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1.
Transplantation ; 108(10): e301-e312, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38578699

RESUMO

BACKGROUND: Liver transplantation (LT) is crucial for end-stage liver disease patients, but organ shortages persist. Donation after circulatory death (DCD) aims to broaden the donor pool but presents challenges. Complications like acute rejection, hepatic artery thrombosis, and biliary issues still impact posttransplant prognosis. Biomarkers, including extracellular vesicles (EVs) and microRNAs (miRNAs), show promise in understanding and monitoring posttransplant events. This study explores the role of EVs and their miRNA cargo in LT, including their potential as diagnostic tools. METHODS: EVs from intrahepatic end-ischemic organ preservation solution (eiOPS) in 79 donated livers were detected using different techniques (nanosight tracking analysis, transmission electron microscopy, and flow cytometry). EV-derived miRNAs were identified by quantitative real time-polymerase chain reaction. Bioinformatics analysis was performed using the R platform. RESULTS: Different-sized and origin-specific EVs were found in eiOPS, with significantly higher concentrations in DCD compared with donation after brain death organs. Additionally, several EV-associated miRNAs, including let-7d-5p , miR-28-5p , miR-200a-3p , miR-200b-3p , miR-200c-3p , and miR-429 , were overexpressed in DCD-derived eiOPS. These miRNAs also exhibited differential expression patterns in liver tissue biopsies. Pathway analysis revealed enrichment in signaling pathways involved in extracellular matrix organization and various cellular processes. Moreover, specific EVs and miRNAs correlated with clinical outcomes, including survival and early allograft dysfunction. A predictive model combining biomarkers and clinical variables showed promise in acute rejection detection after LT. CONCLUSIONS: These findings provide new insights into the use of EVs and miRNAs as biomarkers and their possible influence on posttransplantation outcomes, potentially contributing to improved diagnostic approaches and personalized treatment strategies in LT.


Assuntos
Biomarcadores , Isquemia Fria , Vesículas Extracelulares , Transplante de Fígado , MicroRNAs , Soluções para Preservação de Órgãos , Preservação de Órgãos , Transplante de Fígado/efeitos adversos , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/transplante , MicroRNAs/metabolismo , MicroRNAs/genética , Isquemia Fria/efeitos adversos , Preservação de Órgãos/métodos , Masculino , Feminino , Pessoa de Meia-Idade , Biomarcadores/metabolismo , Adulto , Resultado do Tratamento , Idoso , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/genética , Sobrevivência de Enxerto
2.
Clin Cancer Res ; 29(18): 3744-3758, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37432984

RESUMO

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.


Assuntos
Neoplasias de Bainha Neural , Neurofibrossarcoma , Humanos , Biomarcadores , Linhagem Celular Tumoral , Endoglina/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias de Bainha Neural/tratamento farmacológico , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/metabolismo , Transdução de Sinais
3.
Nat Commun ; 13(1): 7113, 2022 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402789

RESUMO

NRAS-mutated melanoma lacks a specific line of treatment. Metabolic reprogramming is considered a novel target to control cancer; however, NRAS-oncogene contribution to this cancer hallmark is mostly unknown. Here, we show that NRASQ61-mutated melanomas specific metabolic settings mediate cell sensitivity to sorafenib upon metabolic stress. Mechanistically, these cells are dependent on glucose metabolism, in which glucose deprivation promotes a switch from CRAF to BRAF signaling. This scenario contributes to cell survival and sustains glucose metabolism through BRAF-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2/3 (PFKFB2/PFKFB3). In turn, this favors the allosteric activation of phosphofructokinase-1 (PFK1), generating a feedback loop that couples glycolytic flux and the RAS signaling pathway. An in vivo treatment of NRASQ61 mutant melanomas, including patient-derived xenografts, with 2-deoxy-D-glucose (2-DG) and sorafenib effectively inhibits tumor growth. Thus, we provide evidence for NRAS-oncogene contributions to metabolic rewiring and a proof-of-principle for the treatment of NRASQ61-mutated melanoma combining metabolic stress (glycolysis inhibitors) and previously approved drugs, such as sorafenib.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sorafenibe/farmacologia , Linhagem Celular Tumoral , Mutação , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Glicólise/genética , Glucose/metabolismo , Estresse Fisiológico , Fosfofrutoquinase-2/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo
4.
Oncoimmunology ; 11(1): 2067944, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35481283

RESUMO

Nearly 40% of the advanced cancer patients will present brain metastases during the course of their disease, with a 2-year life expectancy of less than 10%. Immune system impairment, including the modulation of both STAT3 and PD-L1, is one of the hallmarks of brain metastases. Liquid biopsy could offer several advantages in brain metastases management, such as the possibility of noninvasive dynamic monitoring. Extracellular vesicles (EVs) have been recently proposed as novel biomarkers especially useful in liquid biopsy due to their secretion in biofluids and their role in cell communication during tumor progression. The main aim of this work was to characterize the size and protein cargo of plasma circulating EVs in patients with solid tumors and their correlation with newly diagnosed brain metastases, in addition to their association with other relevant clinical variables. We analyzed circulating EVs in the plasma of 123 patients: 42 patients with brain metastases, 50 without brain metastases and 31 healthy controls. Patients with newly diagnosed brain metastases had a lower number of circulating EVs in the plasma and a higher protein concentration in small EVs (sEVs) compared to patients without brain metastases and healthy controls. Interestingly, melanoma patients with brain metastases presented decreased STAT3 activation and increased PD-L1 levels in circulating sEVs compared to patients without central nervous system metastases. Decreased STAT3 activation and increased PD-L1 in plasma circulating sEVs identify melanoma patients with brain metastasis.


