RESUMEN
Thousands of long noncoding RNAs (lncRNAs) have been annotated via high-throughput RNA sequencing, yet only a small fraction have been functionally investigated. Genomic knockout is the mainstream strategy for studying the biological function of protein-coding genes and lncRNAs, whereas the complexity of the lncRNA locus, especially the natural antisense lncRNAs (NAT-lncRNAs), presents great challenges. Knocking out lncRNAs often results in unintended disruptions of neighboring protein-coding genes and small RNAs, leading to ambiguity in observing phenotypes and interpreting biological function. To address this issue, we launched LncRNAway, a user-friendly web tool based on the BESST (branchpoint to 3' splicing site targeting) method, to design sgRNAs for lncRNA knockout. LncRNAway not only provides specific and effective lncRNA knockout guidelines but also integrates genotyping primers and quantitative PCR primers designing, thereby streamlining experimental procedures of lncRNA function study. LncRNAway is freely available at https://www.lncrnaway.com.
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Internet , ARN Guía de Sistemas CRISPR-Cas , ARN Largo no Codificante , Programas Informáticos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Humanos , Técnicas de Inactivación de Genes , Sistemas CRISPR-CasRESUMEN
Miscarriage is a common complication during early pregnancy and affects approximately 10%-15% of all pregnant women. Several studies have reported that the abnormal expression of mitochondrial oxidative stress-related genes might be involved in the occurrence and progression of miscarriage. The present study attempted to uncover the role of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in miscarriage chorionic villous tissue. The hypothesis that PGC-1α is crucial for glycolysis and oxidative phosphorylation during early pregnancy was tested. The results showed that the mRNA and protein levels of PGC-1α were significantly increased in the miscarriage chorionic villous tissues compared with the artificial selective abortion group, and that the expression was regulated by mTOR in knockdown and overexpression of mTOR in HTR8 cell lines. PGC-1α also promoted mitochondrion oxidative phosphorylation but inhibited glycolysis process. In addition, PGC-1α could drive ROS production, reduce mitochondrial membrane potential and block NADPH synthesis, resulting in cell cycle arrest and cell apoptosis, eventually leading to miscarriage. These results suggested that the aberrant expression of PGC-1α is involved in the etiology of early miscarriage, providing new perspectives regarding the mechanisms of miscarriage and a potential therapeutic target for miscarriage.
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Aborto Espontáneo , Embarazo , Humanos , Femenino , Aborto Espontáneo/genética , Transducción de Señal/genética , Apoptosis , Estrés Oxidativo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of â¼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.
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Técnicas de Inactivación de Genes , Genoma , Genómica , ARN Largo no Codificante , Animales , Exones/genética , Técnicas de Inactivación de Genes/métodos , Técnicas de Inactivación de Genes/normas , Genoma/genética , Poli A/genética , Poli A/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genéticaRESUMEN
Diabetic cardiomyopathy (DCM) is a myocardial disease independent of other cardiovascular diseases, such as coronary heart disease, hypertension, etc. Lipotoxicity is closely related to DCM. In this study, we investigated the mechanism of lipid metabolism disturbance in DCM in HL-1 cells. Through bioinformatics and Western blotting analysis, we found that canagliflozin (CAN) significantly inhibited the expression of inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Ferroptosis is mediated by lipid peroxidation. We demonstrated the presence of ferroptosis in cardiomyocytes by detecting intracellular Fe2+ content and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), and mitochondrial membrane potential (MMP). CAN could significantly regulate the indicators of ferroptosis. By using specific inhibitors celecoxib (coxib), S-methylisothiourea sulfate (SMT), Ferrostatin-1 (Fer-1), and Compound C, we further found that CAN regulated inflammation and ferroptosis through AMP-activated protein (AMPK), and inflammation interacted with ferroptosis. Our study indicated that CAN attenuated lipotoxicity in cardiomyocytes by regulating inflammation and ferroptosis through activating the AMPK pathway. This study provides a new direction of myocardial lipotoxicity and some new information for the treatment of DCM.
