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Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/µL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.
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While the significance of N6-methyladenosine (m6A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m5C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m5C is more abundant than m6A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m5C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m5C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m5C-MeRIP-seq identified the NSUN2-dependent m5C-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from Nsun2 deficiency mice with low levels of m5C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m5C modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of m5C methylation to SARS-CoV-2 RNA.
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COVID-19 , ARN Viral , SARS-CoV-2 , Replicación Viral , Replicación Viral/genética , Animales , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , COVID-19/virología , COVID-19/patología , Ratones , Humanos , Metilación , Virulencia/genética , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Epigénesis Genética , Ratones Noqueados , Adenosina/análogos & derivados , Adenosina/metabolismo , TranscriptomaRESUMEN
A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16 (nsp16), 2'-O-methyltransferase (2'-O-MTase), to cap their RNAs through ribose 2'-O-methylation modification. This process is crucial for maintaining viral genome stability, facilitating efficient translation, and enabling immune escape. Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10, insights into its molecular mechanism have so far been limited. In this study, we systematically characterized the 2'-O-MTase activity of nsp16 in SARS-CoV-2, focusing on its dependence on nsp10 stimulation. We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface. However, a single residue substitution (K58T) in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16. Furthermore, the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor S-adenosyl-l-methionine (SAM). Mechanistically, His-80, Lys-93, and Gly-94 of nsp10 interacted with Asp-102, Ser-105, and Asp-106 of nsp16, respectively, thereby effectively stabilizing the SAM binding pocket. Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex. By assessing the conformational epitopes of nsp16/nsp10 complex, we further determined the critical residues involved in 2'-O-MTase activity. Additionally, we utilized an in vitro biochemical platform to screen potential inhibitors targeting 2'-O-MTase activity. Overall, our results significantly enhance the understanding of viral 2'-O methylation process and mechanism, providing valuable targets for antiviral drug development.
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Metiltransferasas , SARS-CoV-2 , Proteínas no Estructurales Virales , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Metiltransferasas/metabolismo , Metiltransferasas/genética , Metiltransferasas/química , Humanos , ARN Viral/genética , ARN Viral/metabolismo , COVID-19/virología , Unión Proteica , S-Adenosilmetionina/metabolismo , Metilación , Betacoronavirus/enzimología , Betacoronavirus/genética , Modelos Moleculares , Coronavirus del Síndrome Respiratorio de Oriente Medio/enzimología , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00-1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815.
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Espectrometría de Masas en Tándem , Tolvaptán , Espectrometría de Masas en Tándem/métodos , Humanos , Cromatografía Líquida de Alta Presión/métodos , Reproducibilidad de los Resultados , Tolvaptán/sangre , Tolvaptán/química , Modelos Lineales , Límite de Detección , Benzazepinas/sangre , Benzazepinas/farmacocinética , Benzazepinas/química , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu36 â Ile36, Asn138 â His138, Ile153 â Leu153, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.
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COVID-19 , Metiltransferasas , SARS-CoV-2 , Replicación Viral , Humanos , SARS-CoV-2/genética , SARS-CoV-2/enzimología , SARS-CoV-2/patogenicidad , COVID-19/virología , COVID-19/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Metiltransferasas/química , Replicación Viral/genética , ARN Viral/genética , ARN Viral/metabolismo , ARN Viral/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Animales , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismoRESUMEN
OBJECTIVE: PM2.5 is closely linked to vascular endothelial injury and has emerged as a major threat to human health. Our previous research indicated that exposure to PM2.5 induced an increased release of miR-421 from the bronchial epithelium. However, the role of miR-421 in PM2.5-induced endothelial injury remains elusive. MATERIALS AND METHODS: We utilized a subacute PM2.5-exposure model in mice in vivo and an acute injury cell model in vitro to simulate PM2.5-associated endothelial injury. We also used quantitative real-time polymerase chain reaction, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry to investigate the role of miR-421 in PM2.5-induced endothelial injury. RESULTS: Our findings reveal that inhibition of miR-421 attenuated PM2.5-induced endothelial injury and hypertension. Mechanistically, miR-421 inhibited the expression of angiotensin-converting enzyme 2 (ACE2) in human umbilical vein endothelial cells and upregulated the expression of the downstream molecule inducible nitric oxide synthase (iNOS), thereby exacerbating PM2.5-induced endothelial injury. CONCLUSIONS: Our results indicate that PM2.5 exposure facilitates crosstalk between bronchial epithelial and endothelial cells via miR-421/ACE2/iNOS signaling pathway, mediating endothelial damage and hypertension. MiR-421 inhibition may offer a new strategy for the prevention and treatment of PM2.5-induced vascular endothelial injury.
