These further stimulate the migration and proliferation of vascular endothelial cells and promote the formation of new vessels, ultimately leading to the occurrence and development of hemangioma. Proanthocyanidins are naturally active substance from plants and fruits. They possess multiple functions like antiproliferation, antiangiogenesis, and antitumor. It has been demonstrated that proanthocyanidins effectively work in various diseases via inhibiting the expression of various factors, e.g., HIF-1α, VEGF, PI3K, and Akt. Considering the pathogenesis of hemangioma and the effect of proanthocyanidins, we hold a hypothesis that proanthocyanidins would be applied in hemangioma via downregulating cytokine/pathway expression, suppressing vascular cell proliferation and arrest abnormal angiogenesis. Taken together, proanthocyanidins may be a potential novel way for the treatment of hemangioma.Hemangioma, the most common benign vascular tumor, not only affects the appearance and psychology but also has a life-threatening potential. It is considered that clonal vascular endothelial cell proliferation and excessive angiogenesis are responsible for hemangioma pathogenesis, in which abnormal cytokines/pathways are closely implicated, primarily including high expression of hypoxia-inducible factor-1 It not only occurs on the skin but also involves other organs , 2. Althogenesis –11. Currogenesis . As a fiogenesis ; undoubtmangioma –17. Anotmangioma ; neverthpression –21. Recepression ; howeverRecently, numerous studies have demonstrated that natural extracts, proanthocyanidins in particular, possess potent antiangiogenic, antiproliferative, immuno-suppressive, anti-inflammatory, and antineoplastic activities, accompanied with few side effects and even application in infants and pregnant women , 29. Proα and HIF-1β) is crucial to the regulation of hypoxia-induced genes [Although the etiology of hemangioma remains unknown, it is thought that hemangioma is mostly derived from uncontrolled angiogenesis that is generated by the clonal proliferation of vascular endothelial cells . In thised genes . It is sed genes –38. Numeed genes , 40.α actively takes part in the formation of new vessels and the proliferation of endothelial cells in hemangioma [α is regulated by intracellular oxygen concentration [α protein is rapidly degraded at the sufficient oxygen level. Under hypoxia conditions, however, HIF-1α protein is deposited in the nucleus and forms an active complex with HIF-1β, which further activates the transcription of HIF-1α target genes [α level is directly proportional to the activity and severity of hemangioma, and its overexpression is closely implicated in the increased vascularity, invasion, and progression of tumor [α in the proliferative phase of hemangioma than that in the regress phase; but a decrease of HIF-1α emerged from the serum of hemangioma patients after systemic sclerotherapy [α could induce the expression of a series of genes, VEGF gene in particular, then stimulate the proliferation and migration of vascular endothelial cells, ultimately lead to the formation of new vessels [As a potent proangiogenic molecule, HIF-1mangioma . The expntration . Under pet genes . HIF-1α of tumor , 45. Chaotherapy . More im vessels , 48.α, is a vital signaling protein that stimulates angiogenesis and vasculogenesis [β1 integrin markedly decreases, and VEGF level significantly increases, thereby facilitating VEGFR-2 activation [VEGF, the downstream of HIF-1ogenesis . It playogenesis , 51. As ogenesis , 53. By ogenesis  and furtogenesis . VEGFR1 ogenesis , 56. In tivation . Despitetivation –60. VEGFIt is therefore considered that VEGF/VEGFR2 signaling is a crucial signal transducer in both physiologic and pathologic angiogenesis. Jinnin et al. showed that VEGFR1 reduction in hemangioma facilitated VEGF-dependent VEGFR2 signal activation, further stimulating the downstream pathways  and contributing to the proliferation of vascular endothelial cells and the formation of new vessels , 61. TheThe decreased VEGFR1 expression in hemangioma endothelial cells facilitates VEGFR2 persistent activation, which contributes to the combination of VEGFR-2 with VEGF. HIF-1, meanwhile, is activated in the presence of hypoxia, further exciting its downstream VEGF; then, the activated VEGF combines with its receptor VEGFR2, which in turn activates the downstream pathway PI3K/Akt. PI3K and its downstream Akt, on the one hand, directly provoke HIF-1; on the other, they activate their downstream target mTOR/p70S6K to indirectly promote HIF-1 translation, collectively encouraging VEGF activation. As a result, it forms an autocrine loop of VEGF signaling. Above changes ultimately enhance vascular endothelial cell proliferation, accelerate angiogenesis, and trigger hemangioma formation.Proanthocyanidins, a kind of natural substance, are derived from fruits and plants such as grape, apple, and black currant . They bein vitro and in vivo studies that proanthocyanidins inhibited esophageal adenocarcinoma via inactivation of PI3K/Akt/mTOR signal [α by targeting Akt/mTOR/p70S6K/4E-BP-1 pathway and thereby inhibited angiogenesis [α-dependent manner [α and matrix metalloproteinases (MMPs) to stimulate the invasion and migration of tumor cell; however, the mRNA and protein levels of hypoxia-induced MMPs significantly decreased in the presence of proanthocyanidins [α expression by blocking the activation of PI3K/Akt/mTOR pathways [Proanthocyanidins are commonly absorbed as monomers that are widely bound in the liver and then released, circulating in the body or accumulating in tissues before excretion from the kidney, while some of them return to the intestine through bile , 77. BesR signal . Meanwhiogenesis . Moreovet manner . For exayanidins . Collectpathways . Above fpathways , 95, 96.α and VEGF are central contributors to the pathogenesis of hemangioma [α is initially induced by hypoxia and then activates VEGF, further to promote vascular proliferation and aggravates angiogenesis under a serial of signals, e.g., PI3K/Akt, mTOR, and p70S6K kinase [α or/and VEGF have been found in hemangioma lesion [α can activate the proangiogenic factor VEGF, whereas proanthocyanidins facilitate HIF-1α levels decrease [α activation [α translation and the decrease VEGF expression through mediating the PI3K/Akt and mTOR/p70S6K signaling pathways, thereby arresting the proliferation, invasion, and migration of tumor cell [Given that abnormal angiogenesis and vascular endothelial cell proliferation are primarily involved in hemangioma and proanthocyanidins have the properties of antiangiogenesis and antiproliferation, it is postulated that proanthocyanidins may be greatly available in management of hemangioma. Here, the detailed imaginary idea is formulated as following: (1) hemangioma is a common benign vascular tumor that mainly involves uncontrolled angiogenesis and abnormal vascular endothelial cell proliferation . (2) Hypmangioma –35. (3) K kinase –38. (4) a lesion , 49. HIFdecrease , 88–90. decrease , 29. (6)tivation –85. (7) α activation via directly stopping PI3K/Akt pathway or indirectly blocking mTOR/p70S6K signal, further downregulating the VEGF expression. As a result, significant reductions emerge from the proliferation, invasion, and migration of tumor cell. Ultimately, hemangioma is possible to be controlled effectively.Proanthocyanidins are initially extracted from numerous natural fruits and plants, e.g., grapes, apples, and blackcurrants. They, then, competitively combine with VEGF to prevent HIF-1α, and VEGF and inhibit vascular endothelial cell proliferation, angiogenesis, and tumorigenesis. Last but not least, proanthocyanidins are safe for infants, pregnant woman, and the elderly [Hemangioma has long been a hotspot in the field of dermatology research. Although various medications and physiotherapies are used to treat hemangioma, these vehicles are constricted by serious adverse reactions, high cost, and traumatic influence. Proanthocyanidins, a kind of natural plant extracts with few side effects, are expected to be applied in hemangioma. Owing to their antiangiogenic and antiproliferative activities, proanthocyanidins will likely decrease the levels of PI3K, Akt, HIF-1 elderly , 95, 96.in vitro and in vivo. Firstly, animal experiments will be performed in well-established hemangioma mice models to investigate the effectiveness of dietary proanthocyanidins. Subsequently, the therapeutic mechanism of proanthocyanidins on hemangioma should be explored in a cell model and three-dimensional model. Meanwhile, the related parameters are determined through a serial of molecular biotechnologies. Finally, randomized controlled clinical trials may provide a scientific basis for more research and clinical applications of proanthocyanidins.Herein, we speculate that proanthocyanidins have powerful efficacy on hemangioma. To prove the above hypothesis and clarify the effects of proanthocyanidins on hemangioma, further studies are warranted α, VEGF, and PI3K/Akt. By targeting these crucial points, hemangioma is hopeful to be cured safely and effectively. At present, drug treatments for hemangiomas include propranolol, glucocorticoid, interferon, and others. These vehicles indeed exhibit improved effect on hemangioma, but the cost and side effects stop patients from adopting them. Consequently, cost-effective and safe treatments are required for hemangioma. Here, we expound the pathogenesis of hemangioma and enumerate the clinical application of proanthocyanidins. It indicates that proanthocyanidins would be an ideal alternative for the control of hemangioma. Indeed, we will carry out in vitro and in vivo experiments to validate the proposed signaling pathways in hemangioma and confirm the improvements in hemangioma parameters after proanthocyanidin treatment.Above all, hemangioma is a benign vascular tumor, frequently occurring in children, which has a negative impact on quality of life even threatens life. Although the specific pathogenesis of hemangioma has not been fully elucidated, it is currently regarded as multifactorial condition involving vascular endothelial cell proliferation, uncontrolled angiogenesis, and abnormal cytokines/pathways under hypoxia induction, e.g., HIF-1


