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1.
Nat Commun ; 10(1): 2506, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175295

RESUMO

Although there are many known Mendelian genes linked to epileptic or developmental and epileptic encephalopathy (EE/DEE), its genetic architecture is not fully explained. Here, we address this incompleteness by analyzing exomes of 743 EE/DEE cases and 2366 controls. We observe that damaging ultra-rare variants (dURVs) unique to an individual are significantly overrepresented in EE/DEE, both in known EE/DEE genes and the other non-EE/DEE genes. Importantly, enrichment of dURVs in non-EE/DEE genes is significant, even in the subset of cases with diagnostic dURVs (P = 0.000215), suggesting oligogenic contribution of non-EE/DEE gene dURVs. Gene-based analysis identifies exome-wide significant (P = 2.04 × 10-6) enrichment of damaging de novo mutations in NF1, a gene primarily linked to neurofibromatosis, in infantile spasm. Together with accumulating evidence for roles of oligogenic or modifier variants in severe neurodevelopmental disorders, our results highlight genetic complexity in EE/DEE, and indicate that EE/DEE is not an aggregate of simple Mendelian disorders.


Assuntos
Variação Genética , Espasmos Infantis/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , DNA (Citosina-5-)-Metiltransferases/genética , Epilepsias Mioclônicas/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Lactente , Japão , Síndrome de Lennox Gastaut/genética , Modelos Logísticos , Mutação , Neurofibromina 1/genética , Polimorfismo de Nucleotídeo Único , Análise de Componente Principal , Canais de Cátion TRPM/genética , Sequenciamento Completo do Exoma
2.
Mol Med Rep ; 19(6): 4597-4602, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059032

RESUMO

Ubiquilin­1 (Ubqln), a ubiquitin­like protein, regulates degradation of misfolded proteins and has been reported to have a crucial role in multiple pathologic and physiologic conditions. The current study was undertaken to investigate the expression of Ubqln in the brain of a neonatal hypoxia­ischemic (HI) brain injury model induced using the Rice method with some modifications. Mouse pups at postnatal day 7 day were used in this study. Pups underwent permanent ligation of the left common carotid artery and a consecutive hypoxic challenge (8% O2 and 92% N2 for 120 min). The expression of Ubqln in the brain of pups following HI was analyzed by immunofluorescence staining and western blot analysis. Immunofluorescence staining demonstrated that Ubqln was extensively distributed in the cerebral cortex and hippocampus, and Ubqln was expressed in neurons, astrocytes and microglia in the brains of the HI brain injury model mice. Western blot analyses revealed decreased expression of Ubqln in the HI penumbra of the mouse model compared with Ubqln in the sham control group. The results of this study revealed that HI alters the expression of Ubqln, thus may provide a novel understanding of role of Ubqln in neonatal hypoxic ischemic encephalopathy.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Regulação para Baixo , Hipóxia-Isquemia Encefálica/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Western Blotting , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Neurônios/metabolismo
3.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923196

RESUMO

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-ß (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.


Assuntos
Tolerância Imunológica , Fagocitose/imunologia , Infecções por Pseudomonas/imunologia , Fagos de Pseudomonas/fisiologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/virologia , Infecção dos Ferimentos/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Anticorpos Antivirais/imunologia , Humanos , Interferons/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fagos de Pseudomonas/imunologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/metabolismo
4.
Biomed Res Int ; 2019: 6341967, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881993

