Search Results (131)

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

Dietary protein levels greatly influence gut microbial ecosystems; however, their effects on gut archaea and associated functions in ruminants require further elucidation. This study evaluated the impact of varying dietary protein levels on gut archaeal composition, antimicrobial resistance (AMR) genes, virulence factors, and functional capacities in sheep. Eighteen ewes (Yunnan semi-fine wool breed, uniparous, 2 years old, and averaging 50 ± 2 kg body weight) were randomly assigned to diets containing an 8.5 (low; H_1), 10.3 (medium; H_m), or 13.9% (high; H_h) crude protein level from the 35th day of pregnancy to the 90th day postpartum. The total duration of the experiment was approximately 202 days. A total of nine fecal samples (three from each group) were analyzed via 16S rRNA and metagenomics sequencing. Higher archaeal alpha diversity and richness were observed in the H_m and H_h groups compared to the H_l group (p < 0.05). A Beta diversity analysis revealed the archaeal community’s distinct clustering mode based on protein levels. The methanogenic genera Methanobrevibacter and Methanocorpusculum were dominant across the three groups, and their abundance was influenced by protein intake. A functional prediction analysis indicated moderate changes in amino acid and carbohydrate metabolism, which are particularly associated with methane production, an important source of greenhouse gases. AMR genes (e.g., tetA (60), patA, vat, and Erm methyltransferase) and virulence factors (Bacillibactin, LPS) were significantly enriched when animals were fed high-protein diets. Our results demonstrated that dietary protein levels significantly influence gut archaeal composition, AMR gene enrichment, and related functional pathways. Medium-protein diets promoted greater archaeal diversity, whereas high-protein diets favored resistance gene proliferation and enhanced methanogenic activity. Optimizing dietary protein intake may enhance gut health, mitigate antimicrobial resistance risk, and reduce methane emissions, thereby supporting livestock sustainability and environmental protection. Full article

T-cadherin (CDH13) is an atypical, glycosyl-phosphatidylinositol-anchored cadherin with functions ranging from axon guidance and vascular patterning to adipokine signaling and cell-fate specification. Originally identified as a homophilic cue for migrating neural crest cells, projecting axons, and growing blood vessels, it later emerged as a dual metabolic receptor for cardioprotective high-molecular-weight adiponectin and atherogenic low-density lipoproteins. We recently showed that mesenchymal stem/stromal cells lacking T-cadherin are predisposed to adipogenesis, underscoring its role in lineage choice. Emerging evidence indicates that CDH13 expression and function are fine-tuned by non-coding RNAs (ncRNAs). MiR-199b-5p, miR-377-3p, miR-23a/27a/24-2, and the miR-142 family directly bind CDH13 3′-UTR or its epigenetic regulators, affecting transcription or accelerating decay. Long non-coding RNAs (lncRNAs), including antisense transcripts CDH13-AS1/AS2, brain-restricted FEDORA, and context-dependent LINC00707 and UPAT, either sponge these miRNAs or recruit DNMT/TET enzymes to the CDH13 promoter. Circular RNAs (circRNAs), i.e.circCDH13 and circ_0000119, can add a third level of complexity by sequestering miRNA repressors or boosting DNMT1. Collectively, this ncRNA circuitry regulates T-cadherin across cardiovascular, metabolic, oncogenic, and neurodegenerative conditions. This review integrates both experimentally validated data and in silico predictions to map the ncRNA-CDH13 crosstalk between health and disease, opening new avenues for biomarker discovery and RNA-based therapeutics. Full article

Chromobacterium spp. use a density-dependent cell-to-cell communication mechanism (quorum sensing, QS) to control various traits, including the pigment violacein biosynthesis. Recently, one of the type strains of this genus, previously deposited in the American Type Culture Collection under accession number C. violaceum 31532, was reclassified as C. subtsugae, making the QS data obtained for the first species irrelevant to the second. The goal of this study is to conduct transcriptomic and genomic analyses of the C. subtsugae ATCC 31532 (formerly C. violaceum ATCC 31532) strain to identify density-dependent regulated genes and the mechanisms of their QS control. Whole transcriptome dataset analysis comparing QS-negative mid-log phase and QS-positive early stationary phase samples revealed 35 down-regulated and 261 up-regulated genes, including 44 genes that increased transcription activity the most (log2 (fold change) > 4.0). In addition to the violacein biosynthesis, QS-controlled traits in C. subtsugae ATCC 31532 included the following: (i) cdeAB-oprM efflux pump; (ii) RND efflux transporter; (iii) chuPRSTUV iron acquisition system; (iv) polyamine transport system; (v) carbohydrate (semialdehydes) metabolic pathways; (vi) SAM/SPASM maturase system XYE (predicted); (vii) prophage proteins; and (viii) fucose-binding lectin II. Subsequent screening of the promoter regions of the up-regulated genes and operons in most cases showed the presence of CsuR AHL-receptor/transcriptional regulator binding sites with 56.25–68.75% similarity to the ideal 16-base-pair palindrome 5′-CTGTCCGATAGGACAG-3′ sequence, supporting the concept of QS control in C. subtsugae ATCC 31532 by the csuI-csuR gene pair. Notably, several transcriptional regulators (MarR, TetR/AcrR, HU family DNA-binding protein, helix-turn-helix domain-containing protein) were found to be under QS control. Based on these data, a hierarchical QS regulatory network in C. subtsugae ATCC 31532 was hypothesized that provides direct control of the target genes via a canonical autoinduction mechanism and further dissemination of the effect via the activity of QS-controlled transcriptional regulators. Full article