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A new xanthone, allanxanthone F (1), and 10 known compounds were isolated from the ethanol extract of Garcinia bracteata. The structure of compound 1 was elucidated based on spectroscopic methods (UV, IR, HR-ESI-MS, and NMR). In addition, compounds 1-9 were assessed for their anti-inflammatory activities based on the expression of nitric oxide (NO) levels on lipopolysaccharide (LPS)-induced RAW264.7 macrophages, and compounds 1-3, 4 and 6-9 suggested potential anti-inflammatory activities.
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Titanium alloy with high corrosion resistance, high strength-to-density ratio, and excellent biocompatibility has a wide range of applications in the field of biomedical implants. Polishing experiments of titanium alloy with a small size and complex shapes were investigated using an ultrasonic assisted magnetorheological finishing (UMRF) device excited by a three-pole magnetic field generator. The models of the normal force and the shear force were first proposed based on the Preston equation to analyze the mechanism of material removal in the UMRF process. Subsequently, the single-factor experiments using titanium alloy nuts (M3) and the MR polishing fluid with silicon carbide abrasives were carried out. Furthermore, to improve the surface roughness and the change rate of surface roughness of nuts, orthogonal tests with a standard L9(34) orthogonal array were designed and performed based on the optimized process parameters obtained from the single-factor experiment. The results indicated the effect on surface roughness and change rate of surface roughness as applied current > roller speed > ultrasonic amplitude > spindle speed and applied current > roller speed > spindle speed > ultrasonic amplitude, respectively. Moreover, the surface roughness was improved from an initial 1.247 µm to a final 0.104 µm after the polishing for 80 min under these optimal process parameters.
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The clinical diagnosis and treatment of pituitary neuroendocrine tumors (PitNETs) that invade the cavernous sinus are fraught with difficulties and challenges. Exploring the biological characteristics involved in the occurrence and development of PitNETs that invade the cavernous sinus will help to elucidate the mechanism of cavernous sinus invasion. There are differences between intrasellar tumors (IST) and cavernous sinus-invasion tumors (CST) in ultramicrostructure, tumor microenvironment (TME), gene expression, and signaling pathways. The microvascular endothelial cell is increased in CST. The VEGFR signaling pathway, VEGF signaling pathway, and chemokine signaling pathway are activated in CST. HSPB1 is upregulated in CST and promotes cell proliferation, cell viability, and migration. HSPB1 promotes the release of VEGF from GT1-1 cells and activates the VEGF signaling pathway in bEnd.3 cells. HSPB1 promotes the migration of bEnd.3 cells to GT1-1 cells and promotes the formation of blood vessels of bEnd.3 cells. bEnd.3 cells can release CCL3 and CCL4 and promote the vitality, proliferation, and migration of GT1-1 cells. HSPB1 promotes the formation of blood vessels of bEnd.3 cells and ultimately leads to tumor growth in vivo. HSPB1 acts as a key gene for invasion of the cavernous sinus in PitNETs, remodeling TME by promoting the formation of blood vessels of brain microvascular endothelial cells. The synergistic effect of tumor cells and microvascular endothelial cells promotes tumor progression. The mechanism by which HSPB1 promotes tumor invasion by inducing angiogenesis in PitNETs may be a new target for the treatment of PitNETs invading the cavernous sinus.
