RESUMEN
BACKGROUND: Elevated extracellular matrix (ECM) accumulation is a major contributing factor to the pathogenesis of fibrotic diseases. Recent studies have indicated that N6-methyladenosine (m6A) RNA modification plays a pivotal role in modulating RNA stability and contribute to the initiation of various pathological conditions. Howbeit, the precise mechanism by which m6A influences ECM deposition remains unclear. METHODS: In this study, we used hypertrophic scars (HTSs) as a paradigm to investigate ECM-related diseases. We focused on the role of ALKBH5-mediated m6A demethylation within the pathological progression of HTSs and examined its correlation with clinical stages. The effects of ALKBH5 ablation on ECM components were studied both in vivo and in vitro. Downstream targets of ALKBH5, along with their underlying mechanisms, were identified using integrated high-throughput analysis, RNA-binding protein immunoprecipitation and RNA pull-down assays. Furthermore, the therapeutic potential of exogenous ALKBH5 overexpression was evaluated in fibrotic scar models. RESULTS: ALKBH5 was decreased in fibroblasts derived from HTS lesions and was negatively correlated with their clinical stages. Importantly, ablation of ALKBH5 promoted the expression of COL3A1, COL1A1, and ELN, leading to pathological deposition and reconstruction of the ECM both in vivo and in vitro. From a therapeutic perspective, the exogenous overexpression of ALKBH5 significantly inhibited abnormal collagen deposition in fibrotic scar models. As determined by integrated high-throughput analysis, key ECM components including COL3A1, COL1A1, and ELN are direct downstream targets of ALKBH5. By means of its mechanism, ALKBH5 inhibits the expression of COL3A1, COL1A1, and ELN by removing m6A from mRNAs, thereby decreasing their stability in a YTHDF1-dependent manner. CONCLUSIONS: Our study identified ALKBH5 as an endogenous suppressor of pathological ECM deposition, contributing to the development of a reprogrammed m6A-targeted therapy for HTSs.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB , Matriz Extracelular , Fibrosis , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Matriz Extracelular/metabolismo , Fibrosis/metabolismo , Humanos , Ratones , Animales , Desmetilación , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Masculino , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Fibroblastos/metabolismoRESUMEN
Methylated arsenicals, including highly toxic species, such as methylarsenite [MAs(III)], are pervasive in the environment. Certain microorganisms possess the ability to detoxify MAs(III) by ArsI-catalyzed demethylation. Here, we characterize a bifunctional enzyme encoded by the arsI gene from Acidovorax sp. ST3, which can detoxify MAs(III) through both the demethylation and oxidation pathways. Deletion of the 22 C-terminal amino acids of ArsI increased its demethylation activity while reducing the oxidation activity. Further deletion of 44 C-terminal residues enhanced the MAs(III) demethylation activity. ArsI has four vicinal cysteine pairs, with the first pair being necessary for MAs(III) demethylation, while at least one of the other three pairs contributes to MAs(III) oxidation. Molecular modeling and site-directed mutagenesis indicated that one of the C-terminal vicinal cysteine pairs is involved in modulating the switch between oxidase and demethylase activity. These findings underscore the critical role of the C-terminal region in modulating the enzymatic activities of ArsI, particularly in MAs(III) demethylation. This research reveals the structure-function relationship of the ArsI enzyme and advances our understanding of the MAs(III) metabolism in bacteria.
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Dioxigenasas , Oxidación-Reducción , Dioxigenasas/metabolismo , Dioxigenasas/genética , Desmetilación , Comamonadaceae/enzimología , Comamonadaceae/metabolismoRESUMEN
Rieske-type non-heme iron oxygenases (ROs) are an important family of non-heme iron enzymes. They catalyze a diverse range of transformations in secondary metabolite biosynthesis and xenobiotic bioremediation. ROs typically shuttle electrons from NAD(P)H to the oxygenase component via reductase component(s). This chapter describes our recent biochemical characterization of stachydrine demethylase Stc2 from Sinorhizobium meliloti. In this work, the eosin Y/sodium sulfite pair serves as the photoreduction system to replace the NAD(P)H-reductase system. We describe Stc2 protein purification and quality control details as well as a flow-chemistry to separate the photo-reduction half-reaction and the oxidation half-reaction. Our study demonstrates that the eosin Y/sodium sulfite photo-reduction pair is a NAD(P)H-reductase surrogate for Stc2-catalysis in a flow-chemistry setting. Experimental protocols used in this light-driven Stc2 catalysis are likely to be applicable as a photo-reduction system for other redox enzymes.
