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This study aimed to identify characteristic proteins in infantile epileptic spasm syndrome (IESS) patients' plasma, offering insights into potential early diagnostic biomarkers and its underlying causes. Plasma samples were gathered from 60 patients with IESS and 40 healthy controls. Data-independent acquisition proteomic analysis was utilized to identify differentially expressed proteins (DEPs). These DEPs underwent functional annotation through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Gene set enrichment analysis (GSEA) was employed for both GO (GSEA-GO) and KEGG (GSEA-KEGG) analyses to examine the gene expression profiles. Receiver operating characteristic (ROC) curves assessed biomarkers' discriminatory capacity. A total of 124 DEPs were identified in IESS patients' plasma, mainly linked to pathways, encompassing chemokines, cytokines, and oxidative detoxification. GSEA-GO and GSEA-KEGG analyses indicated significant enrichment of genes associated with cell migration, focal adhesion, and phagosome pathways. ROC curve analysis demonstrated that the combination of PRSS1 and ACTB, PRSS3, ACTB, and PRSS1 alone exhibited AUC values exceeding 0.7. This study elucidated the significant contribution of cytokines, chemokines, oxidative detoxification, and phagosomes to the IESS pathogenesis. The combination of PRSS1 and ACTB holds promise as biomarkers for the early diagnosis of IESS.
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Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%-15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling.
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Esclerose Tuberosa , Humanos , Esclerose Tuberosa/diagnóstico , Esclerose Tuberosa/genética , Esclerose Tuberosa/patologia , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Mosaicismo , MutaçãoRESUMO
Electroencephalogram (EEG) has been an important tool for scientists to study epilepsy and evaluate the treatment of epilepsy for half a century, since epilepsy seizures are caused by the diffusion of excessive discharge of brain neurons. This paper reviews the clinical application of scalp EEG in the treatment of intractable epilepsy with vagus nerve stimulation (VNS) in the past 30 years. It mainly introduces the prediction of the therapeutic effect of VNS on intractable epilepsy based on EEG characteristics and the effect of VNS on EEG of patients with intractable epilepsy, and expounds some therapeutic mechanisms of VNS. For predicting the efficacy of VNS based on EEG characteristics, EEG characteristics such as epileptiform discharge, polarity of slow cortical potential changes, changes of EEG symmetry level and changes of EEG power spectrum are described. In view of the influence of VNS treatment on patients' EEG characteristics, the change of epileptiform discharge, power spectrum, synchrony, brain network and amplitude of event-related potential P300 are described. Although no representative EEG markers have been identified for clinical promotion, this review paves the way for prospective studies of larger patient populations in the future to better apply EEG to the clinical treatment of VNS, and provides ideas for predicting VNS efficacy, assessing VNS efficacy, and understanding VNS treatment mechanisms, with broad medical and scientific implications.
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Epilepsia Resistente a Medicamentos , Estimulação do Nervo Vago , Eletroencefalografia , Humanos , Estudos Prospectivos , Couro Cabeludo , Resultado do TratamentoRESUMO
During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.
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Antígenos Virais/genética , DNA Helicases/genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/metabolismo , Proteínas Imediatamente Precoces/genética , Glicoproteínas de Membrana/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Proteínas Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Replicação do DNA/genética , DNA Viral/genética , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Herpesvirus Humano 4/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.
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Metilação de DNA , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Ativação Viral , Fator de Ligação a CCCTC , Regulação para Baixo , Interações Hospedeiro-Patógeno , Transcrição GênicaRESUMO
UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.