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Proteínas de Choque Térmico , Chaperonas Moleculares , Invasividad Neoplásica , Neovascularización Patológica , Neoplasias Hipofisarias , Humanos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Animales , Chaperonas Moleculares/metabolismo , Ratones , Proteínas de Choque Térmico/metabolismo , Neoplasias Hipofisarias/patología , Neoplasias Hipofisarias/metabolismo , Masculino , Femenino , Ratones Desnudos , Línea Celular Tumoral , Proliferación Celular , Movimiento Celular , AngiogénesisRESUMEN
Atractylodes macrocephala III (ATL III), with anti-inflammatory and antitumor effects, is the main compound of Atractylodes macrocephala. Whether ATL III has an effect on cervical cancer and the specific mechanism are still unclear. Here, we investigated the effects of ATL III on cervical cancer cells at different concentrations and found that ATL III downregulates insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), which was found to be highly expressed in cervical cancer tissue by RNA-Seq. In this study, we found that ATL III promotes apoptosis and regulates epithelial-mesenchymal transition (EMT) in cervical cancer cells (HeLa and SiHa cells) and that IGF2BP3 is a common target gene of ATL III in HeLa and SiHa cells. The expression level of IGF2BP3 in cervical cancer cells was proportional to their migration and invasion abilities. This was verified by transfection of cells with a small interfering RNA and an IGF2BP3 overexpression plasmid. After ATL III treatment, the migration and invasion abilities of cervical cancer cells were obviously reduced, but these effects were attenuated after overexpression of IGF2BP3. In addition, the transcription factor IGF2BP3 was predicted by the JASPAR system. After intersection with our sequencing results, we verified the promotional effect of ETV5 (ETS translocation variant 5) on IGF2BP3 and found that ALT III inhibited ETV5. In general, our research showed that ATL III inhibits the migration and invasion of cervical cancer cells by regulating IGF2BP3 through ETV5.
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Atractylodes , Neoplasias del Cuello Uterino , Femenino , Humanos , Atractylodes/química , Neoplasias del Cuello Uterino/patología , Línea Celular Tumoral , Factores de Transcripción/metabolismo , Transición Epitelial-Mesenquimal/genética , Proliferación Celular/genética , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ADN/genéticaRESUMEN
INTRODUCTION: The aim of this study was to explore the renoprotective effects of Klotho on podocyte injury mediated by complement activation and autoantibodies in idiopathic membranous nephropathy (IMN). METHODS: Rat passive Heymann nephritis (PHN) was induced as an IMN model. Urine protein levels, serum biochemistry, kidney histology, and podocyte marker levels were assessed. In vitro, sublytic podocyte injury was induced by C5b-9. The expression of Klotho, transient receptor potential channel 6 (TRPC6), and cathepsin L (CatL); its substrate synaptopodin; and the intracellular Ca2+ concentration were detected via immunofluorescence. RhoA/ROCK pathway activity was measured by an activity quantitative detection kit, and the protein expression of phosphorylated-LIMK1 (p-LIMK1) and p-cofilin in podocytes was detected via Western blotting. Klotho knockdown and overexpression were performed to evaluate its role in regulating the TRPC6/CatL pathway. RESULTS: PHN rats exhibited proteinuria, podocyte foot process effacement, decreased Klotho and Synaptopodin levels, and increased TRPC6 and CatL expression. The RhoA/ROCK pathway was activated by the increased phosphorylation of LIMK1 and cofilin. Similar changes were observed in C5b-9-injured podocytes. Klotho knockdown exacerbated podocyte injury, while Klotho overexpression partially ameliorated podocyte injury. CONCLUSION: Klotho may protect against podocyte injury in IMN patients by inhibiting the TRPC6/CatL pathway. Klotho is a potential target for reducing proteinuria in IMN patients.
