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1.
Pathobiology ; 87(1): 45-50, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023616

RESUMO

Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) fusion gene rearrangement is a key driver mutation in non-small cell lung cancer (NSCLC). Although Break-Apart ALK fluorescence in situ hybridization (FISH) is a reliable diagnostic method for detecting ALK gene rearrangement, it is also costly and time-consuming to use as a routine screening test. Our aim was to evaluate the clinical utility of a novel one-step, automated, rapid FISH (Auto-RaFISH) method developed to facilitate hybridization. This method takes advantage of the non-contact mixing effect of an alternating-current electric field. Ten representative specimens from 85 patients diagnosed at multiple centers with primary lung cancer with identified ALK-FISH status were collected. The specimens were all tested using FISH, RaFISH, and Auto-RaFISH. With both RaFISH protocols, the ALK test was completed within 4.5 h, as compared to the 20 h needed for the standard protocol. We found 100% agreement between the standard FISH, RaFISH, and new Auto-RaFISH based on the ALK status, and all methods stained equally well. These findings suggest that Auto-RaFISH could potentially serve as an automated clinical tool for prompt determination of ALK status in NSCLC.

2.
J Clin Pathol ; 72(9): 603-608, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31129615

RESUMO

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are effective against HER2-positive breast cancers. However, their lack of survival benefit in HER2-negative patients as well as their toxic effects and high cost highlight the need for accurate assessment of HER2 status. Our aim was to evaluate the clinical utility of a reagent-saving in situ hybridisation (Saving ISH) that facilitates hybridisation and saves HER2/chromosome enumeration probe by taking advantage of the non-contact mixing effect of an alternating current (AC) electric field. METHODS: With a new device, we apply a high-voltage, low-frequency AC electric field to the tissue sections, which mixes the probe within microdroplets as the voltage is switched on and off. Specimens (n=113) from patients with breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+) were used. The specimens were all tested using conventional dual ISH (DISH), DISH with an automated slide stainer (ASS) and Saving ISH (1:1-1:3 dilution). RESULTS: The Saving ISH with 1:2 probe dilution produced stable results with less non-specific staining while using smaller amounts of probe. The accuracy of HER2 status with Saving ISH was equal to standard. We found 96.4% agreement between DISH using ASS and Saving ISH (kappa coefficient=0.912). CONCLUSIONS: These results suggest reagent-saving HER2 ISH could be used as a clinical tool for accurate and stable HER2 assessment, even when reagent concentrations vary.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Eletricidade , Amplificação de Genes , Hibridização In Situ/métodos , Receptor ErbB-2/genética , Biomarcadores Tumorais/análise , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Desenho de Equipamento , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ/instrumentação , Valor Preditivo dos Testes , Receptor ErbB-2/análise , Reprodutibilidade dos Testes
3.
Acute Med Surg ; 6(1): 12-24, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30651993

RESUMO

Aim: To describe the registry design of the Osaka Emergency Information Research Intelligent Operation Network system (ORION) and its profile of hospital information, patient and emergency medical service characteristics, and in-hospital outcomes among all patients transported to critical care centers and emergency hospitals in Osaka Prefecture, Japan. Methods: The Osaka Prefecture Government has developed and introduced an information system for emergency patients (the ORION system) that uses a smartphone application (app) for hospital selection by on-scene emergency medical service personnel and has been accumulating all ambulance records. Since January 2015, medical institutions have obtained information on the diagnosis and outcome of patients transported to medical institutions, and the ORION system merged these data with ambulance records including smartphone app data. Results: From January 2015 to December 2016, 753,301 eligible patients were registered. The mean age was 58.7 years, and 51.5% of patients were male. After hospital arrival, 39.7% were hospitalized, 58.2% were discharged from hospital, 1.1% changed hospital, and 1.0% died. The most common diagnoses were injury, poisoning, and certain other consequences of external causes. Among the hospitalized patients, 29.2% were continuously hospitalized, 59.0% discharged, 5.2% changed hospital, and 5.8% were dead at 21 days after hospitalization. The most common confirmed diagnosis was diseases of the circulatory system. Conclusion: Using the ORION system developed and operated by Osaka Prefecture since January 2015, we described the epidemiological data of all emergency patients transported to emergency hospitals. Analysis using the ORION database in the future could lead to improvements in the emergency transport system and patient outcomes.

