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Adenoviral infections, particularly in children, remain a significant public health issue with no approved targeted treatments. Artemisinin and its derivatives, well-known for their use in malaria treatment, have shown antiviral activities in recent studies. However, their efficacy against human adenovirus (HAdV) remains unexplored. This study aimed to assess the activity of artemisinin and its derivatives against HAdV infection in vitro using cell lines and primary cells. Our data revealed that artemisinin exhibited dose-dependent anti-HAdV activity with no apparent cytotoxicity over a wide concentration range. Mechanistically, artemisinin did not affect viral attachment or entry into target cells, nor the viral genome entry into cell nucleus. Instead, it inhibited HAdV through suppression of viral DNA replication. Comparative analysis with its derivatives, artesunate and artemisone, showed distinct cytotoxicity and anti-adenoviral profiles, with artemisone showing superior efficacy and lower toxicity. Further validation using a primary airway epithelial cell model confirmed the anti-adenoviral activity of both artemisinin and artemisone against different virus strains. Together, our findings suggest that artemisinin and its derivatives may be promising candidates for anti-HAdV treatment.
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Purpose: Plastic bronchitis (PB), a rare complication of respiratory infection characterized by the formation of casts in the tracheobronchial tree, can lead to airway obstruction and severe condition. Adenovirus is one of the common pathogens of PB caused by infection. This study aimed to evaluate the clinical features and risk factors for PB in children with severe adenovirus pneumonia. Methods: A retrospective study of children with severe adenovirus pneumonia with bronchoscopy results at Guangzhou Women and Children's Hospital between January 2018 and January 2020 was performed. Based on bronchoscopy, we divided children with severe adenovirus pneumonia into two groups: PB and non-PB. Binary logistic regression analysis was used to identify independent risk factors for PB in patients with severe adenovirus pneumonia after univariate analysis. Results: Our study examined 156 patients with severe adenovirus pneumonia with bronchoscopy results in hospital. Among them, 18 developed PB and 138 did not. On multivariate analysis, the independent risk factors of PB in children with severe adenovirus pneumonia were history of allergies (OR 10.147, 95% CI 1.727-59.612; P=0.010), diminished breath sounds (OR 12.856, 95% CI 3.259-50.713; P=0.001), and increased proportion of neutrophils (>70%; OR 8.074, 95% CI 1.991-32.735; P=0.003). Conclusion: Children with severe adenovirus pneumonia with a history of allergies, diminished breath sounds, and increased the proportion of neutrophils >70% may show higher risk of PB.
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Introduction: Human adenovirus (HAdV) is a common respiratory virus, which can lead to severe pneumonia in children and immunocompromised persons, and canonical inflammasomes are reported to be involved in anti-HAdV defense. However, whether HAdV induced noncanonical inflammasome activation has not been explored. This study aims to explore the broad roles of noncanonical inflammasomes during HAdV infection to investigate the regulatory mechanism of HAdV-induced pulmonary inflammatory damage. Methods: We mined available data on GEO database and collected clinical samples from adenovirus pneumonia pediatric patients to investigate the expression of noncanonical inflammasome and its clinical relevance. An in vitro cell model was employed to investigate the roles of noncanonical inflammasomes in macrophages in response to HAdV infection. Results: Bioinformatics analysis showed that inflammasome-related genes, including caspase-4 and caspase-5, were enriched in adenovirus pneumonia. Moreover, caspase-4 and caspase-5 expression levels were significantly increased in the cells isolated from peripheral blood and broncho-alveolar lavage fluid (BALF) of pediatric patients with adenovirus pneumonia, and positively correlated with clinical parameters of inflammatory damage. In vitro experiments revealed that HAdV infection promoted caspase-4/5 expression, activation and pyroptosis in differentiated THP-1 (dTHP-1) human macrophages via NF-κB, rather than STING signaling pathway. Interestingly, silencing of caspase-4 and caspase-5 in dTHP-1 cells suppressed HAdV-induced noncanonical inflammasome activation and macrophage pyroptosis, and dramatically decreased the HAdV titer in cell supernatants, by influencing virus release rather than other stages of virus life cycle. Discussion: In conclusion, our study demonstrated that HAdV infection induced macrophage pyroptosis by triggering noncanonical inflammasome activation via a NF-kB-dependent manner, which may explore new perspectives on the pathogenesis of HAdV-induced inflammatory damage. And high expression levels of caspase-4 and caspase-5 may be a biomarker for predicting the severity of adenovirus pneumonia.
