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• Epigenetics, a rapidly expanding field of biology, involves the study of changes in gene expression that don't stem from changes in the DNA sequence, but rather from chemical modifications of DNA and its associated proteins. • Epigenetic mechanisms can significantly influence gene expression, cell differentiation, tissue development, and susceptibility to diseases. • Understanding epigenetic changes is crucial for elucidating the role of environmental and lifestyle factors in health and disease, as well as the intergenerational transmission of phenotypes. • Recent studies indicate that epigenetics may play a vital role in a wide range of diseases, including cardiovascular disease, cancer, neurodevelopmental disorders, and neurodegenerative disorders. • Epigenetic modifications are potentially reversible, offering promising new therapeutic approaches for treating these diseases using epigenetic modulators. • Epigenetics also offers insights into disease pathogenesis and potential biomarkers for disease diagnosis and risk stratification. • However, there are potential risks associated with epigenetic interventions, including unintended consequences like adverse drug reactions, developmental abnormalities, and cancer. • Rigorous studies are necessary to minimize the risks associated with epigenetic therapies and to develop safe and effective interventions to improve human health. • The article provides a synthetic and historical view of the origin of epigenetics and highlights some of the most relevant achievements in the field. Epigenetics is a rapidly growing field of biology that studies the changes in gene expression that are not due to alterations in the DNA sequence but rather the chemical modifications of DNA and its associated proteins. Epigenetic mechanisms can profoundly influence gene expression, cell differentiation, tissue development, and disease susceptibility. Understanding epigenetic changes is essential to elucidate the mechanisms underlying the increasingly recognized role of environmental and lifestyle factors in health and disease and the intergenerational transmission of phenotypes. Recent studies suggest epigenetics may be critical in various diseases, from cardiovascular disease and cancer to neurodevelopmental and neurodegenerative disorders. Epigenetic modifications are potentially reversible and could provide new therapeutic avenues for treating these diseases using epigenetic modulators. Moreover, epigenetics provide insight into disease pathogenesis and biomarkers for disease diagnosis and risk stratification. Nevertheless, epigenetic interventions have the potential for unintended consequences and may potentially lead to increased risks of unexpected outcomes, such as adverse drug reactions, developmental abnormalities, and cancer. Therefore, rigorous studies are essential to minimize the risks associated with epigenetic therapies and to develop safe and effective interventions for improving human health. This article provides a synthetic and historical view of the origin of epigenetics and some of the most relevant achievements. [ABSTRACT FROM AUTHOR]

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32°C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32°C, a significant reduction in the proliferation rate (approx. percnt;50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32°C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli. [ABSTRACT FROM AUTHOR]

