- Research article
- Open Access
Analyses of murine GBP homology clusters based on in silico, in vitro and in vivo studies
- Alexandra Kresse†1,
- Carolin Konermann†1,
- Daniel Degrandi1,
- Cornelia Beuter-Gunia1,
- Jan Wuerthner1,
- Klaus Pfeffer1Email author and
- Sandra Beer1Email author
© Kresse et al; licensee BioMed Central Ltd. 2008
- Received: 13 December 2007
- Accepted: 10 April 2008
- Published: 10 April 2008
The interactions between pathogens and hosts lead to a massive upregulation of antimicrobial host effector molecules. Among these, the 65 kDa guanylate binding proteins (GBPs) are interesting candidates as intricate components of the host effector molecule repertoire. Members of the GBP family are highly conserved in vertebrates. Previous reports indicate an antiviral activity of human GBP1 (hGBP1) and murine GBP2 (mGBP2). We recently demonstrated that distinct murine GBP (mGBP) family members are highly upregulated upon Toxoplasma gondii infection and localize around the intracellular protozoa T. gondii. Moreover, we characterised five new mGBP family members within the murine 65 kDa GBP family. Here, we identified a new mGBP locus named mGbp11. Based on bacterial artificial chromosome (BAC), expressed sequence tag (EST), and RT-PCR analyses this study provides a detailed insight into the genomic localization and organization of the mGBPs. These analyses revealed a 166-kb spanning region on chromosome 3 harboring five transcribed mGBPs (mGbp1, mGbp2, mGbp3, mGbp5, and mGbp7) and one pseudogene (pseudomGbp1), as well as a 332-kb spanning region on chromosome 5 consisting of six transcribed mGBPs (mGbp4, mGbp6, mGbp8, mGbp9, mGbp10, and mGbp11), and one pseudogene (pseudomgbp2). Besides the strikingly high homology of 65% to 98% within the coding sequences, the mGBPs on chromosome 5 cluster also exhibit a highly homologous exon-intron structure whereas the mGBP on chromosome 3 reveals a more divergent exon-intron structure. This study details the comprehensive genomic organization of mGBPs and suggests that a continuously changing microbial environment has exerted evolutionary pressure on this gene family leading to multiple gene amplifications. A list of links for this article can be found in the Availability and requirements section.
- Bacterial Artificial Chromosome
- Basic Local Alignment Search Tool
- Bacterial Artificial Chromosome Sequence
- Guanylate Binding Protein
- CaaX Motif
The guanylate binding proteins (GBPs) were first described in 1979 when Gupta et al. identified a 67 kDa protein induced in human fibroblasts after interferon γ (IFNγ) stimulation . Some years later, it was shown that two orthologous proteins are expressed in murine fibroblasts after stimulation with IFNγ . Besides the strong inducer IFNγ, the GBPs can also be induced by type I interferons [2–6], tumor-necrosis-factor α (TNF-α), interleukin-1β (IL-1β), IL-1α [7, 8], and TLR agonists .
Human and murine GBPs possess the unique ability to bind to agarose-immobilized GMP (guanosine monophosphate), GDP (guanosine diphosphate), and GTP (guanosine triphosphate) with the same affinity, thereby differing from heterotrimeric or Ras-like GTP-binding proteins . In addition, they hydrolyse GTP not only to GDP but also to GMP . Further biochemical properties of the GBPs are the low binding affinity to nucleotides, their stability in the absence of guanine nucleotides and their high turnover GTPase activity . Remarkably, the sequence of the common G4-motif N/TKxD is modified in the GBPs to the unique T(L/V)RD motif . In the case of hGBP1 a nucleotide-dependent oligomerization and concentration-dependent GTPase activity has been observed . These biochemical properties classified the GBPs as distantly related family members of the dynamin superfamily despite the lack of any sequence homology of the primary sequences [11, 12]. The similarity to the dynamin family is further corroborated by the analysis of the crystal structure of hGBP1. It has an amino-terminal globular domain containing the GTP binding region and an elongated carboxy-terminal series of α-helices. The GBPs possess the common 'dynamin domain structure' with a GTPase domain (~300 residues), a 'middle' or 'assembly' domain (150–200 residues) and a GTPase effector domain (~100 residues) .
