scholarly journals Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1

2016 ◽  
Vol 113 (11) ◽  
pp. 3036-3041 ◽  
Author(s):  
Pooja Singhmar ◽  
XiaoJiao Huo ◽  
Niels Eijkelkamp ◽  
Susana Rojo Berciano ◽  
Faiza Baameur ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Molecular Mechanism ◽  
Inflammatory Pain ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Mechanical Hyperalgesia ◽  
Dependent Manner ◽  
Phosphorylation Event

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1−/− mice are protected against inflammatory hyperalgesia in the complete Freund’s adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1−/−, mice. Using sensory neuron-specific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

scholarly journals Bactericidal/Permeability-Increasing Protein Preeminently Mediates Clearance of Pseudomonas aeruginosa In Vivo via CD18-Dependent Phagocytosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Jomkuan Theprungsirikul ◽  
Sladjana Skopelja-Gardner ◽  
Ashley S. Burns ◽  
Rachel M. Wierzbicki ◽  
William F. C. Rigby
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Critical Role ◽  
Chronic Obstructive ◽  
Dependent Manner ◽  
Opsonic Activity ◽  
Obstructive Pulmonary Disease ◽  
Neutrophil Dysfunction ◽  
Chronic Lung Infection

Chronic Pseudomonas aeruginosa infection mysteriously occurs in the airways of patients with cystic fibrosis (CF), bronchiectasis (BE), and chronic obstructive pulmonary disease (COPD) in the absence of neutrophil dysfunction or neutropenia and is strongly associated with autoimmunity to bactericidal permeability-increasing protein (BPI). Here, we define a critical role for BPI in in vivo immunity against P. aeruginosa. Wild type and BPI-deficient (Bpi-/-) mice were infected with P. aeruginosa, and bacterial clearance, cell infiltrates, cytokine production, and in vivo phagocytosis were quantified. Bpi-/- mice exhibited a decreased ability to clear P. aeruginosa in vivo in concert with increased neutrophil counts and cytokine release. Bpi-/- neutrophils displayed decreased phagocytosis that was corrected by exogenous BPI in vitro. Exogenous BPI also enhanced clearance of P. aeruginosa in Bpi-/- mice in vivo by increasing P. aeruginosa uptake by neutrophils in a CD18-dependent manner. These data indicate that BPI plays an essential role in innate immunity against P. aeruginosa through its opsonic activity and suggest that perturbations in BPI levels or function may contribute to chronic lung infection with P. aeruginosa.

Interaction and Regulation of HSC with Osteopontin Is Mediated through α4β1 and α9β1Integrins, Which Are Differentially Expressed in the HSC Pool.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1330-1330
Author(s):  
David N. Haylock ◽  
Genevieve A. Whitty ◽  
Brenda Williams ◽  
Melonie J. Storan ◽  
Susie K. Nilsson
Keyword(s):  
Cord Blood ◽  
Critical Role ◽  
Negative Regulator ◽  
Hemopoietic Stem Cell ◽  
Null Mouse ◽  
Dependent Manner ◽  
Independent Manner ◽  
Hsc Niche

Abstract Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α4β1 and α9β1. HSC are known to express α4β1, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34+CD38+ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α9β1, which also occurs in a differential manner, but mimicking CD34. Expression of α9β1 is highest on cord blood CD34+CD38− cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α4β1 and α9β1. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α4β1 binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α4β1 and α9β1 to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α9β1, which together with α4β1 provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3376-3376
Author(s):  
Romain Gioia ◽  
Cedric Leroy ◽  
Claire Drullion ◽  
Valérie Lagarde ◽  
Serge Roche ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Dependent Manner ◽  
Stable Isotope Labelling ◽  
Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals β-Chemokines Enhance Parasite Uptake and Promote Nitric Oxide-Dependent Microbiostatic Activity in Murine Inflammatory Macrophages Infected with Trypanosoma cruzi

1999 ◽  
Vol 67 (9) ◽  
pp. 4819-4826 ◽  
Author(s):  
Júlio C. S. Aliberti ◽  
Fabiana S. Machado ◽  
Janeusa T. Souto ◽  
Ana P. Campanelli ◽  
Mauro M. Teixeira ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Neutralizing Antibodies ◽  
Specific Inhibitor ◽  
No Production ◽  
Dependent Manner ◽  
Macrophage Infection ◽  
Parasite Replication ◽  
Ifn Γ

ABSTRACT In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of β-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1α, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of β-chemokine MIP-1α, MIP-1β, RANTES, and JE mRNA by macrophages. The expression of the β-chemokine MIP-1α, MIP-1β, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms ofT. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by β-chemokines. Hence, treatment with anti-β-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous β-chemokines MIP-1α, MIP-1β, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. l-NMMA, a specific inhibitor of thel-arginine–NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with β-chemokines. Among the β-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-γ). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-γ. Together, these results suggest that in addition to their chemotactic activity, murine β-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruziinfection.

scholarly journals YBX1 is required for maintaining myeloid leukemia cell survival by regulating BCL2 stability in an m6A-dependent manner