Assuntos
Neoplasias Encefálicas , Vesículas Extracelulares , Melanoma , Antígeno B7-H1 , Vesículas Extracelulares/metabolismo , Humanos
5.
Nat Cancer ; 2(12): 1387-1405, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34957415

RESUMO

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.


Assuntos
Vesículas Extracelulares , Melanoma , Animais , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Linfangiogênese/fisiologia , Metástase Linfática , Melanoma/metabolismo , Camundongos , Proteínas do Tecido Nervoso , Receptores de Fator de Crescimento Neural/genética , Microambiente Tumoral
6.
Nat Commun ; 11(1): 4730, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934237

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

7.
Nat Commun ; 11(1): 4261, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848136

RESUMO

Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.


Assuntos
Neoplasias da Mama/metabolismo , Comunicação Celular/fisiologia , Vesículas Extracelulares/metabolismo , Integrina beta3/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Endocitose , Feminino , Quinase 1 de Adesão Focal/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , Camundongos Nus , Modelos Biológicos , Metástase Neoplásica/patologia , Transplante de Neoplasias
8.
Oncogene ; 39(13): 2756-2771, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015486

RESUMO

Aldehyde dehydrogenases (ALDHs) are multifunctional enzymes that oxidize diverse endogenous and exogenous aldehydes. We conducted a meta-analysis based on The Cancer Genome Atlas and Gene Expression Omnibus data and detected genetic alterations in ALDH1A1, ALDH1A3, or ALDH3A1, 86% of which were gene amplification or mRNA upregulation, in 31% of nonsmall cell lung cancers (NSCLCs). The expression of these isoenzymes impacted chemoresistance and shortened survival times in patients. We hypothesized that these enzymes provide an oxidative advantage for the persistence of NSCLC. To test this hypothesis, we used genetic and pharmacological approaches with DIMATE, an irreversible inhibitor of ALDH1/3. DIMATE showed cytotoxicity in 73% of NSCLC cell lines tested and demonstrated antitumor activity in orthotopic xenografts via hydroxynonenal-protein adduct accumulation, GSTO1-mediated depletion of glutathione and increased H2O2. Consistent with this result, ALDH1/3 disruption synergized with ROS-inducing agents or glutathione synthesis inhibitors to trigger cell death. In lung cancer xenografts with high to moderate cisplatin resistance, combination treatment with DIMATE promoted strong synergistic responses with tumor regression. These results indicate that NSCLCs with increased expression of ALDH1A1, ALDH1A3, or ALDH3A1 may be targeted by strategies involving inhibitors of these isoenzymes as monotherapy or in combination with chemotherapy to overcome patient-specific drug resistance.


Assuntos
Família Aldeído Desidrogenase 1/antagonistas & inibidores , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Oxirredutases/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Retinal Desidrogenase/antagonistas & inibidores , Idoso , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Alcinos/farmacologia , Alcinos/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Amplificação de Genes , Glutationa/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Compostos de Sulfidrila/farmacologia , Compostos de Sulfidrila/uso terapêutico , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Exp Med ; 216(5): 1061-1070, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30975894

RESUMO

Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse.


Assuntos
Progressão da Doença , Vesículas Extracelulares/metabolismo , Exsudatos e Transudatos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Seroma/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Intervalo Livre de Doença , Drenagem , Exossomos/metabolismo , Feminino , Humanos , Metástase Linfática , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Proteômica/métodos , Neoplasias Cutâneas/patologia
11.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30530526

RESUMO

Neuropathic lysosomal storage disorders (LSDs) present with activated pro-inflammatory microglia. However, anti-inflammatory treatment failed to improve disease pathology. We characterise the mechanisms underlying microglia activation in Niemann-Pick disease type A (NPA). We establish that an NPA patient and the acid sphingomyelinase knockout (ASMko) mouse model show amoeboid microglia in neurodegeneration-prone areas. In vivo microglia ablation worsens disease progression in ASMko mice. We demonstrate the coexistence of different microglia phenotypes in ASMko brains that produce cytokines or counteract neuronal death by clearing myelin debris. Overloading microglial lysosomes through myelin debris accumulation and sphingomyelin build-up induces lysosomal damage and cathepsin B extracellular release by lysosomal exocytosis. Inhibition of cathepsin B prevents neuronal death and behavioural anomalies in ASMko mice. Similar microglia phenotypes occur in a Niemann-Pick disease type C mouse model and patient. Our results show a protective function for microglia in LSDs and how this is corrupted by lipid lysosomal overload. Data indicate cathepsin B as a key molecule mediating neurodegeneration, opening research pathways for therapeutic targeting of LSDs and other demyelinating diseases.