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Citoesqueleto de Actina , Catepsina L , Glomerulonefritis Membranosa , Glucuronidasa , Proteínas Klotho , Podocitos , Transducción de Señal , Canal Catiónico TRPC6 , Podocitos/metabolismo , Podocitos/patología , Glomerulonefritis Membranosa/metabolismo , Glomerulonefritis Membranosa/patología , Animales , Glucuronidasa/metabolismo , Ratas , Canal Catiónico TRPC6/metabolismo , Masculino , Citoesqueleto de Actina/metabolismo , Catepsina L/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Humanos , Modelos Animales de Enfermedad , Proteínas de Microfilamentos/metabolismo , Proteinuria/metabolismo , Ratas Sprague-Dawley , Quinasas Asociadas a rho/metabolismo , Canales Catiónicos TRPC/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismoRESUMEN
Exercise can stimulate physiological cardiac growth and provide cardioprotection effect in ischemia/reperfusion (I/R) injury. MiR-210 is regulated in the adaptation process induced by exercise; however, its impact on exercise-induced physiological cardiac growth and its contribution to exercise-driven cardioprotection remain unclear. We investigated the role and mechanism of miR-210 in exercise-induced physiological cardiac growth and explored whether miR-210 contributes to exercise-induced protection in alleviating I/R injury. Here, we first observed that regular swimming exercise can markedly increase miR-210 levels in the heart and blood samples of rats and mice. Circulating miR-210 levels were also elevated after a programmed cardiac rehabilitation in patients that were diagnosed of coronary heart diseases. In 8-week swimming model in wild-type (WT) and miR-210 knockout (KO) rats, we demonstrated that miR-210 was not integral for exercise-induced cardiac hypertrophy but it did influence cardiomyocyte proliferative activity. In neonatal rat cardiomyocytes, miR-210 promoted cell proliferation and suppressed apoptosis while not altering cell size. Additionally, miR-210 promoted cardiomyocyte proliferation and survival in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and AC16 cell line, indicating its functional roles in human cardiomyocytes. We further identified miR-210 target genes, cyclin-dependent kinase 10 (CDK10) and ephrin-A3 (EFNA3), that regulate cardiomyocyte proliferation and apoptosis. Finally, miR-210 KO and WT rats were subjected to swimming exercise followed by I/R injury. We demonstrated that miR-210 crucially contributed to exercise-driven cardioprotection against I/R injury. In summary, this study elucidates the role of miR-210, an exercise-responsive miRNA, in promoting the proliferative activity of cardiomyocytes during physiological cardiac growth. Furthermore, miR-210 plays an essential role in mediating the protective effects of exercise against cardiac I/R injury. Our findings suggest exercise as a potent nonpharmaceutical intervention for inducing miR-210, which can alleviate I/R injury and promote cardioprotection.
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The shear yield stress is an important parameter for the industrial application of magnetorheological (MR) fluids. A test equipment was designed and built to perform investigations on the behaviours of compression and shear after squeeze of MR fluids. Mathematical expression of magnetic flux density was further established. Furthermore, the magnetic field distribution of the test device based on two-coil mode and single-coil mode was simulated and compared using finite element analysis(ANSYS/Multiphysics). An experimental test system was fabricated and modified based on the final conditions and simulation results. The compression and shear after squeeze performances of MR fluids were tested. The results showed that a smaller initial gap distance or a larger compressive strain corresponds to a larger compressive stress under the same external magnetic field strength. The shear yield stress after the squeeze of MR fluids increases quickly with the increasing compression stress and the increasing magnetic flux density. This test equipment was thought to be suitable for studying the compression and shear after squeeze performances of MR fluids.