4.
J Clin Pathol ; 72(1): 25-30, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30228214

RESUMO

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are an effective approach to treating patients with HER2-positive breast cancer. However, the lack of survival benefit in HER2-negative patients, as well as the toxic effects and high cost of the drugs, highlight the need for accurate and prompt assessment of HER2 status. Our aim was to evaluate the clinical utility of a novel reagent-saving immunohistochemistry method (AC-IHC) that saves HER2 antibody by taking advantage of the non-contact mixing effect in microdroplets subjected to an alternating current electric field. METHODS: Ninety-five specimens were used from patients diagnosed with primary breast cancers identified immunohistochemically as HER2 0/1+, 2+ or 3+ using ASCO/CAP guideline-certified standard IHC. The specimens were all tested using the conventional IHC method (1:50 antibody dilution) as well as AC-IHC (1:50 dilution) and reagent-saving AC-IHC (1:100 dilution). RESULTS: The reagent-saving AC-IHC produced stable results with less non-specific staining using smaller amounts of labelled antibody. Moreover, the staining and accuracy of HER2 status evaluated with the reagent-saving AC-IHC method was equal to that achieved with standard IHC. CONCLUSIONS: These results suggest reagent-saving AC-IHC could be used as a clinical tool for accurate and stable HER2 IHC, even when reagent concentrations vary.


Assuntos
Anticorpos/imunologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Testes Diagnósticos de Rotina , Eletricidade , Feminino , Humanos , Imuno-Histoquímica/instrumentação , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
FEBS Open Bio ; 8(6): 940-946, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29928574

RESUMO

Proteins are attractive materials for supramolecular chemistry due to their multifunctionality and self-organization ability. In this work, we synthesized a diheme compound, in which two iron-protoporphyrin IX molecules are associated via a linker chain, and introduced it into a de novo designed four-helix bundle protein with two heme-binding sites. The protein gradually bound the diheme compound by bis-histidyl ligation and formed supramolecular polymers. Polymer formation was observed by atomic force microscopy (AFM), which revealed the highly branched, dendritic forms of the fibrous architecture. The present results may open a pathway toward nanowire construction with de novo heme-proteins.

6.
ESC Heart Fail ; 5(2): 306-310, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29405655

RESUMO

A 52-year-old male visited our hospital with abnormal electrocardiogram and exertional fatigue. The electrocardiogram showed first-degree atrioventricular block, complete right bundle branch block, and inverted T waves in Leads II, III, aVF, V3, and V4. Echocardiography showed biventricular wall thickening involving granular sparkling of the interventricular septum. Late gadolinium enhancement on cardiovascular magnetic resonance (CMR) was found at the circumferential right ventricular wall and patchy regions of the left ventricle. Although these findings strongly suggested cardiac amyloidosis, he was finally diagnosed with systemic sarcoidosis due to the following. First, endomyocardial biopsy revealed non-caseating epithelioid granuloma with giant cells. Second, 18 F-fluorodeoxyglucose positron emission tomography showed uptake in bilateral hilar lymph nodes, para-aortic lymph nodes, and the biventricular wall of the heart. Although echocardiography and CMR are very useful tools for diagnosis of cardiomyopathies, their specificity and accuracy need to be considered.


