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Phosphatidylinositol 3-Kinase Controls Human Intestinal Epithelial Cell Differentiation by Promoting Adherens Junction Assembly and p38 MAPK Activation*

  • Patrick Laprise
    Footnotes
    Affiliations
    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Pierre Chailler
    Affiliations
    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Mathieu Houde
    Affiliations
    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Jean-François Beaulieu
    Affiliations
    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Marie-Josée Boucher
    Affiliations
    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Nathalie Rivard
    Correspondence
    Scholar from the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed. Tel.: 819-564-5271; Fax: 819-564-5320
    Affiliations
    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
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  • Author Footnotes
    * This work was supported by Canadian Institutes of Health Research Grants MT-14405 and GR-15186.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Student Scholar from the Fonds de la Recherche en Santé du Québec.
Open AccessPublished:December 27, 2001DOI:https://doi.org/10.1074/jbc.M110235200
      The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.
      MAPK
      mitogen-activated protein kinase
      PI3K
      phosphatidylinositol 3-kinase
      ZO-1
      zonula occludens-1
      PBS
      phosphate-buffered saline
      GFP
      green fluorescent protein
      PIPES
      1,4-piperazinediethanesulfonic acid
      APC
      adenomatous polyposis coli
      The epithelium of the small intestine is characterized by its rapid and constant renewal. This process involves cell generation and migration from the stem cell population located at the bottom of the crypt to the extrusion of terminally differentiated cells at the tip of the villus (
      • Babyatsky M.W.
      • Podolsky D.K.
      ). Thus, the crypt is mainly composed of proliferative and poorly differentiated cells, whereas the villus is lined with functional absorptive, goblet, and endocrine cells (
      • Babyatsky M.W.
      • Podolsky D.K.
      ). The molecular and cellular mechanisms responsible for the fine coordination between proliferation, migration, and differentiation along the crypt-villus axis are still largely unknown.
      E-cadherin-mediated cell-cell attachment plays an important role in the differentiation, polarization, and homeostasis of many epithelia (
      • Takeichi M.
      ,
      • Larue L.
      • Antos C.
      • Butz S.
      • Huber O.
      • Delmas V.
      • Dominis M.
      • Kemler R.
      ,
      • Vleminckz K.
      • Kemler R.
      ). Cadherins are responsible for cell-cell adhesion through a calcium-dependent interaction of their extracellular domains. Their cytoplasmic tails are linked to the cytoskeleton through a complex of proteins that includes α-, β-, and γ-catenins. This link is involved in the strengthening of cell-cell adhesion and in the cohesion of epithelial tissues (
      • Jou T.S.
      • Stewart D.B.
      • Stappert J.
      • Nelson W.J.
      • Mars J.A.
      ). The importance of cadherins in the renewal of the intestinal epithelium has been demonstrated in vivo in two mouse models. Overexpression of E-cadherin in the crypts of the small intestine reduces cell proliferation and migration (
      • Hermiston M.L.
      • Gordon J.I.
      ). Conversely, expression of a dominant-negative N-cadherin leads to over-proliferation, uncoordinated differentiation, and a Crohn's disease phenotype (
      • Hermiston M.L.
      • Gordon J.I.
      ).
      The intracellular signaling pathways that transmit extracellular cues for epithelial differentiation along the crypt-villus axis of the intestine remain poorly defined. We recently reported that p38 MAPK1 plays a crucial role in intestinal epithelial cell differentiation by enhancing the transactivation capacity of CDX2 (
      • Houde M.
      • Laprise P.
      • Jean D.
      • Blais M.
      • Asselin C.
      • Rivard N.
      ), an intestine-specific homeobox gene product well known for its broad effect on enterocyte differentiation (
      • Traber P.G.
      • Silberg D.G.
      ). However, the upstream signaling pathways activating p38 MAPK in committed intestinal cells induced to differentiate remain to be defined. Interestingly, in vitroexperiments have shown that the establishment of cell-cell contacts in intestinal cell cultures could be a critical step in initiating p38 MAPK action (
      • Houde M.
      • Laprise P.
      • Jean D.
      • Blais M.
      • Asselin C.
      • Rivard N.
      ), cell cycle arrest (
      • Evers B.M.
      • Ko T.C.
      • Li J.
      • Thompson E.A.
      ,
      • Deschênes C.
      • Vézina A.