To detect the development, function and therapeutic potential of epicardial adipose tissue (EAT); analyze a related gene expression dataset, including data from neonates, infants, and children with congenital heart disease (CHD); compare the data to identify the codifferentially expressed (DE) mRNAs and lncRNAs and the corresponding miRNAs; generate a potential competitive endogenous RNA (ceRNA) network; and assess the involvement of immunocyte infiltration in the development of the EAT.Multiple algorithms for linear models for microarray data algorithms (LIMMA), CIBERSORT, gene-set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were used. The miRcode, miRDB, miRTarBase, and TargetScan database were used to construct the ceRNA network. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DE mRNAs were performed.MIR210HG, hsa-miR-449c-5p, and CACNA2D4 was generated and shown to target monocyte infiltration.Thirteen co-DE mRNAs and 47 co-DE lncRNAs were subsequently identified. The related categories included negative regulation of myoblast differentiation, regulation of ion transmembrane transport, and heart development, which were primarily identified for further pathway enrichment analysis. Additionally, the hub ceRNA network in EAT development involving MIR210HG, and CACNA2D4, which were shown to target the RIG-I-like receptor signaling pathway and PPAR signaling pathway, indicating that these factors may be novel regulators and therapeutic targets in EAT development.These findings suggest that the pathways of myoblast differentiation and ion transmembrane transport may be potential hub pathways involved in EAT development in CHD patients. In addition, the network includes monocytes,  EAT is known to have a protective role as a mechanical buffer and a rapid source of free fatty acids (FFAs) in the myocardium and vascular arteries . There apathways . Anatomipathways . Researcpathways , 7, 8. Rpathways . Of note2 [For adult patients, Patel et al. revealed that EAT could secrete multiple adipokines, including apelin, adiponectin, adrenomedullin, angiotensinogen, leptin, and visfatin, coupled with the potential paracrine function of regulating inflammation, immune response, coronary plaques, ventricular remodeling, and vascular smooth muscle cell growth and migration . Sakamot2 . Additio2 . Chechi 2 , 13.IGF1R) was shown to be stimulated after myocardial damage and then promotes the differentiation of epicardial cells into adipocytes to protect cardiomyocytes and ensure the survival of cardiac progenitors [In this scenario, EAT, an immune and endocrine organ, may function as a key inflammatory mediator for cardiovascular system development. Here, the insulin-like growth factor 1 receptor | greater than 1.5. Herein, these genes were identified as having significantly increased (log2FC > 1.5) or decreased (log2FC < − 1.5) expression. Figure We performed a differential expression (DE) analysis based on “affy” package of R software that was curated by data processing principles includes expression data background correction, log2 transformation, data normalization and linear model and DE analyses. Here, the data normalization, missing value procession, and powerful DE analyses were performed by a function of the robust multi-array average (RMA), K-nearest neighbor (KNN), and linear models for microarray data algorithms (LIMMA), respectively . We defihttps://cibersort.stanford.edu/) to estimate the burden of immune infiltration subtypes in the EAT development process, based on a linear support vector regression (SVR) algorithm with gene expression data with systems-level gene expression data in the R toolkit [p-value < 0.05; (4) multiple comparisons among the neonatal, infant and child groups were tested by one-way ANOVA, and the p-value was adjusted by the Bonferroni method.In this routine, we applied the CIBERSORT algorithm ( toolkit . We choshttp://metascape.org/), which is a web-based analytics tool used for gene functional enrichment analysis [p-value = 0.05; (3) the min enrichment score = 1.5.Gene Ontology (GO) term enrichment analyses were carried out separately for the DE genes using the MetaScape database  was performed [p-value < 0.05; (2) the permutation test number = 1000; (3) the gene profile was defined as continuous phenotypes with time series experiments for EAT development and defined as class labels in response to the expression level of hub mRNAs/immunocyte infiltration; (4) enrichment statistic was used for weighting; (5) the metric for ranking genes selected “Pearson” for time series analysis and defined the “Signal2Noise” for categorical analysis.Based on the time series-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and biological process term enrichment, a gene-set enrichment analysis  [http://mirdb.org/) [http://mirtarbase.mbc.nctu.edu.tw/php/index.php) [http://www.targetscan.org/mamm_31/) databases [The ceRNA networks of lncRNAs, miRNAs, and mRNAs were predicted based on the miRcode (de.org/) , miRDB (db.org/) , miRTarBdex.php) , and Taratabases . Here, tatabases ; (2) prePearson statistical method [After grouping by the median expression value of the target gene, lncRNA, and infiltrating immunocytes, we applied the gene set enrichment analysis (GSEA) to detect the downstream pathways. The normalized enrichment score (NES) was obtained via cluster analysis based on the l method . In addil method .Ovis aries. The CIBERSORT algorithm was used to compare the EAT sample immune infiltration between 1 and 4 weeks of age. Furthermore, the candidate pathway variation was assessed by the GSVA package. The mRNA and lncRNA expression values between the two groups were compared with powerful DE analyses with the LIMMA algorithm.Finally, we also verified the landscape of immunocyte infiltration, regulatory networks and hub gene expression related to EAT development based on the GSE115799 dataset for p-value = 0.001), melanogenesis , and hedgehog signaling pathway  were negatively correlated with EAT development, while other categories including the VEGF signaling pathway , glycosaminoglycan biosynthesis heparan sulfate , and renal cell carcinoma  were positively associated with EAT development , cyclic nucleotide binding , and neuronal action potential , which were negatively correlated with EAT development, and negative regulation of receptor mediated endocytosis , acidic amino acid transport , and establishment or maintenance of monopolar cell polarity , which were positively correlated with EAT development . Here, for the comparison among the neonate, infant, and child EAT samples, to illustrate the transcription and expression profiles in response to EAT development, we performed a powerful differential analysis across the comparison groups. The results showing that 270, 889, and 1069 DE mRNAs were detected in the comparisons of infant p-value = 7.96E−05), regulation of ion transmembrane transport , and heart development  , ion channel activity , and substrate-specific channel activity  , regulation of cell proliferation involved in tissue homeostasis , and positive regulation of electron transfer activity  for cellular components  and NK cells activated  were identified  1742 links, were calculated based on the 47 co-DE lncRNAs and targeted miRNAs, (2) and 27 lncRNAs and 7 miRNAs were selected after removing the duplicate nodes, (3) and 4 targeted mRNAs were selected after determination of the miRNAs targeting mRNAs and co-DE mRNAs in multiple group comparisons. As a consequence, the ceRNA network including 27 lncRNAs, 7 miRNAs, and 4 mRNAs was accordingly constructed for EAT development for CHD patients  , CYP4A11 (54%), SLC27A2 (30%), and CYP27A1 (28%), were identified in the PPAR signaling pathway , TRAF2 (64%), IL12A (59%), IFNK (64%), MAPK12 (59%), and TBKBP1 (62%) were detected in the retinoic acid-inducible gene-I (RIG-I) like receptor signaling pathway  in protein-coding mRNAs, microRNAs, long noncoding RNAs, pseudogenic RNAs, and circular RNAs , 26. RecMIR210HG was identified as an important competing endogenous RNA of hsa-miR-449c-5p to regulate the CACNA2D4 expression level and thus regulate monocyte infiltration during the EAT development of CHD patients in early life. The PPAR signaling pathway was positively correlated with MIR210HG, CACNA2D4 expression, and monocyte infiltration, while the RIG-I like receptor signaling pathway was inversely correlated.Based on the complex bioinformatic analysis, our study demonstrated a framework to systematically characterize a regulatory map involving DE mRNAs, DE lncRNAs, immunocyte infiltration, and potentially regulated pathways. In the comparisons among the neonate, infant, and child EAT samples from CHD patients, a total of 13 co-DE mRNAs and 47 co-DE lncRNAs were identified as core regulators that are primarily involved in the pathways of myoblast differentiation, ion transmembrane transport, and heart development. We subsequently constructed a ceRNA regulatory network for these co-DE mRNAs and co-DE lncRNAs as presented above. As a result, CACNA2D4 encodes a protein of the voltage-dependent calcium channel complex that serves as a mediator leading to the influx of calcium ions through the cytomembrane. Based on the PathCards database (https://pathcards.genecards.org/) [CACNA2D4 may be correlated with the pathways of T cell receptor signaling and dilated cardiomyopathy (DCM). Weeke et al. showed that the calcium channel subunit genes CACNB2 and CACNA2D4 were significantly correlated with familial atrial fibrillation [MIR210HG, as reported by recent studies, may function as an oncogenic regulator that promotes tumor metastasis, proliferation, and invasion in breast cancer, glioma, hepatocellular carcinoma, and non-small cell lung cancer [MIR210HG may be involved in the pathway of hypoxic and inflammatory stress of endothelial cells [Functionally, ds.org/) , we founillation . While tillation . The long cancer –34. In aal cells , 36. Hsaal cells –39. Xu eal cells .The PPARs, including PPARα, PPARβ/δ, and PPARγ, were reported to regulate adipogenesis, lipid metabolism, and immunoinflammatory responses, especially in the maintenance of metabolic homeostasis . InteresMIR210HG, hsa-miR-449c-5p, and CACNA2D4 was detected in EAT development and may serve as a primary regulator controlling thermogenesis and immunoinflammatory response. These results might be used in novel strategies to develop effective therapies targeting myocardial energy supply and development in CHD patients. Our findings could further elucidate the molecular mechanisms involved in EAT, thereby enabling the exploration of EAT development and CHD pathophysiology from a new perspective, which may help improve the current therapies.Related studies have proved that EAT development was regulated by the immune-inflammatory microenvironment and that the genetic regulation was complicated. In our study, these results further revealed the potential effects and research value of myoblast differentiation and ion transmembrane transport in EAT development during early childhood, and thus, these related DE genes were further assessed in the follow-up study. Additionally, a network including monocytes, Additional file 1: Table S1. The epicardial adipose tissue (EAT) development was correlated with pathways based on time series-related GSEA.Additional file 2: Table S2. The differentially expressed mRNAs and lncRNAs were identified in multiple comparisons among the EAT samples at different developmental stages.Additional file 3: Table S3. The Gene Ontology enrichment terms for codifferentially expressed mRNAs among the neonatal, infant and child EAT samples.Additional file 4: Table S4. The immunocyte infiltration detection of the GSE82155 and GSE115799 datasets.Additional file 5: Table S5. The competing endogenous RNA (ceRNA) regulatory network construction.Additional file 6: Table S6.Pearson’s correlation coefficient values of hub miRNAs and monocyte infiltration level.Additional file 7: Table S7. The gene set enrichment analysis after grouping by the monocyte infiltration level, CACNA2D4 and MIR210HG expression value.


Chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) is a multifunctional protein participated in diverse cellular processes, including chromosome remodeling, cell differentiation and development. CHD1L is a regulator of chromosomal integrity maintenance, DNA repair and transcriptional regulation through its bindings to DNA. By regulating kinds of complex networks, CHD1L has been identified as a potent anti-apoptotic and pro-proliferative factor. CHD1L is also an oncoprotein since its overexpression leads to dysregulation of related downstream targets in various cancers. The latest advances in the functional molecular basis of CHD1L in normal cells will be described in this review. As the same time, we will describe the current understanding of CHD1L in terms of structure, characteristics, function and the molecular mechanisms underlying CHD1L in tumorigenesis. We inference that the role of CHD1L which involve in multiple cellular processes and oncogenesis is well worth further studying in basic biology and clinical relevance. CHD1L gene (Chromodomain helicase/ATPase DNA binding protein 1-like gene), also called the ALC1 gene (amplified in liver cancer 1), locates on chromosome 1q21 region of human hepatoma cells and is cloned by Guan using the comparative genomic hybridization (CGH) technique .0130018) . Schemathttps://www.ncbi.nlm.nih.gov/homologene/?term=11590). Human CHD1L is more than 87, 73, and 66% similar to mammals, the less-evolved vertebrate chicken and zebrafish, respectively (Table http://www.informatics.jax.org/homology/GOGraph/11590#Annotations). Since CHD1L is expressed in many species including invertebrates, it appears that CHD1L exists in the common ancestor of vertebrates. In human, CHD1L can be found in various tissues and exhibit different expression patterns in space and time . ThThe difference structures between CHD1L and CHD1 are that CHD1 has two tandem chromodomians that affect DNA functions, including transcription, replication, recombination and repair, while CHD1L uses the Marco domain to perform biological functions by binding PAR. The Macro domain proteins also recognize poly-ADP-ribose as a ligand, and the ADP-ribosylation of proteins is an important posttranslational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation .Studies have shown that CHD1 plays an important role in embryonic stem cell differentiation, hematopoietic stem cells emergence and tumorigenesis. Full length CHD1 is required in embryonic stem cell differentiation, and loss of the serine-rich region (SRR) renders CHD1 unable to support normal differentiation of ESCs into the three germ layers . Endothehttps://tcga-data.nci.nih.gov/tcga/) and The Genotype-Tissue Expression (GTEx) project . In TCGA dataset, the expression of CHD1L in BLCA, BACA, CHOL, COAD, ESCA, GBM, HNSC, KIRC, LIHC, LUAD, LUSC, PRAD, READ, SARC, STAD and THCA was higher than the normal tissues, while in KICH was lower than normal tissues, and the differences are statistically significant -ribose (PAR) chains via a C-terminal macro domain, which rapidly recruited to sites of DNA damage by PAR chains synthesized by PARP1/2 . TherefoCHD1L is a multifunctional protein that plays important roles in normal or pathological conditions. However, how does CHD1L manage all those diverse functions? This may be related to its three main domains: the N-terminal domain of SNF2 family, HELICc and Macro domain. In normal cells, CHD1L mainly functions through the Macro domain. First, CHD1L recognizes PAR through the macro domain. PARP activates the chromatin-repositioning enzyme of CHD1L, which in turn activates PAR-dependent chromatin remodeling and promotes DNA repair. In addition, under pathological conditions, CHD1L is usually abnormally expressed in a variety of tumors. The mechanism of CHD1L involved in tumorigenesis may be the activation of certain genes  and/or suppression of other genes . The overexpression of CHD1L is also related to the clinical characteristics and prognosis of patients, so the expression level of CHD1L can be considered as a potential prognostic factor for cancer.In conclusion, CHD1L recognizes several activities that play key roles in many biological processes. The significance of CHD1L in cellular and pathological effects have not been fully understood and remain to be explored. Therefore, this review suggests that CHD1L may be a novel biomarker for cancers and represents a fascinating target for molecular cancer therapy in the future.


QCCs are also therapeutically challenging due to their resistance to most conventional cancer treatments that selectively act on proliferating cells. Considering the significant impact of QCCs on cancer progression and treatment, better understanding of appropriate experimental models, and the evaluation of QCCs are key questions in the field that have direct influence on potential pharmacological interventions. Here, this review focuses on existing and emerging preclinical models and detection methods for QCCs and discusses their respective features and scope for application. By providing a framework for selecting appropriate experimental models and investigative methods, the identification of the key players that regulate the survival and activation of QCCs and the development of more effective QCC-targeting therapeutic agents may mitigate the consequences of QCCs.Quiescent cancer cells (QCCs) are cancer cells that are reversibly suspended in G Quies0 phase ,4 and, darrested . Cells earrested ,6. QuiesThe presence of QCCs is common, and evident in various tumor types ,4,7,8, s0 phase. However, QCCs are distinct than cancer stem cells (CSCs) as CSCs can exist in any phase in the cell cycle, not necessarily in G0 phase. While quiescent CSCs are located at endosteal bone surface, QCCs are evident in tumor masses and everywhere as circulating tumor cells (CTCs) or disseminating tumor cells (DTCs) are frequently in quiescence. Moreover, self-renewal, “stem-ness” markers and specific transcription factors, which are expressed by the CSCs, but are absent in QCCs [0 phase and nonproliferating, other properties of QCCs are having less RNA content [QCCs are similar to quiescent CSCs, in terms that they are both cancer cells in G in QCCs . In addi content ,12 and e content . These c content ,15,16.QCCs are nonproliferating, thus marking them as resistant to most conventional cancer treatments that act preferentially on proliferating cells . Survivi0 state. A rational understanding of these approaches, therefore, requires relevant models recapitulating QCCs behavior, as well as superior methods to evaluate QCCs activity. This review emphasizes the existing and emerging models of studying and measuring QCCs and discusses their respective features and applications.Therapeutic strategies targeting QCCs include blocking QCCs from re-entering the cell cycle, encouraging timing of therapies dependent on cell proliferation to match cell cycle re-entry points, or eradication of QCCs while in the GExperimental models can be established through the use of either a homogenous or a heterogeneous QCC environment . Homogen1 phase, mitogens, such as insulin-like growth factor-1 (IGF-1) [0 phase, compared to 15% of cells cultured with full medium. Serum replenishment for 24 h, after serum withdrawal, released 50% of cells from the G0 phase [Prior to the “restriction point” in early G (IGF-1)  and plat (IGF-1) , are rem (IGF-1) . Serum wG0 phase . A serumG0 phase ,22,23,24The experimental protocol of serum deprivation varies considerably, suggesting that cross-study comparisons may not be valid. Cells are generally serum-deprived for 42 h  to 7 day2α (cPLA2α) [Serum deprivation-induced quiescence involves changes in multiple signaling molecules, including pathways involving retinoblastoma protein (Rb) ,12,19, c(cPLA2α) . Many of0 cells, respectively [To sustain cellular energy, glucose is required by proliferating cancer cells . Therefoectively .0 phase and may return to the G1 phase when oxygen is replenished [2 (concentration ranges from 100 μM to 500 μM) [2 through induction of hypoxia-inducible factor-1 (HIF-1). When CoCl2 solution is withdrawn from the media, the cells reproliferate more slowly than in a hypoxia chamber, as additional time is required to remove the remaining CoCl2 from the cells. Compared to hypoxia chambers, the CoCl2 method offers a more sustainable oxygen-deficient environment and easier administration in in vivo animal models [When the oxygen supply is insufficient, tumor cells survive by entering the Glenished ,15. Canclenished ,15,27,28lenished ,27,28 or 500 μM) ,27,28 or 500 μM)  to 14 daThe hypoxia model is useful to mimic cancer interventions that act through hypoxia or ischemic mechanisms . For exaA quiescent state can also be induced in cultured cells through contact inhibition. When a cell culture ,22,24,30The proportion of QCCs increase when cancer cells are exposed to cisplatin , doxorubThis model closely resembles QCC induction following cancer treatment in an actual clinical setting and is, thus, ideal for screening or testing potential drugs to reduce the high proportion of QCCs following anticancer treatment. However, the number of surviving QCCs in this model can be inadequate for further studies. For example, doxorubicin-treated mouse mammary carcinoma (MMC) cells underwent apoptosis, and only 31% of MMC cells remained viable 3 weeks posttreatment. Of these viable cells, 22% were QCCs compared to 3% QCCs in control MMC cells .On the evidence noted above, the proximity of cancer cells to blood vessels would be expected to contribute to tumor heterogeneity. Quiescence is readily promoted in cells furthest from blood vessels since there is less access to nutrients and oxygen and richer production of acidic metabolites . SimilarA 3D cell model can be constructed through scaffold or non-scaffold techniques. In the scaffold method, cancer cells are seeded on a matrix, such as basement membrane extract (BME) , poly  in serum- and cytokine-free medium (SFM) to establish stable three-dimensional (3D) microvascular networks in SFM. HUVECs were then cocultured with fibroblasts from the lung or with bone marrow mesenchymal stem cells to construct a lung- or bone marrow-like microvascular niche. Thereafter, breast T4-2, MCF-7, and MDA-MB-231 cancer cells were seeded in these niches, and their growth was compared . Mature 0 phase, primarily attributable to passaging of relevant miRNAs through gap junctional intercellular communication (GJIC) between breast cancer cells and bone marrow stroma. Additionally, the quiescence of breast cancer cells is secondarily due to the transfer of exosomes containing miRNAs from bone marrow stroma to breast cancer cells [Besides the organotypic coculture method described above, simple coculture models have been applied to examine the impact of the tumor environment niche on the quiescence or reactivation of prostate cancer cells  and breaer cells . ScrutinWe herein describe some in vivo models that can recapitulate the interactions between cancer cells and the systemic host response during the quiescence process . To examIn investigating the impact of the environmental niche