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Proceedings of the National Academy of... Jun 2024The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N-methyladenosine (mA),...
The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N-methyladenosine (mA), N-methyladenosine (mA), 3-methylthymine (mT), and 3-methyluracil (mU) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the mA, mA, or mT modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Ketoglutaric Acids; Iron; Humans; Substrate Specificity; Adenosine; Protein Conformation; Uracil; Molecular Dynamics Simulation; Thymine
PubMed: 38865275
DOI: 10.1073/pnas.2404457121 -
BMJ (Clinical Research Ed.) Jun 2024To assess the effect of different antiplatelet strategies on clinical outcomes after coronary artery bypass grafting. (Randomized Controlled Trial)
Randomized Controlled Trial
OBJECTIVE
To assess the effect of different antiplatelet strategies on clinical outcomes after coronary artery bypass grafting.
DESIGN
Five year follow-up of randomised Different Antiplatelet Therapy Strategy After Coronary Artery Bypass Grafting (DACAB) trial.
SETTING
Six tertiary hospitals in China; enrolment between July 2014 and November 2015; completion of five year follow-up from August 2019 to June 2021.
PARTICIPANTS
500 patients aged 18-80 years (including 91 (18.2%) women) who had elective coronary artery bypass grafting surgery and completed the DACAB trial.
INTERVENTIONS
Patients were randomised 1:1:1 to ticagrelor 90 mg twice daily plus aspirin 100 mg once daily (dual antiplatelet therapy; n=168), ticagrelor monotherapy 90 mg twice daily (n=166), or aspirin monotherapy 100 mg once daily (n=166) for one year after surgery. After the first year, antiplatelet therapy was prescribed according to standard of care by treating physicians.
MAIN OUTCOME MEASURES
The primary outcome was major adverse cardiovascular events (a composite of all cause death, myocardial infarction, stroke, and coronary revascularisation), analysed using the intention-to-treat principle. Time-to-event analysis was used to compare the risk between treatment groups. Multiple post hoc sensitivity analyses examined the robustness of the findings.
RESULTS
Follow-up at five years for major adverse cardiovascular events was completed for 477 (95.4%) of 500 patients; 148 patients had major adverse cardiovascular events, including 39 in the dual antiplatelet therapy group, 54 in the ticagrelor monotherapy group, and 55 in the aspirin monotherapy group. Risk of major adverse cardiovascular events at five years was significantly lower with dual antiplatelet therapy versus aspirin monotherapy (22.6% 29.9%; hazard ratio 0.65, 95% confidence interval 0.43 to 0.99; P=0.04) and versus ticagrelor monotherapy (22.6% 32.9%; 0.66, 0.44 to 1.00; P=0.05). Results were consistent in all sensitivity analyses.
CONCLUSIONS
Treatment with ticagrelor dual antiplatelet therapy for one year after surgery reduced the risk of major adverse cardiovascular events at five years after coronary artery bypass grafting compared with aspirin monotherapy or ticagrelor monotherapy.
TRIAL REGISTRATION
NCT03987373ClinicalTrials.gov NCT03987373.
Topics: Humans; Coronary Artery Bypass; Platelet Aggregation Inhibitors; Female; Male; Middle Aged; Ticagrelor; Aspirin; Aged; Follow-Up Studies; Adult; Aged, 80 and over; Drug Therapy, Combination; Adolescent; Postoperative Complications; Treatment Outcome; Young Adult; China; Dual Anti-Platelet Therapy
PubMed: 38862179
DOI: 10.1136/bmj-2023-075707 -
Stroke Jul 2024The atherosclerotic sources of embolism are a significant contributor to embolic stroke of undetermined source (ESUS). However, there is limited evidence for the... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
The atherosclerotic sources of embolism are a significant contributor to embolic stroke of undetermined source (ESUS). However, there is limited evidence for the efficacy of intensive dual antiplatelet therapy for ESUS. We conducted an investigation to determine whether gene-directed dual antiplatelet therapy could reduce the risk of recurrent stroke in patients with ESUS.
METHODS
CHANCE-2 (Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events-II) was an investigator-initiated, multicenter, randomized, double-blind, placebo-controlled trial that objectively compared ticagrelor plus aspirin and clopidogrel plus aspirin in patients with minor stroke or transient ischemic attack who carried loss-of-function alleles in China. All study participants were classified into ESUS and non-ESUS groups for the prespecified exploratory analysis. Cox proportional hazards models were used to assess the interaction of the state of ESUS with the effects of dual antiplatelet therapy with ticagrelor-aspirin versus clopidogrel-aspirin, adjusting for sociodemographic and clinical factors.
