78 research outputs found
Patterned expression of neurotrophic factors and receptors in human limbal and corneal regions
PURPOSE: To evaluate the expression patterns of neurotrophic factors (NTFs) and their receptors in the human cornea with the intention of exploring the role of NTFs in maintaining corneal epithelial stem cells in the limbus. METHODS: Fresh human corneoscleral tissues were prepared for frozen sections. Immunofluorescent staining was performed with primary antibodies against six members of three NTF families and their six receptors. To confirm the specificity of NTF primary antibodies, neutralization experiments with their corresponding peptides and western blot analysis were performed. RESULTS: Based on spatial and differential immuno-localization, three patterns of NTF expression were potentially involved in epithelial-mesenchymal interaction on the ocular surface: (1) the epithelial type: nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF); (2) the paracrine type: neurotrophin (NT)-3 and NT-4/5; and (3) the reciprocal type: brain-derived neurotrophic factor (BDNF). The stem cell-enriched basal cells of the limbal epithelium expressed three unique staining patterns for NTFs: (1) exclusively positive for NGF, GDNF, and their corresponding receptors, TrkA and GDNF family receptor alpha (GFR)-1; (2) relatively high levels of BDNF; and (3) negative for NT-3 and NT-4. Additionally, the neurotrophin common low-affinity receptor, p75NTR, was mainly expressed by the basal layer of the entire corneal and limbal epithelia, and TrkB and TrkC were evenly expressed by the entire corneal and limbal epithelia. BDNF, p75NTR, TrkB, and TrkC are also abundantly expressed by limbal stroma cells. No specific immunoreactivity to ciliary neurotrophic factor (CNTF) and its receptor, CNTFR, was detected in cornea tissue in situ. CONCLUSIONS: Our findings revealed patterned expression of NTFs and their receptors in the human ocular surface, suggesting that they may play a vital role in maintaining corneal epithelial stem cells in the limbus. NGF, GDNF, GFR-1, TrkA, and BDNF may serve as new limbal basal cell markers defining the corneal epithelial stem cell phenotype.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000250807800002&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Biochemistry & Molecular BiologyOphthalmologySCI(E)42ARTICLE217-191934-19411
Blood or serum collected on filter paper for detection of antibodies to bovine herpesvirus type 1 (BoHV-1)
Background: The method of collection as well as the packaging conditions in which samples are submitted to laboratories play a critical role on the acquisition of reliable results on diagnostic tests. Alternative methods however have been proposed, as the adsorption of blood or serum in filter paper. In this work, it was evaluated the viability of using serum or whole blood samples from bovines collected in filter paper for serological testing against bovine herpesvirus type 1 (BoHV-1). Materials, Methods & Results: One hundred and seven whole blood and serum samples were collected by standard methods. Serum neutralization test was used as a golden standard method for evaluation of the serum samples. The same samples of both whole blood and sera were also adsorbed on filter paper strips for further comparisons. Optimal conditions for serum and blood elution from filter paper were determined. Adsorbed samples on filter paper disks were eluted in PBS and subsequently diluted further with PBS 5% skimmed milk. The eluates were tested for antibodies to BoHV-1 in an indirect ELISA (iELISA) and matched with the results obtained by serum neutralization of standard serum samples. Comparison between results obtained by serum neutralization of standard serum samples and the ones from the iELISA of serum eluted from paper disks resulted in sensitivity, specificity, positive and negative predictive values of 95, 94, 80, 99%, respectively, and a correlation coefficient (κ) of 0.83. Comparison between the results of serum neutralization of standard serum samples and the ones from the iELISA of blood eluted from paper disks resulted in sensitivity, specificity, positive and negative predictive values of 91, 97, 87, 98% , respectively, and a correlation coefficient (κ) of 0.86. Standard serum samples were also tested in the iELISA and the results compared with those of iELISA from samples eluted from filter paper. Comparison of the results from iELISA between serum samples and serum eluted from filter paper resulted in sensitivity, specificity, positive and negative predictive values of 91, 81, 69, 95%, respectively, and a correlation coefficient (κ) of 0.66. The comparison of