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Epigenetics plays a pivotal role in regulating gene expression and cellular differentiation. DNA methylation, involving the addition of methyl groups to specific cytosine bases, is a well-known epigenetic modification. The recent discovery of 5-hydroxymethylcytosine (5hmC) has provided new insights into cytosine modifications. 5hmC, derived from the oxidation of 5-methylcytosine (5mC), serves as both an intermediate in demethylation and a stable chemical modification in the genome. In this comprehensive review, we summarize the recent research advancements regarding the functions of 5hmC in development and disease. We discuss its implications in gene expression regulation, cellular differentiation, and its potential role as a diagnostic and prognostic marker in various diseases. Additionally, we highlight the challenges associated with accurately detecting and quantifying 5hmC and present the latest methodologies employed for its detection. Understanding the functional role of 5hmC in epigenetic regulation and further advancing our understanding of gene expression dynamics and cellular processes hold immense promise for the development of novel therapeutic strategies and precision medicine approaches. [ABSTRACT FROM AUTHOR]

Conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins leads to the accumulation of 5hmC in the central nervous system; however, the role of 5hmC in the postnatal brain and how its levels and target genes are regulated by TETs remain elusive. We have generated mice that lack all three Tet genes specifically in postnatal excitatory neurons. These mice exhibit significantly reduced 5hmC levels, altered dendritic spine morphology within brain regions crucial for cognition, and substantially impaired spatial and associative memories. Transcriptome profiling combined with epigenetic mapping reveals that a subset of genes, which display changes in both 5hmC/5mC levels and expression patterns, are involved in synapse-related functions. Our findings provide insight into the role of postnatally accumulated 5hmC in the mouse brain and underscore the impact of 5hmC modification on the expression of genes essential for synapse development and function. [ABSTRACT FROM AUTHOR]

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression. [Display omitted] • 5fC forms chromocenters in the perinucleolar compartment during Xenopus ZGA • 5fC colocalizes with RNA Pol III and accumulates on active tRNA tandem repeats • 5fC promotes Pol III recruitment and tRNA transcription at ZGA • 5fC stimulates transcription of a 5fC-modified tRNA transgene An open question in epigenetics is if 5-formylcytosine (5fC) is merely a byproduct of active demethylation or an instructive mark that regulates gene expression on its own. This work demonstrates 5fC chromocenters in early frog embryos and shows that 5fC acts as an instructive mark to promote RNA Pol III transcription during zygotic genome activation. [ABSTRACT FROM AUTHOR]

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription, the epigenome and development. Nevertheless, previous studies using knockdown (KD) or knockout (KO) models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles, as well as compensatory effects among TET family members, which has made the understanding of the enzymatic role of TET not accurate enough. To solve this problem, we successfully generated mice catalytically inactive for specific Tet members (Tetm/m). We observed that, compared with the reported KO mice, mutant mice exhibited distinct developmental defects, including growth retardation, sex imbalance, infertility, and perinatal lethality. Notably, Tetm/m mouse embryonic stem cells (mESCs) were successfully established but entered an impaired developmental program, demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation. Intriguingly, Tet3, traditionally considered less critical for mESCs due to its lower expression level, had a significant impact on the global hydroxymethylation, gene expression, and differentiation potential of mESCs. Notably, there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation. In summary, our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation. [ABSTRACT FROM AUTHOR]

Objective: Significant evidence links epigenetic processes governing the dynamics of DNA methylation and demethylation to an increased risk of syndromic and nonsyndromic cleft lip and/or cleft palate (CL/P). Previously, we characterized mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation in the mouse incisor dental pulp. The main objective of this research was to characterize the transcriptional landscape of regulatory genes associated with DNA methylation and demethylation at a single-cell resolution. Design: We used single-cell RNA sequencing (scRNA-seq) data to characterize transcriptome in individual subpopulations of MSCs in the mouse incisor dental pulp. Settings: The biomedical research institution. Patients/Participants: This study did not include patients. Interventions: This study collected and analyzed data on the single-cell RNA expssion in the mouse incisor dental pulp. Main outcome measure(s): Molecular regulators of DNA methylation/demethylation exhibit differential transcriptional landscape in different subpopulations of osteogenic progenitor cells. Results: scRNA-seq analysis revealed that genes encoding DNA methylation and demethylation enzymes (DNA methyltransferases and members of the ten-eleven translocation family of methylcytosine dioxygenases), methyl-DNA binding domain proteins, as well as transcription factors and chromatin remodeling proteins that cooperate with DNA methylation machinery are differentially expressed within distinct subpopulations of MSCs that undergo different stages of osteogenic differentiation. Conclusions: These findings suggest some mechanistic insights into a potential link between epigenetic alterations and multifactorial causes of CL/P phenotypes. [ABSTRACT FROM AUTHOR]

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This study evaluated the relationship between CL features assessed by ultrasound (luteal tissue area and blood flow, BF) or rectal palpation (size), uterine tone (UT), plasma progesterone (P4) concentration on Day 7 (D7) and subsequent pregnancy outcomes in bovine embryo recipients. A total of 163 cows and heifers were included in this study. The expected day of ovulation after the synchronization protocol was designated as D0. On D7, ovaries and uterus were examined by ultrasonography and rectal palpation, and subjective scores (1–3 scale) were assigned for CL size, area and BF, and for UT. Blood samples were collected for further P4 analysis. Each embryo recipient then received a grade I frozen–thawed in vivo‐produced blastocyst, which was transferred to the uterine horn ipsilateral to the CL. Pregnancy diagnosis was performed on D35, and the results were retrospectively compared with the assigned scores for CL and UT. We observed a significant (p <.02) interaction between CL size and UT, with a progressive increase in the likelihood of pregnancy for recipients bearing a large CL among those with turgid UT. Ultrasound scoring of the CL using B‐mode and Doppler‐mode did not significantly predict pregnancy rates on D35 (p <.6 and p <.5, respectively). However, logistic regression analysis revealed a trend towards a quadratic effect (p <.08 and p <.06) indicating that the probability of pregnancy varied according to the area of luteal tissue and P4 concentrations, respectively. No significant (p >.05) association was found between the probability of pregnancy and the BF area of the CL. In summary, UT before embryo transfer may reflect successful recipient synchronization. Elevated P4 levels, assessed by CL size, may offset uterine contractility, mitigating adverse effects. Additionally, the CL area may be more important than its vascularization area when evaluating recipients D7 after ovulation. [ABSTRACT FROM AUTHOR]

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes.

Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their involvement in adult stem cell regulation remains unclear. Here, we identify a novel enzymatic activity-independent function of Tet in the Drosophila germline stem cell (GSC) niche. Tet activates the expression of Dpp, the fly homologue of BMP, in the ovary stem cell niche, thereby controlling GSC self-renewal. Depletion of Tet disrupts Dpp production, leading to premature GSC loss. Strikingly, both wild-type and enzyme-dead mutant Tet proteins rescue defective BMP signaling and GSC loss when expressed in the niche. Mechanistically, Tet interacts directly with Bap55 and Stat92E, facilitating recruitment of the Polybromo Brahma associated protein (PBAP) complex to the dpp enhancer and activating Dpp expression. Furthermore, human TET3 can effectively substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. Our findings highlight a conserved novel catalytic activity-independent role of Tet as a scaffold protein in supporting niche signaling for adult stem cell self-renewal. Synopsis: Jak-Stat and BMP signaling are co-regulated in the Drosophila ovary niche to maintain germline stem cell (GSC) function, but the molecular mechanisms involved remain unclear. Here, the cytosine modifying enzyme Tet is shown to act as scaffolding factor in ovary somatic cells, integrating chromatin and transcription control to ensure GSC self-renewal. Tet depletion from the ovary niche impairs GSC maintenance. Tet supports GSC self-renewal independently of its dioxygenase activity. Tet functions as a protein scaffold that recruits the PBAP chromatin remodeling complex to Stat92E to activate dpp expression in the niche. Human TET3 can substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. A conserved scaffold function of the cytosine conversion enzyme Tet supports somatic BMP signaling activation and adult stem cell self-revewal in the Drosophila ovary. [ABSTRACT FROM AUTHOR]

During early development, both genome‐wide epigenetic reprogramming and metabolic remodeling are hallmark changes of normal embryogenesis. However, little is known about their relationship and developmental functions during the preimplantation window, which is essential for the acquisition of totipotency and pluripotency. Herein, we reported that glutathione (GSH), a ubiquitous intracellular protective antioxidant that maintains mitochondrial function and redox homeostasis, plays a critical role in safeguarding postfertilization DNA demethylation and is essential for establishing developmental potential in preimplantation embryos. By profiling mitochondria‐related transcriptome that coupled with different pluripotency, we found GSH is a potential marker that is tightly correlated with full pluripotency, and its beneficial effect on prompting developmental potential was functionally conformed using in vitro fertilized mouse and bovine embryos as the model. Mechanistic study based on preimplantation embryos and embryonic stem cells further revealed that GSH prompts the acquisition of totipotency and pluripotency by facilitating ten‐eleven‐translocation (TET)‐dependent DNA demethylation, and ascorbic acid (AsA)‐GSH cycle is implicated in the process. In addition, we also reported that GSH serves as an oviductal paracrine factor that supports development potential of preimplantation embryos. Thus, our results not only advance the current knowledge of functional links between epigenetic reprogramming and metabolic remodeling during preimplantation development but also provided a promising approach for improving current in vitro culture system for assisted reproductive technology. [ABSTRACT FROM AUTHOR]

Research objectives: This study investigates the development of the embassy service in the post-Horde states, with a specific focus on identifying its structure and analyzing its personnel composition. Research materials: The research primarily relies on the analysis of Russian embassy books and chronicles. Results and novelty of the research: The examination of Russian documents reveals that Khan’s embassies followed a three-part structure: Ambassador (elchi)-deputy (tet)-secretary (bakshi). The Russian chancellery was notably reluctant to use the term “tet,” likely because individuals with this title were responsible for collecting specific duties from Russian lands in favor of the Khan. It is possible that the Russian documents referred to the “tet” as a toll collector; in any case, we know that ambassadors could be accompanied by “toll collectors.” After 1475, these duties were abolished. In total, 7 mentions of Crimean tets and 6 Kazan tets were identified. References to baksheys in embassies are better preserved, with a total of 18 Crimean, 8 Kazan, 2 Nogai, and 1 Siberian bakshi identified. One of the primary responsibilities of baksheys was to compile a certificate. Thus, the observation of the embassy mission’s composition relies on source-based evidence. Even when there is no specific information about shoring, the mere presence of a bakshey serves as a reliable indicator that the outcome of the negotiations was expected to involve shoring. The study reveals that we can discuss the process of forming the diplomatic service in the post-Horde Chingisid states.

IntroductionThe clinical successes seen with anti-programmed cell death protein 1/programmed death-ligand 1 (anti-PD-[L]1) agents have galvanized the field of immuno-oncology. We evaluated the landscape and trends in immunotherapy trials involving the PD-(L)1 axis in intrathoracic tumors.MethodsWe identified clinical trials involving anti-PD-(L)1 agents on the ClinicalTrials.gov registry through November 13, 2020 for NSCLC, SCLC, mesothelioma, and thymic epithelial tumor. Clinical trials were indexed according to monotherapy versus combination approaches, PD-(L)1 agents under investigation, clinical settings, trial start date, and partner drug(s). We assessed redundancy among the clinical trials.ResultsWe found 686 clinical trials investigating anti-PD-(L)1 agents for intrathoracic tumors (540 trials in NSCLC, 96 in SCLC, 38 in mesothelioma, and 12 in thymic epithelial tumor). A total of 23 PD-(L)1 inhibitors are undergoing clinical development. A total of 81% of trials assess combination treatment. The number of clinical trials has been growing exponentially in the past decade. PD-(L)1 blockade was frequently combined with chemotherapy or immunomodulatory therapy. Various strategies are in development to overcome resistance to PD-(L)1 blockade in metastatic NSCLC. PD-(L)1 blockade is also increasingly evaluated in neoadjuvant and adjuvant settings. After the U.S. Food and Drug Administration's approval of an anti-PD-(L)1 agent for a specific indication, 14 trials were launched thereafter, which continued to randomize patients to treatments that were inferior to the best available therapy.ConclusionsThe number of clinical trials investigating anti-PD-(L)1 agents in intrathoracic tumors has experienced a steep increase over the past decade with a notable upward trend for combination trials. To reduce duplicative research efforts and accelerate the development of effective immunotherapeutics, improved coordination among key stakeholders and the adoption of innovative trial designs will be vital.

