PAH), tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT), in a sex-specific manner. Furthermore, programmed male offspring displayed reduced antioxidant glutathione peroxidase 1 (Gpx1) expression, increased superoxide dismutase 1 (SOD1) and catalase (CAT) expression, and increased pro-oxidant NADPH oxidase activator 1 (Noxa1) expression in the adrenal glands. In addition, prenatal Dex exposure alters expression of epigenetic regulators histone deacetylase (HDAC) 1, 5, 6, 7, 11, in male and HDAC7 in female offspring. These results suggest that glucocorticoids may mediate the fetal programming of hypertension via alteration of epigenetic machinery and oxidative stress pathways.The field of cardiovascular fetal programming has emphasized the importance of the uterine environment on postnatal cardiovascular health. Studies have linked increased fetal glucocorticoid exposure, either from exogenous sources (such as dexamethasone (Dex) injections), or from maternal stress, to the development of adult cardiovascular pathologies. Although the mechanisms are not fully understood, alterations in gene expression driven by altered oxidative stress and epigenetic pathways are implicated in glucocorticoid-mediated cardiovascular programming. Antioxidants, such as the naturally occurring polyphenol epigallocatechin gallate (EGCG), or the superoxide dismutase (SOD) 4-hydroxy-TEMPO (TEMPOL), have shown promise in the prevention of cardiovascular dysfunction and programming. This study investigated maternal antioxidant administration with EGCG or TEMPOL and their ability to attenuate the fetal programming of hypertension via Dex injections in WKY rats. Results from this study indicate that, while Dex-programming increased blood pressure in male and female adult offspring, administration of EGCG or TEMPOL via maternal drinking water attenuated Dex-programmed increases in blood pressure, as well as changes in adrenal mRNA and protein levels of catecholamine biosynthetic enzymes phenylalanine hydroxylase ( Hypertension is a key risk factor in the pathogenesis of cardiovascular disease (CVD) which, as of 2016, was the leading cause of death accounting for a total of 15.2 million deaths worldwide [Maternal insults, such as malnutrition or stress , can permanently alter tissue structure and function, programming for cardiovascular dysfunction in the fetus . UnderstAdministration of Dex throughout the last trimester of pregnancy has been shown to alter the expression of catecholamine biosynthetic enzymes in adult offspring, leading to elevated plasma epinephrine and contributing to hypertensive programming ,7. ElevaPNMT expression, potentially leading to elevated catecholamines, and glucocorticoids are known to increase ROS [Nox) and reduced expression of glutathione peroxidase (GPx) [2−) and hydrogen peroxide (H2O2), leading to cardiovascular programming in sheep [Reactive oxygen species (ROS) have been shown to impact ease ROS . Adminisse (GPx) . Researcin sheep . ROS mayin sheep .2− and H2O2, it is important to employ an antioxidant that combats these ROS [Research has yet to fully understand the effects of glucocorticoid programming on catecholamine biosynthetic enzyme expression and the protective effect antioxidants may display. Despite strong evidence for a connection, the role of ROS in the glucocorticoid-mediated fetal programming of hypertension is unclear. Some studies have attempted to uncover this connection via the use of maternal antioxidants throughout programming . Dietaryhese ROS . Polyphehese ROS ,18. Furthese ROS ,20.In addition to naturally occurring antioxidants, synthetic antioxidants are useful in targeting specific ROS and investigating their impact on fetal programming. 4-hydroxy-TEMPO (TEMPOL) is a membrane permeable, stable, superoxide dismutase (SOD) mimetic that catalyzes the degradation of superoxide radicals . TEMPOL CAT), NADPH oxidase activator 1 (Noxa1), glutathione peroxidase 1 (Gpx1), and SOD in adrenal tissue [Recent data highlights a connection between epigenetics and oxidative stress in the glucocorticoid-mediated fetal programming of hypertension . Specifil tissue . The aimn = 6) and female (n = 18), were acquired at 6 weeks old from Charles River Laboratories . Animals were fed standard rat chow from Harlan Tekland  ad libitum. Further, the light cycle consisted of a 12/12 light/dark period with light between 6:00 a.m. to 6:00 p.m.Animal care procedures were approved by the Animal Care Committee at Laurentian University (AUP2018-02-01), in agreement with the Canadian Council on Animal Care guidelines. Wistar-Kyoto (WKY) rats; male (n = 6) to specific treatment groups: Control (water), TEMPOL  [Rats were left to acclimate for four weeks until they reached 10 weeks of age. Males were then introduced to females until vaginal plugs were observed. Pregnant females were then singly housed for the remainder of the pregnancy. Following copulation, pregnant rats were randomly assigned  , or 0.1%MO, USA) ,24. EGCGn = 6)  were then further subdivided to make 6 total groups (n = 3). Three dams from each group received a saline vehicle injection , and the remaining received dexamethasone (0.1 mg/kg per day) sub-cutaneous (S.C.) from gestational day (GD: 15–21) as described previously [n = 6) (The three previous groups of dams n =  (Controleviously . Offspri [n = 6) .Pups from each group were sexed and weaned at 3 weeks of age and separated into groups of 2–3. Body weight was recorded from week 3 forward to reduce early life handling stress which has been shown to impact HPA-axis, which may skew results . Blood pAt week 14, offspring were anesthetized via intra-peritoneal (I.P.) injection of a solution of 5 mg/kg xylazine  and 75 mg/kg Ketalean  . Animals2 profiler array (Qiagen). Samples were run using 7.5 ng input cDNA with a total volume of 15 μL reactions. Primers for DBH, TH, PNMT, specificity protein 1 (SP1), early growth response 1 (EGR-1), GR, B-Actin, and RPL29 were purchased from Sigma-Aldrich. Change in expression was quantified using the Ct value for each sample via the Pfaffl method ratio = (targetE) ΔCTtarget(control−sample)/(Eref) ΔCTref(control−sample) [The left adrenal from each animal was homogenized using the Tissuelyser  with Trizol reagent (Sigma-Aldrich) as previously described ,7. Total−sample) . All samWestern blot was performed as described previously . In summg for 20 min as performed previously [Blood was stored in Vacutainer blood collection vials with EDTA  and centrifuged at 1500× eviously . Plasma p ≤ 0.05 reaching significance. Significant interactions were detected and have been included in figure legends. All data is presented as mean ± SEM.GraphPad PRISM software  was employed to assess statistical significance via two-way ANOVA (Fisher’s LSD test) factoring for maternal injections and or antioxidant administration with a As reported previously , Dex-expSaline and Dex control data has been previously reported with the exception of PAH and is provided for reference . Male DeEGCG alone had no effect on gene expression in unprogrammed male or female offspring. However, EGCG attenuated Dex-mediated increases in PAH (1.1-fold), TH (1.2-fold), DBH (1.6-fold), and PNMT (1.1-fold) in males A–H. ThisAdministration of TEMPOL alone did not alter the expression of catecholamine biosynthetic enzymes in males; however, interestingly, females displayed increased TH expression (1.9-fold) compared to Control-Saline D. SimilaSaline and Dex control transcription factor data has been previously reported and has been provided for reference . PrenataTEMPOL alone did not significantly alter the expression of any transcription factors for either sex. Furthermore, similar to EGCG, TEMPOL attenuated the increased expression of SP1 (1.3), EGR1 (1.8-fold), and GR (1.1-fold) in Dex-exposed male offspring and expression levels are comparable to control groups A,C,E. InSaline and Dex control data has been previously reported and is provided for reference . The in-EGCG alone did not significantly alter protein level for catecholamine enzymes in males or females. Interestingly, EGCG attenuated elevated levels of TH (0.7-fold) and DBH (1.7-fold) but not PNMT (2.7-fold) in Dex-exposed males A,C,E. LiTEMPOL alone did not result in increased protein levels of catecholamine enzymes. Like EGCG, TEMPOL prevented Dex-mediated increase in TH (0.6-fold) and DBH (0.9-fold) but also PNMT (1.4-fold) in males A,C,E. InOnce again saline and Dex control data has been provided for reference . InteresOnce again contrary to gene expression data, TEMPOL alone significantly increased SP1 levels in males (2.1-fold) and females (1.8-fold) but no change in GR A–D. In cPlasma catecholamine levels were quantified via ELISA as discussed previously, there were no significant changes in corticosterone levels between groups. . EGCG alPrevious results have shown that in contrast to epinephrine levels, there is a reduction in norepinephrine levels in response to prenatal Dex-exposure in males and females . EGCG alAs shown previously Dex-exposed males display increased levels of CAT, GPx1, NOXA1, and SOD1 . InteresAs discussed previously, epigenetic regulators HDACs 1, 5, 6, 7, 11) displayed increased expression in Dex-exposed males, with females reaching significance in HDAC7 alone [, 11 dispHDACs 1, 5, 6, 7, 11, evidence for alterations in ROS production and antioxidant pathways were discovered, which include CAT, Gpx1, Noxa1, and SOD1 [2O2 production from complex I in programmed sheep [GPx mRNA and increase Nox expression in vitro, further highlighting a role for superoxide in programming [Prenatal exposure to Dex has previously been shown to reduce birth weight and result in increased blood pressure, mRNA and protein levels of catecholamine biosynthetic enzyme levels, as well as circulating levels of epinephrine . Previouand SOD1 ,29. Von gramming ,16. Glucgramming . Once elResults from the current study show that maternal administration of antioxidants, EGCG or TEMPOL, significantly attenuate the development of the hypertensive phenotype, highlighting the role of ROS in glucocorticoid-mediated fetal programming. Specifically, antioxidants were effective particularly in males, in preventing decreased bodyweight at week 3, elevations in blood pressure, and the majority of altered enzyme expression and protein levels, as well as restoring plasma catecholamine levels in response to prenatal Dex-exposure. Low birth weight is indicative of Dex-mediated fetal programming of hypertension ,31. AntiPAH, TH, DBH, and PNMT  between sexes, and has been implicated in the pathogenesis of hypertension [GR expression, and there is a complex interplay between GR and estrogen receptor in promoting gene expression at estrogen response elements in gene promoter regions, further complicating the mechanism behind estrogen’s protective effects in glucocorticoid programming [TEMPOL was particularly effective