Assuntos
Cardiomiopatias/diagnóstico , Ventrículos do Coração/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética/métodos , Miocárdio/patologia , Sarcoidose/diagnóstico , Amiloidose/diagnóstico , Diagnóstico Diferencial , Eletrocardiografia , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons
7.
Sci Rep ; 7(1): 15116, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-29118432

RESUMO

Echinoderm microtubule-associated protein-like 4 gene and anaplastic lymphoma kinase gene (EML4-ALK) rearrangement is a key driver mutation in non-small cell lung cancer (NSCLC). Although Break-Apart ALK fluorescence in situ hybridization (FISH) is a reliable diagnostic method for detecting ALK gene rearrangement, it is too costly and time-consuming for use as a routine screening test. Our aim was to evaluate the clinical utility of a novel rapid FISH (RaFISH) method developed to facilitate hybridization. RaFISH takes advantage of the non-contact mixing effect of an alternating current (AC) electric field. Eighty-five specimens were used from patients diagnosed with NSCLC identified immunohistochemically as ALK 0, (1/2+) or (3+). With RaFISH, the ALK test was completed within 4.5 h, as compared to 20 h needed for the standard FISH. Although RaFISH produced results more promptly, the staining and accuracy of the ALK evaluation with RaFISH was equal to the standard. We found 97.6% agreement between FISH and RaFISH based on the status of the ALK signals. These results suggest RaFISH could be used as a clinical tool to promptly determine ALK status.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Técnicas Eletroquímicas/métodos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/genética , Proteínas Associadas aos Microtúbulos/genética , Serina Endopeptidases/genética , Idoso , Quinase do Linfoma Anaplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Feminino , Rearranjo Gênico , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mutação , Reprodutibilidade dos Testes , Serina Endopeptidases/metabolismo
8.
Parasitol Int ; 66(6): 798-801, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28847744

RESUMO

Leeches belonging to the genus Ozobranchus are ectoparasitic on sea and freshwater turtles. The freshwater species O. jantseanus has been recorded from China and Japan. Ozobranchus jantseanus inhabiting Japan is considered to be a non-indigenous species, because their primary host, the Reeves' pond turtle, Mauremys reevesii, is thought to have been introduced in the last few centuries from adjacent Asian countries. To assess whether the Japanese populations of O. jantseanus were likely to have been introduced, their genetic diversity was investigated using mitochondrial cytochrome c oxidase subunit I sequences. The very low sequence diversity as well as the historical record of this species from Japan suggest that Japanese populations of O. jantseanus may have been artificially introduced along with their host turtles. Molecular phylogenies of COI showed that two marine Ozobranchus species formed a clade together with the freshwater O. jantseanus.


Assuntos
DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Sanguessugas/genética , Animais , Variação Genética , Interações Hospedeiro-Parasita , Espécies Introduzidas , Japão , Filogenia , Tartarugas/parasitologia
9.
Sci Rep ; 7(1): 2810, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28584266

RESUMO

Axillary lymph node status and pathological diagnosis of sentinel lymph nodes (SLNs) is a prognostic factor that influences management of postoperative therapy. Recent reports indicate that one-step nucleic acid amplification and hematoxylin and eosin (HE)-stained frozen sections are effective for intraoperative diagnosis of SLNs. In the present study, we report a rapid-immunohistochemical staining (R-IHC) method that enables intraoperative detection of SLN metastases within 16 min using an anti-cytokeratin antibody. This is the first report on SLN diagnosis using R-IHC in patients with breast cancer. We prospectively examined 160 dissected SLNs from 108 breast cancer patients who underwent surgery at our institute. The dissected SLNs were sectioned and conventionally stained with HE or immunohistochemically labeled with anti-cytokeratin antibody using R-IHC procedures. Intraoperative R-IHC analyses were completed within 16 min, after which diagnoses were made by two pathologists. The total time required for intraoperative diagnosis was about 20 min. In this study series, R-IHC detected four metastatic SLNs that were undetected using conventional HE staining (4/20, 20.0%). Compared with subsequent permanent diagnosis, R-IHC offered 95.2% sensitivity and 100% specificity. These findings indicate R-IHC is a clinically applicable technique that enables precise and quick intraoperative detection of micro- and macrometastasis in breast cancer.