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Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação Viral , Células HeLa , HumanosRESUMO
UNLABELLED: Epstein-Barr virus (EBV) lytic replication involves complex processes, including DNA synthesis, DNA cleavage and packaging, and virion egress. These processes require many different lytic gene products, but the mechanisms of their actions remain unclear, especially for DNA cleavage and packaging. According to sequence homology analysis, EBV BALF3, encoded by the third leftward open reading frame of the BamHI-A fragment in the viral genome, is a homologue of herpes simplex virus type 1 UL28. This gene product is believed to possess the properties of a terminase, such as nucleolytic activity on newly synthesized viral DNA and translocation of unit length viral genomes into procapsids. In order to characterize EBV BALF3, the protein was produced by and purified from recombinant baculoviruses and examined in an enzymatic reaction in vitro, which determined that EBV BALF3 acts as an endonuclease and its activity is modulated by Mg(2+), Mn(2+), and ATP. Moreover, in EBV-positive epithelial cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following induction of the lytic cycle, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Interestingly, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the basis of these results, we suggest that EBV BALF3 is involved simultaneously in DNA synthesis and packaging and is required for the production of mature virions. IMPORTANCE: Virus lytic replication is essential to produce infectious virions, which is responsible for virus survival and spread. This work shows that an uncharacterized gene product of the human herpesvirus Epstein-Barr virus (EBV), BALF3, is expressed during the lytic cycle. In addition, BALF3 mediates an endonucleolytic reaction and is involved in viral DNA synthesis and packaging, leading to influence on the production of mature virions. According to sequence homology and physical properties, the lytic gene product BALF3 is considered a terminase in EBV. These findings identify a novel viral gene with an important role in contributing to a better understanding of the EBV life cycle.
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Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação Viral , Cátions Bivalentes/metabolismo , Ativadores de Enzimas/metabolismo , Magnésio/metabolismo , Manganês/metabolismoRESUMO
Phosphatase and tensin homolog (PTEN) is vital for B cell development, acting as a key negative regulator in the PI3K signaling pathway. We used CD23-cre to generate PTEN-conditional knockout mice (CD23-cKO) to examine the impact of PTEN mutation on peripheral B cells. Unlike mb1-cre-mediated PTEN deletion in early B cells, CD23-cKO mutants exhibited systemic inflammation with increased IL-6 production in mature B cells upon CpG stimulation. Inflammatory B cells in CD23-cKO mice showed elevated phosphatidylinositol 3-phosphate [PI(3)P] levels and increased TLR9 endosomal localization. Pharmacological inhibition of PI(3)P synthesis markedly reduced TLR9-mediated IL-6. Single-cell RNA-sequencing (RNA-seq) revealed altered endocytosis, BANK1, and NF-κB1 expression in PTEN-deficient B cells. Ectopic B cell receptor (BCR) expression on non-inflammatory mb1-cKO B cells restored BANK1 and NF-κB1 expression, enhancing TLR9-mediated IL-6 production. Our study highlights PTEN as a crucial inflammatory checkpoint, regulating TLR9/IL-6 axis by fine-tuning PI(3)P homeostasis. Additionally, BCR downregulation prevents the differentiation of inflammatory B cells in PTEN deficiency.
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Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcinogenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotoxicity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation.
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Herpesvirus Humano 4/efeitos dos fármacos , Isotiocianatos/farmacologia , Neoplasias Nasofaríngeas/virologia , Antivirais/farmacologia , Carcinoma , Suplementos Nutricionais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/prevenção & controle , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Precoces , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 4/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Regiões Promotoras Genéticas , Sulfóxidos , Transativadores/genética , Transativadores/metabolismo , Células Tumorais Cultivadas , Ativação Viral/efeitos dos fármacosRESUMO
Identifying and treating tumors early is the key to secondary prevention in cancer control. At present, prevention of oral cancer is still challenging because the molecular drivers responsible for malignant transformation of the 11 clinically defined oral potentially malignant disorders are still unknown. In this review, we focused on studies that elucidate the epigenetic alterations demarcating malignant and nonmalignant epigenomes and prioritized findings from clinical samples. Head and neck included, the genomes of many cancer types are largely hypomethylated and accompanied by focal hypermethylation on certain specific regions. We revisited prior studies that demonstrated that sufficient uptake of folate, the primary dietary methyl donor, is associated with oral cancer reduction. As epigenetically driven phenotypic plasticity, a newly recognized hallmark of cancer, has been linked to tumor initiation, cell fate determination, and drug resistance, we discussed prior findings that might be associated with this hallmark, including gene clusters (11q13.3, 19q13.43, 20q11.2, 22q11-13) with great potential for oral cancer biomarkers, and successful examples in screening early-stage nasopharyngeal carcinoma. Although one-size-fits-all approaches have been shown to be ineffective in most cancer therapies, the rapid development of epigenome sequencing methods raises the possibility that this nonmutagenic approach may be an exception. Only time will tell.