on quiescent cells, immunocompetent BKAL mice  and immuTumor dormancy was also studied through orthotopic injection of breast MDA-MB-231 cancer cells into NOD-SCID mice . The priIn addition to dormancy of solid tumor, in vivo models can be established for hematological malignancies. To induce dormancy in mouse B cell lymphoma model (BCL1), BALB/c mice were first intraperitoneally injected with spleen cells from BCL1 tumor-bearing mice, followed by injection of IgM on day 28, 38, and 45 post-inoculation. At day 60 post-inoculation, mice immunized with IgM had smaller tumor growth than mice non-immunized with IgM. This model shows that anti-idiotype antibody is produced as response to injected IgM, and this antibody involves in the signaling maintaining dormancy . The bioThe use of labels, such as green fluorescent protein (GFP) ,48,50,51For the typical 2D in vitro culture, the medium contains adequate nutrients, growth factor and oxygen, allowing cancer cells to proliferate faster. However, these elements are less available in in vivo, causing slower tumor growth and higher proportion of QCCs ,60. In vAlert”, midway between deep quiescence and activation. GAlert cells have higher transcriptional activity and mitochondrial metabolism, larger cell size, and faster cell cycle re-entry than deep quiescent cells [Quiescence is not a homogenous phase but instead represents a diversified state . Variousnt cells .Considering that different quiescence induction stimuli induce varying gene profiles , the modAs actual solid tumors develop in vivo in a 3-dimensional manner, the 3D culture model may more closely resemble solid tumor biology, in terms of heterogeneous tumor composition , the mic0 phase from cells at other phases, especially G1 phase, because cells at G0 and G1 phases have the same DNA content [Currently, the identification of Ki-67 negativity and low cellular RNA content are commonly used to define QCCs. Other measurements, such as assessment of DNA content and potential molecular markers for quiescence, are often required as supplementary evidence to facilitate identification of cells at G content   activity, presence of p16 and degradation of MDM2 [0 cells, the Ki-67 marker is regularly combined with other DNA stains  [Ki-67, a nuclear protein present in proliferating cells, has been used as a clinical marker in cancer prognosis to reflect the proliferative index . Ki-67 p1 phase , resulti1 phase . Neverthnegative . Of notenegative , and cane (DAPI)  and PI [e (DAPI) ,15) or ce (DAPI)  in vitro1 cells have 2n DNA [0 from G1 phase cells [Although quiescent and Ge 2n DNA , quiescee 2n DNA ,12, and 0/1 cells from cells at other phases. Pyronin stains RNA and facilitates the identification of G0 cells that have lower RNA than G1 cells [0/1 cells from other S/G2/M phases. However, this necessitates the combined use of RNA staining or other markers to identify G0 cells [Hoechst 33528, pyronin Y ,12, acriG1 cells ,12. CellG1 cells . AO emitG1 cells ,64. HoweG1 cells ; thus, dG1 cells ,65, enabG1 cells . In contG1 cells . PropidiG0 cells . However, the application of DNA and RNA content methodology with stains is more complex than the Ki-67 assay. An unsuitable concentration of AO affects the accuracy of determining the cellular DNA and RNA contents; excessive AO will denature DNA, while overdiluted AO will partially denature RNA . Higher 1 and G0 phase and gradually declines in subsequent phases, allowing mKO2-hCdt1 to mark G0/1 cells with red fluorescence. As geminin accumulates in S/G2/M phases, mAG-hGem labels S/G2/M cells green [++/mAG-, mKO2+/mAG-, mKO2-/mAG-, mKO2+/mAG+, and mKO2-/mAG+, which represent G0, G1, very early G1, G1/S, and S/G2/M phases, respectively [-, which is also instrumental in discerning G0 cells from G1 cells, as G0 cells express high levels of both mVenus-p27K- and mCherry-hCdt1 [FUCCI utilizes the phase-dependent abundance of certain proteins, such as Cdt1 and geminin, to differentiate cells at different phases of the cell cycle. The fluorescent proteins mKO2 and mAG are fused to Cdt1 and geminin, respectively . Cdt1 isls green . Cell phectively . Anotherry-hCdt1 .0 cells; therefore, the combination of these markers with the measurements mentioned in the previous section is advantageous in detecting QCCs. Increased hypoxic conditions within tumors has been well documented [Some proteins that are considerably up- or downregulated in QCCs have been considered QCC biomarkers. However, QCC molecular markers are nonexclusively present in Gcumented ,15,28 ancumented . Hypoxiccumented ,65 and Hcumented ) mark hycumented ,31,65, ihigh/ERKlow [low/H3K9me2low/ HES1high [2 for 72 h. Western blot analysis of both 2D and 3D cells recorded high p38 and p21, low ERK activity and low mRNA levels of c-Myc compared with untreated controls [0/G1 switch gene 2 (GOS2) is another potential QCC biomarker yet to be further explored, as GOS2 is highly expressed in G0 cells. GOS2 promotes quiescence in hematopoietic stem cells [0 phase and induces tumor regression in vivo [QCCs display p38h/ERKlow  and MCM2HES1high , high leHES1high ,19, DYRKHES1high , and lowHES1high  and ROS HES1high . In a stcontrols . G0/G1 sem cells , increas in vivo .® DiD [+ or DiD+ cells [Briefly, the pulse-chase method involves tagging fluorescent compounds onto cells in the pulse phase and chase phase, allowing the fluorescence to be diluted as a result of cell division. In the pulse phase, fluorescent markers, such as the proliferation marker carboxyfluorescein succinimidyl ester (CFSE)  or lipop® DiD , are tag® DiD ,76. The ® DiD  or under® DiD ,76. ThenD+ cells ,76. In cD+ cells . In addiD+ cells ,78. The D+ cells . PrimaryD+ cells . To furtD+ cells .2/M phase arrest [0 phase, hence necessitating coupling this assay with G0 identification methods in identifying QCCs. An adequate culturing time period of up to several weeks is required to differentiate between non- and label-retaining cells [With the pulse-chase method, live cells can be sorted for subsequent functional studies and comparisons with other cell subpopulations . Howevere arrest ,76, theyng cells .0 phase, investigators were able to verify that Ki-67 levels were significantly lower in benign cells and nonatypical hyperplastic cells compared to atypical hyperplastic cells and malignant cells from endometrial samples [Better ways to characterize and detect QCCs are essential to bridge the gap between limited biological knowledge and clinical observation of QCCs. Current insights into QCCs have been largely drawn from static histology of static cytologic examination or retention of fluorescent dye, but a more advanced QCC detection method that allows examination of the dynamic process of QCCs is required. Quantum dot imaging, MRI labeling, and intravital microscopy are some examples of in vivo imaging methods that can be integrated into QCC studies to provide real-time observations on QCCs behavior . Althoug samples . Another samples .1, GAlert and G0 phase, or vice versa. Other markers include the DNA intercalating agents BrdU and EdU, which are absent or poorly retained in quiescent cells. However, both BrdU and EdU are unsuitable for downstream and lengthy analysis because they harm cells and induce mutations [0 and G1 cells. Both nucleic acid-binding dyes and cytoplasmic dyes  are not feasible for prolonged experiments because they disrupt cell proliferation [In brief, Ki-67 alone or in combination with fluorescent dyes or other protein markers may detect and measure QCCs in preclinical studies. Ki-67 has been a reliable marker for QCCs , and detutations . Fluoresutations . Howeverutations  cause diferation  or are hferation . Conversferation ,82, withferation ,78,82. QCCs have been predominantly studied preclinically, and of the discussed QCC biomarkers, only Ki-67 has been clinically assessed and included in clinical practice oncology guidelines . Contrar0 phase [Cancer dormancy is a well-known clinical phenomenon that is attributable to QCCs. Reversibly residing in G0 phase  and bein0 phase . When ex0 phase ,17, and 0 phase . Despite0 cells are diploid, and it is not possible to separate them from G1 cells by DNA content, QCCs are detected and currently quantified through the absence of Ki-67 protein or low RNA content. Harnessing live cancer cells with a fluorescence-labeled G0 specific protein so that quiescence (gain of fluorescence) and cell cycle re-entry (loss of fluorescence) can be monitored under natural conditions without any experimental induction deserves serious consideration for future studies in this field.We have provided a broad framework for assessing the relevance of QCC models, including (i) the suitability of the available models for investigated cancer types, (ii) the drawbacks, and (iii) possible opportunities for overcoming these obstacles. Because G


Staphylococcus aureus and Staphylococcus epidermidis. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole  The World Health Organization (WHO) identified antibiotic-resistant bacteria as one of the greatest threats to human health in the future . There i4(3)) [6(3)) [3(5)) [4(3) cannot penetrate the polarized membrane of living microorganisms, and only enters depolarized cells, displaying enhanced fluorescence and red spectral shift upon binding to intracellular proteins [3(5) accumulate on polarized membranes resulting in self-quenching of fluorescence [4(3) and the carbocyanine dye diSC3(5) have been proposed for kinetic monitoring of changes in membrane polarity of Gram-positive bacteria exposed to antimicrobial compounds [In the past decades, membrane interactions of AMPs have been extensively investigated by using model membrane systems, and on this basis, several models of peptide-membrane interaction have been proposed ,9,10,11.4(3)) , or carb) [6(3))  and 3,3′) [3(5)) . These a) [3(5)) . This improteins ,34. On trescence ,36. Uponrescence ,38, antirescence ,40, and rescence  by flow rescence ,42,43 anrescence ,44,45 mirescence ,46. In trescence ,43,44,45rescence ,46. An aompounds .Staphylococcus aureus and Staphylococcus epidermidis. S. aureus has been ranked by WHO as a high priority pathogen for the development of new antibiotics, mainly due to the high prevalence of methicillin-resistance and increasing resistance to vancomycin [S. epidermidis represents an emerging opportunistic pathogen responsible for foreign-body associated infections due to its ability to form biofilm [S. epidermidis and S. aureus. By combining the potentiometric dye diSC3(5) with the DNA-staining dye PI, it was possible to simultaneously monitor the peptide-induced phenomena of membrane depolarization due to ion movements across the membrane, and membrane permeabilization due to the formation of pores, and thus to distinguish between specific membrane effects due to different modes of peptide-membrane interaction.In the present study, we partially adapted these latter methods to two Gram-positive species representing important human pathogens, namely ncomycin . S. epid biofilm . Hence,  biofilm . The aim biofilm , and the biofilm , with cy4(3) and the carbocyanine diSC3(5), and of the DNA stain PI, were evaluated in our assay