Although, the GBPs have been discovered almost 30 years ago, only little is known about their biological function. It has been suggested that the GBPs are important for cell growth regulation as demonstrated for mGBP2 and hGBP1 [7, 13]. Both, hGBP1 and mGBP2 also alter matrix metalloproteinase (MMP) gene expression and thereby change cellular interactions with the extracellular environment . In addition, hGBP1 was reported to be involved with paclitaxel resistance in ovarian cancer cell lines . Further studies revealed that hGBP1 and mGBP2 exhibit a moderate antiviral activity against vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) [16, 17]. Recently, we have demonstrated that mGBPs are highly upregulated in mice after infection with Listeria monocytogenes or Toxoplasma gondii, and localize around the parasitophorous vacuole of T. gondii, thus suggesting that the mGBPs play a role in the defense against intracellular bacteria and apicomplexa .
Besides humans and mice the GBPs have been found in rats , chicken , fish , and several other vertebrate species. In humans seven orthologs and at least one pseudogene have been identified [21, 22]. In mice five GBPs have been described [2, 23, 24]. Recently, one additional mGBP was discovered by an in silico study . In search of new IFNγ regulated genes using Affymetrix analyses we independently identified mGbp6, mGbp7, and mGbp8 . Further comprehensive genome and sequence analyses yielded two more homologous genes, named mGbp9 and mGbp10 . The subsequent investigation of mGBP gene loci revealed an incorrect assembly concerning the mGbp8 locus in the genome databases (Ensembl, NCBI). Thus, to clarify the genomic organization of mGBP coding genes, we used BAC and EST sequences obtained from NCBI. Based on these sequence analyses we were able to identify another mGBP locus named mGbp11. In this study, we address the genomic organization and localization of each mGBP family member and present a revised assembly of the murine GBP homology clusters on chromosomes 3 and 5.
The mGBP genes are arranged in two clusters located on chromosomes 3 and 5
Further, we used the BACs RP24-63G23, RP23-329M7 and RP24-210D14 for analyses of the region between 105.25 MB and 105.58 MB on chromosome 5 (Fig. 1C). By means of these BAC sequences we were able to determine the precise loci for mGbp4, mGbp6 (formerly mpa2l), mGbp8, mGbp9, mGbp10, mGbp11, and the pseudogene pseudomGbp2. This cluster has approximately twice the size of the cluster on chromosome 3 with an extension of 332 kb. The length of each gene locus on chromosome 5 ranges from 23 kb (mGbp6) to 44 kb (pseudomGbp2). In contrast to the mGBPs located on chromosome 3 the mGBPs on chromosome 5 are all transcribed from the negative strand.
Conserved exon-intron structure in the mGBPs
The size of the first non-coding exon of the mGBPs on chromosome 3 ranges from 65 bp (mGBP1) to 205 bp (mGBP2) (Fig. 2A). For mGBP3 several alternative 5' UTR exons were identified. In the database 26 EST and cDNA sequences which cover the 5' UTR from mGBP3 were found: seven ESTs contain exon 1-Ia upstream of exon 2, three sequences harbor exon 1-Ia and 1-Ib in combination, 16 sequences comprise exon 1-II and only one EST was found having exon 1-III as a 5' non coding exon. For mGBP7 two different splice sites in the 5' UTR of exon 2 were observed as reported previously . In every mGBP locus the exons 3 (128 bp), exons 4 (110 bp), exons 5 (197 bp), exons 7 (281 bp), exons 8 (213 bp), exons 9 (103 bp), and exons 10 (194 bp) cover the same size, except mGBP5 which has an exon 5 with 200 bp instead of 197 bp and an exon 10 with 182 bp instead of 194 bp. Moreover, for mGBP5 an alternative splice form, mGBP5a, which lacks exons 3, exon 4, a part of exon 5, and possesses an exon 10a, has been described . Exons 6 of mGBP1, mGBP2, and mGBP5 consist of 243 bp whereas exons 6 of the other mGBPs span 246 bp.