ABSTRACT Chlamydia, comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis, a close phylogenetic relative of human C. trachomatis, which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin (inv), located within a chromosomal region between the rRNA operon (rrn) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis. These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication (ori) and selection markers but lacked the native chlamydial plasmids or its ori. We first recovered allele-replacement mutants using a vector that targets the tryptophan (trp) operon of C. suis. The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori. Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene. IMPORTANCE The obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

Background5' methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing.ResultsUsing an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions.ConclusionWe suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field.

Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field. [ABSTRACT FROM AUTHOR]

Architectural proteins are essential epigenetic regulators that play a critical role in organizing chromatin and controlling gene expression. CTCF (CCCTC-binding factor) is a key architectural protein responsible for maintaining the intricate 3D structure of chromatin. Because of its multivalent properties and plasticity to bind various sequences, CTCF is similar to a Swiss knife for genome organization. Despite the importance of this protein, its mechanisms of action are not fully elucidated. It has been hypothesized that its versatility is achieved through interaction with multiple partners, forming a complex network that regulates chromatin folding within the nucleus. In this review, we delve into CTCF's interactions with other molecules involved in epigenetic processes, particularly histone and DNA demethylases, as well as several long non-coding RNAs (lncRNAs) that are able to recruit CTCF. Our review highlights the importance of CTCF partners to shed light on chromatin regulation and pave the way for future exploration of the mechanisms that enable the finely-tuned role of CTCF as a master regulator of chromatin. [ABSTRACT FROM AUTHOR]

DNA methylation may be involved in the development of osteosarcomas. Osteosarcomas commonly arise during the bone growth and remodeling in puberty, making it plausible to infer the involvement of epigenetic alterations in their development. As a highly studied epigenetic mechanism, we investigated DNA methylation and related genetic variants in 28 primary osteosarcomas aiming to identify deregulated driver alterations. Methylation and genomic data were obtained using the Illumina HM450K beadchips and the TruSight One sequencing panel, respectively. Aberrant DNA methylation was spread throughout the osteosarcomas genomes. We identified 3146 differentially methylated CpGs comparing osteosarcomas and bone tissue samples, with high methylation heterogeneity, global hypomethylation and focal hypermethylation at CpG islands. Differentially methylated regions (DMR) were detected in 585 loci (319 hypomethylated and 266 hypermethylated), mapped to the promoter regions of 350 genes. These DMR genes were enriched for biological processes related to skeletal system morphogenesis, proliferation, inflammatory response, and signal transduction. Both methylation and expression data were validated in independent groups of cases. Six tumor suppressor genes harbored deletions or promoter hypermethylation (DLEC1, GJB2, HIC1, MIR149, PAX6, and WNT5A), and four oncogenes presented gains or hypomethylation (ASPSCR1, NOTCH4, PRDM16, and RUNX3). Our analysis also revealed hypomethylation at 6p22, a region that contains several histone genes. Copy-number changes in DNMT3B (gain) and TET1 (loss), as well as overexpression of DNMT3B in osteosarcomas provide a possible explanation for the observed phenotype of CpG island hypermethylation. While the detected open-sea hypomethylation likely contributes to the well-known osteosarcoma genomic instability, enriched CpG island hypermethylation suggests an underlying mechanism possibly driven by overexpression of DNMT3B likely resulting in silencing of tumor suppressors and DNA repair genes. [ABSTRACT FROM AUTHOR]

Diabetic kidney disease (DKD) is the leading cause of the end-stage renal disease. Recent studies have shown that epigenetic modifications contribute to alterations in gene expression and the development of DKD. This study aimed to show an expression profile of key DNA (de)methylation enzymes (DNMT, TET proteins) and their differences between sexes under obesity and diabetic condition. Male and female black and tan brachyury (BTBR) ob/ob mice and their corresponding wild-type littermates (BTBR WT) were studied until 16 weeks of age. Metabolic parameters, kidney morphophysiology and the expression of fibrotic markers and epigenetic enzymes were studied in whole kidney tissue or specifically in the glomerulus. The results showed sexual dimorphism in the development of metabolic disease and in kidney morphophysiology. Female mice have a different profile of DNMTs expression in both WT and obese/diabetic condition. Furthermore, metabolic condition negatively modulated the glomerular expression of TET1 and TET3 only in females. To our knowledge, this is the first study that shows a kidney profile of the expression of key (de) methylation enzymes, DNMTs and TETs, in the BTBR ob/ob experimental model of DKD and its association with sex. The knowledge of this epigenetic profile may help future research to understand the pathophysiology of DKD in males and females. [ABSTRACT FROM AUTHOR]

Three forms of methylated cytosines are present in the eukaryotic genome: 3-methylcytosine, 4-methylcytosine and 5-methylcytosine. 3-methylcytosines create methyl lesions, which impair local DNA function and flexibility, resulting in replication and transcription error. On the other hand, 5-methylcytosine is usually present at the gene promoter which blocks transcription and translation. Fe(II)/2OG-dependent nucleic acid-modifying enzymes are the class of enzymes responsible for the demethylation of these modified cytosines. ALKBH2 and 3 remove 3-methylcytosine via a one-step direct demethylation process. On the other hand, active demethylation of 5mC is initiated by Ten-Eleven Translocation (TET)-family dioxygenases. Via oxidative demethylation, TET1-3 converts 5mC into 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Remarkably, recent findings demonstrate that ALKBH2,3 possess oxidative demethylation properties, along with direct demethylation. On the other hand, the TET family of enzymes possess direct demethylation properties along with oxidative demethylation. Here we review the importance of methylated cytosines in human DNA, their origin, function and removal. In addition, we discuss the recent findings of extraordinary flexibility of Fe(II)/2OG-dependent nucleic acid-modifying enzymes ALKBH2,3 and TET family of enzymes in cytosine demethylation, as well as their impact on epigenetics.

