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Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.
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Factor de Crecimiento Epidérmico , Receptores Notch , Humanos , Animales , Ratones , Factor de Crecimiento Epidérmico/química , Células HEK293 , Receptores Notch/metabolismo , Receptor Notch1/química , Acetilglucosamina/metabolismo , Proteínas de Unión al Calcio , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes. In influenza infection, elevating Gb3, either endogenously or exogenously, promoted broadly reactive antibody responses and cross-protection. These data demonstrate that Gb3 determines the affinity and breadth of B cell immunity and has potential as a vaccine adjuvant.
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Anticuerpos Antivirales , Linfocitos B , Centro Germinal , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Trihexosilceramidas , Formación de Anticuerpos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Trihexosilceramidas/metabolismo , Trihexosilceramidas/farmacología , Animales , Ratones , Ratones Noqueados , Humanos , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunologíaRESUMEN
ABSTRACT: Glucosamine (UDP-N-acetyl)-2-epimerase and N-acetylmannosamine (ManNAc) kinase (GNE) is a cytosolic enzyme in de novo sialic acid biosynthesis. Congenital deficiency of GNE causes an autosomal recessive genetic disorder associated with hereditary inclusion body myopathy and macrothrombocytopenia. Here, we report a pediatric patient with severe macrothrombocytopenia carrying 2 novel GNE missense variants, c.1781G>A (p.Cys594Tyr, hereafter, C594Y) and c.2204C>G (p.Pro735Arg, hereafter, P735R). To investigate the biological significance of these variants in vivo, we generated a mouse model carrying the P735R mutation. Mice with homozygous P735R mutations exhibited cerebral hemorrhages as early as embryonic day 11 (E11), which subsequently progressed to large hemorrhages in the brain and spinal cord, and died between E11.5 and E12.5. Defective angiogenesis such as distended vascular sprouts were found in neural tissues and embryonic megakaryocytes were abnormally accumulated in the perineural vascular plexus in mutant mouse embryos. Furthermore, our in vitro experiments indicated that both C594Y and P735R are loss-of-function mutations with respect to de novo sialic acid biosynthesis. Overall, this study reveals a novel role for GNE-mediated de novo sialic acid biosynthesis in mouse embryonic angiogenesis.
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Angiogénesis , Ácido N-Acetilneuramínico , Animales , Niño , Humanos , Ratones , Encéfalo , Mutación , Mutación MissenseRESUMEN
Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling. Abundance of Gb3 amplifies the PI3-kinase/Akt/Foxo1 pathway to drive affinity maturation. Moreover, this lipid regulates MHC-II expression to increase diversity of T follicular helper (Tfh) and GC B cells reactive with subdominant epitopes. In influenza infection, Gb3 promotes broadly reactive antibody responses and cross-protection. Thus, we show that Gb3 determines affinity as well as breadth in B cell immunity and propose this lipid as novel vaccine adjuvant against viral infection. One Sentence Summary: Gb3 abundance on GC B cells selects antibodies with high affinity and broad epitope reactivities, which are cross-protective against heterologous influenza infection.
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BACKGROUND AND AIMS: Accurate risk stratification for hepatocellular carcinoma (HCC) after achieving a sustained viral response (SVR) is necessary for optimal surveillance. We aimed to develop and validate a machine learning (ML) model to predict the risk of HCC after achieving an SVR in individual patients. METHODS: In this multicenter cohort study, 1742 patients with chronic hepatitis C who achieved an SVR were enrolled. Five ML models were developed including DeepSurv, gradient boosting survival analysis, random survival forest (RSF), survival support vector machine, and a conventional Cox proportional hazard model. Model performance was evaluated using Harrel' c-index and was externally validated in an independent cohort (977 patients). RESULTS: During the mean observation period of 5.4 years, 122 patients developed HCC (83 in the derivation cohort and 39 in the external validation cohort). The RSF model showed the best discrimination ability using seven parameters at the achievement of an SVR with a c-index of 0.839 in the external validation cohort and a high discriminative ability when the patients were categorized into three risk groups (P <0.001). Furthermore, this RSF model enabled the generation of an individualized predictive curve for HCC occurrence for each patient with an app available online. CONCLUSIONS: We developed and externally validated an RSF model with good predictive performance for the risk of HCC after an SVR. The application of this novel model is available on the website. This model could provide the data to consider an effective surveillance method. Further studies are needed to make recommendations for surveillance policies tailored to the medical situation in each country. IMPACT AND IMPLICATIONS: A novel prediction model for HCC occurrence in patients after hepatitis C virus eradication was developed using machine learning algorithms. This model, using seven commonly measured parameters, has been shown to have a good predictive ability for HCC development and could provide a personalized surveillance system.