RESUMO

Background: Colon cancer is a heterogeneous disease, differing in clinical symptoms, epigenetics, and prognosis for each individual patient. Identifying the core genes is important for early diagnoses and it provides a more precise method for treating colon cancer. Materials and Methods: In this study, we wanted to pinpoint these core genes so we obtained GSE101502 microRNA profiles from the GEO database, which resulted in 17 differential expressed microRNAs that were identified by GEO2R analysis. Then, 875 upregulated and 2920 downregulated target genes were predicted by FunRich. GO and KEGG pathway were used to do enrich analysis. Results: GO analysis indicated that upregulated genes were significantly enriched in the regulation of cell communication and signaling and in nervous system development, while the downregulated genes were significantly enriched in nervous system development and regulation of transcription from the RNA polymerase II promoter. KEGG pathway analysis suggested that the upregulated genes were enriched in axon guidance, MAPK signaling pathway, and endocytosis, while the downregulated genes existed in pathways in cancer, focal adhesion, and PI3K-Akt signaling pathway. The top four molecules including 82 hub genes were identified from the PPI network and involved in endocytosis, spliceosome, TGF-beta signaling pathway, and lysosome. Finally, NUDT21, GNB1, CLINT1, and COL1A2 core gene were selected due to their correlation with the prognosis of IIA stage colon cancer. Conclusion: this study suggested that NUDT21, GNB1, CLINT1, and COL1A2 might be the core genes for colon cancer that play an important role in the development and prognosis of IIA stage colon cancer.


Assuntos
Neoplasias do Colo/genética , Biologia Computacional , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Colágeno Tipo I/genética , Neoplasias do Colo/patologia , Bases de Dados Genéticas , Intervalo Livre de Doença , Subunidades beta da Proteína de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico
5.
Genome Biol ; 20(1): 33, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760294

RESUMO

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional mechanism mediated by ADAR enzymes that have been recently associated with cancer. RESULTS: Here, we characterize the inosinome signature in normal brain and de novo glioblastoma (GBM) using new metrics that re-stratify GBM patients according to their editing profiles and indicate this post-transcriptional event as a possible molecular mechanism for sexual dimorphism in GBM. We find that over 85% of de novo GBMs carry a deletion involving the genomic locus of ADAR3, which is specifically expressed in the brain. By analyzing RNA editing and patient outcomes, an intriguing gender-dependent link appears, with high editing of Alus shown to be beneficial only in male patients. We propose an inosinome-based molecular stratification of GBM patients that identifies two different GBM subgroups, INO-1 and INO-2, which can identify novel high-risk gender-specific patient groups for which more aggressive treatments may be necessary. CONCLUSIONS: Our data provide a detailed picture of RNA editing landscape in normal brain and GBM, exploring A-to-I RNA editing regulation, disclosing unexpected editing implications for GBM patient stratification and identification of gender-dependent high-risk patients, and suggesting COG3 I/V as an eligible site for future personalized targeted gene therapy.


Assuntos
Adenosina Desaminase/metabolismo , Neoplasias Encefálicas/genética , Glioblastoma/genética , Inosina/metabolismo , Edição de RNA , Proteínas Adaptadoras de Transporte Vesicular/genética , Fatores Etários , Elementos Alu , Astrócitos/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Estudos de Casos e Controles , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Células HEK293 , Humanos , Fatores Sexuais
6.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670552

RESUMO

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)-TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3-/- and TRIF-/- mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-γ), and nitric oxide (NO). Unlike those of T. gondii, N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-α and IFN-ß in the presence of the parasite. Furthermore, TRIF-/- infected macrophages produced lower levels of IL-12p40, while exogenous IFN-α replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Coccidiose/imunologia , Coccidiose/parasitologia , Neospora/fisiologia , RNA de Protozoário/imunologia , Receptor 3 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Coccidiose/genética , Feminino , Interações Hospedeiro-Parasita , Humanos , Interferon gama/genética , Interferon gama/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neospora/genética , Neospora/imunologia , Óxido Nítrico/imunologia , RNA de Protozoário/genética , Células Th1/imunologia , Células Th1/parasitologia , Receptor 3 Toll-Like/genética
7.
Parasitol Res ; 118(2): 539-549, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30643971