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Infecções por Adenoviridae , Infecções por Adenovirus Humanos , Pneumonia Viral , Humanos , Criança , Inflamassomos/metabolismo , Piroptose , Infecções por Adenovirus Humanos/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Caspases/metabolismo , Pneumonia Viral/metabolismo , Infecções por Adenoviridae/complicaçõesRESUMO
BACKGROUND: Human adenovirus (HAdV) is one of the most common viruses causing respiratory infections among young children. Most adenovirus infections are mild and self-limited; however, these infections may occasionally cause severe pneumonia and even death. The mortality risk factors for severe adenovirus pneumonia are not completely clear. This study aimed to evaluate the mortality risk factors in children with severe adenovirus pneumonia. METHODS: A retrospective study of children with severe adenovirus pneumonia hospitalized in Guangzhou Women and Children's Hospital between July 2018 and January 2020 was performed. Binary logistic regression analysis was used to identify independent mortality risk factors for severe adenovirus pneumonia after univariate analysis. RESULTS: Our study included 189 patients (123 males and 66 females). Among them, 13 patients did not survive with a mortality of 6.88%. In multivariate analysis, the independent mortality risk factors in children with severe adenovirus pneumonia were age less than 1 year (OR = 18.513, 95% CI: 2.157-158.883, p = 0.008), hypoxia (OR = 62.335, 95% CI: 2.385-1629.433, p = 0.013), and thrombocytopenia (platelet <100∗10Ë9/L) (OR = 13.324, 95% CI: 1.232-144.075, p = 0.033). CONCLUSIONS: In children with severe adenovirus pneumonia who are younger than one year old, hypoxia and platelet counts less than 100∗10Ë9/L represent mortality risk factors.
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Infecções por Adenoviridae , Adenovírus Humanos , Pneumonia Viral , Pneumonia , Masculino , Criança , Humanos , Feminino , Lactente , Pré-Escolar , Estudos Retrospectivos , Infecções por Adenoviridae/complicações , Pneumonia Viral/complicações , Hipóxia , Fatores de RiscoRESUMO
Background: Human adenovirus (HAdV) lower respiratory tract infections (LRTIs) are prone to severe cases and even cause death in children. Here, we aimed to develop a classification model to predict severity in pediatric patients with HAdV LRTIs using complete blood count (CBC). Methods: The CBC parameters from pediatric patients with a diagnosis of HAdV LRTIs from 2013 to 2019 were collected during the disease's course. The data were analyzed as potential predictors for severe cases and were selected using a random forest model. Results: We enrolled 1,652 CBC specimens from 1,069 pediatric patients with HAdV LRTIs in the present study. Four hundred and seventy-four patients from 2017 to 2019 were used as the discovery cohort, and 470 patients from 2013 to 2016 were used as the validation cohort. The monocyte ratio (MONO%) was the most obvious difference between the mild and severe groups at onset, and could be used as a marker for the early accurate prediction of the severity [area under the subject operating characteristic curve (AUROC): 0.843]. Four risk factors [MONO%, hematocrit (HCT), red blood cell count (RBC), and platelet count (PLT)] were derived to construct a classification model of severe and mild cases using a random forest model (AUROC: 0.931 vs. 0.903). Conclusion: Monocyte ratio can be used as an individual predictor of severe cases in the early stages of HAdV LRTIs. The four risk factors model is a simple and accurate risk assessment tool that can predict severe cases in the early stages of HAdV LRTIs.
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BACKGROUND: Adenovirus pneumonia is prone to severe clinical and imaging manifestations in children. Bronchoscopic alveolar lavage (BAL) is an important adjunctive therapy for patients with severe imaging findings. The study aimed to evaluate the effect of the timing on the efficacy of bronchoalveolar lavage in children with adenovirus pneumonia. METHODS: This study included 134 patients with adenovirus pneumonia treated with BAL at Guangzhou Women and Children's Medical Center from January 2019 to January 2020.They were classified into the severe and mild groups. Based on the timing of BAL, each group was divided into the early BAL layer (received BAL within 1-9 days of the illness course) and the late BAL layer (received BAL within 10-14 days of the illness course). The clinical data of patients with different BAL timings were analyzed in two groups. RESULTS: Among the 134 patients, 70 were categorized into the mild group and 64 were categorized into the severe group. Of the 134 patients, 42 patients received BAL early (mild group: n = 21 and severe group: n = 21) and 92 patients received BAL later (mild group: n = 49 and severe group: n = 43). In the mild group, the fever and hospital duration were shorter in patients who received BAL early than in those who received BAL later (p < 0.05). However, in the severe group, there were no statistically significant differences in the fever and hospital duration between patients who received BAL early and those who received BAL later. However, the need for mechanical ventilation and the incidence of BAL complications, such as new need for oxygen, were higher in patients who received BAL early than in those who received BAL later in the severe group (p < 0.05). CONCLUSION: For mild adenovirus pneumonia, early BAL may shorten the fever and hospital duration. However, early BAL in severe cases might not shorten the course of the disease or improve prognosis and may even increase the risks of mechanical ventilation and BAL complications.
