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Neddylation, an important type of post-translational modification, has been implicated in innate and adapted immunity. But the role of neddylation in innate immune response against RNA viruses remains elusive. Here we report that neddylation promotes RNA virus-induced type I IFN production, especially IFN-α. More importantly, myeloid deficiency of UBA3 or NEDD8 renders mice less resistant to RNA virus infection. Neddylation is essential for RNA virus-triggered activation of Ifna gene promoters. Further exploration has revealed that mammalian IRF7undergoes neddylation, which is enhanced after RNA virus infection. Even though neddylation blockade does not hinder RNA virus-triggered IRF7 expression, IRF7 mutant defective in neddylation exhibits reduced ability to activate Ifna gene promoters. Neddylation blockade impedes RNA virus-induced IRF7 nuclear translocation without hindering its phosphorylation and dimerization with IRF3. By contrast, IRF7 mutant defective in neddylation shows enhanced dimerization with IRF5, an Ifna repressor when interacting with IRF7. In conclusion, our data demonstrate that myeloid neddylation contributes to host anti-viral innate immunity through targeting IRF7 and promoting its transcriptional activity.
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Inmunidad Innata/inmunología , Factor 7 Regulador del Interferón/inmunología , Células Mieloides/inmunología , Infecciones por Virus ARN/inmunología , Virus ARN/inmunología , Animales , Factor 7 Regulador del Interferón/biosíntesis , Ratones , Células Mieloides/metabolismo , Proteína NEDD8/deficiencia , Procesamiento Proteico-Postraduccional , Ubiquitinas/deficienciaRESUMEN
The receptor for activated C kinase 1 (RACK1) adaptor protein has been implicated in viral infection. However, whether RACK1 promotes in vivo viral infection in mammals remains unknown. Moreover, it remains elusive how RACK1 is engaged in antiviral innate immune signaling. In this study, we report that myeloid RACK1 deficiency does not affect the development and survival of myeloid cells under resting conditions but renders mice less susceptible to viral infection. RACK1-deficient macrophages produce more IFN-α and IFN-ß in response to both RNA and DNA virus infection. In line with this, RACK1 suppresses transcriptional activation of type 1 IFN gene promoters in response to virus infection. Analysis of virus-mediated signaling indicates that RACK1 inhibits the phosphorylation of IRF3/7. Indeed, RACK1 interacts with IRF3/7, which is enhanced after virus infection. Further exploration indicates that virus infection triggers AMPK activation, which in turn phosphorylates RACK1 at Thr50 RACK1 phosphorylation at Thr50 enhances its interaction with IRF3/7 and thereby limits IRF3/7 phosphorylation. Thus, our results confirm that myeloid RACK1 promotes in vivo viral infection and provide insight into the control of type 1 IFN production in response to virus infection.
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Proteínas Quinasas Activadas por AMP , Factor 3 Regulador del Interferón , Proteínas Adaptadoras Transductoras de Señales , Animales , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Ratones , Fosforilación , Receptores de Cinasa C Activada , Transducción de SeñalRESUMEN
Neddylation is a ubiquitination-like pathway that controls cell survival and proliferation by covalently conjugating NEDD8 to lysines in specific substrate proteins. However, the physiological role of neddylation in mammalian metabolism remains elusive, and no mitochondrial targets have been identified. Here, we report that mouse models with liver-specific deficiency of NEDD8 or ubiquitin-like modifier activating enzyme 3 (UBA3), the catalytic subunit of the NEDD8-activating enzyme, exhibit neonatal death with spontaneous fatty liver as well as hepatic cellular senescence. In particular, liver-specific UBA3 deficiency leads to systemic abnormalities similar to glutaric aciduria type II (GA-II), a rare autosomal recessive inherited fatty acid oxidation disorder resulting from defects in mitochondrial electron transfer flavoproteins (ETFs: ETFA and ETFB) or the corresponding ubiquinone oxidoreductase. Neddylation inhibition by various strategies results in decreased protein levels of ETFs in neonatal livers and embryonic hepatocytes. Hepatic neddylation also enhances ETF expression in adult mice and prevents fasting-induced steatosis and mortality. Interestingly, neddylation is active in hepatic mitochondria. ETFs are neddylation substrates, and neddylation stabilizes ETFs by inhibiting their ubiquitination and degradation. Moreover, certain mutations of ETFs found in GA-II patients hinder the neddylation of these substrates. Taken together, our results reveal substrates for neddylation and add insight into GA-II.