Assuntos
Neoplasias da Mama/diagnóstico , Linfonodo Sentinela/patologia , Idoso , Biomarcadores , Neoplasias da Mama/cirurgia , Feminino , Humanos , Imuno-Histoquímica/métodos , Período Intraoperatório , Queratinas/metabolismo , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Linfonodo Sentinela/metabolismo , Biópsia de Linfonodo Sentinela
10.
Ann Thorac Surg ; 104(2): 471-476, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28527968

RESUMO

BACKGROUND: Intraoperative pathologic diagnosis of solitary pulmonary tumors to differentiate between metastatic and primary lung cancer is extremely important to determine the appropriate range of excision. Accurate intraoperative pathologic evaluation may be often difficult, however, and needs additional immunohistochemical (IHC) evaluation to support the diagnosis. Although conventional IHC is a powerful tool for diagnosis, its clinical use is limited intraoperatively because of time constraints. To address this issue, we developed a device that enables complete and rapid IHC (R-IHC) analyses within 20 minutes. We aimed to evaluate the discriminative ability of the R-IHC with anti-thyroid transcription factor-1 (TTF-1) antibody, which is a highly specific IHC marker for primary lung adenocarcinoma. METHODS: A total of 61 pulmonary tumors that were resected at our institute from May 2011 to September 2013 were retrospectively examined. The samples were sectioned, labeled with anti-TTF-1 antibody using the R-IHC method, and pathologically evaluated. The standard used for evaluation was conventional IHC with TTF-1. RESULTS: With the R-IHC procedure, analyses were completed within 20 minutes, with a diagnostic accuracy of 96.7% (59 of 61). Among the 47 primary lung adenocarcinomas, the R-IHC detected 31 (66%) tumors that were positive for TTF-1, with a positive predictive value of 100% (31 of 31). CONCLUSIONS: Our newly developed method of R-IHC with anti-TTF-1 antibody was useful for diagnosing and differentiation of solitary pulmonary tumors. This technology may prove to be an important supplement to standard intraoperative pathologic diagnosis in routine practice.


Assuntos
Adenocarcinoma/diagnóstico , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/diagnóstico , Pulmão/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/cirurgia , Adenocarcinoma de Pulmão , Biomarcadores Tumorais/metabolismo , Diagnóstico Diferencial , Humanos , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/cirurgia , Pneumonectomia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos , Cirurgia Torácica Vídeoassistida , Fator Nuclear 1 de Tireoide
12.
Sci Rep ; 6: 30034, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27443187

RESUMO

Human epidermal growth factor receptor 2 (HER2)-targeted agents are an effective approach to treating HER2-positive breast cancer patients. However, the lack of survival benefit in HER2-negative patients as well as the toxic effects and high cost of the drugs highlight the need for accurate and prompt assessment of HER2 status. Our aim was to evaluate the clinical utility of a novel rapid dual in-situ hybridization (RISH) method developed to facilitate hybridization. The method takes advantage of the non-contact mixing effect of an alternating current (AC) electric field. One hundred sixty-three specimens were used from patients diagnosed with primary breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+). The specimens were all tested using conventional dual in-situ hybridization (DISH), DISH with an automated slide stainer, and RISH. With RISH the HER2 test was completed within 6 h, as compared to 20-22 h needed for the standard protocol. Although RISH produced results more promptly using smaller amounts of labeled antibody, the staining and accuracy of HER2 status evaluation with RISH was equal to or greater than with DISH. These results suggest RISH could be used as a clinical tool to promptly determine HER2 status.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Eletricidade , Hibridização In Situ/métodos , Patologia Molecular/métodos , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/análise , Fatores de Tempo
14.
Sci Rep ; 5: 15371, 2015 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-26477850

RESUMO

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen-antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time.