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Infecções por Adenoviridae/terapia , Lavagem Broncoalveolar/métodos , Lavagem Broncoalveolar/estatística & dados numéricos , Pneumonia Viral/terapia , Adenoviridae , Infecções por Adenoviridae/complicações , Lavagem Broncoalveolar/efeitos adversos , Criança , Pré-Escolar , China , Feminino , Humanos , Lactente , Tempo de Internação , Masculino , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The wheat seedlings of 6 days old were daily subjected to ultraviolet irradiation (irradiating for 5, 10, 20, 40, and 60 min/day, respectively), Polyethylene glycol 6000 (5, 10, 15, 20, 25% in 1/2 Hoagland solution, respectively), and salinity solution (10, 25, 50, 100, 200 mM in 1/2 Hoagland solution, respectively), while the control group (CK) was supplied only with the Hoagland solution. The wheatgrass was harvested regularly seven times and the total soluble polysaccharides, ascorbic acid, chlorophyll, total polyphenol, total triterpene, total flavonoid, and proanthocyanins content were tested. The antioxidant capacity was evaluated through 2,2'-azino-bis (3-ethylbenzthia-zoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging ability, and ferric ion reducing power. Technique for order preference by similarity to ideal solution (TOPSIS) mathematical model was adopted to comprehensively assess the functional phytochemicals of the different treatments. The results showed that the accumulation patterns of phytochemicals under abiotic stress were complex and not always upregulated or downregulated. The antioxidant activity and functional phytochemicals content of wheatgrass were significantly affected by both the stress treatments and seedling age, while the latter affected the chemicals more efficiently. The top five highest functional phytochemicals were observed in the 200 mM NaCl treated group on the 21st and 27th day, 25% PEG treated group on the 24th day, 200 mM NaCl treated group on the 24th day, and the group of 40 min/day ultraviolet exposure on 27th day.
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Chemotaxis, which is G protein-coupled receptor (GPCR)-mediated directional cell migration, plays pivotal roles in diverse human diseases, including recruitment of leukocytes to inflammation sites and metastasis of cancer. It is still not fully understood how eukaryotes sense and chemotax in response to chemoattractants with an enormous concentration range. A genetically traceable model organism, Dictyostelium discoideum, is the best-studied organism for GPCR-mediated chemotaxis. Recently, we have shown that C2GAP1 controls G protein coupled receptor-mediated Ras adaptation and chemotaxis. Here, we investigated the molecular mechanism and the biological function of C2GAP1 membrane targeting for chemotaxis. We show that calcium and phospholipids on the plasma membrane play critical roles in membrane targeting of C2GAP1. Cells lacking C2GAP1 (c2gapA -) displayed an improved chemotaxis in response to chemoattractant gradients at subsensitive or low concentrations (<100 nM), while exhibiting impaired chemotaxis in response to gradients at high concentrations (>1 µM). Taken together, our results demonstrate that the membrane targeting of C2GAP1 enables Dictyostelium to sense chemoattractant gradients at a higher concentration range. This mechanism is likely an evolutionarily conserved molecular mechanism of Ras regulation in the adaptation and chemotaxis of eukaryotes.
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Chronic granulomatous disease (CGD) is caused by gene mutations that affect the phagocyte NADPH oxidase. This results in recurrent infections by catalase-positive bacteria or fungi. Here, we report a case of X-linked CGD presenting a mixed infection with Burkholderia cepacia and Aspergillus. A novel mutation was found by bioinformatics analyses of his genealogy (c.1234delG), which perhaps changed the structure and function of the related proteins.
