RESUMO
Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.
Assuntos
Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Treonina/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Células HCT116 , Humanos , Fosforilação , Domínios de Homologia à Plecstrina , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-akt/química , Serina/química , Transdução de Sinais , Treonina/químicaRESUMO
TET proteins, by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are hypothesized, but not directly shown, to protect promoter CpG islands (CGIs) against abnormal DNA methylation (DNAm) in cancer. We define such a protective role linked to DNA damage from oxidative stress (OS) known to induce this abnormality. TET2 removes aberrant DNAm during OS through interacting with DNA methyltransferases (DNMTs) in a "Yin-Yang" complex targeted to chromatin and enhanced by p300 mediated TET2 acetylation. Abnormal gains of DNAm and 5hmC occur simultaneously in OS, and knocking down TET2 dynamically alters this balance by enhancing 5mC and reducing 5hmC. TET2 reduction results in hypermethylation of promoter CGIs and enhancers in loci largely overlapping with those induced by OS. Thus, TET2 indeed may protect against abnormal, cancer DNAm in a manner linked to DNA damage.
Assuntos
Cromatina/metabolismo , Metilação de DNA , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilação , Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Dioxigenases , Proteína p300 Associada a E1A/metabolismo , Células HCT116 , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Neoplasias/genética , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Fatores de Tempo , Transfecção , UbiquitinaçãoRESUMO
Dysregulated neurite outgrowth and synapse formation underlie many psychiatric disorders, which are also manifested by wolfram syndrome (WS). Whether and how the causative gene WFS1 deficiency affects synapse formation remain elusive. By mirroring human brain development with cerebral organoids, WFS1-deficient cerebral organoids not only recapitulate the neuronal loss in WS patients, but also exhibit significantly impaired synapse formation and function associated with reduced astrocytes. WFS1 deficiency in neurons autonomously delays neuronal differentiation with altered expressions of genes associated with psychiatric disorders, and impairs neurite outgrowth and synapse formation with elevated cytosolic calcium. Intriguingly, WFS1 deficiency in astrocytes decreases the expression of glutamate transporter EAAT2 by NF-κB activation and induces excessive glutamate. When co-cultured with wildtype neurons, WFS1-deficient astrocytes lead to impaired neurite outgrowth and increased cytosolic calcium in neurons. Importantly, disrupted synapse formation and function in WFS1-deficient cerebral organoids and impaired neurite outgrowth affected by WFS1-deficient astrocytes are efficiently reversed with Riluzole treatment, by restoring EAAT2 expression in astrocytes. Furthermore, Riluzole rescues the depressive-like behavior in the forced swimming test and the impaired recognition and spatial memory in the novel object test and water maze test in Wfs1 conditional knockout mice. Altogether, our study provides novel insights into how WFS1 deficiency affects synapse formation and function, and offers a strategy to treat this disease.
Assuntos
Células-Tronco Embrionárias Humanas , Síndrome de Wolfram , Animais , Camundongos , Humanos , Síndrome de Wolfram/tratamento farmacológico , Síndrome de Wolfram/genética , Síndrome de Wolfram/metabolismo , Riluzol/farmacologia , Riluzol/metabolismo , Cálcio/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/metabolismo , Camundongos Knockout , Sinapses/metabolismoRESUMO
Ligand 1, a rim-differentiated pillar[5]arene macrocycle modified with five naphthalimide groups through click chemistry, serves as an effective ratiometric fluorescent chemosensor for Cu2+. In contrast to the monomeric naphthalimide control compound 2, which shows only monomer emission, ligand 1 demonstrates dual emission characteristics encompassing both the monomer and excimer of the naphthalimide moieties. The binding properties of ligand 1 toward 15 different metal ions were systematically investigated in CH2Cl2/CH3CN (v/v, 1 : 1) by UV-vis and fluorescence spectroscopy. Remarkably, ligand 1 exhibits exceptional selectivity for Cu2+ ions. Upon complexation with Cu2+, the excimer emission of ligand 1 diminishes, concomitant with an enhancement of its monomer emission. The binding ratio for 1·Cu2+ was determined to be 1 : 1, with an association constant of (3.39 ± 0.40) × 105 M-1 calculated using a nonlinear least-squares curve-fitting method. Furthermore, the limit of detection (LOD) was found to be 185 ± 7 nM. Our results from 1H NMR titration, high-resolution mass spectrometry analysis and density functional theory calculations of 1·Cu2+ suggest synergistic coordination between Cu2+ and the triazole groups on ligand 1.
RESUMO
Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.