Diabetic kidney disease (DKD) is the leading cause of the end-stage renal disease. Recent studies have shown that epigenetic modifications contribute to alterations in gene expression and the development of DKD. This study aimed to show an expression profile of key DNA (de)methylation enzymes (DNMT, TET proteins) and their differences between sexes under obesity and diabetic condition. Male and female black and tan brachyury (BTBR) ob/ob mice and their corresponding wild-type littermates (BTBR WT) were studied until 16 weeks of age. Metabolic parameters, kidney morphophysiology and the expression of fibrotic markers and epigenetic enzymes were studied in whole kidney tissue or specifically in the glomerulus. The results showed sexual dimorphism in the development of metabolic disease and in kidney morphophysiology. Female mice have a different profile of DNMTs expression in both WT and obese/diabetic condition. Furthermore, metabolic condition negatively modulated the glomerular expression of TET1 and TET3 only in females. To our knowledge, this is the first study that shows a kidney profile of the expression of key (de)methylation enzymes, DNMTs and TETs, in the BTBR ob/ob experimental model of DKD and its association with sex. The knowledge of this epigenetic profile may help future research to understand the pathophysiology of DKD in males and females.

Background: There is still debate over the use of regional anesthesia during thyroid surgeries. Objective: This investigation looked at the effectiveness of an ultrasound-guided bilateral superficial cervical plexus block (BSCPB) combined with a tolerable endotracheal tube (TET) for reducing hemodynamic stress response and respiratory issues as well as controlling pain during and after surgery. Methods: This prospective randomized controlled comparative clinical research was performed at the Department of Anesthesia, Surgical Critical Care and Pain Control in Zagazig University Hospitals. Patients were split into two groups: Group I (Control group, "C") where patients received general anesthesia with classic endotracheal tube (ETT) and systemic analgesia. Group II ("BSCPB+TET" "B") patients got ultrasound guided BSCPB then general anesthesia with TET. Results: Postoperative cough, bronchospasm, and sore throat were substantially distinct between the tested groups statistically. Group B had significantly less cough than group C. None of patients in group B had postoperative bronchospasm compared to 22.22% of patients in group C. Group B had significantly lower sore throat than group C Conclusions: The use of BSCPB with TET in thyroid surgeries can achieve intraoperative hemodynamic stability, increase time for 1st analgesic request, decrease total dose of intraoperative and postoperative analgesic requirements and reduce the frequency of postoperative difficulties in patients undergoing elective thyroid surgeries. [ABSTRACT FROM AUTHOR]

In recent years, the continuous development of electronic chips and the increasing integration of devices have led to extensive research on the thermal properties of ultrathin metallic materials. In particular, accurate characterization of their thermal transport properties has become a research hotspot. In this paper, we review the characterization methods of metallic nanomaterials, focusing on the principles of the transient electrothermal (TET) technique and the differential TET technique. By using the differential TET technique, the thermal conductivity, electrical conductivity, and Lorenz number of extremely confined metallic nanostructures can be characterized with high measurement accuracy. At present, we are limited by the availability of existing coating machines that determine the thickness of the metal films, but this is not due to the measurement technology itself. If a material with a smaller diameter and lower thermal conductivity is used as the substrate, much thinner nanostructures can be characterized. [ABSTRACT FROM AUTHOR]

Ten-Eleven Translocation (TET) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) leading to a dynamic epigenetic state of DNA that can influence transcription and chromatin organization. While TET proteins interact with complexes involved in transcriptional repression and activation, the overall understanding of the molecular mechanisms involved in TET-mediated regulation of gene expression still remains limited. Here, we show that TET proteins interact with the chromatin remodelling protein lymphoid-specific helicase (LSH/HELLS) in vivo and in vitro. In mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESCs) knock out of Lsh leads to a significant reduction of 5-hydroxymethylation amount in the DNA. Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh, some regions of the genome gain 5hmC while others lose it, with mild correlation with gene expression changes. We further show that differentially hydroxymethylated regions did not completely overlap with differentially methylated regions indicating that changes in 5hmC distribution upon Lsh knock-out are not a direct consequence of 5mC decrease. Altogether, our results suggest that LSH, which interacts with TET proteins, contributes to the regulation of 5hmC levels and distribution in MEFs and ESCs.

Abstract Background Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA—the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP–TET interplay. Results Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. Conclusions According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine.

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal , SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65 , TLR4 , and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes. [Display omitted] • Reduced hippocampal DNA 5-hydroxymethylation levels were found in the hippocampus of people with temporal lobe epilepsy. • In the rodent experimental model of temporal lobe epilepsy, we recapitulated DNA 5-hydroxymethylation findings in human temporal lobe epilepsy. • Post-status epilepticus, the majority of genomic DNA 5-hydroxymethylation loss in the hippocampus occurs within intergenic regions. • Differential DNA 5-hydroxymethylation was found at gene regions involved in neuronal and synaptic regulation. • Manipulating TET1 expression in the rat hippocampus promotes seizure susceptibility and resiliency. [ABSTRACT FROM AUTHOR]

