Uterine leiomyomas (ULs), frequent benign tumours of the female reproductive tract, are associated with a range of symptoms and significant morbidity. Despite extensive research, there is no consensus on essential points of UL initiation and development. The main reason for this is a pronounced inter- and intratumoral heterogeneity resulting from diverse and complicated mechanisms underlying UL pathobiology. In this review, we comprehensively analyse risk and protective factors for UL development, UL cellular composition, hormonal and paracrine signalling, epigenetic regulation and genetic abnormalities. We conclude the need to carefully update the concept of UL genesis in light of the current data. Staying within the framework of the existing hypotheses, we introduce a possible timeline for UL development and the associated key events—from potential prerequisites to the beginning of UL formation and the onset of driver and passenger changes. Uterine leiomyoma (UL) is the most common tumour of the female reproductive system, with an incidence rate reaching 70–80% . DespiteUL aetiopathogenesis is being actively investigated. Parity, oral contraceptive intake and smoking are among possible protective factors for UL development ,6,7,8,9.The present review analyses the cellular, endocrine, paracrine, genetic and epigenetic aspects of UL tumorigenesis, discusses potential prerequisites and suggests possible key events on the timeline of UL development.ULs represent dense nodules (myomas) of rounded shape, consisting of transformed cells that produce an excess of extracellular matrix (ECM) . The ECMOCT4 (octamer-binding transcription factor 4), NANOG (Nanog homeobox), DNMT3B (DNA methyltransferase 3B), GDF3 (growth differentiation factor-3)) was isolated from a UL as well [Myomas are commonly considered to be monoclonal in nature, with their diverse cellular composition resulting from the expansion and differentiation of a single transformed myometrium cell ,10,23. T as well ,28.MED12 (mediator complex subunit 12) mutations [UL stem cells have several differences from those of the myometrium. In culture conditions, ULs form fewer colonies of non-differentiated cells, and the cells, in turn, have a shorter cell cycle ,29,30. Sutations . At the utations . To grow and proliferate, myometrial and UL stem cells need the presence of differentiated cells with higher levels of hormone receptors. The interaction between stem and mature cells is believed to occur through paracrine signals (WNT (wingless-type) proteins, growth factors, etc.) ,32,33 orThe cellular diversity is a considerable, but not the only, contributor to the intratumoral heterogeneity in ULs. Signalling pathways, gene variants, chromosomal abnormalities and epigenetic regulation are among the other aspects of tumorigenesis that create the unique profile of each UL cell .Sex steroid hormones are the key regulators of UL growth. Not only can UL cells bind oestrogen and progesterone from the blood flow, but they can also convert circulating androgens into oestrogens, which explains higher oestrogen levels in UL tissue as compared to the adjacent myometrium .Testosterone and androstenedione are converted into oestradiol with aromatase and 17β-hydroxysteroid dehydrogenase enzymes. The activity of this enzyme system enables UL cells to produce enough oestrogen to support their growth . TreatmeOestrogens, particularly their most active natural form, 17β-oestradiol, are essential components of many pathways associated with UL development. Oestrogens act through their specific receptors (α and β), which bind to 17β-oestradiol and its natural analogues (estrone and estriol) as well as a variety of xenoestrogens, many of which are considered to be endocrine-disrupting chemicals (EDCs) . OestrogDuring the luteal phase of the menstrual cycle, characterised by an increased progesterone blood level, a UL features a higher expression of proliferation markers and a higher mitotic activity ,47,48. IMED12 mutations are linked, among other factors, to changes in the expression and functioning of various WNT/β-catenin signalling components [Oestrogen and progesterone also have non-genomic mechanisms of action in UL cells ,54, realmponents ,57, whermponents ,59,60,61Non-hormonal UL development regulators are growth factors (fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β)), proinflammatory cytokines (tumour necrosis factor TNF-α) and ECM components . The UL Thus, oestrogen and progesterone synergistically regulate the mechanisms of cell proliferation, differentiation and fibrosis in the UL through their receptors. The UL’s overlapping network of intra- and intercellular signalling goes beyond sex hormones to include a variety of growth factors, receptors, cellular adhesion molecules, cytokines, ECM proteins and kinase cascades associated with them. Not only can the reprogramming of fundamental hormonal, paracrine and immune response mechanisms result from genetic and epigenetic changes, but it can serve as an independent factor triggering genetic instability and aberrant gene expression in UL cells.The Lancet published medical data on four generations of one family, including 15 women over 15 years old [Researchers have theorised about the hereditary nature of ULs for a rather long time, considering the ample evidence of a higher UL frequency among close blood relatives. In the 1950s, ears old . Accordiears old . The rolears old  and seveears old ,69. Women of ethnic Latin American and African ancestry have a considerably higher risk of developing ULs than women of European ancestry . FurtherWNT4 (Wnt family member 4), GREB1 (growth regulating oestrogen receptor binding 1), TERC (telomerase RNA component), TERT (telomerase reverse transcriptase), HMGA1 (high mobility group AT-hook 1), FOXO1 (forkhead box O1), TP53 (tumour protein p53), etc.) [KCNMB2 , FBN2 (fibrillin 2), ESR1 (oestrogen receptor 1) and CELF4 (CUGBP Elav-like family member 4) [COL6A3 , COL13A , ARHGAP26 (Rho GTPase activating protein 26), MAN1C1 , BET1L (Bet1 golgi vesicular membrane trafficking protein like), TNRC6B (trinucleotide repeat containing adaptor 6B) and COMT (catechol-O-methyltransferase) gene variants associated with the accumulation of ECM in the UL tissue, the size, localisation and the multiple form of UL [Modern genomic research (genome-wide association search (GWAS) and genome-wide single-nucleotide polymorphism analysis) has identified variants determining predisposition for UL in over 50 genes , etc.) ,79,80,81ember 4) . Researcrm of UL ,85,86,87Hereditary Leiomyomatosis and Renal Cell Carcinoma), Reed’s syndrome is caused by germline mutations in FH, the gene encoding fumarate hydratase, a tricarboxylic acid cycle enzyme [FH gene inactivation, fumarate accumulation in the cells, changes in cell metabolism, the stabilisation of hypoxia-inducible factors (HIFs), the activation of carcinogenic transcription factor NRF2 (nuclear factor erythroid 2 related factor 2) and changes to the expression profile [FH mutations  [FH mutations are often histologically distinctive, demonstrate hypercellularity, nuclear atypia, inclusion-like nucleoli, stromal oedema, and occur with a high frequency among atypical leiomyomas (37.3% compared to 1.6% in unselected ULs) [Particular hereditary syndromes , which feature a high risk of UL , warrante enzyme ,89. Pati profile . In mosttations) ,91,92,93ted ULs) . COL4A3, COL4A4, COL4A5 ). Patients with this syndrome experience primarily progressive loss of kidney function and hearing loss [COL4A5-COL4A6 locus. Overall, the contribution of FH and COL4A5-COL4A6 mutations in the total UL frequency is low, not exceeding 5% [Alport syndrome is caused by basement membrane damage resulting from germinal mutations in the genes of type IV collagen , FH and COL4A5-COL4A6 genes. Less frequent mutations in MED8 (mediator complex subunit 8), MED18 (mediator complex subunit 18), CDK8 (cyclin-dependent kinase 8), CASC15 (cancer susceptibility 15), COL4A6, DCN (decorin), AHR (aryl hydrocarbon receptor), NRG1 (neuregulin 1), ADAM18 , HUWE1 , FBXW4 (F-box and WD repeat domain containing 4), FBXL13 (F-box and leucine-rich repeat protein 13) and CAPRIN1 (cell cycle-associated protein 1) genes [ULs are characterised by varied chromosomal abnormalities and high heterogeneity in acquired somatic mutations, with the most frequent being harboured in 1) genes  and mito1) genes .MED12 mutations (MED12-ULs) varies from 31% to 85% [MED12 mutations are detected more frequently in multiple ULs and small, mitotically active myomas of normal morphology and subserous  localisation with a high ECM content [MED12 gene [MED12 mutations because women with multiple ULs can have MED12 mutations in some of the myomas but not all of them.In different samples, the frequency of ULs with % to 85% ,104,105.% to 85% . MED12 m content ,110,111.D12 gene . HoweverMED12 gene is located in the Xq13.1 chromosome region and encodes the large mediator complex subunit , which bridges gene-specific regulatory proteins in the promoter region to the RNA polymerase II initiation complex. Pathological variants in MED12 are represented in ULs primarily by heterozygous missense mutations in codon 44 of exon 2; ULs have also been found to feature missense mutations in codons 36 and 43, in-frame deletion plus insertion mutations and in rare cases, exon 1 mutations [MED12 mutations have been detected in UL stem cells [The utations ,112. MEDem cells  but not em cells  and are MED12 is expressed primarily from the mutant allele [MED12 c.131G>A mutant allele in uterine tissue is sufficient for the development of UL-like tumours with chromosomal aberrations [MED12 mutations trigger UL growth remains to be established. MED12 mutations undermine the transduction of several cytoplasmic and transcription signals due to the disrupted ability of the Mediator to activate cyclin C and CDK8/CDK19 cyclin-dependent kinases [MED12 mutations are associated with an increased expression of WNT/β-catenin signalling and changes in the regulation of downstream pathways  linked to cell proliferation, autophagy and ECM accumulation [RANKL) and tryptophan 2,3-dioxygenase (TDO2) genes) [MED12 mutations [In MED12-ULs, t allele , indicatrrations . However kinases ,116,117.mulation ,57,118. ) genes) ,120. Gen) genes) ,121. Nevutations . Smooth utations .MED12 mutations. Within the first several passages in culture, MED12-ULs manifest an elimination of cells with MED12 mutations with the expansion of wild-type cells [MED12 mutations are present in smooth muscle cells but not tumour-associated fibroblasts [MED12 mutations is typical for all three predominant UL populations: fibroblasts, smooth muscle cells and endothelial cells [Importantly, not all cells of a MED12-UL carry pe cells . Wu et aroblasts . Howeveral cells . Variatial cells ,125. In al cells ,128,129.MED12 mutations could either be the only genetic aberration detected in a myoma [HMGA2, FH, COL4A5-COL4A6) [MED12 mutations [ a myoma ,130,131 -COL4A6) ,133. Furutations ,134, whiutations ,134,135.At least 40% of ULs carry chromosomal abnormalities. Initially, they were detected using karyotyping of short-term and long-term tumour cell cultures. Over the last few decades, cytogeneticists have described over 200 UL-specific abnormalities, of which the most frequent are structural rearrangements involving chromosome regions 1p, 6p21, 7q and 12q15 . In a siHMGA2/HMGI-C locus [HMGA2 gene encodes a non-histone protein that regulates chromatin conformation and gene expression via binding with AT-rich DNA sequences of enhancers and promoters. Normally, this gene is active in embryonic tissues, stem cells and many malignant tumours [Region 12q14-15 was among the first to be referred to as a “hot spot” of chromosomal rearrangements in ULs . This re-C locus ,145,146. tumours ,148,149.HMGA2 gene, upregulating its expression [let-7 regulatory microRNA binding sites in truncated or chimeric transcripts of the damaged HMGA2 gene [HMGA2 is typical for a considerable subset of ULs without 12q14-15 rearrangements [HMGA2 hypomethylation or decreased levels of repressor microRNAs: let-7a, miR-26a, miR-26b, miR-93 and miR-106b [HMGA2 overexpression are consolidated into a single pathogenetic subgroup, HMGA2-ULs.In ULs, most 12q14-15 breaks are located upstream of the promoter sequence of the pression ,150,151.GA2 gene ,153. Furngements  and coulmiR-106b ,155. TheHMGA2-positive smooth muscle cells and are characterised by a larger size and higher growth rate, mostly through mitotic activity rather than excessive ECM accumulation [HMGA2 locus are stably maintained throughout multiple passages, but their growth is coupled with a decrease in proliferation level and HMGA2 expression and a parallel increase in cellular senescence markers [HMGA2-ULs contain 90% of mulation ,109,145.mulation . In stan markers ,158,159.HMGA2 variant in myometrial stem cells is sufficient for their transformation into progenitor UL cells [ESR1), the PLAG1 (pleomorphic adenoma gene 1) proto-oncogene, CCND1, CCND2, CCND3 and CDK6 cell cycle regulators, epidermal (EGF) and transforming (TGFA) growth factors, the fibroblast growth factor (FGF2) and the vascular endothelial growth factor (VEGFA) [RAD51B/RAD51L1 (14q24), G2/M cell cycle checkpoint regulator CCNB1IP1/HEI10 (14q11), cytochrome C oxidase subunit COX6C (8q22), cytokine response regulator TRAF3IP2 (6q21) and Golgi complex proteins NAA11 (4q21) and COG5 (7q22-31) [It has been demonstrated in an animal UL model that the expression of a shortened UL cells . HMGA2-U (VEGFA) ,164,165.7q22-31) ,171,172.7q22-31) ,165.HMGA1/HMGI-C gene, which emphasises the contribution of the HMGA protein family to tumorigenesis [HMGA2, increased HMGA1 expression is mediated by the hypomethylation of CpG islands at the promoter gene region and may present in a UL without 6p21 rearrangements [PLAG1 proto-oncogene and PAPPA2 , the insulin-like growth factor bioavailability regulator, and inhibited expression of SMOC2, a gene involved in angiogenesis [As another distinctive trait of ULs, rearrangements in chromosome region 6p21 are associated with the damage and overexpression of the igenesis ,174. Simngements ,175,176.ogenesis ,155. RAD51B gene), chromosome region 10q22 is another frequent translocation partner for regions 12q14-15 and 6p21 [KAT6B/MORF/MYST4 (lysine acetyltransferase 6B), which reflects the involvement of anomalous chromatin regulation in UL development [KAT6B abnormalities and their impact on clinical and morphological UL characteristics have barely been studied, there are data suggesting that chimeric transcripts of KAT6B-KANSL1 (17q21) may be discovered in fast-growing cellular ULs, although they are most typical for malignant uterine tumours [Apart from locus 14q24  are also among the most frequent chromosomal rearrangements in ULs ,138,181.CUX1/CUTL1 (cut-like homeobox 1), which could serve as a haploinsufficient tumour suppressor [ORC5/ORC5L and the transmembrane protein gene LHFPL3 are also presumed to be of significance [LMTK2 (lemur tyrosine kinase 2), COPS6 , CUX1, MLL5/KMT2E (lysine methyltransferase 2E), LHFPL3 (LHFPL tetraspan subfamily member 3), LAMB1 (laminin subunit beta 1) and more [While ULs have been found to feature deletions along the entire long arm of chromosome 7, they most frequently affect region 7q22 ,188,189.ppressor ,190,191.ificance ,193,194.and more ,196.An important characteristic of ULs with deletions in 7q (7q-ULs) is their similarity to karyotypically normal ULs in terms of size and response to gonadotropin-releasing hormone agonist therapy (as opposed to ULs with rearrangements in 12q14-15 region) . The mosVarious chromosome 1 rearrangements, which may occur in both karyotypically normal and karyotypically abnormal cells, also significantly contribute to the clonal UL evolution. ULs are characterised by the formation of ring chromosomes 1 and translocations involving both homologues of chromosome 1 or other chromosomes ,200,201.Chromothripsis is a special case of complex chromosomal rearrangements characterised by multiple breaks and deletions in one or few chromosomes . InitialMED12 or FH mutations. Furthermore, the use of molecular techniques allows for finding chromothripsis in ULs with a previously detected apparently normal karyotype or other chromosomal abnormalities indicating the decrease in the proliferative potential of cells with chromothripsis in vitro [Compared to malignant tumours, chromothripsis in UL cells is characterised by a lower number of breaks (around twenty or more versus tens to hundreds) and a larger number of chromosomes involved (up to four) . Chromotin vitro ,143. Meain vitro . Most liRegions involved in chromosomal rearrangements in ULs are located almost throughout the entire genome. Apart from the chromosomes and chromosomal regions described in this section, research papers on UL cytogenetics often mention rearrangements in regions 2p, 2q, 3p, 3q, 5q, 13q, 19q, 22q, Xp, trisomy 12 and monosomy 10 and 22 ,136,210.MED12 mutations as compared to the “wild-type” ULs [The high diversity of somatic changes in ULs is meaningful not only for fundamental studies of UL pathogenesis but also in the development of treatment approaches. Unlike malignant neoplasms, the correlation between the UL response to pharmacological therapy and its genotype/karyotype has been barely examined. For the moment, ULs with a translocation t have been established to show less shrinkage in response to gonadotropin-releasing hormone agonist therapy than ULs with deletions in 7q or with a normal karyotype ,197. Theype” ULs . Howeverype” ULs .FH mutations [Data on genetic aberrations also serve as an auxiliary tool in the clonal origin analysis of ULs and other tumours. Whereas the majority of multiple myomas in the uterus emerge independently from one another, in some cases, different myomas could be clonally linked and could share the same chromosomal abnormalities ,215,216.utations  and the utations . Pathogeutations ,231,232.Areas with UL-specific histology may be present in uterine leiomyosarcomas, attesting to the possibility of UL malignisation ,234. UteKaryotype abnormalities in UL cells could also lead to false positive results in non-invasive prenatal testing (NIPT), a modern technique for detecting foetal chromosomal abnormalities based on the analysis of circulating cell-free foetal DNA in the mother’s blood ,241,242.Therefore, the genetic alterations should be taken into account not only in the investigation of UL tumorigenesis mechanisms but also when assessing the risk of its benign metastasising and malignisation, developing efficient treatment approaches and diagnosing other diseases and conditions.AR methylation status, which confirmed the non-random inactivation of chromosome X in this group of ULs as well [AR methylation analysis results obtained using varied techniques cast a shadow of doubt on the matter [AR expression (RNA-HUMARA) instead of AR methylation to confirm the monoclonal origin in 23/25 (92%) examined ULs, including for individual tumour cell populations: fibroblasts, smooth muscle cells and endothelial cells [The occurrence mechanisms of genetic abnormalities specific to ULs have been investigated in parallel with the discussion about the clonal origin of myomas. As early as in the 1960s, researchers analysed the expressed alleles of glucose-6-phosphate dehydrogenase and established the non-random X chromosome inactivation in myomas, which attested to possible UL monoclonality . The dis as well . Since te matter . SubsequMED12 mutations in UL stem cells [MED12-associated myometrial cell transformation [MED12 mutation not as a transforming event but in the early stages of tumorigenesis  and HMGA2 (12q14-15) overexpression in some ULs without the rearrangements of corresponding chromosome regions [HMGA2 expression across multiple myomas in one patient, the localised pattern of its expression in the myometrium of UL patients [HMGA2- and 7q-ULs speak in favour of this suggestion [Rearrangements involving region 12q14-15  are presumed to occur in the early stages of UL formation, while deletions in chromosomes 1 and 7 occur in later stages ,200,201. regions ,175,176,patients  and the ggestion .Since UL cell proliferation is not coupled with telomerase or ALT  activity, ULs are characterised by shorter telomeres than the myometrium ,251,252.MED12 mutations and how acquired chromosomal abnormalities can change the “developmental programme” of MED12-ULs.Therefore, we are suggesting that, in the period from the beginning of tumour mass formation to its surgical removal, a UL experiences multiple changes in dominating processes that may cause somatic mutations. In the early stages, the more significant factors are a hereditary predisposition, chromatin-level changes, overexpression of individual genes and abnormal hormonal regulation, whereas in the late stages, they give way to telomere shortening, deterioration in DNA repair systems, general metabolic and functional abnormalities in UL cells, the mechanical influence of ECM and adjacent tissues and finally, hormonal and physiological changes in the female body associated with pregnancy, childbirth, lactation and the late reproductive period. Nevertheless, it remains unclear, considering the intercellular genetic heterogeneity of MED12-ULs, whether cytogenetic changes occur in wild-type cells or cells carrying An epigenome is a record of various chemical markers to the DNA and histone proteins and molecules involved in gene expression regulation on the pre- and post-transcription levels. The functional role of epigenetic factors goes beyond genome regulation to include changes in chromosome compaction throughout the cell cycle, centromere positioning, ensuring DNA availability for its recognition by repair complexes, intercellular communication and more ,256. ErrHistones  are highly conserved DNA-binding proteins whose primary functions are packaging DNA into nucleosomes and chromatin compaction. Not only do the post-translational modifications  of amino-acid residues in N- and C-terminal regions of histone proteins ensure the dynamic structure of chromatin, but they are also involved in gene expression regulation, the interaction of DNA with other proteins and the processes of chromosome replication, repair, recombination and disjunction . AcetylaULs have been found to feature various changes in histone modification patterns and associated enzymatic systems. ULs are characterised by increased histone deacetylase expression levels as compared to the myometrium ,261. In EZH2 (the enhancer of zeste homologue 2) and content of H3K27me3 histone than the myometrium [RAD51B, BRCA1 (BRCA1 DNA repair associated) and MSH2 (mutS homolog 2) genes [Furthermore, around 70% of ULs feature higher expression of histone methyltransferase ometrium . This en2) genes ,265. In 2) genes . Adminis2) genes ,268.YEATS4 and ZNHIT1 encoding chromatin remodelling complex proteins (SRCAP), which predispose to UL development [A recent study has documented constitutional variations in the genes elopment . Somaticelopment .3 group from S-adenosylmethionine to cytosine is catalysed by DNA methyltransferases. DNA methyltransferase 1 (DNMT1) maintains the methylated state of the DNA in dividing cells by attaching the methyl group to cytosine residues in the newly replicated strand using the parental strand as a template. DNA methyltransferases 3A and 3B  catalyse methylation de novo and can methylate cytosine both in hemimethylated and unmethylated DNA [Another gene expression regulation mechanism