Assuntos
Imunidade Inata , Linfócitos , Camundongos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/genética , Fatores de Transcrição/metabolismo , Sirolimo/farmacologia , Mamíferos/metabolismoRESUMO
BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are the most dominant ILCs in heart tissue, and sex-related differences exist in mouse lung ILC2 phenotypes and functions; however, it is still unclear whether there are sex differences in heart ILC2s. RESULTS: Compared with age-matched wild-type (WT) male mice, 8-week-old but not 3-week-old WT female mice harbored an obviously greater percentage and number of heart ILC2s in homeostasis. However, the percentage of killer-cell lectin-like receptor G1 (Klrg1)- ILC2s was higher, but the Klrg1+ ILC2s were lower in female mice than in male mice in both heart tissues of 3- and 8-week-old mice. Eight-week-old Rag2-/- mice also showed sex differences similar to those of age-matched WT mice. Regarding surface marker expression, compared to age-matched male mice, WT female mice showed higher expression of CD90.2 and Ki67 and lower expression of Klrg1 and Sca-1 in heart total ILC2s. There was no sex difference in IL-4 and IL-5 secretion by male and female mouse heart ILC2s. Increased IL-33 mRNA levels within the heart tissues were also found in female mice compared with male mice. By reanalyzing published single-cell RNA sequencing data, we found 2 differentially expressed genes between female and male mouse heart ILC2s. Gene set variation analysis revealed that the glycine, serine and threonine metabolism pathway was upregulated in female heart ILC2s. Subcluster analysis revealed that one cluster of heart ILC2s with relatively lower expression of Semaphorin 4a and thioredoxin interacting protein but higher expression of hypoxia-inducible lipid droplet-associated. CONCLUSIONS: These results revealed greater numbers of ILC2s, higher expression of CD90.2, reduced Klrg1 and Sca-1 expression in the hearts of female mice than in male mice and no sex difference in IL-4 and IL-5 production in male and female mouse heart ILC2s. These sex differences in heart ILC2s might be due to the heterogeneity of IL-33 within the heart tissue.
Assuntos
Coração , Imunidade Inata , Interleucina-33 , Linfócitos , Caracteres Sexuais , Animais , Feminino , Masculino , Camundongos , Interleucina-33/genética , Interleucina-33/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Pulmão/metabolismo , Linfócitos/metabolismo , Camundongos Knockout , Antígenos Thy-1/metabolismoRESUMO
BACKGROUND: Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection. RESULTS: Male C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads at day 7 postinfection, but final MCMV clearance was not affected. The total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice at day 7 postinfection but not day 21. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells at day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice had enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass than control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation levels of CD4+ and CD8+ T cells. CONCLUSIONS: These results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.
Assuntos
Infecções por Citomegalovirus , Muromegalovirus , Animais , Linfócitos T CD8-Positivos , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/metabolismo , Imunidade , Células Matadoras Naturais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/fisiologiaRESUMO
OBJECTIVE: Vascular endothelial growth factor B (VEGFB) was regarded to improve lipid metabolism and reduce obesity-related hyperlipidemia. Whether VEGFB participates in lipid metabolism in nonalcoholic fatty liver disease (NAFLD) has not been clear yet. This study investigated the involvement of VEGFB in lipid metabolism and insulin resistance via the AMPK signaling pathway in NAFLD. METHODS: We constructed the animal and cell model of NAFLD after VEGFB gene knockout to detect liver damage and metabolism in NAFLD. Bioinformatics analysis of VEGFB and the AMPK signaling pathway relative genes to verify the differential proteins. And mRNA levels of NAFLD fatty acid metabolism-related genes were detected. RESULTS: After the systemic VEGFB knockout mice were fed with high fat, the body fat, serum lipoprotein, NAFLD score, and insulin resistance were increased. Animal and cell experiments showed that the expression levels of phosphorylated proteins of CaMKK2 and AMPK decreased, the expression of proteins related to AMPK/ACC/CPT1 signaling pathway decreased, and the target genes CPT1α and Lcad decreased accordingly, reducing fatty acid oxidation in hepatocyte mitochondria; The expression of AMPK/SREBP1/Scd1 signaling pathway relative proteins increased, ACC1 and FAS increased correspondingly, which increased lipid synthesis in the endoplasmic reticulum. CONCLUSION: VEGFB can participate in lipid metabolism and insulin resistance of NAFLD through the AMPK signaling pathway.
Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Fator B de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Transdução de SinaisRESUMO
The aim of this study was to examine the combined effects of sulfated ß-Glucan from Saccharomyces cerevisiae (sGSC) on growth performance, antioxidant ability, nonspecific immunity, and intestinal flora of the red swamp crayfish (Procambarus clarkii). Four experimental diets (sGSC25, sGSC50, sGSC100 and sGSC200) with different levels of sGSC (0.025%, 0.05%, 0.1% and 0.2% in diet, respectively) were fed to juvenile crayfish (average weight: 2.5 ± 0.5 g) for 8 weeks. The control diet was given with 2000 mg/kg GSC (GSC200 group). The based control diet was given without sGSC or GSC (blank group). Each group had 3 parallel test pools, 20 crayfish were reared in each pool. At the end of the growth trial, adding dietary 0.025%-0.1% sGSC could significantly improve the growth performance, antioxidant capacity and immunity of crayfish. Compared with GSC, sGSC had a better effect at lower concentration. Higher concentration of sGSC (>0.1%) would cause some side effects. sGSC also could improve the structure of the intestinal flora and optimize the function of the flora. sGSC would increase the abundances of probiotics such as Hafnia and Acinetobacter, and decreases the abundances of maleficent bacteria such as Enterobacteriaceae. Higher concentration of sGSC (>0.1%) would increase the abundance of Aeromonas. To conclude, 0.025%-0.1% sGSC can be used as a supplement in crayfish feed to increase growth, immunity, and antioxidant capacity and improve the structure of intestinal flora. These results provided a theoretical basis for the application of sGSC instead of GSC in crayfish breeding. It will be necessary to further study the optimal concentration of sGSC in feed additives in different growth stages of crayfish in the future.
Assuntos
Microbioma Gastrointestinal , beta-Glucanas , Animais , Antioxidantes/farmacologia , Astacoidea , Melhoramento Vegetal , Saccharomyces cerevisiae , Sulfatos/farmacologia , beta-Glucanas/farmacologiaRESUMO
BACKGROUND: Tuberous sclerosis complex 1 (Tsc1) is known to regulate the development and function of various cell types, and RORγt is a critical transcription factor in the immune system. However, whether Tsc1 participates in regulating RORγt-expressing cells remains unknown. METHODS: We generated a mouse model in which Tsc1 was conditionally deleted from RORγt-expressing cells (Tsc1RORγt) to study the role of RORγt-expressing cells with Tsc1 deficiency in brain homeostasis. RESULTS: Type 3 innate lymphoid cells (ILC3s) in Tsc1RORγt mice displayed normal development and function, and the mice showed normal Th17 cell differentiation. However, Tsc1RORγt mice exhibited spontaneous tonic-clonic seizures and died between 4 and 6 weeks after birth. At the age of 4 weeks, mice in which Tsc1 was specifically knocked out in RORγt-expressing cells had cortical neuron defects and hippocampal structural abnormalities. Notably, over-activation of neurons and astrogliosis were observed in the cortex and hippocampus of Tsc1RORγt mice. Moreover, expression of the γ-amino butyric acid (GABA) receptor in the brains of Tsc1RORγt mice was decreased, and GABA supplementation prolonged the lifespan of the mice to some extent. Further experiments revealed the presence of a group of rare RORγt-expressing cells with high metabolic activity in the mouse brain. CONCLUSIONS: Our study verifies the critical role of previously unnoticed RORγt-expressing cells in the brain and demonstrates that the Tsc1 signaling pathway in RORγt-expressing cells is important for maintaining brain homeostasis.
Assuntos
Encéfalo , Linfócitos , Proteína 1 do Complexo Esclerose Tuberosa/deficiência , Animais , Encéfalo/imunologia , Encéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.
Assuntos
Anticorpos Monoclonais/imunologia , Análise Serial de Proteínas/métodos , Fatores de Transcrição/metabolismo , Animais , Clonagem Molecular , Bases de Dados Factuais , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos TestesRESUMO
A novel and low-cost DNAzyme, Ni/Fe layered double hydroxide (LDH) nanosheet/G-quadruplex (without hemin) with enhanced peroxidase-mimic activity was designed. The catalytic mechanism was investigated. The detection of Cu(II) in actual serum samples could be realized sensitively via this efficient DNAzyme-based method.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , DNA Catalítico/metabolismo , Hemina , Hidróxidos , Peroxidase , PeroxidasesRESUMO
A new polycyclic polyprenylated acylphloroglucinol (PPAP), hypermonin C (1), along with nine known PPAPs (2-10) were obtained from the leaves and twigs of Hypericum monogynum. The structures of the isolates were determined on the basis of extensive spectroscopic analysis. The neuroprotective effects of the isolates against several chemical-induced injuries in SH-SY5Y and PC12 cells were assessed, and most of the compounds exhibited significant protective effects at 10 µg/ml. Especially, three compounds (1, 3, and 7) showed excellent neuroprotective activity with a cell viability of 92.4% â¼ 95.8% in KCl-induced SH-SY5Y cell injury. Their preliminary structure-activity relationship was also discussed and the configuration of substituent in furohyperforin may be critical for the neuroprotective activity of PPAP derivatives.
