Search Results (369)

DNA methylation, mediated by DNA methyltransferases (DMTs) and demethylases (DMLs), is an important epigenetic modification that maintains genomic stability and regulates gene expression in plant growth, development, and stress responses. However, a comprehensive characterization of these gene families in Populus sect. Turanga remains lacking. In this study, eight PeDMT and two PeDML genes were identified in Populus euphratica, and six PpDMT and three PpDML genes in Populus pruinosa. Phylogenetic analysis revealed that DMTs and DMLs could be classified into four and three subfamilies, respectively. The analysis of cis-acting elements indicated that the promoter regions of both DMTs and DMLs were enriched with elements responsive to growth and development, light, phytohormones, and stress. Collinearity analysis detected three segmentally duplicated gene pairs (PeDMT5/8, PeDML1/2, and PpDML2/3), suggesting potential functional diversification. Transcriptome profiling showed that several PeDMTs and PeDMLs exhibited leaf shape- and developmental stage-specific expression patterns, with PeDML1 highly expressed during early stages and in broad-ovate leaves. Whole-genome bisulfite sequencing revealed corresponding decreases in DNA methylation levels, suggesting that active demethylation may contribute to heteromorphic leaf formation. Overall, this study provides significant insights for exploring the functions and expression regulation of plant DMTs and DMLs and will contribute to future research unraveling the molecular mechanisms of epigenetic regulation in P. euphratica. Full article

Epigenetic regulation, particularly DNA methylation, plays a crucial role in embryonic development by controlling gene expression patterns. The disruption of this regulation by environmental factors can have long-lasting consequences. Opioid drugs, such as morphine, are known to cross the placental barrier and affect the developing central nervous system, yet their precise epigenetic effects during early development remain unclear. This study aimed to elucidate the impact of chronic morphine exposure on the DNA methylation landscape and gene expression in mouse embryonic stem cells (mESCs). mESCs were chronically exposed to morphine (10 μM for 24 h). Genome-wide bisulfite sequencing was performed to identify DNA methylation changes, while RNA sequencing (RNA-Seq) assessed corresponding gene expression alterations. Global levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were quantified using mass spectrometry. Morphine exposure induced global DNA hypomethylation and identified 16,808 differentially methylated genes (DMGs) related to development, cell signalling, metabolism, and transcriptional regulation. Integrative transcriptomic analysis with RNA-Seq data revealed 651 overlapping genes, including alterations in key epigenetic regulators involved on DNA methylation machinery. Specifically, Tet1 was upregulated with promoter hypomethylation, while Dnmt1 was downregulated, without changes in promoter methylation after morphine exposiure. Mass spectrometry results confirmed a global decrease in 5mC levels alongside increased 5hmC, indicating the involvement of both passive and active demethylation pathways. These findings demonstrate for the first time that morphine disrupts the epigenetic homeostasis of mESCs by promoting global and gene-specific DNA demethylation, which might be key to the phenotypic changes that occur in adulthood. This work provides novel mechanistic insights into how opioid exposure during early development may lead to persistent epigenetic alterations, with potential long-term implications for neurodevelopment and disease susceptibility. Full article

Brown spot, caused by Alternaria alternata, is the most important leaf fungal disease threatening kiwifruit production in China, and it is typically controlled through the application of fungicides, such as procymidone and difenoconazole. To date, fungicide resistance development has not yet been systematically reported for the pathogen of kiwifruit. A total of 135 single-conidium A. alternata isolates were collected from different cities in Zhejiang Province, China. Alternaria alternata developed prevailing resistance to procymidone and initial resistance to difenoconazole, with resistance frequencies of 60.7 and 13.3%, respectively. Positive cross-resistance was observed between procymidone and iprodione but not between procymidone and difenoconazole, tebuconazole, prochloraz, pydiflumetofen, pyraclostrobin, or thiophanate-methyl. Moreover, no cross-resistance was observed between difenoconazole and all other tested fungicides, including the two other demethylation inhibitors, tebuconazole and prochloraz. A fitness penalty was not detected in procymidone-resistant (Pro^R) or difenoconazole-resistant (Dif^R) isolates. However, double-resistant (Pro^R Dif^R) isolates had a fitness penalty, showing significantly decreased sporulation, germination, and pathogenicity. The P894L single point mutation, caused by the change from CCA to CTA at the 894th codon of Os1, was detected in Pro^R isolates. Molecular dynamic simulation showed that the P894L mutation significantly decreased the inhibitory activity of procymidone against AaOs1 in A. alternata. These results provide insight into the development and characteristics of fungicide resistance, offering guidance for the study and management of kiwifruit diseases. Full article