is the methylation of cytosine at the fifth position (DNA methylation). For example, 5-methylcytosine blocks the binding of transcriptional factors with DNA directly or indirectly through specific protein complexes . The traated DNA . The 5-mated DNA .FH) [TET enzymes catalyse the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine ,274,275.FH) ,277. DecFH) . The AIDFH) . The excFH) ,281. TheFH) .DNMT3A and DNMT3B [DMNT1 in ULs is either higher or equal to that of the myometrium [FAM9A, CPXCR1, CXORF45, TAF1, NXF5, VBP1, GABRE, DDX53, FHL1, BRCC3, DMD, GJB1, AP1S2 and PCDH11X) [COL4A1, COL4A2 and COL6A3), prolactin, oestrogen receptor α, repair enzyme gene RAD51B and several oncogenes  [EFEMP1, FBLN2, ARHGAP10, HTATIP2, DLEC1 and KRT19), RANKL gene encoding tumour-associated cytokine, glycoprotein Neuregulin 1 NRG1 gene, 14-3-3γ protein YWHAG gene, and transcription factor genes SATB2, KLF4, KLF11 and ATF3 [MED12 mutations [Unlike the myometrium, ULs feature global genome hypomethylation due to decreased expression of de novo DNA methyltransferases d DNMT3B . By contometrium ,284,285.PCDH11X) . Hypomet TFAP2C) ,288,289.and ATF3 ,292,293.utations  and coulutations .The DNA methylation patterns described above are more typical of mature tumour cells as the population of stem/progenitor cells in ULs is relatively limited ,295. RecTET1 and TET3 than the myometrium [TET1 and TET3 in UL stem cells is lower than in differentiated ones, in full alignment with the differences in their DNA methylation levels [H19-let-7-TET3 axis is an important expression regulator for the genes associated with the early stages of UL development, signalling pathways and collagen production. In particular, the TET3 protein has the capacity for binding with the promoters of MED12, TGFBR2 (transforming growth factor beta receptor 2) and TSP1 (thrombospondin 1), thus facilitating their demethylation and increased chromatin availability [TET1 and TET3 knockdown or treatment with specific TET inhibitors result in a decrease in both DNA hydroxymethylation levels and UL cell proliferation [TET3 expression in the UL also depends on sex steroid hormones and increases under combined exposure to oestrogen and progesterone [TET1 expression in UL cells is associated with the risk of tumour recurrence [Global hypomethylation of the UL genome can be also significantly influenced by TET-mediated active DNA demethylation. ULs are characterised by a higher expression of ometrium . Meanwhin levels . The H19lability . TET1 anferation . TET3 exesterone . Furthercurrence .MED12 mutations but depends on the menstrual cycle phase: it is higher in the follicular than in the luteal phase [Indeed, 5-Hydroxymethylcytosine is the first product of TET-mediated 5-methylcytosine oxidation and is most widely represented in the genome . Locatedal phase .Non-coding RNAs (ncRNAs) are involved in transcription, translation, splicing, lengthening of telomeres, genome imprinting, X chromosome inactivation, intercellular communication and more. ULs have been found to feature dysregulation in several RNA classes, namely microRNAs (miRNAs), circular RNAs (circRNAs) and long ncRNAs (lncRNAs).let-7 and miR-29 families. The let-7 miRNA is a negative regulator of HMGA2 mRNA translation, and its expression is reduced in HMGA2-ULs [let-7 miRNA may also serve as a marker of cellular senescence and inflammation in ULs. Downregulation of the miR-29 miRNA is shown to be responsible for TGF-β3- and oestrogen/progesterone-dependent ECM accumulation in UL cells [miR-29 content inhibits both the expression of collagen genes and the mechanisms of cell proliferation, invasion and metastasising [MiRNAs are short single-stranded RNA molecules containing 18-25 nucleotides. Changes in miRNA profiles in a UL are associated with the expression of components of DNA demethylation, immune response and cell cycle proteins, with the tumour cell differentiation, EDC exposure, tumour size and the patient’s ethnicity ,314,315.MGA2-ULs . The letUL cells . High mitasising . Overalltasising .hsa_circ_0083920, hsa_circ_0056686, hsa_circ_0062558, hsa_circ_0020376 and hsa_circ_0043597. In particular, hsa_circ_0056686 circRNA expression is higher in tumour-associated fibroblasts than in fibroblasts isolated from adjacent myometrial tissues and is associated with UL size, the regulation of proliferation, migration and apoptosis and endoplasmic reticulum stress [CircRNAs are stable regulatory RNA molecules covalently closed at the 3′ and 5′ ends. ULs have been found to feature increased expression of 579 and reduced expression of 625 circRNAs as compared to the myometrium . The mosm stress ,320.HOTAIR long ncRNA gene are associated with the risk of UL development [H19, APTR, XIST, CAR10, CASC15, TCONS_l2_00000923, UC.10, HOTTIP, LINC00890, MEG3, LNCRNA-ATB, MAMDC2-AS1, TSIX, HULC, BX640708, UCA1 and AK023096 lncRNAs as compared to the myometrium [H19 lncRNA is the key regulator of HMGA2 and MED12 (“UL drivers”) expression [APTR  and XIST (X-inactive specific transcript) lncRNAs are involved in the hormone-dependent proliferation of UL cells, the activation of the WNT signalling pathway, collagens and fibronectin gene expression [lnc-AL445665.1-4 lncRNA is typical of multiple but not solitary ULs [SRA1 (steroid receptor RNA activator 1) and MIAT  lncRNAs, which regulate steroidogenesis and ECM accumulation, respectively [LncRNAs are RNA molecules containing 200 or more nucleotides. Some variants of the elopment . ULs alsometrium . The H19pression . The APTpression ,324. Inctary ULs . The MEDectively .6.1-6.3 demonstrate that epigenetic changes in ULs are highly diverse and could be specific either to the condition at large or to patients, molecular subgroups of UL or even cell populations. On the one hand, the epigenetic features of a cell are fairly stable and specific. On the other hand, they are characterised by plasticity and are subject to changes under the influence of endogenous and exogenous stimuli.Data presented in Over the last few years, a considerable quantity of experimental and clinical studies have been issued on the role of endocrine-disrupting chemicals (EDCs), in particular, on xenoestrogens  in UL development ,329,330.The search for a universal event that triggers UL development is considerably complicated by the heterogeneity of molecular features across ULs. For a long time, dependence on sex steroid hormones was considered to be the only common trait of all ULs. However, around three decades ago, Bulun et al. demonstrated that over 90% of myomas are also characterised by an increased expression of aromatase, which converts androgens to oestrogens—up to 25 times as high as in the adjacent myometrium. Aromatase expression was also discovered in the myometrium of 75% of women with UL and 0% of women without ULs . A suggeApart from ULs, aromatase overexpression is also observed in other benign uterine neoplasms: endometriosis and adenomyosis ,345. SimBenign neoplasms of the female reproductive tract share a multitude of molecular features that were addressed in detail by Baranov et al. . AccordiEDC exposure in the early stages of ontogenesis and abnormal aromatase activation may result in a possibility of prepubertal emergence of areas with a high concentration of oestrogen in uterine tissue. Normally, oestrogens trigger the activity of multiple genes and signalling cascades; however, hyperproduction of oestrogens and their oxidised metabolites (catechols and quinones) may also serve as the transforming factor ,358,359.UL is a neoplastic disease characterised by a high incidence, diverse clinical presentation, pronounced autoregulation and considerable changeability in the tumour’s genome and epigenome, which surprisingly coincides with a low risk of malignisation. It is generally accepted that a UL develops from a single myometrial cell—presumably a stem cell—as a result of its transformation and clonal expansion. In our opinion, however, the clonal origin of UL once again requires serious investigation in the context of modern data and methodological approaches. The search for an event that triggers UL formation is strongly impeded by the broad range and non-unique nature of predisposing factors, their uneven contribution, on the one hand, and the pronounced variability in clinicopathological, morphological, genetic and epigenetic characteristics of ULs, on the other hand. By the time of their detection and surgical removal, myomas have existed in the uterus for months, years or even decades, undergoing sequential developmental stages and exposure to a variety of endogenous and exogenous factors. As a result, every myoma acquires a unique portrait with a pronounced intercellular heterogeneity, which makes identifying the characteristics of the initially transformed cell highly problematic . UnfortuMED12 and FH mutations, HMGA2 rearrangements and overexpression, COL4A5-COL4A6 rearrangements) and passenger events , with the latter occurring in later stages and making the greatest contribution to the intratumoral heterogeneity (MED12, HMGA2 and other genes appear to be emerging in undifferentiated cells at the early stages of UL tumorigenesis and largely define the fate of each myoma. However, MED12-ULs and HMGA12-ULs have also been demonstrated to feature cells without corresponding driver mutations, which attests to the fact that, instead of emerging in the initial transformed myometrial cell, they appeared later in one of its daughter cells. In light of this, we suggest that the prerequisite of a myometrial cell transformation into a UL cell may be an epigenetic alteration. Furthermore, the prerequisite event and the beginning of UL formation could be separated by a considerable stretch of time. Moreover, we cannot rule out the possibility that the potential prerequisite may occur during gametogenesis (The key role in the neoplastic transformation belongs to genetic and epigenetic alterations, and in this context, the UL is most likely no exception. The wide variety of such aberrations in ULs can be divided into driver events (ogeneity . Driver ogenesis . In this


