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Danielson, U. Helena, ProfessorORCID iD iconorcid.org/0000-0003-2728-0340
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Publications (10 of 166) Show all publications
Xu, X., Makaraviciute, A., Abdurakhmanov, E., Werneling, F., Li, S., Danielson, U. H., . . . Zhang, Z. (2020). Estimating Detection Limits of Potentiometric DNA sensors Using Surface Plasmon Resonance Analyses. ACS Sensors, 5(1), 217-224
Open this publication in new window or tab >>Estimating Detection Limits of Potentiometric DNA sensors Using Surface Plasmon Resonance Analyses
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2020 (English)In: ACS Sensors, E-ISSN 2379-3694, Vol. 5, no 1, p. 217-224Article in journal (Refereed) Published
Abstract [en]

As the signals of potentiometric-based DNA ion-selective field effect transistor (ISFET) sensors differ largely from report to report, a systematic revisit to this method is needed. Herein, the hybridization of the target and the probe DNA on the sensor surface and its dependence on the surface probe DNA coverage and the ionic strength were systematically investigated by surface plasmon resonance (SPR). The maximum potentiometric DNA hybridization signal that could be registered by an ISFET sensor was estimated based on the SPR measurements, without considering buffering effects from any side interaction on the sensing electrode. We found that under physiological solutions (200 to 300 mM ionic strength), the ISFET sensor could not register the DNA hybridization events on the sensor surface due to Debye screening. Lowering the salt concentration to enlarge the Debye length would at the same time reduce the surface hybridization efficiency, thus suppressing the signal. This adverse effect of low salt concentration on the hybridization efficiency was also found to be more significant on the surface with higher probe coverage due to steric hindrance. With the method of diluting buffer, the maximum potentiometric signal generated by the DNA hybridization was estimated to be only around 120 mV with the lowest detection limit of 30 nM, occurring on a surface with optimized probe coverage and in the tris buffer with 10 mM NaCl. An alternative method would be to achieve high-efficiency hybridization in the buffer with high salt concentration (1 M NaCl) and then to perform potentiometric measurements in the buffer with low salt concentration (1 mM NaCl). Based on the characterization of the stability of the hybridized DNA duplexes on the sensor surface in low salt concentration buffer solutions, the estimated maximum potentiometric signal could be significantly higher using the alternative method. The lowest detection limit for this alternative method was estimated to be around 0.6 nM. This work can serve as an important quantitative reference for potentiometric DNA sensors.

National Category
Electrical Engineering, Electronic Engineering, Information Engineering Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-397806 (URN)10.1021/acssensors.9b02086 (DOI)000510079300029 ()31833355 (PubMedID)
Funder
Swedish Foundation for Strategic Research , ICA 12-0047Swedish Foundation for Strategic Research , FFL15-0174Swedish Research Council, VR 2014-5588Wallenberg Foundations
Available from: 2019-11-25 Created: 2019-11-25 Last updated: 2020-03-20Bibliographically approved
Yang, J., Talibov, V. O., Peintner, S., Rhee, C., Poongavanam, V., Geitmann, M., . . . Kihlberg, J. (2020). Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1. ACS OMEGA, 5(8), 3979-3995
Open this publication in new window or tab >>Macrocyclic Peptides Uncover a Novel Binding Mode for Reversible Inhibitors of LSD1
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2020 (English)In: ACS OMEGA, ISSN 2470-1343, Vol. 5, no 8, p. 3979-3995Article in journal (Refereed) Published
Abstract [en]

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (4) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was >40 fold more potent K-i = 2.3 mu M) than 4. Linear and macrocyclic peptides exhibited unexpected differences in structure-activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of 31 in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of 31 suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2020
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-408512 (URN)10.1021/acsomega.9b03493 (DOI)000518851600022 ()32149225 (PubMedID)
Funder
Swedish Research Council, 2016-05160Swedish Research Council, D0571301EU, FP7, Seventh Framework Programme, 283570
Available from: 2020-04-09 Created: 2020-04-09 Last updated: 2020-04-09Bibliographically approved
Xu, X., Makaraviciute, A., Kumar, S., Wen, C., Sjödin, M., Abdurakhmanov, E., . . . Zhang, Z. (2019). Structural Changes of Mercaptohexanol Self-assembled Monolayers on Gold and their Influence on Impedimetric Aptamer Sensors. Analytical Chemistry, 91(22), 14697-14704
Open this publication in new window or tab >>Structural Changes of Mercaptohexanol Self-assembled Monolayers on Gold and their Influence on Impedimetric Aptamer Sensors
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2019 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 22, p. 14697-14704Article in journal (Refereed) Published
Abstract [en]