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Flavoproteínas Transportadoras de Electrones/metabolismo , Ácidos Grasos/metabolismo , Hígado/metabolismo , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/metabolismo , Animales , Flavoproteínas Transportadoras de Electrones/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Oxidación-Reducción , Ubiquitinación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
SARS-CoV-2 nucleocapsid (N) protein has been proposed as a good vaccine target. N-specific T cells were observed in SARS-CoV-2 N immunized mice and COVID-19 convalescents. It is of importance to identify the T cell responses triggered by SARS-CoV-2 N protein. Intradermal immunization with SARS-CoV N protein was demonstrated to elicit non-protective T cell responses which may be avoided by intranasal vaccination. Therefore, we conducted intranasal vaccination of BALB/c mice with recombinant adenovirus type-5 expressing SARS-CoV-2 N protein. Such procedure induced CD8 T cell responses in the lung. Meanwhile CD4 T cell responses were observed in the spleen, which was associated with robust antibody production. Our study further supports the notion that SARS-CoV-2 N protein can work as a target for vaccine development.
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Anticuerpos Antivirales/inmunología , COVID-19/prevención & control , Proteínas de la Nucleocápside de Coronavirus/inmunología , Linfocitos T/inmunología , Vacunas Virales/inmunología , Administración Intranasal , Animales , Proteínas de la Nucleocápside de Coronavirus/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/administración & dosificación , Fosfoproteínas/inmunología , SARS-CoV-2/inmunología , VacunaciónRESUMEN
CD4+ T cells play predominant roles in protective immunity against blood-stage Plasmodium infection, both for IFN-γ-dependent effector mechanisms and providing B cell helper signals. Neddylation, an ubiquitination-like process triggered by covalent conjugation of NEDD8 to specific targets, has emerged as a potential regulator of T cell activities to TCR engagement. However, its contribution to T cell-mediated immunity to blood-stage malaria remains unclear. Here using an experimental model induced by Plasmodium yoelii 17XNL, and conditional knockout mice with T cell-specific deficiency of crucial components of neddylation pathway, we demonstrate activation of neddylation in T cells during blood-stage Plasmodium infection is essential for parasite control and host survival. Mechanistically, we show that apart from promoting CD4+ T cell activation, proliferation, and development of protective T helper 1 (Th1) cell response as suggested previously, neddylation is also required for supporting CD4+ T cell survival, mainly through B-cell lymphoma-2 (Bcl-2) mediated suppression of the mitochondria-dependent apoptosis. Furthermore, we provide evidence that neddylation contributes to follicular helper T (Tfh) cell differentiation, probably via augmenting the ubiquitin ligase Itch activity and proteasomal degradation of FoxO1, thereby facilitating germinal center (GC) formation and parasite-specific antibody production. This study identifies neddylation as a positive regulator of anti-Plasmodium immunity and provides insight into an involvement of such pathway in host resistance to infectious diseases.
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Linfocitos T CD4-Positivos/inmunología , Malaria/inmunología , Proteína NEDD8/fisiología , Inmunidad Adaptativa/inmunología , Animales , Linfocitos B/inmunología , Inmunidad Celular , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL/fisiología , Ratones Noqueados , Proteína NEDD8/metabolismo , Plasmodium yoelii/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Células TH1/inmunologíaRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC), the most common primary cancer of the liver, is one of the most common malignancies and the leading cause of cancer-related death worldwide. Leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1) is an E3 ubiquitin ligase involved in diverse cellular activities, including the regulation of cargo sorting, cell adhesion and antibacterial autophagy. The role of LRSAM1 in HCC remains unknown. METHODS: In this study, we reviewed the TCGA database and then performed gain-of-function and loss-of-function analyses of LRSAM1 in HCC cell lines. RESULTS: We found that the mRNA expression level of LRSAM1 was significantly increased in clinical HCC tissues in the TCGA database. Transient LRSAM1 knockdown in several human HCC cell lines led to reduced growth in conventional culture conditions. Stable LRSAM1 knockdown in HepG2 cells led to impaired anchorage-independent growth whereas its stable ectopic overexpression yielded the opposite effects. LRSAM1 overexpression in HepG2 cells enhanced in vivo tumorigenicity, whereas LRSAM1 knockdown in this cell line significantly impaired tumor growth. CONCLUSIONS: Our data suggest that LRSAM1 promotes the oncogenic growth of human HCC cells, although the underlying mechanisms remain to be explored.
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Optical spike-timing-dependent plasticity (STDP) synapses form the basis of learning in photonic neuromorphic system. In biological neural systems, STDP synapses generally have multiplicative boundary mechanisms, and can be modulated by a third factor such as dopamine. Analogously, we introduce a third factor into optical STDP: The current-injection of semiconductor optical amplifiers can be modified in an adaptive way according to local or global feedback signals. The local one is present synaptic weight, which elicits an optical weight-dependent STDP, while the global one is a reward signal. We demonstrate that the optical weight-dependent STDP can emulate the behavior of biological STDP synapses more closely, and can be seen as an intermediate configuration between additive and multiplicative STDP, which balances stability and competition among synapses. Simulation studies with scalable photonic neurons further show that optical STDP with reward modulation enables reward-based reinforcement learning.