Background: About 30% of Prostate cancer (PCa) patients progress to metastatic PCa that remains largely incurable. This evidence underlines the need for the development of innovative therapies. In this direction, the potential research focus might be on long non-coding RNAs (lncRNAs) like H19, which serve critical biological functions and show significant dysregulation in cancer. Previously, we showed a transcriptional down-regulation of H19 under combined pro-tumoral estrogen and hypoxia treatment in PCa cells that, in turn, induced both E-cadherin and β4 integrin expression. H19, indeed, acts as transcriptional repressor of cell adhesion molecules affecting the PCa metastatic properties. Here, we investigated the role of H19/cell adhesion molecules circuitry on in vivo PCa experimental tumor growth and metastatic dissemination models. Methods: H19 was silenced in luciferase-positive PC-3 and 22Rv1 cells and in vitro effect was evaluated by gene expression, proliferation and invasion assays before and after treatment with the histone lysine demethylase inhibitor, GSK-J4. In vivo tumor growth and metastasis dissemination, in the presence or absence of GSK-J4, were analyzed in two models of human tumor in immunodeficient mice by in vivo bioluminescent imaging and immunohistochemistry (IHC) on explanted tissues. Organotypic Slice Cultures (OSCs) from fresh PCa-explant were used as ex vivo model to test GSK-J4 effects. Results: H19 silencing in both PC-3 and 22Rv1 cells increased: i) E-cadherin and β4 integrin expression as well as proliferation and invasion, ii) in vivo tumor growth, and iii) metastasis formation at bone, lung, and liver. Of note, treatment with GSK-J4 reduced lesions. In parallel, GSK-J4 efficiently induced cell death in PCa-derived OSCs. Conclusions: Our findings underscore the potential of the H19/cell adhesion molecules circuitry as a targeted approach in PCa treatment. Modulating this interaction has proven effective in inhibiting tumor growth and metastasis, presenting a logical foundation for targeted therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Despite the rapid advance in cancer therapies, treatment-resistant relapse remains a significant challenge in cancer treatment. Acquired resistance arises during or after treatment administration, and is usually the main contributor to relapse. For example, prostate cancer, the most frequent type of cancer in the elderly male population, frequently develops into aggressive forms resistant to chemical and hormonal therapies. In this condition, the so-called "cholinic phenotype" that is characterized by the overexpression of choline kinase alpha (CHKA) and increased phosphocholine levels leads to aberrant lipid metabolism. Our work demonstrates that CHKA, which is necessary for membrane phospholipid synthesis, is a target of the long non-coding RNA MALAT1. This study helps to further decipher how MALAT1 affects the regulation of crucial phospholipid/sphingolipid metabolic enzymes, as well as how the androgen receptor pathway is involved in MALAT1-dependent transcriptional regulation. Background. Choline kinase alpha (CHKA), an essential gene in phospholipid metabolism, is among the modulated MALAT1-targeted transcripts in advanced and metastatic prostate cancer (PCa). Methods. We analyzed CHKA mRNA by qPCR upon MALAT1 targeting in PCa cells, which is characterized by high dose-responsiveness to the androgen receptor (AR) and its variants. Metabolome analysis of MALAT1-depleted cells was performed by quantitative High-resolution 1 H-Nuclear Magnetic Resonance (NMR) spectroscopy. In addition, CHKA genomic regions were evaluated by chromatin immunoprecipitation (ChIP) in order to assess MALAT1-dependent histone-tail modifications and AR recruitment. Results. In MALAT1-depleted cells, the decrease of CHKA gene expression was associated with reduced total choline-containing metabolites compared to controls, particularly phosphocholine (PCho). Upon MALAT1 targeting a significant increase in repressive histone modifications was observed at the CHKA intron-2, encompassing relevant AR binding sites. Combining of MALAT1 targeting with androgen treatment prevented MALAT1-dependent CHKA silencing in androgen-responsive (LNCaP) cells, while it did not in hormone-refractory cells (22RV1 cells). Moreover, AR nuclear translocation and its activation were detected by confocal microscopy analysis and ChIP upon MALAT1 targeting or androgen treatment. Conclusions. These findings support the role of MALAT1 as a CHKA activator through putative association with the liganded or unliganded AR, unveiling its targeting as a therapeutic option from a metabolic rewiring perspective. [ABSTRACT FROM AUTHOR]

This symposium was held in Trento, Italy, on June 19-21, 2014, and was focused on advances in biology, physiology, and pathology of neoplasms affected by hormones, especially breast and prostate cancers. The stem cell function, the genetic and epigenetic interactions with hormones, the mechanisms of estrogen receptor transcription, biochemical markers and therapeutic targets in breast cancer, promotion of breast cancer carcinogenesis by progesterone, the basis for prostate cancer progression and the relevance of DNA repair processes, androgen receptor programming during prostate carcinogenesis, the metabolic stress role in tumor survival, and the diagnostic use of imaging in prostate cancer were discussed. [ABSTRACT FROM AUTHOR]

Abstract: Fluorescence in situ hybridization analysis for evaluation of 7, 8, X chromosomes and EGFR, LPL, MYC, AR genes in 79 neoplastic foci from 56 patients with clinically localized prostate cancer was performed. We found aneusomy for chromosome 7, 8 and X in 74/77 (96.1%), 56/76 (73.7%), 26/70 (37.1%) of examined foci respectively. No specimen was amplified for EGFR and AR genes, only 2/71 (2.8%) specimens showed MYC gene amplified. LPL deletion was present in 52/76 (68.4%) specimens. Statistically association between Gleason score and both chromosome 7 aneusomy and 8p21 deletion was present. The frequency of chromosome 7 aneusomy was statistically higher in T3-4 cases than T2c and T2a-T2b ones. We considered as unfavorable a genetic set if aneusomy for at least two chromosomes and one altered gene were present. The percentage of tumors, with unfavorable genetic pattern, increased from 36.4 to 75.0% in those with Gleason >7 and from 40.0 to 73.7% in those with stage T3 or more. These alterations could be considered potent genetic markers adjunctive to conventional prognostic parameters. Our objective was to establish specific genetic profiles which may discriminate favorable and unfavorable genetic prognosis tumors. [Copyright &y& Elsevier]

RNA epigenetics is perhaps the most recent field of interest for translational epigeneticists. RNA modifications create such an extensive network of epigenetically driven combinations whose role in physiology and pathophysiology is still far from being elucidated. Not surprisingly, some of the players determining changes in RNA structure are in common with those involved in DNA and chromatin structure regulation, while other molecules seem very specific to RNA. It is envisaged, then, that new small molecules, acting selectively on RNA epigenetic changes, will be reported soon, opening new therapeutic interventions based on the correction of the RNA epigenetic landscape. In this review, we shall summarize some aspects of RNA epigenetics limited to those in which the potential clinical translatability to cardiovascular disease is emerging [ABSTRACT FROM AUTHOR]