RESUMO

Worldwide approximately 68 million people are infected with lymphatic filariasis (Lf), provoked by Wuchereria bancrofti, Brugia malayi and Brugia timori. This disease can lead to massive swelling of the limbs (elephantiasis) and disfigurement of the male genitalia (hydrocele). Filarial induced immune regulation is characterised by dominant type 2 helper T cell and regulatory immune responses. In vitro studies have provided evidence that signalling via Toll-like receptor-mediated pathways is triggered by filarial associated factors. Nevertheless, until now, less is known about the role of the adapter molecule TRIF during in vivo infections. Here, we used the rodent-specific nematode Litomosoides sigmodontis to investigate the role of TLR signalling and the corresponding downstream adapter and regulatory molecules TRIF, MyD88, IRF1 and IRF3 during an ongoing infection in semi-susceptible C57BL/6 mice. Interestingly, lack of the central adapter molecule TRIF led to higher worm burden and reduced overall absolute cell numbers in the thoracic cavity (the site of infection) 30 days post-infection. In addition, frequencies of macrophages and lymphocytes in the TC were increased in infected TRIF-/- C57BL/6 mice, whereas frequencies of eosinophils, CD4+ and CD8+ T cells were reduced. Nevertheless, cytokine levels and regulatory T cell populations remained comparable between TRIF-deficient and wildtype C57BL/6 mice upon 30 days of L. sigmodontis infection. In summary, this study revealed a crucial role of the adapter molecule TRIF on worm recovery and immune cell recruitment into the site of infection 30 days upon L. sigmodontis infection in C57BL/6 mice.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Filariose/imunologia , Filariose/parasitologia , Filarioidea/crescimento & desenvolvimento , Filarioidea/imunologia , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Citocinas/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Células Th2/imunologia
8.
Pathol Res Pract ; 215(4): 738-747, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30679084

RESUMO

Cervical carcinoma is one of the most universal cancers among women. Recent researches have reported that microRNA-150-5p (miR-150-5p) is up-regulated in diverse carcinomas containing cervical carcinoma. The purpose of this study was to further investigate the potential role of miR-150-5p in the progress of cervical carcinoma cells including proliferation and epithelial-mesenchymal transition (EMT).The ability of miR-150-5p to promote carcinogenesis was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot assays, respectively. Bioinformatics analyses predicted and identified whether SRC kinase signaling inhibitor 1 (SRCIN1) was served as a potential target of miR-150-5p. C-33A and HeLa cells were utilized to determine the function of miR-150-5p through targeting SRCIN1. Among the aberrantly expressed miRNAs, miR-150-5p was significantly revealed differential expression in cervical carcinoma cell lines and was closely relevant to cell growth regulation. Furthermore, we found that SRCIN1 overexpression could obviously inhibit the proliferation and EMT of cervical cancer cells triggered by miR-150-5p mimics as well as accelerated the apoptosis of cervical carcinoma cells. In conclusion, our data demonstrated that miR-150-5p could promote the proliferation and EMT of cervical carcinoma cells via targeting SRCIN1. Thus, miR-150-5p may hold a promise as a prognostic biomarker and potential therapeutic target for cervical carcinoma.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
9.
Acta Diabetol ; 56(4): 457-472, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30603868

RESUMO

AIMS: Macrocalcification and microcalcification present different clinical risks, but the regulatory of their formation was unclear. Therefore, this study explored the underlying mechanisms of macrocalcification and microcalcification in diabetes mellitus. METHODS: Anterior tibial arteries of amputated diabetic feet were collected. According to the calcium content, patients were divided into less-calcification group and more-calcification group. And calcification morphology in plaques was observed. For further study, an in vivo mouse diabetic atherosclerosis model and an in vitro primary mouse aortic smooth muscle cell model were established. After the receptors for AGEs (RAGE) or galectin-3 were silenced, calcified nodule sizes and sortilin expression were determined. Scanning electron microscopy (SEM) was performed to detect the aggregation of matrix vesicles with the inhibition or promotion of sortilin. RESULTS: Both macro- and microcalcification were found in human anterior tibial artery plaques. Macrocalcification formed after the silencing of RAGE, and microcalcification formed after the silencing of galectin-3. In the process of RAGE- or galcetin-3-induced calcification, sortilin played an important role downstream. SEM showed that sortilin promoted the aggregation of MVs in the early stage of calcification and formed larger calcified nodules. CONCLUSION: RAGE downregulated sortilin and then transmitted microcalcification signals, whereas galectin-3 upregulated sortilin, which accelerated the aggregation of MVs in the early stage of calcification and mediated the formation of macrocalcifications, These data illustrate the progression of two calcification types and suggest sortilin as a potential target for early intervention of calcification and as an effective biomarker for the assessment of long-term clinical risk and prognosis.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Galectina 3/fisiologia , Placa Aterosclerótica/genética , Receptor para Produtos Finais de Glicação Avançada/fisiologia , Calcificação Vascular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Amputação , Animais , Aorta/metabolismo , Aorta/patologia , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/cirurgia , Pé Diabético/patologia , Pé Diabético/cirurgia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Placa Aterosclerótica/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estreptozocina , Artérias da Tíbia/metabolismo , Artérias da Tíbia/patologia , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
10.
Mol Pain ; 15: 1744806919830018, 2019 Jan-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30672380