Global water scarcity and antimicrobial resistance (AMR) represent two escalating crises that urgently demand integrated and effective solutions. While wastewater reuse is increasingly promoted as a strategy to alleviate water scarcity, conventional treatment processes often fail to eliminate persistent contaminants and antibiotic-resistant microorganisms. Cold plasma (CP), a non-thermal advanced oxidation process, has demonstrated the strong potential to simultaneously inactivate pathogens and degrade micropollutants. CP generates a diverse mix of reactive oxygen and nitrogen species (ROS and RNS), as well as UV photons and charged particles, capable of breaking down complex contaminants and inducing irreversible damage to microbial cells. Laboratory studies have reported bacterial log reductions ranging from 1 to >8–9 log₁₀, with Gram-negative species such as E. coli and Pseudomonas aeruginosa showing higher susceptibility than Gram-positive bacteria. The inactivation of endospores and mixed-species biofilms has also been achieved under optimized CP conditions. Viral inactivation studies, including MS2 bacteriophage and norovirus surrogates, have demonstrated reductions >99.99%, with exposure times as short as 0.12 s. CP has further shown the capacity to degrade antibiotic residues such as ciprofloxacin and sulfamethoxazole by >90% and to reduce ARGs (e.g., bla, sul, and tet) in hospital wastewater. This perspective critically examines the mechanisms and current applications of CP in wastewater treatment, identifies the operational and scalability challenges, and outlines a research agenda for integrating CP into future water reuse frameworks targeting AMR mitigation and sustainable water management. Full article

Background: The use of antibiotics in beekeeping has potential implications for honeybee health and environmental contamination. Recent research indicates that extensive antibiotic use in beekeeping, especially oxytetracycline, promotes antimicrobial resistance in bee-related bacteria. Honeybees can transport oxytetracycline-resistance genes during foraging, potentially establishing reservoirs of resistance in the colony and facilitating intergeneric gene transfer among various gut bacteria as well as in the microbiome of the flowers and the wider environment, where honeybees can spread antibiotic-resistance genes over a large distance. This study investigates the effects of oxytetracycline hydrochloride (OTC) treatment on honeybees from a One Health perspective, examining antibiotic residues in honey, environmental spread, and the presence of tetracycline-resistance genes (TET-RGs). Methods: In the spring of 2022, two groups of four honeybee hives were placed near an almond grove in Central Italy. One group was treated with 1.68 g of OTC, while the other remained untreated. Samples were collected from bees, honey, hive entrances, and flowers before treatment and at 3 as well as 9 days post-treatment. OTC residues and TET-RGs were analyzed to assess contamination and resistance gene dissemination. Results: OTC residues were detected in honey from both treated (day 3: 263,250.0 ± 100,854.3 µg/kg; day 9: 132,600 ± 146,753.9 µg/kg) and untreated hives (day 3: 20.5 ± 8.2 µg/kg; day 9: 135.8 ± 198.6 µg/kg), suggesting cross-contamination. Residues were also found in almond tree flowers (0.7 ± 0.1 µg/kg), with TET-RGs (tet(K), tet(L), tet(M), tet(B), tet(O), tet(D)) detected pre- and post-treatment. In honeybee gut bacteria, resistance genes (tet(M), tet(A), tet(D), tet(B)) appeared post-treatment in both groups. No significant correlation was observed between hive distance and resistance gene presence in flowers, although the presence of other farms located within the bees’ flight range, in which OTC might have been used in the past, could have influenced the results. Conclusions: These findings highlight the risk of OTC-induced antibiotic cross-contamination and the spread of TET-RG, raising concerns for bee health and environmental safety. Given honeybees’ social nature and the negative effects of antibiotics on their health, an antibiotic-free management approach is recommended for sustainable apiculture. Full article

