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604 Notch Signaling Suppresses the Transcriptional Activity of Hath1 Gene, Resulting in the Undifferentiated Form of Human Intestinal Epithelial Cells
AGA Abstracts
et al, Mol Cell Biol 26:9060;2006). We thus hypothesized that the presence/absence of Gata4 in the small intestine defines the jejunal-ileal junction. To test this hypothesis, we characterized bile acid absorption in mice in which VillinCreERT2-mediated recombination results in inducible, intestine-specific deletion of Gata4, and expression of placental alkaline phosphatase (Ap) under the control of the endogenous Gata4 promoter (Gata4 null). Wholemount AP staining demonstrated an intestinal pattern that mimics that of Gata4 and revealed a sharp demarcation localized at the 85% position along the length of the small intestine. Real-time RT-PCR analyses showed that Asbt mRNA, which is normally undetectable in duodenum and jejunum, was induced in jejunum of Gata4 mutant mice (10% of ileal levels), and duodenum (15% of ileal levels) and jejunum (100% of ileal levels) of Gata4 null mice. Ileal lipid binding protein (Ilbp) mRNA was also induced in jejunum of Gata4 mutant (40% of ileal levels) and null (90% of ileal levels) mice, revealing that Asbt is not the only target repressed by Gata4. Fecal bile acid excretion in Gata4 mutant mice was decreased 90% (P<0.001) as compared to controls, demonstrating a significant increase in bile acid absorption. Expression of fibroblast growth factor-15, a key component of a gut-liver signaling pathway that is activated in absorptive enterocytes by the influx of bile acids, was induced in Gata4 null mice in proximal segments, but reduced in distal segments, consistent with absorption of bile acids in the proximal intestine and a depletion of luminal bile acids in distal intestine. Conclusion: The absence of Gata4 expression in the small intestine defines the region in which bile acid absorption occurs. We propose that the sharp demarcation in Gata4 presence/absence defines the jejunal-ileal junction in mammals.
605 Signaling Through Fibroblast Growth Factor Receptor 3 Modulates Tcf4/βCatenin Activation and Paneth Cell Lineage Allocation in Intestinal Crypt Epithelial Cells Alda Vidrich, Brooks Brodrick, Jenny M. Buzan, Steven M. Cohn Fibroblast growth factor receptors are a family of receptor tyrosine kinases that mediate cellular growth and differentiation in many cell types during normal development and injuryrepair. FGF receptor-3 (FGFR3) expression is restricted to the undifferentiated epithelial cells within the lower portion of the crypt during normal intestinal development. Activation of the Tcf4/β-catenin transcription complex is crucial for maintaining the intestinal stem cell compartment and for the maturation of Paneth cells. Previously we showed that FGFR3-/mice display fewer numbers of crypts and have reduced numbers of clonogenic stem cells per crypt compared to wild-type (wt) mice. FGFR3-/- mice were also deficient in Tcf4/βcatenin activation. Since prior studies indicate that crypt stem cell replication and Paneth cell maturation are dependent on Tcf/β-catenin activation, we examined whether Tcf4/βcatenin activation was linked to FGFR3 signaling and whether secretory cell lineage allocation/ differentiation was perturbed in FRFR3-/- mice. Methods: Regional patterns of epithelial differentiation were examined from birth through day 21 by immunohistochemistry, using a variety of markers that distinguish the various intestinal cell lineages, and by real-time RT-PCR to determine mRNA levels for specific differentiated cell products. Results: Caco2 cells transfected with a constitutively active FGFR3 construct maintain elevated levels of Tcf4 activity after reaching confluence compared to untransfected controls. All of the principal differentiated cell types were present in both the small intestinal epithelium and colon of FGFR3-/- mice. The timing of initial appearance and number of goblet cells and neuroendocrine cells containing PYY, serotonin, and secretin present in the small intestine and colon were similar between FGFR3-/- and wt mice. Paneth cells first appeared at post natal day 14 in both FGFR3-/- and wt mice. However, FGFR3-/- mice displayed a significant reduction in the number of Paneth cells throughout the small intestine by day 21. The mRNA levels of several Paneth cell products were also significantly reduced in FGFR3-/- mice (lysozyme: ko, 2.9±0.36 x10-3 vs wt, 1.2±0.25 x10-2 ; cryptdin 5: ko, 6.1±1.5 x10-4 vs wt, 2.0±0.14 x10-3). There were no detectable differences in the expression of Goblet or endocrine cellspecific markers between FGFR3-/- and wt mice. Conclusion: FGFR3-/- mice have a deficit only in Paneth cell maturation/numbers suggesting that signaling through FGFR3 specifically modulates Paneth cell lineage allocation possibly through the Tcf/β-catenin signaling complex.