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OBJECTIVES: To identify the characteristics of influenza-associated neurologic complications (INCs) in children from a recent H3N2 outbreak in Shenzhen, China during COVID-19 lockdown. METHODS: A retrospective cohort study of INCs in children hospitalized with H3N2 infection was conducted. RESULTS: From June 01, 2022 to July 01, 2022, 513 children with H3N2 infection were hospitalized and 97 developed INCs. Of the 18 patients with encephalopathy/encephalitis, 13 were previously healthy. Three developed acute necrotizing encephalopathy and two died. Of the 63 patients with febrile seizures, 55 (87%) had simple febrile seizures. Of the 14 patients with an exacerbation of seizure with underlying epilepsy, the seizure symptoms occurred mostly within 24 hours of disease onset (13/14). The comparison of the three groups (encephalopathy/encephalitis, febrile seizure and exacerbation of seizure with underlying epilepsy) reported no significant differences in sex, pre-existing neurologic diseases, vaccination rate, white blood cell count, C-reactive protein, procalcitonin, blood glucose, lactic acid, or duration of fever. The influenza vaccination rates were generally low (22% vs 32% vs 21%). Patients with encephalopathy/encephalitis had a higher rate of elevated alanine aminotransferase (28% vs 3% vs 0, P = 0.005). CONCLUSION: H3N2-related neurologic complications in children mainly occur early in the disease course. Most patients were previously healthy and unvaccinated against influenza. Elevated alanine aminotransferase is more common in encephalopathy/encephalitis.
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Encefalopatias , COVID-19 , Encefalite , Influenza Humana , Doenças do Sistema Nervoso , Convulsões Febris , Criança , Humanos , Influenza Humana/complicações , Influenza Humana/epidemiologia , Influenza Humana/diagnóstico , Convulsões Febris/etiologia , Convulsões Febris/complicações , Vírus da Influenza A Subtipo H3N2 , Estudos Retrospectivos , Alanina Transaminase , COVID-19/complicações , COVID-19/epidemiologia , Controle de Doenças Transmissíveis , Doenças do Sistema Nervoso/epidemiologia , Doenças do Sistema Nervoso/etiologia , Encefalopatias/epidemiologia , Encefalopatias/etiologia , China/epidemiologiaRESUMO
NUS1 is responsible for encoding of the Nogo-B receptor (NgBR), which is a subunit of cis-prenyltransferase. Over 25 variants in NUS1 have been reported, and these variants have been found to be associated with various phenotypes, such as congenital disorders of glycosylation (CDG) and developmental and epileptic encephalopathy (DEE). We report on the case of a patient who presented with language and motor retardation, epilepsy, and electroencephalogram abnormalities. Upon conducting whole-exome sequencing, we discovered a novel pathogenic variant (chr6:118024873, NM_138459.5: c.791 + 6T>G) in NUS1, which was shown to cause Exon 4 to be skipped, resulting in a loss of 56 amino acids. Our findings strongly suggest that this novel variant of NUS1 is responsible for the development of neurological disorders, including epilepsy. It is believed that the truncation of Nogo-B receptor results in the loss of cis-prenyltransferase activity, which may be the underlying cause of the disease.