One prevalent neurological disorder is epilepsy. Modulating GABAergic/glutamatergic neurotransmission, Nrf2/HO-1, PI3K/Akt, and TLR-4/NF-B pathways might be a therapeutic strategy for epilepsy. Eight-week-old BALB/c mice were administered 12.5, 25, or 50 mg/kg (-) pseudosemiglabrin orally one hour before inducing epilepsy with an i.p. injection of 360 mg/kg pilocarpine. (-) Pseudosemiglabrin dose-dependently alleviated pilocarpine-induced epilepsy, as revealed by the complete repression of pilocarpine-induced convulsions and 100% survival rate in mice. Furthermore, (-) pseudosemiglabrin significantly enhanced mice’s locomotor activities, brain GABA, SLC1A2, GABARα1 levels, glutamate decarboxylase activity, and SLC1A2 and GABARα1mRNA expression while decreasing brain glutamate, SLC6A1, GRIN1 levels, GABA transaminase activity, and SLC6A1 and GRIN1 mRNA expression. These potentials can be due to the suppression of the TLR-4/NF-κB and the enhancement of the Nrf2/HO-1 and PI3K/Akt pathways, as demonstrated by the reduction in TLR-4, NF-κB, IL-1β, TNF-α mRNA expression, MDA, NO, caspase-3, Bax levels, and Bax/Bcl-2 ratio, and the enhancement of Nrf2, HO-1, PI3K, Akt mRNA expression, GSH, Bcl-2 levels, and SOD activity. Additionally, (-) pseudosemiglabrin abrogated the pilocarpine-induced histopathological changes. Interestingly, the (-) pseudosemiglabrin intervention showed a comparable effect to the standard medication, diazepam. Therefore, (-) pseudosemiglabrin can be a promising medication for the management of epilepsy. Epilepsy is one of the world’s most common neurological disorders, characterized by recurrent seizures caused by sudden abnormal neuronal discharges in the brain that momentarily impair brain function. It affects more than 50 million people worldwide, kills 125,000 people each year, and is the sixth leading cause of disability worldwide; more than 85% of cases occur in low-income populations. As a result, it has a detrimental influence on health outcomes while also increasing healthcare expenses and the psychological, cognitive, social, and economic burden e values ; HRESIMS93.1338) .Pilocarpine administration at a dose of 360 mg/kg produced convulsions and status epilepticus (SE) in all the experimental animals in the pilocarpine-induced convulsion (PIL) group, with a mortality rate of 70%. However, (-) pseudosemiglabrin treatment exhibited significant protection against pilocarpine-induced convulsion in a dose-dependent manner, as revealed by the prolongation of latency to first convulsion, reduction in convulsion, SE percentage, and seizures severity score; meanwhile, it increased the survival rate to 100% in the convulsion-induced group pretreated by pseudosemiglabrin 50 mg/kg (SSH group), compared with the PIL group. Moreover, both the SSH and convulsion-induced group pretreated by diazepam 5 mg/kg (DIZ) groups showed complete protection against pilocarpine-induced convulsion in all mice; however, the survival rate in the SSH group was 100%, but it was 95% in the DIZ group .Pilocarpine injections significantly suppressed mice’s locomotor activities, as indicated by the reduction in mice’s crossing, rearing, and grooming numbers, with an increase in immobility number and latency to initiate locomotion compared with the control group. However, the pretreatment with (-) pseudosemiglabrin significantly enhanced the suppressed mice’s locomotor activities in a dose-dependent manner, as shown by the increase in mice’s crossing, rearing, and grooming numbers, as well as the reduction in immobility number and latency to initiate locomotion compared with the PIL group. In contrast, the pretreatment with diazepam significantly augmented the suppression of the mice’s locomotor activities induced by pilocarpine, as revealed by the further reduction in mice’s crossing, rearing, and grooming numbers, with the further increase in immobility number and latency to initiate locomotion compared with the PIL group .(-) Pseudosemiglabrin dose-dependently and significantly enhanced the decreased brain GABA, solute carrier family 1 member 2 (SLC1A2) levels, glutamate decarboxylase activity, and SLC1A2 mRNA expression, as well as reduced the increased brain glutamate, sodium-and chloride-dependent GABA transporter 1 (SLC6A1) levels, GABA transaminase activity, and SLC6A1 mRNA expression induced by pilocarpine injection. Furthermore, the SSH group showed a more significant enhancement of brain GABA level and a more significant reduction in brain GABA transaminase activity, SLC6A1 level, and SLC6A1 mRNA expression compared with the DIZ group. However, the DIZ group exhibited a more significant reduction in the brain glutamate level and a more significant enhancement of the brain SLC1A2 level and mRNA expression than the SSH group. Nevertheless, there was no significant difference between the SSH and DIZ groups regarding the enhancement of the brain glutamate decarboxylase activity .Parallel to the enhancement of the brain GABA level and the reduction in the brain glutamate level, (-) pseudosemiglabrin dose-dependently and significantly enhanced the reduced brain GABA receptor subunit alpha-1 (GABARα1) level and mRNA expression and reduced the increased glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) level and mRNA expression, induced by pilocarpine. However, the DIZ group showed a more significant enhancement of the reduced brain GABARα1 level and mRNA expression and a more significant reduction in the increased GRIN1 mRNA expression than the SSH group. So far, there has been no significant difference between the DIZ and SHH groups regarding the reduction in the increased GRIN1 level .The brain tissue antioxidant system is significantly disturbed by pilocarpine administration, as exhibited by the raised brain tissue malondialdehyde (MDA) and nitric oxide (NO) levels, and the reduction in brain tissue reduced glutathione (GSH) level, superoxide dismutase (SOD) activity, heme oxygenase-1 (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf-2) mRNA expression compared with the control group. Meanwhile, the (-) pseudosemiglabrin pretreatment significantly and dose-dependently restored the brain tissue antioxidant system, as evinced by the reduction in the raised brain tissue MDA and NO levels, and the enhancement of the reduced brain tissue GSH level, SOD activity, and HO-1 and Nrf-2 mRNA expression compared with the PIL group. Moreover, in contrast to the DIZ group, the (-) pseudosemiglabrin pretreatment in the SSH group reduced the elevated NO level induced by pilocarpine injection .Pilocarpine injection enhanced neuronal apoptosis in the brain tissue, as shown by the increased caspase-3 and BCL2-Associated X Protein (Bax) levels, Bax/B-cell lymphoma two protein (Bcl-2) ratio, and the reduction in Bcl-2 level and the mRNA expression of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) compared with the control group. Meanwhile, the pretreatment with (-) pseudosemiglabrin significantly inhibited the neuronal apoptosis induced by pilocarpine injection in a dose-dependent manner, as evidenced by the reduction in the increased caspase-3 and Bax levels and Bax/Bcl-2 ratio together with the promotion of the diminished Bcl-2 level and the mRNA expression of PI3K and Akt, compared with the PIL group. In contrast, the pretreatment with diazepam showed a non-significant effect on the increased caspase-3 and Bax levels and the decreased Bcl-2 level compared with the PIL group. Furthermore, the SSH group showed a more significant Bax/Bcl-2 ratio reduction and enhancement of PI3K and Akt mRNA expressions than the DIZ group .Pilocarpine administration activated the brain tissues’ neuro-inflammation, as revealed by the raised mRNA expression of TLR-4, NF-kB, IL-1β, and TNF-α compared with the control group. However, the pretreatment with (-) pseudosemiglabrin significantly and dose-dependently suppressed the brain tissues’ neuro-inflammatory signals, as it decreased the enhanced TLR-4, NF-kB, IL-1β, and TNF-α mRNA expression, compared with the PIL group. Moreover, there was no significant difference between the SSH and DIZ groups regarding the brain tissues’ neuro-inflammatory signal suppression .The hematoxylin and eosin (H and E)-stained sections of the control group displayed intact and neatly arranged neurons. However, in the PIL group, there was intense cell damage exhibited by nuclear pyknosis, cytoplasmic vacuolation, and neuronal necrosis, along with cerebral congestion and inflammatory influx throughout various brain areas compared with the control group. (-) Pseudosemiglabrin pretreatment noticeably mitigated the pilocarpine-induced neurodegeneration in a dose-dependent manner, to be nearly like the control group in the SSH group. Statistically, (-) pseudosemiglabrin pretreatment significantly and dose-dependently abrogated the histopathological changes induced by pilocarpine injection, which was revealed by the significantly reduced histopathological score. The SSH group also demonstrated more reduction in the elevated histopathological score than the DIZ group .Additionally, Nissl’s staining showed many pyramid-shaped neurons with dense cytoplasmic granules and Nissl’s bodies in the hippocampal CA1 and CA3 regions of the control group. However, pilocarpine injection induced a significant neuronal loss in the hippocampal CA1 and CA3 regions compared with the control group, and the remaining cells were irregular, swollen, and necrotic. Nevertheless, (-) pseudosemiglabrin pretreatment effectively ameliorated neuronal degeneration in the hippocampal CA1 and CA3 regions, as well as significantly and dose-dependently increasing the neuronal survival of the hippocampal CA1 and CA3 regions compared with the PIL group. Moreover, the SSH group demonstrated a more significant increase in the neuronal survival of the hippocampal CA1 and CA3 regions than the DIZ group .(-) Pseudosemiglabrin used in the present study was identified by comparing the spectroscopic data with those reported in the literature . The curFurthermore, (-) pseudosemiglabrin-inhibited neuronal apoptosis induced by pilocarpine injection in a dose-dependent manner, as evidenced by the reduction in increased caspase-3 and Bax levels, the Bax/Bcl-2 ratio, and the promotion of decreased Bcl-2 level and PI3K and Akt mRNA expression. In addition, (-) pseudosemiglabrin significantly suppressed the neuro-inflammatory signals in the brain tissues, as it decreased the increased TLR-4, NF-kB, IL-1β, and TNF-α mRNA expression, as well it increased the number of surviving hippocampal neurons in the CA1 and CA3 regions, and reversed the H and E histopathological changes caused by pilocarpine injection. Interestingly, the (-) pseudosemiglabrin intervention showed a considerable neuroprotective effect on the treated mice, comparable to the traditional medication diazepam.Curing refractory epilepsy requires the use of the appropriate epilepsy model. Acute animal models, such as the pilocarpine-induced epilepsy model, are believed to be useful for initial anti-epileptic pharmacological screening . The pilA receptors, causes sedation and dizziness by boosting GABA action, thus exacerbating the locomotor activity reduction produced by pilocarpine, which was thought to be related to sedation induction in mice [Traditional anti-epileptic drugs frequently cause sedation and dizziness as adverse effects, and patients with epilepsy are more vulnerable to the side effects of anti-epileptic drugs, potentially due to structural or functional abnormalities . In this in mice ,36. HoweThe current study found that pilocarpine injection decreased brain GABA, SLC1A2, and GABARα1 levels, glutamate decarboxylase activity, and SLC1A2 and GABARα1 mRNA expression while increasing brain glutamate, SLC6A1, GRIN1 levels, GABA transaminase activity, and SLC6A1 and GRIN1 mRNA expression. As a result of these data, epilepsy can be induced by a disruption in GABAergic and glutamatergic neurotransmission. These findings are consistent with earlier findings ,16,37,38Astrocytes are recognized to have a role in regulating neurotransmitter concentrations, such as GABA and glutamate, as well as neuronal homeostasis and immunological response . The priIt is well-recognized that downregulation of the N-methyl-D-aspartate receptors (NMDAR) has neuroprotective properties, suppressing epilepsy development and protecting anti-epileptic treatment refractoriness . FurtherThe brain tissue is susceptible to free radical damage due to its high rate of oxidative metabolism and limited antioxidant defenses . ExcessiThe PIL group in the current study exhibited a disruption of the brain antioxidant system, which was considerably alleviated by (-) pseudosemiglabrin pretreatment, with considerable mitigation of epilepsy-induced reductions in SOD activity and GSH level. Moreover, compared with the control group, pilocarpine injection significantly increased brain MDA and NO levels; however, (-) pseudosemiglabrin pretreatment resulted in a dose-dependent reduction in raised MDA and NO levels compared with the PIL group. These findings support prior research on the antioxidant potentiality of (-) pseudosemiglabrin and Tephrosia species, in which (-) pseudosemiglabrin is the predominant biologically active component ,25,26,27Moreover, the activation of the Nrf2/HO-1 signaling pathway improves endogenous antioxidant defense and provides neuroprotection against pilocarpine-induced seizures ,45,46. TAdditionally, activation