Interestingly, the mGBPs on chromosome 5 show even higher similarities concerning the genomic organisation (Fig. 2B). The first exons of these mGBPs have nearly identical sizes (between 106 and 108 bp). Only for mGBP4 alternative 5' non-coding exons were observed (1-I, 1-II, and 1-III). EST sequences harboring either exon 1-I or exon 1-II as well as all three alternative 5' UTR exons were found in the database. Furthermore, we were able to identify an alternative splice form of mGBP4. Due to a mutation at the splice donor site in intron 2 two different transcripts, named mGBP4 and mGBP4.1, are generated . Except exon 3 of mGBP4, all mGBPs on chromosome 5 share identical sizes of the individual coding exons 3 to 10. Even the intron sequences are highly conserved in these genes. In particular, intron 4, intron 7, intron 8, intron 9, and intron 10 exhibit high similarities. Comparable to all other mGBPs the pseudogene pseudomGbp2 shows a highly conserved exon-intron structure. Yet, an inversion of 'exon 1' and 'exon 2' results in a non-functional locus. Taken together, due to their high structural homologies these loci seem to be duplicated quite recently.
In vitro and in vivo analyses of the new mGBP members
After determination of the precise exon-intron structure of each mGBP we confirmed the predicted sequences by cloning and sequencing the corresponding cDNAs out of IFNγ stimulated macrophages. These studies revealed that mGBP11 has a premature stop codon within exon 8 leading to an ORF sequence with only 1329 bp. The amino acid sequence and the GTP-binding motif are depicted in additional file 1.
Sequence alignments and homologies of the mGBPs
Comparison of the conserved G1, G2, G3, and G4 motifs of the mGBPs. The canonical GTP motifs of all mGBPs and the overall consensus sequence are shown. Amino acid substitutions are highlighted in bold.
GF YH TGKS
Percent identities of the mGBPs based on ORF sequences. For the calculation of percent identities a multiple sequence alignment with the ClustalW algorithm using MegAlign (DNAStar) was performed. Percent identities greater than 80% are shown in bold.
In order to find new IFNγ regulated host effector molecules we were able to identify and characterize five novel members of the mGBP family . Further, we showed that all hitherto identified members of the mGBP family are IFNγ induced and moreover are highly upregulated in mice after infection with L. monocytogene s or T. gondii. Furthermore, we demonstrated that in infected cells most mGBPs surround the parasitophorous vacuole of T. gondii . Consecutively, within this study, comprehensive homology and motif searches against public databases (NCBI, Ensembl) using EST and BAC sequences resolved the genomic organization and localization of the mGBPs in more detail. During these analyses we were able to identify the additional mGBP member mGBP11. The scope of this study was to determine the precise loci of the 11 mGBPs and of the two pseudogenes, to compare the structure and organization of the mGBPs, to compile all cDNA sequences, and to verify the expression of mGBP mRNAs.
Genomic organization of the mGBPs
The combined analyses revealed two mGBP homology clusters on chromosomes 3 and 5. One mGBP cluster is located within the H3 region on chromosome 3 which is in contrast to previously published data where the cluster was mapped to the H1 region . The second cluster is located in the E5 region on chromosome 5 which is in accordance to Olszewski et al. . Furthermore, Olszewski et al. noted that within the mGBP cluster on chromosome 5 the only functional mGBP gene is mGbp4 and that in addition three pseudogenes (pseudomGbp2, pseudomGbp3, and pseudomGbp4) are located on this chromosome. Our in silico and mRNA sequence analyses now clearly demonstrate that besides mGbp4 five expressed mGBPs (mGbp6, mGbp8, mGbp9, mGbp10, and mGbp11) are located on chromosome 5 (this study and ). In a previous report, we have shown that the mGbp4 locus does not encode for a complete mGBP4 protein . For pseudomGbp2 we could not find any corresponding EST sequence and were not able to clone a cDNA corresponding to pseudomGBP2, thus excluding the possibility of an alternative start downstream of exon 2. Based on BAC analyses we could demonstrate that the two pseudogenes pseudomGbp3 and pseudomGbp4 described by Olszewski et al. are both part of mGbp8 which was virtually disrupted by the Abcg3 gene locus in a former incorrect assembly within the public databases (see also ). Further transcript analyses revealed a functional mGbp8 locus and showed an IFNγ dependent upregulation of the mGBP8 mRNA comparable to mGBP6, mGBP9, mGBP10, and mGBP11. Similarly, all these mGBPs were highly induced upon L. monocytogenes infection in C57BL/6 mice (this study and ). The BAC sequences as well as our sequenced cDNA clones of the newly identified mGBP11 contain a premature stop codon within exon 8, leading to an ORF sequence of only 1329 bp. However, in the database (NCBI) one cDNA of mGBP11 without a premature stop was found (Acc. No. BC111039). It might be possible that the presence of different mGBP11 cDNAs is due to allelic variation. Further studies have to clarify, whether from this locus a functional protein can be translated and whether other mouse backgrounds differ in exon 8.