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Oxidación-Reducción , Sinorhizobium meliloti , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/enzimología , Sinorhizobium meliloti/metabolismo , Oxigenasas/metabolismo , Oxigenasas/genética , Oxigenasas/química , Desmetilación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
Nitenpyram, taking the place of imidacloprid, is a widely used neonicotinoid insecticide to control Nilaparvata lugens in Asia. Two P450s, CYP4CE1 and CYP6ER1, are key factors in the metabolic resistance against nitenpyram and imidacloprid. In this study, we found that CYP4CE1 expression was strongly associated with nitenpyram resistance in 8 field-collected populations, whereas CYP6ER1 expression correlated with imidacloprid resistance. Hence, we focused on nitenpyram metabolism by CYP4CE1, due to that imidacloprid metabolism by CYP6ER1 has intensively investigated. Mass spectrometry analysis revealed that recombinant CYP4CE1 metabolized nitenpyram into three products, N-desmethyl nitenpyram, hydroxy-nitenpyram, and N-desmethyl hydroxy-nitenpyram, with a preference for hydroxylation. In contrast, CYP6ER1 metabolized nitenpyram into a single product, N-desmethyl nitenpyram. These results provide new insights into the specific catalytic mechanisms of P450 enzymes in neonicotinoid metabolism and underscore the importance of different catalytic reactions in neonicotinoid insecticide resistance.
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Proteínas de Insectos , Insecticidas , Neonicotinoides , Oxidación-Reducción , Neonicotinoides/metabolismo , Neonicotinoides/química , Insecticidas/metabolismo , Insecticidas/química , Hidroxilación , Animales , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/química , Desmetilación , Hemípteros/metabolismo , Hemípteros/genética , Hemípteros/enzimología , Nitrocompuestos/metabolismo , Nitrocompuestos/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Resistencia a los Insecticidas/genéticaRESUMEN
OBJECTIVE: To analyze the relationship between serum cystatin C (CysC), ß2-microglobulin (ß2-MG) and the efficacy of demethylation therapy in patients with acute myeloid leukemia (AML). METHODS: A prospective cohort study was conducted on 98 AML patients admitted to the Affiliated Hospital of Inner Mongolia Medical University from February 2019 to January 2022. All patients were treated with decitabine (DAC) + HAG regimen, 28 days as a course and treated for 3-4 courses. At the end of each course of treatment, the treatment effect of the patients was evaluated, and the patients who achieved complete remission (CR) transferred to consolidation therapy, while the patients who did not reach CR at the end of the course of treatment were considered as treatment failure. The examination items before treatment include routine blood parameters, serum CysC, and ß2-MG, and general clinical data of the patients were collected. According to the statistical results, logistic regression model was used to analyze the relationship between serum CysC, ß2-MG and the efficacy of demethylation therapy in AML patients. The ROC curves were drawn, and the predictive efficacy of serum CysC, ß2-MG on demethylation therapy in AML patients was evaluated by the area under the curve (AUC). RESULTS: Of the 98 AML patients enrolled in the study, 5 cases were excluded during the treatment period, and 93 cases finally completed the chemotherapy courses. Among them, 23 patients achieved CR after the initial induction chemotherapy (course 1-2), and 11 patients achieved CR after the re-induction chemotherapy (course 3-4). The success rate of demethylation therapy was 36.56 % (34/93). Compared with the patients in treatment success group, patients in treatment failure group had a higher proportion of intermediate- and adverse-risk, lower levels of platelet (PLT) and hemoglobin (Hb), and higher expression levels of serum CysC and ß2-MG, all of which were statistically significant (P < 0.05). Logistic regression analysis showed that high expression of serum CysC, ß2-MG and adverse-risk were independent risk factors for failure of demethylation treatment in AML patients (OR >1, P < 0.05). The ROC curves showed that the AUC values of serum CysC, ß2-MG alone and combined in predicting the efficacy of demethylation therapy in AML patients were 0.788, 0.785 and 0.834, respectively. CONCLUSION: The failure of demethylation therapy in AML patients is related to the high expression of serum CysC and ß2-MG, and detection of serum CysC and ß2-MG before treatment can predict the risk of demethylation therapy failure in AML patients.