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Sodium azide (NaN3), an inhibitor of cytochrome oxidase, induces the release of excitotoxins via an energy impairment and this, in turn, results in neurodegeneration. The present study aimed to investigate the toxic effects NaN3 on apoptosis of PC12 cells and its mechanism of action in peroxisome proliferatoractivated receptor γ coactivator 1α (Pgc1α)associated signaling pathways. To induce apoptosis, PC12 cells were exposed to NaN3 (0, 5, 10, 20, 40 and 80 mM) for 12, 24, 48 and 72 h. Cell viability was determined by CCK8 assay. DAPI staining was employed to additionally examine apoptotic cells and their nuclear changes. Production of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptotic rate were also assessed by flow cytometry. Cellular ATP content was estimated by firefly luciferase assay. In addition, the expression levels of Bcell lymphoma 2 (Bcl2), Bcl2associated X protein (Bax), phosphorylated (p)Ca2+/calmodulindependent protein kinase (CaMK), pp38 mitogenactivated protein kinase (p38 MAPK), Pgc1α, nuclear respiratory factor (Nrf)1, mitochondrial transcription factor A (Tfam), pextracellular signalregulated kinase (Erk)1/2, Nrf2 and complex IV (Cox IV) were determined by western blot analysis. The data suggested that NaN3 may induce PC12 cell injury and dosedependently decrease the cell viability. The expression levels of proapoptotic proteins Bax and cytochrome c were upregulated, while the expression levels of antiapoptotic proteins procaspase3 and Bcl2 were downregulated. In addition, the phosphorylation of MAPK and Ca2+/calmodulindependent protein kinase II (CaMKâ ¡) family members including pancalcineurin A was increased, in particular the ratios of pp38/p38 and pCaMKâ ¡/CaMKâ ¡. However, the expression levels of Pgc1α and its associated proteins, including Nrf1/2, Tfam and pErk1/2 were decreased. In addition, mitochondria were the target organelles of NaN3induced toxicity in PC12 cells, which moderated the dissipation of ΔΨm, preserved the cellular ATP content, promoted the production of ROS and increased the apoptotic rate. These results suggested that NaN3 induced cell death in PC12 cells via Pgc1αassociated signaling pathways and provided a theoretical basis for additional investigation of the neurotoxic mechanism of NaN3, with applications in neurodegenerative disorders.
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Mitocôndrias/efeitos dos fármacos , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Azida Sódica/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Azida Sódica/toxicidadeRESUMO
Chemotaxis, which is chemoattractant-guided directional cell migration, plays major roles in recruitment of neutrophils, the metastasis of cancer cells, and the development of the model organism Dictyostelium discoideum. These cells share remarkable similarities in the signaling pathways by which they control chemotaxis. They all use a G protein-coupled receptor (GPCR)-mediated signal transduction pathway to sense the chemotactic gradient to guide cell migration. Diverse chemokines activate Rac through conserved GPCR signaling pathways. ELMO proteins are an evolutionarily conserved, essential component of the ELMO/Dock complex, which functions as a guanine nucleotide exchange factor (GEF) for small G protein Rac activation. The linkages between the GPCR-initiated gradient sensing compass and the Rac-mediated migrating machinery have long been missing. Here, we summarize recent findings on ELMO proteins that directly interact with G protein and transduce GPCR signaling to control the reorganization of actin-based cytoskeleton through regulating Rac activation during chemotaxis, first in D. discoideum and then in mammalian cancer cells. This represents an evolutionarily conserved signaling shortcut from GPCR to the actin cytoskeleton.
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Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Eucarióticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Quimiotaxia , Dictyostelium/metabolismo , Humanos , Mamíferos , Transdução de SinaisRESUMO
Aconitine, a diterpenoid alkaloids derived from Aconitum plants, is widely employed to treat various diseases. The aim of the present study was to investigate the apoptotic effect of aconitine in H9c2 cardiac cells. H9c2 cell apoptosis induced by aconitine was detected by a Cell Counting kit8 assay, DAPI staining, Annexin VFITC/propidium iodide double staining and western blotting. The effects of aconitine on reactive oxygen species levels and mitochondrial membrane potential were confirmed by fluorescence microscopy and flow cytometry. In addition, ATP contents were determined using a ATPdependent bioluminescence assay kit. The levels of peroxisome proliferator activated receptor γ coactivator 1α (PGC1α) expression and apoptosisassociated proteins including Caspase3, Bcell lymphoma 2 (Bcl2), Bcl2associated X protein (Bax) and Cytochrome c were also assessed. Taken together, the results indicated that aconitine may inhibit cell viability, decrease PGC1α expression, induce mitochondrial dysfunctions, upregulate Cytochrome c, Bax and Caspase3, and downregulate Bcl2, suggesting that aconitine may induce apoptosis through mitochondriamediated signaling pathways in H9c2 cells.