In a recently published report, some subtle differences to our extensive genomic analyses have been described . Firstly, in this report no mGBP7 gene is presented. Secondly, mGBP6 in  is termed mGBP7 based on our NCBI database submission in 2006 (BK005760, ). Thirdly, mGBP12 has been deposited in 2007 as mGBP11 by us (EU304258, this study). To keep consistency between database and nomenclature we propose to refer to the mGBP assignment in Figure 1. This is also in accordance with the extensive protein sequence and functional analyses which were provided previously . mGBP13 has been described as a pseudogene by Olszewski and here (see Fig. 1 pseudoGbp1). Unfortunately, no description of the methods used for the identification of the mGBP13 locus is given in . However, further studies are required to confirm whether this is a functional mGBP locus.
Besides the chromosomal localization of the mGBPs we also elucidated the exon-intron structure of these genes. Interestingly, all mGBPs consist of eleven exons with an impressively similar gene organization, with only one exception in mGbp8 which lacks exon 6. In addition, the translation of all mGBPs starts in exon 2. For mGbp3 and mGbp4 alternative non-coding exons were found in the 5' regions. The usage of different 5' exons may influence the stability of the mRNAs . Indeed, the frequencies of ESTs of mGBP3 and mGBP4 with the different alternative 5' exons are quite variable, so their functional significance has to be validated. It has been reported that mRNAs of genes with alternative 5' coding or non-coding exons are often expressed in a tissue-specific manner . Further studies will be necessary to verify a potential tissue-specific expression/regulation of the different mRNA isoforms of mGBP3 and mGBP4.
Evolution of the mGBPs
Recent data indicate that the GBPs are host effector molecules involved in pathogen defense [5, 16, 17]. Defense against pathogens requires permanent adaptation to the changes in pathogen virulence strategies [31, 32]. We suggest that evolutionary pressure led to gene duplication events which have resulted in the current mGBP clusters on chromosomes 3 and 5. It is most likely, that these gene duplications started with one primordial mGBP. We suppose that this ancestor mGBP is located on chromosome 3 because these mGBPs are more divergent among each other as compared to the mGBPs on chromosome 5. Interestingly, on chromosome 3 two homology clusters have evolved. One homology cluster with mGbp1, mGbp2, and mGbp5 is characterized by a C-terminal CaaX motif for isoprenylation and a 'TLRD' G4 motif. In contrast, the second homology cluster with mGbp3 and mGbp7 lacks the CaaX motif and possess a 'TVRD' G4 motif. This finding leads to the hypothesis, that mGbp3 or mGbp7 is the ancestor for all mGBPs on chromosome 5 which also lack a CaaX motif and have a "TVRD" G4 motif. This is further corroborated by the high cDNA sequence identities (around 70%) of mGBP3 and mGBP7 with mGBP4, mGBP6, mGBP8, mGBP9, mGBP10, and mGBP11 on chromosome 5. On this chromosome also the most recent duplication event occurred, where mGbp6 emanated from mGbp10 or vice versa. This is supported by the high homology of 98.4% between these two GTPases. Moreover, we suggest that the mGBP cluster on chromosome 5 is a "genomic hot spot" permanently exposed to genetic recombination events. Consistent with this suggestion, we detected a transposon-like element (>1000 bp) which is integrated several times in this mGBP cluster (data not shown). Further studies have to clarify whether these genes evolved due to evolutionary pressure and whether these genes have redundant or non-redundant functions during pathogen defense.
We have shown that mGBPs are highly induced upon IFNγ stimulation and infection with intracellular bacteria or protozoa indicating an important role as effector molecules in host defense. Now, we describe and characterize the genomic loci of the mGBPs on chromosomes 3 and 5. These data will be very important for the analyses of evolutionary gene amplifications required in host defense as well as for functional studies by the generation of gene targeted mice which are under way.