The epigenetic enzyme p300/CBP-associated factor (PCAF) belongs to the GCN5-related N-acetyltransferase (GNAT) family together with GCN5. Although its transcriptional and post-translational function is well characterized, little is known about its properties as regulator of cell metabolism. Here, we report the mitochondrial localization of PCAF conferred by an 85 aa mitochondrial targeting sequence (MTS) at the N-terminal region of the protein. In mitochondria, one of the PCAF targets is the isocitrate dehydrogenase 2 (IDH2) acetylated at lysine 180. This PCAF-regulated post-translational modification might reduce IDH2 affinity for isocitrate as a result of a conformational shift involving predictively the tyrosine at position 179. Site-directed mutagenesis and functional studies indicate that PCAF regulates IDH2, acting at dual level during myoblast differentiation: at a transcriptional level together with MyoD, and at a post-translational level by direct modification of lysine acetylation in mitochondria. The latter event determines a decrease in IDH2 function with negative consequences on muscle fiber formation in C2C12 cells. Indeed, a MTS-deprived PCAF does not localize into mitochondria, remains enriched into the nucleus, and contributes to a significant increase of muscle-specific gene expression enhancing muscle differentiation. The role of PCAF in mitochondria is a novel finding shedding light on metabolic processes relevant to early muscle precursor differentiation. [ABSTRACT FROM AUTHOR]

Organ-specific mesenchymal cells naturally reside in the stroma, where they are exposed to some environmental variables affecting their biology and functions. Risk factors such as diabetes or aging influence their adaptive response. In these cases, permanent epigenetic modifications may be introduced in the cells with important consequences on their local homeostatic activity and therapeutic potential. Numerous results suggest that mesenchymal cells, virtually present in every organ, may contribute to tissue regeneration mostly by paracrine mechanisms. Intriguingly, the heart is emerging as a source of different cells, including pericytes, cardiac progenitors, and cardiac fibroblasts. According to phenotypic, functional, and molecular criteria, these should be classified as mesenchymal cells. Not surprisingly, in recent years, the attention on these cells as therapeutic tools has grown exponentially, although only very preliminary data have been obtained in clinical trials to date. In this review, we summarized the state of the art about the phenotypic features, functions, regenerative properties, and clinical applicability of mesenchymal cells, with a particular focus on those of cardiac origin. [ABSTRACT FROM AUTHOR]

Aims: Beyond the control of nuclear-cytoplasmic trafficking nucleoporins regulate gene expression and are involved in cardiac diseases. Notably, a number of cardiovascular disorders have been linked to alterations in epigenetic mechanisms. Here we aimed to determine the contribution of Nup153 to the epigenetic alterations occurring in cardiomyopathy of dystrophin-deficient mdx mice (C57BL/10ScSn-Dmdmdx/J). Methods and results: Nup153 was lysine-acetylated and its expression was significantly increased at protein level in mdx hearts compared with controls. Accordingly, lysine acetyl transferase (KAT) activity associated with Nup153 was higher in mdx hearts paralleling increased binding with the lysine acetylases P300/CBP-associated factor (PCAF) and p300. Interestingly, Nup153 silencing in mdx organotypic heart tissue slices caused a reduction in PCAF- and p300-specific activities. Remarkably, the level of nitric oxide (NO), which is reduced in mdx mice, was important for KAT-dependent regulation of Nup153. In fact, treatment of mdx heart tissue with an NO donor or the KAT inhibitor anacardic acid normalized Nup153 protein expression. Nup153 was recruited to chromatin and regulated the transcription of genes involved in cardiac remodelling, including the actin-binding protein nexilin. Accordingly, nexilin protein expression was abrogated by Nup153 silencing in mdx organotypic cultures. Electrophysiological and molecular experiments revealed that Nup153 overexpression in normal cardiomyocytes increases Cav1.2 calcium channel expression and function. Alterations in Nup153 protein expression and intracellular localization were also found in dystrophic cardiomyocytes derived from patient-specific induced pluripotent stem cells. Importantly, Nup153 up-regulation and increased acetylation were also found in the heart of Duchenne muscular dystrophy patients. Conclusions: Our data indicate that Nup153 is an epigenetic regulator which, upon altered NO signalling, mediates the activation of genes potentially associated with early dystrophic cardiac remodelling. [ABSTRACT FROM AUTHOR]