603 Notch Signaling in Pancreatic Islet Precursor Cells Inhibits Endocrine Differentiation and Favors a Duct Cell Fate Archana Kapoor, Maryann Giel-Moloney, Guido Rindi, Andrew B. Leiter Notch signaling plays an important role in enabling adjacent cells to adopt alternate cell fates. The basic helix loop helix (bHLH) transcription factor neurogenin3 (Ngn3) appears to initiate islet cell fate determination in the pancreas and subsequently activates expression of a second bHLH protein, NeuroD, which is required for terminal differentiation. Increased notch signaling blocks endocrine differentiation early in pancreagenesis by inhibiting the function of both Ngn3 and NeuroD, whereas, loss of Notch is associated with expanded endocrine differentiation from undifferentiated embryonic foregut. The aim of the present study was to determine at what stages of pancreatic islet differentiation endocrine precursor cells respond to notch signals. We crossed mice expressing Cre recombinase under control of either the Ngn3 or NeuroD gene with ROSA-Notch mice, a line containing a bicistronic transgene with the Notch 1 intracellular domain (NICD) and EGFP inserted into the ROSA26 locus. Transcription read through of the transgene was prevented by an upstream stop sequence flanked by loxP sites. Expression of Cre recombinase results in excision of the stop sequence, allowing conditional expression of the effector intracellular domain of Notch 1 and EGFP in islet precursor cells at either an early (Ngn3) or late (NeuroD) stage of differentiation. Ngn3-Cre;ROSA-Notch mice failed to develop pancreatic islets and died within 2 days of birth with severe diabetes in contrast to an earlier study showing lethality at embryonic day 13.5. Further examination revealed an increased number of small ducts, as well as rare single endocrine cells, most of which expressed the Notch transgene. As we previously reported, activation of the notch pathway at a later developmental stage in NeuroD+ cells of mice did not result in diabetes. NeuroD-Cre ;ROSA26-Notch mice showed mislocalization of non-beta cells to the islet core and arrested differentiation of islet cells with loss of endocrine marker expression. Mice showed increased numbers of large and small ducts expressing the Notch transgene in close association with islets. Since lineage tracing indicates that NeuroD+ cells are exclusively committed to become endocrine cells, Notch misexpression appears to induce cell fate switching of endocrine precursors to duct cells in the pancreas. We conclude that Notch signaling has striking effects on the differentiation of the pancreatic endocrine lineages that depend on the timing of the notch signal and confirm the importance of timing observed in invertebrates.
