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Publications (10 of 30) Show all publications
Guerriero, G., Silvestrini, L., Obersriebnig, M., Hausman, J.-F., Strauss, J. & Ezcurra, I. (2016). A WDR gene is a conserved member of a chitin synthase gene cluster and influences the cell wall in Aspergillus nidulans. International Journal of Molecular Sciences, 17(7), 1031
Open this publication in new window or tab >>A WDR gene is a conserved member of a chitin synthase gene cluster and influences the cell wall in Aspergillus nidulans
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2016 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 17, no 7, p. 1031-Article in journal (Refereed) Published
Abstract [en]

WD40 repeat (WDR) proteins are pleiotropic molecular hubs. We identify a WDR gene that is a conserved genomic neighbor of a chitin synthase gene in Ascomycetes. The WDR gene is unique to fungi and plants, and was called Fungal Plant WD (FPWD). FPWD is within a cell wall metabolism gene cluster in the Ascomycetes (Pezizomycotina) comprising chsD, a Chs activator and a GH17 glucanase. The FPWD, AN1556.2 locus was deleted in Aspergillus nidulans strain SAA.111 by gene replacement and only heterokaryon transformants were obtained. The re-annotation of Aspergilli genomes shows that AN1556.2 consists of two tightly linked separate genes, i.e., the WDR gene and a putative beta-flanking gene of unknown function. The WDR and the beta-flanking genes are conserved genomic neighbors localized within a recently identified metabolic cell wall gene cluster in genomes of Aspergilli. The heterokaryons displayed increased susceptibility to drugs affecting the cell wall, and their phenotypes, observed by optical, confocal, scanning electron and atomic force microscopy, suggest cell wall alterations. Quantitative real-time PCR shows altered expression of some cell wall-related genes. The possible implications on cell wall biosynthesis are discussed.

Place, publisher, year, edition, pages
MDPI AG, 2016
Keywords
Aspergillus nidulans; WDR gene; beta-flanking gene; chitin synthase; collinear genes; cell wall
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-199949 (URN)10.3390/ijms17071031 (DOI)000381500900046 ()2-s2.0-84976547527 (Scopus ID)
Note

QC 20170123

Available from: 2017-01-19 Created: 2017-01-19 Last updated: 2017-11-29Bibliographically approved
Wang, Y., Azhar, S., Gandini, R., Divne, C., Ezcurra, I. & Aspeborg, H. (2015). Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana. Plant Science, 241, 151-163
Open this publication in new window or tab >>Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana
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2015 (English)In: Plant Science, ISSN 0168-9452, E-ISSN 1873-2259, Vol. 241, p. 151-163Article in journal (Refereed) Published
Abstract [en]

Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5_7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50 kDa, a pH optimum of 5.5-6.0 and a temperature optimum of 25 degrees C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5_7 subfamily.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
Glycoside hydrolase, GH5, endo-β-1, 4-Mannan hydrolase, Cell wall, Mannan polysaccharides/oligosaccharides
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-178198 (URN)10.1016/j.plantsci.2015.10.002 (DOI)000367487500015 ()2-s2.0-84945291912 (Scopus ID)
Funder
Swedish Foundation for Strategic Research VINNOVASwedish Research Council Formas
Note

QC 20160104. QC 20160201

Available from: 2015-12-07 Created: 2015-12-07 Last updated: 2017-12-01Bibliographically approved
Ratke, C., Pawar, P.-A. M., Balasubramanian, V. K., Naumann, M., Duncranz, M. L., Derba-Maceluch, M., . . . Mellerowicz, E. J. (2015). Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification. Plant Biotechnology Journal, 13(1), 26-37
Open this publication in new window or tab >>Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification
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2015 (English)In: Plant Biotechnology Journal, ISSN 1467-7644, E-ISSN 1467-7652, Vol. 13, no 1, p. 26-37Article in journal (Refereed) Published
Abstract [en]

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.

Keywords
secondary wall, xylan biosynthesis, poplar, genetic engineering, transgenic trees, wood development
National Category
Other Biological Topics
Identifiers
urn:nbn:se:kth:diva-159364 (URN)10.1111/pbi.12232 (DOI)000346915000004 ()2-s2.0-84919847910 (Scopus ID)
Funder
Swedish Research Council FormasSwedish Research CouncilVINNOVASwedish Foundation for Strategic Research Bio4Energy
Note

QC 20150130

Available from: 2015-01-30 Created: 2015-01-29 Last updated: 2017-12-05Bibliographically approved
Derba-Maceluch, M., Awano, T., Takahashi, J., Lucenius, J., Ratke, C., Kontro, I., . . . Mellerowicz, E. J. (2015). Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood. New Phytologist, 205(2), 666-681
Open this publication in new window or tab >>Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood
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2015 (English)In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 205, no 2, p. 666-681Article in journal (Refereed) Published
Abstract [en]

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremulaxtremuloides).PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen.PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

Keywords
endotransglycosylase, growth stresses, hybrid aspen, Populus, secondary cell wall, wood formation, xylan, xylanase
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-160058 (URN)10.1111/nph.13099 (DOI)000346584600024 ()2-s2.0-84919632563 (Scopus ID)
Funder
Swedish Research Council FormasSwedish Research CouncilVINNOVAKnut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research
Note