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Dystroglycan (DG), a muscular transmembrane protein, plays a critical role in transducing extracellular matrix-derived signals to the cytoskeleton and provides physical strength to skeletal muscle cell membranes. The extracellular domain of DG, α-DG, displays unique glycosylation patterns. Fully functional glycosylation is required for this domain to interact with components of extracellular matrices, including laminin. One of the unique sugar compositions found in such functional glycans on DG is two ribitol phosphates that are transferred by the sequential actions of fukutin (FKTN) and fukutin-related protein (FKRP), which use CDP-ribitol as a donor substrate. These are then further primed for matriglycan biosynthesis. A recent in vitro study reported that glycerol phosphate could be similarly added to α-DG by FKTN and FKRP if they used CDP-glycerol (CDP-Gro) as a donor substrate. However, the physiological relevance of these findings remains elusive. Imae et al. addressed the knowledge gap regarding whether CDP-Gro is present in mammals and how CDP-Gro is synthesized and functions in mammals.
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Distroglicanos , Pentosiltransferasa , Animales , Distroglicanos/metabolismo , Glicerol , Glicosilación , Pentosiltransferasa/metabolismo , Ribitol/metabolismo , Ribitol/farmacologíaRESUMEN
Background: Previous research revealed a lack of comfort and knowledge regarding nuclear and radiological events among medical staff. We investigated the awareness and knowledge of radiological and nuclear events among the Japanese medical staff by comparing differences by occupation (doctors, nurses, and other medical specialists). Methods: We conducted a cross-sectional questionnaire survey among trainees undergoing Japanese disaster medical training courses between July 2014 and February 2016. The differences by occupation were evaluated for all questions on awareness and knowledge concerning disasters or radiological and nuclear events and demographics. Results: Among the occupations, there were significant differences in the willingness to work onsite based on the types of disaster, familiarity with the national disaster medical response system, the accuracy rate of some knowledge about medical practice and the risk, and demographic characteristics such as practical experience and educational degree. The accuracy rates of responses to some questions on knowledge were very low in all occupations. Conclusion: There were significant differences in awareness and knowledge of radiological and nuclear events by occupation. We believe that the results can be used to develop and modify the content of training courses on radiological and nuclear events to make such courses beneficial for each healthcare worker.
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Planificación en Desastres , Estudios Transversales , Personal de Salud , Humanos , Japón , Encuestas y CuestionariosRESUMEN
Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.
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Glucolípidos , Neoplasias , Animales , Antígenos de Carbohidratos Asociados a Tumores , Biomarcadores de Tumor , Humanos , Transducción de SeñalRESUMEN
Microvascular thrombosis in patients with thrombotic thrombocytopenic purpura (TTP) is initiated by GPIbα-mediated platelet binding to von Willebrand factor (VWF). Binding of VWF to GPIbα causes activation of the platelet surface integrin αIIbß3. However, the mechanism of GPIbα-initiated activation of αIIbß3 and its clinical importance for microvascular thrombosis remain elusive. Deletion of platelet C-type lectin-like receptor 2 (CLEC-2) did not prevent VWF binding to platelets but specifically inhibited platelet aggregation induced by VWF binding in mice. Deletion of platelet CLEC-2 also inhibited αIIbß3 activation induced by the binding of VWF to GPIbα. Using a mouse model of TTP, which was created by infusion of anti-mouse ADAMTS13 monoclonal antibodies followed by infusion of VWF, we found that deletion of platelet CLEC-2 decreased pulmonary arterial thrombosis and the severity of thrombocytopenia. Importantly, prophylactic oral administration of aspirin, an inhibitor of platelet activation, and therapeutic treatment of the TTP mice with eptifibatide, an integrin αIIbß3 antagonist, reduced pulmonary arterial thrombosis in the TTP mouse model. Our observations demonstrate that GPIbα-mediated activation of integrin αIIbß3 plays an important role in the formation of thrombosis in TTP. These observations suggest that prevention of platelet activation with aspirin may reduce the risk for thrombosis in patients with TTP.