in attenuating Dex programming, providing further evidence that fetal Dex-exposure drives oxidative stress via superoxide generation as TEMPOL was able to prevent these shifts. In females, TEMPOL had less of an effect on reducing catecholamine producing enzyme expression, especially to males D,F. Thisrtension ,39. Sex rtension . Thus, irtension . This martension . The rolrtension . Estradigramming .Dex-programming has been shown to lead to elevated plasma epinephrine in adult offspring as a result of increased catecholamine biosynthetic enzymes . Indeed,Overall, both antioxidants were effective in reducing circulating catecholamine levels, likely a result of significant attenuations in altered catecholamine enzyme levels. Further, antioxidants prevent Dex-mediated changes in these enzymes differently based on sex, indicating ROS differences based on sex in the programming of adrenal catecholamine enzymes.2 qPCR data indicate that significant changes occur in antioxidant defense enzymes, as well as epigenetic regulators in programmed male offspring, compared to unprogrammed (Noxa1)  A further (Noxa1) A. Other pression C, which ffspring , which lnd sheep ,29, whicHDACs 1, 5, 6, 7, and 11 in males, may contribute to ROS generation and altered antioxidant defense enzymes, leading to programming. Females, on the other hand, displayed only increased HDAC7 and seemed to be protected from altered antioxidant enzyme expression, likely contributing to less programming of catecholamine production [As described previously, programming has increased the expression of epigenetic regulators, including Nox levels, leading to increased superoxide production [CAT, NOXA1, and SOD1 [There are likely numerous links between oxidative stress enzymes and epigenetic regulators which have remained undiscovered. However, research has described increased HDACs driving increased oduction . Furtheroduction . Indeed,and SOD1 .PAH, TH, DBH, PNMT). However, compared to females, males disproportionately showed severe dysregulation of the antioxidant defense enzyme (SOD1), increased expression of pro-oxidant activator gene (Noxa1) and altered HDAC 7 expression levels. Altered epigenetic regulators, such as HDACs, have been implicated in glucocorticoid programming, and there is evidence they alter expression levels of enzymes in the antioxidant pathway, increasing ROS, and affecting glucocorticoid programming [It is evident that many factors are implicated in the development of cardiovascular disease. The fetal environment is extremely fragile and susceptible to insult from excess glucocorticoids due to maternal stress. Increasing evidence points to epigenetics and ROS in the glucocorticoid programming of cardiovascular dysfunction. Our findings support studies that show maternal antioxidant therapy is a useful tool in the prevention of cardiovascular programming . Many an


Regulatory T (Treg) cells are indispensable for immune homeostasis due to their roles in peripheral tolerance. As the master transcription factor of Treg cells, Forkhead box P3 (Foxp3) strongly regulates Treg function and plasticity. Because of this, considerable research efforts have been directed at elucidating the mechanisms controlling Foxp3 and its co-regulators. Such work is not only advancing our understanding on Treg cell biology, but also uncovering novel targets for clinical manipulation in autoimmune diseases, organ transplantation, and tumor therapies. Recently, many studies have explored the post-translational regulation of Foxp3, which have shown that acetylation, phosphorylation, glycosylation, methylation, and ubiquitination are important for determining Foxp3 function and plasticity. Additionally, some of these targets have been implicated to have great therapeutic values. In this review, we will discuss emerging evidence of post-translational regulations on Foxp3 in Treg cells and their exciting therapeutic applications. Thanks to the precious gift inherited from jawed fish ancestors thought to have lived some 500 million years ago, our immune defenses are equipped with a powerful adaptive arm with the ability to mount responses to a near-infinite diversity of targets . However+ suppressive T cells  condition without pathogen challenge), and loss of these cells led to severe autoimmune diseases   or thymic Treg (tTreg) and the latter is known as peripheral Treg (pTreg) or inducible Treg (iTreg), especially when generated v cells) . While bv cells) –36, TGF-v cells) , 37. Addv cells) –46. Lastv cells) , 48, whiv cells) –51. Despv cells) –58, a cov cells) , 60.+Foxp3int resting Treg (rTreg) or CD45RA-Foxp3high effector Treg (eTreg) cells. Although both rTreg and eTreg are immunosuppressive in vitro, rTreg cells have a lower suppressive activity than eTreg cells. However, rTreg cells can be activated to proliferate and differentiate into eTreg cells, but eTreg cells have been described as anergic and prone to apoptosis  family, subfamily P. In mammals, there are four members in this subfamily, namely Foxp1–4 . Among tFoxp3 protein is about 431 amino acids long and 47.24 kDa in molecular weight. It includes four functional domains, namely the repressor (aa 1–190), zinc finger , leucine zipper , and Fork-head domain   Figure. The zinAs the master transcription factor of Treg cells, Foxp3 can form large protein complexes with other co-factors. These complexes vary from 300 to 1,200 kDa in size and may involve up to 361 different potential partners , 76. SomFoxp3 is tightly regulated by a network of different mechanisms with certain redundancy. Epigenetically, Foxp3 can be regulated DNA methylation, histone modification, and nucleosome positioning . For exaFoxp3 can be regulated by a number of cis-acting elements, which are located on the promoter and the enhancer regions  of the Foxp3 locus , 94. TheUpon T cell stimulation, TCR-induced NF-κB pathway plays an important role in regulating Foxp3 expression and Treg development. Mutating or deleting many key enzymes within the pathway, including PKC-θ , Bcl-10 Another important pathway is the PLCγ–NFAT/AP-1 pathway. Upon TCR engagement, PLCγ induces calcium influx, which leads to the activation of NFAT1 . NFAT1 n+ T cells in mice  and IL-2Rγ (CD132) are essential for Foxp3 expression, as losing them leads to no detectable Foxp3 in mice –110. Add in mice , 112. In in mice . Then, d in mice . This si in mice . It also in mice , 116.TGF-β is an important cytokine for both Treg induction and maintenance . Recent Post-translational modification (PTM) refers to enzymatic processes that alter a protein after its synthesis. These modifications will influence the characteristics of the protein, including its localization, interaction, turnover, etc. Given the importance of this factor and the T cell lineage it defines, Foxp3 is, not surprisingly, tightly regulated by a number of mechanisms involving post-translational modifications. In this section, we will review some recent findings on post-translational modifications of Foxp3 Figure 2Phosphorylation is a mechanism by which a protein kinase attaches a covalently bound phosphate group to the serine (S), threonine (T), or tyrosine (Y) residue of a protein. It is a reversible process as the protein can be dephosphorylated by phosphatases. These two modifications are important for protein function and stability. For Foxp3, more than 15 sites have been documented to be targets of phosphorylation. Among these, kinases CDK2, PIM1, and PIM2 negatively regulate Foxp3 functions; kinase NLK and phosphatase PP1 positively regulate Foxp3 function  . This process can be reversed by histone deacetylases (HDACs). In regulatory T cells, acetylation and deacetylation of Foxp3 protein are mostly regulated by three HATs and nine HDACs with certain redundancy  , these authors showed that loss of O-GlcNAcylation destabilizes Foxp3 protein in Treg cells ex vivo. Furthermore, using liquid chromatography with tandem mass spectrometry (LC-MS/MS) through electron transfer dissociation (ETD), these authors further found the above O-GcNAc sites using HEK293 cells expressing Foxp3. Although O-GlcNAc-deficient Treg cells develop normally in mice (Foxp3YFP-Cre/YOgtfl/Y), their Foxp3 expression level is moderately decreased and their suppressive function is significantly impacted partially due to attenuated IL-2/STAT5 and Notch signaling  at multiple sites, including Thr-38, Ser-57, Ser-58, Ser-270, and Ser-273 . Using Tignaling , 157. Beignaling , 159 FF+Ogtfl/YUbiquitination is a type of post-translational modification that attaches ubiquitin molecules to the target protein . It is aIn Treg cells, RNF31, Stub1, and TRAF6 are the three E3 ligases that directly ubiquitinate Foxp3 . It is aUnlike RNF31, which mediates monoubiquitination, Stub1 and TRAF6 regulate polyubiquitination on Foxp3. Stub1 is a U-box type E3 ligase that interacts with HSP70 . When chAs the Janus side of ubiquitination, deubiquitination also plays important roles in regulating Foxp3 function and plasticity. Among nearly 100 DUBs reported so far , USP7, UBesides the natural post-translational modifications reviewed above, Foxp3 binding peptides may function as artificial PTMs and are worth discussing. These binding peptides can be either pieces of the Foxp3 amino acid sequence competing for the binding partners and co-factors of Foxp3, or random peptides that block critical Foxp3 functions. So far, two peptides have been carefully studied regarding their roles in regulating Foxp3 functions. First, Foxp3 393–403 peptide can interact with the RHR domain of NFAT1, disrupting of the Foxp3/NFAT1 interaction—a molecular pairing that is thought to regulate Treg-associated genes. Such inhibition reduces Treg suppression and may be useful for tumor immunotherapies . AnotherIn addition to post-translational regulations of Foxp3, modified Foxp3 can play a critical role in regulating other PTMs, for example, SUMOylation. Similar to ubiquitination, SUMOylation tags small ubiquitin-like modifiers (SUMO) to the substrate for multiple functions, including cell signaling and protein turnover . As the As a rising field in its own right, the therapeutic use and modulation of Treg cells can have major clinical applications in the treatment of autoimmune diseases, transplantation, cancer, as well as colitis and other inflammatory diseases. So far, 1,010 clinical trials involving Treg cells have been documented at the NIH. In addition, hundreds of small molecules drugs, antibodies, and other nanoparticles have been designed to target Treg cells directly or indirectly, indicating the broad range of potential applications involving Treg cells . In thisIn brief, autoimmune diseases occur when the immune system mistakenly targets self. Some common autoimmune diseases include Lupus, Celiac disease, Sjogren’s syndrome, Multiple sclerosis, Polymyalgia rheumatica, Type 1 diabetes (T1D), and rheumatoid arthritis (RA) . Of