of the PI3K/Akt pathway is essential for neuronal survival as it promotes cell survival while blocking apoptosis . Akt, a +2 channel signals and lowering GABA receptor responsiveness [The critical factor driving degenerative changes to neural tissue following seizures is the inflammatory response of brain tissue caused by seizures . TLRs arsiveness ,11. Moresiveness ,46.In the current study, (-) pseudosemiglabrin effectively suppressed TLR-4, NF-κB, IL-1β, and TNF-α mRNA expressions, which were induced by pilocarpine injection, implying that it has the potential to prevent the development of epilepsy by suppressing astrocyte activation, inflammatory cytokine production, and their upstream transcription factors, TLR-4 and NF-κB, and was involved in neuroinflammation control. As a result, it improved the brain’s excitability-inhibitability balance, which explains its neuroprotective action against pilocarpine-induced neuronal damage. Similarly, in a rat inflammation model, (-) pseudosemiglabrin demonstrated substantial anti-inflammatory activity by reducing IL-1, TNF-, and NO secretion .Furthermore, noncommunicable diseases, such as cardiovascular diseases, are the primary cause of mortality in the epileptic population. This is most likely due to the progressive emergence of atherosclerosis-accelerating factors, such as obesity and significant alterations in metabolic components, often known as metabolic syndrome (MS). Several studies indicate that the prevalence of MS in the epileptic population ranges from 30.6% to 52.6%. It may be connected with metabolic abnormalities caused by seizures, long-term antiepileptic medication use, a sedentary lifestyle, and other behavioral risk factors in the epileptic population. The higher risk of MS in people with epilepsy may also be attributed to a decline in quality of life, including worse mental function and the presence of psycho-emotional stress ,52. TherIn human epilepsy and animal models of epilepsy, the hippocampus is regarded as the principal seizure starting zone, as evidenced by partial removal of the hippocampus, including the neuronally damaged area, resulting in seizure-free patients ,38,39. F3), acetone, glutamic acid, formalin-buffered saline, hematoxylin, and eosin stains. However, Nissl’s stain was bought from VitroVivoBiotech, Gaithersburg, MD, USA, Tris-HCl buffer was supplied from Research Organics, Cleveland, OH, USA, and nuclear lysis buffer was purchased from ThermoFisher Scientific, Waltham, MA, USA.The following chemicals and reagents were bought commercially from Sigma, St. Louis, MO, USA, pilocarpine, methyl scopolamine, phosphate-buffered saline (PBS), diazepam, dimethyl sulfoxide (DMSO), I-oxoglutarate, pyridoxal phosphate, methanol, trichloroacetic acid, ferric chloride (FeClTephrosia purpurea L. (Pers.) as previously described [(-) Pseudosemiglabrin, used in the present study, was extracted and isolated from escribed . Plant mad libitum access to water and a standard laboratory diet. Mice were acclimated for seven days before the experimental procedure. All experiments were approved by the Research Ethics Committee on the Care and Use of Laboratory Animals at Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia (Registration Reference No. SCBR-056-2022), which conforms to ARRIVE guidelines and National Institutes of Health guidelines for the care and use of laboratory animals. Every effort was made to minimize animal suffering, and all animal handling occurred between 8 a.m. and 8 p.m.Eight-week-old BALB/c mice weighing 20–25 g were used in the present study. Mice were obtained from the Animal Care Unit, Prince Sattam bin Abdulaziz University, Faculty of Pharmacy, Saudi Arabia, and housed in ventilated polypropylene cages at a temperature of 22 ± 2 °C with a 12 h light/dark cycle, with The mouse model of epilepsy was induced by intraperitoneal (i.p.) injection of 360 mg/kg pilocarpine in PBS 30 min after the i.p. injection of 1 mg/kg of methyl scopolamine to reduce the peripheral cholinergic effects of pilocarpine . The negw/v homogenate, centrifuged at 5000 rpm at 4 °C for 10 min, and the supernatant collected. However, the pellet was resuspended in ice-cold nuclear lysis buffer, the suspension was centrifuged at 5000 rpm at 4 °C for 10 min, and the supernatant was collected. The remaining half was homogenized in ice-cold lysis buffer to obtain a 10% w/v homogenate. The brain tissue homogenate and supernatants were stored at −80 °C and used to evaluate the brain tissue biochemical, nuclear, and quantitative real-time polymerase chain reaction (qRT-PCR) parameters.In the present study, 110 mice were used. They were randomly and blindly allocated into six groups. Group I , normal mice were administered PBS intraperitoneally (i.p.) in a volume equivalent to pilocarpine. Group II  was convulsion-induced mice by an i.p. injection of 360 mg/kg pilocarpine . Group IImmediately after pilocarpine injection  pseudosemiglabrin and diazepam), mice’s locomotor activities were evaluated to assess the sedative effect of (-) pseudosemiglabrin using the methods described by Kandeda et al., (2022). The open maze consists of a wooden box with a length of 20 cm, a width of 20 cm, and a height of 45 cm. The exploration area was divided into 16 squares (5 cm × 5 cm). Mice were placed in the center of the experimental device. The behavior of each mouse was monitored for 5 min. In addition, several behavioral parameters were evaluated, including the number of crossings , rearings (times the mouse stood on its hind legs), groomings (mouse self-cleaning behavior), and immobility, as well as latency to initiate locomotion .The total protein content of brain tissue was determined according to the manufacturer’s instructions using a colorimetric assay kit available from AssayGenei, Dublin, Ireland (Cat. # BA0168), with a minimum detection limit of 0.06 mg/mL. Samples absorbances were analyzed using a Stat Fax 2100 automated plate reader, Fisher Bioblock Scientific, BP, Illkirch Cedex, France, and results were expressed as mg protein/mL brain tissue homogenate.Brain GABA and glutamate levels were determined according to the manufacturer’s instructions using colorimetric assay kits available from Antibodies-Online GmbH, Limerick, PA, USA (Cat. # ABIN6966944) and LifeSpan BioSciences, Inc., Seattle, WA, USA (Cat. # LS-F55498), with minimum detection limits of 18.75 pg/mL or 48.2 ng/mL. The absorbances of the samples were analyzed on a Stat Fax 2100 automated plate reader, and the results were expressed as ng/g brain tissue.3 (pH 2), followed by 5 min addition of 4 mL of acetone and evaluated after 30 and 90 s at 610 nm using a Biosystems semi-automated analyzer  against the blank. Brain tissue GABA transaminase activity was expressed in units/g brain tissue, where one unit corresponds to the production of 1 nM succinic semialdehyde/g wet tissue/min [GABA transaminase and glutamate decarboxylase activities of brain tissue were analyzed colorimetrically as defined by Kandeda et al., (2021) and Kandeda et al., (2022) respectively. Briefly, GABA transaminase activity was assessed by adding 15 mM I-oxoglutarate, 15 mM GABA, 10 mg pyridoxal phosphate, and 0.1 mL brain tissue homogenate supernatant to 10 mL tubes, and the volume was made up to 3 mL by addition of Tris-HCl buffer . The mixture was then incubated for 30 min at 37 °C. The blank was prepared by adding 0.1 mL of 5% methanol instead of the homogenate supernatant. The reaction was stopped by adding 0.5 mL of 20% trichloroacetic acid. Then, the color complex of succinic semialdehyde and 3-methyl-2-benzothiazole-2-hydrazone was developed in the presence of 1 mL of 12% FeClssue/min . HoweverThe brain GRIN1, GABARα1, SLC6A1, and SLC1A2 were evaluated by measuring their brain protein levels and mRNA expressions. The brain GRIN1, GABARα1, SLC6A1, and SLC1A2 protein levels were assessed using the manufacturer’s instructions of ELISA kits bought from MyBioSource Inc., San Deigo, CA, USA, Abbexa LLC., Sugar Land, TX, USA, and Antibodies-Online GmbH, Aachen, Germany , respectively, with minimum detection limits of 0.122 ng/mL for the GRIN1 kit, 0.1 μmol/L for the GABARα1 kit, and 0.054 ng/mL for the SLC6A1 and SLC1A2 kits. A Stat Fax 2100 automated plate reader was used to detect brain GRIN1, SLC6A1, SLC1A2, and GABARα1 levels, and expressed as pg and nmol/mg tissue protein.Oxidative stress indicators were assessed by determining brain tissue MDA, GSH, NO levels, SOD activity, HO-1, and Nrf-2 mRNA expression. MDA, GSH, NO levels, and SOD activity in brain tissue were assessed using the manufacturer’s instructions of colorimetric assay kits bought from MyBioSource Inc., San Deigo, CA, USA . Brain tissue MDA, GSH, and NO levels were expressed as nmol/mg tissue protein, and SOD activity was expressed as U/mg tissue protein. Brain tissue oxidative stress markers’ absorbances were analyzed using a Stat Fax 2100 automated plate reader.Brain tissue apoptosis was assessed by measuring caspase-3, Bax, and Bcl-2 protein levels, the mRNA expression of PI3K, and Akt. Caspase-3, Bax, and Bcl-2 protein levels were measured using the manufacturer’s instructions of sELISA kits purchased from MyBioSource . Brain tissue apoptosis markers’ levels were analyzed by an automated ELISA plate reader, Stat Fax 2100, and were expressed as pg and ng/mg tissue protein, and Bax/Bcl-2 ratio.Brain tissue inflammatory signals were assessed by evaluating brain tissue TLR-4, NF-κB, IL-1β, and TNF-α mRNA expressions.The quantitative real-time polymerase chain reaction was used to quantify the mRNA expressions of brain GRIN1, GABARα1, SLC6A1, SLC1A2, HO-1, Nrf2, PI3K, AKT, NF-κB, IL-1β, TNF-α, and TLR-4. The total RNA was extracted from brain tissues utilizing a total RNA extraction kit  according to the manufacturer’s instructions. The iScript cDNA Synthesis Kit was used for reverse transcription . The cDNA obtained was amplified with a real-time PCR system , with the thermal cycling conditions as follows, the initial denaturation and enzyme activation at 95 °C for 3 min (1 cycle), and denaturation at 95 °C for 15 s (sec), annealing at 55 °C for 30 s, and extension at 72 °C for 30 s (40 cycles) using iQ SYBR Green Supermix . The melting curve was performed after the cycle to ensure no non-specific products were present (1 cycle). Relative mRNA expression was normalized to β-actin or GADPH as an internal control to standardize the difference, and the primers’ sequences are shown in Brain tissues were processed as described by Suvarna et al. (2018). Briefly, left hemispheres were fixed in 10% formalin-buffered saline, embedded in paraffin, cut into 5 μm sections, and stained with H and E and Nissl’s stain for further evaluation of the histopathological changes . Sectionp-value < 0.05 is considered significant.The present study’s data were assessed using the Statistical Package for the Social Sciences Statistics Software for Windows , and the data were expressed as mean ± SD. The groups’ data were compared with a one-way analysis of variance (ANOVA) followed by Dunnett’s T3 multiple comparison post-hoc test to determine significant differences between individual groups. A Data from the current study revealed that (-) pseudosemiglabrin therapy successfully alleviates pilocarpine-induced epilepsy. This potentiality may be partly mediated by enhancing GABAergic neurotransmission while decreasing glutamatergic neurotransmission and its antioxidant, anti-apoptotic, anti-inflammatory, and neuroprotective properties. Furthermore, (-) pseudosemiglabrin displayed anti-epileptic efficacy comparable to diazepam, the standard anti-epileptic medication, in this animal model without sedation induction. As a result, (-) pseudosemiglabrin can be a feasible and safer medication for the management of epilepsy.