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Aconitina/farmacologia , Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficient c2gapA- cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects of c2gapA- cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis.
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Quimiotaxia/genética , Dictyostelium/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas de Protozoários/genética , Proteínas ras/genética , Actinas/genética , Actinas/metabolismo , Adaptação Fisiológica , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Dictyostelium/ultraestrutura , Proteínas Ativadoras de GTPase/deficiência , Regulação da Expressão Gênica , Transporte Proteico , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Proteínas ras/metabolismoRESUMO
The aim of the current study was to investigate the effect of mitochondrial division inhibitor 1 (Mdivi1) in sodium azideinduced cell death in H9c2 cardiac muscle cells. Mdivi1 is a key inhibitor of the mitochondrial division protein dynaminrelated protein 1 (Drp1). Mdivi1 was added to H9c2 cells for 3 h, after which, the cells were treated with sodium azide for 24 h. Cell viability was measured by Cell Counting kit8 assay. DAPI staining was used to observe nuclear morphology changes by microscopy. To further investigate the role of mitochondria in sodium azideinduced cell death, mitochondrial membrane potential (ΔΨm) and the cellular ATP content were determined by JC1 staining and ATPdependent bioluminescence assay, respectively. Reactive oxygen species (ROS) production was also assessed by use of the specific probe 2',7'dichlorodihydrofluorescein diacetate. In addition, the expression of Drp1 and of the apoptosisrelated proteins BCL2 apoptosis regulator (Bcl2), and BCL2 associated X (Bax) was determined by western blotting. The present findings demonstrated that pretreatment with Mdivi1 attenuated sodium azideinduced H9c2 cell death. Mdivi1 pretreatment also inhibited the sodium azideinduced downregulation of Bcl2 expression and upregulation of Bax and Drp1 expression. In addition, the mitochondrion was revealed to be the target organelle of sodium azideinduced toxicity in H9c2 cells. Mdivi1 pretreatment moderated the dissipation of ΔΨm, preserved the cellular ATP contents and suppressed the production of ROS. The results suggested that the mechanism of sodium azideinduced cell death in H9c2 cells may involve the mitochondriadependent apoptotic pathway. The present results indicated that Mdivi1 may have a cardioprotective effect against sodium azideinduced apoptosis in H9c2 cardiac muscle cells.
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Apoptose/efeitos dos fármacos , Cardiotônicos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Quinazolinonas/farmacologia , Azida Sódica/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Animais , Apoptose/genética , Linhagem Celular , Dinaminas/genética , Dinaminas/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Azida Sódica/toxicidade , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Viral myocarditis is a common cardiovascular disease, which seriously endangers the health of people and even leads to sudden unexpected death. MicroRNAs play very important roles in various physical and pathological processes including cardiogenesis and heart diseases. In recent years, miR-20b has been implicated in various diseases such as breast cancer, gastric cancer, hepatocellular carcinoma, cardiovascular diseases. However, the function of miR-20b in the pathological progress of viral myocarditis has not been reported. In this study, we found that miR-20b was up-regulated in mouse heart tissues post Coxsackievirus B3 (CVB3) infection. Bioinformatics analysis identified ZFP-148, a transcription factor that plays essential roles in the regulation of virus replication, is one of the predicted targets of miR-20b. MiR-20b expression was found to be up-regulated and ZFP-148 protein level was markedly repressed during viral myocarditis. Further studies demonstrated that miR-20b directly binds to the 3'-UTR of ZFP-148 and suppresses its translation. Moreover, aberrant expression of miR-20b promoted the expression of anti-apoptosis proteins Bcl-2 and Bcl-xL, suggesting that altered gene expression might promote cardiomyocytes survival in viral myocarditis. Our findings indicated that miR-20b might be a potential therapeutic target for CVB3-induced viral myocarditis and a useful marker for the diagnosis of viral myocarditis.
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Infecções por Coxsackievirus/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Miocardite/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Animais , Apoptose , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Regulação para Cima , Replicação ViralRESUMO
Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.