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Introduction: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a common autoimmune encephalitis, and it is associated with psychosis, dyskinesia, and seizures. Anti-NMDAR encephalitis (NMDARE) in juveniles and adults presents different clinical charactreistics. However, the pathogenesis of juvenile anti-NMDAR encephalitis remains unclear, partly because of a lack of suitable animal models. Methods: We developed a model of juvenile anti-NMDAR encephalitis using active immunization with an amino terminal domain peptide from the GluN1 subunit (GluN1356 - 385) against NMDARs in 3-week-old female C57BL/6J mice. Results: Immunofluorescence staining suggested that autoantibody levels in the hippocampus increased, and HEK-293T cells staining identified the target of the autoantibodies as GluN1, suggesting that GluN1-specific immunoglobulin G was successfully induced. Behavior assessment showed that the mice suffered significant cognition impairment and sociability reduction, which is similar to what is observed in patients affected by anti-NMDAR encephalitis. The mice also exhibited impaired long-term potentiation in hippocampal CA1. Pilocarpine-induced epilepsy was more severe and had a longer duration, while no spontaneous seizures were observed. Conclusion: The juvenile mouse model for anti-NMDAR encephalitis is of great importance to investigate the pathological mechanism and therapeutic strategies for the disease, and could accelerate the study of autoimmune encephalitis.
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Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2'-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation.
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Proteínas 14-3-3/genética , Pontos de Checagem do Ciclo Celular , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Transativadores/metabolismo , Proteínas 14-3-3/metabolismo , Linhagem Celular , Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/fisiopatologia , Infecções por Vírus Epstein-Barr/virologia , Fase G1 , Herpesvirus Humano 4/genética , Humanos , Proteínas Imediatamente Precoces/genética , Fase S , Transativadores/genética , Ativação TranscricionalRESUMO
The switch between the latency and lytic cycles of Kaposi's sarcoma-associated herpesvirus (KSHV) is accompanied by specific alterations of histone codes. Recently, comprehensive analysis of histone modifications of KSHV showed the deposition of H3K27me3 across the KSHV genome with two specific regions occupied by the heterochromatin marker H3K9me3. Here, we show that knockdown of JMJD2A, an H3K9me3 demethylase, attenuates viral titers, whereas its overexpression increases KSHV reactivation. JMJD2A is localized in regions of latent viral chromosomes that are deficient in the H3K9me3 mark, indicating that JMJD2A may be responsible for the low level of this mark on viral chromatin. The presence of JMJD2A on the latent genome maintains H3K9 in unmethylated form and signals the readiness of specific sets of viral genes to be reactivated. The demethylase activity of JMJD2A is important for KSHV reactivation, because a demethylase-deficient mutant cannot restore the JMJD2A knockdown phenotype. Interestingly, we found that the KSHV encoded K-bZIP associated with JMJD2A, resulting in the inhibition of demethylase activity of JMJD2A both in vivo and in vitro. Inhibition of JMJD2A by K-bZIP is likely due to a physical interaction which blocks substrate accessibility. A consequence of such an inhibition is increasing global levels of H3K9me3 and gene silencing. Consistently, K-bZIP overexpression resulted in a repression of â¼80% of the ≥2-fold differentially regulated genes compared to results for the uninduced control cells. The consequences of K-bZIP targeting JMJD2A during viral replication will be discussed. To our knowledge, this is the first description of a viral product shown to be a potent inhibitor of a host cellular histone demethylase.
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Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Latência Viral , Replicação Viral , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Carga ViralRESUMO
BACKGROUND: The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a molecular switch that initiates a productive replication of latent KSHV genomes. KSHV RTA (K-RTA) is composed of 691 amino acids with high Ser and Thr content (17.7%), but to what extent these Ser and Thr are modified in vivo has not been explored. METHODS: By using tandem mass spectrometric analysis of affinity-purified FLAG tagged K-RTA, we sought to identify Ser and Thr residues that are post-translationally modified in K-RTA. RESULTS: We found that K-RTA is an O-GlcNAcylated protein and Thr-366/Thr-367 is the primary motif with O-GlcNAcylation in vivo. The biological significance of O-GlcNAc modified Thr-366 and Thr-367 was assessed by site-specific amino acid substitution. Replacement of Thr with Ala at amino acid 366 or 367 caused a modest enhancement of K-RTA transactivation activity in a luciferase reporter assay and a cell model for KSHV reactivation. By using co-immunoprecipitation coupled with western blot analysis, we showed that the capacity of K-RTA in associating with endogenous PARP1 was significantly reduced in the Thr-366/Thr-367 O-GlcNAc mutants. PARP1 is a documented negative regulator of K-RTA that can be ascribed by the attachment of large negatively charged polymer onto K-RTA via PARP1's poly (ADP-ribose) polymerase activity. In agreement, shRNA-mediated depletion of O-GlcNAc transferase (OGT) in KSHV infected cells augmented viral reactivation and virus production that was accompanied by diminished K-RTA and PARP1 complexes. CONCLUSIONS: KSHV latent-lytic switch K-RTA is modified by cellular O-GlcNAcylation, which imposes a negative effect on K-RTA transactivation activity. This inhibitory effect involves OGT and PARP1, two nutritional sensors recently emerging as chromatin modifiers. Thus, we speculate that the activity of K-RTA on its target genes is continuously checked and modulated by OGT and PARP1 in response to cellular metabolic state.