conditions by measuring fluorescence with a microplate reader by using the reported wavelength values for excitation and emission maxima [4(3) was combined with PI, we observed strong interference of PI towards DiBAC4(3) (4(3) + PI in our fluorescence assay. On the other hand, the fluorescence of diSC3(5) was not affected by PI  A,B. Thised by PI C,D and ved by PI E,F, thusBacillus brevis [S. aureus and S. epidermidis, which are the principal target of the present study. These two peptides were thus selected for further experiments.The next step was to find the antimicrobial peptide (AMPs) candidates as positive controls for membrane depolarization and/or membrane permeabilization events. Based on scientific literature, four membrane-active AMPs were selected: gramicidin D, cecropin A, magainin 2, and melittin. The insect peptide cecropin A , the fros brevis ,59. It is brevis . The pris brevis . The ant3(5) or PI, the fluorescence of both probes combined together was monitored kinetically in the presence of each AMP. Two widely used uncouplers, i.e., carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), to be possibly used as controls of complete depolarization, were also included in these experiments. As shown in 3(5) fluorescence, in line with the report of te Winkel et al. [3(5) fluorescence precluded their use in this assay.To verify whether the selected peptides could affect the fluorescence of diSCl et al. , whereasS. epidermidis ATCC 35984 or S. aureus ATCC 25923 were incubated with the ion channel-forming gramicidin D, or with the pore-forming melittin, in PBS-glc containing 400 nM diSC3(5) and 5 µg/mL PI at 37 °C in 96-well black microtiter plates . Fluorescence was measured throughout the assay and, at 30 min incubation, aliquots were taken to determine bacterial viability by colony forming units (CFU) counts.To monitor the phenomena of membrane depolarization and membrane permeabilization simultaneously, Staphylococcus spp. gramicidin D induced a significant increase of diSC3(5) (3(5) fluorescence kinetics (3(5) and PI per se were not toxic to bacteria because in the absence of gramicidin D neither of the two probes showed an increase of fluorescence (diSC3(5) A,D, but diSC3(5) B,E. The diSC3(5) C,F), whikinetics A,C,D,F, kinetics B,E. In arescence A,B,D,E, 3(5) fluorescence increased only at bactericidal melittin concentrations (0.25–1 μM) (The results obtained with melittin gave a completely different picture . This po25–1 μM) A,D.To investigate whether the distinct membrane effects induced by melittin and gramicidin resulted in different morphology of treated bacteria, at the end of fluorescence kinetics  bacteria were processed for analysis by Field Emission Scanning Electron Microscopy (FE-SEM). The micrographs revealed clear differences in bacterial morphology and an evident decrease in bacterial density in treated samples compared to untreated controls, in line with the CFU determinations performed in parallel. Images of untreated bacteria A,B show On the contrary, images of peptide-treated samples show, besides cells with normal appearance, bacteria with remarkably altered morphology. Upon treatment with gramicidin, several cells presented indents, similar to what observed by Hartmann et al. with gramicidin S , or lookS. aureus and S. epidermidis. For the simultaneous monitoring of membrane depolarization and membrane permeabilization, we combined the membrane-impermeable nucleic acid stain PI with a potential-sensitive dye. We wanted to understand whether these two phenomena were clearly distinct or could be causally and temporally correlated, as suggested by other authors in the case of synthetic AMPs acting on Pseudomonas aeruginosa [The bacterial cytoplasmic membrane represents a valid drug target for the development of novel antimicrobial agents. The interactions of membrane-active AMPs with target membranes have been extensively studied in artificial systems, usually by using a relatively high peptide-to-lipid ratio. However, knowing the complexity of living microbial membranes, there is a need for a deeper characterization of AMP-induced effects on membranes of live bacteria. Therefore, in the present study, our goal was to set up a fluorescence assay for real-time monitoring of changes in membrane permeability induced by active peptide concentrations in whole bacteria. Specifically, we focused on Gram-positive microorganisms of medical relevance such as ruginosa . Thereforuginosa ,64.S. aureus and S. epidermidis collected at their mid-log phase, washed and resuspended in physiological salt solution supplemented with 25 mM glucose as a carbon and energy source [To investigate the complex phenomena occurring at the membrane level and related to membrane integrity, bacteria should be in an optimal growth phase with fully activated oxidative metabolism. Experiments were performed with y source . Given t3(5) fluorescence in conventional and low-binding black microtiter plates).At difference with several published methods based on flow cytometry ,66,67, o4(3), at difference with the report of Clementi et al. [3(5), which has clearly distinct fluorescence parameters and did not show any interference with PI. By using the combination diSC3(5) + PI we were able to simultaneously monitor the kinetics of membrane depolarization and membrane permeabilization induced by well-characterized membrane-targeting AMPs at bactericidal and sub-inhibitory concentrations. It is important to note, however, that PI fluorescence could only be detected with a sufficiently high bacterial density. In the case of the Gram-positive S. epidermidis and S. aureus, this corresponds to 108 CFU/mL. We found that for the detection of both dyes together this suspension density was optimal, although a slightly lower density, i.e., 107 CFU/mL, would be sufficient for the detection of diSC3(5) only  fluorescence reached the same level, indicating complete membrane depolarization also at lower gramicidin concentrations. In this latter case, the delay could be due to an initial compensation by the microorganism that could not be pursued over a certain point resulting in a delayed, yet lethal effect. However, PI fluorescence did increase in any of the cases, even at the highest peptide concentrations. It is important to note in this regard that the diameter of gramicidin D-induced channels is estimated to measure about ≈4 Å, what is sufficient to accommodate the passage of monovalent cations [S. epidermidis and S. aureus by membrane depolarization without formation of larger pores that could enable PI uptake. As confirmed by CFU counts, the membrane depolarization induced by gramicidin D was the bactericidal event. Hence, any increase in PI fluorescence, recorded after several hours of incubation, as reported by some authors [Concerning gramicidin D, our data are in line with those reported in the literature which reveals that gramicidin D causes a rapid and full dissipation of membrane potential by forming small cation specific channels ,59,70. I cations , but not authors , should 3(5) fluorescence did not indicate a mode of action primarily based on membrane depolarization but was rather a consequence of irreversible membrane permeabilization, which appears as the key event. Curiously, by FE-SEM analysis we rarely noticed macroscopic membrane lesions. Instead, the appearance of the deflated or emptied bag would suggest that the emptying of cell content occurred due to osmotic imbalance or, alternatively, that membrane perturbation elicited an autolytic action. Melittin has long been considered as a paradigmatic pore-forming peptide [3(5) fluorescence  only, we would conclude that both peptides, gramicidin D and melittin, act by causing membrane depolarization. On the contrary, if we had used only PI, we would speculate that gramicidin D, at the difference with melittin, did not cause membrane permeabilization and, perhaps killed bacteria by acting on some internal target(s).The findings obtained thanks to the combination of both fluorescent dyes contribute to the advancement of our knowledge concerning the mode of action of gramicidin D and melittin. If for instance the assay would have been performed by using diSC3(5) and PI, we measured the phenomena of membrane depolarization and permeabilization on the same bacteria population simultaneously. This assay thus enables us to distinguish between these two phenomena and contributes to shedding light on the mode of action of membrane-active agents such as the AMPs. This study confirmed that gramicidin D causes membrane depolarization and melittin induces membrane permeabilization. We also provide additional evidence that melittin at low concentrations probably creates transient pores causing membrane permeabilization which is reversible to some extent.Conversely, by using the combination of diSCS. aureus and S. epidermidis.We believe that this assay could be applied in investigations aimed at unraveling the mode of action of membrane-active AMPs, as well as for screening of novel compounds targeting the cytoplasmic membrane of Fluorometric measurements were performed with a Multimode Plate Reader  by using low-binding surface 96-black polystyrene microtiter plates  to prevent unspecific binding of the molecules used in the study.LAVVVWLWLWLWG-NHCH2CH2OH), cecropin A (KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH2), magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS-OH), and melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) were purchased from Sigma-Aldrich® . Cecropin A and gramicidin D were dissolved in DMSO and magainin 2 and melittin in pyrogenic water. The stock solutions were kept at −20 °C. The uncouplers carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the membrane potential-sensitive fluorescent distributional probes bis-trimethine oxonol (DiBAC4(3)) and 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)), and the membrane-impermeable fluorescent dye propidium iodide (PI) were purchased from Sigma-Aldrich. Stock solutions were prepared as follows: 400 µM diSC3(5) in 100% DMSO, 50 µM DiBAC4(3) in 100% DMSO, and 1 mg/mL PI in ddH2O (PI would precipitate in a more concentrated solution). All stocks, were protected from light by aluminum foil and were stable at −20 °C for at least 6 months.The selected membrane-active peptides gramicidin D . Bacteria were maintained on Mueller–Hinton  agar plates. For antimicrobial assays, bacteria were cultured in liquid Brain Heart Infusion (BHI) overnight, 1:50-diluted in fresh medium and allowed to grow in an orbital shaker at 37 °C. Mid-log phase bacteria were harvested after 10 min centrifugation at 1000× g and resuspended in phosphate-buffered solution (PBS) to optimal density assessed by turbidity at 600 nm, with reference to previously determined standards. For fluorescence kinetics, mid-log phase bacteria were collected by centrifugation at 1000× g, washed two times with PBS (pH 7.4), and finally resuspended in PBS supplemented with 25 mM glucose (PBS-glc) at the desired density.Two Gram-positive, 5 CFU/mL, as previously reported [The minimum inhibitory concentration (MIC) of selected peptides was determined by a broth