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Canagliflozina , Cardiomiopatías Diabéticas , Ferroptosis , Peroxidación de Lípido , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Proteínas Quinasas Activadas por AMP , Canagliflozina/uso terapéutico , Cardiomiopatías Diabéticas/tratamiento farmacológico , Ferroptosis/efectos de los fármacos , Inflamación/tratamiento farmacológico , Miocitos Cardíacos , Especies Reactivas de Oxígeno , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéuticoRESUMEN
Diabetic foot infection (DFI) is a common complication in diabetes patients, with foot infections being the leading cause of amputations. Staphylococcus aureus is frequently found in diabetic foot infections, of which methicillin-resistant Staphylococcus aureus (MRSA) has become a major clinical and epidemiological challenge. Since MRSA strains are resistant to most ß-lactam antibiotics, and also partially resistant to other antibiotics, treatment is difficult and costly. The emergence of drug-resistant bacteria often arises from overuse or misuse of antibiotics. Clinically, canagliflozin is commonly used for the treatment of type 2 diabetes. On this basis, we investigated the antibacterial activity and mechanism of canagliflozin against MRSA, with the aim to discover novel functions of canagliflozin and provide new insights for the treatment of MRSA. Using the microbroth dilution method to determine the half maximal inhibitory concentration of drugs, we found that canagliflozin not only can inhibit the growth of methicillin-sensitive Staphylococcus aureus (MSSA) but also exhibits antibacterial activity against MRSA. The IC50 values, at approximately 56.01 µM and 57.60 µM, were almost the same. At 12 h, canagliflozin showed a significant antibacterial effect against MRSA at and above 30 µM. In addition, its combined use with penicillin achieved better antibacterial effects, which were increased by about three times. Additive antibacterial activity (FICI = 0.69) was found between penicillin and canagliflozin, which was better than that of doxycycline and canagliflozin (FICI = 0.95). Canagliflozin also affected bacterial metabolic markers, such as glucose, ATP, and lactic acid. The results of crystal violet staining indicate that canagliflozin disrupted the formation of bacterial biofilm. Our electron microscopy results showed that canagliflozin distorted the bacterial cell wall. The results of RT-PCR suggest that canagliflozin down-regulated the expressions of biofilm-related gene (clfA, cna, agrC, mgrA, hld) and methicillin-resistance gene (mecA), which was related to MRSA. Molecular docking also indicated that canagliflozin affected some interesting targets of MRSA, such as the sarA, crtM and fnbA proteins. In conclusion, canagliflozin exhibits antibacterial activity against MRSA by affecting bacterial metabolism, inhibiting its biofilm formation, distorting the bacterial cell wall, and altering the gene expression of biofilm formation and its virulence. Our study reveals the antibacterial activity of canagliflozin against MRSA, providing a new reference for treating diabetic foot infections.
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Endometrium decidualization is a complex biological process, which includes the interplay of transcription factors, cytokines, cell cycle regulators, and other signaling pathways. However, the underlying molecular mechanisms of this process are not fully elucidated to date. In this study, we aimed to investigate the possible association between autophagy and recurrent implantation failure (RIF). A total of 81 genes were downregulated and 231 genes were upregulated in the RIF group compared with the control group, and the differences were statistically significant (p < 0.05). Further, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway were analyzed, and we found that some autophagy markers, for example, LC3-II, LAMP2, and HIF-1α were significantly increased, whereas P62 was drastically downregulated in the RIF group. Similar results were observed in proteins level; and the autophagy puncta were also markedly enhanced in the endometrial tissues of RIF patients. Autophagy is closely associated with the RIF occurs and may be involved in the pathogenesis of RIF.
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Implantación del Embrión , Endometrio , Femenino , Humanos , Implantación del Embrión/genética , Endometrio/metabolismo , Factores de Transcripción/genética , Genes Reguladores , Autofagia/genéticaRESUMEN
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) has been identified as a group of enzymes that catalyze cytosine deamination in single-stranded (ss) DNA to form uracil, causing somatic mutations in some cancers. We analyzed the APOBEC3 family in 33 TCGA cancer types and the results indicated that APOBEC3s are upregulated in multiple cancers and strongly correlate with prognosis, particularly in low grade glioma (LGG). Then we constructed a prognostic model based on family expression in LGG where the APOBEC3 family signature is an accurate predictive model (AUC of 0.85). Gene mutation, copy number variation (CNV), and a differential gene expression (DEG) analysis were performed in different risk groups, and the weighted gene co-expression network analysis (WGCNA) was employed to clarify the role of various members in LGG; CIBERSORT algorithm was deployed to evaluate the landscape of LGG immune infiltration. We found that upregulation of the APOBEC3 family expression can strengthen Ras/MAPK signaling pathway, promote tumor progression, and ultimately reduce the treatment benefits of Raf inhibitors. Moreover, the APOBEC3 family was shown to enhance the immune response mediated by myeloid cells and interferon gamma, as well as PD-L1 and PD-L2 expression, implying that they have immunotherapy potential. Therefore, the APOBEC3 signature enables an efficient assessment of LGG patient survival outcomes and expansion of clinical benefits by selecting appropriate individualized treatment strategies.