      • Beaulieu J.-F.
      • Rivard N.
      ), and induction of the differentiation process (
      • Houde M.
      • Laprise P.
      • Jean D.
      • Blais M.
      • Asselin C.
      • Rivard N.
      ,
      • Pinto M.
      • Robine-Leon S.
      • Appay M.D.
      • Kedinger M.
      • Triadou N.
      • Bussaulx N.
      • Lacroix B.
      • Simon-Assman P.
      • Haffen K.
      • Fogh J.
      • Zweibaum A.
      ,
      • Vachon P.H.
      • Beaulieu J.-F.
      ,
      • Peterson M.D.
      • Bement W.M.
      • Mooseker M.S.
      ,
      • Aliaga J.C.
      • Deschênes C.
      • Beaulieu J.-F.
      • Calvo E.L.
      • Rivard N.
      ).
      An important role for p38 MAPK in various mammalian cell differentiation processes has recently been proposed (
      • Nebrada A.R.
      • Porras A.
      ). For instance, differentiation of C2C12 and L8 myoblasts into myotubes has been found to be mediated by p38 activation (
      • Cuenda A.
      • Cohen P.
      ,
      • Zetser A.
      • Gredinger E.
      • Bengal E.
      ). Although this skeletal muscle differentiation requires phosphatidylinositol 3-kinase (PI3K), it is not yet clear whether PI3K and p38 MAPK act in a common pathway (
      • Chun Y.K.
      • Kim J.
      • Kwon S.
      • Choi S.H.
      • Hong F.
      • Moon K.
      • Kim J.M.
      • Choi S.L.
      • Kim B.M.
      • Kim S.S.
      ,
      • Li Y.
      • Jiang B.
      • Ensign W.Y.
      • Vogt P.K.
      • Han J.
      ). The PI3K family members are lipid kinases that phosphorylate phosphoinositides at position 3 of the inositol ring (
      • Rameh L.E.
      • Cantley L.C.
      ), acting as membrane anchors that locate and activate pleckstrin homology domain-containing effectors such as the well characterized serine/threonine kinase Akt (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). Class I PI3Ks are generally composed of a p85 regulatory subunit and a p110 catalytic subunit (
      • Rameh L.E.
      • Cantley L.C.
      ). This class of PI3Ks can be activated by a wide variety of extracellular stimuli, including those elicited by E-cadherin-mediated cell-cell adhesion (
      • Pece S.
      • Chiariello M.
      • Murga C.
      • Gutkind J.S.
      ).
      In this study, the role and regulation of PI3K in intestinal epithelial cells were investigated. Using several approaches, we demonstrated that PI3K is necessary for the functional and morphological differentiation of intestinal epithelial cells. We also found that PI3K is recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cultures and that this activation appears to be essential for the integrity of the adherens junctions and the association of their components with the cytoskeleton. Finally, we have provided evidence that the assembly of the adherens junctions stimulates Akt and p38 MAPK in a PI3K-dependent manner.

      DISCUSSION

      Little is known regarding the mechanisms involved in the regulation of cell growth and differentiation in the human intestinal epithelium. The data presented in this report suggest that PI3K plays a crucial role in the control of differentiation events in this tissue. Indeed, we have demonstrated for the first time that PI3K is part of a signaling pathway necessary for functional and morphological differentiation of intestinal epithelial cells. Inhibition of PI3K decreased the expression of enterocyte markers, viz.sucrase-isomaltase and villin, and reduced cell polarization and brush-border formation. Furthermore, we demonstrated that E-cadherin engagement triggers the recruitment of PI3K and activates its signaling at the sites of cell-cell contact, and this activation appears to be essential for the assembly of adherens junctions and the association of their components with the cytoskeleton. Finally, we have provided evidence that one of the molecular events resulting from E-cadherin-mediated cellular aggregation is the activation of Akt and p38 MAPK in a PI3K-dependent manner.
      A key question in intestinal development is what triggers the differentiation process. In this regard, it has been demonstrated that cell-cell junction systems, particularly adherens junctions, play an important role in the control of cell differentiation during intestinal ontogeny as well as during the continuous epithelial cell renewal in the mature organ. For instance, studies with E-cadherin knockout mice have revealed that E-cadherin-mediated cell adhesion is essential for the compaction of mesenchymal cells and their transition to a polarized epithelium (
      • Larue L.
      • Antos C.