Background: Sirolimus (SRL, rapamycin) is a clinically important mTOR inhibitor used in immunosuppression, oncology, and cardiovascular drug-eluting devices. Despite its long-standing FDA approval, the human metabolic profile of SRL remains incompletely characterized. SRL is primarily metabolized by CYP3A enzymes in the liver and intestine, but the diversity, pharmacokinetics, and biological activity of its metabolites have been poorly explored due to the lack of structurally identified standards. Methods: To investigate SRL metabolism, we incubated SRL with pooled human liver microsomes (HLM) and isolated the resulting metabolites. Structural characterization was performed using high-resolution mass spectrometry (HRMS) and ion trap MSⁿ. We also applied Density Functional Theory (DFT) calculations to assess the energetic favorability of metabolic transformations and conducted molecular dynamics (MD) simulations to model metabolite interactions within the CYP3A4 active site. Results: We identified 21 unique SRL metabolites, classified into five major structural groups: O-demethylated, hydroxylated, didemethylated, di-hydroxylated, and mixed hydroxylated/demethylated derivatives. DFT analyses indicated that certain demethylation and hydroxylation reactions were energetically preferred, correlating with metabolite abundance. MD simulations further validated these findings by demonstrating the favorable orientation and accessibility of key sites within the CYP3A4 binding pocket. Conclusions: This study provides a comprehensive structural map of SRL metabolism, offering mechanistic insights into the formation of its metabolites. Our integrated approach of experimental and computational analyses lays the groundwork for future investigations into the pharmacodynamic and toxicodynamic effects of SRL metabolites on the mTOR pathway. Full article

The enzyme ABH2, one of nine human DNA dioxygenases of the AlkB family, belongs to the superfamily of Fe(II)/α-ketoglutarate-dependent dioxygenases and plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases. ABH2 has broad substrate specificity, directly oxidizing DNA damages such as N¹-methyladenine, N³-methylcytosine, 1,N⁶-ethenoadenine, 3,N⁴-ethenocytosine, and a number of others. In our investigation, we sought to uncover the subtleties of the mechanisms governing substrate specificity in ABH2 by focusing on several critical amino acid residues situated in its active site. To gain insight into the function of this enzyme, we performed a functional mapping of its active site region, concentrating on pivotal residues, participating in forming a damaged binding pocket of the enzyme (Val99 and Ser125), as well as the residues directly involved in interactions with damaged bases, namely Arg110, Phe124, Arg172, and Glu175. To support our experimental data, we conducted a series of molecular dynamics simulations, exploring the interactions between the ABH2 mutant forms, bearing corresponding substitutions and DNA substrates, and harboring various types of methylated bases, specifically N¹-methyladenine or N³-methylcytosine. The comparative studies revealed compelling data indicating that alterations in most of the studied amino acid residues significantly influence both the binding affinity of the enzyme for DNA and its catalytic efficiency. Intriguingly, the findings suggest that the mutations impact the catalytic activity of ABH2 to a greater extent than its ability to associate with DNA strands. Collectively, these results show how changes to the active site affect molecular dynamics and reaction kinetics, improving our understanding of the substrate recognition process in this pivotal enzyme. Full article

Epigenetic modifications act as crucial regulators of gene activity and are influenced by both internal and external environmental factors, with diet being the most impactful external factor. On the other hand, cellular metabolism encompasses a complex network of biochemical reactions essential for maintaining cellular function, and it impacts every cellular process. Many metabolic cofactors are critical for the activity of chromatin-modifying enzymes, influencing methylation and the global acetylation status of the epigenome. For instance, dietary nutrients, particularly those involved in one-carbon metabolism (e.g., folate, vitamins B12 and B6, riboflavin, methionine, choline, and betaine), take part in the generation of S-adenosylmethionine (SAM), which represents the main methyl donor for DNA and histone methylation; α-ketoglutarate and ascorbic acid (vitamin C) act, respectively, as a co-substrate and cofactor for Ten-eleven Translocation (TET), which is responsible for DNA demethylation; and metabolites such as Acetyl-CoA directly impact histone acetylation, linking metabolism of the TCA cycle to epigenetic regulation. Further, bioactive compounds, such as polyphenols, modulate epigenetic patterns by affecting methylation processes or targeting epigenetic enzymes. Since diet and nutrition play a critical role in shaping epigenome functions and supporting human health, this review offers a comprehensive update on recent advancements in metabolism, epigenetics, and nutrition, providing insights into how nutrients contribute to metabolic balance, epigenome integrity maintenance and, consequently, disease prevention. Full article

