jungmann R, Rizzoli SO, Opazo F.

Nanoscale. 2020 May 14;12(18):10226-10239. doi: 10.1039/d0nr00227e.

Singlet-filtered NMR spectroscopy.

Mamone S, Rezaei-Ghaleh N, Opazo F, Griesinger C, Glöggler S.

Science Advances 21 Feb 2020:Vol. 6, no. 8, eaaz1955, DOI: 10.1126/sciadv.aaz1955.

View at ScienceAdvances

2019

The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications.

Götzke H, Kilisch M, Martínez-Carranza M, Sograte-Idrissi S, Rajavel A, Schlichthaerle T, Engels N, Jungmann R, Stenmark P, Opazo F, Frey S.

Nat Commun. 2019 Sep 27;10(1):4403. doi: 10.1038/s41467-019-12301-7.

A MICOS-TIM22 Association Promotes Carrier Import into Human Mitochondria.

Callegari S, Müller T, Schulz C, Lenz C, Jans DC, Wissel M, Opazo F, Rizzoli SO, Jakobs S, Urlaub H, Rehling P, Deckers M.

J Mol Biol. 2019 May 17. pii: S0022-2836(19)30284-0. doi: 10.1016/j.jmb.2019.05.015. [Epub ahead of print]

Fluorinated Nanobodies for Targeted Molecular Imaging of Biological Samples using Nanoscale Secondary Ion Mass Spectrometry.

Kabatas S, Agüi-Gonzalez P, Hinrichs R, Jähne S, Opazo F, Diederichsen U, Rizzoli SO, Phan NTN.

J. Anal. At. Spectrom., 2019 Apr 24; 34, 1083. DOI: 10.1039/c9ja00117d.

Boron-Containing Probes for Non-Optical High-Resolution Imaging of Biological Samples.

Kabatas S, Agüi-Gonzales P, Saal KA, Jähne S, Opazo F, Rizzoli SO, Phan TNN.

Angew Chem Int Ed Engl. 2019 Mar 11;58(11):3438-3443. doi: 10.1002/anie.201812032. Epub 2019 Feb 6.

Boron-Containing Probes for Non-Optical High-Resolution Imaging of Biological Samples.

Kabatas S, Agüi-Gonzales P, Saal KA, Jähne S, Opazo F, Rizzoli SO, Phan TNN.

Angew Chem 2019 Mar 11;131(11):3476-3481. doi:10.1002/anie.201812032. Epub 2019 Jan 7.

Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons.

Maidorn M, Olichon A, Rizzoli SO, Opazo F.

MAbs. 2019 Feb/Mar;11(2):305-321. doi: 10.1080/19420862.2018.1551675. Epub 2018 Dec 28.

Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane.

Gomes de Castro MA, Wildhagen H, Sograte-Idrissi S, Hitzing C, Binder M, Trepel M, Engels N, Opazo F.

Nat Commun. 2019 Feb 18;10(1):820. doi: 10.1038/s41467-019-08677-1.

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors.

Sograte-Idrissi S, Oleksijevets N, Isbaner S, Eggert-Martinez M, Enderlein J, Tsukanov R, Opazo F.

Cells. 2019 Jan 14;8(1). pii: E48. doi: 10.3390/cells8010048.

2018

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

Fornasiero EF, Mandad S, Wildhagen H, Alevra M, Rammner B, Keihani S, Opazo F, Urban I, Ischebeck T, Sakib MS, Fard MK, Kirli K, Centeno TP, Vidal RO, Rahman RU, Benito E, Fischer A, Dennerlein S, Rehling P, Feussner I, Bonn S, Simons M, Urlaub H, Rizzoli SO.

Nat Commun. 2018 Oct 12;9(1):4230. doi: 10.1038/s41467-018-06519-0.

View at PubMed

The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain.

Mandad S, Rahman RU, Centeno PT, Vidal RO, Wildhagen H, Rammner B, Keihani S, Opazo F, Urban I, Ischebeck T, Kirli K, Benito E, Fischer A, Yousefi RY, Dennerlein S, Rehling P, Feussner I, Urlaub H, Bonn S, Rizzoli SO, Fornasiero EF.

Sci Rep. 2018 Nov 15;8(1):16913. doi: 10.1038/s41598-018-35277-8.

The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression.

Vanshylla K, Opazo F, Gronke K, Wienands J, Engels N.

Eur J Immunol. 2018 Mar;48(3):441-453. doi: 10.1002/eji.201747196. Epub 2017 Dec 1.

2017

Aptamers provide superior stainings of cellular receptors studied under super-resolution microscopy.

Gomes de Castro MA, Höbartner C,Opazo F.