microdilution assay in 96-well microtiter plates, using MH broth with logarithmic-phase microorganisms at 5 × 10reported , followi4(3) was studied at 125, 250, and 500 nM  [ex = 490 nm, λem = 617 nm) [3(5) alone  [3(5) plus 5, 10, and 20 µg/mL PI, were measured in PBS-glc.To verify the correct excitation and emission wavelengths of each fluorescent dye, their excitation and emission spectra were measured in our assay conditions. DiBAC 516 nm)  plus PI  617 nm)  in PBS s 672 nm) , and 4003(5) or with PI, both fluorescent dyes (400 nM diSC3(5) and 5 µg/mL PI) combined together in the same wells of a black 96-well plate (Optiplate) were monitored kinetically for 30 minutes at 37 °C in PBS-glc in the presence of each selected molecule.To verify whether uncouplers, peptides, and solvents have any interference with diSCS. epidermidis and S. aureus, resuspended at 1 × 108 CFU/mL in PBS-glc (prepared as described above) were incubated in the orbital shaker at 37 °C for 15 min. Thereafter, diSC3(5) and PI were added at final concentrations of 400 nM and 5 µg/mL, respectively. These concentrations were established in preliminary experiments and were optimal in our conditions (data not shown). The suspension was mixed by short vortexing and 200 μL was added to the wells of a black 96-well plate (Optiplate). Samples were preincubated at 37 °C with fluorescence measurements every minute for 5 min, or until readings were stabilized. After this time, the plate was ejected. Depolarizing agents were added in duplicate wells at the desired final concentrations. The plate was rapidly placed back into the reader to continue monitoring diSC3(5) and PI (diSC3(5); λex = 652, λem = 672 nm and PI; λex = 535, λem = 617 nm) [Mid-log phase  617 nm) , every 0S. epidermidis, deposited on polycarbonate filters, were studied by Field Emission Scanning Electron Microscopy (FE-SEM) (JEOL model JSM-7610FPlus). Briefly, upon 30 min incubation, as described above, all samples were collected by centrifugation at 1000× g for 10 min and fixed with 2.5% (v/v) glutaraldehyde in PBS for 1 h at 4 °C. Fixed bacteria were extensively rinsed with filtered sterile PBS and dehydrated in graded series of ethanol solutions (20 min each) by centrifuging at each step. Finally, samples were deposited on 0.2 µm Isopore polycarbonate membrane filters  and sputter-coated with a thin gold layer prior to FE-SEM analysis. The images were collected in the secondary electron detection mode. The working distance was set to 8 mm, the acceleration voltage to 5 keV, and the probe current to 11 to decrease the interaction depth and obtain more detailed information on the surface. FE-SEM was performed in duplicate for each sample.The morphology of


N-acetyl-l-cysteine (NAC), a ROS scavenger was used to verify the role of ROS in NEFA induced apoptosis of GCs. NAC pretreatment reversed the NEFA-induced ERS-related protein and apoptosis-related protein levels. Meanwhile, NAC pretreatment also blocked the phosphorylation of ERK1/2 and p38 induced by NEFA, and the nucleation of Nrf2 and p53, suggesting that ROS plays a crucial role in regulating the NEFA-induced apoptosis of GCs. Together, these findings provide an improved understanding of the mechanisms underlying GCs apoptosis, which could potentially be useful for improving ovarian function.Negative energy balance (NEB) during the perinatal period can affect dairy cow follicular development and reduce the fecundity. Non-esterified fatty acid (NEFA) concentration is elevated during NEB, and is known to be toxic for multiple cell types. In the ovary, NEB increased NEFA, and may influences follicular growth and development. However, the effect and mechanism of NEFA on granulosa cells (GCs) in vitro remains unknown. In this study, we found that NEFA dose-dependently induced apoptosis in primary cultured granulosa cells. Mechanistically, our data showed that NEFA significantly increased reactive oxygen species (ROS) levels, resulting in the activation of endoplasmic reticulum stress (ERS) and eventually cell apoptosis in GCs. Moreover, NEFA also increased the phosphorylation levels of ERK1/2 and p38MAPK pathways, upregulated the expression of p53 and potentially promoted its translocation to the nuclear, thus transcriptionally activated Bax, a downstream gene of this pathway. NEFA also promoted nuclear factor E2 (Nrf2) expression and its level in the nuclear. To elucidate the mechanism of NEFA action,  Due to the integrative outcome of reduced intake and higher demand for maintenance and production, cows in the perinatal period are in the state of negative energy balance (NEB). Non-esterified fatty acid (NEFA) is an important energy metabolite that is used as an indicator of NEB during the perinatal period. The elevation of NEFA is known to result in decreased reproductive potential, especially in high-yielding dairy cows . It was Lipid mobilization is usually accompanied by oxidative stress. The high concentration of NEFA can increase the intracellular production of reactive oxygen species (ROS) and RNS, thus initiating the mechanism of oxidative stress . At the Dulbecco’s modified eagle medium (DMEM)/F-12 medium and fetal bovine serum (FBS) were obtained from Gibco Life Technologies . Oleic acid, linoleic acid, palmitic acid, palmitoleic acid, and stearic acid with 99% purity were from Sigma-Aldrich . N-acetyl cysteine (NAC), RIPA lysate, PMSF, and BCA assay were from Beyotime Biotechnology . Anti-ERK1/2, anti-pERK1/2, and anti-α-Tubulin were from Cell Signaling Technology . Anti-Nrf2, anti-HO-1, anti-Bax, -Bcl-2, -cleaved caspase-3, -SOD2, -Histone, -CHOP, -GRP78, -p-PERK, -GAPDH antibodies were purchased from Proteintech . The anti-p53 antibody was from Abcam . The secondary antibody and ECL were from Biosharp . Annexin V- Alexa Fluor 647/PI double-staining kit was purchased from Fumaisi . The concentration of stock NEFA solution was 50 mM, containing 21.75 mM oleic acid, 15.95 mM palmitic acid, 7.2 mM stearic acid, 2.65 mM palmitoleic acid, and 2.45 mM linoleic acid.2.The study was approved by the animal protection and utilization committee  2017-0027), Nanjing Agricultural University, Nanjing, China. Briefly, bovine ovaries obtained from slaughterhouses  were placed in saline at about 37 °C and returned to the laboratory within 2 h. After washing the ovaries with saline, we used a syringe to extract follicular fluid into saline solution in a sterile environment, centrifuged and suspended cells in complete medium. Subsequently, granulosa cells (GCs) were cultured in DMEM/F-12 medium supplemented with 10% FBS, at 37 °C in a humidified atmosphere of 95% air and 5% COg for 5 min at 4 °C, the supernatant was aspirated as cytoplasmic protein. Next, 50 μL nuclear extraction buffer was added to the sediment. In the next 30 min, vortexing for 30 s every 2 min on the ice took place, then the lysates were centrifuged at 16,000× g for 10 min at 4 °C, the supernatant was aspirated as a nuclear protein.The Nuclear and Cytoplasm Protein Extraction Kit  was used to extra the nuclear and cytoplasm proteins. Briefly, the cells were collected and 200 μL ice-cold cytoplasm extraction buffer A with 1 mM PMSF was added. After incubation with cytoplasm extraction buffer B for 1 min, cell lysates were centrifuged at 12,000× According to the instructions for Pierce Co-Immunoprecipitation Kit , the cells cultured in T75 culture bottle were used for immunoprecipitation. For immunoblots, after incubation with NAC and NEFA, the cells were harvested and lysed with RIPA buffer. The protein content was measured by a BCA assay (Beyotime). Equal amounts of protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to PVDF membranes. Then, 5% skimmed milk was used to block the membranes, the primary antibodies were incubated at 4 °C overnight. The next day, washing membranes three times in TBST for each 10 min and then incubated with the corresponding secondary antibodies for 1 h at room temperature. Antibody detection was accomplished using enhanced chemiluminescence reagent. Densitometry analysis was detected using ImageJ software .6/mL). After 15 min in the dark, the apoptosis rates were analyzed by flow cytometry . The data analysis was using Flowjo software .The cells Annexin V-Alexa Fluor 647/PI Apoptosis Assay Kit was used to measure the extent of apoptosis according to the manufacturer’s instructions. Briefly, cells were washed twice with PBS then gently resuspended in binding buffer containing Annexin V/Alexa Fluor 647 (5 uL) and propidium iodide (10 uL) (the cell concentration is 1 × 10The cells were seeded on six-well plates with cover glasses in each well and then treated with NAC for 2 h before treated with NEFA for 24 h. The intracellular levels of ROS were measured by loading the cells with the Dihydroethidium (DHE)  ,20. The All data were expressed as mean ± SEM and the results at least three independent experiments. T-test was used to analyze statistical, using the GraphPad Prism5 software . P less than 0.05 was considered significant.To determine the correlation between NEFA and the accumulation of ROS in vitro, granulosa cells were cultured in the absence and presence of 0, 0.6, 1.2, 1.8 mM NEFA for 24 h, and DHE staining was performed. The imaging data showed that ROS signals were upregulated NEFA concentration-dependent manner A. At theThe incidence of apoptosis was increased in NEFA treated GCs detected via Annexin V-Alexa Fluor 647 staining and flow cytometry A,B. In tTo determine whether supplementation of NAC can decrease ROS, GCs were cultured in the absence and presence of NEFA, and in addition to minor with NAC (5 mM) for 2 h, the levels of the ROS were assessed. As to be expected, immunostaining revealed that NAC substantially reduced the NEFA induced ROS signals A. To furThe occurrences of apoptosis in the NAC-treated GCs were also significantly decreased as compared with those in the NEFA groups A,E. ConsERK1/2 and p38 are important signaling pathways in mitogen-activated protein kinases, which regulate cell proliferation and apoptosis. To investigate the role of these pathways in NEFA induced apoptosis mediated by ROS, cells were treated with NEFA, and Western blots were performed. As shown in Nrf2 is an important transcription factor in the regulation of oxidative stress and a central regulator in the maintenance of intracellular redox homeostasis. Heme oxygenase-1 (HO-1) is thought to be a major antioxidant enzyme scavenging cellular ROS , and itsThe tumor suppressor, p53 was an important transcription factor in response to a diverse number of cellular stress including cell death, oxidative stress, and hypoxia . We nextRecent studies had also shown that when p53 is stimulated, it not only activates cell apoptosis but also interacts with Bcl-2 family members in mitochondria ,23. SincRecent studies have demonstrated that the follicular fluid total NEFA, ammonia, and urea exceeding the physiological concentration are considered as nutrition and metabolic stressors. Refs. ,25, whicThe apoptosis response of ovarian granulosa cells to NEFA is concentration-dependent. Some studies showed that oxidative stress and endoplasmic reticulum stress are two processes that cause pathological cell death ,28. Our Mitogen-activated protein kinases (MAPKs) are important signaling pathways in eukaryotic cell biology, which are involved in the regulation of cell proliferation, differentiation, and apoptosis. ERK1/2 and p38 are members of the MAPK family, their roles in regulating cell survival and other actives were well documented ,30,31. IThe signal transduction process is a very complex network. ROS induced by NEFA can activate ERK1/2 and p38 signal pathways in ovarian granulosa cells, but there is still a lack of clear understanding of the specific process of signal transduction and which upstream and downstream signal molecules are involved. Many studies have shown that the MAPK signaling pathway can activate Nrf2 ,34,35. NIn summary, our study provides further insights on the molecular mechanisms of NEFA-induced apoptosis of GCs. We found that NEFA induced apoptosis and stimulated an increase in ROS production in a dose-dependent manner in GCs. The accumulation of ROS induced by NEFA results in the up-regulation of ERS related proteins such as p-PERK, GRP78, and CHOP. Importantly, the blockage of ROS production by NAC has significantly reversed NEFA-induced ROS, ERS, and cell apoptosis. NEFA also activated ERK1/2 and p38 pathways, and the effects of NEFA were reversed by pretreatment with NAC. In addition, NAC can also partially reverse NEFA-induced Nrf2 and p53 nucleation and the increased expression of downstream genes HO-1 and Bax to regulate cell apoptosis. Based on our findings, a model of how NEFA induces apoptosis through ROS mediated signaling pathway is proposed . The res