noteex vivo, and then infuse them back to patients. Many trials have been performed using this approach to treat patients with T1D and lupus, including, NCT02772679, NCT02932826, NCT02704338, and NCT02428309  is to use immunosuppressants to down-regulate normal immune functions, including using anti-proliferative agents, steroids, mTOR inhibitors, and Calcineurin inhibitors . Howevernull.IL2rgnull (NRG) mice receiving A2-CAR-Treg cells showed much stronger tolerance induction than those receiving polyclonal nTreg or controls  therapy attracts a lot of attentions , 198. Sicontrols . TherefoWhile important for maintaining immune homeostasis, Treg cells are known to play a pathological role in cancer patients by impeding desirable anti-tumor immunity . They rein vivo (As a proven weapon in the anti-cancer arsenal, checkpoint inhibitor immunotherapies are being used to maintain and boost the function of cytotoxic T cells . From thin vivo . Additioin vivo , 217.Drugs specifically targeting Foxp3 PTM also represent high potential strategies for combating immune suppression in cancer as they are not likely to affect the function of Tconv cells and CTLs. As reviewed in the post-translational modification sections, preventing the acetylation, DNA binding, or dimerization of Foxp3 are potentially useful approaches to suppress Treg cells. Thus, preclinical studies using USP7 inhibitors , p300 inWe are challenged by a variety of potential pathogens at all times, including bacteria, viruses, and fungi . To mainSome factors are known to restrict Treg functions, including chronic exposure to inflammatory cytokines and deprivation of stabilizing factors. Chronic exposure of Tregs to pro-inflammatory cytokines like IL-1β, IL-6, TNFα, which can antagonize Foxp3 expression and skews the gene expression profile to that of Th17 or Th1 –225. Redin vivo, high-throughput target screening assay for agents capable of disrupting the posttranslational means of supporting Foxp3 expression and function and the stronger suppressive potency of Treg cells, and second, a high-throughput and cost-efficient pre-clinical drug examination platform for Treg-based therapies. While the former requires more accurate and efficient CRISPR-based gene-editing techniques to be applied to Treg cells, the latter aspect will benefit from new screening systems. On the one hand, murine disease models are still very different from human cases due to evolutionary heterogeneities (ex vivo (in situ interactions (ex vivo culture systems showed great promise in drug development.After decades of intensive studies, our knowledge of Treg cells has improved significantly. With increasing regulatory mechanisms of Foxp3 being brought to light, the potential of manipulating Foxp3 at the post-translational level has increased dramatically. At this stage, we believe two aspects will be important for future drug discovery: first, an eneities . On the eneities . Besideseneities , making (ex vivo . Moreoveractions . Thus, 3in vivo (Another equally important issue is how to improve Treg-specific targeting. Currently, most strategies targeting Treg cells use monoclonal antibodies or ligand-directed toxins to deplete Treg cells systemically or locally . Howeverin vivo , a speciin vivo . Moreovein vivo –240, whiAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the start-up package from SIAT, no. 1105161001.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Liver hepatocellular carcinoma (LIHC) is a malignance with high incidence and recurrence. Pyroptosis is a programed cell death pattern which both activates the effective immune response and causes cell damage. However, their potential applications of pyroptosis-related genes (PRGs) in the prognostic evaluation and immunotherapy of LIHC are still rarely discussed.  Comprehensive bioinformatics analyses based on TCGA-LIHC dataset were performed in the current study.  A total of 33 PRGs were selected to perform the current study. Of these 33 PRGs, 26 PRGs with upregulation or downregulation in LIHC tissues were identified. We also summarized the related genetic mutation variation landscape. GO and KEGG pathway analysis demonstrated that these 26 PRGs were primarily associated with pyroptosis, positive regulation of interleukin-1 beta production, and NOD-like receptor signaling pathway. An unfavorable OS appeared in LIHC patients with high CASP3, CASP4, CASP6, CASP8, GPX4, GSDMA, GSDME, NLRP3, NLRP7, NOD1, NOD2, PLCG1, and SCAF11 expression and low NLRP6 expression. A prognostic signature constructed by the above 14 prognostic PRGs had moderate to high accuracy to predict LIHC patients' prognosis. And risk score was correlated with the expression of CASP6, CASP8, GPX4, GSDMA, GSDME, NLRP6, and NOD2. Of these 7 genes, CASP8 was identified as the core gene in PPI network. Moreover, lncRNA MIR17HG/hsa-miRNA-130b-3p/CASP8 regulatory axis in LIHC was also detected.  The current study indicated the crucial role of PRGs in the prognostic evaluation of LIHC patients and their correlations with tumor microenvironment in LIHC. Globally, liver cancer accounted for over 800000 new cases and caused over 700000 cancer-related death in 2018. Of these new cases, 85% were diagnosed as liver hepatocellular carcinoma (LIHC), and over 40% were diagnosed at advanced stage [Shigella flexneri was dependent on CASP1 [Pyroptosis is one of programmed cell death patterns and both stimulates effective immune responses and causes tissue damage . The proon CASP1 . With thon CASP1 –11. And on CASP1 , 13. Witon CASP1 –16. HoweWith the rapid development of molecular biology and the enrichment of genomic data, comprehensive analysis on PRG signatures becomes feasible. In the present study, bioinformatic data was utilized to explore the expression profiles, prognostic performance, and related regulation axis of PRG signatures in LIHC. Moreover, we also explored the association between immune cell infiltration and PRG signatures in LIHC. The above findings may offer novel insights on the prognostic evaluation and therapy of LIHC.N = 374) was selected to perform the analyses. The data of copy number variation (CNV) and somatic mutation in LIHC were also extracted from TCGA database and UCSC Xena, respectively. Using R software V4.0.3, statistical analyses were performed. In addition, the P value cutoff was set as 0.05.The genomic data of LIHC patients and related clinical data of these patients, including gender, age, tumor grade, and survival outcome, were downloaded from the Cancer Genome Atlas (TCGA) database on August 1, 2021. The TCGA-LIHC dataset  values before subsequent process.Based on previous studies –11, a tohttp://metascape.org/gp/index.html#/main/step1) [Mutation categories and mutation frequency of the 33 PRGs as well as their waterfall plots were constructed by “maftools” package in R. And the location of variation of these genes on 23 chromosomes was shown by “RCircos” package. Functional enrichment analysis, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, was performed to explore the potential molecular mechanisms and biological functions of target genes by Metascape (n/step1) . MoreoveSetting the threshold as follows: iteration = 100 and resample rate = 80%, consensus cluster analysis was conducted by “ConsensusClusterPlus” package in R. “Survival” package was utilized to evaluate the difference of each cluster in LIHC. Next, the difference of clinical paraments in each cluster was explored by “pheatmap” package. Furthermore, the ImmuneScore, ESTIMATEScore, StromalScore, and abundance of immune cell in each cluster were calculated by the ESTIMATE algorithm . The comThe prognostic value of differently expressed PRGs was assessed by the “survival” package in R and was expressed as prognostic forest maps. The differently expressed PRGs with OS difference in low-expression and high-expression group were defined as prognostic PRGs. According to the above prognostic PRGs, LASSO Cox regression analysis in “glmnet” package was used to construct prognostic model. Those PRGs that constituted prognostic model were selected for further analyses. According to the median risk score, patients were divided into low- and high-risk group, and the OS curves of the two groups were compared. The predictive accuracy of this prognostic model was assessed by time ROC analysis. The univariate and multivariate Cox analysis was performed to excavate the influence of clinical factors, including age, gender, tumor grade, clinical stage and TNM stage, and risk score on the prognosis of LIHC patients. Subgroup analyses on the influence of clinical factors on the prognosis of low-risk and high-risk group were also performed. We were also interested in the difference of risk score in different subgroups, including age, gender, cluster, tumor grade, clinical stage, ImmuneScore, and TNM stage. Besides, the correlation between abundance of immune cells and risk score was also analyzed.https://string-db.org/), a PPI network was constructed to illustrate the association among these genes and seek for the core gene [The PRGs that made up risk score were selected via above analyses. Using STRING (ore gene . Furtherhttp://timer.cistrome.org/) [TIMER (me.org/)  is a biohttp://www.mirdb.org/) [http://mirwalk.umm.uni-heidelberg.de/) [https://starbase.sysu.edu.cn/starbase2/index.php) [https://diana.e-ce.uth.gr/lncbasev3) [A mRNA-miRNA-lncRNA network was constructed for finding out the potential regulatory axis of CASP8 in LIHC. The miRNA targets binding to CASP8 in LIHC were excavated by miRDB (db.org/) , miRWalkerg.de/) , and Stadex.php)  databasecbasev3) , and theAccording to the RNA-sequencing data from TCGA-LIHC dataset, the mRNA levels of 33 pyroptosis-related genes in LIHC tissues and normal liver tissues were shown in The 26 PRGs with aberrant expression in LIHC tissues were selected to perform GO and KEGG pathway analysis. To capture the relationship between the enriched terms, the networks of GO analysis and KEGG pathway analysis were presented in Figures k value = 2 . Moreover, we also studied the association between clinical parameters and gene expression of cluster 1 and cluster 2 and found that age (>60 years or not) is a factor that influence the gene expression of cluster 1 and cluster 2 .Based on the above 26 PRGs with aberrant expression, we consensus clustering categorized LIHC patients. According to the similarity displayed by expression level and the proportion of ambiguous clustering measure, optimal clustering stability appeared when  Figures . The cumP value = 0.0035). However, there was no significant difference in the ESTIMATEScore  and ImmuneScore  between the two clusters. We next explored the difference between abundance of different immune cells in the two clusters. Unexpectedly, there was no significant difference in the abundance of naive B cells , memory B cells , plasma cells , CD8+ T cells , naive CD4+ T