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PURPOSE: Mitogen-activated protein kinases (MAPK), specifically the c-Jun N-terminal kinase (JNK)-MAPK subfamily, play a crucial role in the development of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of JNK1/2 and their upstream regulators, MKK4/7, in HCC carcinogenesis remain unclear. METHODS: In this study, we performed differential expression analysis of JNK-MAPK components at both the transcriptome and protein levels using TCGA and HPA databases. We utilized Kaplan-Meier survival plots and receiver operating characteristic (ROC) curve analysis to evaluate the prognostic performance of a risk scoring model based on these components in the TCGA-HCC cohort. Additionally, we conducted immunoblotting, apoptosis analysis with FACS and soft agar assays to investigate the response of JNK-MAPK pathway components to various death stimuli (TRAIL, TNF-α, anisomycin, and etoposide) in HCC cell lines. RESULTS: JNK1/2 and MKK7 levels were significantly upregulated in HCC samples compared to paracarcinoma tissues, whereas MKK4 was downregulated. ROC analyses suggested that JNK2 and MKK7 may serve as suitable diagnostic genes for HCC, and high JNK2 expression correlated with significantly poorer overall survival. Knockdown of JNK1 enhanced TRAIL-induced apoptosis in hepatoma cells, while JNK2 knockdown reduced TNF-α/cycloheximide (CHX)-and anisomycin-induced apoptosis. Neither JNK1 nor JNK2 knockdown affected etoposide-induced apoptosis. Furthermore, MKK7 knockdown augmented TNF-α/CHX- and TRAIL-induced apoptosis and inhibited colony formation in hepatoma cells. CONCLUSION: Targeting MKK7, rather than JNK1/2 or MKK4, may be a promising therapeutic strategy to inhibit the JNK-MAPK pathway in HCC therapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Carcinoma Hepatocelular/genética , Factor de Necrosis Tumoral alfa , Etopósido , Anisomicina , MAP Quinasa Quinasa 7/genética , MAP Quinasa Quinasa 7/metabolismo , Neoplasias Hepáticas/genética , ApoptosisRESUMEN
Objective To establish a novel hepatocyte injury model induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in vitro. Methods Freshly isolated mouse primary hepatocytes were cultured in vitro and treated with different doses of tumor necrosis factor-α (TNF-α) and 5 mg/mL of D-GalN. The supernatants from hepatocyte culture were detected for alanine aminotransferase (ALT) activity by chemiluminescence assay. Bone marrow-derived macrophages (BMDMs) were stimulated with 1 µg/mL of LPS and the level of TNF-α in supernatants were detected by ELISA. Primary hepatocytes were treated with the BMDM supernatants combined with 5 mg/mL D-GalN or 50 ng/mL actinomycin D (ActD) for 24 hours. The level of ALT from hepatocyte supernatant was detected and morphology of hepatocytes was observed with microscopy. BMDMs and hepatocytes were co-cultured and treated with 1 µg/mL of LPS combined with D-GalN or ActD for 24 hours. Hepatocyte injury was reflected by the ALT activity and hepatocyte morphology. Results The ALT activity was significantly increased in the supernatants of hepatocytes treated with TNF-α and D-GalN, indicating the obvious hepatocyte injury. Co-treatment with LPS-primed BMDM supernatants and D-GalN or ActD could cause hepatocyte injury, as reflected by markedly increased ALT activity and the deformed and cracked hepatocytes. In the context of co-culture of BMDM and hepatocytes, treatment with LPS and D-GalN led to obvious hepatocyte injury as expected. LPS combined with ActD could not cause hepatocyte injury, since the BMDMs started to die earlier than they could secret TNF-α to destruct hepatocytes. Hepatocytes with normal morphology and deformed BMDMs were observed. Conclusion LPS/D-GalN can be used to induce hepatocyte injury in vitro. D-GalN, rather than ActD, should be used as a transcriptional inhibitor when the TNF-α -induced hepatocyte injury is evaluated in a co-culture system of BMDMs and hepatocytes.
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Galactosamina , Lipopolisacáridos , Alanina Transaminasa , Animales , Galactosamina/toxicidad , Hepatocitos , Ratones , Factor de Necrosis Tumoral alfaRESUMEN
We present a long-term chip scale stabilization scheme for optoelectronic oscillators (OEOs) based on a rubidium coherent population trapping (CPT) atomic resonator. By locking a single mode of an OEO to the (85)Rb 3.035-GHz CPT resonance utilizing an improved phase-locked loop (PLL) with a PID regulator, we achieved a chip scale frequency stabilization system for the OEO. The fractional frequency stability of the stabilized OEO by overlapping Allan deviation reaches 6.2 ×10(-11) (1 s) and â¼ 1.45 ×10 (-11) (1000 s). This scheme avoids a decrease in the extra phase noise performance induced by the electronic connection between the OEO and the microwave reference in common injection locking schemes. The total physical package of the stabilization system is [Formula: see text] and the total power consumption is 400 mW, which provides a chip scale and portable frequency stabilization approach with ultra-low power consumption for OEOs.