Cardiovascular disease (CVD) prevalence, pathogenesis, and manifestation is differentially influenced by biological sex. Berry polyphenols target several signaling pathways pertinent to CVD development, including inflammation, oxidative stress, and cardiac and vascular remodeling, and there are innate differences in these pathways that also vary by sex. There is limited research systematically investigating sex differences in berry polyphenol effects on these pathways, but there are fundamental findings at this time that suggest a sex-specific effect. This review will detail mechanisms within these pathological pathways, how they differ by sex, and how they may be individually targeted by berry polyphenols in a sex-specific manner. Because of the substantial polyphenolic profile of berries, berry consumption represents a promising interventional tool in the treatment and prevention of CVD in both sexes, but the mechanisms in which they function within each sex may vary. There are sex differences in the prevalence and pathogenesis of cardiovascular diseases (CVD). These differences are likely related to endogenous estrogen effects, as epidemiological data suggest the menopausal transition, which occurs at approximately 50 years of age, is associated with significant changes in cardiovascular health and CVD diagnoses in females. For example, between ages 20–34 and ages 35–44, 13% and 32% of females are diagnosed with hypertension compared to 26% and 43% of males, respectively; however, 65% of females and 66% of males are diagnosed with hypertension between ages 55–64, and diagnoses in females exceed that of age-matched males in higher age brackets [Inflammation, oxidative stress, and pathological remodeling of the cardiovascular system are consistent across a number of CVD-related pathologies and are major drivers of disease ,8,9. BioPolyphenol intake is greater in females compared to males, especially polyphenols from fruits and vegetables . In a loPolyphenols are subdivided into four major classes: flavonoids, phenolic acids, lignans, and stilbenes. In berries, lignans are relatively absent  and stilPhenolic acids are the most diverse class of polyphenols in berries and are derived in a number of ways. While phenolic acids can be unbound and free in the fruit, they can be derived from (1) metabolic and microbial degradation of flavonoids and (2) hydrolytic liberation of phenolic acid polymers  during digestion . PhenoliPolyphenol parent compounds are rarely detected in serum following consumption ,30, as tPolyphenols almost certainly exert their effects intracellularly due to changes in gene and protein expression (discussed below). However, berry-derived polyphenols are typically hydrophilic due to numerous hydroxyl groups. As such, crossing lipid bilayers poses a challenge. It has been observed that as hydroxyl group count decreases, hydrophobicity for membrane vesicles inversely increases . PolypheBacteriodetes  versus Firmicutes (tend to unfavorable) in both males and females [Prevotella, while switching to a low-fiber animal-based diet reduced Prevotella within four days [Prevotella corresponded with increased short-chain fatty acid synthesis, potent anti-inflammatory compounds which can attenuate CVD pathogenesis [The gut microbiota is associated with cardiovascular function and disease , 30 mg/kg/d of quercetin for six weeks significantly decreased the Firmicutes/Bacteroidetes ratio, which corresponded with increased intestinal short-chain fatty acid production of acetate, propionate, and butyrate [Lactobacillus and Bifidobacterium microbes compared to control animals [Faecalibacterium, Butyricicoccus, and Butyricimonas, representing a positive microbial shift [Lactobacillus and Bifidobacterium populations, while Clostridium significantly decreased [Firmicutes/Bacteroidetes ratio and increased Faecalibacterium, Lactobacillus, and Bifidobacterium [Indeed, a number of berry polyphenols can positively improve gut microbiota diversity in animal models. For example, in male C57BL/6J mice fed a high-fat diet, supplementation of 200 mg/kg/d of blueberry extract for twelve weeks resulted in the reduction of  animals . Peculiabutyrate . In male animals , and the animals ,67. Posial shift . Additioecreased . In a seacterium . Lastly,acterium . ConsideThere are physiological differences between males and females that impact many processes, including CVD pathogenesis, in a sex-specific manner . Althougn = 23), premenopausal females , and males (n = 18) [There appears to be nearly equivalent ER concentration across the cardiovascular system of male and female rats  and in m(n = 18) . Serum p(n = 18) . This coSex-specific polyphenolic action is likely related to ER mediation, possibly due to dynamic competitive binding interactions with E2, though there is evidence of beneficial effects of polyphenols that are ER-independent (discussed in detail below). Non-isoflavone polyphenols, such as ellagic acid, kaempferol, myricetin, epicatechin, and quercetin, may act as phytoestrogens, as they can act as ER ligands. However, it is unclear how ER concentration between sexes may affect polyphenolic actions in humans. Due to the cardio-protective effects of ER, it is possible that polyphenols may be of particular importance in the prevention of CVD in males who tend to be at greater risk. Polyphenol binding to ER occurs due to the structure of the ER binding pocket, in which there is a requirement for a phenolic ring to facilitate binding . The hydIn the context of CVD, berry polyphenols target a variety of cellular pathways that drive these disease processes forward , includiIn CVD, inflammation is a major driver of atherosclerosis, endothelial dysfunction, hypertension, and HF. In particular, activation of toll-like receptor (TLR)-4 and tumor necrosis factor receptor (TNFR) and their downstream signaling of mitogen activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are primarily responsible for these deleterious effects ,92,93,94TLR-4 is found ubiquitously in the cardiovascular system and has a number of ligands. While classically activated by exogenous lipopolysaccharide (LPS), including low concentrations of LPS due to a high-fat meal , a varie1R). Ang II may interact with glycoprotein MD2 on the TLR-4 receptor independent of AT1R, leading to its activation [1R, and reduced angiotensin converting enzyme (ACE) [While TLR-4 deficient animals are protected from atherosclerosis and HF ,110, thetivation . While Ttivation . It appetivation . Inhibitme (ACE) . Howeverme (ACE) . ACE2 came (ACE) ,115.Tumor necrosis factor (TNF)-α is an inflammatory cytokine, which is elevated in those with CVD . TNF-α cl-arginine, a co-substrate of eNOS [In addition to the detrimental effects of inflammation, TNF-α has direct effects on endothelial nitric oxide synthase (eNOS), a nitric oxide (NO) producing enzyme critical to vasodilation and vascular health. TNF-α decreases eNOS mRNA in endothelial cells in a time-dependent manner , as well of eNOS . NF-κB s of eNOS .n = 47) and females (n = 17) with at least one CVD risk factor . They consumed water or 330 mL of bilberry juice for four weeks, and bilberry juice consumption resulted in a reduction of serum IL-6 and C-reactive protein (CRP), but not TNF-α, all of which are NF-κB transcriptional products [hs-CRP), in a separate investigation of females with metabolic syndrome [hs-CRP [Clinically, berries tend to elicit anti-inflammatory effects. For example, Karlsen et al.  studied products . Howeverproducts . This suproducts  were mosproducts , which fsyndrome . Howeversyndrome . Strawbesyndrome . Bilberrsyndrome , while ssyndrome ; these dsyndrome . Followisyndrome . In 72 d [hs-CRP . Further [hs-CRP . FurtherBerries likely exert their anti-inflammatory effects by acting as regulators of TLR-4 and TNFR signaling at multiple protein targets. For example, in a pilot study conducted by Nair et al. , nine maThese findings highlight the multiple targets of polyphenols in inflammatory signaling, and the protective effects of polyphenols in macrophages are replicated in models of the cardiovascular system. For example, in human umbilical vein endothelial cells (HUVECs), pre-treatment with 25 μM quercetin for 1 h followed by 50 μM oxidized LDL, a TLR-4 ligand, for 24 h significantly reduced TLR-4 protein expression compared to oxidized LDL treatment alone and also decreased p65 nuclear translocation . ResveraThis theme is replicated in several in vitro models, as berry polyphenols reduce p65 nuclear translocation induced by Ang II  and TNF-2−) and hydrogen peroxide (H2O2), which contribute significantly to pathological processes in the cardiovascular system, including endothelial dysfunction, hypertension, atherosclerosis, and HF [Similar to inflammation, oxidative stress is a characteristic feature of CVD pathogenesis. A balance between ROS production and antioxidant enzyme activity is necessary to maintain normal cellular function. Oxidative stress occurs when the formation of ROS surpasses antioxidant capacity, facilitating NF-κB and MAPK signaling due to upstream redox-sensitive IKK and MEK activation, respectively . Major s, and HF ,166,167., and HF . In gene, and HF , central, and HF ,171 unde, and HF , demonst2− generation and, in the case of Nox4, H2O2 generation [2− cannot freely pass between lipid bilayers, H2O2 is able to do so [2− to H2O2. Thus, cellular localization of each Nox determines whether O2− or H2O2 will be the predominant end-product. A variety of stimuli can lead to Nox transcription