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Cistatina C , Leucemia Mieloide Aguda , Microglobulina beta-2 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/sangre , Estudios Prospectivos , Microglobulina beta-2/sangre , Cistatina C/sangre , Desmetilación , Inducción de Remisión , Decitabina , Masculino , Femenino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Persona de Mediana EdadRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide, facing increasing challenges in terms of prevention and treatment. The methylation of lysine and arginine residues on histone proteins is dynamically controlled by histone methyltransferases (HMTs) and histone demethylases (HDMs), regulating chromatin structure and gene transcription. Mutations, genetic translocations, and altered gene expression involving HMTs and HDMs are frequently observed in NAFLD. HMTs and HDMs are receiving increasing attention in regulating NALFD. Targeting specific HMTs and HDMs for drug development is becoming a new strategy for treating NAFLD. This review provides a comprehensive summary of the regulatory mechanism of histone methylation/demethylation in NAFLD. Additionally, we discuss the potential applications of HMTs and HDMs inhibitors in preventing NAFLD, which may provide a scientific basis for the treatment of NAFLD.
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Histonas , Enfermedad del Hígado Graso no Alcohólico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Humanos , Metilación , Histonas/metabolismo , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/metabolismo , Desmetilación , Animales , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Histona Metiltransferasas/metabolismo , Histona Metiltransferasas/antagonistas & inhibidores , Estructura MolecularRESUMEN
Monomethylmercury (MMHg) plays a crucial role in the accumulation of mercury (Hg) within aquatic food chains. Since ambient levels of methylmercury are governed by the balance of simultaneous methylation and demethylation processes, determining in situ methylation and demethylation rates is critically important to understand the dynamics of methylmercury in the environment. This is especially important in the Wabigoon River system in Ontario, Canada, which is severely contaminated with Hg by a chlor-alkali facility operating in the 1960s, and still exhibits some of the highest recorded fish mercury concentrations in Canada. This work used a simultaneous addition of isotope enriched Hg and MMHg tracers to ascertain Hg methylation and MMHg demethylation potentials. At the locations investigated for this study, the most favourable conditions for Hg methylation were found at the Hydroelectric dam, being able to transform 4.2 % and 4.4 % of added Hg in water and sediments per day, respectively, to MMHg. This could correspond to 1.9 ng/L and 29 ng/g of new MMHg being produced from current ambient Hg. Clay Lake, which is considered a sink for mercury and exhibiting a seasonal anoxic environment at its bottom waters, also demonstrated significant MMHg generation, being able to produce 2.7 ng/L and 13 ng/g of MMHg per day, respectively. Demethylation rates in sediments of riverbed and wetland locations showed an average half-life for methylmercury of 2.1 days, indicating a rapid turnover of MMHg in the Wabigoon River. However, significantly lower demethylation rates were also measured near the inflow of Clay Lake, where it took up to 144 days for MMHg to decrease by 50 %. Generally, most of the investigated locations downstream of the pollution source displayed the potential to generate methylmercury, which could be distributed throughout the Wabigoon River system and therefore require attention with respect to future remediation activities.