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Hipertensión Pulmonar , Púrpura Trombocitopénica Trombótica , Trombosis , Aspirina , Plaquetas/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Activación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Púrpura Trombocitopénica Trombótica/metabolismo , Trombosis/etiología , Factor de von Willebrand/metabolismoRESUMEN
The strong fascination exerted by the binary compound of FeSe demands reliable engineering protocols and more effective approaches toward inducing superconductivity in FeSe thin films. Our study addresses the peculiarities in pulsed laser deposition that determine FeSe thin-film growth and focuses on the film/substrate interface, which has only been considered hypothetically in the past literature. The FeSe/MgO interface has been assumed (1) to be clean and (2) to obey lattice-matching epitaxy. Our studies reveal that both assumptions are misleading and demonstrate the tendency for domain-matching epitaxial growth, which accompanies the problem of chemical heterogeneity. We propose that homogenization of the film/substrate interface by an Fe buffer can improve the control of stoichiometry and nanostrain in a way that favors superconductivity even in ultrathin FeSe films. We will also show that on a chemically homogenized FeSe/Fe interface, the control of film texture with preparation conditions is still possible.
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BACKGROUND AND AIMS: It remains unclear whether obesity increases the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C who achieved a sustained virological response (SVR) with antiviral therapy. METHODS: In this multicenter cohort study, we enrolled patients with chronic hepatitis C who achieved SVR with interferon (IFN)-based therapy (IFN group) or direct-acting antiviral (DAA) therapy (DAA group) between January 1, 1990, and December 31, 2018. The patients underwent regular surveillance for HCC. Cumulative incidence of and the risk factors for HCC development after SVR were assessed using the Kaplan-Meier method and Cox proportional hazard regression analysis, respectively. RESULTS: Among 2,055 patients (840 in the IFN group and 1,215 in the DAA group), 75 developed HCC (41 in the IFN group and 34 in the DAA group) during the mean observation period of 4.1 years. The incidence rates of HCC at 1, 2, and 3 years were 1.2, 1.9, and 3.0%, respectively. Multivariate analysis revealed that in addition to older age, lower albumin level, lower platelet count, higher alpha-fetoprotein level, and absence of dyslipidemia, obesity (body mass index ≥25 kg/m2) and heavy alcohol consumption (≥60 g/day) were independent risk factors for HCC development, with adjusted hazard ratio (HR) of 2.53 (95% confidence interval [CI]: 1.51-4.25) and 2.56 (95% CI: 1.14-5.75), respectively. The adjusted HR was not significant between the 2 groups (DAA vs. IFN; HR 1.19, 95% CI: 0.61-2.33). CONCLUSIONS: Obesity and heavy alcohol consumption increased the risk of HCC development after SVR.
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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a pandemic. Severe disease is associated with dysfunction of multiple organs, but some infected cells do not express ACE2, the canonical entry receptor for SARS-CoV-2. Here, we report that the C-type lectin receptor L-SIGN interacted in a Ca2+-dependent manner with high-mannose-type N-glycans on the SARS-CoV-2 spike protein. We found that L-SIGN was highly expressed on human liver sinusoidal endothelial cells (LSECs) and lymph node lymphatic endothelial cells but not on blood endothelial cells. Using high-resolution confocal microscopy imaging, we detected SARS-CoV-2 viral proteins within the LSECs from liver autopsy samples from patients with COVID-19. We found that both pseudo-typed virus enveloped with SARS-CoV-2 spike protein and authentic SARS-CoV-2 virus infected L-SIGN-expressing cells relative to control cells. Moreover, blocking L-SIGN function reduced CoV-2-type infection. These results indicate that L-SIGN is a receptor for SARS-CoV-2 infection. LSECs are major sources of the clotting factors vWF and factor VIII (FVIII). LSECs from liver autopsy samples from patients with COVID-19 expressed substantially higher levels of vWF and FVIII than LSECs from uninfected liver samples. Our data demonstrate that L-SIGN is an endothelial cell receptor for SARS-CoV-2 that may contribute to COVID-19-associated coagulopathy.