606 KLF4 Is a Downstream Target of the Notch Signaling Pathway That Controls Goblet Cell Differentiation in the Mouse GI Tract Xiangdong Yang, David M. Pritchard, Shigeo Takaishi, shuiping Tu, Frédéric Marrache, Shanisha A. Gordon, Timothy C. Wang, Walden Ai Introduction: In KLF4 deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. Aim: To examine whether Notch represses KLF4 gene expression to regulate goblet cell differentiation. Methods: A transgenic mouse model was generated (KLF4/EGFP mice) where EGFP is under the control of large pieces of 5' (~119kb) and 3' (~82kb) fragments of the KLF4 gene using a KLF4 gene containing bacterial artificial chromosome (RP23-322L22). A gamma-secretase inhibitor (CompE) was used to inhibit the Notch signaling pathway In Vivo. Tissue sections, RNA and protein samples were prepared from transgenic mice, followed by immunohistochemical, quantitative RT-PCR and Western blotting analyses. Promoter activities of KLF4 were assessed by transient transfection and dual luciferase assays. Electrophoretic mobility shift assays (EMSAs) were used to examine direct binding of FoxD3 with the KLF4 promoter region. Results: EGFP signal in KLF4/EGFP transgenic mice was detected in the stomach, small intestine, colon, and other tissues. Specificity of EGFP signal was confirmed by immunostaining with both anti-KLF4 and anti-GFP antibodies. Inhibition of the Notch signaling pathway by 5 day-CompE treatment (10µmol/kg) significantly increased goblet cells and EGFP positive cells in KLF4/GFP mice. One day CompE treatment, that did not induce significant morphological changes in mouse small intestine or colon, increased both EGFP and KLF4 positive cells in the crypts, accompanied by elevated mRNA levels of KLF4 and EGFP in mouse small intestine and colon. In a colon cancer cell line (HCT116), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1). An ICN1 responsive element was then mapped in human KLF4 promoter. Online search of transcription factor database and EMSAs suggested that FoxD3 directly binds this element. Further mutation analysis and reporter assays indicated that an intact FoxD3 binding site was required for ICN1 to inhibit KLF4 promoter activity. In a FoxD3 expressing cell line (N-Tera2 cells), overexpression of FoxD3 increased KLF4 mRNA levels. Consistently, downregulation of FoxD3 by siRNA decreased KLF4 mRNA levels. Conclusions: KLF4 is inhibited by the Notch signaling pathway that regulates the development of mouse goblet cells. FoxD3 may function as an important mediator of KLF4 inhibition by the Notch signaling pathway.
604 Notch Signaling Suppresses the Transcriptional Activity of Hath1 Gene, Resulting in the Undifferentiated Form of Human Intestinal Epithelial Cells Kiichiro Tsuchiya, Kazunari Inoue, Mikayo Arakaki, Ryuichi Okamoto, Tetsuya Nakamura, Takanori Kanai, Mamoru Watanabe Background & Aims: Notch-HES1 signaling regulates cell fate decision and proliferation of progenitor cells in intestine. In mice model, Notch signaling and Hes1 results in the decrease of Math1 gene expression inducing the decrease of differentiated cells. However, it remains unknown how Notch-HES1 signaling suppresses Atoh1 gene expression, moreover it remains unknown whether Notch signaling suppresses the gene expression of Atoh1 human homolog; Hath1 in human intestine. Methods: Immunohistochemical analysis using surgically ressected human intestinal tissues was performed to determine expression and localization of cleaved Notch1(NICD) and Hath1. Cell-based functional assays were performed using human IECderived cells in which forced expression of NICD or HES1 could be induced by Tet-on system. The transcriptional activity of Hath1 was analyzed using 5' region of Hath1 genome constructed into luciferase plasmid. Results: Immunohistochemical examination of normal human intestinal tissues showed reciprocal expression between NICD and Hath1 in the epithelial cells. NICD was expressed in Ki67 positive cells at crypt and Hath1 was expressed in MUC2 positive cells at villus respectively. The expression of either NICD or HES1 in LS174T cells significantly decreased the transcriptional activity and gene expression of Hath1, causing the decrease of MUC2 expression. Furthermore, only the treatment with Hath1 siRNA significantly down-regulated MUC2 gene expression without Notch activation in LS174T cells. Conclusions: These results suggest that Notch activation within human intestinal epithelial cells functions as a transcriptional repressor of Hath1 gene, which has a central role in keeping the undifferentiated form of intestinal epithelial cells during the proliferation status.