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Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/genética , N-Acetilglucosaminiltransferases/genética , Transativadores/genética , Acilação , Alanina/química , Substituição de Aminoácidos , Western Blotting , Cromatografia Gasosa-Espectrometria de Massas , Células HEK293 , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Imunoprecipitação , N-Acetilglucosaminiltransferases/metabolismo , Oligopeptídeos , Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Espectrometria de Massas em Tandem , Treonina/química , Transativadores/metabolismoRESUMO
BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) antibodies mediate inflammatory demyelinating diseases of the central nervous system. This study aimed to understand the clinical characteristics of MOG antibody-associated aseptic meningitis (MOGAM). METHODS: Here, we report the cases of two children with MOGAM. A systematic literature review was conducted and included patients who had MOGAM only, without neurological parenchymal lesions. The clinical characteristics that may have affected the outcome were statistically analyzed. RESULTS: We reviewed 12 cases of MOGAM; male: female = 9: 3. Prolonged fever lasting over 7 days (11/12) was the most frequent symptom, followed by headache (10/12), vomiting (5/12), and seizures (4/12). None of the patients had focal neurological manifestations or parenchymal lesions on imaging. Cerebrospinal fluid (CSF) leukocytosis was observed in all patients (12/12), and blood leukocytosis and elevated CSF pressure was observed in all patients who had corresponding results (9/9 and 4/4, respectively). Seizures occurrence was lower than that of MOG antibody-associated cortical encephalitis. Seven cases progressed to other MOG antibody-associated diseases (MOGADs) in the later phase of MOGAM. Patients who did not progress to other MOGADs had a shorter disease duration from onset to the initiation of intravenous methylprednisolone than those who did. All the patients achieved full recovery after steroid treatment. One patient had relapses. CONCLUSIONS: MOGAM without inflammatory demyelination is a rare but distinct phenotype of MOGAD, with fewer clinical manifestations mimicking bacterial or viral meningitis/encephalomeningitis. Delayed diagnosis and treatment may induce the progression to other severe MOGADs. Early recognition of this unique autoimmune aseptic meningitis may contribute to early diagnosis, treatment, and better outcomes.
Assuntos
Encefalite , Meningite Asséptica , Feminino , Humanos , Masculino , Autoanticorpos , Glicoproteína Mielina-Oligodendrócito , Convulsões , CriançaRESUMO
We evaluated the efficacy and safety of vigabatrin in focal epilepsy associated with tuberous sclerosis complex by retrospectively reviewing patients with focal epilepsy and tuberous sclerosis complex treated with vigabatrin at a pediatric epilepsy center over an 8-year period. Of 85 patients, 20 (23.5%) were seizure-free for >12 months, 45 (52.9%) were responders (≥50% seizure reduction), and 20 (23.5%) were nonresponders. The median age (in months) at seizure onset in the seizure-free group (median, 15; interquartile range [IQR], 6-23.3) was higher than that of responders (median, 5; IQR, 3-14) and nonresponders (median, 6; IQR, 2-12). Fewer patients in the seizure-free group had calcification in their largest tubers, but the presence of tuber calcification did not differ among groups. Vigabatrin is more likely to result in seizure freedom in children with tuberous sclerosis complex who have later infantile onset of focal seizures and no calcification in their largest tuber.