Aerobic composting is widely used for the degradation of organic matter, simultaneously reducing the presence of antibiotic resistance genes (ARGs) in swine manure. However, the phenomenon of abundance rebound or even enrichment of ARGs is still a problem. The effect and mechanism of humus soil (Hs) on ARG reduction by adding it into the piles (0% for the control group (CK); 10% for S1 group; 20% for S2 group; and 30% for S3 group) after the thermophilic phase of composting was investigated. The results indicated that Hs promoted organic matter degradation and nitrogen loss. During days 15–36, the greatest reduction of 69.91% in total ARG abundance was observed in S2, while the abundance rebounded by 222.75% in CK and decreased only 13.71% in S3. With the 20% Hs addition, 85.42% abundance reduction for mobile genetic elements (MGEs) and 100% removal rates for aadA5, aadA9, sul1, sul2, and tetX were achieved. Moreover, the addition of Hs immediately changed the bacterial community structure of the substrate and varied the bacterial community successional direction in the treatments. Additionally, significantly positive correlations (|r| > 0.6; p < 0.05) were found between the top 20 genera and ARGs. The potential host bacteria for ARGs changed from Lactobacillus, Fermentimonas, Pusillimonas, and Ruminofilibacter in CK to Lactobacillus, Romboutsia, and Streptococcus in S2, highlighting the shift and reduction in host bacteria driven by Hs, which, in turn, influenced the abundance variations in ARGs. This study verified the feasibility of inhibiting the rebound of ARG abundance effectively by influencing the microecological niche in the pile, offering an approach for promoting a reduction in ARGs in animal wastes. Full article

Background/Objectives: Aberrant hypermethylation in the promoter regions of tumor suppressor genes facilitates the pathogenesis and progression of cancer. Therefore, inhibitors targeting DNA methyltransferase (DNMT) have been tested in clinical studies. However, the current monotherapy of DNMT inhibitors shows limited efficacy. Furthermore, the mechanism of action of DNMT inhibitors is DNA replication-dependent. To address these limitations, we developed a novel core–shell-type “epigenetics control (EpC) nanocarrier” that encapsulated decitabine (5-aza-dC) in the PLGA core nanoparticle and hybridized TET1 gene-encoding pDNA on the lipid shell surface. This study aimed to evaluate whether the dual delivery of DNMT inhibitors and pDNA of TET1 could synergistically enhance tumor suppressor gene expression and induce cell cycle arrest and/or apoptosis in cancer cells. Herein, we demonstrate the potential of the EpC carrier in HCT116 human colon cancer cells to upregulate tumor suppressor gene expression and rapidly achieve cell cycle arrest. Methods: PLGA core nanoparticles were prepared by the W/O/W double emulsion method. The formation of core–shell nanoparticles and complexation with pDNA were investigated and optimized by dynamic light scattering, zeta potential measurement, and agarose gel electrophoresis. The cellular uptake and transfection efficiency were measured by confocal laser scanning microscopy and a luciferase assay, respectively. The expression of p53 protein was detected by Western blotting. The anti-tumor effects of the EpC nanocarrier were evaluated by cell cycle analysis and an apoptosis assay. Results: The EpC nanocarrier delivered the DNMT inhibitor and TET gene-encoding pDNA into HCT116 cells. It promoted the expression of the tumor suppressor protein p53 and induced rapid cell cycle arrest in the G2/M phase in HCT116 cells. Conclusions: Our findings suggest that the dual-targeting of DNMT and TET enzymes effectively repairs aberrant DNA methylation and induces growth arrest in cancer cells, and the dual-targeting strategy may contribute to the advancement of epigenetic cancer therapy. Full article

Technology-enhanced teaching (TET) is most effective when integrated meaningfully into classroom settings. Teachers require technological pedagogical knowledge (TPK) to achieve this integration. This study details the development and evaluation of an online professional development (OPD) course aimed at enhancing teachers’ TPK for the effective use of technology in science and language subjects. The course was created to rethink OPD, i.e., to overcome known shortcomings of OPD design consciously. It was based on the Interactive–Constructive–Active–Passive (ICAP) framework, which promotes interactive and constructive learning activities. Thus, the OPD course incorporated text-based and video-based interactive learning modules (learning nuggets), discussions about staged video vignettes showing classroom situations with conventional vs. good use of technology by the teacher, professional learning communities, and practical teaching trials with group reflections. The evaluation study of this OPD followed a pre–post design involving 76 in-service teachers. Participants completed surveys and a test of their ability to apply ICAP knowledge to teaching situations before and after the OPD. The evaluation revealed that teachers perceived the course as beneficial, though no statistically significant improvement in applied ICAP knowledge was observed. These findings highlight opportunities and challenges in designing and measuring effective OPDs that support the meaningful integration of technology into classroom practice. Full article