Nitrogen (N) is a crucial nutrient that plays a significant role in enhancing crop yield. Its availability, including both supply and deficiency, serves as a crucial signal for plant development. However, excessive N use in agriculture leads to environmental and economic issues. Enhancing nitrogen use efficiency (NUE) is, therefore, essential to minimize negative impacts. Prior studies have investigated the genetic factors involved in N responses and the process of low-nitrogen (LN) adaptation. In this review, we discuss recent advances in understanding how epigenetic modifications, including DNA methylation, histone modification, and small RNA, participate in the regulation of N response and LN adaptation. We highlight the importance of decoding the epigenome at various levels to accelerate the functional study of how plants respond to N availability. Understanding the epigenetic control of N signaling and adaptation can lead to new strategies to improve NUE and enhance crop productivity sustainably. N is a primary macronutrient for plant growth, and it is a vital component of DNA, RNA, and proteins. Conventionally, farmers have used synthetic fertilizers to provide sufficient N levels to the crops . HoweverPCF (TCP)-DOMAIN FAMILY PROTEIN 20 (TCP20) [TGACG MOTIF-BINDING FACTOR 1 (TGA1) [The uptake and assimilation of N are essential processes that ensure optimal nutrient acquisition, storage, and metabolism in plants ,5. These (TCP20)  and TGAC1 (TGA1) . The int1 (TGA1) . This guTranscriptional regulation in plants is not only controlled by genetic factors, but also in an epigenetic manner. Epigenetics refers to the process where chemical or structural modifications alter the chromatin state, impacting gene expression without altering the underlying DNA sequence. DNA methylation, histone modifications, enhancer-promoter 3D structural interactions, and noncoding RNA-mediated regulation are examples of epigenetic mechanisms that can affect gene expression ,15. In rIn this review, we summarize current knowledge of epigenetic regulation under different N conditions and propose decoding the epigenome at various levels provides an opportunity to accelerate the functional study of how plants respond to N availability, and thus improving NUE in crops.Plants possess the remarkable ability to promptly perceive nutrient availability, initiating a cascade of responses to maintain nutrient homeostasis . N, a viArabidopsis [N metabolism in plants encompasses processes such as uptake, transportation, and assimilation, all of which are subject to precise and dynamic regulation by NMGs ,10. Notabidopsis . Alterinbidopsis ,33,34,35bidopsis ,23,36.Arabidopsis, the H3K36me3 ‘writer’ SET DOMAIN GROUP8 (SDG8) plays a significant role in regulating genes related to energy metabolism, including NMGs and photosynthesis, through H3K36me3 modification [SDG8 is induced by N treatment in roots, particularly in the lateral root cap and pericycle [SDG8 mediates N-triggered gene-expression reprogramming, including NRT2.1, NRT1.5, and GLUTAMINE SYNTHETASE 1;4  [sdg8 mutants have shown lower nitrate acquisition than wild-type plants under high N conditions [Solanum lycopersicum), the homologs of SDG8, namely SlSDG33 and SlSDG34, regulate SlNRT1.1 in an N-dependent manner that is associated with the root response to N treatments [AtNRT2.1 expression in Arabidopsis [AtNRT2.1 locus to repress its transcription [AtHNI9 control reactive oxygen species (ROS) homeostasis under conditions of excess N. The mutation of AtHNI9 leads to elevated ROS levels and ROS-dependent upregulation of AtNRT2.1, indicating the role of HNI9 in balancing N uptake and ROS levels in response to N supply [In dicots, histone modification, such as tri-methylation of lysine 36 or lysine 27 on histone H3  are implicated in the regulation of N uptake process. In fication . SDG8 isericycle . Recent    encodes a subunit of the ATP-dependent chromatin remodeling complex known as the SWITCH/SUCROSE NONFERMENTING (SWI/SNF) complex [ZmCHB101 has been found to govern the expression of nitrate transport genes, including ZmNRT2.1 and ZmNRT2.2, in response to nitrate supply [ZmCHB101-RNAi lines showed significantly reduced nucleosome densities at the −1 and +1 nucleosome regions of ZmNRT2.1 and ZmNRT2.2 as compared to WT under nitrate treatment. These low nucleosome densities resulted in enhanced ZmNLP3.1 binding to the promoter regions of ZmNRT2.1 and ZmNRT2.2, further regulating their expression [In monocots, N uptake was regulated by changes in chromatin rearrangement.  complex ,41. In me supply   on key TFs involved in root development in monocots. One such TF is ARABIDOPSIS NITRATE REGULATED 1 (ANR1), a member of the MIKC-type MADS (MCM1/AGAMOUS/DEFICIENS/SRFl) box family of TFs, which mediates the root response to external N . It stimANR1   [AUXIN RESPONSE FACTOR 8 (ARF8) in pericycle cells in Arabidopsis [T5120, a long non-coding RNA modulated by NLP7 and NRT1.1, promotes nitrate assimilation and root growth  in Arabidopsis [ARF8 being induced by an N metabolite produced during N reduction, coordinating plant growth and development in response to both external and internal N availability [In dicots, in particular 3 (AFB3) . Similarbidopsis   and SQUAMOSA-promoter binding protein-like 14 (SPL14)) through H3K27me3 modification [TEOSINTE BRANCHED1 (OsTB1), which is a branching-inhibitory gene, by recruiting PRC2 and promoting bud outgrowth [N supply exerts an epigenetic influence not only on root development but also on plant architecture, particularly for tillering in monocots. In rice, the application of N fertilizer induces changes in the genome-wide H3K27me3 pattern through NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5 (NGR5). This mechanism facilitates the recruitment of PRC2, thus promoting the repression of branching-inhibitory genes  through interactions with auxin transport ,64. Notabidopsis . Furtherbidopsis  , coupled with a reduction in H3K27me3 modifications, work in synergy to enhance the expression of genes associated with root development under LN conditions , the auxin biosynthesis gene [TAR2 has also been reported to be a target of RNA-directed DNA methylation (RdDM) in Arabidopsis [Regulation of auxin biosynthesis in the roots involves DNA methylation. Under LN conditions, the biosynthesis of auxin in the roots of eedlings  and wheaeedlings  is inducsis gene . Moreovebidopsis   to inherit adaptive traits to cope with N-deficiency stress. In rice, for instance, modified cytosine methylation patterns have been identified as the key mediators enabling the stable transmission of adaptive traits under N-deficiency stress to the progeny . The offArabidopsis. The differential expression of specific miRNAs, such as miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857, has been observed in Arabidopsis in response to N starvation [Arabidopsis in response to N starvation [NUCLEAR FACTOR Y A-B1 (NFYA-B1) has been identified as playing a pivotal role in promoting root development and enhancing the expression of nitrate transporters in response to LN conditions [TaNFYA-B1 by cleaving its mRNA [For adaptation to LN environment, recent studies have shed light on the intricate involvement of miRNAs in the regulation of root architecture in arvation . Notablyarvation . In wheanditions . Interesits mRNA  , and DNA methylations, to orchestrate morphological developmental changes and adapt their N metabolism in response to environmental N availability. Recent evidence emphasizes the functional significance of PRC2-TF regulatory modules as potential targets for enhancing NUE in crops. Nevertheless, several questions remain unanswered. Firstly, it is crucial to elucidate how N signaling triggers chromatin-level responses. Additionally, understanding the specific “mediators,” including bjective .Both plants and animals heavily rely on nutrients and energy for their developmental processes, with nutrient signaling directly impacting the epigenome ,74. A recis-elements within these regions, the trans-factors involved can be identified through DNA-protein interaction assays. Additionally, protein interaction assays, as demonstrated in Arabidopsis [Rather than inducing global alterations in chromatin modification, the availability of N tends to affect specific genomic regions and subsets of genes, playing a crucial role in shaping the developmental programs of particular tissues ,67. Thisbidopsis , can helFurthermore, the response of different root parts to N availability exhibits cellular specificity, suggesting unique functions for each cell type . Single-The use of GWAS analysis is widespread in identifying factors that regulate important agronomic traits and mining elite allelic variations in key regulators . HoweverIn maize, a distal enhancer located approximately 63.9 kb upstream of the transcription start site (TSS) regulates the expression of teosinte branched1 (TB1), influencing shoot branching . MoreoveUnderstanding the molecular connections between N signaling and chromatin response can enhance our knowledge of developmental regulation. By targeting specific chromatin modifiers and factors involved in the response to N availability, we can precisely control gene expression, ultimately increasing crop yields. Exploring the differential DNA methylation region, identification of distal regulatory regions and their interactions with genes through population genetics, and 3D genomics provides valuable insights for optimizing N utilization in crops. With these advancements, CRISPR-based genetic and epigenetic editing that targets genes, regulatory regions, or epigenetic marks have the potential to revolutionize crop improvement, promoting sustainable agriculture practices and minimizing environmental impacts.