Ulva prolifera (Chlorophyta), a pivotal species in green tide generation, is particularly vulnerable to abiotic stressors, including variations in temperature and light intensity, requiring specific regulatory frameworks for survival. Epigenetic modification is recognized as a molecular mechanism contributing to the flexible adaptability to environmental alterations. In this study, using DNA methylation pattern analysis, we investigated abiotic stress responsive methylation events, as well as gene and pathway expression patterns, in green macroalgae U. prolifera cultured under elevated temperature–light stress (30 °C and 300 µmol photons m⁻² s⁻¹⁾ and identified a negative correlation between CG methylation and gene expression patterns which indicated that abiotic stress caused CG demethylation and afterwards provoked the transcription response. CHG and CHH methylation exhibited an increased mutability and were preeminently found in transposable elements and intergenic regions, possibly contributing to genetic stability by restricting transposon activity. Furthermore, a rapid regeneration through spore ejection and the formation of new thalli was observed, which emphasized its tenacity capacity for stress memory. Our study also revealed an upregulation of genes associated with the glycolysis pathway and highlighted the critical roles of hexokinase, 6-phosphofructokinase-1, and fructose-6-phosphate in triggering glycolysis as a significant stress-adaptive pathway. Overall, these findings suggested that DNA methylation functions as a potential regulatory mechanism, maintaining environmental adaptability, genomic integrity, and underpinning regenerative capacity in U. prolifera. The findings elucidated the molecular resilience of U. prolifera, highlighting its feasibility for sustainable development and biotechnological applications. Full article

As one kind of renewable aromatic polymer, lignin is severely underused due to its chemical recalcitrance. Lignin can endure demethylation modification to improve its activation by releasing more active functional groups. However, the process suffers from expensive, corrosive, and toxic issues by employing halogen-containing reagents, which has become an obstacle to industrial applications. Herein, a series of dicarboxylic acid-based protic ionic liquids (DAPILs) systems composed of ethanolamine and dibasic organic acids (e.g., aspartic acid (Asp), glutamic acid (Glu), succinic acid (SA), and glutaric acid (GA)) with 1~2:1 stoichiometric ratio, have been finely designed for the demethylation of industrial lignin. With [EOA][GA] treatment, the polyphenol content in lignin was favorably increased beyond 1.58 times. The structural tailoring and variation were fully characterized by 2D HSQC and ¹H NMR. The analysis results indicated that, with the increase of phenolic hydroxyl content in lignin, the β-O-4′ bond was broken and the content of structural units (S, G) and the S/G ratio of lignin decreased accordingly. After the treatment, the used IL and tailored lignin can be recovered over 95%. This novel, halogen-free and environmentally friendly lignin-cutting strategy not only opens avenues for high-value utilization of lignin but also expands the field of application of dicarboxylic acid-based protic ionic liquids. Full article

DNA methylation plays a pivotal role in the epigenetic regulation of gene expression and holds promise for enhancing livestock meat production. In this study, we analyzed the DNA methylome and transcriptome of the longissimus dorsi muscle (LDM) in Duroc pigs with varying growth rates. Our results reveal that DNA methylation suppressed the expression of key muscle development markers (MYOD, MYOG, MHC1) and proliferation markers (PI67, PCNA), as well as the protein expression and phosphorylation of PI3K and AKT (p < 0.05). Dual-luciferase reporter assays and EMSA showed that SP1 overexpression enhanced the luciferase activity of the wild-type LPAR1 promoter, an effect amplified by the demethylating agent 5-AZA (p < 0.05). The EMSA further demonstrates the relationship between SP1 and the LPAR1 promoter region. Overexpression of SP1 upregulated LPAR1 expression at both the mRNA and protein levels (p < 0.05). Knockdown of LPAR1 reduced muscle marker gene expression and delayed myotube formation, while silencing SP1 disrupted the expression of LPAR1, MEF2C, and MHC1 (p < 0.05), and the demethylation induced by 5-AZA partially reversed these effects. These findings suggest that the DNA methylation/SP1/LPAR1 axis is critical for skeletal muscle growth and development, underscoring the regulatory role of DNA methylation in muscle formation. Full article