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Monitoreo del Ambiente , Sedimentos Geológicos , Mercurio , Compuestos de Metilmercurio , Ríos , Contaminantes Químicos del Agua , Compuestos de Metilmercurio/análisis , Contaminantes Químicos del Agua/análisis , Ríos/química , Mercurio/análisis , Sedimentos Geológicos/química , Metilación , Ontario , DesmetilaciónRESUMEN
BACKGROUND: Mercury (Hg), especially methylmercury (MeHg) as a most toxic format of Hg in the environment, has been paid widely concern due to its high bioaccumulative capability and great risk to humans. Great efforts have been made to develop ethylation-purge and trap-gas chromatography-inductively coupled plasma mass spectrometry system for MeHg analysis and Hg biogeochemical cycling investigation. However, the generally manual operation limits the analytical efficiency, and the lack of applications in the real environmental samples restricts the future study. There is a great need for a rapid and accurate method to determine MeHg and Hg methylation/demethylation processes in environmental samples. RESULTS: Herein, an automatic ethylation-purge and trap-GC-ICP-MS system based on isotope dilution method for MeHg analysis was developed. The results showed that the limit of detection of the developed method was 0.01 ng L-1, the MeHg can be analyzed within 6 min with a relative standard deviation of 4.3 %. The accuracy of this proposed method was verified by the satisfying recoveries of certified reference materials (99.0 ± 0.35 % in ECM-CC580, sediment; 98.0 ± 0.67 % in DORM-4, Fish protein). In addition, comparable concentrations of MeHg in natural water were measured using both of the developed and classical distillation methods. Subsequently, the developed method was adapted for measuring concentrations of MeHg in the water, sediment, and fish muscle collected from the coastal and freshwater systems. Finally, the photic demethylation and biotic methylation/demethylation rate constants in natural surface water and sediment were determined using isotope dilution/tracing methods by automatic ethylation-purge and trap-GC-ICP-MS. SIGNIFICANCE AND NOVELTY: The developed automatic ethylation-purge and trap-GC-ICP-MS system is promising for accurate and convenient MeHg analysis and Hg biogeochemical cycling investigation in real environmental samples with isotope dilution and tracing methods.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Mercurio , Compuestos de Metilmercurio , Compuestos de Metilmercurio/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Metilación , Mercurio/análisis , Animales , Desmetilación , Contaminantes Químicos del Agua/análisis , Monitoreo del Ambiente/métodosRESUMEN
The ocean's mercury (Hg) content has tripled due to anthropogenic activities, and although the dark ocean (>200 m) has become an important Hg reservoir, concentrations of the toxic and bioaccumulative methylmercury (MeHg) are low and therefore very difficult to measure. As a consequence, the current understanding of the Hg cycle in the deep ocean is severely data-limited, and the factors controlling MeHg, as well as its transformation rates, remain largely unknown. By analyzing 52 globally distributed bathypelagic deep-ocean metagenomes and 26 new metatranscriptomes from the Malaspina Expedition, our study reveals the widespread distribution and expression of bacterial-coding genes merA and merB in the global bathypelagic ocean (â¼4000 m depth). These genes, associated with HgII reduction and MeHg demethylation, respectively, are particularly prevalent within the particle-attached fraction. Moreover, our results indicate that water mass age and the organic matter composition shaped the structure of the communities harboring merA and merB genes living in different particle size fractions, their abundance, and their expression levels. Members of the orders Corynebacteriales, Rhodobacterales, Alteromonadales, Oceanospirillales, Moraxellales, and Flavobacteriales were the main taxonomic players containing merA and merB genes in the deep ocean. These findings, together with our previous results of pure culture isolates of the deep bathypelagic ocean possessing the metabolic capacity to degrade MeHg, indicated that both methylmercury demethylation and HgII reduction likely occur in the global dark ocean, the largest biome in the biosphere.