Assuntos
Catepsina B/metabolismo , Microglia/patologia , Doença de Niemann-Pick Tipo A/patologia , Esfingomielina Fosfodiesterase/genética , Animais , Linhagem Celular , Pré-Escolar , Modelos Animais de Doenças , Progressão da Doença , Humanos , Recém-Nascido , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Camundongos Knockout , Microglia/metabolismo , Doença de Niemann-Pick Tipo A/genética , Fenótipo , Esfingomielinas/metabolismo
13.
Endocrinology ; 153(2): 847-60, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22166974

RESUMO

Dieldrin is an endocrine disruptor that accumulates in mammalian adipose tissue and brain. It induces convulsions due to its antagonism of the γ-aminobutyric acid A receptor (GABA(A)R). We have previously reported that long-term exposure to dieldrin causes the internalization of the N-methyl-D-aspartate receptor (NMDAR) as a result of persistent GABA(A)R inhibition. Because the neurosteroids 17ß-estradiol (E2) and allopregnanolone are known to modulate the function and trafficking of GABA(A)R and NMDAR, we examined the effects of E2 and allopregnanolone on dieldrin-induced GABA(A)R inhibition, NMDAR internalization, and neuronal death in cortical neurons. We found that 1 nM E2 increased the membrane expression of NR1/NR2B receptors and postsynaptic density 95 but did not induce their physical association. In contrast, 10 nM E2 had no effect on these proteins but reduced NR2A membrane expression. We also found that exposure to 60 nM dieldrin for 6 d in vitro caused the internalization of NR1 and NR2B but not NR2A. Treatment with either 1 nM E2 or 10 µM allopregnanolone prevented the dieldrin-induced reduction in membrane levels of the NR1/NR2B receptors. Furthermore, prolonged exposure to 200 nM dieldrin down-regulated the expression of NR2A; this was inhibited only by allopregnanolone. Although both hormones restored NMDAR function, as measured by the NMDA-induced rise in intracellular calcium, allopregnanolone (but not E2) reversed the inhibition of GABA(A)R and neuronal death caused by prolonged exposure to dieldrin. Our results indicate that allopregnanolone protects cortical neurons against the neurotoxicity caused by long-term exposure to dieldrin by maintaining GABA(A)R and NMDAR functionality.


Assuntos
Dieldrin/efeitos adversos , Neurônios/efeitos dos fármacos , Pregnanolona/farmacologia , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Anestésicos/farmacologia , Animais , Membrana Celular , Córtex Cerebral/citologia , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos/citologia , Disruptores Endócrinos/efeitos adversos , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neurônios/metabolismo , Gravidez , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Transmissão Sináptica/fisiologia
14.
Toxicol Sci ; 120(2): 413-27, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21278053

RESUMO

The organochlorine chemicals endosulfan, dieldrin, and γ-hexachlorocyclohexane (lindane) are persistent pesticides to which people are exposed mainly via diet. Their antagonism of the γ-aminobutyric acid-A (GABA(A)) receptor makes them convulsants. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER). Here, we study the effects of dieldrin, endosulfan, and lindane on ERs in primary cultures of cortical neurons (CN) and cerebellar granule cells (CGC). All the compounds tested inhibited the binding of [(3)H]-estradiol to the ER in both CN and CGC, with dieldrin in CGC showing the highest affinity. We also determined the effects of the pesticides on protein kinase B (Akt) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Dieldrin and endosulfan increased Akt phosphorylation in CN, which was inhibited by the ERß antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Instead, Akt and ERK1/2 phosphorylation induced by dieldrin in CGC was mediated by multiple activation of ERα, ERß, and G protein-coupled receptor 30. Lindane did not activate these pathways, but it inhibited estradiol-mediated Akt and ERK1/2 activation. In CN, all the chemicals activated ERK1/2 through a mechanism involving GABA(A) and glutamate receptors. Long-term exposure to these pesticides reduced the levels of ERα, but not of ERß. Moreover, extracts of CN treated with endosulfan, dieldrin, or lindane induced cell proliferation in MCF-7 human breast cancer-derived cells, whereas only extracts of CGC treated with dieldrin induced MCF-7 cell proliferation. Overall, the observed alterations on ER-mediated signaling and ER levels in neurons might contribute to the neurotoxicity of these organochlorine pesticides.


Assuntos
Disruptores Endócrinos/toxicidade , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Hidrocarbonetos Clorados/toxicidade , Neurônios/efeitos dos fármacos , Praguicidas/toxicidade , Animais , Animais Recém-Nascidos , Western Blotting , Técnicas de Cultura de Células , Células Cultivadas , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Dieldrin/química , Dieldrin/toxicidade , Disruptores Endócrinos/química , Endossulfano/química , Endossulfano/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hexaclorocicloexano/química , Hexaclorocicloexano/toxicidade , Humanos , Hidrocarbonetos Clorados/química , Camundongos , Neurônios/metabolismo , Praguicidas/química , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de GABA-A/metabolismo
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