Clonal hematopoiesis of indeterminate potential (CHIP) is increasingly recognized as a significant contributor to ischemic stroke and other cardiovascular diseases due to its association with somatic mutations in hematopoietic cells. These mutations, notably in genes like DNMT3A, TET2, and JAK2, induce pro-inflammatory and pro-atherosclerotic processes, promoting vascular damage and stroke risk. With the prevalence of CHIP rising with age, its presence correlates with higher mortality and morbidity rates in ischemic stroke patients. This article explores the mechanisms through which CHIP influences vascular aging and stroke, emphasizing its potential as a biomarker for early risk stratification and a target for therapeutic intervention. The findings highlight the necessity of integrating CHIP status in clinical evaluations to better predict outcomes and personalize treatment strategies in stroke management. Full article

Background: Antibiotics were extensively used in the pigeon breeding industry previously to promote growth and prevent disease, leading to the spread of antibiotic-resistant genes (ARGs) in gut microbes, which has become a major public health concern. Methods: A metagenomic analysis was performed to investigate the gut microbial communities and ARGs in young and older pigeons in Nanjing, Jiangsu Province, China. Results: There were obviously distinct gut microbiota and functional compositions between young and older pigeons. Both Pseudomonadota and Uroviricota were dominant in young and older pigeons. Although sharing 24 gut microbiota phyla between young and older pigeons, Bacillota and Pseudomonadota were the dominant microbial phyla in them, respectively. Besides the shared metabolic pathways and biosynthesis of secondary metabolites, biosynthesis of amino acids was the most abundant Kyoto Encyclopedia of Genes and Genomes (KEGG) function in young pigeons, while microbial metabolism in diverse environments was abundant in older pigeons. A total of 142 ARGs conferring multidrug resistance, tetracycline, and aminoglycoside resistance were identified; the most abundant gene in young pigeons was tetracycline-tetW, while in older pigeons, it was multidrug-acrB. Conclusions: Our findings revealed significant differences in the gut microbial communities and ARGs between young and older pigeons. This study enhances our understanding of pigeon gut microbiota and antibiotic resistomes, contributing to knowledge-based sustainable pigeon meat production. Full article

DNA methylation and demethylation are key epigenetic events that regulate gene expression and cell fate. DNA demethylation via oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is typically mediated by TET (ten-eleven translocation) enzymes. The 5hmC modification is considered an intermediate state of DNA demethylation; it is particularly prevalent in the brain and is believed to play a role in the development of many cell types in the brain. Our previous studies have identified that vitamin C (Vc) and MEK inhibitor PD0325901 could significantly promote OPC (oligodendrocyte progenitor cell)-to-OL (oligodendrocyte) differentiation. Here we discovered that Vc and PD0325901 may promote OPC-to-OL differentiation by inducing DNA demethylation via hydroxymethylation. Blocking 5hmC formation almost totally blocked Vc- and PD0325901-stimulated OPC-to-OL differentiation. In addition, TET1 is not involved in Vc,- and PD0325901-promoted OL generation. We also found a synergistic effect between the two compounds in inducing OL generation, suggesting the possibility of a combination therapy for demyelination diseases in the future. Full article