      • Butz S.
      • Huber O.
      • Delmas V.
      • Dominis M.
      • Kemler R.
      ,
      • Riethmacher D.
      • Brinkman V.
      • Birchmeier C.
      ). In a chimeric-transgenic animal model, expression of a dominant-negative N/E-cadherin mutant in villous enterocytes resulted in perturbation of cell-cell adhesion associated with an increased enterocyte migration rate along the crypt-villus axis, loss of the differentiated polarized phenotype, and increased apoptosis (
      • Hermiston M.L.
      • Gordon J.I.
      ). However, signaling components that relay the signal from adherens junction proteins to the nuclear targets for the control of intestine-specific gene expression remain elusive. Our observations showing that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in intestinal epithelial cells suggest that it may be one of these signaling components. Such stimulation of PI3K by E-cadherin-mediated cell-cell contacts has recently been reported in other epithelial cell types (
      • Pece S.
      • Chiariello M.
      • Murga C.
      • Gutkind J.S.
      ,
      • Bergin E.
      • Levine J.S.
      • Koh J.S.
      • Lieberthal W.
      ). Furthermore, inhibition of PI3K activity by ectopic expression of a dominant-negative form of the regulatory p85 subunit (Δp85) or by use of the LY294002 inhibitor repressed the expression of intestine-specific genes and delayed functional and morphological epithelial differentiation. Inhibition of PI3K was also found to alter adherens junction integrity in newly confluent monolayers of Caco-2/15 cells by reducing the amount of cytoskeleton-associated E-cadherin and β-catenin at the site of cell-cell contact. Thus, we believe that E-cadherin-dependent PI3K activation acts as an intermediate in the formation of adherens junctions, suggesting a bidirectional regulation between PI3K activity and adherens junction assembly. Such bidirectional regulation has been recently described for E-cadherin and Rac/Cdc42 (
      • Braga V.M.
      ,
      • Kim S.H.
      • Zhigang L.
      • Sacks D.B.
      ,
      • Nakagawa M.
      • Fukata M.
      • Yamaga M.
      • Itoh N.
      • Kaibuchi K.
      ). Taken together, these data indicate that PI3K plays an important role in regulating the integrity of adherens junctions, which in turn seems to be crucial for the efficient differentiation of intestinal epithelial cells. The recent demonstration that PI3K is involved in three-dimensional morphogenesis and tissue-specific differentiation in the mammary gland (
      • Somasiri A.
      • Colleen W.
      • Ellchuk T.
      • Turley S.
      • Rosekelley C.D.
      ) strengthens this hypothesis.
      The mechanism by which PI3K inhibition impairs adherens junctions is unknown. In mammary epithelial cells, this response appears to be mediated by changes at the level of the actin cytoskeleton (
      • Somasiri A.
      • Colleen W.
      • Ellchuk T.
      • Turley S.
      • Rosekelley C.D.
      ). In this regard, our data indeed suggest that PI3K activity regulates the recruitment of F-actin at the site of cell-cell contact. It is known that phosphatidylinositol 3,4,5-triphosphate, a lipid product of PI3K, can recruit and activate the GTP exchange factor for Rac, which is required for adherens junction formation in Madin-Darby canine kidney cells and keratinocytes (
      • Braga V.M.
      • Machesky L.M.
      • Hall A.
      • Hotchin N.A.
      ,
      • Takaishi K.
      • Sasaki T.
      • Kotani H.
      • Nishioka H.
      • Takai Y.
      ), whereas Rac promotes the recruitment of F-actin to these junctions (
      • Takaishi K.
      • Sasaki T.
      • Kotani H.
      • Nishioka H.
      • Takai Y.
      ). These observations raise the possibility that a PI3K/Rac signaling pathway controls the integrity of adherens junctions in intestinal epithelial cells. However, in well polarized Caco-2/15 cells with mature intercellular junctions, we have observed that PI3K inhibition no longer interfered with the association of E-cadherin and β-catenin with the cytoskeleton. It is noteworthy that PI3K inhibition has no effect on the expression of differentiation markers such as sucrase-isomaltase and villin in well polarized Caco-2/15 cells.
      P. Laprise and N. Rivard, unpublished data.
      PI3K may thus act locally to control the early establishment of E-cadherin-mediated cell-cell contact and the initiation of the differentiation program rather than their maintenance. These observations are consistent with the fact that properties of E-cadherin-mediated cell-cell contact appear to be modulated by junction maturation (
      • Braga V.M.