RESULTS
The subgroup analysis comprised 5796 participants (90.4% of the total 6412 participants) in the CHANCE-2 trial, with a median age of 64.9 years (range, 57.0-71.4 years), of whom 1964 (33.9%) were female. These participants underwent diffusion-weighted imaging as part of the study protocol. After systematic evaluation, 15.2% of patients (881/5796) were deemed to have ESUS. The incidence of stroke recurrence in patients with ESUS was found to be 5.6% in the ticagrelor-aspirin group and 9.2% in the clopidogrel-aspirin group (hazard ratio, 0.57 [95% CI, 0.33-0.99]; =0.04). In patients without ESUS, the respective incidence rates were 5.6% and 7.5% (hazard ratio, 0.72 [95% CI, 0.58-0.90]; <0.01). The value was 0.56 for the treatment × ESUS status interaction effect.
CONCLUSIONS
In this prespecified exploratory analysis, ticagrelor with aspirin was superior to clopidogrel with aspirin for preventing stroke at 90 days in patients with acute ischemic stroke or transient ischemic attack who carried loss-of-function alleles and were classified as ESUS.
REGISTRATION
URL: https://www.clinicaltrials.gov; Unique identifier: NCT04078737.
Topics: Humans; Middle Aged; Female; Male; Platelet Aggregation Inhibitors; Aged; Clopidogrel; Aspirin; Ticagrelor; Double-Blind Method; Dual Anti-Platelet Therapy; Embolic Stroke; Cytochrome P-450 CYP2C19; Stroke
PubMed: 38860396
DOI: 10.1161/STROKEAHA.124.046834 -
Cell Death & Disease Jun 2024Diabetic cardiomyopathy (DCM) is a prevalent myocardial microvascular complication of the myocardium with a complex pathogenesis. Investigating the pathogenesis of DCM...
Diabetic cardiomyopathy (DCM) is a prevalent myocardial microvascular complication of the myocardium with a complex pathogenesis. Investigating the pathogenesis of DCM can significantly contribute to enhancing its prevention and treatment strategies. Our study revealed an upregulation of lysine acetyltransferase 2 A (Kat2a) expression in DCM, accompanied by a decrease in N6-methyladenosine (m6A) modified Kat2a mRNA levels. Our study revealed an upregulation of lysine acetyltransferase 2 A (Kat2a) expression in DCM, accompanied by a decrease in N6-methyladenosine (m6A) modified Kat2a mRNA levels. Functionally, inhibition of Kat2a effectively ameliorated high glucose-induced cardiomyocyte injury both in vitro and in vivo by suppressing ferroptosis. Mechanistically, Demethylase alkB homolog 5 (Alkbh5) was found to reduce m6A methylation levels on Kat2a mRNA, leading to its upregulation. YTH domain family 2 (Ythdf2) played a crucial role as an m6A reader protein mediating the degradation of Kat2a mRNA. Furthermore, Kat2a promoted ferroptosis by increasing Tfrc and Hmox1 expression via enhancing the enrichment of H3K27ac and H3K9ac on their promoter regions. In conclusion, our findings unveil a novel role for the Kat2a-ferroptosis axis in DCM pathogenesis, providing valuable insights for potential clinical interventions.
Topics: Diabetic Cardiomyopathies; Animals; Ferroptosis; Humans; Heme Oxygenase-1; Mice; Histone Acetyltransferases; Male; Mice, Inbred C57BL; Myocytes, Cardiac; Membrane Proteins; Adenosine
PubMed: 38858351
DOI: 10.1038/s41419-024-06771-x -
Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells.Journal of Visualized Experiments : JoVE May 20248-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression...
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H2O2). We determined the concentration of H2O2 that induced oxidative damage in MCF-7 cells by detecting its IC50 value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H2O2 for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H2O2. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.
Topics: Humans; DNA Damage; 8-Hydroxy-2'-Deoxyguanosine; MCF-7 Cells; Enzyme-Linked Immunosorbent Assay; Hydrogen Peroxide; Oxidative Stress; Deoxyguanosine
PubMed: 38856223
DOI: 10.3791/66888 -
Redox Biology Aug 2024Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a...
Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a leading cause of poor prognosis in clinical patients. However, the underlying mechanisms of lactate's involvement in sepsis-associated ALI remain unclear. In this study, we demonstrate that lactate regulates N6-methyladenosine (m6A) modification levels by facilitating p300-mediated H3K18la binding to the METTL3 promoter site. The METTL3-mediated m6A modification is enriched in ACSL4, and its mRNA stability is regulated through a YTHDC1-dependent pathway. Furthermore, short-term lactate stimulation upregulates ACSL4, which promotes mitochondria-associated ferroptosis. Inhibition of METTL3 through knockdown or targeted inhibition effectively suppresses septic hyper-lactate-induced ferroptosis in alveolar epithelial cells and mitigates lung injury in septic mice. Our findings suggest that lactate induces ferroptosis via the GPR81/H3K18la/METTL3/ACSL4 axis in alveolar epithelial cells during sepsis-associated ALI. These results reveal a histone lactylation-driven mechanism inducing ferroptosis through METTL3-mediated m6A modification. Targeting METTL3 represents a promising therapeutic strategy for patients with sepsis-associated ALI.
Topics: Ferroptosis; Methyltransferases; Animals; Sepsis; Mice; Humans; Coenzyme A Ligases; Adenosine; Lung Injury; Acute Lung Injury; Male; Disease Models, Animal; Lactic Acid
PubMed: 38852200
DOI: 10.1016/j.redox.2024.103194 -
Biology Direct Jun 2024The molecular mechanisms of osteosarcoma (OS) are complex. In this study, we focused on the functions of melanoma cell adhesion molecule (MCAM), methyltransferase 3...
BACKGROUND
The molecular mechanisms of osteosarcoma (OS) are complex. In this study, we focused on the functions of melanoma cell adhesion molecule (MCAM), methyltransferase 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in OS development.
METHODS
qRT-PCR assay and western blot assay were performed to determine mRNA and protein expression of MCAM, METTL3, IGF2BP1 and YY1. MTT assay and colony formation assay were conducted to assess cell proliferation. Cell apoptosis, invasion and migration were evaluated by flow cytometry analysis, transwell assay and wound-healing assay, respectively. Methylated RNA Immunoprecipitation (MeRIP), dual-luciferase reporter, Co-IP, RIP and ChIP assays were performed to analyze the relationships of MCAM, METTL3, IGF2BP1 and YY1. The functions of METTL3 and MCAM in tumor growth were explored through in vivo experiments.
RESULTS
MCAM was upregulated in OS, and MCAM overexpression promoted OS cell growth, invasion and migration and inhibited apoptosis. METTL3 and IGF2BP1 were demonstrated to mediate the m6A methylation of MCAM. Functionally, METTL3 or IGF2BP1 silencing inhibited OS cell progression, while MCAM overexpression ameliorated the effects. Transcription factor YY1 promoted the transcription level of METTL3 and regulated METTL3 expression in OS cells. Additionally, METTL3 deficiency suppressed tumor growth in vivo, while MCAM overexpression abated the effect.
CONCLUSION
YY1/METTL3/IGF2BP1/MCAM axis aggravated OS development, which might provide novel therapy targets for OS.
Topics: Osteosarcoma; Methyltransferases; Humans; RNA-Binding Proteins; Adenosine; Cell Line, Tumor; Animals; Mice; Cell Proliferation; Bone Neoplasms; Disease Progression; Mice, Nude; Apoptosis; Cell Movement; Gene Expression Regulation, Neoplastic
PubMed: 38849910
DOI: 10.1186/s13062-024-00486-x -
Nature Communications Jun 2024N-methyladenosine (mA) plays critical roles in regulating mRNA metabolism. However, comprehensive mA methylomes in different plant tissues with single-base precision...
N-methyladenosine (mA) plays critical roles in regulating mRNA metabolism. However, comprehensive mA methylomes in different plant tissues with single-base precision have yet to be reported. Here, we present transcriptome-wide mA maps at single-base resolution in different tissues of rice and Arabidopsis using mA-SAC-seq. Our analysis uncovers a total of 205,691 mA sites distributed across 22,574 genes in rice, and 188,282 mA sites across 19,984 genes in Arabidopsis. The evolutionarily conserved mA sites in rice and Arabidopsis ortholog gene pairs are involved in controlling tissue development, photosynthesis and stress response. We observe an overall mRNA stabilization effect by 3' UTR mA sites in certain plant tissues. Like in mammals, a positive correlation between the mA level and the length of internal exons is also observed in plant mRNA, except for the last exon. Our data suggest an active mA deposition process occurring near the stop codon in plant mRNA. In addition, the MTA-installed plant mRNA mA sites correlate with both translation promotion and translation suppression, depicting a more complicated regulatory picture. Our results therefore provide in-depth resources for relating single-base resolution mA sites with functions in plants and uncover a suppression-activation model controlling mA biogenesis across species.
