RESUMO
The CACNA1A gene, encoding the voltage-gated calcium channel subunit α1A, is involved in pre- and postsynaptic Ca(2+) signaling, gene expression, and several genetic neurological disorders. We found that CACNA1A coordinates gene expression using a bicistronic mRNA bearing a cryptic internal ribosomal entry site (IRES). The first cistron encodes the well-characterized α1A subunit. The second expresses a transcription factor, α1ACT, which coordinates expression of a program of genes involved in neural and Purkinje cell development. α1ACT also contains the polyglutamine (polyQ) tract that, when expanded, causes spinocerebellar ataxia type 6 (SCA6). When expressed as an independent polypeptide, α1ACT-bearing an expanded polyQ tract-lacks transcription factor function and neurite outgrowth properties, causes cell death in culture, and leads to ataxia and cerebellar atrophy in transgenic mice. Suppression of CACNA1A IRES function in SCA6 may be a potential therapeutic strategy.
Assuntos
Canais de Cálcio/genética , Ataxias Espinocerebelares/genética , Fatores de Transcrição/genética , Animais , Canais de Cálcio/metabolismo , Morte Celular , Linhagem Celular Tumoral , Cerebelo/embriologia , Cerebelo/fisiopatologia , Regulação da Expressão Gênica , Humanos , Camundongos , Neuritos/metabolismo , Peptídeos/genética , Células de Purkinje/metabolismo , Ratos , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/fisiopatologia , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
BACKGROUND AND OBJECTIVES: Comprehensive analysis of multi-omics data is crucial for accurately formulating effective treatment plans for complex diseases. Supervised ensemble methods have gained popularity in recent years for multi-omics data analysis. However, existing research based on supervised learning algorithms often fails to fully harness the information from unlabeled nodes and overlooks the latent features within and among different omics, as well as the various associations among features. Here, we present a novel multi-omics integrative method MOSEGCN, based on the Transformer multi-head self-attention mechanism and Graph Convolutional Networks(GCN), with the aim of enhancing the accuracy of complex disease classification. MOSEGCN first employs the Transformer multi-head self-attention mechanism and Similarity Network Fusion (SNF) to separately learn the inherent correlations of latent features within and among different omics, constructing a comprehensive view of diseases. Subsequently, it feeds the learned crucial information into a self-ensembling Graph Convolutional Network (SEGCN) built upon semi-supervised learning methods for training and testing, facilitating a better analysis and utilization of information from multi-omics data to achieve precise classification of disease subtypes. RESULTS: The experimental results show that MOSEGCN outperforms several state-of-the-art multi-omics integrative analysis approaches on three types of omics data: mRNA expression data, microRNA expression data, and DNA methylation data, with accuracy rates of 83.0% for Alzheimer's disease and 86.7% for breast cancer subtyping. Furthermore, MOSEGCN exhibits strong generalizability on the GBM dataset, enabling the identification of important biomarkers for related diseases. CONCLUSION: MOSEGCN explores the significant relationship information among different omics and within each omics' latent features, effectively leveraging labeled and unlabeled information to further enhance the accuracy of complex disease classification. It also provides a promising approach for identifying reliable biomarkers, paving the way for personalized medicine.
Assuntos
Doença de Alzheimer , Multiômica , Humanos , Metilação de DNA , Algoritmos , BiomarcadoresRESUMO
Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Bactérias/genética , Ilhas Genômicas , Transferência Genética Horizontal , Plasmídeos/genética , Sequências Repetitivas DispersasRESUMO
The plant hormone ethylene plays a critical role in fruit defense against Botrytis cinerea attack, but the underlying mechanisms remain poorly understood. Here, we showed that ethylene response factor SlERF.C1 acts as a key regulator to trigger the ethylene-mediated defense against B. cinerea in tomato fruits without compromising ripening. Knockout of SlERF.C1 increased fruit susceptibility to B. cinerea with no effect on ripening process, while overexpression enhanced resistance. RNA-Seq, transactivation assays, EMSA and ChIP-qPCR results indicated that SlERF.C1 activated the transcription of PR genes by binding to their promoters. Moreover, SlERF.C1 interacted with the mitogen-activated protein kinase SlMPK8 which allowed SlMPK8 to phosphorylate SlERF.C1 at the Ser174 residue and increases its transcriptional activity. Knocking out of SlMPK8 increased fruit susceptibility to B. cinerea, whereas overexpression enhanced resistance without affecting ripening. Furthermore, genetic crosses between SlMPK8-KO and SlERF.C1-OE lines reduced the resistance to B. cinerea attack in SlERF.C1-OE fruits. In addition, B. cinerea infection induced ethylene production which in turn triggered SlMPK8 transcription and enhanced the phosphorylation of SlERF.C1. Overall, our findings reveal the regulatory mechanism of the 'Ethylene-MPK8-ERF.C1-PR' module in resistance against B. cinerea and provide new insight into the manipulation of gray mold disease in fruits.