      • Del Maschio A.
      • Machesky L.
      • Dejana E.
      ). For instance, the regulation of E-cadherin function by Rac is progressively lost as the E-cadherin junction matures (
      • Braga V.M.
      • Del Maschio A.
      • Machesky L.
      • Dejana E.
      ,
      • Akhtar N.
      • Hotchin N.A.
      ).
      In addition to its participation in E-cadherin-mediated epithelial cell adhesion, β-catenin is a key player in the APC/Wnt signaling pathway. In normal colonic epithelial cells, APC, in combination with glycogen-synthase kinase-3β and axin, regulates free cytoplasmic β-catenin levels by binding to and targeting β-catenin for degradation by ubiquitination-dependent proteolysis. This regulates the availability of free β-catenin for binding with the T-cell factor/lymphoid enhancer family of transcription factors (
      • Mimori-Kiyosue Y.
      • Tsukita S.
      ). In the absence of a Wnt signal, APC promotes the degradation of cytoplasmic β-catenin, whereas in the presence of a Wnt signal, β-catenin accumulates in the cytoplasm, translocates to the nucleus, and coordinates with the T-cell factor/lymphoid enhancer to activate gene transcription (
      • Ikeda S.
      • Kishida S.
      • Yamamoto H.
      • Murai H.
      • Koyama S.
      ). Our observation that the inhibition of PI3K led to a decreased association of E-cadherin and β-catenin with the cytoskeleton fraction could suggest a possible regulation of the APC/Wnt/β-catenin signaling pathway by PI3K. However, inhibition of PI3K activity by ectopic expression of a dominant-negative form of p85 (Δp85) or by use of the LY294002 inhibitor did not enhance pTOPFLASH activity, a T-cell factor promoter/luciferase reporter plasmid that directly assays β-catenin/T-cell factor activity (
      • Korinek V.
      • Barker N.
      • Morin P.J.
      • van Wichen D.
      • de Weger R.
      • Kinzler K.W.
      • Vogelstein B.
      • Clevers H.
      ) (data not shown). Furthermore, our data shown in Fig. 4 A clearly indicate that the total cellular amounts of β-catenin protein remained comparable following PI3K inhibition, suggesting that β-catenin was not targeted for degradation in the proteasome. Taken together, these data suggest that PI3K does not regulate the Wnt/β-catenin signaling pathway in newly confluent Caco-2/15 cells. However, future studies will be needed to further clarify the involvement of PI3K in the regulation of this pathway in intestinal epithelial cells.
      Another interesting finding from this work is the demonstration that E-cadherin engagement led to a PI3K-dependent activation of Akt. Akt stimulation has been involved in skeletal muscle differentiation (
      • Jiang B.H.
      • Aoki M.
      • Zheng J.Z.
      • Li J.
      • Vogt P.K.
      ), which also depends on PI3K (
      • Chun Y.K.
      • Kim J.
      • Kwon S.
      • Choi S.H.
      • Hong F.
      • Moon K.
      • Kim J.M.
      • Choi S.L.
      • Kim B.M.
      • Kim S.S.
      ,
      • Li Y.
      • Jiang B.
      • Ensign W.Y.
      • Vogt P.K.
      • Han J.
      ). Such a contribution of Akt to intestinal epithelial cell differentiation would be interesting to explore. However, Akt has been implicated in the control of cell survival and thereby could mediate the protective action of E-cadherin-mediated cell-cell contacts against apoptosis (
      • Hermiston M.L.
      • Gordon J.I.
      ,
      • Bergin E.
      • Levine J.S.
      • Koh J.S.
      • Lieberthal W.
      ,
      • Peluso J.J.
      • Pappalardo A.
      • Fernandez G.
      ).
      Our study also provides evidence for the first time that E-cadherin-mediated cell-cell contact triggers p38 MAPK cascade activation. The p38α MAPK pathway was demonstrated previously to be an important modulator of enterocyte differentiation (
      • Houde M.
      • Laprise P.
      • Jean D.
      • Blais M.
      • Asselin C.
      • Rivard N.