Topics: Oryza; Arabidopsis; Adenosine; Gene Expression Regulation, Plant; RNA, Messenger; Transcriptome; RNA, Plant; 3' Untranslated Regions; Gene Expression Profiling; RNA Stability; Exons; Codon, Terminator
PubMed: 38849358
DOI: 10.1038/s41467-024-48941-7 -
Virus Research Aug 2024The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to...
The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to proteome diversity and regulation in metazoans including humans. In addition to its transcriptome-regulating role, ADARs also play a major part in immune response to viral infection, where an interferon response activates interferon-stimulated genes, such as ADARp150, in turn dynamically regulating host-virus interactions. A previous report has shown that infection from reoviruses, despite strong activation of ADARp150, does not influence the editing of some of the major known editing targets, while likely editing others, suggesting a potentially nuanced editing pattern that may depend on different factors. However, the results were based on a handful of selected editing sites and did not cover the entire transcriptome. Thus, to determine whether and how reovirus infection specifically affects host ADAR editing patterns, we analyzed a publicly available deep-sequenced RNA-seq dataset, from murine fibroblasts infected with wild-type and mutant reovirus strains that allowed us to examine changes in editing patterns on a transcriptome-wide scale. To the best of our knowledge, this is the first transcriptome-wide report on host editing changes after reovirus infection. Our results demonstrate that reovirus infection induces unique nuanced editing changes in the host, including introducing sites uniquely edited in infected samples. Genes with edited sites are overrepresented in pathways related to immune regulation, cellular signaling, metabolism, and growth. Moreover, a shift in editing targets has also been observed, where the same genes are edited in infection and control conditions but at different sites, or where the editing rate is increased for some and decreased for other differential targets, supporting the hypothesis of dynamic and condition-specific editing by ADARs.
Topics: Animals; Mice; Fibroblasts; Transcriptome; RNA Editing; Inosine; Adenosine Deaminase; Adenosine; Reoviridae Infections; Host-Pathogen Interactions; RNA-Binding Proteins; Reoviridae
PubMed: 38848818
DOI: 10.1016/j.virusres.2024.199413 -
Critical Reviews in Immunology 2024Sustained expression of the long noncoding RNA (lncRNA) LINC01106 in tumors is crucial for the malignant phenotype of tumor cells. Nevertheless, the mechanisms and...
BACKGROUND
Sustained expression of the long noncoding RNA (lncRNA) LINC01106 in tumors is crucial for the malignant phenotype of tumor cells. Nevertheless, the mechanisms and clinical effects of LINC01106 in lung adenocarcinoma (LUAD) are limited. This study shows the effect of vir-like m6A methyltransferase-associated (KIAA1429)-mediated N6-methyladenosine (m6A) modification on steady LINC01106 expression on LUAD progression.
METHODS
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine LINC01106 and KIAA1429 levels in LUAD tissues. Transwell, 5-ethynyl-2'-deoxyuridine (EdU), and cell counting kit-8 (CCK-8) assays were used to analyze the functional roles of LINC01106. A xenograft was constructed to verify the function of silencing LINC01106 in tumor growth. The regulatory role of LINC01106 was investigated using methylated RNA immunoprecipitation (MeRIP), qRT-PCR, and the actinomycin D assay. Western blotting was used to identify key proteins in the JAK/STAT3 (JAK2, STAT3) pathway.
RESULTS
LINC01106 and KIAA1429 were highly expressed in LUAD, and LINC01106 was interconnected with high tumor grade, stage, and poor prognosis. Data revealed that LINC01106 inhibition reduced LUAD cell proliferation, invasion, and migration and restrained LUAD cell tumorigenicity. In addition, LINC01106 silencing reduced phosphorylated JAK2 and STAT3 levels. KIAA1429-mediated LINC01106 enhances its m6A modification and expression in LUAD cells. Moreover, KIAA1429 promotion eliminated the malignant phenotypic suppression induced by low expression in LUAD cells.
CONCLUSION
This study showed that KIAA1429 enhanced LINC01106 m6A modification to promote LUAD development. These results may lead to a better understanding of the mechanism of KIAA1429-m6A-LINC01106 in LUAD and offer a valuable therapeutic target for LUAD.
Topics: Humans; RNA, Long Noncoding; STAT3 Transcription Factor; Adenocarcinoma of Lung; Animals; Lung Neoplasms; Mice; Signal Transduction; Cell Proliferation; Gene Expression Regulation, Neoplastic; Cell Line, Tumor; Adenosine; Methyltransferases; Mice, Nude; Janus Kinase 2; Cell Movement; Female; Janus Kinases; Male; RNA-Binding Proteins
PubMed: 38848293
DOI: 10.1615/CritRevImmunol.2024052728