Assuntos
Frutas , Solanum lycopersicum , Frutas/metabolismo , Solanum lycopersicum/genética , Etilenos/metabolismo , Botrytis/fisiologia , Doenças das Plantas/genética , Resistência à Doença/genética , Regulação da Expressão Gênica de PlantasRESUMO
Kiwifruit ripening is a complex and highly coordinated process that occurs in conjunction with the formation of fruit edible quality. The significance of epigenetic changes, particularly the impact of N6-methyladenosine (m6A) RNA modification on fruit ripening and quality formation, has been largely overlooked. We monitored m6A levels and gene expression changes in kiwifruit at four different stages using LC-MS/MS, MeRIP, RNA-seq, and validated the function of AcALKBH10 through heterologous transgenic expression in tomato. Notable m6A modifications occurred predominantly at the stop codons and the 3' UTRs and exhibited a gradual reduction in m6A levels during the fruit ripening process. Moreover, these m6A modifications in the aforementioned sites demonstrated a discernible inverse relationship with the levels of mRNA abundance throughout the ripening process, suggesting a repression effect of m6A modification in the modulation of kiwifruit ripening. We further demonstrated that AcALKBH10 rather than AcECT9 predominantly regulates m6A levels in ripening-related genes, thereby exerting the regulatory control over the ripening process and the accumulation of soluble sugars and organic acids, ultimately influencing fruit ripening and quality formation. In conclusion, our findings illuminate the epi-regulatory mechanism involving m6A in kiwifruit ripening, offering a fresh perspective for cultivating high-quality kiwifruit with enhanced nutritional attributes.
Assuntos
Actinidia , Adenosina , Frutas , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , RNA Mensageiro , Actinidia/genética , Actinidia/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Genes de PlantasRESUMO
MicroRNAs, a class of small RNA regulators, function throughout neurodevelopment, from neural stem cell neurogenesis to neuronal maturation, synaptic formation, and plasticity. α1ACT, a transcription factor (TF), plays a critical role in neonatal cerebellar development by regulating an ensemble of genes.â¯Of these, ChIP-seq analysis matched near 50% genes directly regulated by α1ACT. Yet, more than half the regulated transcripts lacked direct interaction with α1ACT. To investigate whether α1ACT acts through a microRNA network, we studied α1ACT-associated simultaneous miRNA:mRNA transcriptome profiles, usingâ¯miRNA-seq paired with RNA-seq. Thirty-one differentially expressed miRNAs (DEMs) associated withâ¯α1ACT-regulated differentially expressed genes (DEGs) were profiled in α1ACT-overexpressing PC12 cells and were further validated in neonatal transgenic mouse cerebellum overexpressing α1ACT in a context-dependent manner. Here, we also demonstrated that α1ACT facilitates neurogenesis and development of dendritic synapses and is partially a result of the downregulation of the miR-99 cluster, miR-143, miR-23, miR-146, miR-363, and miR-484. On the other hand, the miR-181, miR-125, and miR-708 clusters were upregulated by α1ACT, which inhibit MAPK signaling and cell death pathways by targeting Ask1, Odc1, Atf4, and Nuf2 for decreased expression. MiR-181a-5p was verified as the most abundant DEM in neonatal cerebellum, which was further induced by α1ACT. Overall, under α1ACT modulation, up-/downregulated miRNA clusters with their paired target genes may form a regulatory network controlling the balance between the neuronal proliferation, differentiation, and cell death in the cerebellum to promote neonatal development. Our findings concerning the α1ACT-related miRNA/mRNA expression profiles in neonatal cerebellum may inform future investigations for cerebellar development.
Assuntos
MicroRNAs , Camundongos , Ratos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/genética , Cerebelo/metabolismo , Neurogênese , Camundongos Transgênicos , RNA Mensageiro , Perfilação da Expressão GênicaRESUMO
Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1, encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BKG354S) in KCNMA1, in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BKG354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BKG354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BKG354S channel, but not the BKWT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.
