Background Aicardi-Goutières syndrome (AGS) is a severe infant or juvenile-onset autoimmune disease characterized by inflammatory encephalopathy with an elevated type 1 interferon-stimulated gene (ISG) expression signature in the brain. Mutations in seven different protein-coding genes, all linked to DNA/RNA metabolism or sensing, have been identified in AGS patients, but none of them has been demonstrated to activate the IFN pathway in the brain of an animal. The molecular mechanism of inflammatory encephalopathy in AGS has not been well defined. Adenosine Deaminase Acting on RNA 1 (ADAR1) is one of the AGS-associated genes. It carries out A-to-I RNA editing that converts adenosine to inosine at double-stranded RNA regions. Whether an AGS-associated mutation in ADAR1 activates the IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined. Methods Mutations in the ADAR1 gene found in AGS patients were introduced into the mouse genome via CRISPR/Cas9 technology. Molecular activities of the specific p.K999N mutation were investigated by measuring the RNA editing levels in brain mRNA substrates of ADAR1 through RNA sequencing analysis. IFN pathway activation in the brain was assessed by measuring ISG expression at the mRNA and protein level through real-time RT-PCR and Luminex assays, respectively. The locations in the brain and neural cell types that express ISGs were determined by RNA in situ hybridization (ISH). Potential AGS-related brain morphologic changes were assessed with immunohistological analysis. Von Kossa and Luxol Fast Blue staining was performed on brain tissue to assess calcification and myelin, respectively. Results Mice bearing the ADAR1 p.K999N were viable though smaller than wild type sibs. RNA sequencing analysis of neuron-specific RNA substrates revealed altered RNA editing activities of the mutant ADAR1 protein. Mutant mice exhibited dramatically elevated levels of multiple ISGs within the brain. RNA ISH of brain sections showed selective activation of ISG expression in neurons and microglia in a patchy pattern. ISG-15 mRNA was upregulated in ADAR1 mutant brain neurons whereas CXCL10 mRNA was elevated in adjacent astroglia. No calcification or gliosis was detected in the mutant brain. Conclusions We demonstrated that an AGS-associated mutation in ADAR1, specifically the p.K999N mutation, activates the IFN pathway in the mouse brain. The ADAR1 p.K999N mutant mouse replicates aspects of the brain interferonopathy of AGS. Neurons and microglia express different ISGs. Basal ganglia calcification and leukodystrophy seen in AGS patients were not observed in K999N mutant mice, indicating that development of the full clinical phenotype may need an additional stimulus besides AGS mutations. This mutant mouse presents a robust tool for the investigation of AGS and neuroinflammatory diseases including the modeling of potential “second hits” that enable severe phenotypes of clinically variable diseases.
Background Aicardi–Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous. Methods Via CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms. Results We established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5. Conclusions The Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.
Background: Identification of novel biomarkers for silicosis could be helpful for disease diagnosis and pathophysiological mechanism exploration. Our study aims to investigate the Clara cell secretory 16-kd protein (CC16) and interleukin-12 (IL-12) levels in bronchoalveolar lavage fluid (BALF) in patients with silicosis at various stages. Methods:The enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method was used to determine the CC16 and IL-12 in BALF levels from 79 patients with silicosis of various stages.Correlation analyses were performed between CC16 and IL-12 levels, and lung function and cytological counts in patients with silicosis at various stages.Results: There were no significant differences in the BALF recovery volume, the number of cells, percentages of macrophages and lymphocytes in the alveolar lavage fluid of patients with silicosis in different stages (P>0.05); the percentage of neutrophils in stage I and stage II were higher than the control group (P<0.05) with statistically significant differences. The CC16 in BALF levels in stage I and II silicosis groups lower than the control group and stage III silicosis group with statistically significant differences (P<0.05), whereas CC16 levels in stage II silicosis group are higher than the stage I group (P<0.01). The IL-12 levels were higher than the control group (P<0.01), and the IL-12 levels in stage II and III silicosis group was higher than the stage I silicosis group (P<0.01). With the increase of the length of dust service, the CC16 and IL-12 levels decreased and showed a positive correlation between these indexes (correlation coefficient r=0.559, P<0.01). In addition, CC16 silicosis patient levels were positively correlated with FEV1/FVC and VCmax (r=0.242, 0.257; both P<0.05); IL-12 levels were negatively correlated with FEV1 and VC max (r=−0.250, −0.483; both P<0.05). Conclusions:The CC16 and IL-12 levels may have a specific reference value for the early diagnosis of silicosis and the assessment of lung function.
Gracilaria lemaneiformis, an edible alga, with various bioactivities is a traditional dish and a favorite food. In the present study, its n-hexane extract showed strong protein tyrosine phosphatase 1B inhibitory activity after screening. To understand the activity composition, nineteen compounds were identified from this extract by GC-MS. The protein tyrosine phosphatase 1B inhibitory activity of the identified compounds was further screened by means of molecular docking individually. As a result, 2,2'-methylenebis-6-(1,1-dimethylethyl)-4-methyl-phenol with the lowest binding energy of -6.70 kcal mol was discovered from the complex extract, and its inhibitory activity against protein tyrosine phosphatase 1B was proved in vitro. The IC value was 53.27 ± 0.54 μM. Therefore, this compound and its source Gracilaria lemaneiformis were suggested to be utilized as a functional food with potential protein tyrosine phosphatase 1B inhibitory activity.
The calibration strategy for synthetic aperture interferometric radiometers based on noise injection has been successfully demonstrated in the Microwave Imaging Radiometer with Aperture Synthesis (MIRAS) instrument onboard the Soil Moisture and Ocean Salinity (SMOS) mission of the European Space Agency (ESA). With the emerging demands of increasing both array size and frequency bands, the internal calibration hardware becomes more difficult to implement. Although several external calibration methods have been proposed, the so-called phase ambiguity problem was not carefully considered. In this paper, a novel calibration strategy for both phase and amplitude calibration based on redundant space calibration is proposed, which utilizes the unique structure of equispaced circular array and rotational sampling strategy. The original irregular phase ambiguity degenerates to the fixed π-ambiguity, where only two possible solutions to phases are introduced. Both phase and amplitude of visibility can be simultaneously calibrated by the proposed strategy, and no cooperative calibration target is needed.
The RNA-sensing signaling pathway has been well studied as an essential antiviral mechanism of innate immunity. However, its role in non-infected cells is yet to be thoroughly characterized. Here, we demonstrated that the RNA sensing signaling pathway also reacts to the endogenous cellular RNAs in endothelial cells (ECs), and this reaction is regulated by the RNA-editing enzyme ADAR1. Cellular RNA sequencing analysis showed that EC RNAs endure extensive RNA editing, especially in the RNA transcripts of short interspersed nuclear elements. The EC-specific deletion of ADAR1 dramatically reduced the editing level on short interspersed nuclear element RNAs, resulting in newborn death in mice with damage evident in multiple organs. Genome-wide gene expression analysis revealed a prominent innate immune activation with a dramatically elevated expression of interferon-stimulated genes. However, blocking the RNA sensing signaling pathway by deletion of the cellular RNA receptor MDA-5 prevented interferon-stimulated gene expression and rescued the newborn mice from death. This evidence demonstrated that the RNA-editing/RNA-sensing signaling pathway dramatically modulates EC function, representing a novel molecular mechanism for the regulation of EC functions.
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