Blood ◽  
2021 ◽  
Author(s):  
Mengdie Feng ◽  
Xueqin Xie ◽  
Guoqiang Han ◽  
Tiantian Zhang ◽  
Yashu Li ◽  
...  
Keyword(s):  
Cell Survival ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Leukemia Cell ◽  
Dependent Manner ◽  
Myeloid Leukemia Cell

RNA-binding proteins (RBPs) are critical regulators of transcription and translation that are often dysregulated in cancer. Although RBPs are increasingly appreciated as being important for normal hematopoiesis and for hematological malignancies as oncogenes or tumor suppressors, essential RBPs for leukemia maintenance and survival remain elusive. Here we show that YBX1 is specifically required for maintaining myeloid leukemia cell survival in an m6A-dependent manner. We found that expression of YBX1 is significantly upregulated in myeloid leukemia cells, and deletion of YBX1 dramatically induces apoptosis, promotes differentiation, coupled with reduced proliferation and impaired leukemic capacity of primary human and mouse acute myeloid leukemia (AML) cells in vitro and in vivo. Loss of YBX1 does not obviously affect normal hematopoiesis. Mechanistically, YBX1 interacts with IGF2BPs and stabilizes m6A-tagged RNA. Moreover, YBX1 deficiency dysregulates the expression of apoptosis-related genes, and promotes mRNA decay of MYC and BCL2 in an m6A-dependent manner, which contributes to the defective survival due to YBX1 deletion. Thus, our findings uncover a selective and critical role of YBX1 in maintaining myeloid leukemia survival that might provide a rationale for the therapeutic targeting of YBX1 in myeloid leukemia.

scholarly journals Streptococcus gallolyticus Increases Expression and Activity of Aryl Hydrocarbon Receptor-Dependent CYP1 Biotransformation Capacity in Colorectal Epithelial Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Rahwa Taddese ◽  
Rian Roelofs ◽  
Derk Draper ◽  
Xinqun Wu ◽  
Shaoguang Wu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Aryl Hydrocarbon Receptor ◽  
Specific Inhibitor ◽  
Intestinal Bacteria ◽  
Opportunistic Pathogen ◽  
Dependent Manner ◽  
Aryl Hydrocarbon ◽  
Streptococcus Gallolyticus

ObjectiveThe opportunistic pathogen Streptococcus gallolyticus is one of the few intestinal bacteria that has been consistently linked to colorectal cancer (CRC). This study aimed to identify novel S. gallolyticus-induced pathways in colon epithelial cells that could further explain how S. gallolyticus contributes to CRC development.Design and ResultsTranscription profiling of in vitro cultured CRC cells that were exposed to S. gallolyticus revealed the specific induction of oxidoreductase pathways. Most prominently, CYP1A and ALDH1 genes that encode phase I biotransformation enzymes were responsible for the detoxification or bio-activation of toxic compounds. A common feature is that these enzymes are induced through the Aryl hydrocarbon receptor (AhR). Using the specific inhibitor CH223191, we showed that the induction of CYP1A was dependent on the AhR both in vitro using multiple CRC cell lines as in vivo using wild-type C57bl6 mice colonized with S. gallolyticus. Furthermore, we showed that CYP1 could also be induced by other intestinal bacteria and that a yet unidentified diffusible factor from the S. galloltyicus secretome (SGS) induces CYP1A enzyme activity in an AhR-dependent manner. Importantly, priming CRC cells with SGS increased the DNA damaging effect of the polycyclic aromatic hydrocarbon 3-methylcholanthrene.ConclusionThis study shows that gut bacteria have the potential to modulate the expression of biotransformation pathways in colonic epithelial cells in an AhR-dependent manner. This offers a novel theory on the contribution of intestinal bacteria to the etiology of CRC by modifying the capacity of intestinal epithelial or (pre-)cancerous cells to (de)toxify dietary components, which could alter intestinal susceptibility to DNA damaging events.

scholarly journals Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

1996 ◽  
Vol 109 (6) ◽  
pp. 1479-1495 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Rodriguez-Paris ◽  
J.M. Bush ◽  
L. Zhang ◽  
J.A. Cardelli
Keyword(s):  
Morphological Changes ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Contractile Vacuole ◽  
Dependent Manner ◽  
Concanamycin A ◽  
Latex Beads ◽  
And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

scholarly journals PPARβ/δ Agonist GW501516 Inhibits Tumorigenesis and Promotes Apoptosis of the Undifferentiated Nasopharyngeal Carcinoma C666-1 Cells by Regulating miR-206

2019 ◽  
Vol 27 (8) ◽  
pp. 923-933 ◽  
Author(s):  
Linglan Gu ◽  
Yi Shi ◽  
Weimin Xu ◽  
Yangyang Ji
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Critical Role ◽  
Current Data ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Promoting Effect ◽  
Apoptotic Pathway ◽  
Peroxisome Proliferator Activated Receptor