Assuntos
Cerebelo/patologia , Clorzoxazona/administração & dosagem , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Mitocôndrias/patologia , Degenerações Espinocerebelares/genética , Adolescente , Animais , Animais Recém-Nascidos , Linhagem Celular , Cerebelo/citologia , Análise Mutacional de DNA , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mutação com Perda de Função , Camundongos , Oócitos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/tratamento farmacológico , Degenerações Espinocerebelares/patologia , Transfecção , Sequenciamento do Exoma , XenopusRESUMO
BACKGROUND: In China, adolescents account for about a quarter of those treated for mental disorders each year, and adolescent mental health issues have become a social hotspot. Although several epidemiological surveys of mental disorders have been conducted in China, no study has yet focused on the prevalence of mental disorders among adolescents in a certain region of Zhejiang. METHODS: In the first stage, 8219 middle school students aged 12-18 years in a city of Zhejiang Province (Shaoxing) were screened with the mental health screening checklist. In the second stage, participants who screened positive were tested with the Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID) and the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). Then, the prevalence of mental disorders were calculated. RESULTS: The overall prevalence in this population was 12.4%, with prevalence rates exceeding 20% in both the 17- and 18-year-old age groups. The most common mental disorders were obsessive-compulsive disorder (OCD) (9.1%) and major depressive disorder (MDD) (8.9%). CONCLUSIONS: Mental disorders are common among middle school students, and girls are at higher risk than boys. As the most prevalent mental disorders, OCD and MDD should receive timely attention, especially for upper grade students.
RESUMO
BACKGROUND AND AIM: Pyrrolizidine alkaloids (PA) induced hepatic sinusoidal obstruction syndrome (HSOS) occurred worldwide and the mortality rate remained high because there were no specific therapies. Defibrotide was effective for HSOS following hematopoietic stem cell transplantation. But the pathogenesis of the two types of HSOS were not equivalent. The purpose of this study was to see if defibrotide was also effective in PA induced rat HSOS. METHODS: First we improved rat HSOS model by using higher dose (230 mg/kg) of monocrotaline (a kind of PA) as the dose of median lethal dose. So drug effectiveness could be assessed by survival time. Next, male SD rats were divided into 5 groups. They were control group, model group, low dose low molecular weight heparin (LMWH) treatment group, high dose LMWH treatment group and defibrotide treatment group. Rats' survival time, liver function, white blood cell count and cytokines were compared among the groups. The DeLeve score was used to assess the severity of liver pathology. RESULTS: The model group exhibited typical liver pathology of HSOS, such as hepatic sinus dilation, congestion, endothelial injury of central lobular vein, coagulative necrosis of hepatocytes and fibrin deposition in the subendothelial. The pathologic characteristics indicated that the model was built up successfully. The survival rate was significantly higher in defibrotide group (81.8%) than model group (43.7%), while the survival rates were similar in the two LMWH groups (62.5% and 75%) and model group. The survival time only be prolonged by defibrotide (P=0.028) but not LMWH (P>0.05). DeLeve score was improved most in the defibrotide group than the two LMWH groups (both P<0.01). Changes in DeLeve score, liver function, plasma level of tumor necrosis factor α and plasminogen activator inhibitor-1 exhibited the same trends. CONCLUSION: Defibrotide could improve the outcome of monocrotaline-induced rat HSOS indicating that defibrotide might be a better choice than LMWH in clinical practice.
Assuntos
Hepatopatia Veno-Oclusiva , Alcaloides de Pirrolizidina , Masculino , Ratos , Animais , Hepatopatia Veno-Oclusiva/induzido quimicamente , Hepatopatia Veno-Oclusiva/tratamento farmacológico , Hepatopatia Veno-Oclusiva/patologia , Monocrotalina/efeitos adversos , Ratos Sprague-Dawley , Alcaloides de Pirrolizidina/efeitos adversos , Anticoagulantes/uso terapêuticoRESUMO
The HER2/neu signaling pathway is one of the most frequently mutated in human cancer. Although therapeutics targeting this pathway have good efficacy, cancer cells frequently develop resistance. The HER2 gene encodes the full-length HER2 protein, as well as smaller c-terminal fragments (CTFs), which have been shown to be a cause of resistance. Here, we show that HER2 CTFs, exclusive from the full-length HER2 protein, are generated via internal translation of the full-length HER2 mRNA and identify regions which are required for this mechanism to occur. These regions of the HER2 mRNA may present novel sites for therapeutic intervention via small molecules or antisense oligonucleotides (ASOs).