We divided included patients into four groups based on their use and type; Group 1 included patients without a prescription of β-blockers, Group 2 included patients with a prescription of dialyzable and cardioselective β-blockers, Group 3 included patients with a prescription of non-dialyzable and non-cardioselective β-blockers, and Group 4 included patients with prescription of non-dialyzable and cardioselective β-blockers. The number of patients in Groups 1, 2, 3, and 4 were 34,514, 2789, 15,808, and 1021, respectively. The 5-year survival rates in Groups 1, 2, 3, and 4 were 69.3%, 66.0%, 68.8%, and 69.2%, respectively. Univariate Cox regression analyses showed the hazard ratios to be 1.10  in Group 2 and 1.05  in Group 3 compared to Group 1. However, multivariate Cox regression analyses did not show statistical significance among the four groups. Our study showed that there was no significant difference in patient survival based on the use or types of β-blockers.Previous results regarding the association between types of β-blockers and outcomes in patients on hemodialysis (HD) were inconsistent. Our study aimed to evaluate patient survival according to the type of β-blockers administered using a large sample of patients with maintenance HD. Our study included patients on maintenance HD patients from a national HD quality assessment program ( The end-stage renal disease requires renal replacement therapy. Among the three known renal replacement therapies, namely hemodialysis (HD), peritoneal dialysis, and kidney transplantation, HD is the most commonly used modality ,2. Patieβ-blockers are the most commonly used antihypertensive drugs in patients with HD. They are known to have survival benefits in patients with myocardial infarction (MI) or heart failure beyond simple blood pressure control . HoweverThis retrospective study utilized data from a national HD quality assessment program, as well as information from claims data provided by the Health Insurance Review and Assessment (HIRA) of the Republic of Korea ,20. The The total number of participants in the 4th, 5th, and 6th HD quality assessment programs were 21,846, 35,538, and 31,294, respectively .From this pool, we excluded individuals who had participated in multiple assessments or had incomplete datasets, those who underwent HD via non-cuffed or cuffed tunneled catheter, and those who were prescribed two or more β-blockers or received them for less than 90 days within each six-month assessment period. Finally, our study included a total of 54,132 patients. The study received approval from the institutional review board (IRB) of the Yeungnam University Medical Center . Informed consent was not obtained from the patients as all records and participant information had been anonymized and de-identified before the analysis. urea, serum albumin concentration (measured in g/dL), serum calcium levels (measured in mg/dL), serum phosphorus levels (measured in mg/dL), serum creatinine levels (measured in mg/dL), predialysis systolic blood pressure readings (measured in mmHg), predialysis diastolic blood pressure readings (measured in mmHg), and ultrafiltration volume per session (measured in liters). Data collection occurred monthly, with all laboratory values averaged across the readings. Kt/Vurea was calculated using Daugirdas’ equation [We gathered a comprehensive set of data variables, including demographic information such as age, sex, primary cause of end-stage renal disease, duration of HD treatment (in months), and types of vascular access used. Our dataset also incorporated laboratory measurements taken during the assessment, such as hemoglobin levels (measured in g/dL), Kt/Vequation .The codes for medication are shown in The presence of comorbidities was evaluated for a year before the evaluation of the HD quality assessment program. Comorbidity was defined using the codes utilized by Quan et al. ,23. The We assessed all-cause mortality as the primary outcome and cardiac-cerebrovascular events (CVE) as the secondary outcome in our study. CVE was defined as interventions related to revascularization, which were evaluated using medical treatment, procedure, or operation codes listed in We tracked the outcomes continuously until April 2022. In cases where a patient transferred to peritoneal dialysis or underwent kidney transplantation, the date of transfer or transplantation was marked as the endpoint of follow-up, and the data were censored accordingly. Throughout the follow-up period, electronic records were used to define clinical outcomes, excluding cases of death. Censoring codes applied were O7072, O7071, or O7061 for peritoneal dialysis and R3280 for kidney transplantation. Information regarding patient mortality was extracted from the HIRA database. 2 test or Fisher’s exact test was employed. Continuous variable comparisons were conducted using a one-way analysis of variance, followed by the Tukey post hoc test. The data were subjected to analysis using the SAS Enterprise Guide version 7.1  or R version 3.5.1 . Categorical variables were expressed in terms of numbers and percentages, while continuous variables were presented as means with accompanying standard deviations. To analyze categorical variables, Pearson’s χp-values for the comparison of survival curves. In our multivariate Cox regression analyses, adjustments were made for various factors, including age, gender, type of vascular access, the underlying cause of end-stage renal disease, CCI score, HD vintage, ultrafiltration volume, Kt/Vurea, hemoglobin levels, serum albumin levels, serum creatinine levels, serum phosphorus levels, serum calcium levels, predialysis systolic blood pressure, predialysis diastolic blood pressure, and the use of medications such as aspirin, renin–angiotensin system blockers, clopidogrel, ticlopidine, or statins, and the presence of MI or congestive heart failure (CHF). These multivariate Cox regression analyses were conducted using the enter mode. Statistical significance was set at p < 0.05.Survival estimates were computed using the Kaplan–Meier curve, and Cox regression analyses were applied. The log-rank test was employed to determine The numbers of patients in Groups 1, 2, 3, and 4 were 34,514, 2789, 15,808, and 1021, respectively . urea and lower phosphorus and creatinine levels than those in Group 2 or 3. Among the four groups, arteriovenous fistulas were commonest in Group 3. Hemoglobin levels in Group 1 or 2 were greater than those in Group 3. Male predominance was lower in Group 1 than in the other three groups. The patients in Group 1 had lower incidences of diabetes mellitus (DM), use of renin–angiotensin system blockers, statin, aspirin, clopidogrel, or ticlopidine, and MI or CHF than those in the other three groups. They had lower CCI scores, SBP, and ultrafiltration volume than those in the other groups. Moreover, the patients in Group 1 had greater Kt/Vp = 0.113).The respective numbers of patients in the survivor, death, peritoneal dialysis, or kidney transplantation subgroup at the endpoint of follow-up were 18,407 (53.3%), 13,380 (38.8%), 119 (0.3%), and 2608 (7.6%) in Group 1; 1449 (52.0%), 1120 (40.2%), 15 (0.5%), and 205 (7.4%) in Group 2; 8285 (52.4%), 6249 (39.5%), 56 (0.4%), and 1218 (7.7%) in Group3; and 565 (55.3%), 388 (38.0%), 0, and 68 (6.7%) in Group 4  for Group 2 and 1.05  for Group 3 compared to Group 1 . However, multivariate Cox regression analyses did not show statistical significance among the four groups. We also performed Cox regression analyses based on the characteristics of β-blockers . Univariate and multivariate Cox regression analyses revealed consistent patterns, as observed in the analysis of the four distinct groups.Subsequently, we conducted subgroup analyses stratified by sex, age, and the presence of DM and heart disease (MI or CHF) . In thesFor CVE analyses of CVE, there were 31,080 patients in Group 1, 2353 patients in Group 2, 14,129 patients in Group 3, and 887 patients in Group 4. The 5-year survival rates in Groups 1, 2, 3, and 4 were 86.3%, 83.6%, 85.1%, and 85.1%, respectively. On univariate and multivariate analyses, Group 2 had poorer CVE-free survival rates than Group 1 or 3 . Group 2We analyzed 54,132 patients who underwent an HD quality assessment program in the Republic of Korea. Our results revealed that the use of β-blockers was not associated with mortality in patients on maintenance HD. In univariate Cox regression analyses, Group 2 or 3 had poorer patient survival than Group 1. However, in multivariate Cox regression analyses, statistical significance was not seen across all four groups. Subgroup analyses using patients with old age, DM, or heart diseases showed a favorable patient survival in Group 3 than in Group 2. Some hypotheses are associated with favorable outcomes regarding cardioselective β-blockers compared to non-cardioselective ones. Cardioselective β-blockers lower blood pressure via a decrease in cardiac output rather than vasodilation; however, drugs with affinity to α- and β-receptors can attenuate compensatory vasoconstriction, which is associated with hemodynamic instability during ultrafiltration ,9. β1-seSome studies showed better clinical outcomes with dialyzable and/or cardioselective β-blockers than with non-dialyzable and/or non-cardioselective ones, despite overall inconsistent results between the types of β-blockers and the clinical outcomes ,16,17,18In subgroups with old age, DM, or heart diseases, non-dialyzable and non-cardioselective β-blockers were associated with better patient survival than dialyzable and cardioselective ones. The risk of hyperkalemia is greater in such patients, and they are more susceptible to changes in pharmacokinetics during HD sessions. Therefore, non-dialyzable and non-cardioselective β-blockers would be helpful in maintaining stable blood pressure via stable pharmacokinetics during dialysis in stable patients on HD; the low risk of bradycardia with non-cardioselective β-blockers may be useful in attenuating heart blocks or bradycardias due to electrolyte abnormalities.The prognosis of patients with HD can be significantly influenced by various comorbidities; therefore, adjustment for these factors is essential in multivariate analyses. In our study, instead of adjusting individually for each comorbidity, we chose a simplified approach. Specifically, we utilized a merged score from the Charlson Comorbidity Index (CCI) as a confounding factor in multivariate analyses and independently analyzed DM and heart diseases (MI or CHF). The CCI in our study included 17 comorbidities: MI, CHF, peripheral vascular disease, cerebrovascular disease, dementia, chronic pulmonary disease, rheumatologic disease, peptic ulcer disease, mild liver disease, DM without chronic complications, hemiplegia, renal disease, DM with chronic complications, any malignancy, moderate to severe liver disease, metastatic tumor, and acquired immune deficiency syndrome. Among these comorbidities, only heart disease (MI or CHF) was considered an independent confounding factor or utilized in subgroup analysis. For DM, it was only used in the subgroup analysis. However, for other comorbidities, we chose to include them solely as confounding factors in multivariate analyses, utilizing the CCI scores derived from them. While it is possible that each comorbidity included in the CCI could individually affect the prognosis of patients with HD, our study aimed to primarily examine differences in overall mortality rates based on types of β-blockers. Therefore, we selected a simpler model. Analyzing the individual risks associated with each comorbidity is an interesting research topic but beyond the scope of our study’s main objectives.In the analyses of patients not receiving treatment for recent cardiac or cerebrovascular events, the CVE-free survival rate was poorer in Group 2 than in Group 1 or 3, even though there was no association with all-cause mortality. Further, Group 2 had a poorer CVE-free survival trend than did Group 4. The lack of statistical significance between Group 2 and Group 4 may be due to the small sample size in Group 4. These findings are consistent with Weir’s study, which demonstrated favorable outcomes with non-dialyzable β-blockers compared to dialyzable β-blockers . These sWe conducted subgroup analyses to investigate the effects of β-blockers and potential differences among β-blocker types in patients with coexisting conditions, such as MI, CHF, and atrial fibrillation. However, our study did not demonstrate a mortality benefit associated with β-blocker use or a significant difference among β-blocker types. Based on previous research and considering the specific limitations and characteristics of our study, we expected that β-blocker use in patients on HD with conditions like MI, CHF, and atrial fibrillation would likely benefit patient prognosis. The lack of such findings in our study may be attributed to certain limitations and patient characteristics. First, we acknowledge that using ICD-10 codes for diagnostic accuracy might have introduced inaccuracies since accurate diagnosis usually involves essential tests such as coronary angiography, electrocardiography, and echocardiography. Moreover, the high incidence of comorbidities and the relatively advanced