Determination of the genomic sequences and localization of the mGBPs
To analyse the chromosomal localization of the mGBPs the mouse genome research page on the Ensembl website http://www.ensembl.org, version 38 was used. The genomic sequences of the appropriate chromosomal segments were determined using bacterial artificial chromosome (BAC) sequences. To display all available BACs on the contig view the corresponding field was activated using the 'decorations' function. For this study we have chosen the sequences of the BAC clones RP23-100J23 and RP24-314I8 for chromosome 3 and RP24-63G23, RP23-329M7, RP23-152O10, and RP24-210D14 for chromosome 5. We obtained the BACs' genomic sequences from the website of the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov. The sequences were downloaded [Accession numbers: AC102108 (RP23-100J23), AC115865 (RP24-314I8), AC113980 (RP24-63G23), AC123697 (RP23-329M7), AC144914 (RP24-210D14), and AC162798 (RP23-152O10)] and imported into EditSeq (DNAStar, Madison, WI) for assembly into contigs using the SeqMan II program (DNAStar, Madison, WI). For further analyses expressed sequence tags (ESTs) of mGBPs were obtained with the Basic Local Alignment Search Tool (BLAST) 'blastn'  from the NCBI website and mapped onto the respective genomic area.
Identification of the exon-intron structure
The transcript sequences from mGBP4 (mpa-2) and mGBP6 (mpa-2l) were downloaded from the Ensembl website and imported into EditSeq. We then determined the exon-intron structure by using BLAST "align two sequences" using standard parameters on the NCBI website. The sequences of single exons from mGBP4 and mGBP6 were aligned with the corresponding BAC sequences. If the exons mapped to multiple regions on the BAC with high homology the regions were retained and imported into EditSeq. The equivalent sequences from the resulting BLAST hits on the BACs were subsequently aligned with MeqAlign (DNAStar, Madison, WI) using standard parameters. The 3' and 5' splice sites were identified manually by inspecting the alignment and confirmed by comparing the genomic sequences to the corresponding exon sequences. Once all exons were mapped on the genome the exons indicating a new gene locus were assembled and potential cDNAs were created. Finally, we determined the open reading frames by using the ORF search tool from EditSeq.
Alignment and phylogenetic tree
The alignment of mGBP cDNAs was created with the software ClustalW  and the subsequent layout was done with JalView http://www.jalview.org. The phylogenetic analysis was accomplished using the maximum likelihood method and treepuzzle http://www.tree-puzzle.de for construction of the phylogenetic tree. The treepuzzle software was run with the option for exact parameter estimates using the neighbor joining method. Finally, we used the software drawtree from the phylip package http://www.phylip.com to plot the tree data. All software was run on a Linux PC workstation.
Cell culture and stimulation
The macrophage cell line ANA-1  was cultured in very low endotoxin RPMI (Biochrom, Berlin, Germany) supplemented with 10% heat inactivated, low endotoxin fetal calf serum (Cambrex, Veniers, Belgium) and 50μM 2-β-Mercaptoethanol (Invitrogen, Karlsruhe, Germany).
For stimulation of ANA-1 cells we used 100 U/ml recombinant mouse IFNγ (R&D Systems, Mainz, Germany). After 16 h of IFNγ stimulation the cells were harvested for RNA preparation.
Infection with Listeria monocytogenes
C57BL/6N mice were purchased from Charles River (Sulzfeld, Germany) and maintained in the animal facility of the Medical Faculty of the Heinrich-Heine-University under SPF conditions. All procedures performed on animals in this study have been approved by the Animal Care and Use Committee of the local government of Duesseldorf and have been in accordance with the German animal laws. C57BL/6N mice were intraperitoneally infected with 0.1 × LD50 L. monocytogenes (American type culture collection strain 43251), and organs were removed 48 h after infection.
Amplification and cloning of mGBPs
Total RNA from cells and tissues was isolated using Trizol Reagent (Invitrogen) according to the manufacture's instructions. First-strand cDNA synthesis was performed using 1 μg of total RNA with M-MLV reverse transcriptase and oligo dT primer (Invitrogen). The subsequent PCR reactions were accomplished using specific forward and reverse primers (additional file 3), and sequencing for both DNA strands was done by GATC Biotech AG (Konstanz, Germany).