BiVO 4 /Cu 2 O/g-C 3 N 4 ternary heterojunction as a double Z-scheme sonocatalyst was effectively synthesized by the solvothermal and thermal decomposition method. The sonocatalysts' structure, morphology, chemical makeup, and physicochemical characteristics were assessed by using XRD, SEM, TEM, XPS, DRS, and BET. Tetracycline (TET) was utilized for the modeled chemical contaminant to investigate the sonocatalytic capabilities of the sonocatalysts. The findings showed that BiVO 4 /Cu 2 O/g-C 3 N 4 had remarkable sonocatalytic activity, and the degradation rate reached 93.80±0.33 % within 30 min. By further studying the electron separation and migration behavior, a feasible sonocatalytic mechanism of BiVO 4 /Cu 2 O/g-C 3 N 4 is proposed. This research will pave the practicable way for the new double Z-scheme sonocatalytic system. [Display omitted] • BiVO 4 /Cu 2 O/g-C 3 N 4 was a good sonocatalyst with 93.80±0.33 % of tetracycline removal rate in 30 min. • The construction mechanism of double Z-scheme heterojunction in BiVO 4 /Cu 2 O/g-C 3 N 4 was proposed. • The mechanism and pathway of sonocatalytic degradation of TC by BiVO 4 /Cu 2 O/g-C 3 N 4 was proposed. • BiVO 4 /Cu 2 O/g-C 3 N 4 composite showed good stability and reusability in four reuses. [ABSTRACT FROM AUTHOR]

Abstract DNA methylation is one of the most important epigenetic mechanisms to regulate gene expression, which is highly dynamic during development and specifically maintained in somatic cells. Aberrant DNA methylation patterns are strongly associated with human diseases including cancer. How are the cell-specific DNA methylation patterns established or disturbed is a pivotal question in developmental biology and cancer epigenetics. Currently, compelling evidence has emerged that long non-coding RNA (lncRNA) mediates DNA methylation in both physiological and pathological conditions. In this review, we provide an overview of the current understanding of lncRNA-mediated DNA methylation, with emphasis on the roles of this mechanism in cancer, which to the best of our knowledge, has not been systematically summarized. In addition, we also discuss the potential clinical applications of this mechanism in RNA-targeting drug development.

DNA methylation is a highly conserved epigenetic modification that plays essential roles inmammalian gene regulation, genome stability and development. Despite being primarily considered a stable and heritable epigenetic silencing mechanism at heterochromatic and repetitive regions, whole genome methylome analysis reveals that DNA methylation can be highly cell-type specific and dynamic within proximal and distal gene regulatory elements during early embryonic development, stem cell differentiation and reprogramming, and tissue maturation. In this Review, we focus on the mechanisms and functions of regulated DNA methylation and demethylation, highlighting how these dynamics, together with crosstalk between DNA methylation and histone modifications at distinct regulatory regions, contribute to mammalian development and tissue maturation. We also discuss how recent technological advances in single-cell and long-read methylome sequencing, along with targeted epigenome-editing, are enabling unprecedented high-resolution and mechanistic dissection of DNA methylome dynamics. [ABSTRACT FROM AUTHOR]

A reduction in myometrial contractile activity can lead to inadequate cleaning of the uterine lumen, resulting in persistent endometritis and potentially endometrosis in mares. Oxytocin (OXT) is a key hormonal regulator of myometrial contraction. While epigenetic regulation of myometrial gene expression has been studied in humans, there is limited information on the expression of DNA methyltransferases (DNMTs) and ten-eleven translocation enzymes (TETs) in the myometrium of mares. This study aimed to evaluate the mRNA transcription of these enzymes and the potential role of DNA methylation in the expression of the OXT receptor (OXTR) gene in the myometrium of mares with endometrosis. Myometrial samples were collected post-mortem during the mid-luteal (n = 23) and follicular (n = 20) phases of the estrous cycle and assessed according to Kenney and Doig endometrial category (I, IIA, IIB, III). mRNA transcription of OXTR, DNMT1, -3A, -3B and TET1, -2, -3 were determined using qPCR. DNA methylation analysis at CpG islands of OXTR exons 1 and 2 was performed using bisulfite pyrosequencing. Myometrial OXTR mRNA transcription and DNA methylation in its promoter region showed no significant differences between categories, although increased methylation was observed at CpG island position 6 in exon 2. DNMT1, TET2, and TET3 mRNA transcription was altered in the equine myometrium depending on the phase of the estrous cycle and the severity of endometrosis (P < 0.05). These findings indicate that DNMTs and TETs were expressed in myometrium in a manner specific to the severity of endometrosis and phases of the estrous cycle, suggesting a potential regulatory role in DNA methylation of myometrial gene expression., Competing Interests: Declaration of Competing Interest No., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The micronutrient vitamin C is essential for the maintenance of skeletal muscle health and homeostasis. The pro-myogenic effects of vitamin C have long been attributed to its role as a general antioxidant agent, as well as its role in collagen matrix synthesis and carnitine biosynthesis. Here, we show that vitamin C also functions as an epigenetic compound, facilitating chromatin landscape transitions during myogenesis through its activity as an enzymatic cofactor for histone H3 and DNA demethylation. Utilizing C2C12 myoblast cells to investigate the epigenetic effects of vitamin C on myogenesis, we observe that treatment of cells with vitamin C decreases global H3K9 methylation and increases 5-hmC levels. Furthermore, vitamin C treatment enhances myoblast marker gene expression and myotube formation during differentiation. We identify KDM7A as a histone lysine demethylase markedly upregulated during myogenesis. Accordingly, knockdown of Kdm7a prevents the pro-myogenic effects of vitamin C. Chromatin immunoprecipitation analysis showed that KDM7A occupies the promoter region of the myogenic transcription factor MyoD1 where it facilitates histone demethylation. We also confirm that the methylcytosine dioxygenases TET1 and TET2 are required for myogenic differentiation and that their loss blunts stimulation of myogenesis by vitamin C. In conclusion, our data suggest that an epigenetic mode of action plays a major role in the myogenic effects of vitamin C., (© 2024 Wiley Periodicals LLC.)