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Mercurio , Compuestos de Metilmercurio , Compuestos de Metilmercurio/metabolismo , Mercurio/metabolismo , Agua de Mar/microbiología , Océanos y Mares , Desmetilación , Contaminantes Químicos del Agua/metabolismo , Bacterias/metabolismoRESUMEN
BACKGROUND: The aim of this study was to construct a prognostic model of colon cancer based on demethylation-related genes. An in-depth understanding of the relationship between the set of demethylated genes and colon cancer not only assists in revealing the pathogenesis of colon cancer but also provides strong support for future therapeutic strategies and individualized medicine. METHODS: Data were obtained from the TCGA database and the GEO-GSE39582 cohort. A risk score model for demethylation-related genes was developed using univariate Cox regression analysis and LASSO regression analysis. The accuracy and reliability of the model were confirmed using K-M survival analysis and ROC curve analysis. Additionally, a nomogram was created by integrating the risk score and clinicopathological variables. Finally, the biological function of the RCOR2 gene was verified by performing qPCR, MTT, colony formation, Transwell, and subcutaneous tumor formation assays in nude mice. RESULTS: We constructed a risk score model containing 30 demethylation-related genes for predicting the survival risk of patients with colon cancer. COAD patients were categorized into high-risk and low-risk groups, and Kaplan-Meier (KM) curve analysis revealed that the high-risk group was associated with a worse prognosis. Univariate and multivariate Cox regression analyses validated the risk score as an independent prognostic factor for COAD. We also analyzed the differences in the sensitivity to nine chemotherapeutic agents and small molecule targeted drugs between the high-risk and low-risk groups. Moreover, we performed experiments in COAD cell lines and nude mice to verify that RCOR2 was differentially expressed between tumor tissues and normal tissues and that high RCOR2 expression promoted a malignant phenotype of colon cancer. CONCLUSION: This study demonstrated the potential roles of demethylation-related genes in colon cancer by conducting a comprehensive analysis and constructing a risk score. These findings also highlight the ability of these genes to indicate patient prognosis and tumor immune microenvironment. Furthermore, this study provides a reliable predictive tool that can assist in guiding the treatment and management of colon cancer patients.
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Biomarcadores de Tumor , Neoplasias del Colon , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Humanos , Neoplasias del Colon/genética , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Neoplasias del Colon/diagnóstico , Animales , Pronóstico , Masculino , Femenino , Biomarcadores de Tumor/genética , Ratones , Línea Celular Tumoral , Desmetilación , Nomogramas , Persona de Mediana Edad , Metilación de ADN , Ratones Endogámicos BALB C , AncianoRESUMEN
Histone demethylation by PHF8 initiates innate immune signaling in acute myeloid leukemia, elucidating a novel therapeutic strategy.
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Histonas , Inmunidad Innata , Leucemia Mieloide Aguda , Inmunidad Innata/inmunología , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/genética , Histonas/inmunología , Histonas/metabolismo , Animales , Desmetilación , Factores de Transcripción/inmunología , Transducción de Señal/inmunología , Histona Demetilasas/inmunología , Histona Demetilasas/metabolismoRESUMEN
N6-methyladenosine (m6A) methylation is a vital epigenetic mechanism associated with drug addiction. However, the relationship between m6A modification and oxycodone rewarding is less well explored. Based on an open field test, the present study evaluated oxycodone rewarding using chromatin immunoprecipitation PCR, immunofluorescence, and RNA sequencing. A marked increase in METTL14 protein and a decrease in PP1α protein due to oxycodone abundance in the striatal neurons were observed in a dose- and time-dependent manner. Oxycodone markedly increased LSD1 expression, and decreased H3K4me1 expression in the striatum. In the open field test, intra-striatal injection of METTL14 siRNA, HOTAIR siRNA, or LSD1 shRNA blocked oxycodone-induced increase in locomotor activity. The downregulation of PP1α was also inhibited after treatment with METTL14/HOTAIR siRNA and LSD1 shRNA. Enhanced binding of LSD1 with CoRest and of CoRest with the PP1α gene induced by oxycodone was also reversed by LSD1 shRNA. In addition, H3K4me1 demethylation was also blocked by the treatment. In summary, the investigation confirmed that METTL14-mediated upregulation of HOTAIR resulted in the repression of PP1α, which in turn facilitated the recruitment of LSD1, thus catalyzing H3K4me1 demethylation and promoting oxycodone addiction.