cells , resting memory CD4+T cells , activated memory CD4+T cells , follicular helper T cells , Tregs , gamma delta T cells , resting NK cells , activated NK cells , monocytes , M0 macrophages , M1 macrophages , M2 macrophages , resting dendritic cells , activated dendritic cells , resting mast cells , activated mast cells , eosinophils  and neutrophils  between the two clusters , CASP4 , CASP6 , CASP8 , GPX4 , GSDMA , GSDME , NLRP3 , NLRP7 , NOD1 , NOD2 , PLCG1 , and SCAF11  had worse OS than those with low expression of these genes, while LIHC patients with high NLRP6 expression had better OS than those with low NLRP6 expression . No significant difference was found in OS between LIHC patients with high and low PRKACA, GSDMB, PJVK, CASP9, NLRP1, TIRAP, GSDMD, GSDMC, PYCARD, IL-1B, AIM2, and IL-6 expression . Thus, a total of 14 genes were consider as prognostic PRGs and were selected for further analyses. For better predicting the prognosis of LIHC patients, the 14 prognostic PRGs were selected to construct prognostic model by LASSO Cox analysis. The partial likelihood deviance and coefficients of prognostic model were presented in Figures P value < 0.001, P value = 0.014, P value = 0.006, P value = 0.003), male patients , patients with grades 1-2 , patients with stages I-II , patients with stages III-IV , patients with T1-2 , patients with T3-4 , patients with N0 , patients with N1 , patients with M0 , and patients with M1 , high-risk group showed worse OS than low-risk group.We also investigated the prognostic performance of above 26 PRGs. LIHC patients with high CASP3 . LIHC patients with grades III-IV showed higher risk score than those with grades I-II , and LIHC patients with high ImmuneScore showed higher risk score than those with low ImmuneScore . However, no significant difference was detected in the subgroups of gender, age, clinical stage, T stage, N stage, and M stage . In addition, the association between immune cell infiltration and risk score was also explored. However, only the correlation between abundance of activated memory CD4+ T cells and risk score was detected with a P value of 0.27 , CD8+ T cell , CD4+ T cell , macrophage , neutrophil , and dendritic cell  . In TMB analysis, similar result was presented in P value = 0.745).We also constructed PPI network to visualize the association among the genes that constituted risk score  . Next, w.48E-12) . Tumor mP value = 7E-08), and LIHC patients with high hsa-miRNA-130b-3p expression had worse OS than those with low hsa-miRNA-130b-3p expression . Thus, hsa-miRNA-130b-3p was considered as the most promising miRNA target of CASP8 in LIHC. The upstream lncRNA targets interacting to the promising miRNA targets were also explored via LncBase and StarBase database. A total of 3 lncRNA targets were identified based on the data in above databases, including H19, KCNQ1OT1, and MIR17HG , while the expression level of KCNQ1OT1  and MIR17HG  were upregulated in LIHC tissues. Nevertheless, only MIR17HG was correlated with the prognosis of LIHC patients, which showed a better OS in high expression group . Thus, MIR17HG was considered as the most promising lncRNA target of CASP8 in LIHC. According the above results, lncRNA MIR17HG/hsa-miRNA-130b-3p/CASP8 regulatory axis was detected and might influence the occurrence and progression of LIHC.The above analyses indicated that CASP8 was correlated with immune cell infiltration in LIHC. And CASP8 acted as the core gene in the PPI network constructed by PRGs associated with risk score. Thus, CASP8 was selected to construct a mRNA-miRNA-lncRNA network, which might uncover the potential CASP8-related regulatory axis in LIHC. First of all, CASP8 targeting miRNAs in miRDB, StarBase, and miRWalk database were compared to identify the common ground, which included hsa-miR-519a-3p, hsa-miR-105-5p, and hsa-miR-130b-3p . Next, w MIR17HG . The expPyroptosis is defined as a type of programed cell death that is dependent on the gasdermin protein family, and its occurrence is often as a result of activation of inflammatory CASP protein . With thβ and IL-18, and GSDMD pore could mediate the release of IL-1 via electrostatic filtering [β [κB signaling pathway [We firstly explored the expression level of 33 PRGs in LIHC tissues and normal liver tissues. Of these 33 PRGs, 26 genes with aberrant expression in LIHC tissues were selected to perform functional enrichment analysis and consensus cluster analysis. Programed cell death pathway, mainly including apoptosis and pyroptosis, protects mammals from infection. Outer membrane vesicles of Gram-negative bacteria delivered various bacterial molecules to host cells; outer membrane vesicle-associated molecules participated in the activation of apoptosis and pyroptosis . CASP1 ciltering . He et aering [β . And the pathway , 32–34. Prognostic model was widely utilized in prognostic evaluation of cancer patients, which provided epidemiological evidence for clinicians. In the current study, a total of 14 prognostic PRGs were enrolled to build up a prognostic model. Through further screening, the risk score consisted of the seven PRGs, including CASP6, CASP8, GPX4, GSDMA, GSDME, NLRP6, and NOD2. And the LIHC patients in high-risk score group generally showed worse OS compared to low-risk score group. Other three bioinformatics analyses, respectively, constructed prognostic model to explore the correlation between prognosis of lung adenocarcinoma, skin cutaneous melanoma and glioblastoma, and PRGs, which indicated a worse prognosis in high-risk group . The aboIn the current study, CASP8 was detected as the core gene in the PPI network constructed by PRGs associated with risk score. Thus, we were interested on the role of CASP8 in LIHC. Previous studies reported that CASP8 not only induced apoptosis of LIHC cells but also had a nonapoptotic function in proliferation-related DNA damage response through CASP8/RIPK1/FADD/cFLIP complex in LIHC cells . And KosA potential mRNA-miRNA-lncRNA regulatory axis in LIHC, lncRNA MIR17HG/hsa-miRNA-130b-3p/CASP8 regulatory axis, was also detected in the present study. MIR17HG participated in the generation of miRNA-17-92 cluster, which included miRNA-17, miRNA-18a, miRNA-19a, miRNA-20a, miRNA-19b, and miRNA-92a . These sSome limitations still exist in the current study. Firstly, most of the analyses in the current study are conducted at the transcription level; some results may not apply to the studies based on the protein level. Secondly, related fundamental and clinical studies that focus on the clinical value and molecule mechanisms of PRGs in LIHC are rare. Third, genetic background and etiology of LIHC patients are influenced by many factors, such as patients' race, patients' gender, and patients' age. Thus, more in-depth studies are necessary to validate the results.In summary, the present study built up a pyroptosis-related gene signature to predict LIHC patients' prognosis and their correlations with immune infiltration, which indicated that pyroptosis-related genes were of great significance in the LIHC patients' prognosis and microenvironment of LIHC. However, in-depth studies are needed to validate our results.


Ebolavirus, family Filoviridae, have a non-segmented, single-stranded RNA that contains seven genes: (a) nucleoprotein (NP), (b) viral protein 35 (VP35), (c) VP40, (d) glycoprotein (GP), (e) VP30, (f) VP24, and (g) RNA polymerase (L). All genes encode for one protein each except GP, producing three pre-proteins due to the transcriptional editing. These pre-proteins are translated into four products, namely: (a) soluble secreted glycoprotein (sGP), (b) Δ-peptide, (c) full-length transmembrane spike glycoprotein (GP), and (d) soluble small secreted glycoprotein (ssGP). Further, shed GP is released from infected cells due to cleavage of GP by tumor necrosis factor α-converting enzyme (TACE). This review presents a detailed discussion on various functional aspects of all EBOV proteins and their residues. An introduction to ebolaviruses and their life cycle is also provided for clarity of the available analysis. We believe that this review will help understand the roles played by different EBOV proteins in the pathogenesis of the disease. It will help in targeting significant protein residues for therapeutic and multi-protein/peptide vaccine development.Ebola virus (EBOV), member of genus  Ebolavirus belongs to the family Filoviridae and consists of six identified species. Schematic taxonomy classification of Filoviridae according to the International Committee on Taxonomy of Viruses (ICTV) is presented in Dianlovirus, has been proposed by Yang and co-workers, including the virus circulating in Chinese bats, named Měnglà virus (MLAV)  and Bombali virus (BOMV), cause severe and frequently fatal hemorrhagic fever in humans and non-human primates (NHPs). In contrast, BOMV infects bats exclusively while RESTV is known to be pathogenic for humanized mice, though no human infection has been detected as yet . A,4. AEbold as yet ,2,5.EBOV has a thread-like shape virion, which can be changed to circular or filamentous . The filIn the last 45 years, ebolavirus outbreaks with varying fatalities have been documented mainly in Africa, resulting in over 15,200 deaths . The risA schematic depiction of the viral life cycle is presented in To enter the host cell, EBOV can use several attachment factors, such as human folate receptor- α , β1 inteUpon binding to the receptor, EBOV enters the host cells via three mechanisms: (a) Macropinocytosis , (b) ClaEBOV GP consists of two subunits, GP1 and GP2 (discussed later). After uptake, proteolysis of EBOV GP1 appears significant for viral entry . The mecNP encapsidates both, filoviral genome, as well as anti-genome . NP asso0 and pre-ssGP, respectively . V. V105]. To conclude, VP35 plays a chief role in host immune evasion by blocking viral dsRNA recognition by host immune receptors, inhibiting IRF-3 and TBK-1/ IKKε complex formation, increasing IRF-7 SUMOylation, preventing RIG-I ATPase activation and inactivating PKR . MoreoveVP40, the most abundantly expressed protein , is esseVP40 is classified as a transformer protein ,150, as NTD aa 127, 129 and 130  and aa 2In addition to dimers, filaments, and hexamers, VP40 can form an octameric ring configuration where NTD plays the leading role . This coThese data indicate that the main function of VP40 is in viral assembly and budding . TherefoGP is a type I transmembrane fusogenic protein, which is translated only 20% of time by the GP gene as it can be cytotoxic to certain cell types ,31. As tGP1 mediates attachment to host cells using the receptor-binding site (RBS) located at aa 54–201  122]. S. S122]. GP2 contributes to the fusion of viral and host cell membranes . An inte+ T cell recognition ,18033,18The impact of phosphorylation on VP30-NP interaction was extensively debated over the past two decades ,181,182.The