Three forms of methylated cytosines are present in the eukaryotic genome: 3-methylcytosine, 4-methylcytosine and 5-methylcytosine. 3-methylcytosines create methyl lesions, which impair local DNA function and flexibility, resulting in replication and transcription error. On the other hand, 5-methylcytosine is usually present at the gene promoter which blocks transcription and translation. Fe(II)/2OG-dependent nucleic acid-modifying enzymes are the class of enzymes responsible for the demethylation of these modified cytosines. ALKBH2 and 3 remove 3-methylcytosine via a one-step direct demethylation process. On the other hand, active demethylation of 5mC is initiated by Ten-Eleven Translocation (TET)-family dioxygenases. Via oxidative demethylation, TET1-3 converts 5mC into 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Remarkably, recent findings demonstrate that ALKBH2,3 possess oxidative demethylation properties, along with direct demethylation. On the other hand, the TET family of enzymes possess direct demethylation properties along with oxidative demethylation. Here we review the importance of methylated cytosines in human DNA, their origin, function and removal. In addition, we discuss the recent findings of extraordinary flexibility of Fe(II)/2OG-dependent nucleic acid-modifying enzymes ALKBH2,3 and TET family of enzymes in cytosine demethylation, as well as their impact on epigenetics. [ABSTRACT FROM AUTHOR]

Background: Cannabis use is a component risk factor for the manifestation of schizophrenia. The biological effects of cannabis include effects on epigenetic systems, immunological parameters, in addition to changes in cannabinoid receptors 1 and 2, that may be associated with this risk. However, there has been limited study of the effects of smoked cannabis on these biological effects in human peripheral blood cells. We analyzed the effects of two concentrations of tetrahydrocannabinol (THC) vs. placebo in lymphocytes of a subset of participants who enrolled in a double-blind study of the effects of cannabis on driving performance (outcome not the focus of this study). Methods: Twenty four participants who regularly use cannabis participated in an experiment in which they smoked cannabis cigarettes (5.9 or 13.4% THC) or placebo (0.02%) ad libitum. Blood samples were drawn at baseline and several times after smoking. Lymphocytes were separated and stored at -80°C for further analysis. Samples were analyzed for mRNA content for cannabinoid receptors 1 (CB1) and 2 (CB2), methylation and demethylating enzymes (DNMT, TET), glucocorticoid receptor (NRC3) and immunological markers (IL1B, TNFa) by qPCR using TaqMan probes. The results were correlated with THC whole blood levels during the course of the day, as well as THCCOOH baseline levels. Statistical analyses used analysis of variance and covariance and t-tests, or non-parametric equivalents for those values which were not normally distributed. Results: There were no differences in background baseline characteristics of the participants except that the higher concentration THC group was older than the low concentration and placebo groups, and the low concentration THC group had higher baseline CB2 mRNA levels. Both the 5.9 and 13.4% THC groups showed increased THC blood levels that then decreased toward baseline within the first hour. However, there were no significant differences between THC blood levels between the 5.9 and 13.4% groups at any time point. At the 4-h time point after drug administration the 13.4% THC group had higher CB2 (P = 0.021) and DNMT3A (P = 0.027) mRNA levels than the placebo group. DNMT1 mRNA levels showed a trend in the same direction (P = 0.056). The higher 13.4% THC group had significantly increased CB2 mRNA levels than the 5.9% concentration group at several post drug administration time points and showed trends for difference in effects for between 5.9 and 13.4% THC groups for other mRNAs. TET3 mRNA levels were higher in the 13.4% THC group at 55 min post-cannabis ingestion. When the high and lower concentration THC groups were combined, none of the differences in mRNA levels from placebo remained statistically significant. Changes in THC blood levels were not related to changes in mRNA levels. Conclusion: Over the time course of this study, CB2 mRNA increased in blood lymphocytes in the high concentration THC group but were not accompanied by changes in immunological markers. The changes in DNMT and TET mRNAs suggest potential epigenetic effects of THC in human lymphocytes. Increases in DNMT methylating enzymes have been linked to some of the pathophysiological processes in schizophrenia and, therefore, should be further explored in a larger sample population, as one of the potential mechanisms linking cannabis use as a trigger for schizophrenia in vulnerable individuals. Since the two THC groups did not differ in post-smoking blood THC concentrations, the relationship between lymphocytic changes and the THC content of the cigarettes remains to be determined. [ABSTRACT FROM AUTHOR]

Abstract Background Phosphoinositide-3 kinase (PI3K)/AKT signaling participates in cellular proliferation, survival and tumorigenesis. The activation of AKT signaling promotes the cellular reprogramming including generation of induced pluripotent stem cells (iPSCs) and dedifferentiation of primordial germ cells (PGCs). Previous studies suggested that AKT promotes reprogramming by activating proliferation and glycolysis. Here we report a line of evidence that supports the notion that AKT signaling is involved in TET-mediated DNA demethylation during iPSC induction. Methods AKT signaling was activated in mouse embryonic fibroblasts (MEFs) that were transduced with OCT4, SOX2 and KLF4. Multiomics analyses were conducted in this system to examine the effects of AKT activation on cells undergoing reprogramming. Results We revealed that cells undergoing reprogramming with artificially activated AKT exhibit enhanced anabolic glucose metabolism and accordingly increased level of cytosolic α-ketoglutarate (αKG), which is an essential cofactor for the enzymatic activity of the 5-methylcytosine (5mC) dioxygenase TET. Additionally, the level of TET is upregulated. Consistent with the upregulation of αKG production and TET, we observed a genome-wide increase in 5-hydroxymethylcytosine (5hmC), which is an intermediate in DNA demethylation. Moreover, the DNA methylation level of ES-cell super-enhancers of pluripotency-related genes is significantly decreased, leading to the upregulation of associated genes. Finally, the transduction of TET and the administration of cell-permeable αKG to somatic cells synergistically enhance cell reprogramming by Yamanaka factors. Conclusion These results suggest the possibility that the activation of AKT during somatic cell reprogramming promotes epigenetic reprogramming through the hyperactivation of TET at the transcriptional and catalytic levels.