Assuntos
Ovos , Imunofluorescência , Mamíferos , Animais , Camundongos , Microscopia de Fluorescência , Coloração e Rotulagem
15.
Thromb Haemost ; 95(4): 637-42, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16601834

RESUMO

Plasminogen activator inhibitor type 1 (PAI-1), the primary inhibitor of plasminogen activators, also forms high molecular weight complexes with either thrombin or factor Xa (FXa) in the presence of heparin, resulting in the loss of mutual activities of enzyme and inhibitor. We have proposed that the inactivation of PAI-1 by these activated coagulation factors is one of the mechanisms responsible for coagulation-associated enhancement of fibrinolysis. In the present study, we compared the effects of low molecular weight (LMW)- and unfractionated-heparin on the interaction between PAI-1 and either thrombin or FXa. Both types of heparin enhanced the inhibition of thrombin activity by PAI-1 with a bell-shaped pattern, though the magnitude of the enhancement was significantly weaker with LMW-heparin. In FXa inhibition by PAI-1, only unfractionated-heparin enhanced the inhibition. In the presence of vitronectin (Vn), the inhibition of thrombin and FXa by PAI-1 was further promoted by both types of heparin but to a significantly lesser extent with LMW-heparin. We then analyzed the possible enhancing effect of heparin on tissue plasminogen activator (tPA)-induced fibrinolysis. As a consequence of thrombin-dependent inactivation of PAI-1, tPA-induced fibrin clot lysis time in the presence of PAI-1 was shortened by unfractionated-heparin as well as by LMW-heparin with lesser extent, which was further enhanced by Vn. Less pronounced enhancement of complex formation between thrombin and PAI-1 by LMW-heparin appeared to be directly related to the weaker potential of LMW-heparin in enhancing fibrinolysis and accelerating hemorrhagic tendency via neutralization of PAI-1 activity.


Assuntos
Heparina de Baixo Peso Molecular/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Trombina/química , Coagulação Sanguínea , Cálcio/metabolismo , Fator Xa/metabolismo , Fibrinólise , Heparina/metabolismo , Heparina de Baixo Peso Molecular/metabolismo , Humanos , Proteínas Recombinantes/metabolismo , Trombina/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Vitronectina/metabolismo
16.
Brain Res ; 964(1): 116-20, 2003 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-12573519

RESUMO

Although stroke is a major cause of death and disability in the elderly, the inhibitory effects of neuroprotectants in acute stroke have been investigated using experimental cerebral ischemic models of young animals. Recent clinical trials have found that few neuroprotectants are effective. These observations indicate that effects in the clinical setting do not always reflect data from young animals. Thus, we compared the effects of the NMDA receptor antagonist MK-801 and of the AMPA receptor antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinixaline] on ischemic cerebral damage in the photothrombosis model of aged and young rats. MK-801 administered immediately after MCA occlusion significantly (P<0.05) reduced the extent of cerebral damage in young, but not in aged, rats and the effects of NBQX were similar. In separate experiments, we evaluated brain damage after microinjecting NMDA or kainic acid into the cortex using a stereotaxic apparatus. We found no significant differences in focal cerebral damage caused by NMDA between young and aged rats. On the other hand, kainic acid caused all of the aged rats tested to die, but none of the young rats. Our observations indicate that NMDA and AMPA receptor antagonists are less effective in aged, than in young, rats and that cerebral damage by receptor agonists depends on the type of receptor, such as NMDA and AMPA.


Assuntos
Envelhecimento/metabolismo , Isquemia Encefálica/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Receptores de AMPA/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/metabolismo , Infarto Cerebral/fisiopatologia , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Ácido Caínico/farmacologia , Masculino , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Telencéfalo/efeitos dos fármacos , Telencéfalo/metabolismo , Telencéfalo/fisiopatologia
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