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Zieba, Agata
Alternative names
Publications (10 of 17) Show all publications
Zieba, A., Ponten, F., Uhlen, M. & Landegren, U. (2018). In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation. Modern Pathology, 31(2), 253-263
Open this publication in new window or tab >>In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation
2018 (English)In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 31, no 2, p. 253-263Article in journal (Refereed) Published
Abstract [en]

Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Immunology in the medical area Other Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-347648 (URN)10.1038/modpathol.2017.102 (DOI)000424761400003 ()28937142 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2008:0143EU, FP7, Seventh Framework Programme, 222635 241481Swedish Research CouncilVINNOVAEU, European Research Council, 294409
Available from: 2018-04-06 Created: 2018-04-06 Last updated: 2018-04-06Bibliographically approved
Blokzijl, A., Zieba, A., Hust, M., Schirrmann, T., Helmsing, S., Grannas, K., . . . Landegren, U. (2016). Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens. Molecular & cellular proteomics (online), 15(6), 1848-1856
Open this publication in new window or tab >>Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
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2016 (English)In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 15, no 6, p. 1848-1856Article in journal (Refereed) Published
Abstract [en]

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-299590 (URN)10.1074/mcp.M115.055756 (DOI)000377822900006 ()26929218 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationEU, European Research Council, 222635; 241481; 294409Swedish Research CouncilSwedish Research Council, NT-E0383401 MH-K2013-66x-14436-10-5
Available from: 2016-07-22 Created: 2016-07-22 Last updated: 2017-11-28Bibliographically approved
Raja, E., Tzavlaki, K., Vuilleumier, R., Edlund, K., Kahata, K., Zieba, A., . . . Moustakas, A. (2016). The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling. OncoTarget, 7(2), 1120-1143
Open this publication in new window or tab >>The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling
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2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 2, p. 1120-1143Article in journal (Refereed) Published
Abstract [en]

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease.

Keywords
BMP; differentiation; Drosophila; LKB1; lung cancer; Pathology Section
National Category
Clinical Laboratory Medicine Basic Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-278888 (URN)10.18632/oncotarget.6683 (DOI)000369951100005 ()26701726 (PubMedID)
Funder
Swedish Research Council, K2007-66X-14936-04-3Swedish Research Council, K2010-67X-14936-07-03Swedish Research Council, K2013-66X-14936-10-5EU, European Research Council, MRTN-2005-005428
Available from: 2016-02-26 Created: 2016-02-26 Last updated: 2018-02-01Bibliographically approved
Grannas, K., Arngården, L., Lönn, P., Mazurkiewicz, M., Blokzij, A., Zieba Wicher, A. & Söderberg, O. (2015). Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes. Journal of Molecular Biology, 427(21), 3407-3415
Open this publication in new window or tab >>Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
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2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 21, p. 3407-3415Article in journal (Refereed) Published
Abstract [en]

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-268434 (URN)10.1016/j.jmb.2015.04.015 (DOI)000363823200006 ()25937570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568Swedish Research Council
Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2017-12-01Bibliographically approved
Zieba, A., Sjöstedt, E., Olovsson, M., Fagerberg, L., Hallström, B. M., Oskarsson, L., . . . Pontén, F. (2015). The Human Endometrium-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling. Omics, 19(11), 659-668
Open this publication in new window or tab >>The Human Endometrium-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling
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2015 (English)In: Omics, ISSN 1536-2310, E-ISSN 1557-8100, Vol. 19, no 11, p. 659-668Article in journal (Refereed) Published
Abstract [en]

The human uterus includes the complex endometrial mucosa, the endometrium that undergoes dynamic, hormone-dependent alterations throughout the life of fertile females. Here we have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to analyze gene expression patterns in the normal endometrium. Human endometrial tissues from five women were used for deep sequencing (RNA-Seq). The mRNA and protein expression data from the endometrium were compared to 31 (RNA) and 44 (protein) other normal tissue types, to identify genes with elevated expression in the endometrium and to localize the expression of corresponding proteins at a cellular resolution. Based on the expression levels of transcripts, we could classify all putative human protein coding genes into categories defined by expression patterns and found altogether 101 genes that showed an elevated pattern of expression in the endometrium, with only four genes showing more than five-fold higher expression levels in the endometrium compared to other tissues. In conclusion, our analysis based on transcriptomics and antibody-based protein profiling reports here comprehensive lists of genes with elevated expression levels in the endometrium, providing important starting points for a better molecular understanding of human reproductive biology and disease.

National Category
Medical Genetics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-269256 (URN)10.1089/omi.2015.0115 (DOI)000364547500001 ()26488136 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation
Available from: 2015-12-15 Created: 2015-12-15 Last updated: 2018-01-10Bibliographically approved
Dahl, M., Maturi, V., Lönn, P., Papoutsoglou, P., Zieba, A., Vanlandewijck, M., . . . Moustakas, A. (2014). Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling. PLoS ONE, 9(8), e103651
Open this publication in new window or tab >>Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 8, p. e103651-Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.

METHODS:

A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.

RESULTS:

TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.