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Metiltransferasas , Oxicodona , ARN Largo no Codificante , Animales , Masculino , Ratones , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de los fármacos , Desmetilación , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Histonas/metabolismo , Lisina/análogos & derivados , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones Endogámicos C57BL , Oxicodona/farmacología , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/genética , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Regulación hacia ArribaRESUMEN
N-methyladenosine (m6A) represents a prevalent RNA modification observed in colorectal cancer. Despite its abundance, the biological implications of m6A methylation on the lncRNA CARMN remain elusive in colorectal cancer, especially for mutant p53 gain-of-function. Here, we elucidate that CARMN exhibits diminished expression levels in colorectal cancer patients with mutant p53, attributed to its rich m6A methylation, which promotes cancer proliferation, invasion and metastasis in vitro and in vivo. Further investigation illustrates that ALKBH5 acts as a direct demethylase of CARMN, targeting 477 methylation sites, thereby preserving CARMN expression. However, the interaction of mutant p53 with the ALKBH5 promoter impedes its transcription, enhancing m6A methylation levels on CARMN. Subsequently, YTHDF2/YTHDF3 recognise and degrade m6A-modified CARMN. Concurrently, overexpressing CARMN significantly suppressed colorectal cancer progression in vitro and in vivo. Additionally, miR-5683 was identified as a direct downstream target of lncRNA CARMN, exerting an antitumour effect by cooperatively downregulating FGF2 expression. Our findings revealed the regulator and functional mechanism of CARMN in colorectal cancer with mutant p53, potentially offering insights into demethylation-based strategies for cancer diagnosis and therapy. The m6A methylation of CARMN that is prime for mutant p53 gain-of-function-induced malignant progression of colorectal cancer, identifying a promising approach for cancer therapy.
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Desmetilasa de ARN, Homólogo 5 de AlkB , Neoplasias Colorrectales , MicroARNs , ARN Largo no Codificante , Proteína p53 Supresora de Tumor , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Animales , Ratones , Progresión de la Enfermedad , Desmetilación , Línea Celular Tumoral , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Ratones Desnudos , Regulación Neoplásica de la Expresión GénicaRESUMEN
Maxillofacial bone defects exhibit intricate anatomy and irregular morphology, presenting challenges for effective treatment. This study aimed to address these challenges by developing an injectable bioactive composite microsphere, termed D-P-Ak (polydopamine-PLGA-akermanite), designed to fit within the defect site while minimizing injury. The D-P-Ak microspheres biodegraded gradually, releasing calcium, magnesium, and silicon ions, which, notably, not only directly stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) but also activated sensory nerve cells to secrete calcitonin gene-related peptide (CGRP), a key factor in bone repair. Moreover, the released CGRP enhanced the osteogenic differentiation of BMSCs through epigenetic methylation modification. Specifically, inhibition of EZH2 and enhancement of KDM6A reduced the trimethylation level of histone 3 at lysine 27 (H3K27), thereby activating the transcription of osteogenic genes such as Runx2 and Osx. The efficacy of the bioactive microspheres in bone repair is validated in a rat mandibular defect model, demonstrating that peripheral nerve response facilitates bone regeneration through epigenetic modification. These findings illuminated a novel strategy for constructing neuroactive osteo-inductive biomaterials with potential for further clinical applications.
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Regeneración Ósea , Células Madre Mesenquimatosas , Microesferas , Osteogénesis , Animales , Ratas , Células Madre Mesenquimatosas/metabolismo , Regeneración Ósea/genética , Regeneración Ósea/efectos de los fármacos , Regeneración Ósea/fisiología , Osteogénesis/fisiología , Osteogénesis/genética , Diferenciación Celular , Desmetilación , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Cerámica , Histonas/metabolismo , Histonas/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Masculino , Materiales Biocompatibles/metabolismoRESUMEN
The human alkylation B (AlkB) homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair enzymes that remove 1-methyladenine (1meA) and 3-methylcytosine (3meC) lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1koALKBH3ko cells showed enhanced resistance toward SN1- and SN2-type methylating agents, whereas MLH1koALKBH2ko cells were only resistant to SN1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH2ko3ko) rendered cells sensitive to SN1- and SN2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH2ko3ko cells have an ATR-dependent arrest at the G2/M checkpoint, increased apoptotic signaling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1koALKBH2ko3ko cells. Finally, in MLH1koALKBH2ko3ko cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1.