iELISA results between standard serum and blood samples eluted from filter paper resulted, in sensitivity, specificity, positive and negative predictive values of 78, 92, 82, 89%, respectively, and a correlation coefficient (κ) of 0.70. Fifty samples collected on filter paper were retested eight months later in order to determine whether those would retain its viability; both sensitivity and specificity remained unaltered. Discussion: Sampling on filter paper has been successfully described for antibody detection in a number of diseases such as Aujeszky’s disease virus and Newcastle disease virus. In this work, it has been demonstrated that both blood and sera collected in filter paper can be used for submission of samples aiming detection of antibodies to BoHV-1 in an iELISA, without significant loss of sensitivity and specificity. Submission of samples on filter paper is a practical and economical alternative as no special conditions are required for storaging and transporting. This method also enables the collection of samples from distant places assuring its quality for serological test
Effect of Antioxidant Treatment on Fibrogenesis in Rats with Carbon Tetrachloride-Induced Cirrhosis
Aim. This study aimed to assess the antioxidant activity of quercetin (Q) in an experimental model of cirrhosis induced by CCl4 inhalation. Materials and Methods. We used 25 male Wistar rats (250 g) that were divided into 3 groups: control (CO), CCl4, and CCl4 + Q. The rats were subjected to CCl4 inhalation (2x/week) for 16 weeks, and they received phenobarbital in their drinking water at a dose of 0.3 g/dL as a P450 enzyme inducer. Q (50 mg/Kg) was initiated intraperitoneally at 10 weeks of inhalation and lasted until the end of the experiment. Statistical analysis was by ANOVA Student Newman-Keuls (mean ± SEM), and differences were considered statistically significant when P < 0.05. Results. After treatment with quercetin, we observed an improvement in liver complications, decreased fibrosis, as analyzed by picrosirius for the quantification of collagen, and decreased levels of matrix metalloproteinase 2 (MMP-2) compared with the CCl4 group. It also reduced oxidative stress, as confirmed by the decrease of substances reacting to thiobarbituric acid (TBARS), the increased activity of antioxidant enzymes, and the reduced glutathione ratio and glutathione disulfide (GSH/GSSG). Conclusion. We suggest that the use of quercetin might be promising as an antioxidant therapy in liver fibrosis
Chaperoned amyloid proteins for immune manipulation: A-synuclein/hsp70 shifts immunity toward a modulatory phenotype
a-Synuclein (aSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson’s disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective aSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical ‘‘chaperone’’ activities and their recently discovered and diverse ‘‘immunoactive’’ properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an aSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the aSyn/Hsp70 complex, including a restrained aSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4 CD25 Foxp3 ) and Teff (CD4 Foxp3 ) cell populations, as opposed to significant changes in mice immunized with aSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against aSyn challenge for the ‘‘aSyn/Hsp70’’ experimental group as measured by IFN-g and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-g and immunomodulatory IL-10 indicated a unique shift toward an immunomodulato-ry/immunoprotective phenotype in mice immunized with the aSyn/Hsp70 complex. Overall, we propose the use of functional ‘‘HSP-chaperoned amyloid/ aggregating proteins’’ generated with appropriate HSP-substrate protein combinations, such as the aSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or ‘‘misfolding’’ neurodegenerative disorders.Financial support was provided by the Carlos III Institute
of Health of Spain (Spanish Ministry of Economy and
Competitiveness) according to the Strategic Action in
Health (CP10/00527 to CR; PI14-01600 to DP) with
co-funding by FEDER funds, the Spanish Ministry of
Economy and Competitiveness (SAF-2012/39720 to CR),
the Andalusian Ministry of Economy, Science and Innovation
(P10-CTS-6928 and P11-CTS-8161 to DP) and the
PAIDI Program from the Andalusian Government (CTS-
677 to DP). ALG holds a FPU Predoctoral Fellowship from
the Spanish Ministry of Education (AP-2009/3816). The
works of EJDG and CMD are supported by the Wellcome
Trust, and the UK Medical, and Biotechnological and
Biological Sciences Research Councils
IL-13 Stimulates Proliferation and Expression of Mucin and Immunomodulatory Genes in Cultured Conjunctival Goblet Cells