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Desaminasas APOBEC , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma , Modelos Biológicos , Inhibidores de Proteínas Quinasas/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Quinasas raf , Desaminasas APOBEC/biosíntesis , Desaminasas APOBEC/genética , Supervivencia sin Enfermedad , Femenino , Glioma/tratamiento farmacológico , Glioma/enzimología , Glioma/genética , Glioma/mortalidad , Humanos , Masculino , Tasa de Supervivencia , Quinasas raf/antagonistas & inhibidores , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
RNA-binding proteins are frequently dysregulated in human cancer and able to modulate tumor cell proliferation as well as tumor metastasis through post-transcriptional regulation on target genes. Abnormal DNA damage response and repair mechanism are closely related to genome instability and cell transformation. Here, we explore the function of the RNA-binding protein muscleblind-like splicing regulator 2 (MBNL2) on tumor cell proliferation and DNA damage response. Transcriptome and gene expression analysis show that the PI3K/AKT pathway is enriched in MBNL2-depleted cells, and the expression of cyclin-dependent kinase inhibitor 1A (p21CDKN1A) is significantly affected after MBNL2 depletion. MBNL2 modulates the mRNA and protein levels of p21, which is independent of its canonical transcription factor p53. Moreover, depletion of MBNL2 increases the phosphorylation levels of checkpoint kinase 1 (Chk1) serine 345 (S345) and DNA damage response, and the effect of MBNL2 on DNA damage response is p21-dependent. MBNL2 would further alter tumor cell fate after DNA damage, MBNL2 knockdown inhibiting DNA damage repair and DNA damage-induced senescence, but promoting DNA damage-induced apoptosis.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ARN/genética , Apoptosis/genética , Proliferación Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HCT116 , Células HeLa , Humanos , Fosforilación , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The microenvironment plays a vital role in tumor progression, and hypoxia is a typical microenvironment feature in nearly all solid tumors. In this study, we focused on elucidating the effect of canagliflozin (CANA), a new class of antidiabetic agents, on hepatocarcinoma (HCC) tumorigenesis under hypoxia, and demonstrated that CANA could significantly inhibit hypoxia-induced metastasis, angiogenesis, and metabolic reprogramming in HCC. At the molecular level, this was accompanied by a reduction in VEGF expression level, as well as a reduction in the epithelial-to-mesenchymal transition (EMT)-related proteins and glycolysis-related proteins. Next, we focused our study particularly on the modulation of HIF-1α by CANA, which revealed that CANA decreased HIF-1α protein level by inhibiting its synthesis without affecting its proteasomal degradation. Furthermore, the AKT/mTOR pathway, which plays an important role in HIF-1α transcription and translation, was also inhibited by CANA. Thus, it can be concluded that CANA decreased metastasis, angiogenesis, and metabolic reprogramming in HCC by inhibiting HIF-1α protein accumulation, probably by targeting the AKT/mTOR pathway. Based on our results, we propose that CANA should be evaluated as a new treatment modality for liver cancer.
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Canagliflozina/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Glucólisis/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Proteínas Proto-Oncogénicas c-akt/genética , Serina-Treonina Quinasas TOR/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.