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Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Reparación de la Incompatibilidad de ADN , Homólogo 1 de la Proteína MutL , Humanos , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Homólogo 1 de la Proteína MutL/metabolismo , Homólogo 1 de la Proteína MutL/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , DesmetilaciónRESUMEN
Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates â¼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms, yielding a platform strain for efficient O-demethylation of lignin-related aromatic compounds to value-added products.
Asunto(s)
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ingeniería Metabólica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desmetilación , Evolución Molecular DirigidaRESUMEN
Follicle selection in chicken refers to the process of selecting a follicle to enter hierarchy from a cohort of small yellow follicles (SY) with a diameter of 6 to 8 mm. The follicle being selected will develop rapidly and ovulate. Follicle selection is a key stage affecting chicken egg-laying performance. Our previous study showed that the phosphorylation level of lysine (K)-specific demethylase 1A (LSD1) at serine 54 (LSD1Ser54p) was significantly increased in F6 follicles compared to prehierarchal SY follicles, but its function was unclear. Here, the mechanism of this modification, the effect of LSD1Ser54p dephosphorylation on gene expression profile of chicken hierarchal granulosa cells and the function of fibroblast growth factor 9 (FGF9) that is regulated by LSD1Ser54p were further investigated. The modification of LSD1Ser54p was predicted to be mediated by cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). Treatment of chicken hierarchal granulosa cells with CDK5 inhibitor significantly decreased LSD1Ser54p level (P < 0.05) and LSD1Ser54p interacted with CDK5, suggesting that, in the granulosa cells of chicken hierarchal follicles, LSD1Ser54p modification was carried out by CDK5. When the LSD1Ser54p level decreased in the granulosa cells of chicken hierarchal follicles, both the mRNA expression of FGF9 and α-actinin 2 (ACTN2) and the H3K4me2 level in their promoter regions significantly increased (P < 0.05), indicating that this phosphorylation modification enhanced the demethylation activity of LSD1. Moreover, in chicken hierarchal granulosa cells, overexpression of chicken FGF9 stimulated their proliferation and increased the mRNA expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b) and steroidogenic acute regulatory protein (StAR). This study collectively revealed that phosphorylation of LSD1 at serine 54 by CDK5 enhanced its demethylation activity in chicken ovarian granulosa cells and regulated genes including FGF9 that is engaged in chicken follicle selection.
Asunto(s)
Proteínas Aviares , Pollos , Células de la Granulosa , Histona Demetilasas , Folículo Ovárico , Animales , Femenino , Células de la Granulosa/metabolismo , Pollos/genética , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Folículo Ovárico/metabolismo , Fosforilación , Proteínas Aviares/metabolismo , Proteínas Aviares/genética , Desmetilación , Regulación de la Expresión Génica/efectos de los fármacos , Serina/metabolismoRESUMEN
The N6-methyladenosine (m6A) mRNA modification is crucial for plant development and stress responses. In rice, the male sterility resulting from the deficiency of OsFIP37, a core component of m6A methyltransferase complex, emphasizes the significant role of m6A in male fertility. m6A is reversible and can be removed by m6A demethylases. However, whether mRNA m6A demethylase regulates male fertility in rice has remained unknown. Here, we identify the mRNA m6A demethylase OsALKBH9 and demonstrate its involvement in male fertility regulation. Knockout of OsALKBH9 causes male sterility, dependent on its m6A demethylation activity. Cytological analysis reveals defective tapetal programmed cell death (PCD) and excessive accumulation of microspores exine in Osalkbh9-1. Transcriptome analysis of anthers shows up-regulation of genes involved in tapetum development, sporopollenin synthesis, and transport pathways in Osalkbh9-1. Additionally, we demonstrate that OsALKBH9 demethylates the m6A modification in TDR and GAMYB transcripts, which affects the stability of these mRNAs and ultimately leads to excessive accumulation of pollen exine. Our findings highlight the precise control of mRNA m6A modification and reveal the pivotal roles played by OsALKBH9-mediated m6A demethylation in tapetal PCD and pollen exine accumulation in rice.