Morning-time heart attacks are associated with an ablation in the sleep-time dip in blood pressure, the mechanism of which is unknown. The epigenetic changes are the hallmark of sleep and circadian clock disruption and homocystinuria (HHcy). The homocystinuria causes ablation in the dip in blood pressure during sleep. Interestingly, HHcy is generated during the epigenetic gene turning off and turning on (i.e., imprinting) by methylation of the DNA promoter. The mitochondrial sulfur metabolism by 3-mercaptopyruvate sulfur transferase (3MST), ATP citrate lyase (ACYL), and epigenetic rhythmic methylation are regulated by folate 1-carbon metabolism (FOCM), i.e., the methionine (M)-SAM-SAH-Hcy, adenosine, and uric acid cycle. Epigenetic gene writer (DNMT), gene eraser (TET/FTO), and editor de-aminase (ADAR) regulate the rhythmic, i.e., reversible methylation/demethylation of H3K4, H3K9, H4K20, m⁶A, and m⁵C. The mitochondrial ATP citrate cycle and creatine kinase (CK) regulate chromatin transcription, maturation, and accessibility as well as muscle function. The transcription is regulated by methylation. The maturation and accessibility are controlled by acetylation. However, it is unclear whether a high fat dysbiotic diet (HFD) causes dysrhythmic expression of the gene writer, eraser, and editor, creating hyperuricemia and cardiac and renal dysfunction. We hypothesized that an HFD increases the gene writer (DNMT1) and editor (ADAR), decreases the eraser (TET/FTO), and increases uric acid to cause chronic diseases. This increases the levels of H3K4, H3K9, H4K20, m⁶A, and m⁵C. Interestingly, the DNMT1KO mitigates. Further, the DNMT1KO and ADAR inhibition attenuate HFD-induced NGAL/FGF23/TMPRSS2/MMP2, 9, 13, and uric acid levels and improve cardiac and renal remodeling. Although the novel role of nerve endings by the Piezo channels (i.e., the combination of ENaC, VDAC, TRPV, K⁺, and Mg²⁺ channels) in the interoception is suggested, interestingly, we and others have shown mechanisms independent of the nerve, by interoception, such as the cargo of the exosome in denervation models of heart failure. If proper and appropriate levels of these enzymes are available to covert homocysteine to hydrogen sulfide (H₂S) during homocystinuria, then the H₂S can potentially serve as a newer form of treatment for morning heart attacks and renal sulfur transsulfuration transport diseases. Full article

The retinoblastoma gene product (Rb1), a master regulator of the cell cycle, plays a prominent role in cell differentiation. Previously, by analyzing the differentiation of cells transiently overexpressing the ΔS/N DN Rb1 mutant, we demonstrated that these cells fail to differentiate into mature adipocytes and that they constitutively silence Pparγ2 through CpG methylation. Here, we demonstrate that the consequences of the transient expression of ΔS/N DN Rb1 are accompanied by the retention of Cebpa promoter methylation near the TSS under adipogenic differentiation, thereby preventing its expression. The CGIs of the promoters of the Rb1, Ezh2, Mll4, Utx, and Tet2 genes, which are essential for adipogenic differentiation, have an unmethylated status regardless of the cell differentiation state. Moreover, Dnmt3a, a de novo DNA methyltransferase, is overexpressed in undifferentiated ΔS/N cells compared with wild-type cells and, in addition to Dnmt1, Dnmt3a is significantly upregulated by adipogenic stimuli in both wild-type and ΔS/N cells. Notably, the chromatin modifier Ezh2, which is also involved in epigenetic reprogramming, is highly induced in ΔS/N cells. Overall, we demonstrate that two major genes, Pparγ2 and Cebpa, which are responsible for terminal adipocyte differentiation, are selectively epigenetically reprogrammed to constitutively silent states. We hypothesize that the activation of Dnmt3a, Rb1, and Ezh2 observed in ΔS/N cells may be a consequence of a stress response caused by the accumulation and malfunctioning of Rb1-interacting complexes for the epigenetic reprogramming of Pparγ2/Cebpa and prevention of adipogenesis in an inappropriate cellular context. The failure of ΔS/N cells to differentiate and express Pparγ2 and Cebpa in culture following the expression of the DN Rb1 mutant may indicate the creation of epigenetic memory for new reprogrammed epigenetic states of genes. Full article

Previous studies have demonstrated that when the cyclin D2 (CCND2), a cell-cycle regulatory protein, is overexpressed in human-induced pluripotent stem cells (hiPSCs), cardiomyocytes (CMs) differentiated from these CCND2-overexpressing hiPSCs can proliferate after transplantation into infarcted hearts, which significantly improves the cells’ potency for myocardial regeneration. However, persistent CM proliferation could lead to tumor growth or the development of arrhythmogenic complications; thus, the goal of the current study was to generate a line of hiPSCs in which CCND2 overexpression could be tightly controlled. First, we transfected hiPSCs with vectors coding for a doxycycline-inducible Tet-On transactivator and S. pyogenes dCas9 fused to the VPR activation domain; then, the same hiPSCs were engineered to express guide RNAs targeting the CCND2 promotor. Thus, treatment with doxycycline (dox) activated dCas9-VPR expression, and the guide RNAs directed dCas9-VPR to the CCND2 promoter, which activated CCND2 expression. Subsequent experiments confirmed that CCND2 expression was dox-dependent in this newly engineered line of hiPSCs (^doxCCND2-hiPSCs): CCND2 protein was abundantly expressed after 48 h of treatment with dox and declined to near baseline level ~96 h after dox treatment was discontinued. Full article