Ethylene-insensitive 3/ethylene-insensitive 3-like (EIN3/EIL) transcription factors are central regulators of ethylene signaling and stress adaptation in plants. However, their roles in Osmanthus fragrans, a globally cherished ornamental and aromatic plant with significant economic value, remain poorly characterized. Here, we identified nine OfEIL genes across eight chromosomes in the O. fragrans “Liuye Jingui” genome. Conserved motif analysis revealed core domains (Motif1/2/4/7), and promoter cis-elements highlighting hormone-related, stress-related, and growth-related regulatory potential. During late flowering stages, six OfEILs (3/4/5/6/7/9) were significantly upregulated. Under 5-azacytidine (AZA, a DNA demethylation agent), OfEIL2 and OfEIL7 were downregulated, whereas the ETH treatment activated OfEIL3/7/8/9. Strikingly, OfEIL7 exhibited dual regulatory roles, correlating strongly with natural flowering progression, AZA-induced demethylation, and ETH responses. Functional divergence was observed in petal senescence, with OfEIL2–5 and OfEIL7–9 showing stage-specific and tissue-specific expression patterns. These results position OfEIL7 as a key hub integrating epigenetic and hormonal signals to modulate floral longevity and stress adaptation. Our study provides the first genome-wide characterization of the EIL family in O. fragrans, offering critical insights for molecular breeding aimed at enhancing ornamental traits and environmental resilience in this economically significant species. Full article

Carbonaceous materials (CMs) have gained great attention as heterogeneous catalysts in water treatment because of their high efficiency and potential contribution to achieving carbon neutrality. Expanded graphite (EG) is ideal for studying CMs because the reactivity in CMs largely depends on graphitic structures, and most surface of EG is exposed, minimizing mass transfer resistance. However, EG is poor in adsorption and catalysis. In this study, EG was modified by simple thermal treatment to investigate the effects of characteristics of graphitic structures on reactivity. Tetracycline (TC) removal rate via activating peroxydisulfate (PDS) by the EG treated at 550 °C (EG550) was more than 10 times that of EG. The thermal modification did not significantly increase surfaces but led to increases in damaged, rough surfaces, graphitization degree, C content, defects, and C=O. Radical and non-radical pathways, such as SO₄^•−, O₂^•−, ¹O₂, and electron transfer, were involved in TC removal in EG550+PDS. TC degradation in EG550+PDS was initiated by hydroxylation, followed by demethylation, dehydroxylation, decarbonylation, and ring-opening. The ions ubiquitous in water systems did not significantly affect the performance of EG550+PDS, except for H₂PO₄⁻ and HCO₃⁻, suggesting the high potential of practical applications. This study demonstrated that graphitic structure itself and surface area are not detrimental in the catalytic reactivity of CMs, which is different from previous studies. Rather, the reactivity is governed by the characteristics, i.e., defects and functional groups of the graphitic structure. It is thought that this study provides valuable insights into the development of highly reactive CMs and the catalytic systems using them. Full article

In this study, droplet digital PCR and deep bisulfite sequencing were used to study the absolute and active rDNA copy number (CN) and the effect of paternal age on human and mouse sperm. The absolute CN ranged from 98 to 404 (219 ± 47) in human and from 98 to 177 (133 ± 14) in mouse sperm. Methylation of the human upstream control element/core promoter (UCE/CP) region and the 5′ external transcribed spacer, as well as that of the mouse CP, the spacer promoter, and 28S rDNA, significantly increased with donor age and absolute CN. Overall, rDNA hypomethylation was much more pronounced in mouse sperm, with 101.7 ± 11.4 copies showing a completely (0%) unmethylated and 11.3 ± 2.8 (8.5%) a slightly methylated (1–10%) CP region, compared to humans with 25.7 ± 9.5 (12%) completely unmethylated and 83.0 ± 19.8 slightly methylated UCE/CP regions. Although the absolute CN was much higher in human sperm, the number of copies with a hypomethylated (0–10%) promoter was comparable in humans (108.7 ± 28.3) and mice (113.0 ± 12.2). However, in mice, the majority (77%) of all copies were completely unmethylated, whereas in humans a high percentage (38%) showed one or two single CpG methylation errors. These different germline methylation dynamics may be due to species differences in reproductive strategies and lifespan. Complete demethylation of the sperm rDNA promoter in mice may be essential for embryonic genome activation, which already occurs at the 2-cell stage in mice and at the 4–8-cell stage in humans. The paternal age effect has been conserved between humans and mice with some notable differences. In humans, the number of hypomethylated (0–10%) copies decreased with age, whereas in mice only the completely unmethylated copies decreased with age. The number of methylated rDNA copies (>1% in mice and >10% in humans) significantly increased with age. Full article