Despite a large number of publications describing biosensors based on electrochemical impedance spectroscopy (EIS), little attention has been paid to the stability and reproducibility issues of the sensor interfaces. In this work, the stability and reproducibility of faradaic EIS analyses on the aptamer/mercaptohexanol (MCH) self-assembled monolayer (SAM) functionalized gold surfaces in ferri- and ferrocyanide solution were systematically evaluated prior to and after the aptamer-probe DNA hybridization. It is shown that the EIS data exhibited significant drift, and this significantly affected the reproducibility of the EIS signal of the hybridization. As a result, no significant difference between the charge transfer resistance (RCT) changes induced by the aptamer-target DNA hybridization and that caused by the drift could be identified. A conditioning of the electrode in the measurement solution for more than 12 hours was required to reach a stable RCT baseline prior to the aptamer-probe DNA hybridization. The monitored drift in RCT and CDL during the conditioning suggests that the MCH SAM on the gold surface reorganized to a thinner but more closely packed layer. We also observed that the hot binding buffer used in the following aptamer-probe DNA hybridization process could induce additional MCH and aptamer reorganization thus further drift in RCT. As a result, the RCT change caused by the aptamer-probe DNA hybridization was less than that caused by the hot binding buffer (blank control experiment). Therefore, it is suggested that the use of high temperature in the EIS measurement should be carefully evaluated or avoided. This work provides practical guidelines for the EIS measurements. Moreover, since SAM functionalized gold electrodes are widely used in biosensors, e.g., DNA sensors, an improved understanding of the origin of the observed drift is very important for the development of well-functioning and reproducible biosensors.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-397690 (URN)10.1021/acs.analchem.9b03946 (DOI)000498280100072 ()31650834 (PubMedID)
Funder
Swedish Foundation for Strategic Research , ICA 12-0047Swedish Foundation for Strategic Research , FFL15-0174Swedish Research Council, VR 2014-5588Knut and Alice Wallenberg Foundation, Wallenberg Academy Fellow Program
Available from: 2019-11-22 Created: 2019-11-22 Last updated: 2020-01-13Bibliographically approved
Fabini, E., Talibov, V. O., Mihalic, F., Naldi, M., Bartolini, M., Bertucci, C., . . . Danielson, U. H. (2019). Unveiling the Biochemistry of the Epigenetic Regulator SMYD3. Biochemistry, 58(35), 3634-3645
Open this publication in new window or tab >>Unveiling the Biochemistry of the Epigenetic Regulator SMYD3
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2019 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 35, p. 3634-3645Article in journal (Refereed) Published
Abstract [en]

SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K-260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-394688 (URN)10.1021/acs.biochem.9b00420 (DOI)000484643700003 ()31389685 (PubMedID)
Funder
EU, Horizon 2020EU, FP7, Seventh Framework Programme, 283570Swedish Research Council, D0571301
Available from: 2019-10-24 Created: 2019-10-24 Last updated: 2019-10-24Bibliographically approved
Linkuviene, V., Talibov, V. O., Danielson, U. H. & Matulis, D. (2018). Introduction of Intrinsic Kinetics of Protein-Ligand Interactions and Their Implications for Drug Design. Journal of Medicinal Chemistry, 61(6), 2292-2302
Open this publication in new window or tab >>Introduction of Intrinsic Kinetics of Protein-Ligand Interactions and Their Implications for Drug Design
2018 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 6, p. 2292-2302Article in journal (Refereed) Published
Abstract [en]