Asunto(s)
Desmetilación , Regulación de la Expresión Génica de las Plantas , Oryza , Proteínas de Plantas , Polen , Oryza/genética , Oryza/metabolismo , Polen/genética , Polen/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Apoptosis/genética , Infertilidad Vegetal/genéticaRESUMEN
Inorganic mercury (HgII) can be transformed into neurotoxic methylmercury (MeHg) by microorganisms in paddy soils, and the subsequent accumulation in rice grains poses an exposure risk for human health. Warming as an important manifestation of climate change, changes the composition and structure of microbial communities, and regulates the biogeochemical cycles of Hg in natural environments. However, the response of specific HgII methylation/demethylation to the changes in microbial communities caused by warming remain unclear. Here, nationwide sampling of rice paddy soils and a temperature-adjusted incubation experiment coupled with isotope labeling technique (202HgII and Me198Hg) were conducted to investigate the effects of temperature on HgII methylation, MeHg demethylation, and microbial mechanisms in paddy soils along Hg gradients. We showed that increasing temperature significantly inhibited HgII methylation but promoted MeHg demethylation. The reduction in the relative abundance of Hg-methylating microorganisms and increase in the relative abundance of MeHg-demethylating microorganisms are the likely reasons. Consequently, the net Hg methylation production potential in rice paddy soils was largely inhibited under the increasing temperature. Collectively, our findings offer insights into the decrease in net MeHg production potential associated with increasing temperature and highlight the need for further evaluation of climate change for its potential effect on Hg transformation in Hg-sensitive ecosystems.
Asunto(s)
Mercurio , Compuestos de Metilmercurio , Oryza , Contaminantes del Suelo , Suelo , Contaminantes del Suelo/metabolismo , Contaminantes del Suelo/análisis , Mercurio/metabolismo , Mercurio/análisis , Metilación , Suelo/química , Microbiología del Suelo , Cambio Climático , Desmetilación , Monitoreo del AmbienteRESUMEN
Myocardial ischemiaâreperfusion injury (MI/RI) is closely related to pyroptosis. alkB homolog 5 (ALKBH5) is abnormally expressed in the MI/RI models. However, the detailed molecular mechanism of ALKBH5 in MI/RI has not been elucidated. In this study, rats and H9C2 cells served as experimental subjects and received MI/R induction and H/R induction, respectively. The abundance of the targeted molecules was evaluated using RT-qPCR, Western blotting, immunohistochemistry, immunofluorescence, and enzyme-linked immunosorbent assay. The heart functions of the rats were evaluated using echocardiography, and heart injury was evaluated. Cell viability and pyroptosis were determined using cell counting Kit-8 and flow cytometry, respectively. Total m6A modification was measured using a commercial kit, and pri-miR-199a-5p m6A modification was detected by Me-RNA immunoprecipitation (RIP) assay. The interactions among the molecules were validated using RIP and luciferase experiments. ALKBH5 was abnormally highly expressed in H/R-induced H9C2 cells and MI/RI rats. ALKBH5 silencing improved injury and inhibited pyroptosis. ALKBH5 reduced pri-miR-199a-5p m6A methylation to block miR-199a-5p maturation and inhibit its expression. TNF receptor-associated Factor 3 (TRAF3) is a downstream gene of miR-199a-5p. Furthermore, in H/R-induced H9C2 cells, the miR-199a-5p inhibitor-mediated promotion of pyroptosis was reversed by ALKBH5 silencing, and the TRAF3 overexpression-mediated promotion of pyroptosis was offset by miR-199a-5p upregulation. ALKBH5 silencing inhibited pri-miR-199a-5p expression and enhanced pri-miR-199a-5p m6A modification to promote miR-199a-5p maturation and enhance its expression, thereby suppressing pyroptosis to alleviate MI/RI through decreasing TRAF3 expression.