RESUMO

Elevated excitability of primary afferent neurons underlies chronic pain in patients with functional or inflammatory bowel diseases. Recent studies have established an essential role for an enhanced transient receptor potential vanilloid subtype 1 (TRPV1) signaling in mediating peripheral hyperalgesia in inflammatory conditions. Since colocalization of Toll-like receptor 4 (TLR4) and TRPV1 has been observed in primary afferents including the trigeminal sensory neurons and the dorsal root ganglion neurons, we test the hypothesis that TLR4 might regulate the expression and function of TRPV1 in primary afferent neurons in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis using the TLR4-deficient and the wild-type C57 mice. Despite having a higher disease activity index following administration of 2,4,6-trinitrobenzene sulfate, the TLR4-deficient mice showed less inflammatory infiltration in the colon than the wild-type mice. Increased expression of TLR4 and TRPV1 as well as increased density of capsaicin-induced TRPV1 current was observed in L4-S2 dorsal root ganglion neurons of the wild-type colitis mice till two weeks post 2,4,6-trinitrobenzene sulfate treatment. In comparison, the TLR4-deficient colitis mice had lower TRPV1 expression and TRPV1 current density in dorsal root ganglion neurons with lower abdominal withdrawal response scores during noxious colonic distensions. In the wild type but not in the TLR4-deficient dorsal root ganglion neurons, acute administration of the TLR4 agonist lipopolysaccharide increased the capsaicin-evoked TRPV1 current. In addition, we found that the canonical signaling downstream of TLR4 was activated in 2,4,6-trinitrobenzene sulfate-induced colitis in the wild type but not in the TLR4-deficient mice. These results indicate that TLR4 may play a major role in regulation of TRPV1 signaling and peripheral hyperalgesia in inflammatory conditions.


Assuntos
Colite/patologia , Gânglios Espinais/patologia , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Canais de Cátion TRPV/metabolismo , Receptor 4 Toll-Like/deficiência , Regulação para Cima/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Capsaicina/farmacologia , Colite/induzido quimicamente , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/metabolismo , Neurônios/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Ácido Trinitrobenzenossulfônico/toxicidade
11.
J Mol Neurosci ; 67(4): 495-503, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30610591