QC 20150305

Available from: 2015-03-05 Created: 2015-02-13 Last updated: 2017-12-04Bibliographically approved
Guerriero, G., Hausman, J.-F. & Ezcurra, I. (2015). WD4O-Repeat Proteins in Plant Cell Wall Formation: Current Evidence and Research Prospects. Frontiers in Plant Science, 6, Article ID 1112.
Open this publication in new window or tab >>WD4O-Repeat Proteins in Plant Cell Wall Formation: Current Evidence and Research Prospects
2015 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, article id 1112Article in journal (Refereed) Published
Abstract [en]

The metabolic complexity of living organisms relies on supramolecular protein structures which ensure vital processes, such as signal transduction, transcription, translation and cell wall synthesis. In eukaryotes WD40-repeat (WDR) proteins often function as molecular "hubs" mediating supramolecular interactions. WDR proteins may display a variety of interacting partners and participate in the assembly of complexes involved in distinct cellular functions. In plants, the formation of lignocellulosic biomass involves extensive synthesis of cell wall polysaccharides, a process that requires the assembly of large transmembrane enzyme complexes, intensive vesicle trafficking, interactions with the cytoskeleton, and coordinated gene expression. Because of their function as supramolecular hubs, WDR proteins could participate in each or any of these steps, although to date only few WDR proteins have been linked to the cell wall by experimental evidence. Nevertheless, several potential cell wall-related WDR proteins were recently identified using in silico approaches, such as analyses of co-expression, interactome and conserved gene neighborhood. Notably, some WDR genes are frequently genomic neighbors of genes coding for GT2-family polysaccharide synthases in eukaryotes, and this WDR-GT2 collinear microsynteny is detected in diverse taxa. In angiosperms, two WDR genes are collinear to cellulose synthase genes, CesAs, whereas in ascomycetous fungi several WDR genes are adjacent to chitin synthase genes, chs. In this Perspective we summarize and discuss experimental and in silico studies on the possible involvement of WDR proteins in plant cell wall formation. The prospects of biotechnological engineering for enhanced biomass production are discussed.

Place, publisher, year, edition, pages
FRONTIERS MEDIA, 2015
Keywords
WDR proteins, protein-protein interaction, plant cell wall, genomic collinearity, lignocellulose
National Category
Plant Biotechnology
Identifiers
urn:nbn:se:kth:diva-180485 (URN)10.3389/fpls.2015.01112 (DOI)000366911800001 ()2-s2.0-84954070875 (Scopus ID)
Note

QC 20160118

Available from: 2016-01-18 Created: 2016-01-14 Last updated: 2017-11-30Bibliographically approved
Kaewthai, N., Gendre, D., Eklöf, J. M., Ibatullin, F. M., Ezcurra, I., Bhalerao, R. P. & Brumer, H. (2013). Group III-A XTH Genes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth. Plant Physiology, 161(1), 440-454
Open this publication in new window or tab >>Group III-A XTH Genes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth
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2013 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 161, no 1, p. 440-454Article in journal (Refereed) Published
Abstract [en]

The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin beta-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.

Keywords
Plant-Cell Walls, Pea Stem Segments, Enzymatic-Properties, Structural-Analysis, Beta-Galactosidase, Crystal-Structures, Flowering Plants, Hypocotyl Growth, Pichia-Pastoris, Hybrid Aspen
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-108074 (URN)10.1104/pp.112.207308 (DOI)000312964000035 ()2-s2.0-84871817930 (Scopus ID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilFormas
Note

QC 20130110

Available from: 2012-12-19 Created: 2012-12-19 Last updated: 2017-12-06Bibliographically approved
Guerriero, G., Spadiut, O., Kerschbamer, C., Giorno, F., Baric, S. & Ezcurra, I. (2012). Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: Partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A. Journal of Experimental Botany, 63(16), 6045-6056
Open this publication in new window or tab >>Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: Partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A
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2012 (English)In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 63, no 16, p. 6045-6056Article in journal (Refereed) Published
Abstract [en]

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus-domestica was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.-xdomestica are discussed.

Place, publisher, year, edition, pages
Oxford University Press, 2012
Keywords
Bicistronic mRNA, cellulose synthase, collinear genes, Malus-domestica, WD40 repeats, zinc motif
National Category
Botany
Identifiers
urn:nbn:se:kth:diva-105259 (URN)10.1093/jxb/ers255 (DOI)000309919500026 ()2-s2.0-84867543200 (Scopus ID)
Funder
VINNOVA
Note

QC 20121120

Available from: 2012-11-20 Created: 2012-11-19 Last updated: 2017-12-07Bibliographically approved
Ezcurra, I., Johansson, C., Tamizhselvan, P., Winzell, A. & Aspeborg, H. (2011). An AC-type element mediates transactivation of secondary cell wall carbohydrate-active enzymes by PttMYB021, the Populus MYB46 orthologue. BMC Proceedings, 5
Open this publication in new window or tab >>An AC-type element mediates transactivation of secondary cell wall carbohydrate-active enzymes by PttMYB021, the Populus MYB46 orthologue
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2011 (English)In: BMC Proceedings, ISSN 1753-6561, E-ISSN 1753-6561, Vol. 5Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-51358 (URN)10.1186/1753-6561-5-S7-O40 (DOI)
Note