Assuntos
Epilepsias Parciais , Esclerose Tuberosa , Anticonvulsivantes/uso terapêutico , Criança , Epilepsias Parciais/complicações , Epilepsias Parciais/tratamento farmacológico , Humanos , Lactente , Estudos Retrospectivos , Convulsões/complicações , Convulsões/etiologia , Resultado do Tratamento , Esclerose Tuberosa/complicações , Esclerose Tuberosa/tratamento farmacológico , Vigabatrina/uso terapêuticoRESUMO
PURPOSE: To evaluate the retention rate, efficacy, and safety of ketogenic diet therapy for drug-resistant epilepsy in children and compare the results with those of a previous cohort at our institution. METHODS: A total of 634 children with drug-resistant epilepsy were included in this retrospective study. Patients were categorized into two groups. The previous cohort was included as a control group and included 317 children assessed between 2004 and 2011, whereas the current group included 317 children assessed between 2015 and 2019. The control group was provided care as usual, and the current group additionally adopted the goal and long-term management strategy. Outcomes were measured with respect to retention rate, seizure reduction, and adverse reaction. RESULTS: Patient demographics were consistent between both cohorts. Compared to the past ten years, the retention rate significantly increased over time (3 months: 62.8% vs. 82.0%, p <0.001; 6 months: 42.0% vs. 60.6%, p <0.001; 12 months: 24.3% vs. 34.1%, p = 0.007), and the response rate was significantly improved (3 months: 35.0% vs. 55.5%, p <0.001; 6 months: 26.2% vs. 43.2%, p <0.001; 12 months: 18.6% vs. 31.5%, p <0.001). Constipation (n = 79, 24.9%) was the most common side effect in the current cohort. Food refusal and hypoproteinaemia reduced to 3.5% and 0.9%, respectively. CONCLUSION: Goal and long-term management is effective for ketogenic diet therapy, which significantly improved the ketogenic diet retention rate, efficacy, and incidence of adverse reactions. This strategy has promising applicability in ketogenic diet therapy. CLINICAL REGISTRATION: ChiCTR-IIR-16,008,342.
Assuntos
Dieta Cetogênica , Epilepsia Resistente a Medicamentos , Preparações Farmacêuticas , Criança , Humanos , Estudos Retrospectivos , Convulsões , Resultado do TratamentoRESUMO
OBJECTIVE: It has been suggested that asymmetric hypsarrhythmia is associated with structural etiology. We devised the Hypsarrhythmic Asymmetric Scoring Scheme (HASS) to quantify the degree of hypsarrhythmic asymmetry in a retrospective series of patients who underwent surgical treatment at our center. The present study aimed to investigate the role of HASS in predicting the postsurgical seizure outcomes. METHODS: We retrospectively analyzed the records of 46 children with hypsarrhythmia who underwent resective epilepsy surgery between 2018 and 2020 and were followed up for at least 1 year after surgery. Hypsarrhythmia severity in each hemisphere was quantified and scored. The HASS score was calculated as the difference between the two hemispheres. Univariate results were submitted to logistic regression models to identify independent predictors for favorable surgical outcomes. RESULTS: Of the 46 patients who underwent resective surgery, Engel's class I-â ¡ outcomes were achieved in 34 (73.9%). The Engel I-â ¡ group had a significantly higher HASS score than the Engel â ¢-â £ group (p<0.001). Multivariate analysis showed that the HASS score was the only significant predictor of good outcomes (p = 0.011). Further receiver operating characteristic analysis showed that a threshold of 7 yielded a better seizure outcome with a sensitivity of 97.06% and specificity of 83.33%. SIGNIFICANCE: As the first hypsarrhythmia scoring system specially designed for presurgical evaluation, the HASS score may contribute to predicting the postsurgical seizure outcome from the electroencephalography perspective.