      ). Thus, it seems that PI3K and p38α MAPK act in a common pathway in intestinal epithelial cells toward the regulation of their differentiation. We provided several indications that PI3K-dependent activation of p38α MAPK relies on the ability of PI3K to promote the assembly and integrity of adherens junctions. First, a good correlation of the kinetics of adherens junction disruption with a decrease in p38α MAPK activity was observed following PI3K inhibition. Second, in well polarized cells, PI3K inhibition did not alter adherens junction integrity or affect p38α activity. And third, PI3K inhibition, which inhibited adherens junction but not tight junction assembly, also attenuated E-cadherin-dependent activation of p38α. Therefore, the ability of PI3K to control adherens junction formation and p38α activity, known to control intestine-specific gene transcription (
      • Houde M.
      • Laprise P.
      • Jean D.
      • Blais M.
      • Asselin C.
      • Rivard N.
      ), confers to this kinase a central role in the promotion of intestinal epithelial cell differentiation. However, the mechanism relating PI3K to adherens junction integrity and p38α MAPK activation remains to be elucidated.
      The complexity of the PI3K-dependent pathways is further emphasized by the recent work of Wang et al. (
      • Wang Q.
      • Wang X.
      • Hernandez A.
      • Kim S.
      • Evers B.M.
      ), who showed that PI3K inhibition through overexpression of PTEN (which directly dephosphorylates the D3 phosphate group of the lipid products of PI3K) or wortmannin treatment results in an increase in alkaline phosphatase and sucrase-isomaltase enzymatic activities, suggesting a regulatory effect of this pathway on intestinal cell differentiation. Although it cannot be excluded that these contradictory effects are the result of the use of distinct cell lines, and they are not without precedent (
      • Aliaga J.C.
      • Deschênes C.
      • Beaulieu J.-F.
      • Calvo E.L.
      • Rivard N.
      ,
      • Taupin D.
      • Podolsky D.K.
      ), the fact that PTEN effects were observed mostly in the presence of butyrate is a good indication that alternative pathways may exist.
      In conclusion, migration of intestinal epithelial cells from the crypts to the villus tips involves programming for proliferation- and differentiation-related events. A key question in intestinal development is what triggers cell cycle arrest and the differentiation process. In vitro cell culture experiments have shown that cell-cell contact can trigger differentiation and therefore substitute the in vivo signal. The data presented herein indicate that PI3K signaling plays an important role in initiating intestinal epithelial cell differentiation. In this model (Fig.8), E-cadherin engagement recruits and activates PI3K signaling. This activation promotes the assembly of adherens junction components with the cytoskeleton, which in turn activates the p38 MAPK cascade, enhancing the transactivation capacity of CDX2. Rac is a likely candidate as a signaling intermediate because, as reported in other cell types, it could be a downstream effector of PI3K (
      • Braga V.M.
      • Machesky L.M.
      • Hall A.
      • Hotchin N.A.
      ) and an upstream activator of the p38 MAPK module (
      • Zhang S.
      • Han J.
      • Sells M.A.
      • Chernoff J.
      • Knaus U.G.
      • Ulevitch R.J.
      • Bokoch G.M.
      ). However, regardless of the exact mechanism, the ability of PI3K to control adherens junction formation and p38 MAPK activity confers to PI3K a central role in the promotion of intestinal epithelial cell differentiation.
      Figure thumbnail gr8
      Figure 8Schematic of proposed pathways involved in the regulation of functional and morphological differentiation of intestinal epithelial cells based on the findings of this study.In this model, E-cadherin engagement recruits and activates PI3K signaling. This activation of PI3K might increase the amount of F-actin at the sites of cell-cell contact (through Rac?) and thereby promote the assembly of adherens junction components with the cytoskeleton. The ability of PI3K to promote the assembly and integrity of adherens junctions allows the activation of p38α MAPK, which enhances the transactivation capacity of CDX2 for intestine-specific genes.TJ, tight junctions; AJ, adherens junctions;D, desmosomes; BB, brush border; W, terminal web of actin; SI, sucrase-isomaltase.

      Acknowledgments

      We acknowledge the expert technical assistance of Pierre Magny, Denis Martel, Anne Vézina, Dominique Jean, and Claude Deschênes. We thank Elizabeth Herring and Amy Svotelis for critical reading of the manuscript. We also acknowledge Dr. Jacques Landry for kindly providing rabbit anti-p38α MAPK antibody. Special thanks go to Dr. Claude Asselin for constant encouragement, judicious comments, and excellent collaboration in the course of this work.

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