Accession numbers of EST and cDNA sequences
NCBI accession numbers of EST and cDNA sequences for all mGBPs are listed. The cDNA sequence numbers are shown in bold. In the case of mGBP6 and mGBP10 some ESTs could not be assigned to one individual GBP due to sequence identities and are therefore grouped together.
AW476703, BB033108, BI853322, BQ126171, BY575536, CA894196, CJ141369, EF494422, NM_010259
AV337765, BB840634, BI249610, BI790719, BM933217, BM935622, BQ550593, BQ550594, CB574409, CF583081, CF583082, CJ055311, CJ056573, CJ057752, NM_010260
AA162247, AA170007, AA276469, AA276918, AA289618, AA289780, AA839370, AW228655, BB862339, BE282356, BE847132, BG861498, BG861928, BG862944, BG864332, BG865051, BG914515, BG915574, BG973623, BI105618, BI653801, BI653963, BI654072, BI654363, BI656878, BI656930, BI567265, BI658478, BI661510, BM120304, BM206838, BM210659, BM221385, BM222892, BM240875, BM244805, BP760734, BQ552945, BQ552946, BX528767, BY215748, BY329622, BY483332, BY487480, BY562117, CA541279, CA544763, CA546244, CA573999, CA577912, CF910901, CK329532, CK331660, CK389037, CX205022, NM_018734, U44731, BC019195
BB859245, BI661547, BY225616, CJ141442, CJ141546, EF494424, NM_008620
BF138720, BI659458, BY749043, EF494423
AA170248, AA866719, AW228052, BE227153, BE336008, BI659458, BG864747, BG865054, BM244843, BM244986, CF911085
AI021670, AV329198, BB022173, BB617729, BB634786, BF163382, BG863163, BI558563, BI662495, BY212549, BY214522, BY220452, BY221363, BY224819, NM_153564
AK128993, BC057969, BC115768, BF015742, BF015763, BK005759
BI853721, BG915468, BI853566, DQ985743
mGBP6 + mGBP10
AA140542, AA267762, BE687038, BG915105, BG916153, BG916251, BM239887, BM241485, BM242358, BM243209, BP764987, BX525828, BY765347, CA572886, CD351560, CJ062488, CK331259, CN660654
AA200741, AI226719, AW106727, BB554853, BB666410, BF452604, BG914537, BI657558, BK005760, BP766023, BX513589, BX522404, BY016011, BY178861, BY197520, BY215691, BY221698, BY221980, BY223566BY742706, BY 761161, BY761419, BY765425, BY765812, CA574325, CA576100, CA885708, CF899303, CJ137202, CJ140108, CK342680, CK342922, CK343278, CX207094, CX219108, DV057094
AA155498, BE686748, BM245207, BM245316, BM246467, BQ552741, DQ295175, BY717902, BI657260, CJ141546, CJ141512, NM_029509
AA122564, BE650518, BE692183, BE849117, BG915970, BY747136, CA535632, CJ165022, CJ183419, CJ164864, BY060909, BY064961, DQ985742, NM_172777
AA087907, BC111039, BI145047, BI655333, BF470780, BI660636
To analyse the chromosomal localization of the mGBPs the mouse genome research page on the Ensembl website http://www.ensembl.org/Multi/newsview?rel=38 was used. We obtained the BACs' genomic sequences from the website of the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov. The alignment of mGBP cDNAs was created with the software ClustalW  and the subsequent layout was done with JalView http://www.jalview.org. The phylogenetic analysis was accomplished using the maximum likelihood method and treepuzzle http://www.tree-puzzle.de for construction of the phylogenetic tree. The treepuzzle software was run with the option for exact parameter estimates using the neighbor joining method. Finally, we used the software drawtree from the phylip package http://www.phylip.com to plot the tree data. All software was run on a Linux PC workstation.
We thank Gerrit Praefcke for helpful discussions. This work was supported by grants PF259/3-3, FOR729 and Leibniz (to K.P.) and the GRK1045/1 (to S.B. and K.P.) of the Deutsche Forschungsgemeinschaft (DFG) and MUGEN (to K.P.). The authors declare that they have no financial conflict of interests that might be construed to influence the results or interpretation of the manuscript.
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