QC 20111227

Available from: 2011-12-12 Created: 2011-12-12 Last updated: 2017-12-08Bibliographically approved
Winzell, A., Guerriero, G., Aspeborg, H., Wang, Y., Rajangam, A. S., Teeri, T. T. & Ezcurra, I. (2010). Biochemical characterization of family 43 glycosyltransferases in the Populus xylem: challenges and prospects. Paper presented at Colloquium on Green Chemistry in Plants and Microorganisms Japanese Soc Promot Sci, Stockholm, SWEDEN, MAY 25, 2009. Plant Biotechnology, 27(3), 283-288
Open this publication in new window or tab >>Biochemical characterization of family 43 glycosyltransferases in the Populus xylem: challenges and prospects
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2010 (English)In: Plant Biotechnology, ISSN 1342-4580, Vol. 27, no 3, p. 283-288Article in journal (Refereed) Published
Abstract [en]

Wood formation is a biological process of great economical importance. Genes active during the secondary cellwall formation of wood fibers from Populus tremulatremuloides were previously identified by expression profilingthrough microarray analyses. A number of these genes encode glycosyltransferases (GTs) with unknown substratespecificities. Here we report heterologous expression of one of these enzymes, PttGT43A, a putative IRREGULARXYLEM9 (IRX9) homologue. Expression trials in Pichia pastoris and insect cells revealed very low levels of accumulationof immunoreactive PttGT43A, whereas transient expression in Nicotiana benthamiana leaves by Agrobacterium infiltration(agroinfiltration) using a viral vector produced substantial amounts of protein that mostly precipitated in the crude pellet.Agroinfiltration induced weak endogenous xylosyltransferase activity in microsomal extracts, and transient PttGT43Aexpression further increased this activity, albeit only to low levels. PttGT43A may be inactive as an individual subunit,requiring complex formation with unknown partners to display enzymatic activity. Our results suggest that transient coexpressionin leaves of candidate subunit GTs may provide a viable approach for formation of an active xylanxylosyltransferase enzymatic complex.

Keywords
GT43 glycosyltransferase, IRX9, populus xylem, xylan, xylosyltransferase.
National Category
Botany
Identifiers
urn:nbn:se:kth:diva-14265 (URN)000280085800010 ()2-s2.0-77955322528 (Scopus ID)
Conference
Colloquium on Green Chemistry in Plants and Microorganisms Japanese Soc Promot Sci, Stockholm, SWEDEN, MAY 25, 2009
Funder
Swedish Foundation for Strategic Research
Note

QC 20110404

Available from: 2010-07-29 Created: 2010-07-29 Last updated: 2016-04-18Bibliographically approved
Winzell, A., Aspeborg, H., Wang, Y. & Ezcurra, I. (2010). Conserved CA-rich motifs in gene promoters of Pt x tMYB021-responsive secondary cell wall carbohydrate-active enzymes in Populus. Biochemical and Biophysical Research Communications - BBRC, 394(3), 848-853
Open this publication in new window or tab >>Conserved CA-rich motifs in gene promoters of Pt x tMYB021-responsive secondary cell wall carbohydrate-active enzymes in Populus
2010 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 394, no 3, p. 848-853Article in journal (Refereed) Published
Abstract [en]

In order to understand gene regulation during wood formation, we cloned a MYB46-like gene in hybrid aspen. Populus tremula x tremuloides, called PtxtMYB021 Phylogenetic and paired identity analysis of MYB46-like genes in Populus and Arabidopsis reveals relationships between paralogous pairs of Populus MYB46-like proteins and their Arabidopsis counterparts MYB46 and MYB83, and suggest that PtxtMYB021 is the ortholog of MYB46 Ptxt-MYB021 is expressed mainly in xylem tissues, and transiently expressed PtxtMYB46 transactivates gene promoters of xylan-active CAZymes GT43A, GT43B and Xyn10A Analysis of conserved motifs within these promoters identify the sequence CCACCAAC, called ACTYP, which is similar to the AC elements mediating transactivation by MYB transcription factors during lignin biosynthesis Further analysis by Motif Finder identifies four 6 bp CA-rich motifs overlapping ACTYP, and we show that these motifs are enriched in xylem-specific promoters We propose that AC-type regulatory elements mediate xylem-specific MYB46-dependent expression of secondary cell wall carbohydrate-active enzymes (CAZymes), besides activating gene expression of lignin biosynthesis enzymes.

Keywords
Populus, Xylem, Xylan, Transcription factor, MYB46, Regulatory elements
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-28291 (URN)10.1016/j.bbrc.2010.03.101 (DOI)000276785900070 ()2-s2.0-77950492955 (Scopus ID)
Funder
Formas
Note
QC 20110113Available from: 2011-01-13 Created: 2011-01-12 Last updated: 2017-12-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-9080-3031

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