Assuntos
Neoplasias , Receptor ErbB-2 , Humanos , Oligonucleotídeos Antissenso/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
We have discovered that the P/Q-type voltage-gated Ca2+ channel (VGCC) gene, CACNA1A, encodes both the α1A (Cav2.1) subunit and a newly recognized transcription factor, α1ACT, by means of a novel internal ribosomal entry site (IRES) within the α1A C-terminal coding region. α1ACT, when mutated with an expansion of the polyglutamine tract in the C-terminus, gives rise to spinocerebellar ataxia type 6 (SCA6). Because silencing of the entire CACNA1A gene would result in the loss of the essential Cav2.1 channel, the IRES controlling α1ACT expression is an excellent target for selective silencing of α1ACT as a therapeutic intervention for SCA6. We performed a high-throughput screen of FDA-approved small molecules using a dual luciferase reporter system and identified ten hits able to selectively inhibit the IRES. We identified four main candidates that showed selective suppression of α1ACT relative to α1A in HEK cells expressing a native CACNA1A vector. We previously pursued another avenue of molecular intervention through miRNA silencing. We studied three human miRNAs (miRNA-711, -3191-5p, -4786) that would potentially bind to sequences within the CACNA1A IRES region, based on an miRNA prediction program. Only miRNA-3191-5p was found to selectively inhibit the translation of α1ACT in cells. We developed a hyperacute model of SCA6 in mice by injecting a pathogenic form of the IRES-mediated α1ACT (AAV9-α1ACTQ33). Finally, we tested the effectiveness of the miRNA therapy by co-expressing either control miRNA or miRNA-3191-5p and found that miRNA-3191-5p decreased the levels of α1ACTQ33 and prevented the hyperacute disease in mice. These studies provide the proof of principle that a therapy directed at selectively preventing α1ACT expression could be used to treat SCA6.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Sítios Internos de Entrada Ribossomal/fisiologia , Ataxias Espinocerebelares/tratamento farmacológico , Ajmalina/farmacologia , Animais , Canais de Cálcio/genética , Canais de Cálcio Tipo L/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação/genética , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Ataxias Espinocerebelares/genética , Transfecção , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Spinocerebellar ataxia (SCA) type 6 is an autosomal dominant disease affecting cerebellar degeneration. Clinically, it is characterized by pure cerebellar dysfunction, slowly progressive unsteadiness of gait and stance, slurred speech, and abnormal eye movements with late onset. Pathological findings of SCA6 include a diffuse loss of Purkinje cells, predominantly in the cerebellar vermis. Genetically, SCA6 is caused by expansion of a trinucleotide CAG repeat in the last exon of longest isoform CACNA1A gene on chromosome 19p13.1-p13.2. Normal alleles have 4-18 repeats, while alleles causing disease contain 19-33 repeats. Due to presence of a novel internal ribosomal entry site (IRES) with the mRNA, CACNA1A encodes two structurally unrelated proteins with distinct functions within an overlapping open reading frame (ORF) of the same mRNA: (1) α1A subunit of P/Q-type voltage gated calcium channel; (2) α1ACT, a newly recognized transcription factor, with polyglutamine repeat at C-terminal end. Understanding the function of α1ACT in physiological and pathological conditions may elucidate the pathogenesis of SCA6. More importantly, the IRES, as the translational control element of α1ACT, provides a potential therapeutic target for the treatment of SCA6.