Abstract Background Thymic epithelial tumour (TET) is the most common tumour affecting the anterior mediastinum in adults. The cardiac extension is often limited to the pericardium, and intracardiac extension is rare. We present a unique case of encasement and displacement of the left anterior descending coronary artery by the large mediastinal tumour leading to myocardial ischemia. Case presentation Our patient is a 28-year-old lady with stage 4 TET. She presented with acute chest pain associated with 12-lead ECG changes and a significant rise in serial troponin I. Multimodality cardiac imaging revealed encasement and displacement of the left anterior descending coronary artery by the large mediastinal tumour. CT-FFR demonstrates evidence of ischemia which would account for her acute presentation. Following detailed MDT discussions between cardiologists, oncologists and cardiothoracic surgeons, the decision was made to treat this lady with palliative chemotherapy. Given the extent of the tumour invasion and failure of the initial therapy, her prognosis and the outcome were poor. Conclusions TET could cause atrial compression, myocardial infiltration, and invasion of the pulmonary and caval veins; however, to the best of our knowledge, this is the first case reported of coronary artery displacement and encasement by TET.

Ascorbate (vitamin C) is an essential micronutrient in humans. The severe chronic deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. From a micronutrient analysis, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique as other antioxidants failed to promote plasma cell differentiation. Ascorbate is especially critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. As a cofactor for epigenetic enzymes, ascorbate facilitates TET2/3-mediated DNA modification and demethylation of multiple elements at the Prdm1 locus. DNA demethylation augments STAT3 association at the Prdm1 promoter and a downstream enhancer, thus ensuring efficient gene expression and plasma cell differentiation. The results suggest that an adequate level of ascorbate is required for antibody response and highlight how micronutrients may regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.

DNA methylation at the fifth position of cytosine (5mC) is one of the most studied epigenetic mechanisms essential for the control of gene expression and for many other biological processes including genomic imprinting, X chromosome inactivation and genome stability. Over the last years, accumulating evidence suggest that DNA methylation is a highly dynamic mechanism driven by a balance between methylation by DNMTs and TET-mediated demethylation processes. However, one of the main challenges is to understand the dynamics underlying steady state DNA methylation levels. In this review article, we give an overview of the latest advances highlighting DNA methylation as a dynamic cycling process with a continuous turnover of cytosine modifications. We describe the cooperative actions of DNMT and TET enzymes which combine with many additional parameters including chromatin environment and protein partners to govern 5mC turnover. We also discuss how mathematical models can be used to address variable methylation levels during development and explain cell-type epigenetic heterogeneity locally but also at the genome scale. Finally, we review the therapeutic implications of these discoveries with the use of both epigenetic clocks as predictors and the development of epidrugs that target the DNA methylation/demethylation machinery. Together, these discoveries unveil with unprecedented detail how dynamic is DNA methylation during development, underlying the establishment of heterogeneous DNA methylation landscapes which could be altered in aging, diseases and cancer.

BackgroundCannabis use is a component risk factor for the manifestation of schizophrenia. The biological effects of cannabis include effects on epigenetic systems, immunological parameters, in addition to changes in cannabinoid receptors 1 and 2, that may be associated with this risk. However, there has been limited study of the effects of smoked cannabis on these biological effects in human peripheral blood cells. We analyzed the effects of two concentrations of tetrahydrocannabinol (THC) vs. placebo in lymphocytes of a subset of participants who enrolled in a double-blind study of the effects of cannabis on driving performance (outcome not the focus of this study).MethodsTwenty four participants who regularly use cannabis participated in an experiment in which they smoked cannabis cigarettes (5.9 or 13.4% THC) or placebo (0.02%) ad libitum. Blood samples were drawn at baseline and several times after smoking. Lymphocytes were separated and stored at –80°C for further analysis. Samples were analyzed for mRNA content for cannabinoid receptors 1 (CB1) and 2 (CB2), methylation and demethylating enzymes (DNMT, TET), glucocorticoid receptor (NRC3) and immunological markers (IL1B, TNFα) by qPCR using TaqMan probes. The results were correlated with THC whole blood levels during the course of the day, as well as THCCOOH baseline levels. Statistical analyses used analysis of variance and covariance and t-tests, or non-parametric equivalents for those values which were not normally distributed.ResultsThere were no differences in background baseline characteristics of the participants except that the higher concentration THC group was older than the low concentration and placebo groups, and the low concentration THC group had higher baseline CB2 mRNA levels. Both the 5.9 and 13.4% THC groups showed increased THC blood levels that then decreased toward baseline within the first hour. However, there were no significant differences between THC blood levels between the 5.9 and 13.4% groups at any time point. At the 4-h time point after drug administration the 13.4% THC group had higher CB2 (P = 0.021) and DNMT3A (P = 0.027) mRNA levels than the placebo group. DNMT1 mRNA levels showed a trend in the same direction (P = 0.056). The higher 13.4% THC group had significantly increased CB2 mRNA levels than the 5.9% concentration group at several post drug administration time points and showed trends for difference in effects for between 5.9 and 13.4% THC groups for other mRNAs. TET3 mRNA levels were higher in the 13.4% THC group at 55 min post-cannabis ingestion. When the high and lower concentration THC groups were combined, none of the differences in mRNA levels from placebo remained statistically significant. Changes in THC blood levels were not related to changes in mRNA levels.ConclusionOver the time course of this study, CB2 mRNA increased in blood lymphocytes in the high concentration THC group but were not accompanied by changes in immunological markers. The changes in DNMT and TET mRNAs suggest potential epigenetic effects of THC in human lymphocytes. Increases in DNMT methylating enzymes have been linked to some of the pathophysiological processes in schizophrenia and, therefore, should be further explored in a larger sample population, as one of the potential mechanisms linking cannabis use as a trigger for schizophrenia in vulnerable individuals. Since the two THC groups did not differ in post-smoking blood THC concentrations, the relationship between lymphocytic changes and the THC content of the cigarettes remains to be determined.