above discussion suggests that VP30 chiefly plays a role in viral transcription initiation via its zinc-binding, NP interaction and phosphorylation characteristics . AbrogatVP24 makes up approximately 7.5% of total EBOV proteins and is one of the five proteins involved in EBOV NC formation. VP24 aa 169–173  interactLike VP35, VP24 inhibits host immune response using several mechanisms . VP24 inIt appears that the central role of VP24 is in host immune evasion, carried out by inhibiting p38 MAPK and NF-κB pathway activation. VP24 also seems critical to viral replication and is involved in budding viral initiation. Therefore, targeting these residues  during vL, a part of the RNP complex , is the Mononegavirales members [L protein is essential for virus replication, yet little is known about its other functions, mainly due to its large size and lack of specific antibodies ,193. The members ,194,195.A GDNQ motif has been reported as a catalytic center in several negative sense RNA polymerases. In 2017, The EBOV RdRp domain was also reported to consist of the GDNQ motif within aa residues 741–744 , which iIt is imperative to understand proteins functions with respect to the EBOV life cycle and pathogenesis to identify practical and specific protein therapeutic targets. This review summarizes data on aa residues of EBOV proteins involved in essential functions, such as viral entry, host immune evasion, replication, transcription, and budding. A cross-reactive, multi-protein/peptide vaccine candidate developed by utilizing the information presented in this review could help curb various characteristic EVD symptoms and affect different stages of the EVD life cycle.Multiple proteins and aa sites were highlighted in our review, which have a high potential for vaccine development. For instance, NC formation and viral replication could be hindered by collectively targeting NP aa 110–400 and VP24 aa 169–173. Replication and its regulation could also be affected by targeting VP35 aa 210 and L aa 741–744. Viral transcription could be affected by targeting NP aa 562–567, NP aa 600–617, and VP30 aa 140–266, while transcription regulation can be blocked by collectively targeting NP aa 240–383, VP35 aa 20–48, and VP30 aa 29–183. Moreover, targeting NP aa stretch 550–600 might disable multiple NP-host-protein interactions significant for transcription and regulation. Viral ingress may be blocked by targeting GP aa 54–213 as it affects GP stability, interaction with obligate host receptor (NPC1) and membrane-fusion mediated conformational changes. A critical step for viral spread is host immune evasion, accomplished with the help of VP35, sGP, shed GP and VP24 proteins. This could be prevented by targeting specific regions of these proteins, such as VP30 aa 221–340 and VP24 aa 120–190. Moreover, as aa 1–295 are identical in GP, sGP, and ssGP; therefore, therapeutic or vaccine candidates against GP aa 54–213 could prove effective against sGP (primary GP gene product), as well as ssGP. This will be effective in preventing viral entry, as well as in curbing host immune evasion. Three proteins contribute to viral egress: VP40, GP, and VP24. GP2 displays anti-tetherin activity, while VP24 causes favorable structural changes in NC to signal viral egress initiation. As VP40 is primarily involved in assembly and budding, targeting aa 212–275, a region significant for localization to and interaction with the plasma membrane , may hinMultiple studies have analyzed EBOV proteins viz., NP ,201,202,


Cardiovascular (CV) and renal diseases are increasingly prevalent in the United States and globally. CV-related mortality is the leading cause of death in the United States, while renal-related mortality is the 8th. Despite advanced therapeutics, both diseases persist, warranting continued exploration of disease mechanisms to develop novel therapeutics and advance clinical outcomes for cardio-renal health. CV and renal diseases increase with age, and there are sex differences evident in both the prevalence and progression of CV and renal disease. These age and sex differences seen in cardio-renal health implicate sex hormones as potentially important regulators to be studied. One such regulator is G protein-coupled estrogen receptor 1 (GPER1). GPER1 has been implicated in estrogen signaling and is expressed in a variety of tissues including the heart, vasculature, and kidney. GPER1 has been shown to be protective against CV and renal diseases in different experimental animal models. GPER1 actions involve multiple signaling pathways: interaction with aldosterone and endothelin-1 signaling, stimulation of the release of nitric oxide, and reduction in oxidative stress, inflammation, and immune infiltration. This review will discuss the current literature regarding GPER1 and cardio-renal health, particularly in the context of aging. Improving our understanding of GPER1-evoked mechanisms may reveal novel therapeutics aimed at improving cardio-renal health and clinical outcomes in the elderly. Cardiovascular (CV)-related events are the leading causes of mortality in the United States (US) and worldwide ,2. RenalThe overall life expectancy globally has increased by more than 6 years, from 66.8 in 2000 to 73.4 years in 2019 , phosphoinositide 3-kinase (PI3Ks), extracellular signal-related kinases (ERKs) and cyclic adenosine monophosphate (cAMP) . St. St72]. e (cAMP) ,77.2)-mediated Ca2+ mobilization and blocked activation of PI3K signaling through GPER1, but, despite slight binding, not ERα and ERβ i acutely [2+]i [2+ spikes in VSMC [2+]i increase, suggesting that arteries lacking GPER1 are more sensitive to VSMC [Ca2+]i [A and ETB expression levels are indifferent in GPER1 KO carotid arteries [2+]i in order to balance the vasoconstrictive effects of ET-1.ET-1 signaling system has been implicated in GPER1-induced vascular effects. G-1 activation of GPER1 attenuates ET-1-mediated vasoconstriction and causes acute vasodilation in porcine coronary arteries . G-1 inc acutely  while GPly [2+]i . Additio in VSMC . GPER1 K [Ca2+]i . ETA andarteries , which pB receptor-stimulated NO bioactivity [B and ECE-2 gene expression in myocardial tissue [In aortic tissue of male mice, acute inhibition of GPER1 by G-15 decreased ETactivity . Overallactivity ,137. An A and ETB, compared to other parts of the kidney [A and ETB collaborate to mediate GPER1-evoked natriuresis and diuresis [A, and ETB mRNA expression in kidney tissues [The renal ET-1 system promotes natriuresis ,139 and e kidney . More soe kidney ,70. We re kidney ,143. Spediuresis ,143. Fur tissues . These dNO is a well-known vasodilator. Three isoforms of NO synthase (NOS) have been identified including: neuronal , inducible , and endothelial  . These tSeveral studies have suggested that NO production and release are reduced with age in rodents ,156. UriNO bioavailability and activity are reduced in human populations with aging ,160,161.Evidence suggests that GPER1 stimulates NO production and promotes relaxation in multiple vascular beds ,165,166.2−), hydroxyl radicals and hydrogen peroxide, leads to cellular toxicity in various disease states such as atherosclerosis, coronary ischemia, cardiac hypertrophy, and heart failure [ROS constitute essential components of aerobic respiration and cell signaling and are delicately balanced with antioxidant systems under physiological conditions. Nevertheless, excess production of ROS, including superoxide  or pharmacologic (G-36) inactivation of GPER1 in male mice suggests that GPER1 may play a role in cardiovascular aging and disease through promotion of ROS . Vasculalaxation . Meyer edeletion . Furtherdeletion . AlthougIn the last decades, chronic inflammation has been acknowledged as a major driver of cardiovascular and renal disease ,193,194.GPER1 signaling appears to have an important role in regulation of inflammatory response in various systems, including the vasculature ,201, adiGPER1 appears to mediate anti-inflammatory effects in the vasculature and cardiac tissue. G-1 was shown to ameliorate tumor necrosis factor-induced upregulation of pro-inflammatory mediators in the endothelium in vitro . Pre-tre2 or G-1 reduces the high levels of interleukin-1β and interleukin-6 induced by methotrexate [GPER1 activation appears to have anti-inflammatory effects in the renal system as well . Estrogeotrexate . Howeverotrexate . These rOverall, GPER1 signaling has a clear role in RAAS, ET-1, NO, ROS, mitochondria, immunity, and inflammation signaling pathways that play critical roles in cardio-renal health . These iSeveral lines of research implicate an important role for GPER1 signaling pathways in different cardiorenal disease pathologies. In the current section, we will highlight the potential role of GPER1 in hypertension, cardiac and kidney diseases.Hypertension, also known as the “silent killer”, is a leading risk factor for cardiovascular disease. It is responsible for about 9.5 million deaths per year worldwide ,215. AccStudies point to GPER1 as a novel regulator for blood pressure and sodium homeostasis. GPER1 activation with G-1 acutely reduces blood pressure in male Sprague Dawley rats . Chronic2-induced spike in Ca2+ in the distal renal tubules [Recently, we showed that renal medullary GPER1 activation promotes ET-1 dependent natriuresis in female, but not male, rats, which may also contribute to GPER1 blood pressure lowering actions ,143. GPE tubules . Importa tubules .In women, GPER1 hypofunctional variant is associated with increased blood pressure  and incrHeart disease is the number one leading cause of death in the US . CDC datPrevious studies have provided evidence for the cardioprotective role of GPER1 activation against heart failure in male mice  and myocEstrogen-induced regulation of heart rate appears to be linked to GPER1 rather than ERα and Erβ ,235. In Acute kidney injury (AKI) is a pathological condition characterized primarily by a rapid increase in serum creatinine over a short period of time . AKI cau2 signaling in the regulation of eNOS expression in rat kidneys, which provides protection against ischemia reperfusion-induced AKI [2 accelerates the regeneration of renal tubules in male rats after ischemia reperfusion-induced AKI through reducing inflammation [Previous studies have reported an important role of Euced AKI . Moreoveammation . Furtherammation ,232,247.Damage in the structure and/or function of the kidney often leads to CKD, which is characterized by its irreversibility and slow progression . An estiInterestingly, GPER1 activation has been shown to reduce kidney damage in mRen2.Lewis rats that were fed a high-salt diet . EarlierCollectively, evidence suggests GPER1 is protective in hypertension, heart, and kidney disease . A more Future studies aimed at better understanding the impact of GPER1 on cardio-renal disease mechanisms during aging could reveal novel therapeutics aimed at improving clinical outcomes. RAAS, NO, ET-1, ROS, inflammation, and immune infiltration have all been implicated in CV and renal disease. Furthermore, there have been described changes in each of these pathways with age and sex bolstering support for understanding mechanisms that could be impacted by sex hormones. GPER1 is one such mechanism that could provide protection against age-related CV and renal disease development and progression. Recently Sharma et al. investigated the preclinical potential of G-1 in mouse models of obesity and diabetes . While t


VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis-elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that  PAL, CHS, DFR, ANS, F3H and CHI encoding key enzymes directly catalyzing anthocyanin biosynthesis have been isolated and functionally identified. Many transcription factors have also been proved to contribute to the anthocyanin biosynthesis regulation. Among them, the MBW complex, composed of R2R3-MYB protein, bHLH protein of MYC family and transcription factors of the WD40 protein family, is believed to play a key role in regulating tissue specific expression of typical anthocyanins [Anthocyanins, a kind of natural polyphenols widely exist in many plants, are known to have great health-promoting effects mostly due to their high antioxidant activity. In recent years, a large number of studies on anthocyanin metabolism have been carried out ,2. To exocyanins ,6.R gene was the first bHLH identified in plants, which could regulate at least two anthocyanin biosynthesis structural genes [Arabidopsis thaliana and tobacco have been reported to have the ability to improve anthocyanin accumulations [DFR and BAN gene expression and plays an important role in controlling flavonoid metabolism in Arabidopsis seed coat [NnTT8 is a homologous gene of AtTT8, and its overexpression can restore the anthocyanin and proanthocyanin accumulation of the AtTT8 mutant [BjTT8 in Brassica juncea contributed greatly to the formation of purple leaves [MdbHLH3 gene is low temperature inducible, and its encoded protein interacts with MdMYB1, a transcription factor controlling apple anthocyanin biosynthesis. In addition, MdbHLH3 can bind to promotors of MdDFR, MdUFGT and MdMYB1 to regulate their expression, and play an important role in anthocyanin accumulation and fruit coloring triggered by low temperature [Neomi activated the expression of proanthocyanidin synthesis-related genes and improved proanthocyanidin content in citrus callus [ThMYC4E is the candidate bHLH gene controlling the blue grain trait from Th. Ponticum, its overexpression in common wheat JW1 activated the anthocyanin biosynthesis in transgenic plants and led to much higher anthocyanin accumulation in grains, leaves and glumes [DRF and MYB genes [The bHLH transcription factors comprise the second largest plant transcription factor superfamily, and many members have been inferred to play roles in anthocyanin biosynthesis ,8. The Lal genes . Its oveulations . Arabidoeed coat . Lotus N8 mutant . The up-e leaves . The expperature . Citrus s callus ,16. The s callus . ThMYC4Ed glumes . The PetYB genes . All theVaccinium corymbosum L.) is native to North America and East Asia. It has only been cultivated for a little more than 100 years, but its annual yield ranks only second to strawberry among all the berries. Blueberries are rich in nutrients, especially anthocyanins [MYB genes [Blueberry . The results obtained in this study will provide basis for the future function analysis of bHLH transcription factors and for research and applications of bHLH genes in high anthocyanin blueberry breeding.The publications of the draft blueberry genome data ,31 will In total, six candidate blueberry anthocyanin biosynthesis-related bHLH proteins were identified from blueberry genome data through homologous protein sequence alignment using reported anthocyanin biosynthesis-related bHLHs from Arabidopsis, apple, kiwifruit, and some other plant species ,37,38,39Populus alba PalbHLH1 (PAYT030711.1) shared the highest similarity with VcbHLH1-1 and VcbHLH1-2 . The similarity between VcbHLH1-1 and Petunia PPLS1 (SeITa.7G195400) was 57.12%, and the similarity of VcbHLH1-1 with SmelJAF13 (Smel_008G3192001.01), AtEGL3 (At1G63650), AtGL3 (At5G41315) and ThMYC4E (KX914905) was 53.85%, 48.23%, 47. 77% and 37.54%, respectively. Moreover, the similarity of VcbHLH1-2 with eggplant SmelJAF13, Arabidopsis AtEGL3 and AtGL3 was 48.55%, 46.95% and 46.42%, respectively. The large similarities between these blueberry bHLHs and bHLHs from other plant species indicated that they might have similar functions as their corresponding homologous proteins.The eggplant SmelAN1 (Smel_009G3266401.01) shared the highest similarity with VcAN1, of up to 77.12%. The similarities of kiwifruit AcbHLH42 (MH643775), apple MdbHLH3 (ADL36597.1) and citrus Neomi (Cs5g31400) with VcAN1 were all higher than 60%, which was 74.42%, 65.08% and 63.08%, respectively. The similarity of AcbHLH42 with VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 was 79.10%, 79.37% and 79.23%, respectively. Arabidopsis AtTT8 (At4G09820), apple MdbHLH3 (ADL36597.1) and citrus Neomi (Cs5g31400) all shared more than 60% similarity with VcbHLH42-1, which was 73.21%, 69.03% and 67.25%, respectively. Apple MdbHLH3 (ADL36597.1) and citrus Neomi (Cs5g31400) shared more than 60% similarity with both VcbHLH42-2 and VcbHLH42-3. The CDS length of the six blueberry bHLH genes ranges from 1803 bp to 2328 bp, and their encoded proteins contain 600~775 aa, with molecular weight ranging from 67,581.48 to 85,474.08 Da, and the isoelectric point (PI) ranging from 5.45 to 6.25. According to the PI, instability index and grand average of hydropathicity (GRAVY) values, all thes VcbHLHs were predicted to be acidic, unstable hydrophilic proteins . SubcellbHLH genes, it was found that VcAN1 gDNA contained eight introns, while other bHLH genes contained only seven introns. Moreover, the total intron lengths of VcbHLH1-1 and VcbHLH1-2 were found to be much shorter than the other four blueberry bHLHs  and bHLH_SF super domain. In addition, the N-terminus of bHLH_SF super domain of all the bHLH proteins contained DNA binding sites and dimerization interfaces B.Conserved motif analysis showed that VcAN1 and VcbHLH42-1~3 contained a total of 13 motifs (Motif1~Motif13). VcbHLH1-1 and VcbHLH1-2 did not contain Motif6, Motif9 and Motif10, but they contained an extra Motif14 C. Motif2Phylogenetic analysis showed that the six blueberry anthocyanin biosynthesis-related bHLHs could be divided into two groups, which was consistent with the results of protein similarity, gene structure, and conserved motif analysis. VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, and their relationship with kiwifruit AcbHLH42 was the closest. VcbHLH1-1 and VcbHLH1-2 were clustered into the other group, and they showed close relationship with SmelJAF13 and PalbHLH1 .VcbHLH genes showed that they were separately located on six different scaffolds. VcAN1, VcbHLH42-1, VcbHLH42-2, VcbHLH42-3, VcbHLH1-1 and VcbHLH1-2 was located on Vaccdscaffolds11, 24, 15, 19, 28 and 44, respectively . The VcbHLH1-2 promoter specifically contained a gibberellin-responsive element p-box. Moreover, the promoter of VcbHLH42-2, VcbHLH1-1 and VcbHLH1-2 respectively contained 1, 2 and 2 ethylene response element EREs, while the other three genes’ promoters did not contain this element.Many auxin-, salicylic acid (SA)-, abscisic acid (ABA)-, methyl jasmonate (MeJA)- and some other hormone-responsive elements were identified in the six VcbHLH1-1 and VcbHLH1-2, the promoters of all the other four genes contained the drought induction response element MBS, and the VcbHLH42-1 promoter contained two of the elements. The promoters of VcbHLH42-2, VcbHLH42-3, VcbHLH1-1 and VcbHLH1-2 each contained one low temperature response element, LTR. The VcbHLH1-2 promoter specifically contained a GC-motif specific hypoxia induction-related element. All the promoters contained anaerobic induction-related element ARE, and promoters of VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 contained two of this element, while the other two genes’ promoters contained three.Except VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 promoters contained the same types and amounts of A-box, O2-site and GCN4_motif, while VcbHLH1-1 promoter had no A-box and GCN4_motif elements and the VcbHLH1-2 promoter had no O2-site and GCN4_motif components.For the plant growth and development-related elements, all promoters contained the meristem expression element, CAT-box. bHLH genes  prediction result showed that there were binding sites for 13 kinds of transcription factors, including AP2/ERF, B3, BFR-BPC, BES1, bHLH, C2H2, Dof, ERF, GRAS, MIKC_MADS, MYB_related, NAC and Trihelix, in the promoters of the six blueberry LH genes . The typBased on the Arabidopsis protein database, the protein–protein interactions of the six blueberry bHLHs were predicted using STRING software. As shown in bHLHs belonging to the same group shared very high similarity, in this study, gene specific primers for three of the six bHLH genes were successfully designed according to their nucleotide sequences deposited in the blueberry genome data hub. By using gene-specific primers, cDNA sequences of VcAN1, VcbHLH1-1 and VcbHLH1-2, respectively with lengths of 2885 bp, 2043 bp and 2415 bp, were amplified , and its expression in PF and BF was significantly higher than that in GF, which was about 2.86 times and 2.75 times compared to GF, respectively. The expression of VcbHLH1-1 in GP and PP was similar, both accounting for about 20% of YL. VcbHLH1-2 showed the highest expression in S and the lowest expression in RF, and its expression in S was about 206 times of that in RF. Its expression in YL is about 58.7% in S, but is about 3.32 times of that in OL. Its expression in PP was higher than in GP, and its expression in the two kinds of podetiums are both significantly higher than that in fruits. The expression level of VcbHLH1-2 in fruits at different developmental stages followed the order: GF > BF > PF > PiF > RF. In addition, its expression in GF was about 4.24, 36.94, 2.72 and 2.29 times of that in PiF, RF, PF and BF, respectively.To reveal their expression patterns in young leaf (YL), old leaf (OL), stem (S), green podetium (GP) and purple podetium (PP) and fruits at green (GF), pink (PiF), red (RF), purple (PF) and blue (BF) developmental stages, qRT-PCR experiments were performed. Results showed that the expression of l stages . VcAN1 sbHLH genes in secondary metabolites biosynthesis in blueberry fruits, the correlation between the bHLH genes’ expression and secondary metabolites contents in blueberry fruits were investigated. Results showed that VcAN1 expression level was very significantly negatively correlated with total phenolic content and flavonoid content , and very