AGA Abstracts
607 PDX1 Inactivation Restricted to the Intestinal Epithelium in Mice Alters Duodenal Gene Expression in Enterocytes, Paneth and Enteroendocrine Cells Chin Chen, Rixun Fang, Corrine Davis, Eric Sibley Background: Null mutant mice lacking transcription factor pancreatic and duodenal homeobox 1 (Pdx1) are apancreatic and survive only a few days after birth. The role of Pdx1 in regulating intestinal gene expression has therefore yet to be determined in viable mice with normal pancreatic development. Objective: We aimed to characterize In Vivo the effects of Pdx1 inactivation restricted to intestinal epithelium on duodenal morphology and gene expression. Methods: Pdx1flox/flox;VilCre mice with intestine-specific Pdx1 inactivation were generated by crossing a transgenic mouse strain (VilCre) expressing Cre recombinase driven by a mouse villin 1 gene promoter fragment, with a mutant mouse strain (Pdx1flox/flox)
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et al, Mol Cell Biol 26:9060;2006). We thus hypothesized that the presence/absence of Gata4 in the small intestine defines the jejunal-ileal junction. To test this hypothesis, we characterized bile acid absorption in mice in which VillinCreERT2-mediated recombination results in inducible, intestine-specific deletion of Gata4, and expression of placental alkaline phosphatase (Ap) under the control of the endogenous Gata4 promoter (Gata4 null). Wholemount AP staining demonstrated an intestinal pattern that mimics that of Gata4 and revealed a sharp demarcation localized at the 85% position along the length of the small intestine. Real-time RT-PCR analyses showed that Asbt mRNA, which is normally undetectable in duodenum and jejunum, was induced in jejunum of Gata4 mutant mice (10% of ileal levels), and duodenum (15% of ileal levels) and jejunum (100% of ileal levels) of Gata4 null mice. Ileal lipid binding protein (Ilbp) mRNA was also induced in jejunum of Gata4 mutant (40% of ileal levels) and null (90% of ileal levels) mice, revealing that Asbt is not the only target repressed by Gata4. Fecal bile acid excretion in Gata4 mutant mice was decreased 90% (P<0.001) as compared to controls, demonstrating a significant increase in bile acid absorption. Expression of fibroblast growth factor-15, a key component of a gut-liver signaling pathway that is activated in absorptive enterocytes by the influx of bile acids, was induced in Gata4 null mice in proximal segments, but reduced in distal segments, consistent with absorption of bile acids in the proximal intestine and a depletion of luminal bile acids in distal intestine. Conclusion: The absence of Gata4 expression in the small intestine defines the region in which bile acid absorption occurs. We propose that the sharp demarcation in Gata4 presence/absence defines the jejunal-ileal junction in mammals.
605 Signaling Through Fibroblast Growth Factor Receptor 3 Modulates Tcf4/βCatenin Activation and Paneth Cell Lineage Allocation in Intestinal Crypt Epithelial Cells Alda Vidrich, Brooks Brodrick, Jenny M. Buzan, Steven M. Cohn Fibroblast growth factor receptors are a family of receptor tyrosine kinases that mediate cellular growth and differentiation in many cell types during normal development and injuryrepair. FGF receptor-3 (FGFR3) expression is restricted to the undifferentiated epithelial cells within the lower portion of the crypt during normal intestinal development. Activation of the Tcf4/β-catenin transcription complex is crucial for maintaining the intestinal stem cell compartment and for the maturation of Paneth cells. Previously we showed that FGFR3-/mice display fewer numbers of crypts and have reduced numbers of clonogenic stem cells per crypt compared to wild-type (wt) mice. FGFR3-/- mice were also deficient in Tcf4/βcatenin activation. Since prior studies indicate that crypt stem cell replication and Paneth cell maturation are dependent on Tcf/β-catenin activation, we examined whether Tcf4/βcatenin activation was linked to FGFR3 signaling and whether secretory cell lineage allocation/ differentiation was perturbed in FRFR3-/- mice. Methods: Regional patterns of epithelial differentiation were examined from birth through day 21 by immunohistochemistry, using a variety of markers that distinguish the various intestinal cell lineages, and by real-time RT-PCR to determine mRNA levels for specific differentiated cell products. Results: Caco2 cells transfected with a constitutively active FGFR3 construct maintain elevated levels of Tcf4 activity after reaching confluence compared to untransfected controls. All of the principal differentiated cell types were present in both the small intestinal epithelium and colon of FGFR3-/- mice. The timing of initial appearance and number of goblet cells and neuroendocrine cells containing PYY, serotonin, and secretin present in the small intestine and colon were similar between FGFR3-/- and wt mice. Paneth cells first appeared at post natal day 14 in both FGFR3-/- and wt mice. However, FGFR3-/- mice displayed a significant reduction in the number of Paneth cells throughout the small intestine by day 21. The mRNA levels of several Paneth cell products were also significantly reduced in FGFR3-/- mice (lysozyme: ko, 2.9±0.36 x10-3 vs wt, 1.2±0.25 x10-2 ; cryptdin 5: ko, 6.1±1.5 x10-4 vs wt, 2.0±0.14 x10-3). There were no detectable differences in the expression of Goblet or endocrine cellspecific markers between FGFR3-/- and wt mice. Conclusion: FGFR3-/- mice have a deficit only in Paneth cell maturation/numbers suggesting that signaling through FGFR3 specifically modulates Paneth cell lineage allocation possibly through the Tcf/β-catenin signaling complex.