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Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , ARN Largo no Codificante/metabolismo , Acetilcoenzima A/metabolismo , Acetilación/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacología , Animales , Caveolina 2/antagonistas & inhibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Modelos Animales de Enfermedad , Epigénesis Genética , Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Ratones , Ratones Transgénicos , Neuroglía/citología , Neuroglía/metabolismo , Fragmentos de Péptidos/farmacología , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Crecimiento Transformador beta2/antagonistas & inhibidores , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Autophagy dysregulation has emerged in age-related neurological diseases (Ulland et al.; Matheoud et al.; Ashkenazi et al.). Alzheimer Disease (AD), the most common progressive neurodegenerative disorder, is characterized by the accumulation of amyloid-ß (Aß) plaques caused by aberrant Aß metabolism (Qiang et al.; Sevigny et al.; Ittner et al.). Glia constitute the brain immune system and ingest extracellular Aß for degradation via the autophagy-lysosome machinery (Ries and Sastre; Cho et al.). Here, we model the molecular rationale for this clearance process in glioma cells by showing that miR34a inhibits autophagy-mediated disposal of Aß fibrils and identifying two novel direct targets of miR34a, endophilin-3 and cathepsin B (CTSB, a previously reported enzyme for Aß degrading (Sun et al.)). Bioinformatics analyses revealed that endophilin-3 expresses at a significantly lower level in neurodegenerative diseases. Its gain-of-function substantially promotes both uptake and degradation of Aß while small interfering RNA (siRNA)-mediated endophilin-3 knockdown slowed down Aß clearance and blocked autolysosome formation. Mechanistically, gene ontology (GO) analysis of the endophilin-3 interactome identified by mass spectrometry uncovered enriched components involved in actin binding (with the highest score). Importantly, we validated that the actin-binding protein phostensin interacted with endophilin-3. Phostensin knockdown restored endophilin-3-mediated up-regulation of Aß clearance. Thus, our findings indicate that miR34a inhibits Aß clearance by targeting endophilin-3 and CTSB at multiple steps including uptake and autophagy-mediated degradation.
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Long noncoding RNAs (lncRNA) are reported to be potential cancer biomarkers. This study aims to find new lncRNA biomarker relevant to lung adenocarcinoma. Gene expression profile and clinical data of lung adenocarcinoma and lung squamous cell carcinoma patients were downloaded from the UCSC Xena database. These data were analyzed to identify potential lncRNA prognostic biomarkers, and the candidate lncRNAs were analyzed and verified with association analysis, meta-analysis, survival analysis, gene ontology analysis, gene set enrichment analysis, and other statistical methods. A group of 5 lncRNAs was identified from the 1965 differentially expressed (fold-change >2) genes. Four of these 5 lncRNAs were expressed at a lower level in lung adenocarcinoma tissues and the other one at a higher level (P < .0001). A risk score model was constructed using a linear combination of the expression levels of these lncRNAs. High-risk patients showed poorer overall survival (hazard ratio [HR] = 2.14; 95% confidence interval [CI], 1.67-3.06, P < .0001), disease-free survival (HR = 1.84; 95% CI, 1.26-2.35, P = .0007), and recurrence-free survival (HR = 1.51; 95% CI, 1.02-2.40, P = .04). The 5-fold cross-validation and subsequent meta-analysis further verified that patients in the low-risk group had better survival (95% CI, 0.74-1.79, Z = 4.72, P < .00001). Furthermore, both univariate and multivariate Cox regression analyses revealed that the prognostic value of these 5 lncRNAs was independent of other clinical prognostic factors. Further analysis indicated that these 5 lncRNAs might be associated with tumor metastasis. Taken together, our study suggests new prognostic lncRNA biomarkers for lung adenocarcinoma.
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Adenocarcinoma del Pulmón/genética , Biomarcadores de Tumor/genética , Minería de Datos/métodos , Neoplasias Pulmonares/genética , ARN Largo no Codificante/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Pronóstico , Análisis de Regresión , Análisis de SupervivenciaRESUMEN
Nuclear paraspeckle assembly transcript 1 (NEAT1) is the crucial structural platform of paraspeckles, which is one type of nuclear bodies. As a stress-induced lncRNA, the expression of NEAT1 increases in response to viral infection, but little is known about the role of NEAT1 or paraspeckles in the replication of herpes simplex virus-1 (HSV-1). Here, we demonstrate that HSV-1 infection increases NEAT1 expression and paraspeckle formation in a STAT3-dependent manner. NEAT1 and other paraspeckle protein components, P54nrb and PSPC1, can associate with HSV-1 genomic DNA. By binding with STAT3, PSPC1 is required for the recruitment of STAT3 to paraspeckles and facilitates the interaction between STAT3 and viral gene promoters, finally increasing viral gene expression and viral replication. Furthermore, thermosensitive gel containing NEAT1 siRNA or STAT3 siRNA effectively healed the skin lesions caused by HSV-1 infection in mice. Our results provide insight into the roles of lncRNAs in the epigenetic control of viral genes and into the function of paraspeckles.