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COVID-19 , Capilares , Moléculas de Adhesión Celular/metabolismo , Células Endoteliales , Lectinas Tipo C/metabolismo , Hígado/irrigación sanguínea , Vasos Linfáticos , Receptores de Superficie Celular/metabolismo , SARS-CoV-2/fisiología , COVID-19/metabolismo , COVID-19/patología , COVID-19/virología , Capilares/metabolismo , Capilares/patología , Capilares/virología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/virología , Perfilación de la Expresión Génica/métodos , Humanos , Hígado/patología , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Vasos Linfáticos/virología , Glicoproteína de la Espiga del Coronavirus , Internalización del VirusRESUMEN
Eradication of hepatitis C virus (HCV) using direct acting antiviral agents (DAAs) has been reported to alter liver function and reduce the recurrence rate after curative treatment in naïve hepatocellular carcinoma (HCC) patients. However, it is not well known whether administration of DAAs had favourable effect on HCC patients with multiple courses of recurrence. We retrospectively extracted 146 HCV-related HCC (C-HCC) patients who received curative treatment using radiofrequency ablation (RFA) followed by eradication treatment with DAA between 1 January 2015 and 31 December 2017. We also extracted 184 C-HCC patients who were curatively treated using RFA without HCV eradication treatment between 1 January 2009 and 31 July 2014 as controls. We used propensity score matching method and adjusted following factors between the 2 groups: age, sex, liver function, number of recurrence times, tumour diameter and tumour numbers. We finally enrolled 47 C-HCC patients with eradication of HCV, and 47 C-HCC patients without HCV eradication as controls. Primary end point was time to curative treatment failure. We defined time to curative treatment failure as the interval from curative treatment initiation to premature discontinuation of this type of therapy. Their clinical data, time to curative treatment failure and overall survival were compared. We also assessed the prognostic values of time to curative treatment failure and overall survival using multivariate Cox proportional hazard models. The median age was 74.8 years, 60 patients (63.8%) were male, and 81 patients (86.2%) were Child-Pugh class A. The median tumour number was 1, tumour diameter was 20 mm, and frequency of recurrence was 3 times. There were no significant differences about patients' backgrounds between the 2 groups. The cumulative time to curative treatment failure rates of patients who received DAA were 93.6% and 73.2% at 1 and 3 years, respectively; those of controls were 72.5%, and 37.1% (p < .01). Multivariate analysis indicated that eradication with DAAs (HR 0.23, 95% CI; 0.12-0.43, p < .01) and DCP >50 mAU/ml (HR 2.62, 95% CI; 1.45-4.74, p < .01) as independent factors contributed to time to curative treatment failure. The cumulative overall survival rates of patients who received DAAs were 93.6% and 72.6% at 1 and 3 years, respectively; those of controls were 72.8% and 37.4% (p < .01). Multivariate analysis indicated that eradication with DAAs (HR 0.32, 95% CI; 0.17-0.60, p < .01) and frequency of recurrence times (HR 1.20 per 1 time, 95% CI; 1.01-1.42, p = .038) as independent factors related to overall survival. Eradication of HCV using DAAs prolonged not only time to curative treatment failure but also overall survival even in C-HCC patients with multiple courses of recurrence.