PLoS One. 2017 Feb 24;12(2):e0173050. doi: 10.1371/journal.pone.0173050. eCollection 2017.

Correction: ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones.

Kawabe H, Mitkovski M, Kaeser PS, Hirrlinger J, Opazo F, Nestvogel D, Kalla S, Fejtova A, Verrier SE, Bungers SR, Cooper BH, Varoqueaux F, Wang Y, Nehring RB, Gundelfinger ED, Rosenmund C, Rizzoli SO, Südhof TC, Rhee JS, Brose N.

J Cell Biol. 2017 Apr 3;216(4):1205. doi: 10.1083/jcb.20160608603092017c. Epub 2017 Mar 13. No abstract available.

ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones.

Kawabe H, Mitkovski M, Kaeser PS, Hirrlinger J, Opazo F, Nestvogel D, Kalla S, Fejtova A, Verrier SE, Bungers SR, Cooper BH, Varoqueaux F, Wang Y, Nehring RB, Gundelfinger ED, Rosenmund C, Rizzoli SO, Südhof TC, Rhee JS, Brose N.

J Cell Biol. 2017 Apr 3;216(4):1143-1161. doi: 10.1083/jcb.201606086. Epub 2017 Mar 6.

2016

Aptamer Stainings for Super-resolution Microscopy.

de Castro MA, Rammner B, Opazo F.

Methods Mol Biol. 2016;1380:197-210. doi: 10.1007/978-1-4939-3197-2_17.

Tools and limitations to study the molecular composition of synapses by fluorescence microscopy.

Maidorn M, Rizzoli SO, Opazo F

Biochem J. 2016 Oct 15;473(20):3385-3399. Review.

2015

Super-resolution imaging for cell biologists: concepts, applications, current challenges and developments.

Fornasiero EF, Opazo F.

Bioessays. 2015 Apr;37(4):436-51. doi: 10.1002/bies.201400170. Epub 2015 Jan 12.

Secondary-ion mass spectrometry of genetically encoded targets.

Vreja IC, Kabatas S, Saka SK, Kröhnert K, Höschen C, Opazo F, Diederichsen U, Rizzoli SO.

Angew Chem Int Ed Engl. 2015 May 4;54(19):5784-8. doi: 10.1002/anie.201411692. Epub 2015 Mar 17.

Secondary-ion mass spectrometry of genetically encoded targets.

Vreja IC, Kabatas S, Saka SK, Kröhnert K, Höschen C, Opazo F, Diederichsen U, Rizzoli SO.

Angew Chem 2015 May 4;127(19):5876-5880. doi: 10.1002/anie.201411692. Epub 2015 Mar 17.

A contamination-insensitive probe for imaging specific biomolecules by secondary ion mass spectrometry.

Kabatas S, Vreja IC, Saka SK, Höschen C, Kröhnert K, Opazo F, Rizzoli SO, Diederichsen U.

Chem Commun (Camb). 2015 Aug 6;51(67):13221-4. doi: 10.1039/c5cc03895b.

Resolving bundled microtubules using anti-tubulin nanobodies.

Mikhaylova M, Cloin BM, Finan K, van den Berg R, Teeuw J, Kijanka MM, Sokolowski M, Katrukha EA, Maidorn M, Opazo F, Moutel S, Vantard M, Perez F, van Bergen en Henegouwen PM, Hoogenraad CC, Ewers H, Kapitein LC.

Nat Commun. 2015 Aug 11;6:7933. doi: 10.1038/ncomms8933.

Mapping molecules in scanning far-field fluorescence nanoscopy.

Ta H, Keller J, Haltmeier M, Saka SK, Schmied J, Opazo F, Tinnefeld P, Munk A, Hell SW.

Nat Commun. 2015 Aug 13;6:7977. doi: 10.1038/ncomms8977.

Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer.

Opazo F, Eiden L, Hansen L, Rohrbach F, Wengel J, Kjems J, Mayer G.

Mol Ther Nucleic Acids. 2015 Sep 1;4:e251. doi: 10.1038/mtna.2015.25.

2014

Fluorescent in situ hybridization of synaptic proteins imaged with super-resolution STED microscopy.

Zhang WI, Röhse H, Rizzoli SO, Opazo F.

Microsc Res Tech. 2014 Apr 10. doi: 10.1002/jemt.22367.

Cellular and Molecular Applications of Super-resolution Microscopy.

Opazo F, Rizzoli SO.

Fluorescence Microscopy: Super-Resolution and Other Novel Techniques. Academic Press 133-152. ISBN: 978-0-12-409513-7

Link to elsevier

Probing Biological Samples in High-Resolution Microscopy: Making Sense of Spots.

Opazo F.