Age is the most important risk factor for metabolic alterations and cardiovascular accidents. Although class III histone deacetylases, alias Sirtuins, have been appealed as “the fountain of youth” their role in longevity control and prevention of aging-associated disease is still under debate. Indeed, several lines of evidence indicate that sirtuin activity is strictly linked to metabolism and dependent on NAD + synthesis both often altered as aging progresses. During aging the cardiovascular system is attacked by a variety of environmental stresses, including those determined by high blood glucose and lipid levels, or by the presence of oxidized lipoproteins which, among others, determine important oxidative stress signals. In such a milieu, heart and vessels develop a functional impairment leading to atherosclerosis, ischemia, heart insufficiency and failure. Sirtuins, which are believed to have a positive impact on cardiovascular physiology and physiopathology, are distributed in different subcellular compartments including the nucleus, the cytoplasm and the mitochondria, where they regulate expression and function of a large variety of target genes and proteins. Remarkably, experimental animal models indicate resveratrol, the first natural compound described to positively regulate the activity of sirtuins, as able to protect the endothelium and the heart exposed to a variety of stress agents. This review will focus on the regulation and function of mammalian sirtuins with special attention paid to their role as cardiovascular “defenders” giving indication of their targets of potential relevance for the development of future therapeutics. This article is part of a Special Issue entitled CV Aging. [ABSTRACT FROM AUTHOR]

Different ChIP-Seq protocolsmay have a significant impact on the final outcome in terms of quality, number and distribution of called peaks. Sample DNA undergoes a long procedure before the final sequencing step, and damaged DNA can result in excessive mismatches in the alignment with reference genome. In this letter, we present the effect of well-defined modifications (timing of formaldehyde crosslink reversal, brand of the sonicator) of standard ChIP-Seq protocol on parallel samples derived from the same cell line correlating the initial DNA quality control metrics to the final bioinformatics analysis results. [ABSTRACT FROM AUTHOR]

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a down-regulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients. [ABSTRACT FROM AUTHOR]

Histone deacetylase inhibitors (DIs) are promising drugs for the treatment of several pathologies including ischemic and failing heart where they demonstrated efficacy. However, adverse side effects and cardiotoxicity have also been reported. Remarkably, no information is available about the effect of DIs during tissue regeneration following acute peripheral ischemia. In this study, mice made ischemic by femoral artery excision were injected with the DIs MS275 and MC1568, selective for class I and IIa histone deacetylases (HDACs), respectively. In untreatedmice, soon after damage, class IIa HDAC phosphorylation and nuclear export occurred, paralleled by dystrophin and neuronal nitric-oxide synthase (nNOS) down-regulation and decreased protein phosphatase 2A activity. Between 14 and 21 days after ischemia, dystrophin and nNOS levels recovered, and class IIa HDACs relocalized to the nucleus. In this condition, the MC1568 compound increased the number of newly formedmuscle fibers but delayed their terminal differentiation, whereas MS275 abolished the early onset of the regeneration process determining atrophy and fibrosis. The selective DIs had differential effects on the vascular compartment: MC1568 increased arteriogenesis whereasMS275 inhibited it. Capillaro-genesis did not change. Chromatin immunoprecipitations revealed that class IIaHDAC complexes bind promoters of proliferation-associated genes and of class I HDAC1 and 2, highlighting a hierarchical control between class II and I HDACs during tissue regeneration. Our findings indicate that class-selective DIs interfere with normal mouse ischemic hindlimb regeneration and suggest that their use could be limited by alteration of the regeneration process in peripheral ischemic tissues. [ABSTRACT FROM AUTHOR]

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa. [ABSTRACT FROM AUTHOR]

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF- 10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair. [ABSTRACT FROM AUTHOR]

Adult human cardiac mesenchymal-like stromal cells (CStC) represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS) in the presence of 5 μM all-trans Retinoic Acid (ATRA), 5 μM Phenyl Butyrate (PB), and 200 μM diethylenetriamine/nitric oxide (DETA/NO), to create a novel epigenetically active cocktail (EpiC). Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker If current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors. [ABSTRACT FROM AUTHOR]

Background: Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of ''enhancement'' strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit. Principal Findings: Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34+ were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction. Conclusions: Our results show that HDAC blockade leads to phenotype changes in CD34+ cells with enhanced self renewal and cardioprotection. [ABSTRACT FROM AUTHOR]