RESUMO

Soluble amyloid beta (Aß) oligomers are the most common forms of Aß in the early stage of Alzheimer's disease (AD). They are highly toxic to the neurons but their capability to activate microglia remains controversial. Microglia develop two distinct phenotypes, classic (M1) and alternative (M2). Tuning of microglia to the alternative (anti-inflammatory) state is of major interest in treatment of neuroinflammatory disease. This study aimed to assess tuning the microglia to produce interferon beta (IFN-ß) as an anti-inflammatory cytokine through TLR4 pathway in a rat model of AD. Microglial BV-2 cells were treated with 1 µg/ml lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), or vehicles for 24 h, and then incubated with Aß oligomer. After 24 h, cell pellets were harvested and TIR-domain-containing adapter-inducing interferon-ß (TRIF), interferon regulatory factor 3 (IRF3), and IFN-ß levels were measured. The ligands/vehicle were microinjected into the right ventricle of male Wistar rats every 3 days. Two weeks later, an osmotic pump filled with oligomeric Aß/vehicle was implanted in the left ventricle. After 2 weeks, TRIF, IRF3, and IFN-ß levels were measured in the hippocampal tissue. TNF-α and IFN-ß levels were assessed in the hippocampus using immunohistochemistry. The oligomeric Aß did not change TRIF, IRF3, and IFN-ß levels in both cell culture and hippocampal tissue. However, pretreatment with LPS or MPL increased the level of these proteins. BV-2 cells morphologically express M1 state in presence of higher dose of Aß oligomer (10 µM). Pretreatment with LPS or MPL decreased the TNF-α and increased the number of IFN-ß positive cells in the hippocampus of Aß-treated rats. In conclusion, pretreatment with low dose TLR4 agonists could induce microglia to produce neuroprotective cytokines including IFN-ß which may be considered as a potential strategy to combat neuronal degeneration in AD.


Assuntos
Doença de Alzheimer/metabolismo , Interferon beta/genética , Microglia/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Hipocampo/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
FEBS J ; 286(3): 523-535, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30536547

RESUMO

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNß signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.


Assuntos
Proteína de Domínio de Morte Associada a Fas/genética , Interferon beta/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor fas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Anticorpos/farmacologia , Proteína de Domínio de Morte Associada a Fas/imunologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Interferon beta/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Células Jurkat , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Células RAW 264.7 , Transdução de Sinais , Células THP-1 , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Receptor fas/antagonistas & inibidores , Receptor fas/imunologia
13.
Proc Natl Acad Sci U S A ; 116(2): 556-565, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30584088

RESUMO

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.


Assuntos
Autofagossomos/metabolismo , Doença de Depósito de Glicogênio Tipo IIb/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Miócitos Cardíacos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Técnicas de Inativação de Genes , Doença de Depósito de Glicogênio Tipo IIb/genética , Doença de Depósito de Glicogênio Tipo IIb/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/genética , Lisossomos/patologia , Miócitos Cardíacos/patologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo
14.
Pancreatology ; 19(1): 149-157, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30583980

RESUMO

BACKGROUND: Acute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown. METHODS: To uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function. RESULTS: After the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice. CONCLUSIONS: TLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Pancreatite/patologia , Receptor 3 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Ceruletídeo/toxicidade , Regulação da Expressão Gênica , Humanos , Camundongos Knockout , Camundongos Transgênicos , Pancreatite/induzido quimicamente , Pancreatite/genética , Receptor 3 Toll-Like/genética
15.
Mol Med ; 24(1): 66, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30587103

RESUMO

BACKGROUND: Caspase-11, a cytosolic receptor of bacterial endotoxin (lipopolysaccharide: LPS), mediates immune responses and lethality in endotoxemia and experimental sepsis. However, the upstream pathways that regulate caspase-11 activation in endotoxemia and sepsis are not fully understood. The aim of this study is to test whether TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling is critical for caspase-11-dependent immune responses and lethality in endotoxemia. METHODS: Mice of indicated genotypes were subjected to endotoxemia or cecum ligation and puncture (CLP) and monitored daily by signs of a moribund state for lethality. Serum interleukin (IL)-1α, IL-1ß, IL-6 and tumor necrosis factor (TNF) were measured by ELISA. Data were analyzed by using student's t-test or one-way ANOVA followed by post-hoc Bonferroni test. Survival data were analyzed by using the log-rank test. RESULTS: Blockade of type 1 interferon signaling or genetic deletion of TRIF or guanylate-binding proteins (GBPs) prevented caspase-11-dependent immune responses, organ injury and lethality in endotoxemia and experimental sepsis. In vitro, deletion of GBPs blocked cytosolic LPS-induced caspase-11 activation in mouse macrophages. CONCLUSIONS: These findings demonstrate that TRIF signaling is required for caspase-11-dependent immune responses and lethality in endotoxemia and sepsis, and provide novel mechanistic insights into how LPS induces caspase-11 activation during bacterial infection.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Caspases/imunologia , Endotoxemia/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Endotoxemia/induzido quimicamente , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Interferon Tipo I/imunologia , Lipopolissacarídeos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout
16.
BMC Genomics ; 19(1): 907, 2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541452