Assuntos
Canais de Cálcio , Cromossomos Humanos Par 19 , Éxons , Células de Purkinje , Ataxias Espinocerebelares , Expansão das Repetições de Trinucleotídeos , Animais , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 19/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal/genética , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologiaRESUMO
Spinocerebellar ataxia type 6 (SCA6) belongs to the family of CAG/polyglutamine (polyQ)-dependent neurodegenerative disorders. SCA6 is caused by abnormal expansion in a CAG trinucleotide repeat within exon 47 of CACNA1A, a bicistronic gene that encodes α1A, a P/Q-type calcium channel subunit and a C-terminal protein, termed α1ACT. Expansion of the CAG/polyQ region of CACNA1A occurs within α1ACT and leads to ataxia. There are few animal models of SCA6. Here, we describe the generation and characterization of the first Drosophila melanogaster models of SCA6, which express the entire human α1ACT protein with a normal or expanded polyQ. The polyQ-expanded version of α1ACT recapitulates the progressively degenerative nature of SCA6 when expressed in various fly tissues and the presence of densely staining aggregates. Additional studies identify the co-chaperone DnaJ-1 as a potential therapeutic target for SCA6. Expression of DnaJ-1 potently suppresses α1ACT-dependent degeneration and lethality, concomitant with decreased aggregation and reduced nuclear localization of the pathogenic protein. Mutating the nuclear importer karyopherin α3 also leads to reduced toxicity from pathogenic α1ACT. Little is known about the steps leading to degeneration in SCA6 and the means to protect neurons in this disease are lacking. Invertebrate animal models of SCA6 can expand our understanding of molecular sequelae related to degeneration in this disorder and lead to the rapid identification of cellular components that can be targeted to treat it.
Assuntos
Canais de Cálcio/genética , Proteínas de Drosophila/genética , Proteínas de Choque Térmico HSP40/genética , Ataxias Espinocerebelares/genética , alfa Carioferinas/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP40/biossíntese , Humanos , Degeneração Neural/genética , Degeneração Neural/patologia , Neurônios/patologia , Ataxias Espinocerebelares/patologia , Expansão das Repetições de Trinucleotídeos/genética , alfa Carioferinas/biossínteseRESUMO
To observe the effect of polydatin on proliferation and apoptosis of cervical cancer HeLa cells and explore its possible mechanism. The growth inhibitory effect was detected with MTT assay. After HeLa cells were treated with different concentrations (50, 100, 150 µmolâ¢L⻹) of polydatin, MTT assay was used to detect the inhibitory effect of polydatin on proliferation of HeLa cells; Acridine orange/ethidium bromide staining was used for morphological changes in apoptotic HeLa cellsï¼ Annexin/propidium iodide staining was applied to detect HeLa cell apoptotic rate. In addition, flow cytometry was employed to analyze apoptosis and cell cycle distributionï¼ RT-PCR and Western blot assay were used to detect PI3K, AKT, mTOR, and P70S6K mRNA and protein expression levels. The results showed that polydatin significantly inhibited HeLa cells proliferation in a dose-dependent manner. Polydatin can cause S phase arrest for HeLa cells, promote cell apoptosis and decrease the mRNA and protein expression levels of PI3K, AKT, mTOR and P70S6K. It indicated that polydatin could inhibit proliferation and induce apoptosis of cervical cancer HeLa cells, and the mechanism may be associated with inhibiting the PI3K/AKT/mTOR signaling pathway and suppressing downstream gene expression.
Assuntos
Apoptose , Glucosídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células , Feminino , Células HeLa , HumanosRESUMO
BACKGROUND & AIMS: Currently, routine antiviral treatment is not recommended for immune-tolerant subjects with chronic HBV infection. In this study, we assessed the treatment efficacy of combining Peg IFN α-2a with Adefovir (CPIA) in chronic HBV infected pregnant women with normal levels of ALT and high levels of HBV after delivery. METHODS: Chronic hepatitis B pregnant women with normal levels of ALT and high levels of HBV DNA were treated with Telbivudine during the third trimester of their pregnancy. After childbirth, based on serological and virological parameters, the patients were either switched to CPIA treatment for 96 weeks or stopped Telbivudine treatment and followed for 48 weeks. RESULTS: A total of 68 patients were enrolled in this study. Thirty (30/68) of them were switched to CPIA treatment after childbirth, 93.3% (28/30) of them achieved virological response, 56.7% (17/30) achieved HBeAg seroclearance and 26.7% (8/30) cleared HBsAg. The HBV DNA and HBeAg levels before CPIA treatment were negatively associated with HBeAg seroclearance. HBsAg and HBeAg levels in week 12 and week 24 after CPIA treatment were negatively associated with HBsAg seroclearance. Thirty-eight (38/68) patients did not receive antiviral treatment after childbirth, and none of them had HBeAg or HBsAg clearance. CONCLUSION: High rates of viral response and clearance were achieved in chronic hepatitis B pregnant woman with normal levels of ALT and high levels of HBV DNA treated by CPIA after childbirth. (231 words).
Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , DNA Viral/sangue , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Organofosfonatos/uso terapêutico , Polietilenoglicóis/uso terapêutico , Adenina/uso terapêutico , Adulto , Quimioterapia Combinada , Feminino , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B , Humanos , Período Pós-Parto , Gravidez , Estudos Prospectivos , Proteínas Recombinantes/uso terapêutico , Telbivudina , Timidina/análogos & derivados , Timidina/uso terapêutico , Resultado do TratamentoRESUMO
The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders exhibiting cerebellar atrophy and Purkinje cell degeneration whose subtypes arise from 31 distinct genetic loci. Our group previously published the locus for SCA26 on chromosome 19p13.3. In this study, we performed targeted deep sequencing of the critical interval in order to identify candidate causative variants in individuals from the SCA26 family. We identified a single variant that co-segregates with the disease phenotype that produces a single amino acid substitution in eukaryotic elongation factor 2. This substitution, P596H, sits in a domain critical for maintaining reading frame during translation. The yeast equivalent, P580H EF2, demonstrated impaired translocation, detected as an increased rate of -1 programmed ribosomal frameshift read-through in a dual-luciferase assay for observing translational recoding. This substitution also results in a greater susceptibility to proteostatic disruption, as evidenced by a more robust activation of a reporter gene driven by unfolded protein response activation upon challenge with dithiothreitol or heat shock in our yeast model system. Our results present a compelling candidate mutation and mechanism for the pathogenesis of SCA26 and further support the role of proteostatic disruption in neurodegenerative diseases.
Assuntos
Sequência Conservada/genética , Quinase do Fator 2 de Elongação/genética , Ataxias Espinocerebelares/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Western Blotting , Codificação Clínica , Quinase do Fator 2 de Elongação/metabolismo , Loci Gênicos , Predisposição Genética para Doença , Genótipo , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/genética , Conformação Proteica , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA , Ataxias Espinocerebelares/metabolismo , Transfecção , Leveduras/genéticaRESUMO
BACKGROUND: Magnetic resonance elastography (MRE) is recognized as the most precise imaging technology for assessing liver fibrosis in individuals with metabolic dysfunction-associated steatotic liver disease (MASLD). We aimed to investigate the clinical factors and pathological characteristics that may impact LSM in MASLD patients. METHODS: This cross-sectional study recruited 124 patients who concurrently performed MRE, MRI-PDFF, and biopsy-proven MASLD. Linear regression models, Spearman's correlation, and subgroup analysis were employed to identify the variables affecting LSM. RESULTS: The AUROC (95 % CI) of MRE for diagnosing fibrosis stage ≥ 1, 2, 3, and 4 was 0.80 (0.70-0.90), 0.76 (0.66-0.85), 0.92 (0.86-0.99), and 0.99 (0.99-1.00), with corresponding cutoffs of 2.56, 2.88, 3.35, and 4.76 kPa, respectively. Multivariate analyses revealed that AST was the only independent clinical variable significantly correlated with LSM. Furthermore, LSM exhibited a notable association with the grade of lobular inflammation and hepatocellular ballooning. Subgroup analysis showed that when AST ≥ 2 ULN or inflammation grade ≥ 2, LSM of patients with early fibrosis stages showed a slight but significant increase. CONCLUSION: MRE demonstrates significant diagnostic accuracy in predicting liver fibrosis stages for MASLD patients, especially for advanced liver fibrosis and cirrhosis. However, elevated AST and the severity of liver inflammation may impact its accuracy in staging early liver fibrosis.
RESUMO
The plant cell wall is a dynamic structure that plays an essential role in development, but the mechanism regulating cell wall formation remains poorly understood. We demonstrate that two transcription factors, SlERF.H5 and SlERF.H7, control cell wall formation and tomato fruit firmness in an additive manner. Knockout of SlERF.H5, SlERF.H7, or both genes decreased cell wall thickness, firmness, and cellulose contents in fruits during early development, especially in double-knockout lines. Overexpressing either gene resulted in thicker cell walls and greater fruit firmness with elevated cellulose levels in fruits but severely dwarf plants with lower gibberellin contents. We further identified that SlERF.H5 and SlERF.H7 activate the cellulose biosynthesis gene SlCESA3 but repress the gibberellin biosynthesis gene GA20ox1. Moreover, we identified a conserved LPL motif in these ERFs responsible for their activities as transcriptional activators and repressors, providing insight into how bifunctional transcription factors modulate distinct developmental processes.