Our understanding of epigenetic mechanisms important for embryonic vascular development and cardiovascular differentiation is still in its infancy. Although molecular analyses, including massive genome sequencing and/or in vitro/in vivo targeting of specific gene sets, has led to the identification of multiple factors involved in stemness maintenance or in the early processes of embryonic layers specification, very little is known about the epigenetic commitment to cardiovascular lineages. The object of this review will be to outline the state of the art in this field and trace the perspective therapeutic consequences of studies aimed at elucidating fundamental epigenetic networks. Special attention will be paid to the emerging role of nitric oxide in this field. [ABSTRACT FROM AUTHOR]

This review is based on novel observations from our laboratory on the nuclear translocation and functional role of endothelial nitric oxide synthase (eNOS) in endothelial and prostate cancer (PCa) epithelial cells. Nitric oxide (NO), the product of eNOS, is a free radical involved in the physiology and pathophysiology of living organisms and in a variety of biological processes including the maintenance of vascular homeostasis. Of relevance in this context is the role that estrogens play in the apoptotic process and the migration of endothelial cells through the regulation of target genes such as eNOS itself. It has been shown that both estrogen and NO signaling, mediated respectively by the estrogen receptors (ERs) and eNOS, can strongly counteract endothelial senescence through a common effector, the catalytic subunit of human telomerase. Therefore, this protein has been identified as a key molecule in the aging process which, intriguingly, is considered the only risk factor in the development of PCa and one of the major determinants of cardiovascular diseases. Indeed, in both these contexts we have defined a molecular mechanism involving activation of eNOS and hypoxia-inducible factors in association with ERβ that characterizes the most aggressive form of PCa or influences endothelial cell differentiation. Altogether these data led us to postulate that activation of eNOS is a crucial requirement for the delaying of endothelial senescence as well as for the acquisition of androgen-independence and for tumor progression in the prostate microenvironment. [ABSTRACT FROM AUTHOR]

Background: Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the ''angiogenic switch'' during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo. Methodology/Principal Findings: Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression. Conclusions/Significance: These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies. ~ [ABSTRACT FROM AUTHOR]

Abstract: Histone deacetylases (HDACs) are enzymes with a pleiotropic range of intracellular localizations and actions. They are principally involved in the withdrawal of acetyl-groups from a large number of nuclear and cytoplasmic proteins including nuclear core histones as well as cytoskeletal proteins and metabolically relevant enzymes. Initial findings indicated that HDAC inhibitors (DIs) could be successfully applied in a variety of cancer treatment protocols as a consequence of their anti-proliferative and pro-apoptotic properties. Recent observations, however, enlightened the important therapeutic effects of DIs in experimental animal models for arthritis, neurodegenerative and neuromuscular disorders, heart ischemia, cardiac hypertrophy, heart failure and arrhythmias. A small number of clinical trials are now open or planned for the near future to verify the therapeutic properties of DIs in non-cancer-related diseases. This review summarizes some of the most important observations and concepts aroused by the most recent experimental application of DIs to neuromuscular and cardiac diseases. [Copyright &y& Elsevier]

Abstract: Purpose: To confirm our previously obtained results, we genetically characterized prostate cancer from patients undergo radical prostatectomy in a retrospective study. Materials and methods: Histological sections were evaluated for 106 patients treated with surgery from 1991 to 2004. With fluorescence in situ hybridization (FISH) method, the status of LPL (8p22), c-MYC (8q24) genes and 7, 8, X chromosomes was evaluated. Results: Chromosomes 7, 8, X aneusomy was demonstrated in 91.5%, 78.3%, and 51.9% of the samples, respectively, whereas LPL deletion and MYC amplification were found in 76.0% and 1.6%. A genetic profile was considered as unfavorable when at least two aneusomic chromosomes and one altered gene were present. Tumors with an adverse genetic profile were more frequently present in patients with higher stages (P = 0.02), biochemical/clinical progression (P = 0.03), and Gleason grade 4 + 3 (P = 0.02). Multiple correspondence analysis identified one tumor group characterized by chromosome 8 aneusomy, X polysomy, LPL gene deletion, Gleason > 7 and 4 + 3 associated with progression. Conclusions: In this study, we recognized the predictive power of previously identified cytogenetic profiles. Assessment of genetic set may characterize each patient and have influence on postoperative therapeutic strategies. [Copyright &y& Elsevier]

Abstract: The discovery of nitric oxide (NO) revealed its ambiguous nature, which is related to its pleiotropic activities that control the homeostasis of every organism from bacteria to mammals in several physiological and pathological situations. The wide range of action of NO basically depends on two features: 1) the variety of chemical reactions depending on NO, and 2) the differential cellular responses elicited by distinct NO concentrations. Despite the increasing body of knowledge regarding its chemistry, biology and NO-dependent signaling pathways, little information is available on the nuclear actions of NO in terms of gene expression regulation. Indeed, studies of a putative role for this diatomic compound in regulating chromatin remodeling are still in their infancy. Only recently has the role of NO in epigenetics emerged, and some of its putative epigenetic properties are still only hypothetical. In the present review, we discuss the current evidence for NO-related mechanisms of epigenetic gene expression regulation. We link some of the well known NO chemical reactions and metabolic processes (e.g., S-nitrosylation of thiols, tyrosine nitration, cGMP production) to chromatin modification and address the most recent, striking hypothesis about NO and the control of chromosomes structure. [Copyright &y& Elsevier]