Assuntos
Hypericum , Fármacos Neuroprotetores , Animais , Estrutura Molecular , Fármacos Neuroprotetores/farmacologia , Células PC12 , Floroglucinol/farmacologia , Folhas de Planta , RatosRESUMO
Cancer stem-like cells are rare immortal cells within tumor, which are thought to play important roles in ionizing radiation (IR) therapy-resistance. Quercetin is a natural flavonoid with potential anti-cancer properties without significant cytotoxicity in normal tissues. In this study, we demonstrated that quercetin-IR combination treatment exhibited more dramatic anti-cancer effect than either quercetin or IR treatment alone via targeting colon cancer stem cells (CSCs) and inhibiting the Notch-1 signaling. These effects were further verified by in vivo studies which showed remarkable decrease of the CSCs markers and the expression of Notch-1 signaling proteins in human colon cancer xenografts in nude mice. Co-treatment with quercetin and low dose of radiation significantly reduced the expressions of all five proteins of γ-secretase complex in HT-29 and DLD-1 cells. In addition, ectopic expression of the Notch intracellular domain (NICD) partly reversed the inhibition effects by the combination therapy. In conclusion, our results indicated that the combination of quercetin (20 µM) and IR (5Gy) might be a promising therapeutic strategy for colon cancer treatment by targeting colon cancer stem-like cells and inhibiting the Notch-1 signaling. In future studies, we intend to further explore the potential therapeutic efficacy of the quercetin-radiation combination treatment in clinical trials.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/radioterapia , Quercetina/uso terapêutico , Tolerância a Radiação/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Quercetina/farmacologia , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Utilizing nanoparticles to deliver subunit vaccine is considered to be a promising strategy to improve immune response. However, currently reported systems suffered from one or more points, for example, delicate design on molecular structures and elaborate synthesis process, low antigen and/or adjuvant encapsulation efficiency, involvement of toxic materials, and denaturing of bioactivity of antigen and/or adjuvant. To address these issues, here, for the first time, we developed a one-pot method to produce a subunit vaccine by using hexa-histidine metal assembly (HmA) to codeliver tumor-associated antigens (GP100, a peptide KTWGQYWQV) and adjuvant (CpG). The generation of subunit vaccines was detailedly characterized by various techniques, including dynamic scatter, scanning electron microscopy, transmission electron microscopy, UV-visible spectroscopy, agarose gel electrophoresis, etc. HmA displayed high efficiency on encapsulating both subunits (GP100 and CpG) under mild conditions, and the generated subunit vaccine showed a pH-dependent release profile of loaded subunits. In the cellular tests, these subunit vaccines behaved with a quick endocytosis into immune cells and a fast endo/lysosomes escape, inducing maturation of antigen presentative cells and stimulating a potent cellular immune response. These results suggested that HmA is a robust platform for fabricating subunit vaccine, with immense potential for the immunotherapy of various diseases.
Assuntos
Metais/química , Compostos Organometálicos/química , Vacinas/imunologia , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Sistemas de Liberação de Medicamentos , Regulação da Expressão Gênica , Imunidade Celular , Camundongos , Nanopartículas , Subunidades Proteicas , Vacinas Sintéticas/imunologiaRESUMO
To explore the strategy of acute cerebral artery embolism after radiofrequency catheter ablation (RFA) for atrial fibrillation (AF). Reporting two cases with acute cerebral infarction after RFA for AF. Two patients were both with AF, and intracardiac thrombus was excluded through transesophageal echocardiogram (TEE) before procedure. Approach of ablation: circumferential pulmonary vein ablation in left atrium to isolate pulmonary vein plus linear ablation in the top and bottom of left atrium (BOX procedure). They both received Dabigatran Etexilate 110 mg twice daily, starting 6 hr after ablation. Symptoms of major stroke appeared 30 hr after ablation in Case 1. Occlusion was detected in M1 segment of the left middle cerebral artery by MRI 2 hr after symptoms onset. Intravenous thrombolysis was given immediately. In Case 2, the patient presented symptoms of major stroke 34 hr after ablation and occlusion in the basilar artery was confirmed by MRI 4.5 hr after symptoms onset. Although it was beyond the thrombolysis time window, mechanical thrombectomy was taken 7 hr after the symptoms onset. The culprit artery was successfully revascularized in both cases. In Case 1, NIHSS score was reduced from 8 (before thrombolysis) to 0 (24 hr after thrombolysis). In Case 2, NIHSS score decreased from 18 (before embolectomy) to 3 (24 hr after embolectomy). Both of the patients live a normal life without brain function impairment and hemorrhage until the last follow-up. Timely recanalization could attained a good cure effect when acute stoke was happened after RFA for AF.