Structure kinetic relationship analyses and identification of dominating interactions for optimization of lead compounds should ideally be based on intrinsic rate constants instead of the more easily accessible observed kinetic constants, which also account for binding linked reactions. The intrinsic rate constants for sulfonamide inhibitors and pharmacologically relevant isoforms of carbonic anhydrase were determined by a novel surface plasmon resonance (SPR) biosensor-based approach, using chemodynamic analysis of binding-linked pH-dependent effects. The observed association rates (k(a)(obs)) were pH-dependent and correlated with the fraction of deprotonated inhibitor and protonated zinc-bound water molecule. The intrinsic association rate constants (k(a)(intr)) were pH independent and higher than k(a)(obs). By contrast, the observed and intrinsic dissociation rate constants were identical and pH-independent, demonstrating that the observed association and dissociation mechanisms are inherently different. A model accounting for the differences between intrinsic and observed rate constants was developed, useful also for other interactions with binding-linked protonation reactions.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-354359 (URN)10.1021/acs.jmedchem.7b01408 (DOI)000428356600008 ()29466001 (PubMedID)
Funder
Swedish Research Council, D0571301Carl Tryggers foundation
Available from: 2018-06-26 Created: 2018-06-26 Last updated: 2018-10-16Bibliographically approved
Pandya, N. J., Seeger, C., Babai, N., Gonzalez-Lozano, M. A., Mack, V., Lodder, J. C., . . . Smit, A. B. (2018). Noelin1 Affects Lateral Mobility of Synaptic AMPA Receptors. Cell reports, 24(5), 1218-1230
Open this publication in new window or tab >>Noelin1 Affects Lateral Mobility of Synaptic AMPA Receptors
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2018 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 24, no 5, p. 1218-1230Article in journal (Refereed) Published
Abstract [en]

Lateral diffusion on the neuronal plasma membrane of the AMPA-type glutamate receptor (AMPAR) serves an important role in synaptic plasticity. We investigated the role of the secreted glycoprotein Noelin1 (Olfactomedin-1 or Pancortin) in AMPAR lateral mobility and its dependence on the extracellular matrix (ECM). We found that Noelin1 interacts with the AMPAR with high affinity, however, without affecting rise- and decay time and desensitization properties. Noelin1 co-localizes with synaptic and extra-synaptic AMPARs and is expressed at synapses in an activity-dependent manner. Single-particle tracking shows that Noelin1 reduces lateral mobility of both synaptic and extra-synaptic GluA1-containing receptors and affects short-term plasticity. While the ECM does not constrain the synaptic pool of AMPARs and acts only extrasynaptically, Noelin1 contributes to synaptic potentiation by limiting AMPAR mobility at synaptic sites. This is the first evidence for the role of a secreted AMPAR-interacting protein on mobility of GluA1-containing receptors and synaptic plasticity.

Keywords
AMPAR-associated protein, glutamate receptor, receptor mobility, synapse function, synaptic plasticity
National Category
Biological Sciences Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-361395 (URN)10.1016/j.celrep.2018.06.102 (DOI)000440377500012 ()30067977 (PubMedID)
Funder
Swedish Research Council, D0571301EU, FP7, Seventh Framework Programme, 242167EU, FP7, Seventh Framework Programme
Available from: 2018-09-24 Created: 2018-09-24 Last updated: 2018-10-29Bibliographically approved
Belfrage, A. K., Abdurakhmanov, E., Åkerblom, E., Brandt, P., Alogheli, H., Neyts, J., . . . Johansson, A. (2018). Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold. European Journal of Medicinal Chemistry, 148, 453-464
Open this publication in new window or tab >>Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold
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2018 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 148, p. 453-464Article in journal (Refereed) Published
Abstract [en]

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R-3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on beta-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the beta-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype la, both wild-type (K-i=30 nM) and R155K (K-i=2 nM), and genotype 3a (K-i=5 nM).

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Hepatitis C, NS3, Genotype 3, Resistance, Pyrazinone
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-340862 (URN)10.1016/j.ejmech.2018.02.032 (DOI)000428824700036 ()
Funder
Swedish Research Council, D0571301
Available from: 2018-02-04 Created: 2018-02-04 Last updated: 2018-05-31Bibliographically approved
Seeger, C., Talibov, V. O. & Danielson, U. H. (2017). Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin-dependent kinase II. Journal of Molecular Recognition, 30, 1-11, Article ID e2621.
Open this publication in new window or tab >>Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin-dependent kinase II
2017 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 30, p. 1-11, article id e2621Article in journal (Refereed) Published
Abstract [en]