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BACKGROUND: The treatment of dopamine agonists (DA) resistant prolactinomas remains a formidable challenge, as the mechanism of resistance is still unclear, and there are currently no viable alternative drug therapies available. This study seeks to investigate the mechanism of DA resistance in prolactinomas and identify new potentially effective drugs. METHODS: To explore the mechanism of DA resistance in prolactinomas, this study conducted transcriptome sequencing analysis on 27 cases of DA-resistant prolactinomas and 10 cases of sensitive prolactinomas. In addition, single-cell sequencing analysis was performed on 3 cases of DA-resistant prolactinomas and 3 cases of sensitive prolactinomas. Furthermore, to screen for potential therapeutic drugs, the study successfully established an organoids model for DA-resistant prolactinomas and screened 180 small molecule compounds using 8 organoids. The efficacy of the identified drugs was verified through various assays, including CCK-8, colony formation, CTG, and flow cytometry, and their mechanisms of action were confirmed through WB and IHC. The effectiveness of the identified drugs was evaluated both in vitro and in vivo. RESULTS: The results of transcriptome sequencing and single-cell sequencing analyses showed that DA resistance in prolactinomas is associated with the upregulation of the Focal Adhesion (FA) signaling pathway. Additionally, immunohistochemical validation revealed that FAK and Paxillin were significantly upregulated in DA-resistant prolactinomas. Screening of 180 small molecule compounds using 8 organoids identified Genistein as a potentially effective drug for DA-resistant prolactinomas. Experimental validation demonstrated that Genistein inhibited the proliferation of pituitary tumor cell lines and organoids and promoted apoptosis in pituitary tumor cells. Moreover, both the cell sequencing results and WB validation results of the drug-treated cells indicated that Genistein exerts its anti-tumor effect by inhibiting the FA pathway. In vivo, experiments also showed that Genistein can inhibit subcutaneous tumor formation. CONCLUSION: DA resistance in prolactinomas is associated with upregulation of the Focal Adhesion (FA) signaling pathway, and Genistein can exert its anti-tumor effect by inhibiting the expression of the FA pathway.
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Tumores Neuroendocrinos , Neoplasias Hipofisarias , Prolactinoma , Humanos , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Agonistas de Dopamina/farmacología , Agonistas de Dopamina/uso terapéutico , Prolactinoma/tratamiento farmacológico , Prolactinoma/genética , Prolactinoma/metabolismo , Prolactina/metabolismo , Prolactina/uso terapéutico , Genisteína/uso terapéutico , Tumores Neuroendocrinos/tratamiento farmacológico , Resistencia a Antineoplásicos/genéticaRESUMEN
Pyroptosis is an inflammatory programmed cell death (PCD) and is reported to be associated with N6-methyladenosine (m6A) modification. This study aimed to investigate the mechanism of m6A demethylase AlkB homolog 5 (ALKBH5) in pyroptosis in the process of chronic actinic dermatitis (CAD). Changes of m6A-related genes were evaluated between CAD and normal samples using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Human keratinocytes (HaCaT cells) exposed to ultraviolet B (UVB; 10, 20, and 30 mJ/cm2), followed by evaluation of cell proliferation, cell apoptosis, inflammatory cytokines (interleukin (IL)-1ß, IL-18, and tumor necrosis factor (TNF-α)), and pyroptosis-related proteins (gasdermin D (GSDMD), Caspase-1, and Caspase-4). Small interfering RNA (siRNA) targeting ALKBH5 was transfected into HaCaT cells to assess the effect of si-ALKBH5 on CAD. A CAD mice model was induced after exposure to UVB (250 mJ/cm2 per day) to confirm the role of ALKBH5 in CAD. AKKBH5 was highly expressed in CAD patients. UVB also promoted ALKBH5 expression, increased cell apoptosis, and induced the release of inflammatory cytokines (IL-1ß, IL-18, and TNF-α) as well as pyroptosis-related proteins (GSDMD, Caspase-1, and Caspase-4). Silencing ALKBH5 repressed cell apoptosis and suppressed UVB-induced pyroptosis and inflammatory response. Meanwhile, silencing ALKBH5 attenuated UVB-induced skin damage of CAD mice, accompanied with the reduction in expression of inflammatory cytokines and pyroptosis-related proteins. This study helps to further understand the mechanism of ALKBH5 in CAD-induced pyroptosis and provides novel ideas for the research and management of CAD.