Androgens play a central role in prostate development and prostate cancer proliferation. Induction of telomerase is an early event in prostate carcinogenesis and is considered as a marker for both primary tumors and metastases. Interestingly, several reports suggest that telomerase activity is regulated by androgens in vivo. Here, we show that the wild-type (WT) human androgen receptor (AR) inhibits the expression of the human telomerase reverse transcriptase (hTERT) and telomerase activity via inhibition of hTERT promoter activity in the presence of androgen receptor agonists. However, pure androgen antagonists failed to repress hTERT transcription. The androgenmediated repression of hTERT is abrogated in a human prostate cancer cell line exhibiting hormone-dependent growth, which expresses a mutant AR (T877A) frequently occurring in prostate cancer. We reveal that this single amino acid exchange is sufficient for the lack of transrepression. Interestingly, chromatin immunoprecipitation data suggest that, in contrast to the WT AR, the mutant AR is recruited less efficiently to the hTERT promoter in vivo, indicating that loss of transrepression results from reduced chromatin recruitment. Thus, our findings suggest that the WT AR inhibits expression of hTERT, which is indicative of a protective mechanism, whereas the T877A mutation of AR not only broadens the ligand spectrum of the receptor but abrogates this inhibitory mechanism in prostate cancer cells. This novel role of AR mutations in prostate cancer development suggests the benefit to a search for new AR antagonists that inhibit transactivation but allow transrepression. [ABSTRACT FROM AUTHOR]

Objectives: We evaluated telomerase activity in circulating polymorphonuclear neutrophils (PMN) and in PMN isolated from coronary atherosclerotic plaques by a novel approach. Background: Delayed apoptosis of PMN have been demonstrated in unstable angina (UA). These cells have a finite lifespan with low telomerase activity, a polymerase that extends telomeres, structures essential for cell aging. Reactivation of telomerase has been associated with resistance to apoptosis. Methods: We studied 20 patients with UA and 6 patients with chronic stable angina (SA), undergoing a percutaneous coronary intervention. Circulating PMN were isolated from venous blood and PMN derived from coronary plaque were isolated from washing medium of angioplasty balloons. Results: Telomerase activity was higher in coronary plaque PMN of UA patients than in coronary plaque PMN of SA patients (122.7, range 20.5 to 3,696; and 47.7, range 16 to 212.6, respectively, p = 0.001) and higher than in peripheral PMN of SA patients (122.7, range 20.5 to 3,696 vs. 59, range 16.5 to 132.5, p = 0.001). We found a statistically significant difference between venous and coronary plaque PMN telomerase activity in UA patients (z = −2.875; p = 0.004). Among UA patients, a shorter time interval from symptom onset to coronary PMN sampling was the only independent predictor of high telomerase activity in coronary plaque PMN (p < 0.001, R2 = 0.75). Conclusions: In UA patients, telomerase activity is high in coronary plaque PMN, while it is low in peripheral PMN. Telomerase reactivation in resident PMN resulting in a prolonged lifespan might play a key role in the early phases of instability. [Copyright &y& Elsevier]

Telomere dysfunction contributes to reduced cell viability, altered differentiation, and impaired regenerative/proliferative responses. Recent advances indicate that telomerase activity confers a pro-angiogenic phenotype to endothelial cells and their precursors. We have investigated whether telomerase contributes to tissue regeneration following hind limb ischemia and vascular endothelial growth factor 165 (VEGF165) treatment. VEGF delivery induced angiogenesis and increased expression of the telomerase reverse transcriptase (TERT) and telomerase activity in skeletal muscles and satellite end endothelial cells. Adenovirus-mediated transfer of wild type TERT but not of a dominant negative mutant, TERTdn, significantly induced capillary but not arteriole formation. However, when co-delivered with VEGF, TERTdn abrogated VEGF-dependent angiogenesis, arteriogenesis, and blood flow increase. This effect was paralleled by in vitro evidence that telomerase inhibition by 3′-azido-3′-deoxythymidine in VEGF-treated endothelial cells strongly reduced capillary density and promoted apoptosis in the absence of serum. Similar results were obtained with adenovirus-mediated expression of TERTdn and AKTdn, both reducing endogenous TERT activity and angiogenesis on Matrigel. Mechanistically, neo-angiogenesis in our system involved: (i) VEGF-dependent activation of telomerase through the nitric oxide pathway and (ii) telomerase-dependent activation of endothelial cell differentiation and protection from apoptosis. Furthermore, detection of TERT in activated satellite cells identified them as VEGF targets during muscle regeneration. Because TERT behaves as an angiogenic factor and a downstream effector of VEGF signaling, telomerase activity appears required for VEGF-dependent remodeling of ischemic tissue at the capillaries and arterioles level. [ABSTRACT FROM AUTHOR]