603 Notch Signaling in Pancreatic Islet Precursor Cells Inhibits Endocrine Differentiation and Favors a Duct Cell Fate Archana Kapoor, Maryann Giel-Moloney, Guido Rindi, Andrew B. Leiter Notch signaling plays an important role in enabling adjacent cells to adopt alternate cell fates. The basic helix loop helix (bHLH) transcription factor neurogenin3 (Ngn3) appears to initiate islet cell fate determination in the pancreas and subsequently activates expression of a second bHLH protein, NeuroD, which is required for terminal differentiation. Increased notch signaling blocks endocrine differentiation early in pancreagenesis by inhibiting the function of both Ngn3 and NeuroD, whereas, loss of Notch is associated with expanded endocrine differentiation from undifferentiated embryonic foregut. The aim of the present study was to determine at what stages of pancreatic islet differentiation endocrine precursor cells respond to notch signals. We crossed mice expressing Cre recombinase under control of either the Ngn3 or NeuroD gene with ROSA-Notch mice, a line containing a bicistronic transgene with the Notch 1 intracellular domain (NICD) and EGFP inserted into the ROSA26 locus. Transcription read through of the transgene was prevented by an upstream stop sequence flanked by loxP sites. Expression of Cre recombinase results in excision of the stop sequence, allowing conditional expression of the effector intracellular domain of Notch 1 and EGFP in islet precursor cells at either an early (Ngn3) or late (NeuroD) stage of differentiation. Ngn3-Cre;ROSA-Notch mice failed to develop pancreatic islets and died within 2 days of birth with severe diabetes in contrast to an earlier study showing lethality at embryonic day 13.5. Further examination revealed an increased number of small ducts, as well as rare single endocrine cells, most of which expressed the Notch transgene. As we previously reported, activation of the notch pathway at a later developmental stage in NeuroD+ cells of mice did not result in diabetes. NeuroD-Cre ;ROSA26-Notch mice showed mislocalization of non-beta cells to the islet core and arrested differentiation of islet cells with loss of endocrine marker expression. Mice showed increased numbers of large and small ducts expressing the Notch transgene in close association with islets. Since lineage tracing indicates that NeuroD+ cells are exclusively committed to become endocrine cells, Notch misexpression appears to induce cell fate switching of endocrine precursors to duct cells in the pancreas. We conclude that Notch signaling has striking effects on the differentiation of the pancreatic endocrine lineages that depend on the timing of the notch signal and confirm the importance of timing observed in invertebrates.
606 KLF4 Is a Downstream Target of the Notch Signaling Pathway That Controls Goblet Cell Differentiation in the Mouse GI Tract Xiangdong Yang, David M. Pritchard, Shigeo Takaishi, shuiping Tu, Frédéric Marrache, Shanisha A. Gordon, Timothy C. Wang, Walden Ai Introduction: In KLF4 deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. Aim: To examine whether Notch represses KLF4 gene expression to regulate goblet cell differentiation. Methods: A transgenic mouse model was generated (KLF4/EGFP mice) where EGFP is under the control of large pieces of 5' (~119kb) and 3' (~82kb) fragments of the KLF4 gene using a KLF4 gene containing bacterial artificial chromosome (RP23-322L22). A gamma-secretase inhibitor (CompE) was used to inhibit the Notch signaling pathway In Vivo. Tissue sections, RNA and protein samples were prepared from transgenic mice, followed by immunohistochemical, quantitative RT-PCR and Western blotting analyses. Promoter activities of KLF4 were assessed by transient transfection and dual luciferase assays. Electrophoretic mobility shift assays (EMSAs) were used to examine direct binding of FoxD3 with the KLF4 promoter region. Results: EGFP signal in KLF4/EGFP transgenic mice was detected in the stomach, small intestine, colon, and other tissues. Specificity of EGFP signal was confirmed by immunostaining with both anti-KLF4 and anti-GFP antibodies. Inhibition of the Notch signaling pathway by 5 day-CompE treatment (10µmol/kg) significantly increased goblet cells and EGFP positive cells in KLF4/GFP mice. One day CompE treatment, that did not induce significant morphological changes in mouse small intestine or colon, increased both EGFP and KLF4 positive cells in the crypts, accompanied by elevated mRNA levels of KLF4 and EGFP in mouse small intestine and colon. In a colon cancer cell line (HCT116), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1). An ICN1 responsive element was then mapped in human KLF4 promoter. Online search of transcription factor database and EMSAs suggested that FoxD3 directly binds this element. Further mutation analysis and reporter assays indicated that an intact FoxD3 binding site was required for ICN1 to inhibit KLF4 promoter activity. In a FoxD3 expressing cell line (N-Tera2 cells), overexpression of FoxD3 increased KLF4 mRNA levels. Consistently, downregulation of FoxD3 by siRNA decreased KLF4 mRNA levels. Conclusions: KLF4 is inhibited by the Notch signaling pathway that regulates the development of mouse goblet cells. FoxD3 may function as an important mediator of KLF4 inhibition by the Notch signaling pathway.
604 Notch Signaling Suppresses the Transcriptional Activity of Hath1 Gene, Resulting in the Undifferentiated Form of Human Intestinal Epithelial Cells Kiichiro Tsuchiya, Kazunari Inoue, Mikayo Arakaki, Ryuichi Okamoto, Tetsuya Nakamura, Takanori Kanai, Mamoru Watanabe Background & Aims: Notch-HES1 signaling regulates cell fate decision and proliferation of progenitor cells in intestine. In mice model, Notch signaling and Hes1 results in the decrease of Math1 gene expression inducing the decrease of differentiated cells. However, it remains unknown how Notch-HES1 signaling suppresses Atoh1 gene expression, moreover it remains unknown whether Notch signaling suppresses the gene expression of Atoh1 human homolog; Hath1 in human intestine. Methods: Immunohistochemical analysis using surgically ressected human intestinal tissues was performed to determine expression and localization of cleaved Notch1(NICD) and Hath1. Cell-based functional assays were performed using human IECderived cells in which forced expression of NICD or HES1 could be induced by Tet-on system. The transcriptional activity of Hath1 was analyzed using 5' region of Hath1 genome constructed into luciferase plasmid. Results: Immunohistochemical examination of normal human intestinal tissues showed reciprocal expression between NICD and Hath1 in the epithelial cells. NICD was expressed in Ki67 positive cells at crypt and Hath1 was expressed in MUC2 positive cells at villus respectively. The expression of either NICD or HES1 in LS174T cells significantly decreased the transcriptional activity and gene expression of Hath1, causing the decrease of MUC2 expression. Furthermore, only the treatment with Hath1 siRNA significantly down-regulated MUC2 gene expression without Notch activation in LS174T cells. Conclusions: These results suggest that Notch activation within human intestinal epithelial cells functions as a transcriptional repressor of Hath1 gene, which has a central role in keeping the undifferentiated form of intestinal epithelial cells during the proliferation status.
AGA Abstracts
607 PDX1 Inactivation Restricted to the Intestinal Epithelium in Mice Alters Duodenal Gene Expression in Enterocytes, Paneth and Enteroendocrine Cells Chin Chen, Rixun Fang, Corrine Davis, Eric Sibley Background: Null mutant mice lacking transcription factor pancreatic and duodenal homeobox 1 (Pdx1) are apancreatic and survive only a few days after birth. The role of Pdx1 in regulating intestinal gene expression has therefore yet to be determined in viable mice with normal pancreatic development. Objective: We aimed to characterize In Vivo the effects of Pdx1 inactivation restricted to intestinal epithelium on duodenal morphology and gene expression. Methods: Pdx1flox/flox;VilCre mice with intestine-specific Pdx1 inactivation were generated by crossing a transgenic mouse strain (VilCre) expressing Cre recombinase driven by a mouse villin 1 gene promoter fragment, with a mutant mouse strain (Pdx1flox/flox)
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