DNA methylation is an important epigenetic modification with a plethora of effects on cells, ranging from the regulation of gene transcription to shaping chromatin structure. Notably, DNA methylation occurs thanks to the activity of DNA methyltransferases (DNMTs), which covalently add a methyl group to the cytosine in position 5′ in CpG dinucleotides. Different strategies have been developed to study the effects of DNA methylation in cells, involving either DNMTs inhibition (passive DNA demethylation) or the use of Ten-eleven translocation protein (TET) family enzymes, which directly demethylate DNA (active DNA demethylation). In this manuscript, we will briefly cover the most commonly used strategies in the last two decades to achieve DNA demethylation, along with their effects on cells. We will also discuss some of the newest inducible ways to inhibit DNMTs without remarkable side effects, as well as the effect of non-coding RNAs on DNA methylation. Lastly, we will briefly examine the use of DNA methylation inhibition in biomedical research. Full article

Histone demethylases (HDMs) play a pivotal role in colorectal cancer (CRC) progression through dynamic epigenetic regulation. This review summarizes the role and therapeutic potential of HDM in CRC. HDMs primarily target lysine (K) for demethylation (lysine demethylase, KDM). The KDM family is divided into the lysine-specific demethylase family and the Jumonji C domain-containing family. HDMs play complex roles in CRC cell proliferation, invasion, migration, stemness, epithelial–mesenchymal transition, immune response, and chemoresistance through epigenetic regulation of different histone demethylation sites. Increasing evidence suggests that KDM may interact with certain factors and regulate CRC tumorigenesis by modulating multiple signaling pathways and affecting the transcription of target genes. These processes may be regulated by upstream genes and thus form a complex epigenetic regulatory network. However, the potential roles and regulatory mechanisms of some HDMs in CRC remain understudied. Preclinical studies have revealed that small-molecule inhibitors targeting HDM impact the activity of specific genes and pathways by inhibiting specific HDM expression, thereby reshaping the tumorigenic landscape of CRC. However, the clinical translational potential of these inhibitors remains unexplored. In conclusion, HDMs play a complex and critical role in CRC progression by dynamically regulating histone methylation patterns. These HDMs shape the malignant behavior of CRC by influencing the activity of key pathways and target genes through epigenetic reprogramming. Targeting HDM may be a promising direction for CRC treatment. Further exploration of the role of specific HDMs in CRC and the therapeutic potential of HDM-specific inhibitors is needed in the future. Full article

The thiazole heterocycle is one of the most common moieties found in various drugs. Using 2-aminothiazole as the core structure, the amino group was functionalized with an amide. As a result, 30 trisubstituted 2-amino-4, 5-diarylthiazole derivatives were synthesized, with different substitutions introduced at the C2, C4, and C5 positions. The anti-Candida albicans biological activities of these synthetic compounds on five kinds of Candida albicans at different concentrations were detected by the microdilution method. In the first round, four derivatives of 2-amino-4, 5-diarylthiazole exhibited moderate anti-Candida albicans activity. Among them, 4a8 was chosen to be subjected to a demethylation process. Thus, 5a8 was synthesized successfully, giving anti-Candida albicans activity (MIC₈₀ = 9 μM) similar to that of a typical antifungal drug, fluconazole. To understand the mechanism of anti-Candida albicans, molecular docking of the most active 5a8 against four target proteins of anti-Candida albicans, such as glutamine-fructose-6-phosphoamidamitransferase (GFAT), protein kinase (Yck2), heat-shock protein 90 (Hsp90), and lanosterol 14a-demethylase (CYP51) was carried out. Our research will provide an experimental basis and theoretical guidance for the further design of a new aminothiazole-leading pharmaceutical molecule. Full article