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Genes Virales , Herpesvirus Humano 1/fisiología , ARN Largo no Codificante/metabolismo , Transcripción Genética , Replicación Viral/genética , Animales , Secuencia de Bases , Proteínas de Unión al ADN , Regulación Viral de la Expresión Génica , Células HeLa , Herpes Simple/virología , Herpesvirus Humano 1/genética , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Ratones , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.
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Autofagia , Reparación del ADN/genética , Genes Letales , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Recombinación Homóloga , Ratones , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: To evaluate the association of two common methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with recurrent miscarriage (RM) and repeated implantation failure (RIF) METHODS: The study comprised of 521 patients, with a history of RM (n = 370) or RIF (n = 151). One hundred forty-four women with fallopian tube blockages who had successfully conceived after the first in vitro fertilization embryo transfer treatment served as the control group. The MTHFR alleles, genotypes, and haplotypes were assessed in different groups. RESULTS: There was no difference in allele frequency and distribution of MTHFR polymorphisms between case and control patients. The 1298AA genotype was represented in a higher frequency, and 1298AC genotype was significantly lower in subfertile group when compared to the control group. A significant relationship was found between the 1298AC genotype and the RIF subgroup. The haplotype 677CC/1298AA was overrepresented in the RM subgroup (> 2 times) and haplotype 677CC/1298AC was underrepresented in the RIF subgroup (P < 0.05). Nevertheless, these two haplotypes were not connected to fertilization and embryo cleavage rates. CONCLUSION: Our findings indicate that the MTHFR gene polymorphism might play a role in the etiology of patients with RM or RIF. No adverse effects of different MTHFR haplotypes on embryo development were detected. Further studies on the biological role are needed to better understand the susceptibility to pregnancy complications.
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Aborto Habitual/genética , Implantación Tardía del Embrión/genética , Implantación del Embrión/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Aborto Habitual/fisiopatología , Adulto , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Humanos , Polimorfismo de Nucleótido Simple , EmbarazoRESUMEN
Insulin injection relies on strict blood glucose monitoring. However, existing techniques and algorithms for blood glucose monitoring cannot be completed in a timely way. In this study, we have developed a new intelligent glucose-sensitive insulin delivery system to stabilize blood glucose levels in the body. This system does not require real-time detection of blood glucose. First, we successfully synthesized a nanoscale material called PAM-PAspPBA-b-PEG by using chemical methods. We then conducted TEM, DLS, and ¹H-NMR analyses to characterize the physicochemical properties, such as size, molecular composition, and configuration of the nanomaterial. We verified the glucose responsibility of the insulin loading nanoscale material in vitro and evaluated its safety and effect on mice in vivo. Results showed that insulin-loaded PAM-PAspPBA-b-PEG is glucose-sensitive, safer and more effective than regular insulin injection. This study provides a basis for future development of smart insulin delivery systems.
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Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Insulina/administración & dosificación , Nanopartículas , Glucemia/efectos de los fármacos , Ácidos Borónicos/química , Dendrímeros , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/química , Insulina/química , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la Partícula , Péptidos/química , Poliaminas/químicaRESUMEN
Obesity and nonalcoholic fatty liver disease (NAFLD) are highly prevalent and cause numerous metabolic diseases. However, drugs for the prevention and treatment of obesity and NAFLD remain unavailable. In this study, we investigated the effects of mogrosides (luo han guo, LH) in Siraitia grosvenorii saponins on high-fat-diet-induced obesity and NAFLD in mice. We found that compared with the negative control, LH reduced body and liver weight. LH also decreased fat accumulation and increased AMP-activated protein kinase (AMPK) phosphorylation (pAMPK) levels in mouse livers. We also found that high-purity mogroside V upregulated pAMPK expression in HepG2 cells. In addition, high-purity mogroside V inhibited reactive oxygen species production and upregulated sequestosome-1 (SQSTM1, p62) expression in THP-1 cells. These results suggest that LH may affect obesity and NAFLD by enhancing fat metabolism and antioxidative defenses. Mogroside V may be a main component of LH. However, the exact molecular mechanisms and active components responsible for the inhibitory effects of LH on obesity and NAFLD require further investigation.