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INTRODUCTION: Therapeutic potentials of mesenchymal stem cells (MSCs) from different sources have been evaluated in pre-clinical and clinical settings. Although MSCs from different sources share MSC-specific characteristics and functions, inconsistent or controversial results of pre-clinical and clinical applications of such cells are frequently reported. This may be partially due to the fact that MSCs isolated from different origins may differentially express some functions not typical for MSCs, and hence have different therapeutic potentials. The aim of this study is to investigate the differences in human placental MSCs (P-MSCs) of fetal and maternal origins in the aspects of clinical importance. METHODS: P-MSCs of fetal and maternal origins isolated from normal term placentas were characterized for their typical phenotype as well as their expression of receptors and growth factors of clinic interests. P-MSCs that preferentially express hepatocyte growth factor (HGF) and CD200 were evaluated for their therapeutic potentials in models of angiogenesis and allogeneic skin transplantation, in comparison with their HGF and CD200 negative partners. RESULTS: Although all P-MSCs express typical MSC phenotype, fetal but not maternal P-MSCs express high levels of CD200 and HGF. Compared with HGF and CD200 negative P-MSCs, HGF and CD200 positive cells demonstrated significantly high potentials in promoting angiogenesis in vitro and increasing immunosuppressive function in vivo. These therapeutic potentials were at least in part due to their differences in HGF and CD200 expression, respectively. CONCLUSIONS: We conclude that MSC origins may have significant impact on the therapeutic potentials of such cells, and should be taken into consideration in clinical applications.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Placenta/citología , Animales , Antígenos CD/biosíntesis , Proliferación Celular/fisiología , Femenino , Factor de Crecimiento de Hepatocito/biosíntesis , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , EmbarazoRESUMEN
OBJECTIVE: To investigate the effect of human placental-derived mesenchymal stem cells (PMSCs) on immunological rejection in mouse allogeneic skin transplantation. METHODS: The placenta fetal tissues from voluntary donors were used to isolate and culture the PMSCs, and the 3rd passage PMSCs were used in the experiment. Thirty Vr: CD1 (ICR) mice at age of 1-2 days were used as skin donors for allogeneic skin transplantation. Thirty C57BL/6 mice at age of 6-8 weeks as recipients were made back skin defect of 12 mm in diameter and were randomly divided into 3 groups (n=10): group A, autograft; group B, allogeneic graft + PBS tail vein injection; and group C, allogeneic graft + human PMSCs (1 x 10(5) cells/mouse) tail vein injection. The flap survival was observed. At 7 days after skin transplantation, blood leukocyte counting, abdominal fluid macrophage activation, and the expression levels of interleukin 4 (IL-4), interleukin 17 (IL-17), and interferon gamma (INF-gamma) in blood and spleen were detected by ELISA and RT-PCR, respectively. RESULTS: The flap survival time was significantly longer in group A [(58.33 +/- 4.04) days] than in groups B and C [(3.80 +/- 0.92) days and (6.80 +/- 0.82) days] (P < 0.05), and in group C than in group B (P < 0.05). At 7 days after transplantation, the blood leukocyte number was (6.32 +/- 0.45) x 10(9)/L in group A, (7.45 +/- 0.52) x 10(9)/L in group B, and (6.35 +/- 0.39) x 10(9)/L in group C, and it was significantly more in group B than in groups A and C (P < 0.05). The macrophage activation rate of the abdominal fluid was 6.87% +/- 2.40% in group A, 7.84% +/- 0.44% in group B, and 15.98% +/- 2.87% in group C; group C was significantly higher than groups A and B (P < 0.01). ELISA results showed that there was no significant difference in the concentrations of IL-4 among 3 groups (P > 0.05). Compared with group B, the concentrations of IL-17 and IFN-gamma were significantly reduced in group C (P < 0.05), while the concentration of IFN-gamma was significantly increased in group B when compared with group A (P < 0.05). RT-PCR results showed that there were significant differences in the expressions of IL-4, IL-17, and IFN-gamma mRNA between groups B, C and group A (P < 0.05); the expressions of IL-4 and IFN-gamma mRNA were significantly lower in group C than in group B (P < 0.05). CONCLUSION: Human PMSCs transplantation can suppress the acute immunological rejection in allogeneic skin transplantation. The possible mechanism may be partially related to the inhibitory effect on the secretion of IL-17 and IFN-gamma.