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Carcinoma Hepatocelular , Hepatitis C Crónica , Hepatitis C , Neoplasias Hepáticas , Anciano , Antivirales/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/epidemiología , Hepacivirus , Hepatitis C/complicaciones , Hepatitis C/tratamiento farmacológico , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Masculino , Recurrencia Local de Neoplasia , Estudios RetrospectivosRESUMEN
The liver has recently been identified as a major organ for destruction of desialylated platelets. However, the underlying mechanism remains unclear. Kupffer cells, which are professional phagocytic cells in the liver, comprise the largest population of resident tissue macrophages in the body. Kupffer cells express a C-type lectin receptor, CLEC4F, that recognizes desialylated glycans with an unclear in vivo role in mediating platelet destruction. In this study, we generated a CLEC4F-deficient mouse model (Clec4f-/-) and found that CLEC4F was specifically expressed by Kupffer cells. Using the Clec4f-/- mice and a newly generated platelet-specific reporter mouse line, we revealed a critical role for CLEC4F on Kupffer cells in mediating destruction of desialylated platelets in the liver in vivo. Platelet clearance experiments and ultrastructural analysis revealed that desialylated platelets were phagocytized predominantly by Kupffer cells in a CLEC4F-dependent manner in mice. Collectively, these findings identify CLEC4F as a Kupffer cell receptor important for the destruction of desialylated platelets induced by bacteria-derived neuraminidases, which provide new insights into the pathogenesis of thrombocytopenia in disease conditions such as sepsis.
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Receptor de Asialoglicoproteína/metabolismo , Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Modelos Animales de Enfermedad , RatonesRESUMEN
During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.
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Encéfalo/irrigación sanguínea , Aneurisma Intracraneal/etiología , Megacariocitos/patología , Glicoproteínas de Membrana/deficiencia , Aneurisma Roto/embriología , Aneurisma Roto/etiología , Angiopoyetina 1/metabolismo , Animales , Encéfalo/embriología , Células Cultivadas , Hemorragia Cerebral/embriología , Hemorragia Cerebral/etiología , Colágeno/farmacología , Diencéfalo/irrigación sanguínea , Diencéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Aneurisma Intracraneal/embriología , Aneurisma Intracraneal/genética , Aneurisma Intracraneal/patología , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Lectinas Tipo C/fisiología , Megacariocitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Activación Plaquetaria , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptor TIE-2/metabolismoRESUMEN
Sialic acid is a common terminal residue of glycans on proteins and acidic sphingolipids such as gangliosides and has important biological functions. The sialylation process is controlled by more than 20 different sialyltransferases, many of which exhibit overlapping functions. Thus, it is difficult to determine the overall biological function of sialylation by targeted deletion of individual sialyltransferases. To address this issue, we established a mouse line with the Slc35a1 gene flanked by loxP sites. Slc35a1 encodes the cytidine-5'-monophosphate (CMP)-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation. Here we report our study regarding the role of sialylation on megakaryocytes and platelets using a mouse line with significantly reduced sialylation in megakaryocytes and platelets (Plt Slc35a1 /). The major phenotype of Plt Slc35a1/ mice was thrombocytopenia. The number of bone marrow megakaryocytes in Plt Slc35a1/ mice was reduced, and megakaryocyte maturation was also impaired. In addition, an increased number of desialylated platelets was cleared by Küpffer cells in the liver of Plt Slc35a1/ mice. This study provides new insights into the role of sialylation in platelet homeostasis and the mechanisms of thrombocytopenia in diseases associated with platelet desialylation, such as immune thrombocytopenia and a rare congenital disorder of glycosylation (CDG), SLC35A1-CDG, which is caused by SLC35A1 mutations.
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Proteínas de Transporte de Nucleótidos , Trombocitopenia , Plaquetas , Humanos , Hígado , Ácido N-Acetilneuramínico , Proteínas de Transporte de Nucleótidos/genética , Trombocitopenia/genética , TrombopoyesisRESUMEN
Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.