age of patients on HD significantly affected the potential side effects of β-blockers, potentially diminishing their overall benefits. Hence, when contemplating the use of β-blockers in patients with HD with suitable indications, it is crucial to thoroughly evaluate and, if necessary, adjust their usage considering associated complications.Summarizing our study’s findings in the context of β-blocker use, type differences, analyses across various subgroups, and existing literature, we conclude that patients on HD exhibit unique variations. However, there is currently insufficient evidence to conclusively demonstrate the superiority of a specific β-blocker type. Therefore, decisions regarding β-blocker use should be based on pharmacodynamics and a personalized approach. While β-blockers, particularly cardioselective ones, are beneficial in conditions such as MI, CHF, and atrial fibrillation, it is important to consider factors like blood pressure fluctuations, heart rate, and the timing of these changes when deciding whether to continue using or switch β-blocker types. Furthermore, if the primary goal is blood pressure control, non-cardioselective β-blockers could be prioritized. In patients on HD, where the benefits may not be significantly greater than those in non-dialysis patients due to various underlying conditions, it is reasonable to not be overly cautious about discontinuing β-blockers if they cause side effects that could potentially worsen prognosis. However, we did observe differences in mortality rates related to the type of β-blockers in specific subgroups, especially in older adults, those with DM, and heart diseases. In these groups, non-dialyzable non-cardioselective β-blockers were associated with a more favorable prognosis compared to dialyzable β-blockers. This may be due to lower hemodynamic changes.Our study had some limitations. First, the study was designed retrospectively, and there existed a substantial disparity in sample size and baseline characteristics across the four groups. The retrospective nature of the study may lead to selection bias, data quality concerns, and challenges in establishing causal relationships. Therefore, caution should be exercised when generalizing findings or interpreting the results. Second, comorbidities, including heart diseases and the use of β-blockers, were evaluated using claims data. Our study lacks data on precise medication dosages, timing of administration related to dialysis sessions, and medication self-adjustment based on blood pressure changes. There may be discrepancies between the prescribed medication amount and the actual dosage taken, particularly among dialysis patients who may take medications after dialysis due to intradialytic hypotension or immediately before dialysis in cases of intradialytic hypertension. Patients may voluntarily discontinue medications when blood pressure is low or take additional doses when it is high. We should consider these factors as significant confounding variables that could potentially affect our results, but the lack of detailed information on medication administration practices is a limitation of our study. In addition, the etiology of use of β-blockers drug intake can influence patient outcomes. Effects of β-blockers on patient survival may be different between patients taking medications for MI or CHF and those taking medications for simple blood pressure control. Furthermore, the timing of drug intake, such as before or after an HD session or cessation of medication on the day of HD, can influence the association between β-blockers types and patient outcomes. Third, our study lacks data on the cause of death, the occurrence of intradialytic hypotension, and supplementary measurements related to heart function, such as ejection fraction, left ventricular hypertrophy, heart rate, or cardiac mass. β-blockers are known to be effective in managing cardiovascular diseases, and having information on cardiovascular-related mortality or heart function would be highly valuable in understanding the specific advantages and potential risks associated with different types of β-blockers types. This information goes beyond just assessing overall mortality. The presence of intradialytic hypotension is especially considered one of the main hazardous effects of non-dialyzable and/or non-cardioselective β-blockers and data in that regard would be useful to understand the cause-relationship between β-blocker types and patient death. Additionally, another important limitation of our study is that we did not consider the cause and severity of heart failure. We lacked data on these aspects, which is inherent to our study design. Our study diagnoses the disease using a different code-based approach instead of relying on chart review, laboratory, and imaging data. Our approach to heart failure in this study was limited in terms of detailed cause assessment and the ability to assess the degree of functional impairment. Heart failure has various potential causes, such as ischemia, blood pressure, arrhythmia, or amyloidosis, and can be categorized into three groups of heart failure with reduced, moderately reduced, and preserved rejection fractions. These factors can impact prognosis and clinical outcomes differently depending on the type of β-blockers used. We acknowledge that this limitation is significant in our study. We believe that addressing these limitations will be crucial in future research by conducting comprehensive subgroup analyses and utilizing other methods.Our study showed that there was no significant difference in patient survival based on the use or types of β-blockers. Subgroups with old age, DM, or heart diseases had survival benefits with non-dialyzable and non-cardioselective β-blockers than with dialyzable and cardioselective ones. In addition, patients who did not receive interventions associated with revascularization had a better CVE-free survival trend with non-dialyzable than with dialyzable ones. Our results should be carefully interpreted, considering the aforementioned limitations of this study. Additional randomized prospective investigations are necessary to establish definitive conclusions regarding the impact of β-blocker types on outcomes among patients undergoing HD.


The study first identifies a proper 3D design, confines the most relevant process parameters by means of primary electron energy and beam currents, and evaluates post-growth procedures as well. That way, highly crystalline nano-tips with minimal surface contamination and apex radii in the sub-15 nm regime are fabricated and benchmarked against commercial products. The results not only reveal a very high performance during MFM operation but in particular demonstrate virtually loss-free behavior after almost 8 h of continuous operation, thanks to the all-metal character. Even after more than 12 months of storage in ambient conditions, no performance loss is observed, which underlines the high overall performance of the here-introduced FEBID-based Co3Fe MFM nano-probes.Magnetic force microscopy (MFM) is a powerful extension of atomic force microscopy (AFM), which mostly uses nano-probes with functional coatings for studying magnetic surface features. Although well established, additional layers inherently increase apex radii, which reduce lateral resolution and also contain the risk of delamination, rendering such nano-probes doubtful or even useless. To overcome these limitations, we now introduce the additive direct-write fabrication of magnetic nano-cones via focused electron beam-induced deposition (FEBID) using an HCo Magnetic Force Microscopy (MFM) is an advanced Atomic Force Microscopy (AFM) operation mode that can be used to map local magnetic fields at sample surfaces, e.g., for studying magnetic thin film materials ,2, vortiWithin the small pool of relevant fabrication technologies, focused electron beam-induced deposition (FEBID) is a potential candidate due to the strong progress made in recent years . In prinm regime . With apm regime ,13. By tm regime , sensingm regime ,16, plasm regime , nano-mam regime ,19, and m regime . Concernm regime ,21; (2) m regime  can be rm regime , scanninm regime , or elecm regime ; and (3)m regime ,23 on a m regime . Concernm regime ,26,27,283Fe(CO)12 [In this work, the focus lies on the development of specially designed MFM tips from a heterometallic precursor, HCoFe(CO)12 , which rFe(CO)12 ,29 and eFe(CO)12 ,30,31. TFe(CO)12 .3Fe(CO)12 (denoted as Co3Fe) precursor, as shown in The demands for ideal MFM tips are listed in the introduction and require a novel design of the probe, as discussed in the following. In this special case, we aim on the fabrication on flat, tip-less self-sensing cantilever (SS-CL) platforms ,33,34 Fa, which the past ,7,35. Al3Fe precursor, although vertical growth rates were much lower than for Pt-based materials  tips on the same SS-CL platforms , which prameters ). This lrameters ,36. As lrameters . The prorameters  and, by rameters ,38; and 3Fe α-pillars, deposited with the same design  with a metal-oxide sheath is assumed for optimized conditions at 20 keV [Besides the shape criteria, discussed before, the material properties are decisive when applied for MFM. The degree of precursor dissociation, which impacts the chemistry as well, depends on the process parameters ,44. Thert 20 keV ,30. Fortr system ,46, whicNext, beam currents were varied from For higher currents of aterials . A detai as well b. By tha3Fe composition [For a more detailed structural and chemical analysis, transmission electron microscopy (TEM) was conducted for position ,29,48.3Fe composition by the application of lower currents than applied here. For completeness, we want to comment on the right-hand side of Although a closer look at this peculiarity needs to be done in the future, we currently propose that the high areal current density at the very center of the e-beam leads to a different composition, where local beam heating can play a role as well ,50. In oAs discussed before, α-pillar properties can be tuned by fabrication parameters 2O environments was considered, as it is known to effectively remove remaining carbon contents when working with platinum (MeCpPtIVMe3) [2Au(acac), Me2Au(tfa)) [2Ru) [3Fe precursor provides more than 80 at.% metal content and for 3D structures close to pure metal after deposition [3Fe nanoprobes were pre-characterized and then subjected to EBC at First, thermal annealing was applied as it is a simple post-processing route, which partially led to significant improvements ,53,54. FPtIVMe3) , gold (MAu(tfa)) ,56, and )) [2Ru) . Althougposition ,29,48, tposition  were useposition , which mposition ,59, whicposition ,61, whilposition , which . Both aspects become even more evident by a direct 3D comparison of MFM phase images, as shown in (b), for which a 3Fe α-pillars.After deriving a design and identifying parameters for sensitive and stable MFM operation see , FEBID-b3Fe α-pillars were subjected to long-time measurements and long-time storage experiments. First, a slow tip velocity of 3Fe α-pillars reveal low wear and high reliability due to their coating-free, all-metal character. Even if a tip gets blunt, the magnetic functionality is still present and can be laterally correlated with the height, which is one of the main problems for functionally coated standard tips.When comparing commercial products, a closer look at wear and long-time stability is indispensably needed. Wear is a complicated quantity, as it strongly depends on sample surfaces and AFM operation conditions. To nevertheless provide quantitative information, the Co3Fe α-pillars. Hence, MFM experiments were repeated after exactly 12 months with the very same AFM system and identical scan parameters on the same CoPt sample. 3Fe α-pillars, which reveals not only excellent MFM performance but also high wear resistance and long-time stability, as relevant for commercial applications.As aging effects on functional properties have been observed for some FEBID materials ,62, we s3Fe nano-probes for AFM-based magnetic force microscopy, fabricated by focused electron beam induced deposition. To provide mechanical rigidity, we introduce a special conical design, which is realized by gradual electron beam blurring. While the cones , reveal overall heights of 2O environments did not improve the overall performance, which simplifies the initial fabrication routes. In comparison with well-established commercial products, optimized Co3Fe α-pillars reveal slightly lower absolute phase shifts, however, with very low noise and high lateral resolution for clear imaging of magnetic nanofeatures, which cannot be observed with commercial MFM probes. An expanded wear study revealed virtually no quality loss after almost This work introduces all-metal Co3Fe(CO)12) was loaded in a standard gas injection system  and heated to FEBID experiments and SEM analyses were conducted in an SEM/focused ion beam (FIB) dual beam microscope . The precursor material   without na, AUT)  , operated at TEM was performed on (i) a Tecnai F20 , operated at MFM measurements were performed on a FastScanBio AFM Microscope  operated by a Nanoscope V controller. For benchmark measurements, commercially available MESP and MESP-V2  (Bruker,