and over-expression, including changes in shear stress [Oxidative enzymes, such as Nox and XO, are major producers of ROS within the cardiovascular system. Nox1, Nox2, Nox4, and Nox5 are the dominant Nox isoforms in the cardiovascular system, in which all isoforms are present ubiquitously in endothelial cells, VSMCs, and cardiomyocytes  at diffeneration . Nox is to do so . The micr stress , Ang II r stress , endother stress , TNF-α [r stress , and α-ar stress . In cerer stress . Howeverr stress ,182,183,2− or H2O2. XO is nearly absent from the human heart [2− and peroxynitrite (ONOO−) generation and upregulate XO activity through AR-dependent mechanisms [In urea metabolism, nucleotide-derived purines are converted to hypoxanthine (derived from adenosine) and then oxidized by XO to xanthine (derived from guanosine) . XO can an heart ; howeveran heart . Males wan heart . Interesan heart . Despitean heart , testostchanisms . Upregulchanisms , and crochanisms . Collectchanisms ,196,197,2− instead of NO [l-arginine. This can occur when there is reduced BH4 synthesis [l-arginine availability can be reduced via diminished argininosuccinate synthase activity [l-arginine to l-ornithine and urea in the urea cycle [NOS isoforms can also contribute to oxidative stress. NOS produce NO, which is largely cardioprotective when produced constitutively by the endothelium through eNOS ,199. Endad of NO . eNOS unynthesis  or when ynthesis . Furtheractivity ,207,208,activity ,209, or activity . Argininea cycle ,209, andea cycle .2− production, potentially by affecting l-arginine and BH4 availability [l-arginine deficiency and increased markers of l-arginine metabolism [The production of NO and eNOS coupling are known to be affected by E2. The binding of E2 to ERα increases stimulatory phosphorylation sites Ser1177 and Ser635 on eNOS, and E2 binding to ERα or ERβ reduces inhibitory phosphorylation site Thr495 on eNOS , which alability ,214. In tabolism , and FMD− [− oxidize numerous molecules, including sGC. sGC oxidation increases VSMC intracellular Ca2+ flux and depletes BH4, which consequently results in endothelial dysfunction [− can oxidize low density lipoprotein, which is linked to atherogenic processes, including foam cell formation and leukocyte adherence to endothelial cells [−10 M and 10−8 M showed less iNOS protein compared to those treated with 10−12 M testosterone, but iNOS protein treated with 10−6 M testosterone was similar to that of 10−12 M [− generation.When NO is overproduced, such as through activation of inducible NOS (iNOS), it can directly contribute to oxidative stress. iNOS is activated through MAPK and NF-κB signaling under pathological conditions ,216 and − ,219. Noxfunction . In addial cells . iNOS maal cells . Testostal cells . Macroph 10−12 M . This in2− into H2O2 and O2 [2O2 to form H2O and O2. SOD, GPx, and catalase activity are all inversely associated with CVD risk [2− and H2O2, likely related to estrogenic increases in SOD expression [Antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, also contribute to the redox balance by dissociating ROS. SOD1 (a cytoplasmic enzyme) and SOD2  catalyze the dismutation of O2 and O2 . GPx andCVD risk ,225,226.CVD risk . Both SOCVD risk ,229. FurCVD risk ,230. EstCVD risk . Estrogepression ,232 and pression . At thispression ,232. Suppression , but tespression .2+), and biliverdin [2− to H2O2 [Other components of the antioxidant system include heme oxygenase (HO)-1 and NQO1. HO-1 catalyzes free heme, a pro-oxidant  and majoliverdin . CO itseliverdin  and actsliverdin . There iliverdin , but sex to H2O2 . Further to H2O2 , and hum to H2O2 . The pri to H2O2  and Sirt to H2O2 .2O2, reactive cysteine residues on KEAP1 undergo reduction, leading to conformational shifts in Keap1, preventing Nrf2 binding [Nrf2 is continually synthesized, but it is sequestered in the cytosol by the enzyme Keap1, which undergoes continuous ubiquitination and degradation when the Keap1-Nrf2 complex is formed. Under conditions of oxidative stress, such as excessive H binding . As such binding . Because binding . Under c binding . Thus, u binding . For exa binding . ConversIn addition to Nrf2, Sirt1, a redox sensitive deacetylase, has multiple cellular effects, including antioxidant, anti-inflammatory, and anti-apoptotic effects. A direct transcriptional product of Sirt1 activity is SOD2, and Sirt1 increases eNOS, Nrf2, GPx, and catalase expression and inhibits NF-κB activity and apoptotic transcription factor p53 . In animBerries appear efficacious in mediating antioxidant status in humans . In a nu2− compared to placebo. Further, in pre- and stage 1-hypertensive postmenopausal females, 50 g/d of freeze-dried strawberry for eight weeks significantly increased NO metabolites compared to baseline, although these did not correspond with changes in blood pressure at this dose [Berry polyphenols tend to reduce vascular oxidative stress in a number of chronic studies. For example, in an eight-week trial, 48 postmenopausal females with pre- and stage 1-hypertension were assigned to consume 22 g/d of freeze-dried blueberry powder or placebo . Serum chis dose .Sex differences in the efficacy of berry polyphenols likely exist with regard to reductions in oxidative stress. For example, 26 hyperlipidemic males and females consumed 10 g of freeze-dried strawberry for six weeks . OxidizeBerry polyphenols exert cellular antioxidant effects, not merely due to exogenous ROS scavenging, but also by exerting cytoprotective effects, as polyphenols act as effectors for multiple proteins. For example, under basal conditions, quercetin increases Sirt1 mRNA in a dose-dependent manner in HUVECs . AdditioFor example, p62, a scaffolding protein involved in a variety of cellular processes, such as autophagy, can directly bind to the binding pocket of Keap1 that associates with Nrf2, and thus prevent Nrf2 from binding to Keap1 . In an I2− was significantly reduced and cGMP was significantly increased due to anthocyanin supplementation [1R expression, compared to control SHRs [l-NG-monomethyl-arginine [Berry polyphenols may also improve endothelial function mediated by reduced oxidative stress. In endothelial cells, exposure to the anthocyanin cyanidin-3-glucoside  for 2 h followed by 1 μM Ang II for 24 h resulted in a significant attenuation of ROS, reduction of iNOS, and increase in nuclear Nrf2 translocation, which coincided with increased HO-1 and SOD in a dose-dependent manner compared to Ang II alone . These pentation . The antentation . Preserventation . In malerol SHRs . Additiorol SHRs . After targinine .A number of berries have clinically demonstrated improved vascular function in humans. In healthy males, the consumption of wild blueberries increased postprandial FMD . InteresAlthough sex differences in vascular function in response to berry polyphenols have not been fully elucidated, it is likely that sex differences exist. For example, red wine polyphenols  induced endothelium-dependent vasodilation in aortas from female and male rats, but relaxation was more pronounced in female aortic rings than those from males . This ef2O2, myricetin, and Nrf2 siRNA as indicated by increased 4-hydroxynonenal expression, a metabolite of lipid peroxidation, compared to control. It is likely that myricetin prevented MAPK and NF-κB activation at multiple upstream sites, namely reduced TRAF6 degradation, preventing TAK1 activation. Nonetheless, these results do not preclude oxidative modification of IKK and MEK, suggesting that this polyphenol may also inhibit these enzymes independent of ROS. Further experimental details for some of the investigations cited in this section are displayed in While ROS can independently mediate inflammatory signaling (MAPKs and NF-κB), such as by modifying redox sensitive IKK and MEK , polypheCardiac and vessel architecture and plasticity largely reflect function; thus, pathological remodeling of the vasculature can result in CVDs, including HF, hypertension, and atherosclerosis ,293,294.Apoptosis involves either the extrinsic pathway due to cytokine signaling  or the intrinsic pathway due to intracellular damage, such as DNA or mitochondrial damage . Under pThere are marked sex differences in cell survival and cell death, where males show significantly more age-related  and HF-rIn an inflammatory state, the endothelium and heart release chemoattractant molecules, which guide leukocytes, such as monocytes and neutrophils, to infiltrate these tissues ,334. MonERs and ARs sex-specifically regulate fibrosis, collagen and MMP synthesis . Sex dif2O2 for 2 h [2O2-induced apoptosis, although their combination was more effective. Berry polyphenols likely target p53, upstream from Bax, to reduce apoptosis. For example, in rat VSMCs (sex unspecified), 200 μg/mL of blackberry, raspberry, and black raspberry polyphenolic extract for 24 h followed by 100 nM of Ang II for 72 h resulted in a significant reduction in p53 protein expression, O2− and H2O2 production, and p38MAPK and ERK1/2 phosphorylation [2O2 displayed significant decreases in DNA fragmentation, Bax, and cleaved caspase-3 compared to treatment with H2O2 alone [2O2 decreased Bcl-2 and was restored with quercetin. In neonatal rat cardiomyocytes (donor sex unspecified), 3 h of anoxia (95% N2 and 5% CO2) significantly increased caspase-3 activity and apoptosis but was markedly reduced by resveratrol in a dose-dependent manner [Berries are likely efficacious in the reduction of CVD-associated pathologic remodeling. For example, in a 2% blueberry diet in male Fischer-344 rats, ischemic HF was induced by coronary artery ligation . The nec for 2 h . Single rylation . As suchO2 alone . Furthert manner . Thus, it manner . Additiot manner . This sut manner , but polt manner .1R blocker), 1 mg/kg/day carvedilol (β-blocker), or 3 mg/kg/day furosemide (3 mg/kg/day), for two weeks following confirmed HF resulted in a reduction of cardiac collagen I and α-smooth muscle actin to a large extent with gallic acid treatment but minimally with medications [In addition to apoptotic signaling, berry polyphenols also mediate fibrotic processes. For example, in male mice that underwent trans-aortic constriction to induce HF (pressure-overload model), 100 mg/kg/day of gallic acid or medications used in the treatment of HF, including 3 mg/kg/day of losartan  the effects of estrogen and estrogen-signaling in the human cardiovascular system and (2) the effects of berry-derived polyphenols and their cellular targets, which appear to mimic that of endogenous estrogen. Considering the increased risk of CVD in males versus females, increased berry consumption in males should be encouraged for cardio-protection. However, the complex nature of androgens and cardiovascular health remains incompletely understood, yielding this as an unresolved question. Continued research into the sex-specific effects of sex hormones, ERs, and ARs in cardiovascular health is warranted alone and in conjunction with polyphenol interventions. Regardless of a sex-specific effect, berry consumption, due to their rich polyphenolic profile, represents a promising interventional tool in the treatment and prevention of CVD.