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Colon/metabolismo , Colon/microbiología , Microbioma Gastrointestinal , Mucina 2/metabolismo , Moco/metabolismo , Animales , Heces/microbiología , Glicosilación , Ratones , Ratones Noqueados , Mucina 2/genética , Transcripción GenéticaRESUMEN
Stage-specific embryonic antigens (SSEA-1, 3, and 4) are carbohydrate antigens that have been used as markers of embryonic stem (ES) cells. However, the roles of these antigens in the establishment and maintenance of stemness of ES and induced pluripotent stem (iPS) cells are still poorly understood. This study investigated the biological and functional significance of globo-series glycolipids such as SSEA-3 and 4 in mouse iPS cells induced from tail-tip fibroblasts (TTFs) of α1,4Gal-T-knockout mice (lacking SSEA-3 and 4). These iPS cells were induced by retroviral transduction of four factors (Oct3/4, Sox2, Klf4, and c-Myc) into TTFs, and colonies were picked up. Morphologically, the colonies resembled ES cells and were positive for alkaline phosphatase and ES cell markers. Furthermore, in vitro-differentiated induction experiments after embryoid body formation revealed that some colonies derived from α1, 4Gal-T knockout mice were able to differentiate into three germ layers. Three germ layers were also observed in teratomas from iPS cells derived from α1,4Gal-T-knockout mice. These results suggest that SSEA-3 and 4 are not essential, at least for the establishment and maintenance of stemness of mouse iPS cells.
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Células Madre Embrionarias , Células Madre Pluripotentes Inducidas , Animales , Antígenos de Carbohidratos Asociados a Tumores , Diferenciación Celular , Células Cultivadas , Fibroblastos , Factor 4 Similar a Kruppel , Ratones , Antígenos Embrionarios Específico de EstadioRESUMEN
BACKGROUND: High inflammation status despite an absence of known infection characterizes a subpopulation of people with schizophrenia who suffer from more severe cognitive deficits, less cortical grey matter, and worse neuropathology. Transcripts encoding factors upstream of nuclear factor kappa B (NF-κB), a major transcriptional activator for the synthesis of pro-inflammatory cytokines, are increased in the frontal cortex in schizophrenia compared to controls. However, the extent to which these changes are disease-specific, restricted to those with schizophrenia and high-neuroinflammatory status, or caused by loss of a key NF-κB inhibitor (HIVEP2) found in schizophrenia brain, has not been tested. METHODS: Post-mortem prefrontal cortex samples were assessed in 141 human brains (69 controls and 72 schizophrenia) and 13 brains of wild-type mice and mice lacking HIVEP2 (6 wild-type, 7 knockout mice). Gene expression of pro-inflammatory cytokines and acute phase protein SERPINA3 was used to categorize high and low neuroinflammation biotype groups in human samples via cluster analysis. Expression of 18 canonical and non-canonical NF-κB pathway genes was assessed by qPCR in human and mouse tissue. RESULTS: In humans, we found non-canonical upstream activators of NF-κB were generally elevated in individuals with neuroinflammation regardless of diagnosis, supporting NF-κB activation in both controls and people with schizophrenia when cytokine mRNAs are high. However, high neuroinflammation schizophrenia patients had weaker (or absent) transcriptional increases of several canonical upstream activators of NF-κB as compared to the high neuroinflammation controls. HIVEP2 mRNA reduction was specific to patients with schizophrenia who also had high neuroinflammatory status, and we also found decreases in NF-κB transcripts typically induced by activated microglia in mice lacking HIVEP2. CONCLUSIONS: Collectively, our results show that high cortical expression of pro-inflammatory cytokines and low cortical expression of HIVEP2 in a subset of people with schizophrenia is associated with a relatively weak NF-κB transcriptional signature compared to non-schizophrenic controls with high cytokine expression. We speculate that this comparatively milder NF-κB induction may reflect schizophrenia-specific suppression possibly related to HIVEP2 deficiency in the cortex.
Asunto(s)
Encéfalo/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN/biosíntesis , FN-kappa B/metabolismo , Esquizofrenia/metabolismo , Factores de Transcripción/biosíntesis , Adulto , Animales , Encéfalo/patología , Citocinas/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/genética , Esquizofrenia/genética , Esquizofrenia/patología , Factores de Transcripción/genéticaRESUMEN
The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.