CONCLUSION:

Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-231920 (URN)10.1371/journal.pone.0103651 (DOI)000341302700014 ()25133494 (PubMedID)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2018-10-23Bibliographically approved
Gremel, G., Grannas, K., Sutton, L. A., Pontén, F. & Zieba, A. (2013). In situ Protein Detection for Companion Diagnostics. Frontiers in Oncology, 3, Article 271
Open this publication in new window or tab >>In situ Protein Detection for Companion Diagnostics
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2013 (English)In: Frontiers in Oncology, ISSN 2234-943X, E-ISSN 2234-943X, Vol. 3, p. Article 271-Article in journal (Refereed) Published
Abstract [en]

The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests. Assays developed in recent years are aimed at determining both the effectiveness and safety of specific drugs for a defined group of patients, thus, enabling the more efficient design of clinical trials and also supporting physicians when making treatment-related decisions. Immunohistochemistry (IHC) is a widely accepted method for protein expression analyses in human tissues. Immunohistochemical assays, used to localize and quantitate relative protein expression levels within a morphological context, are frequently used as companion diagnostics during clinical trials and also following drug approval. Herein, we describe established immunochemistry-based methods and their application in routine diagnostics. We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests. Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-221320 (URN)10.3389/fonc.2013.00271 (DOI)24199171 (PubMedID)
Available from: 2014-03-28 Created: 2014-03-28 Last updated: 2017-12-05Bibliographically approved
Leuchowius, K.-J., Clausson, C.-M., Grannas, K., Erbilgin, Y., Botling, J., Zieba, A., . . . Söderberg, O. (2013). Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue. Molecular & Cellular Proteomics, 12(6), 1563-1571
Open this publication in new window or tab >>Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue
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2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 6, p. 1563-1571Article in journal (Refereed) Published
Abstract [en]

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-203549 (URN)10.1074/mcp.O112.023374 (DOI)000319865000007 ()
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2013-07-15 Created: 2013-07-15 Last updated: 2017-12-06Bibliographically approved
Sundqvist, A., Zieba, A., Vasilaki, E., Herrera Hidalgo, C., Söderberg, O., Koinuma, D., . . . van Dam, H. (2013). Specific interactions between Smad proteins and AP-1 components determine TGFβ-induced breast cancer cell invasion. Oncogene, 32(31), 3606-3615
Open this publication in new window or tab >>Specific interactions between Smad proteins and AP-1 components determine TGFβ-induced breast cancer cell invasion
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2013 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 32, no 31, p. 3606-3615Article in journal (Refereed) Published
Abstract [en]

Deregulation of the transforming growth factor β (TGFβ) signal transduction cascade is functionally linked to cancer. In early phases, TGFβ acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFβ responses, but relatively little is known about the Smad-containing complexes that are important for epithelial-mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFβ signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFβ-induced invasion and found that various mesenchymal and invasion-associated TGFβ-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFβ signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFβ stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFβ-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFβ-induced invasion program.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-180124 (URN)10.1038/onc.2012.370 (DOI)000322638400005 ()22926518 (PubMedID)
Note

Agata Zieba & Eleftheria Vasilaki contributed equally to this work.

Available from: 2012-08-30 Created: 2012-08-30 Last updated: 2017-12-07Bibliographically approved
Zieba, A. (2012). Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Application of Proximity Ligation Assay for Multidirectional Studies on Transforming Growth Factor-β Pathway
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A comprehensive understanding of how the body and all its components function is essential when this knowledge is exploited for medical purposes. The achievements in biological and medical research during last decades has provided us with the complete human genome and identified signaling pathways that governs the cellular processes that facilitates the development and maintenance of higher order organisms. This has brought about the realization that diseases such as cancer is a consequence of genomic aberrations that effects these signaling pathways, endowing cancer cells with the capacity to circumvent homeostasis by acquiring features like self-sustained proliferation and insensitivity to apoptosis. The increased understanding of biology and medicine has been made possible by the development of advanced methods to carry out biological and clinical analyses. The demands of a method often differ regarding in what context it will be applied. It may be acceptable for method to be laborious and time consuming if it is used in basic research, but for medical purposes molecular methods need to be fast and straightforward to perform. Innovative technologies should preferentially address the demands of both researchers and clinicians and provide data not possible to obtain by other methods. An example of such a method is the in situ proximity ligation assay (in situ PLA). In this thesis I have used this method to determine the activity status, at the single-cell level, of the transforming growth factor-β (TGF-β) signaling pathway and activating protein-1 (AP-1) family of transcription factors.  Both of these pathways are frequently involved in cancer development and progression. In addition to this research I herein also present further modifications of in situ PLA, and analyses thereof, to increase the utility and resolution of this assay.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. p. 43
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 763
Keywords
proximity ligation, TGF-β signaling, Smad, single cell analysis, cell cycle, context-dependent signaling, CRC
National Category
Cell and Molecular Biology Cancer and Oncology Biomedical Laboratory Science/Technology
Research subject
Molecular Cellbiology; Medicinal Chemistry; Molecular Biology
Identifiers
urn:nbn:se:uu:diva-171952 (URN)978-91-554-8336-4 (ISBN)
Public defence
2012-05-25, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-05-02 Created: 2012-03-29 Last updated: 2018-01-12Bibliographically approved
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