Rescue of embryonic lethality in MDM4-/- mice through concomitant loss of p53 has revealed a functional partnership between the two proteins. Biochemical studies have suggested that MDM4 may act as a negative regulator of p53 levels and activity. On the other hand, MDM4 overexpression has been reported to stabilize p53 levels and to counteract MDM2-degradative activity. We have investigated the functional role of MDM4 overexpression on cell behavior. In both established and primary cells cultured under stress conditions, overexpression of MDM4 significantly increased p53-dependent cell death, in correlation with enhanced induction of the endogenous p53 protein levels. This phenomenon was associated with induced p53 transcriptional activity and increased levels of the proapoptotic protein, Bax. Further, p53 stabilization was accompanied by decreased association of the protein to its negative regulator, MDM2. These findings reveal a novel role for MDM4 by demonstrating that in non-tumor cells under stress conditions it may act as a positive regulator of p53 activity, mainly by controlling p53 levels. They also indicate a major distinction between the biological consequences of MDM4 and MDM2 overexpression. [ABSTRACT FROM AUTHOR]

Presents a study which investigated whether estrogen receptor signaling may be responsible for telomerase activation in prostate epithelial cells. Link between maintenance of functional telomeres and human cancer; Description of the human telomerase expression and telomerase activity in normal and transformed prostate epithelial cells after 17Β-estradiol treatment; Effects of the selective aromatase inhibitor on telomerase activity.

The SARS-CoV-2 infection determines the COVID-19 syndrome characterized, in the worst cases, by severe respiratory distress, pulmonary and cardiac fibrosis, inflammatory cytokine release, and immunosuppression. This condition has led to the death of about 2.15% of the total infected world population so far. Among survivors, the presence of the so-called persistent post-COVID-19 syndrome (PPCS) is a common finding. In COVID-19 survivors, PPCS presents one or more symptoms: fatigue, dyspnea, memory loss, sleep disorders, and difficulty concentrating. In this study, a cohort of 117 COVID-19 survivors (post-COVID-19) and 144 non-infected volunteers (COVID-19-free) was analyzed using pyrosequencing of defined CpG islands previously identified as suitable for biological age determination. The results show a consistent biological age increase in the post-COVID-19 population, determining a DeltaAge acceleration of 10.45 ± 7.29 years (+5.25 years above the range of normality) compared with 3.68 ± 8.17 years for the COVID-19-free population (p < 0.0001). A significant telomere shortening parallels this finding in the post-COVID-19 cohort compared with COVID-19-free subjects (p < 0.0001). Additionally, ACE2 expression was decreased in post-COVID-19 patients, compared with the COVID-19-free population, while DPP-4 did not change. In light of these observations, we hypothesize that some epigenetic alterations are associated with the post-COVID-19 condition, particularly in younger patients (< 60 years). [ABSTRACT FROM AUTHOR]

The WHO estimated around 41 million deaths worldwide each year for age-related non-communicable chronic diseases. Hence, developing strategies to control the accumulation of cell senescence in living organisms and the overall aging process is an urgently needed problem of social relevance. During aging, many biological processes are altered, which globally induce the dysfunction of the whole organism. Cell senescence is one of the causes of this modification. Nowadays, several drugs approved for anticancer therapy have been repurposed to treat senescence, and others are under scrutiny in vitro and in vivo to establish their senomorphic or senolytic properties. In some cases, this research led to a significant increase in cell survival or to a prolonged lifespan in animal models, at least. Senomorphics can act to interfere with a specific pathway in order to restore the appropriate cellular function, preserve viability, and to prolong the lifespan. On the other hand, senolytics induce apoptosis in senescent cells allowing the remaining non–senescent population to preserve or restore tissue function. A large number of research articles and reviews recently addressed this topic. Herein, we would like to focus attention on those chemical agents with senomorphic or senolytic properties that perspectively, according to literature, suggest a potential application as senotherapeutics for chronic diseases. [ABSTRACT FROM AUTHOR]