Assuntos
Fibrilação Atrial/cirurgia , Artérias Cerebrais/diagnóstico por imagem , Embolia/terapia , Complicações Pós-Operatórias/terapia , Ablação por Radiofrequência , Terapia Trombolítica/métodos , Doença Aguda , Angiografia Cerebral/métodos , Embolia/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico por imagem , Resultado do TratamentoRESUMO
Trehalose plays a crucial role in the diapause process of many insects, serving as an energy source and a stress protectant. Trehalose accumulation has been reported in diapause pupae of Antheraea pernyi; however, trehalose metabolic regulatory mechanisms associated with diapause termination remain unclear. Here, we showed that the enhanced trehalose catabolism was associated with an increase in endogenous 20-hydroxyecdysone (20E) in hemolymph of A. pernyi pupae during their diapause termination and posttermination period. Injection of 20E increased the mRNA level of trehalase 1A (ApTre-1A) and trehalase 2 (ApTre-2) of A. pernyi diapause pupae in a dose-dependent manner but did not affect the mRNA level of trehalase 1B (ApTre-1B). Meanwhile, exogenous 20E increased the enzyme activities of soluble and membrane-bound trehalase, leading to a decline in hemolymph trehalose. Conversely, the expression of ApTre-1A and ApTre-2 were down-regulated after the ecdysone receptor gene (ApEcRB1) was silenced by RNA interference or by injection of an ecdysone receptor antagonist cucurbitacin B (CucB), which inhibits the 20E pathway. Moreover, CucB treatment delayed adult emergence, which suggests that ApEcRB1 might be involved in regulating pupal-adult development of A. pernyi by mediating ApTre-1A and ApTre-2 expressions. This study provides an overview of the changes in the expression and activity of different trehalase enzymes in A. pernyi in response to 20E, confirming the important role of 20E in controlling trehalose catabolism during A. pernyi diapause termination and posttermination period.
Assuntos
Ecdisterona , Mariposas/metabolismo , Animais , Diapausa de Inseto/efeitos dos fármacos , Ecdisterona/metabolismo , Ecdisterona/farmacologia , Metabolismo Energético/efeitos dos fármacos , Genes de Insetos , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Estágios do Ciclo de Vida/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Pupa/efeitos dos fármacos , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Interferência de RNA , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Trealase/efeitos dos fármacos , Trealase/metabolismo , Trealose/metabolismo , TriterpenosRESUMO
The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full-length BRAFV600E is available. Herein, we show the successful purification of active, full-length BRAFV600E from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAFV600E together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell-based assays demonstrate that BRAFV600E has several unique features that contribute to its tumorigenesis. Firstly, steady-state kinetic analyses reveal that purified BRAFV600E is more active than fully activated wild-type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAFV600E has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side-to-side dimer interface. Thirdly, BRAFV600E bypasses inhibitory P-loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAFV600E , complementing the understanding of BRAF regulation under normal and disease conditions.
Assuntos
Proteínas Proto-Oncogênicas B-raf/química , Ativadores de Enzimas/metabolismo , Células HEK293 , Humanos , Imidazóis/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutação , Oximas/metabolismo , Fosforilação/genética , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Vemurafenib/metabolismoRESUMO
In organic-inorganic hybrid perovskite solar cells, though the current density-voltage (J-V) hysteresis phenomenon is accepted to be caused by ion migration coupled with charge carrier recombination, there are still rich hysteresis characteristics (various J-V hysteresis loops) remaining to be explained. Here, a systematic drift-diffusion simulation study is conducted to explore the effect of interfacial recombination lifetime (τinterface), bulk charge carrier lifetime (τbulk) and mobility (µ) on J-V hysteresis behaviors. The simulation results show that, for devices with only interfacial recombination, the decrease of τinterface will lead to J-V hysteresis loops with a large gap on the open circuit side. For devices with only bulk recombination, the drop of τbulk will lead to J-V hysteresis loops with a large gap on the short circuit side. Meanwhile, in both cases, the decrease of µ aggravates the effect of interfacial and bulk recombination, while it has no effect on VOC. Our simulations reveal the effect of decreased τinterface, τbulk and µ on the J-V characteristics and explain the hysteresis loops with specific shapes, which have been reported in the literature.
RESUMO
Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/.