Calmodulin (CaM) functions depend on interactions with CaM-binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM-binding region of Ca2+/calmodulin-dependent kinase II (CaMKII290-309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity (KDSPR=3M). Calmodulin-CaMKII290-309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity (KDSPR7.1nM). The CaM-Ng interaction had higher affinity under Ca2+-depleted (KDSPR480nM,3.4x105M-1s-1 and k(-1) = 1.6 x 10(-1)s(-1)) than Ca2+-saturated conditions (KDSPR19M). The IQ motif of Ng (Ng(27-50)) had similar affinity for CaM as Ng under Ca2+-saturated conditions (KDSPR=14M), but no interaction was seen under Ca2+-depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng (KDMST890nM) and CaMKII290-309(KDMST190nM) interactions. Although CaMKII290-309 showed expected interaction characteristics, they may be different for full-length CaMKII. The data for full-length Ng, but not Ng(27-50), agree with the current model on Ng regulation of Ca2+/CaM signaling.

Keywords
calmodulin, calmodulin-dependent kinase, surface plasmon resonance, microscale thermophoresis, neurogranin
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-329023 (URN)10.1002/jmr.2621 (DOI)000405095400006 ()
Funder
Swedish Research Council, D0571301
Available from: 2017-09-06 Created: 2017-09-06 Last updated: 2018-10-16Bibliographically approved
Abdurakhmanov, E., Solbak, S. O. & Danielson, U. H. (2017). Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase. Viruses, 9(6), Article ID 151.
Open this publication in new window or tab >>Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase
2017 (English)In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 9, no 6, article id 151Article in journal (Refereed) Published
Abstract [en]

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

Keywords
hepatitis C virus (HCV), non-structural protein (NS) polymerase, genotypes, allosteric inhibitor, surface plasmon resonance (SPR)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-331940 (URN)10.3390/v9060151 (DOI)000405832000026 ()
Funder
Swedish Research Council, D0571301
Available from: 2017-10-24 Created: 2017-10-24 Last updated: 2017-11-29Bibliographically approved
Solbak, S. M. .., Abdurakhmanov, E., Vedeler, A. & Danielson, U. H. (2017). Characterization of interactions between hepatitis C virus NS5B polymerase, annexin A2 and RNA - effects on NS5B catalysis and allosteric inhibition. Virology Journal, 14, Article ID 236.
Open this publication in new window or tab >>Characterization of interactions between hepatitis C virus NS5B polymerase, annexin A2 and RNA - effects on NS5B catalysis and allosteric inhibition
2017 (English)In: Virology Journal, ISSN 1743-422X, E-ISSN 1743-422X, Vol. 14, article id 236Article in journal (Refereed) Published
Abstract [en]

Background: Direct acting antivirals (DAAs) provide efficient hepatitis C virus (HCV) therapy and clearance for a majority of patients, but are not available or effective for all patients. They risk developing HCV-induced hepatocellular carcinoma (HCC), for which the mechanism remains obscure and therapy is missing. Annexin A2 (AnxA2) has been reported to co-precipitate with the non-structural (NS) HCV proteins NS5B and NS3/NS4A, indicating a role in HCC tumorigenesis and effect on DAA therapy.

Methods: Surface plasmon resonance biosensor technology was used to characterize direct interactions between AnxA2 and HCV NS5B, NS3/NS4 and RNA, and the subsequent effects on catalysis and inhibition.

Results: No direct interaction between AnxA2 and NS3/NS4A was detected, while AnxA2 formed a slowly dissociating, high affinity (K D = 30 nM), complex with NS5B, decreasing its catalytic activity and affinity for the allosteric inhibitor filibuvir. The RNA binding of the two proteins was independent and AnxA2 and NS5B interacted with different RNAs in ternary complexes of AnxA2:NS5B:RNA, indicating specific preferences.

Conclusions: The complex interplay revealed between NS5B, AnxA2, RNA and filibuvir, suggests that AnxA2 may have an important role for the progression and treatment of HCV infections and the development of HCC, which should be considered also when designing new allosteric inhibitors.

Keywords
Hepatitis C virus, NS5B, Annexin A2, RNA, allosteric inhibition, filibuvir, surface, plasmon resonance, Hepatocellular carcinoma
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-337575 (URN)10.1186/s12985-017-0904-4 (DOI)000417867100001 ()29228983 (PubMedID)
Funder
Swedish Research Council, D0571301Carl Tryggers foundation
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2018-03-02Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2728-0340

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