RESUMO

BACKGROUND: Swine streptococcosis has caused great economic loss in the swine industry, and the major pathogen responsible for this disease is Streptococcus Suis serotype 2 (SS2). Disease resistance breeding is a fundamental way of resolving this problem. With the development of GWAS and transcriptomic microarray technology, we now have powerful research tools to identify SS2 resistance genes. RESULTS: In this research, we generated an F2 generation of SS2 resistant C57BL/6 and SS2 susceptive A/J mice. With the F2 generation of these two mice strains and GWAS analysis, we identified 286 significant mouse genome SNPs sites associated with the SS2 resistance trait. Gene expression profiles for C57BL/6 and A/J were analyzed under SS2 infection pressure by microarray. In total, 251 differentially expressed genes were identified between these two mouse strains during SS2 infection. After combining the GWAS and gene expression profile data, we located two genes that were significantly associated with SS2 resistance, which were the UBA domain containing 1 gene (Ubac1) and Epsin 1 gene (Epn 1). GO classification and over-representation analysis revealed nine up-regulated related to immune function, which could potentially be involved in the C57BL/6 SS2 resistance trait. CONCLUSION: This is the first study to use both SNP chip and gene express profile chip for SS2 resistance gene identification in mouse, and these results will contribute to swine SS2 resistance breeding.


Assuntos
Resistência à Doença/genética , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Streptococcus suis/patogenicidade , Transcriptoma , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Sorogrupo , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/mortalidade , Infecções Estreptocócicas/veterinária , Streptococcus suis/metabolismo , Taxa de Sobrevida , Complexos Ubiquitina-Proteína Ligase/genética
17.
Cell Physiol Biochem ; 48(6): 2517-2527, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30121664

RESUMO

BACKGROUND/AIMS: The purpose of our experiments was to investigate the targeting relationship of linc00515, miR-140-5p and ATG14 and to explore the roles of linc00515, miR-140-5p and ATG14 in autophagy and chemoresistance of melphalan-resistant multiple myeloma cells. METHODS: Plasmids that could interfere with the expression of linc00515 and ATG14 were loaded into myeloma cells, which were cultured with melphalan. MTT assay and flow cytometry analysis were utilized to investigate the effect of linc00515, miR-140-5p and ATG14 on the resistance of myeloma cells. QRT-PCR was used to determine the levels of mRNAs. Western blot was utilized to explore the level of ATG14 and autophagy-related proteins. Dual luciferase assay was utilized to explore the targeting relationship between linc00515, miR-140-5p and ATG14. GFP LC3 fluorescence assay was conducted to study the autophagy of cells. RESULTS: The expression of linc00515 and ATG14 were significantly higher in melphalan-resistant myeloma cells. Knockdown of linc00515 and ATG14 led to decreased autophagy and chemoresistance of melphalan-resistant myeloma cells. The forced expression of miR-140-5p suppressed autophagy and chemoresistance of melphalan-resistant myeloma cells. CONCLUSION: Linc00515 enhanced autophagy and chemoresistance of melphalan-resistant myeloma by directly inhibiting miR-140-5p, which elevated ATG14 level.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antagomirs/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Masculino , Melfalan/farmacologia , MicroRNAs/genética , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Regulação para Cima
18.
J Immunol Res ; 2018: 6249085, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29977930