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Trastornos por Fotosensibilidad , Piroptosis , Animales , Humanos , Ratones , Adenosina , Desmetilasa de ARN, Homólogo 5 de AlkB , Caspasa 1 , Citocinas , Interleucina-18 , Interleucina-1beta , ARN Interferente Pequeño , Factor de Necrosis Tumoral alfaRESUMEN
Although microglial activation is induced by an increase in chemokines, the role of mitophagy in this process remains unclear. This study aimed to elucidate the role of microglial mitophagy in CKLF/CKLF1 (chemokine-like factor 1)-induced microglial activation and neuroinflammation, as well as the underlying molecular mechanisms following CKLF treatment. This study determined that CKLF, an inducible chemokine in the brain, leads to an increase in mitophagy markers, such as DNM1L, PINK1 (PTEN induced putative kinase 1), PRKN, and OPTN, along with a simultaneous increase in autophagosome formation, as evidenced by elevated levels of BECN1 and MAP1LC3B (microtubule-associated protein 1 light chain 3 beta)-II. However, SQSTM1, a substrate of autophagy, was also accumulated by CKLF treatment, suggesting that mitophagy flux was reduced and mitophagosomes accumulated. These findings were confirmed by transmission electron microscopy and confocal microscopy. The defective mitophagy observed in our study was caused by impaired lysosomal function, including mitophagosome-lysosome fusion, lysosome generation, and acidification, resulting in the accumulation of damaged mitochondria in microglial cells. Further analysis revealed that pharmacological blocking or gene-silencing of mitophagy inhibited CKLF-mediated microglial activation, as evidenced by the expression of the microglial marker AIF1 (allograft inflammatory factor 1) and the mRNA of proinflammatory cytokines (Tnf and Il6). Ultimately, defective mitophagy induced by CKLF results in microglial activation, as observed in the brains of adult mice. In summary, CKLF induces defective mitophagy, microglial activation, and inflammation, providing a potential approach for treating neuroinflammatory diseases.Abbreviation: 3-MA: 3-methyladenine; AIF1: allograft inflammatory factor 1; ANOVA: analysis of variance; BAF: bafilomycin A1; BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CKLF/CKLF1: chemokine-like factor 1; CNS: central nervous system; DMEM: Dulbecco's Modified Eagle Medium; DNM1L: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; IRF3: interferon regulatory factor 3; IgG: immunoglobulin G; LAMP1: lysosomal-associated membrane protein 1; LAPTM4A: lysosomal-associated protein transmembrane 4A; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; mRFP: monomeric red fluorescent protein; mtDNA: mitochondrial DNA; MTORC1: mechanistic target of rapamycin kinase complex 1; OPTN: optineurin; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PINK1: PTEN induced putative kinase 1; PLL: poly-L-lysine; PRKN: parkin RBR E3 ubiquitin protein ligase; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; VDAC: voltage-dependent anion channel.
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Enfermedades Mitocondriales , Mitofagia , Ratones , Animales , Mitofagia/genética , Autofagia , Proteína Sequestosoma-1/metabolismo , Microglía/metabolismo , ADN Mitocondrial/metabolismo , Enfermedades Mitocondriales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Quimiocinas/metabolismoRESUMEN
Extracellular vesicles (EVs) have emerged as novel diagnostic and therapeutic approaches for cardiovascular diseases. EVs derived from various origins exhibit distinct effects on the cardiovascular system. However, the application of native EVs is constrained due to their poor stabilities and limited targeting capabilities. Currently, targeted modification of EVs primarily involves genetic engineering, chemical modification (covalent, non-covalent), cell membrane modification, and biomaterial encapsulation. These techniques enhance the stability, biological activity, target-binding capacity, and controlled release of EVs at specific cells and tissues. The diverse origins of cardioprotective EVs are covered, and the applications of cardiac-targeting EV delivery systems in protecting against cardiovascular diseases are discussed. This review summarizes the current stage of research on the potential of EV-based targeted therapies for addressing cardiovascular disorders.
Asunto(s)
Enfermedades Cardiovasculares , Vesículas Extracelulares , Humanos , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/terapia , Enfermedades Cardiovasculares/metabolismo , Vesículas Extracelulares/metabolismo , Corazón , Membrana CelularRESUMEN
Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the "fake" m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.