Citation: Tukler Henriksson J, Coursey TG, Corry DB, De Paiva CS, Pflugfelder SC. IL-13 stimulates proliferation and expression of mucin and immunomodulatory genes in cultured conjunctival goblet cells. Invest Ophthalmol Vis Sci. 2015;56:4186-4197. DOI:10.1167/iovs.14-15496 PURPOSE. To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes. METHODS. Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 lL per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array. RESULTS. The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-b at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment. CONCLUSIONS. This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined. Keywords: conjunctiva, goblet cells, interleukin-13, cell culture T he conjunctiva covers two-thirds of the ocular surface and functions as a support tissue for cornea. 2,3 Conjunctival goblet cells are surrounded by lymphocytes and dendritic cells and their density has been found to change in certain ocular surface immune/inflammatory conditions. 4 Goblet cell density has been reported to decrease in aqueous tear deficiency, a condition where T helper 1 (Th1) and Th17 cells infiltrate the conjunctiva, and increase in atopic keratoconjunctivitis and vernal keratoconjunctivitis, predominantly Th2-mediated diseases. 5-8 The mucus stimulating activity of the Th2 cytokine IL-13 has been reported to have a defensive role in the intestines by eliminating helminthic parasites and in the airways by protecting from particles or allergens. 9,10 However, excessive IL-13 expression is associated with goblet cell hyperplasia and mucous hypersecretion, both in the gut and in the airways where it can result in airway obstruction. The purpose of the present study was to investigate whether the Th2 cytokine IL-13 can modulate proliferation, differentiation, and expression of mucin and immunomodulatory gene
Autochthonous Canine Visceral Leishmaniasis in Urban Area of Porto Alegre, RS, Brazil
Background: Leishmaniasis is a chronic infectious disease caused by intracellular protozoan Leishmania that affects canine and human. The visceral leishmaniasis (VL) is caused by the Leishmania donovani complex, in which canines are the main reservoir for human VL. In Southern Brazil, case reports of this disease have increased, especially when canines are infected in endemic areas in the country. Canines usually present a systemic disease, characterized by cutaneous lesions, weight loss, generalized lymphadenopathy, hepatomegaly and splenomegaly. This report aims to describe the clinicalpathological features of a case of autochthonous VL in a canine of an urban area of Porto Alegre.Case: A male, mixed breed, dog , 7 year-old, resident at the East Side of Porto Alegre, living together with two other canines, presented at clinical examination progressive weight loss, associated with hyporexia and hyperthermia, evolving to lateral recumbency, paralisys, and death. At necropsy, the dog was cachectic with diffusely pale mucosae. Gross findings included liver enlarged, with multifocal firm brownish areas, spleen enlarged, showing multifocal firm round dark-red areas, and kidneys diffusely pale with evidentiated tranversal striations on cortical zone. At microscopic examination, there was on the spleen diffuse inflammatory infiltrate of macrophages with large cytoplasm containing multiple amastigotes. The liver, exhibited atrophy of hepatocytes and moderate multifocal inflammatory infiltrate in sinusoids of macrophages containing multiple amastigotes. These features were also observed moderately on lymph nodes and lamina propria of large intestine. Immunohistochemistry examination showed marked positive staining for Leishmania spp. in amastigotes located whitin the cytoplasm of macrophages of spleen, liver, lymph nodes and large intestine.Discussion: Canine leishmaniasis is a disease that affects both internal organs and skin. However, the condition is called VL mostly because it is associated with the same species that cause the visceral form in humans. In this case the canine presented liver, spleen and lymph node lesions; however no skin lesion was observed during the clinical examination. A serological evaluation of canids in East Side of Porto Alegre performed in 2005 showed that 3.5% of these dogs were positive for Leishmania; yet neither of these animals presented clinical signs. This demonstrates that the agent was already present, however only 3-10% of the canines infected develop clinical disease. In non-endemic areas VL is related to the migration of canids from endemic areas, where VL is common. However the canine here described was born, raised, and was living in Porto Alegre. Lutzomyia