significantly positively correlated with anthocyanin content (correlation coefficient was 0.940). The expression level of VcbHLH1-1 was very significantly negatively correlated with contents of total phenolic and flavonoids , and somewhat positively correlated with anthocyanin content (correlation coefficient was 0.533). However, the expression level of VcbHLH1-2 was significantly positively correlated with total phenolic content and flavonoid content , and slightly negatively correlated with anthocyanin content (correlation coefficient was 0.144).In our previous study, we measured the contents of total phenolic, falvonoid and anthocyanin of blueberry fruits at the same five different developmental stages . To uncoBlueberry is popular and famous for its anthocyanin-rich characteristics. However, as a young fruit tree species that has been cultivated for only a little more than 100 years, research on the molecular mechanism of blueberry anthocyanin biosynthesis and regulation are very limited. Recently, the regulation roles of several blueberry MYB transcription factors have been clarified ,10,27,28All the six bHLH proteins contained the conserved bHLH-MYC_N domain PF14215.5), the bHLH_SF super domain, DNA binding sites and dimerization interfaces. The length of the conserved bHLH domain usually contained approximately 50~60 amino acids , and the15.5, theSmelAN1 was expressed in all eggplant tissues containing anthocyanins, and showed the highest expression level in fruits [AcbHLH42, also a homologous gene of AtTT8, was highly expressed in the inner pericarp of kiwifruit with the highest anthocyanin content, and its encoding protein was necessary for the activation of AcANS and AcF3GT promoters. The co-expression of AcMYB123 and AcbHLH42 was reported to be an important prerequisite for anthocyanin synthesis, indicating that AcbHLH42 is a key factor in the spatiotemporal regulation of anthocyanin synthesis in kiwifruit [DFR and BAN [Populus alba PalbHLH1 and was closely related to SmeJAF13 and PalbHLH1; they were also identified as homologous proteins of Arabidopsis AtGL3. The overexpression of the PalbHLH1 gene in poplar enhanced the pathogen resistance of transgenic poplar, and this effect was reported to be achieved by increasing the flavonoid accumulation [SmelAN1, SmelJAF13 was also expressed in all eggplant tissues containing anthocyanins, but it showed the highest expression level in flower organs [bHLH genes in different organs varied greatly. VcAN1 showed high expression in OL, S and BF. Its expression in fruit increased as the blueberry fruit ripened and was very significantly positively correlated with the anthocyanin content in fruit, implying that it regulates the anthocyanin biosynthesis in blueberry fruit. Moreover, it was also worth noting that its expression in OL was significantly higher than in YL. The expression level of VcAN1 in podetium was the lowest. Notably, its expression in PP was significantly higher than in GP. This evidence indicated that VcAN1 might play important positive roles in the anthocyanin accumulation in different blueberry organs. VcbHLH1-1 expressed the highest in YL and S, and the lowest in GF. Its expression in YL was significantly higher than in OL. Moreover, the expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in BF was significantly higher than in GF. Similar to VcbHLH1-1, VcbHLH1-2 showed high expression in YL and S, and its expression in YL was also significantly higher than in OL, suggesting that VcbHLH1-1 and VcbHLH1-2 might play similar roles in blueberry leaf and stem. Unlike VcbHLH1-1, however, VcbHLH1-2 showed very low expression in blueberry fruit, and its expression in fruits at different developmental stages followed the order: GF > BF > PF > PiF > RF, suggesting that their functions in blueberry fruit varied. Thus, it was indicated that the anthocyanin biosynthesis regulatory roles of these VcbHLHs were spatially and temporally different [According to the results of protein phylogenetic analysis, the six blueberry bHLHs could be further divided into two groups. One group included VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3. This group of VcbHLHs showed the highest similarity to well-known anthocyanin biosynthesis-related eggplant SmelAN1  or kiwifn fruits . The kiwiwifruit . Arabido and BAN . The othmulation . Similarr organs . Moreover organs . It was ifferent ,33,47.bHLHs can be affected by many envrionmental fators, transcription factors and so on [bHLHs varied greatly between the two groups. This suggested that the light responses of bHLHs belonging to different groups varied.The expression of nd so on . The antnd so on . In thisbHLH genes. Consistent with the protein phylogenetic analysis result, the hormone-responsive element types and numbers in promoters of blueberry bHLHs belonging to the different groups varied greatly. Accumulated evidence has proved that many bHLHs display functions via influencing ABA- and JA- signaling pathways [bHLH promoters were the largest among all hormone-responsive elements. It was thus indicated that these two hormones greatly affected the anthocyanin accumulation by regulating bHLH expression in blueberries. The numbers of ABA- and MeJA-responsive elements in promoters of VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were greater than the other two genes’ promoters. Interestingly, we identified two ERE elements in promoters of VcbHLH1-1 and VcbHLH1-2, indicating that these two bHLHs might play their roles through responding to or regulating ethylene signaling [The anthocyanin biosynthesis and accumulation are also greatly influenced by phytohormones ,49,50. Ipathways ,52. In apathways ,54. Consignaling . MoreovebHLH genes have been increasingly proved to be involved in plant abiotic stress responses, including osmotic stress, drought, low temperature and so on [bHLHs contained anaerobic induction-related elements, and the VcbHLH1-2 promoter specially harbored an anoxic specific inducibility-related element, indicating that these bHLHs might all be osmotic stress responsive. The promoters of VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 contained drought inducibility-related elements that did not exist in promoters of members belonging to the other group, indicating that the expression of two blueberry bHLH groups differed in response to drought. Moreover, the promoter of VcbHLH42-2, VcbHLH42-3, VcbHLH1-1 and VcbHLH1-2 each contained a low-temperature-related element, LTR, suggesting that their expression was regulated by low temperature.nd so on . All theVcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3. VcbHLH1-1 and VcbHLH1-2 promoters, however, specifically possess several binding sites for NAC transcription factors. In sweet potato, a MYB340-bHLH2-NAC56 complex was reported to regulate anthocyanin biosynthesis [NAC gene was identified to be involved in anthocyanin accumulation in blueberry [VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3, indicating that the expression and function of different blueberry bHLH groups was regulated by different transcription factors.Transcription factors also greatly influenced the transcriptional activities of bHLHs . Many bHynthesis . Similarlueberry . TherefoThe young leaf (YL), old leaf (OL), stem (S), green podetium (GP), and purple podetium (PP), and fruits at green (GF), pink (PiF), red (RF), purple (PF), and blue (BF) developmental stages of blueberry ‘FLS03’ used in this study were collected in Guyue mountain farm, Dongfu Town, Jimei District, Xiamen City, Fujian Province, China. Blueberries at green, pink, red, purple, and blue developmental stages were collected according to the standard of Sun et al. . Collecthttps://www.vaccinium.org/analysis/49 (accessed on 1 June 2020)). In addition, the recently published anthocyanin biosynthesis-related bHLH protein sequences were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 20 June 2020)), and were used for local blast searches against the blueberry protein data by using the Blastp program under the criterium of 0.00001. Sequences with an e-value of 0 or the highest bit score were selected as candidate blueberry anthocyanin biosynthesis-related bHLH members. After the bHLH-MYC_N domain (PF14215.5) verification using CDD, the retained blueberry bHLHs were named according to their homologous proteins.The blueberry gDNA, cDNA, and protein sequences were downloaded from the blueberry genome website (https://web.expasy.org/protparam/ (accessed on 24 June 2020)), SignalP ), TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/ (accessed on 24 June 2020)) and WoLFPSORT (https://wolfpsort.hgc.jp/ (accessed on 24 June 2020)) were used for the physiochemical property, signal peptide, transmembrane structure and subcellular localization prediction analysis of blueberry bHLH proteins, respectively [https://www.ebi.ac.uk/Tools/psa/emboss_needle/ (accessed on 24 June 2020)) was applied for the protein sequence alignment and similarity comparison analysis of blueberry bHLHs. GSDS (http://gsds.cbi.pku.edu.cn/ (accessed on 24 June 2020)) was used to show the gene structures of blueberry bHLHs gDNAs. MEME (https://meme-suite.org/meme/tools/meme (accessed on 2 October 2020)) was applied for the conserved motif identification using parameters set as follows: the minimum length = 6, the maximum length = 100, and e-value < 0.00001. The Amazing Simple SeqLogo program embedded in TBtools was used to draw logos for the conserved bHLH domain [bHLHs chromosomal locations were visualized using the Gene Location Visualize (Advanced) program in TBtools [https://string-db.org/ (accessed on 2 October 2020)) with the interaction score set as high confidence (0.700).The online software EXPASY (ectively . The gloH domain . The Mus TBtools . Based obHLHs start codon were extracted from the blueberry genome database and used as promoter sequences. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 2 October 2020)) and PlantTFDB (http://planttfdb.cbi.pku.edu.cn/ (accessed on 2 October 2020)) were used to investigate the existence and distribution of cis-acting elements and transcription factor binding sites on each promoter, respectively.The 2000 bp sequences upstream of the blueberry bHLHs related to anthocyanin synthesis showed very high sequence similarity [VcAN1, VcbHLH1-1 and VcbHLH1-2 genes were successfully designed according to their cDNA sequences in the blueberry genome  for sequencing verification.Total RNA of all the blueberry samples was isolated using Trizol RNA Extraction Kit (TaKaRa). High quality RNA from all samples was equal-weight mixed and used as template for cDNA synthesis using RevertAid First-strand cDNA synthesis Kit (Thermo Scientific). As milarity , in thisy genome . The 25 VcAN1, VcbHLH1-1 and VcbHLH1-2 genes used for qRT-PCR were designed according to our sequencing results  fluorescent dye, 7.4 μL ddH2O, 0.8 μL of each upstream and downstream primers, and 1 μL cDNA template. Their relative expression levels in different samples were calculated using the 2−ΔΔCt method using GAPDH (Genbank ID: AY123769) as the internal reference gene [TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  kit was used to separately synthesize cDNA of different samples. Gene specific primers for  results . qRT-PCRnce gene . Excel wnce gene .bHLH genes related to anthocyanin synthesis were identified from the blueberry genome. Their encoded proteins can be further divided into two groups with significant differences in nucleotide and protein sequences, promoter cis-acting elements and TFBS types and numbers, and gene expression patterns. Of note, qRT-PCR analysis revealed that VcAN1 plays positive roles in anthocyanin biosynthesis regulation not only in fruit, but also in podetium and leaf. Therefore, the exploration of its regulatory roles in blueberry anthocyanin biosynthesis is of great significance. Our study can lay a foundation for the blueberry anthocyanin biosynthesis-related bHLHs in the future, and provide a basis for high anthocyanin blueberry breeding.In this study, six