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Fármacos Antiobesidad/farmacología , Anticolesterolemiantes/farmacología , Momordica/química , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Triterpenos/farmacología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Fármacos Antiobesidad/química , Fármacos Antiobesidad/aislamiento & purificación , Anticolesterolemiantes/química , Anticolesterolemiantes/aislamiento & purificación , Peso Corporal/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Tamaño de los Órganos/efectos de los fármacos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Saponinas/química , Saponinas/aislamiento & purificación , Saponinas/farmacología , Proteína Sequestosoma-1/agonistas , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Células THP-1 , Triterpenos/química , Triterpenos/aislamiento & purificaciónRESUMEN
Numerous herbal-derived natural products are excellent anti-inflammatory agents. Several studies have reported that aloin, the major anthraquinone glycoside obtained from the Aloe species, exhibits anti-inflammatory activity. However, the molecular mechanism of this activity is not well understood. In this report, we found that aloin suppresses lipopolysaccharide-induced pro-inflammatory cytokine secretion and nitric oxide production, and downregulates the expression of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Aloin inhibits the phosphorylation and acetylation of the NF-κB p65 subunit by suppressing the upstream kinases p38 and Msk1, preventing LPS-induced p65 translocation to the nucleus. We have also shown that aloin inhibits LPS-induced caspase-3 activation and apoptotic cell death. Collectively, these findings suggest that aloin effectively suppresses the inflammatory response, primarily through the inhibition of NF-κB signaling.
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Antiinflamatorios/química , Apoptosis/efectos de los fármacos , Emodina/análogos & derivados , Inflamación/etiología , Inflamación/metabolismo , FN-kappa B/antagonistas & inhibidores , Animales , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Emodina/farmacología , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/efectos adversos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Modelos Biológicos , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Transporte de Proteínas , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismoRESUMEN
Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression.
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Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 1/fisiología , Precursores del ARN/biosíntesis , Empalme del ARN/fisiología , ARN Viral/biosíntesis , Factores de Empalme Serina-Arginina/metabolismo , Transcripción Genética/fisiología , Replicación Viral/fisiología , Animales , Chlorocebus aethiops , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Precursores del ARN/genética , ARN Viral/genética , Factores de Empalme Serina-Arginina/genética , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genética , Células VeroRESUMEN
BACKGROUND: Chlamydia, caused by the bacterium Chlamydia trachomatis(C. trachomatis), is the most common sexually transmitted disease. The incidence is not clear due to the asymptomatic nature of early stage of infections. The incidence of Chlamydia has not been fully investigated in the Chinese Han population. Since chronic infection with can C. trachomatis can lead to infertility in males and females, it is important to determine the impact of infection on clinical outcomes. The aim of this study is to explore the epidemiology of C. trachomatis in subfertile couples and to determine whether infections will adversely affect clinical outcomes after assisted reproduction technique (ART) treatment. METHODS: Subfertile patients (n = 30760) were screened in the research for C. trachomatis in our center from January 2010 to December 2014. C. trachomatis-specific DNA was detected by Taq-man PCR from semen or swabs from the urethral, endocervix or vaginal. The control group consisted of 1140 subfertile patients without C. trachomatis infection. The prevalence and characteristics of C. trachomatis were identified for subfertile couples and clinical outcomes were collected and analyzed. A retrospective study was performed. RESULTS: Nine hundred and seventy patients were diagnosed with C. trachomatis infection, and the overall prevalence was 3.15% in the most recent five years, with a yearly increasing. The incidence was a higher in the second half of the year (3.40%) compared to the first half (2.69%). The age group with the highest-risk of infection with C. trachomatis was between 26 to 35 years old, and in about one third of the couples, both partners were infected. The basic parameters and clinical outcomes were not statistically significant between different the groups (P > 0.05), even though some minor data were different (P < 0.05). CONCLUSIONS: C. trachomatis is a common infection in subfertile people and it is essential to test for this organism in ART couples' screening. This study identified no adverse on clinical outcomes after successful treatment of C. trachomatis infection, regardless of gender, age and number of C. trachomatis copies.