Abstract Background Cirrhosis is a recognized risk factor for developing hepatocellular carcinoma (HCC). Few studies have reported the expression profile of circRNAs in HCC samples compared to paratumour dysplastic nodule (DN) samples. Methods The Arraystar Human circRNA Array combined with laser capture microdissection (LCM) was used to analyse the expression profile of circRNAs in HCC samples compared to paratumour DN samples. Then, both in vitro and in vivo HCC models were used to determine the role and mechanism of key circRNA in HCC progression and treatment sensitivity. Results We found that circMEMO1 was significantly downregulated in HCC samples and that the level of circMEMO1 was closely related to the OS and disease-free survival (DFS) of HCC patients. Mechanistic analysis revealed that circMEMO1 can modulate the promoter methylation and gene expression of TCF21 to regulate HCC progression by acting as a sponge for miR-106b-5p, which targets the TET family of genes and increases the 5hmC level. More importantly, circMEMO1 can increase the sensitivity of HCC cells to sorafenib treatment. Conclusion Our study determined that circMEMO1 can promote the demethylation and expression of TCF21 and can be considered a crucial epigenetic modifier in HCC progression.

Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates. Metavirome mining revealed thousands of biosynthetic gene clusters containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a platform for high-throughput screening of GT-containing pathways, relying on the Escherichia coli metabolome as a substrate pool. We successfully reconstituted several pathways and isolated diverse sugar modifications appended to cytosine, including mono-, di-, or tri-saccharides comprised of hexoses, N -acetylhexosamines, or heptose. These findings expand our knowledge of hypermodifications on nucleic acids and the origins of corresponding sugar-installing enzymes. [Display omitted] • Phage-encoded cytosine hypermodification enzymes install diverse sugars on DNA polymers • Cytosine sugar modifications can include hexoses, N -acetylhexosamines, heptose, or Kdo • Phage GTs appear to be structurally and functionally related to cellular and viral enzymes • Thousands of related pathways in the metavirome suggest abundant DNA hypermodifications Pyle, Lund, et al. develop a cell-based system for high-throughput characterization of DNA hypermodifications. Using this approach, they discover diverse sugar modifications installed on cytosine by bacteriophage glycosyltransferases. Sequence- and structure-guided searches reveal thousands of related pathways in the metavirome, suggesting that abundant DNA hypermodification pathways are present in the biosphere. [ABSTRACT FROM AUTHOR]

Thymic epithelial tumors (TETs) are rare and the major symptoms are not obvious until the tumor progresses to a relatively large size and compresses the surrounding organs. As its growth is aggressive and it metastasizes to distant organs, it is important to find novel effective therapies. Lenvatinib, a vascular endothelial growth factor receptor (VEGFR) inhibitor, is approved as a drug therapy for thymic carcinoma (TC); however, although it is a molecular targeted therapy, there are no obvious predictors of therapeutic efficacy. The present study aimed to assess the association between clinicopathological factors and the protein expression of VEGFR, which is associated with tumor aggressiveness and the efficacy of VEGFR inhibitors. The VEGFR-2 protein expression was evaluated in 144 patients with TETs who underwent surgical resection. The present study assessed whether the expression of VEGFR-2 protein was associated with TET classification and pathological stage, progression-free survival and overall survival (OS). A total of 94 cases (65.2%) were positive for VEGFR-2 protein. The expression of VEGFR-2 was higher in the more aggressive type B3 thymoma and TC (88.5%) than in types A, AB, B1 and B2 thymoma (60.2%). The 5-year OS rate for the overall population was 53.1%. The 5-year OS rates of patients with negative VEGFR-2 staining score values (66.5%) were significantly longer than in patients with positive VEGFR-2 staining score values (42.5%; P=0.000078). Furthermore, the pathological stage was the only factor significantly associated with OS in multivariate analysis. The results of the present study suggest the possibility that the indications for VEGF inhibitor therapy could be extended to type B3 thymoma. [ABSTRACT FROM AUTHOR]

5-Hydroxymethylcytosine (5hmC) has recently been found that plays an important role in many diseases; however, there are still few studies in the field of stroke. The purpose of this review is to introduce the influence and function of 5hmC in stroke, in order for more people can study it. In this review, we introduced the role of 5hmC in ischemia and hemorrhage stroke, and summarized the possible therapeutic prospects of 5hmC in stroke. In conclusion, we suggest that 5hmC may serve as a biomarker or therapeutic target for the treatment of stroke. [ABSTRACT FROM AUTHOR]

Background: Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA—the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP–TET interplay. Results: Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. Conclusions: According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine. [ABSTRACT FROM AUTHOR]

The Coefficients H technique (also called the H-technique), developed by Patarin circa 1991, is a tool used to obtain the upper bounds on distinguishing advantages. This tool is known to provide relatively simple and (in some cases) tight bound proofs in comparison to some other well-known tools, such as the game-playing technique and random systems methodology. In this systematization of knowledge (SoK) paper, we aim to provide a brief survey on the H-technique. The SoK is presented in four parts. First, we redevelop the necessary nomenclature and tools required to study the security of any symmetric-key design, especially in the H-technique setting. Second, we provide a full description of the H-technique and some related tools. Third, we present (simple) H-technique-based proofs for some popular symmetric-key designs, across different paradigms. Finally, we show that the H-technique can actually provide optimal bounds on distinguishing advantages. [ABSTRACT FROM AUTHOR]

Ten-Eleven Translocation (TET) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) leading to a dynamic epigenetic state of DNA that can influence transcription and chromatin organization. While TET proteins interact with complexes involved in transcriptional repression and activation, the overall understanding of the molecular mechanisms involved in TET-mediated regulation of gene expression still remains limited. Here, we show that TET proteins interact with the chromatin remodelling protein lymphoid-specific helicase (LSH/HELLS) in vivo and in vitro. In mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESCs) knock out of Lsh leads to a significant reduction of 5-hydroxymethylation amount in the DNA. Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh, some regions of the genome gain 5hmC while others lose it, with mild correlation with gene expression changes. We further show that differentially hydroxymethylated regions did not completely overlap with differentially methylated regions indicating that changes in 5hmC distribution upon Lsh knock-out are not a direct consequence of 5mC decrease. Altogether, our results suggest that LSH, which interacts with TET proteins, contributes to the regulation of 5hmC levels and distribution in MEFs and ESCs. [ABSTRACT FROM AUTHOR]