In previous investigations, we reported that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activation by GW501516 inhibits proliferation and promotes apoptosis in the undifferentiated C666-1 nasopharyngeal carcinoma (NPC) cells by modulating caspase-dependent apoptotic pathway. In the present study, the mechanism by which GW501516 induces apoptosis was explored from the perspective of microRNA (miRNA) expression. Among the assayed miRNAs that were involved in regulating the expression of antiapoptotic protein Bcl-2, miR-206 was increased significantly and specifically by GW501516 in C666-1 cells at both the in vitro level and at the in vivo xenograft samples. The induction on miR-206 expression caused by GW501516 was capable of being antagonized by the PPARβ/δ antagonist GSK3787 and AMPK antagonist dorsomorphin in C666-1 cells. GW501516’s suppression on the growth and apoptosis of C666-1 cells was found to be dependent on the presence of miR-206. miR-206 overexpression resulted in suppressed proliferation and colony formation ability, and further triggered increased apoptosis in C666-1 cells in a caspase-dependent manner. The expression of cleaved caspase 3 and caspase 9, and the ratio of Bax to Bcl-2 were elevated remarkably by miR-206. Consistent with the in vitro result, miR-206 was corroborated to suppress the ectopic NPC xenograft tumorigenesis that derived from the C666-1 cells in BALB/c nu/nu mice. Taken together, the current data demonstrated that miR-206 plays a critical role in the direct apoptosis-promoting effect induced by GW501516 in C666-1 cells. Furthermore, the emphasized tumor-suppressive role of miR-206 in the C666-1 cells indicates that it has the potential to provide a new therapeutic approach for the undifferentiated NPC.

scholarly journals Essential role of p21/waf1 in the mediation of the anti-proliferative effects of GHRH antagonist JMR-132

2008 ◽  
Vol 41 (5) ◽  
pp. 389-392 ◽  
Author(s):  
Aspasia-Athina Volakaki ◽  
Daniel Lafkas ◽  
Eva Kassi ◽  
Andrew V Schally ◽  
Athanasios G Papavassiliou ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Molecular Mechanism ◽  
A549 Cells ◽  
Significant Inhibition ◽  
Human Lung Cancer ◽  
Dependent Manner ◽  
Ghrh Antagonists ◽  
P21 Waf1

GHRH, besides its neuroendocrine action in controlling the release of GH from the pituitary, stimulates the growth of various cancers in vivo and in vitro by direct mechanism(s). However, the molecular mechanism that mediates these proliferative effects of GHRH in extrapituitary tissues remains poorly characterized. In the present study, we investigated whether the tumor suppressor p21/waf1 is involved in the mediation of the proliferative effects of GHRH in A549 human lung cancer epithelial cells. Exposure of A549 cells to the GHRH antagonist JMR-132 caused a significant inhibition in the rate of cell proliferation. In A549 cells, GHRH suppressed while JMR-132 increased the levels of p21 expression in a dose-dependent manner. This suggests that GHRH could regulate p21 levels. We then evaluated whether p21 is required in A549 cells for the regulation of cell proliferation by GHRH. To this end, we knocked-down p21 expression in A549 cells by siRNA and assessed the effects of antagonist JMR-132 on cell proliferation. We found that the loss of p21 expression abolished the anti-proliferative effects of JMR-132. Suppression of p21 expression by siRNA in human HT29 colon cancer cells and non-transformed mouse osteoblasts KS483 also blocked the anti-proliferative effects of JMR-132 suggesting that the regulation of cell proliferation by GHRH is p21 dependent. These results shed light on the molecular mechanism of action of GHRH antagonists in tumor tissues and suggest that the antineoplastic activity of GHRH antagonists could be considered for the treatment of cancers expressing p21.

scholarly journals Blimp-1-Dependent Repression of Pax-5 Is Required for Differentiation of B Cells to Immunoglobulin M-Secreting Plasma Cells

2002 ◽  
Vol 22 (13) ◽  
pp. 4771-4780 ◽  
Author(s):  
Kuo-I Lin ◽  
Cristina Angelin-Duclos ◽  
Tracy C. Kuo ◽  
Kathryn Calame
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Critical Role ◽  
Cell Lineage ◽  
Immunoglobulin M ◽  
Dependent Manner ◽  
Plasmacytic Differentiation

ABSTRACT B-cell lineage-specific activator protein (BSAP), encoded by the Pax-5 gene, is critical for B-cell lineage commitment and B-cell development but is not expressed in terminally differentiated B cells. We demonstrate a direct connection between BSAP and B-lymphocyte-induced maturation protein 1 (Blimp-1), a transcriptional repressor that is sufficient to drive plasmacytic differentiation. Blimp-1 binds a site on the Pax-5 promoter in vitro and in vivo and represses the Pax-5 promoter in a binding-site-dependent manner. By ectopically expressing Blimp-1 or a competitive inhibitor of Blimp-1, we show that Blimp-1 is both necessary and sufficient to repress Pax-5 during plasmacytic differentiation of primary splenic B cells. Blimp-1-dependent repression of Pax-5 is sufficient to regulate BSAP targets CD19 and J chain and is necessary but not sufficient to induce XBP-1. We further show that repression of Pax-5 is required for Blimp-1 to drive differentiation of splenocytes to immunoglobulin M-secreting cells. Thus, repression of Pax-5 plays a critical role in the Blimp-1-dependent program of plasmacytic differentiation.

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