longipalpis is the main vector of VL and despite its wide distribution it was identified only in 2009 in Rio Grande do Sul State. Autochthonous cases of VL were reported in canines and humans in this state, confirming the hypothesis that the vector was widely distributed and it is now present in Porto Alegre, keeping the epidemiological cycle of that disease active. Canine VL occurs prior in humans, since canines are the main domestic reservoirs and are critical for the maintenance of this disease cycle. This is the first autochthonous VL in a canine of an urban area of Porto Alegre; therefore authorities should be alert, and new control measures must be taken to avoid the canine leishmaniasis outbreak due to its potential for zoonotic transmission.Keywords: leishmaniasis, canine, protozoan, immunohistochemistry
Structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation
This is the author accepted manuscript. The final version is avialble via PNAS at http://www.pnas.org/content/112/16/E1994.long#ack-1.We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.We thank Dr. Katherine Stott, from the Biophysics Facility, Department of Biochemistry, University of Cambridge, for her assistance in using these facilities. This work was supported by the Agency for Science, Technology and Research, Singapore (S.W.C.), the “La Caixa” foundation (S.D.), Wellcome/MRC (Medical Research Council) Parkinson’s Disease Consortium Grant WT089698 (to E.D. and N.W.W.), National Institute for Health Research Biomedical Research Centres funding at University College London (to N.W.W.), the BBSRC through Grants BB/H003843/1 (to M.O.) and BB/E019927/1 (to C.M.D.), the Spanish Ministry of Economy and Competitiveness through Grants SAF 2012-39720 (to C.R.), BFU2013-44202 (to J.M.V.), and BIO2011-28941-C03-03 (to C.A. and G.R.), the Spanish Ministry of Health with cofunding by The European Regional Development Fund through Grant CP10/00527 (to C.R.), the Madrid Regional Government through Grant S2013/MIT-2807 (to J.M.V.), Parkinson’s UK through Grant H-0903 (to T.G.), the Wellcome Trust, the Leverhulme Trust, the European Commission through project LSHM-CT-2006-037525 (to C.M.D.), the Medical Research Council through Grant MRC G1002272 (to E.J.D.-G. and C.M.D.), and the Engineering and Physical Sciences Research Council (C.M.D.). A.Y.A. was a Parkinson’s UK Senior Research Fellow. N.C. is a Royal Society Research Fellow and also acknowledges financial support by the Human Frontier Science Program from Long-Term Fellowship LT000795/2009
TFOS DEWS II Report Executive Summary
This article presents an Executive Summary of the conclusions and recommendations of the 10-chapter TFOS DEWS II report. The entire TFOS DEWS II report was published in the July 2017 issue of The Ocular Surface. A downloadable version of the document and additional material, including videos of diagnostic and management techniques, are available on the TFOS website: www.TearFilm.org
TAXA DE NOTIFICAÇÃO DE DOENÇAS TRANSMITIDAS POR ALIMENTOS EM 2021 E 2022 NO CEARÁ
Foodborne Diseases (FBD) are diseases caused by microorganisms or toxins linked to contaminated food, resulting in gastrointestinal disorders and in some cases hospitalization and death. Outbreak is defined when two or more people manifest similar clinical symptoms after eating food in an equivalent period and space. The objective of this work was to trace the profile of FBD in Ceará from data extracted from the Central Laboratory of Public Health (Lacen-CE). The methodology used was the compilation of data issued in outbreak reports in the Laboratory Sample Management System (HARPYA) in 2021 and 2022. During this period, 7 cities reported 11 outbreaks of FBD. The pathogens found were Escherichia coli, coagulase-positive Staphylococci and Bacillus cereus. Knowing the profile of FBD and notifying the outbreaks is important for the sanitary inspection bodies to take protection, promotion and control measures, ensuring a better quality of life for the population.As Doenças Transmitidas por Alimentos (DTA) são doenças causadas por microrganismos ou toxinas ligadas a alimentos contaminados, resultando em distúrbios gastrointestinais e, em alguns casos, hospitalização e morte. O surto é definido quando duas ou mais pessoas manifestam sintomas clínicos semelhantes após consumirem alimentos em período e espaço equivalentes. O objetivo deste trabalho foi traçar o perfil das DTA no Ceará a partir de dados extraídos do Laboratório Central de Saúde Pública (Lacen-CE). A metodologia utilizada foi a compilação de dados emitidos em relatórios de surtos no Sistema de Gerenciamento de Amostras Laboratoriais (HARpya) em 2021 e 2022. Nesse período, 7 cidades notificaram 11 surtos de DTA. Os patógenos encontrados foram Escherichia coli, Staphylococci coagulase positiva e Bacillus cereus. Conhecer o perfil das ETA e notificar os focos é importante para que os órgãos de fiscalização sanitária tomem medidas de proteção, promoção e controle, garantindo melhor qualidade de vida à população