Super-Resolution Microscopy Techniques in the Neurosciences.Neuromethods, Vol 86:369-386. ISBN: 978-1-62703-982-6

Link to SpringerLink

2012

Aptamers as potential tools for super-resolution microscopy.

Opazo F, Levy M, Byrom M, Schäfer C, Geisler C, Groemer TW, Ellington AD, Rizzoli SO.

Nat Methods. 2012 Oct;9(10):938-9. doi: 10.1038/nmeth.2179. No abstract available.

An RNA alternative to human transferrin: a new tool for targeting human cells.

Wilner SE, Wengerter B, Maier K, de Lourdes Borba Magalhães M, Del Amo DS, Pai S, Opazo F, Rizzoli SO, Yan A, Levy M.

Mol Ther Nucleic Acids. 2012 May 15;1:e21. doi: 10.1038/mtna.2012.14.

2010

Endosomal sorting of readily releasable synaptic vesicles.

Hoopmann P, Punge A, Barysch SV, Westphal V, Bückers J, Opazo F, Bethani I, Lauterbach MA, Hell SW, Rizzoli SO.

Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):19055-60. doi: 10.1073/pnas.1007037107. Epub 2010 Oct 18.

The fate of synaptic vesicle components upon fusion.

Opazo F, Rizzoli SO.

Commun Integr Biol. 2010 Sep;3(5):427-9. doi: 10.4161/cib.3.5.12132.

PKC links Gq-coupled receptors to DAT-mediated dopamine release.

Opazo F, Schulz JB, Falkenburger BH.

J Neurochem. 2010 Jul;114(2):587-96. doi: 10.1111/j.1471-4159.2010.06788.x. Epub 2010 Apr 29.

Aged Tgfbeta2/Gdnf double-heterozygous mice show no morphological and functional alterations in the nigrostriatal system.

Heermann S, Opazo F, Falkenburger B, Krieglstein K, Spittau B.

J Neural Transm. 2010 Jun;117(6):719-27. doi: 10.1007/s00702-010-0406-7. Epub 2010 May 11.

Limited intermixing of synaptic vesicle components upon vesicle recycling.

Opazo F, Punge A, Bückers J, Hoopmann P, Kastrup L, Hell SW, Rizzoli SO.
Traffic. 2010 Jun;11(6):800-12. doi: 10.1111/j.1600-0854.2010.01058.x. Epub 2010 Mar 9.

Studying synaptic vesicle pools using photoconversion of styryl dyes.

Opazo F, Rizzoli SO.

J Vis Exp. 2010 Feb 15;(36). doi:pii: 1790. 10.3791/1790.

2009

Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson’s disease models.

Karpinar DP, Balija MB, Kügler S, Opazo F, Rezaei-Ghaleh N, Wender N, Kim HY, Taschenberger G, Falkenburger BH, Heise H, Kumar A, Riedel D, Fichtner L, Voigt A, Braus GH, Giller K, Becker S, Herzig A, Baldus M, Jäckle H, Eimer S, Schulz JB, Griesinger C, Zweckstetter M.

EMBO J. 2009 Oct 21;28(20):3256-68. doi: 10.1038/emboj.2009.257. Epub 2009 Sep 10.

Rapid microtubule bundling and stabilization by the Streptococcus pneumoniae neurotoxin pneumolysin in a cholesterol-dependent, non-lytic and Src-kinase dependent manner inhibits intracellular trafficking.

Iliev AI, Djannatian JR, Opazo F, Gerber J, Nau R, Mitchell TJ, Wouters FS.

Mol Microbiol. 2009 Jan;71(2):461-77. doi: 10.1111/j.1365-2958.2008.06538.x. Epub 2008 Nov 19.

2008

Accumulation and clearance of alpha-synuclein aggregates demonstrated by time-lapse imaging.

Opazo F, Krenz A, Heermann S, Schulz JB, Falkenburger BH.

J Neurochem. 2008 Jul;106(2):529-40. doi: 10.1111/j.1471-4159.2008.05407.x. Epub 2008 Apr 10.

Membrane-permeable Bcl-xL prevents MPTP-induced dopaminergic neuronal loss in the substantia nigra.

Dietz GP1, Stockhausen KV, Dietz B, Falkenburger BH, Valbuena P, Opazo F, Lingor P, Meuer K, Weishaupt JH, Schulz JB, Bähr M.

J Neurochem. 2008 Feb;104(3):757-65. Epub 2007 Nov 6.

2004

Effects of natural flavones and flavonols on the kinase activity of Cdk5.

Zapata-Torres G, Opazo F, Salgado C, Muñoz JP, Krautwurst H, Mascayano C, Sepúlveda-Boza S, Maccioni RB, Cassels BK.

J Nat Prod. 2004 Mar;67(3):416-20.

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