RESUMO

Toll/IL-1R-domain-containing adaptor-inducing IFN-ß (TRIF) is an important adaptor for TLR3- and TLR4-mediated inflammatory signaling pathways. Recent studies have shown that TRIF plays a key role in vessel inflammation and atherosclerosis; however, the precise mechanisms are unclear. We investigated the mechanisms of the TRIF-regulated inflammatory response in RAW264.7 macrophages under oxidized low-density lipoprotein (ox-LDL) stimulation. Our data show that ox-LDL induces TRIF, miR-155, and BIC expression, activates the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways, and elevates the levels of IL-6 and TNF-α in RAW264.7 cells. Knockdown of TRIF using TRIF siRNA suppressed BIC, miR-155, IL-6, and TNF-α expression and inhibited the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways. Inhibition of ERK1/2 signaling also suppressed BIC and miR-155 expression. These findings suggest that TRIF plays an important role in regulating the ox-LDL-induced macrophage inflammatory response and that TRIF modulates the expression of BIC/miR-155 and the downstream SOCS1-STAT3-NF-κB signaling pathway via ERK1/2. Therefore, TRIF might be a novel therapeutic target for atherosclerosis.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Lipoproteínas LDL/farmacologia , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , MicroRNAs/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Inativação Gênica , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/fisiologia , Camundongos , MicroRNAs/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , NF-kappa B/metabolismo , Células RAW 264.7 , Precursores de RNA/metabolismo , RNA Interferente Pequeno , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo
19.
Dev Cell ; 45(5): 621-636.e7, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29870720

RESUMO

The extensive subcellular network of membrane contact sites plays central roles in organelle biogenesis and communication, yet the precise contributions of the involved machineries remain largely enigmatic. The yeast vacuole forms a membrane contact site with mitochondria, called vacuolar and mitochondrial patch (vCLAMP). Formation of vCLAMPs involves the vacuolar Rab GTPase Ypt7 and the Ypt7-interacting Vps39 subunit of the HOPS tethering complex. Here, we uncover the general preprotein translocase of the outer membrane (TOM) subunit Tom40 as the direct binding partner of Vps39 on mitochondria. We identify Vps39 mutants defective in TOM binding, but functional for HOPS. Cells that cannot form vCLAMPs show reduced growth under stress conditions and impaired survival upon starvation. Unexpectedly, our mutant analysis revealed the existence of two functionally independent vacuole-mitochondria MCSs: one formed by the Ypt7-Vps39-Tom40 tether and a second one by Vps13-Mcp1, which is redundant with ER-mitochondrial contacts formed by ERMES.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Fenômenos Fisiológicos Celulares , Fusão de Membrana , Proteínas de Transporte da Membrana Mitocondrial/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
20.
Anim Sci J ; 89(7): 946-955, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29708631

RESUMO

The hypothalamus plays a central role in controlling poultry endocrine and reproductive activities. So far there is limited information focused on the proteome profiles of the hypothalamus from geese during different stages of the egg-laying cycle. In order to identify proteins regulating the egg-laying process of Huoyan geese, we investigated the proteome profiles of the hypothalamus from Huoyan geese during the laying period and pre-laying period by applying an isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic technology. A total number of 3,337 were identified and quantified, of which 18 were significantly up-regulated and 16 were significantly down-regulated. These differentially expressed proteins were subjected to bioinformatics analyses based on the Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway. Some of these were revealed to be involved in hormone and neurotransmitter secretion, exocytosis, calcium ion transport and synaptic transmission. Subsequently, excitatory amino acid transporter 2, complexin-1 and inositol 1,4,5-trisphosphate receptor, type 3 were confirmed at the messenger RNA level using quantitative real-time RT-PCR. Then, the abundance change of these proteins was verified further using Western blotting analysis. These data may aid in elucidating the molecular mechanism of higher laying performance in Huoyan geese.


Assuntos
Proteínas Aviárias/genética , Proteínas Aviárias/fisiologia , Gansos/fisiologia , Hipotálamo/química , Oviparidade/genética , Proteoma/genética , Proteômica/métodos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Regulação para Baixo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/fisiologia , Feminino , Hipotálamo/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Proteoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
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