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Quimiotaxia , Neutrófilos , Receptores Acoplados a Proteínas G , Bioensaio/métodos , Dictyostelium , Células HL-60 , Humanos , Modelos Biológicos , Transdução de SinaisRESUMO
Intracerebral hemorrhage (ICH) results in inflammation, and glucocorticoids have been proven to be effective inhibitors of ICHinduced inflammation. However, the precise underlying mechanisms of ICHinduced inflammation and glucocorticoid function remain largely undefined. Using a mouse ICH model, the present study demonstrated that the short noncoding RNA molecule microRNA155 (miR155) is involved in the inflammatory process initiated by ICH in mice. Increased mRNA expression levels of miR155, as well as the proinflammatory cytokines interferonß (IFNß), tumor necrosis factorα (TNFα) and interleukin6 (IL6), were observed in vivo following ICH. By contrast, the expression level of suppressor of cytokine signaling 1 (SOCS1) protein was reduced in the ICH group compared with control mice. Similar results were observed in vitro using astrocytes, the primary effector cells in ICH. Compared with wild type astrocytes, astrocytes overexpressing miR155 exhibited significant inhibition of SOCS1 protein expression levels. These results suggest that miR155 contributes to the development of ICHinduced inflammation in mice by downregulating SOCS1 protein expression levels and promoting proinflammatory cytokine (IFNß, TNFα and IL6) production. Expression levels of miR155 and proinflammatory cytokines in the ICH group were significantly decreased following dexamethasone administration. This suggests that glucocorticoids attenuate ICHinduced inflammation by targeting the miR155/SOCS1 signaling pathway in mice. In conclusion, the results of the present study demonstrated that the miR155/SOCS1 signaling pathway is required for ICHinduced inflammation, and glucocorticoids inhibit this process by targeting the miR155/SOCS1 signaling pathway.
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Anti-Inflamatórios/uso terapêutico , Hemorragia Cerebral/complicações , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Inflamação/tratamento farmacológico , MicroRNAs/metabolismo , Animais , Células Cultivadas , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/imunologia , Hemorragia Cerebral/metabolismo , Inflamação/imunologia , Masculino , Camundongos Endogâmicos ICR , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Supressora da Sinalização de Citocina/imunologiaRESUMO
Diverse chemokines bind to G protein-coupled receptors (GPCRs) to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. ELMO and Dock proteins form complexes that function as guanine nucleotide exchange factors (GEFs) for Rac activation. However, the linkage between GPCR activation and the ELMO/Dock-mediated Rac activation is not fully understood. In the present study, we show that chemoattractants induce dynamic membrane translocation of ELMO1 in mammalian cells. ELMO1 plays an important role in GPCR-mediated chemotaxis. We also reveal that ELMO1 and Dock1 form a stable complex. Importantly, activation of chemokine GPCR promotes the interaction between ELMO1 and Gßγ. The ELMO1-Gßγ interaction is through the N-terminus of ELMO1 protein and is important for the membrane translocation of ELMO1. ELMO1 is required for Rac1 activation upon chemoattractant stimulation. Our results suggest that chemokine GPCR-mediated interaction between Gßγ and ELMO1/Dock1 complex might serve as an evolutionarily conserved mechanism for Rac activation to regulate actin cytoskeleton for chemotaxis of human cells.
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Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).
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Microscopia Confocal , Imagem Molecular , Canais de Cátion TRPP/metabolismo , Proteínas ras/metabolismo , Linhagem Celular Tumoral , Fatores Quimiotáticos/metabolismo , Fatores Quimiotáticos/farmacologia , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Genes Reporter , Células HL-60 , Humanos , Microscopia Confocal/métodos , Fosforilação , Canais de Cátion TRPP/genética , Proteínas ras/genéticaRESUMO
Eukaryotic phagocytes search and destroy invading microorganisms via chemotaxis and phagocytosis. The social amoeba Dictyostelium discoideum is a professional phagocyte that chases bacteria through chemotaxis and engulfs them as food via phagocytosis. G-protein-coupled receptors (GPCRs) are known for detecting chemoattractants and directing cell migration, but their roles in phagocytosis are not clear. Here, we developed a quantitative phosphoproteomic technique to discover signaling components. Using this approach, we discovered the long sought after folic acid receptor, fAR1, in D. discoideum. We showed that the seven-transmembrane receptor fAR1 is required for folic acid-mediated signaling events. Significantly, we discovered that fAR1 is essential for both chemotaxis and phagocytosis of bacteria, thereby representing a chemoattractant GPCR that mediates not only chasing but also ingesting bacteria. We revealed that a phagocyte is able to internalize particles via a chemoattractant-mediated engulfment process. We propose that mammalian phagocytes may also use this mechanism to engulf and ingest bacterial pathogens.