Transfusion practices in brazilian Intensive Care Units (pelo FUNDO-AMIB)
BACKGROUND AND OBJECTIVES: Anemia of critical illness is a multifactorial condition caused by blood loss, frequent phlebotomies and inadequate production of red blood cells (RBC). Controversy surrounds the most appropriate hemoglobin concentration trigger for transfusion of RBC. We aimed to evaluate transfusion practices in Brazilian ICUs. METHODS: A prospective study throughout a 2-week period in 19 Brazilian ICUs. Hemoglobin (Hb) level, transfusion rate, organ dysfunction assessment and 28-day mortality were evaluated. Primary indication for transfusion and pretransfusion hemoglobin level were collected for each transfusion. RESULTS: Two hundred thirty-one patients with an ICU length of stay longer than 48h were included. An Hb level lower than 10 g/dL was found in 33% on admission in the ICU. A total of 348 RBC units were transfused in 86 patients (36.5%). The mean pretransfusion hemoglobin level was 7.7 ± 1.1 g/dL. Transfused-patients had significantly higher SOFA score (7.9 ± 4.6 vs 5.6 ± 3.8, p < 0.05, respectively), days on mechanical ventilation (10.7 ± 8.2 vs 7.2 ± 6.4, p < 0.05) and days on vasoactive drugs (6.7 ± 6.4 vs 4.2 ± 4.0, p < 0.05) than non-transfused patients despite similar APACHE II scores (15.2 ± 8.1 vs 14.2 ± 8.1, NS). Transfused patients had higher mortality rate (43.5%) than non-transfused patients (36.3%) (RR 0.60-1.15, NS). Only one patient (0.28%) had febrile non-hemolytic transfusion and urticarial reactions. CONCLUSIONS: Anemia is common in critically ill patients.It seems from the present study that transfusion practices in Brazil have had a more restrictive approach with a lower limit transfusion trigger.JUSTIFICATIVA E OBJETIVOS: A anemia é uma condição comum em pacientes graves. A transfusão de hemoderivados aumenta de forma significativa o risco de transmissão de agentes infecciosos e afeta o perfil imunológico. O objetivo deste estudo foi avaliar a incidência de anemia e a prática de transfusão de hemácias em UTI brasileiras. MÉTODO: Estudo prospectivo, multicêntrico, realizado em 19 UTI em um período de duas semanas. A presença de anemia, as indicações e a utilização de concentrados de hemácias, foram avaliadas diariamente. As complicações que ocorreram durante a internação na UTI e após a transfusão da primeira unidade de concentrado de hemácias foram registradas. RESULTADOS: Um total de 33% apresentava anemia na admissão na UTI e esta proporção aumentou para 55% no final de sete dias de internação. Um total de 348 unidades de concentrado de hemácias foi transfundido em 86 pacientes (36,5%). A média de suas unidades por paciente foi 4,1 ± 3,3 U. O nível de hemoglobina limiar para a transfusão de CH foi 7,7 ± 1,1 g/dL. Pacientes transfundidos tinham mais disfunções orgânicas avaliadas pelo escore SOFA (7,9 ± 4,6 versus 5,6 ± 3,8, transfundidos versus não transfundidos, p < 0,05). As taxas de mortalidade foram 43,5% e 36,3% em pacientes transfundidos e não transfundidos, respectivamente (RR 0,61-11,7, NS). Pacientes transfundidos tiveram número maior de complicações (1,58 ± 0,66 versus 1,33 ± 0,49, p = 0,0001). CONCLUSÕES: A anemia é comum em UTI brasileiras. O limiar transfusional de hemoglobina foi menor do que o observado em outros paises.Faculdade de Medicina de São José do Rio PretoUniversidade de São PauloUFRGS Departamento de Medicina Interna HC de Porto AlegreUniversidade Paris VIUFRJ CTI dos Hospitais Cardiotrauma Ipanema e São LucasAMIBUniversidade Estadual de LondrinaUFRGS FAMED HCPAFaculdade de Medicina de CatanduvaUNIFESP-EPMFundação Padre Albino UTI do Complexo HospitalarUniversidade Federal de São Paulo (UNIFESP) Disciplina de Anestesiologia, Dor e Terapia Intensiva Setor de TerapiaSanta Casa de Misericórdia de São PauloHospital Unimed de LimeiraUTI do Hospital Regional de AssisAMIB Departamento de MedicinaAmerican CollegeFundação Getúlio VargasHospital Pró CardíacoUNIRIOFGVHospital Santa Helena de GoiâniaHospital evangélico de Cachoeiro de Itapemirim Unidade coronarianaSBNHospital Evangélico Cachoeiro de Itapemirim UTI Adulto e CoronarianaUFRJUFRN Hospital Onofre Lopes UTIHospital Novo AtibaiaUNIFESP, EPMUNIFESP, Disciplina de Anestesiologia, Dor e Terapia Intensiva Setor de TerapiaSciEL
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