Around the world there are 33.5 million patients suffering from atrial fibrillation (AF) with an annual increase of 5 million cases. Most AF patients have an established form of an atrial cardiomyopathy. The concept of atrial cardiomyopathy was introduced in 2016. Thus, therapy of underlying diseases and atrial tissue changes appear as a cornerstone of AF therapy. Furthermore, therapy or prevention of atrial endocardial changes has the potential to reduce atrial thrombogenesis and thereby cerebral stroke. The present manuscript will summarize the underlying pathophysiology and remodeling processes observed in the development of an atrial cardiomyopathy, thrombogenesis, and atrial fibrillation. In particular, the impact of oxidative stress, inflammation, diabetes, and obesity will be addressed. In 1995, Allessie’s group introduced the term “atrial electrical remodeling”. His fundamental study showed that atrial fibrillation (AF) causes shortening of the atrial action potential and that, thereby, AF begets AF . In theiAtrial cardiomyopathy is defined as any complex of structural, architectural, contractile or electrophysiological changes affecting the atria with the potential to produce clinically-relevant manifestations . The ter“Oxidative stress” has been implicated in the pathogenesis of multiple cardiac/cardiovascular disease conditions among them myocardial infarction, myocardial ischemia/reperfusion, heart failure, diabetic cardiomyopathy, and cardiotoxicity ,11,12,13For decades, “oxidative stress” has taken center stage in its role as a main contributor to development and progression of AF and as a mediator of AF-induced atrial remodeling processes . A numbe2O2, for instance, preferentially oxidizes the thiol side chain of well-defined cysteinyl residues in targeted functional motifs [Of note, reactive oxygen species (ROS) are formed to some extent under physiological conditions where they are indispensable in maintaining cell signaling, redox homeostasis, proliferation, differentiation, and cell viability . ROS canl motifs . The antl motifs ,17,18.AF is the most common arrhythmia in clinical practice and has been repeatedly associated with increased “oxidative stress” . EvidencMihm et al.  were the2+-handling have been identified as key phenomena contributing already to the onset of AF [2+-handling myocyte hypertrophy, and increased apoptosis [It has been shown that the cardiac excitation-contraction coupling components are particularly sensitive to oxidative stress and, thus, contribute to AF substrate formation ,26,27,28et of AF . Along tet of AF . Mitochoet of AF ,32,33,34et of AF . The recpoptosis . AF itsepoptosis  describepoptosis . A recenpoptosis .Increased production of ROS/RNS occurs immediately after new-onset AF as has been shown in patients with lone recurrent AF and for rapid atrial pacing models ,39,40,41Gene expression profiling of atrial tissue samples from patients with SR and AF revealed a decreased expression of anti-oxidative genes, whereas that of several ROS-producing genes was increased . A recenRedox-regulation and mounting of anti-oxidative response mechanisms involves three important groups of enzymes, the glutaredoxins (GRX), peroxiredoxins (PRX), and thioredoxins . In the setting of AF, not much is known on their expression, activity, and specific function. Available data indicate decreased expression of PRX3  levels) in atrial tissue of dogs subjected to two weeks of ventricular tachypacing . In acut2+ prevented the oxidation of cellular constituents and showed cytoprotective effects [Several mechanisms and sources contribute to elevated ROS/RNS levels in cardiovascular disease, and during AF in particular . Mitocho effects .2+-release events contribute to increased spontaneous ectopy [Similar to AF, ischemia also provokes alterations in cellular ionic homeostasis, in particular of calcium and sodium ions. Thereby, ischemia creates a substrate for AF maintenance ,53. Incrs ectopy . In pulms ectopy .Besides mitochondria, a family of seven NADPH-dependent enzymes consisting of the “NADPH oxidases” Nox 1–5 and Duox 1–2 represent a second important source of ROS during AF . DiffereA shift from NADPH oxidase to other cellular sources of ROS which include xanthine oxidase, , monoamil-arginine to NO and l-citrullin. For this reaction, tetrahydrobiopterin (BH4) and Ca2+-activated calmodulins are essential cofactors [− instead [eNOS is a homodimeric enzyme that oxidises ofactors . BH4 facofactors . In the  instead . Reduced instead  which ar instead ,76.2+ handling and contractility [Furthermore, AF is associated with increased levels of ADMA, an endogenous inhibitor of eNOS ,78. Uponactility ,80. AF oactility . Similaractility . Howeveractility .2O2 is formed as a by-product in this reaction. MAO plays a causal role in cardiac dysfunction in response to pressure overload due to oxidative stress [In right atrial appendages (RAA), monoamine oxidase (MAO) has been identified as a substantial source of ROS . MAO, a e stress ,85. MAO e stress .2+ and calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating the AngII/RAP-dependent induction of AF [2+-leak from the sarcoplasmic reticulum via enhanced phosphorylation of RyR2 [2+ concentrations are responsible for the resulting increase in AF inducibility and duration. In this way, CaMKII functions as a cellular sensor for ROS that links the important pathogenetic factors AngII, Nox, and Ca2+ with AF.Although the excessive production of ROS/RNS contributes to cellular damage, physiological levels rather induce reversible and site-specific protein modifications that define “redox signaling” processes which are subject to stringent spatial and temporal regulation . Covalenon of AF . AngII, on of AF ,88. Thison of AF . Mechani of RyR2 . ElevateNuclear factor-κB (NF-κB) is a key transcription regulator coupling redox state to the transcriptional regulation in various pathophysiological settings, including AF . ElevateThe typical target genes of NF-κB comprise pro-inflammatory cytokines such as interleukin-8 and TNFα, but also endothelial adhesion molecules ICAM-1 and VCAM-1, the cardiac sodium channel SCN5A, and the endothelial oxidized low-density lipoprotein (lectin-like) receptor, LOX-1 ,90,91,92RAP and AF not only contribute to structural, electrical, and endocardial/endothelial remodeling in the atria; rapid atrial pacing has been shown to activate the NF-κB pathway also in the left ventricle . In lineThe Nrf2 signaling pathway is closely linked to the development of cardiac diseases such as AF, diabetic cardiomyopathy, myocarditis, heart failure, and ischemic heart disease . Recent 2-dependent protein stability being the best characterized regulatory mechanism [2 is used by prolyl-hydroxylase domain-containing enzymes (PHD) for the corresponding modification of Hif1α, which then becomes a target for ubiquitinoylation by von Hippel-Lindau complex and proteasomal degradation [Hypoxia-inducible factors (Hifs) are heterodimeric factors that control a hypoxia-induced gene expression profile aimed at counteracting adverse cellular and systemic effects of limitations in oxygen supply. Of the three mammalian isoforms, Hif1α probably is the most intensive studied one. Hif1α levels are regulated by oxygen tension at transcriptional, translational, and post-translational level, with Oechanism . Under nradation . Hypoxiaradation . Both faradation ,123. Undradation . Thus, b2O2 are generated. Catalytic forms of LOX have been also detected in nuclei and cytosol, but their function remains to be elucidated fully [2O2 generated by LOX contributes to atrial redox signaling and the ROS-dependent generation of AF substrate is not clear, yet (2O2 are potent chemoattractants for monocytes and VSMC [Lysyl oxidase (LOX) is another established target gene of HIF. Accordingly, LOX expression was found to be induced in response to hypoxia/ischemia. Recent work established a role of LOX in cardiovascular function and disease, including AF ,126,127.ed fully ,130. Inced fully . AngII aed fully . If and ear, yet . Howeverand VSMC ,132, whiand VSMC ,134.N-terminal kinase (JNK) and induction of TNFα [The pathogenesis of several cardiovascular diseases including myocarditis, heart failure and myocardial infarction involves inflammation and inflammatory cytokines . This ha of TNFα ,143. CRP of TNFα  and elev of TNFα . CRP lev of TNFα ,148,149.Obesity and metabolic syndrome, including diabetes, represent major risk factors for AF ,153,154.Comorbidities induce changes in the mitochondrial DNA (mtDNA) and oxLDL, which activates the atrial NLRP3 inflammasome. Reactive oxygen species (ROS) stimulate the NLRP3 inflammasome, which causes stimulation of caspase-1 maturates interleukin (IL)-1β. Thereby, IL-6 and CRP (C-reactive protein) are increased. IL-1β amplifies the NLRP3 inflammatory signaling. NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells .Total and LA EAT volumes as well as left periatrial EAT thickness are greater in persistent AF versus paroxysmal or no AF ,167,168.2+ levels, could be alternatively activated by oxidation  [According to the meta-analysis presented by Huxley et al. , diabetiial ROS) ,188. Advial ROS) . In addiial ROS) . This prial ROS) . The actial ROS) ,194,195.ial ROS) ,197,198.ial ROS) ,201,202.ial ROS) ,207,208.The concept of ‘endocardial remodelling’ in AF was introduced by the consensus document on atrial cardiomyopathies . In accoROS/RNS and redox signaling have been implicated as the central component in the process of atrial remodeling. Redox-signaling contributes to tissue remodeling and self-perpetuation of AF. Nevertheless, so far antioxidant/redox-modifying therapies have not been shown to prevent, terminate, or reverse AF-induced atrial remodeling.