Epigenetics is a relatively new field of science that studies the genetic and non-genetic aspects related to heritable phenotypic changes, frequently caused by environmental and metabolic factors. In the host, the epigenetic machinery can regulate gene expression through a series of reversible epigenetic modifications, such as histone methylation and acetylation, DNA/RNA methylation, chromatin remodeling, and non-coding RNAs. The coronavirus disease 19 (COVID-19) is a highly transmittable and pathogenic viral infection. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which emerged in Wuhan, China, and spread worldwide, causes it. COVID-19 severity and consequences largely depend on patient age and health status. In this review, we will summarize and comparatively analyze how viruses regulate the host epigenome. Mainly, we will be focusing on highly pathogenic respiratory RNA virus infections such as coronaviruses. In this context, epigenetic alterations might play an essential role in the onset of coronavirus disease complications. Although many therapeutic approaches are under study, more research is urgently needed to identify effective vaccine or safer chemotherapeutic drugs, including epigenetic drugs, to cope with this viral outbreak and to develop pre- and post-exposure prophylaxis against COVID-19. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is a sex-steroid hormone-dependent cancer in which estrogens play a critical role in both initiation and progression. Recently, several long non-coding RNAs (lncRNAs) have been associated with PCa and are supposedly playing a pivotal role in the biology and progression of this type of cancer. In this review, we focused on some lncRNAs that are known for their androgen and estrogen transcriptional responsiveness in PCa. Specifically, we summarized recent pieces of evidence about lncRNAs NEAT1, H19, MALAT1, and HOTAIR, in estrogen signaling, emphasizing their role in PCa progression and the acquisition of a castration-resistant phenotype. Here, the reader will find information about lncRNAs present in estrogen-dependent transcriptional complexes. The potential role of lncRNA/estrogen signaling as a novel pathway for PCa treatment will be discussed. [ABSTRACT FROM AUTHOR]

Neuroendocrine prostate cancer (NEPC) can arise de novo, but much more commonly occurs as a consequence of a selective pressure from androgen deprivation therapy or androgen receptor antagonists used for prostate cancer (PCa) treatment. The process is known as neuroendocrine transdifferentiation. There is little molecular characterization of NEPCs and consequently there is no standard treatment for this kind of tumors, characterized by highly metastases rates and poor survival. For this purpose, we profiled 54 PCa samples with more than 10-years follow-up for gene and miRNA expression. We divided samples into two groups (NE-like vs. AdenoPCa), according to their clinical and molecular features. NE-like tumors were characterized by a neuroendocrine fingerprint made of known neuroendocrine markers and novel molecules, including long non-coding RNAs and components of the estrogen receptor signaling. A gene expression signature able to predict NEPC was built and tested on independently published datasets. This study identified molecular features (protein-coding, long non-coding, and microRNAs), at the time of surgery, that may anticipate the NE transformation process of prostate adenocarcinoma. Our results may contribute to improving the diagnosis and treatment of this subgroup of tumors for which traditional therapy regimens do not show beneficial effects. [ABSTRACT FROM AUTHOR]

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules' expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells' metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both β3 and β4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and β integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of β integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a β integrin-mediated invasion. [ABSTRACT FROM AUTHOR]

In the complex network of nuclear hormone receptors, the long non-coding RNAs (lncRNAs) are emerging as critical determinants of hormone action. Here we investigated the involvement of selected cancer-associated lncRNAs in Estrogen Receptor (ER) signaling. Prior studies by Chromatin Immunoprecipitation (ChIP) Sequencing showed that in prostate cancer cells ERs form a complex with the endothelial nitric oxide synthase (eNOS) and that in turn these complexes associate with chromatin in an estrogen-dependent fashion. Among these associations (peaks) we focused our attention on those proximal to the regulatory region of HOTAIR and MALAT1. These transcripts appeared regulated by estrogens and able to control ERs function by interacting with ERα/ERβ as indicated by RNA-ChIP. Further studies performed by ChIRP revealed that in unstimulated condition, HOTAIR and MALAT1 were present on pS2, hTERT and HOTAIR promoters at the ERE/eNOS peaks. Interestingly, upon treatment with17β-estradiol HOTAIR recruitment to chromatin increased significantly while that of MALAT1 was reduced, suggesting an opposite regulation and function for these lncRNAs. Similar results were obtained in cells and in an ex vivo prostate organotypic slice cultures. Overall, our data provide evidence of a crosstalk between lncRNAs, estrogens and estrogen receptors in prostate cancer with important consequences on gene expression regulation. [ABSTRACT FROM AUTHOR]

The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)-the catalyst of the reaction-and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy. [ABSTRACT FROM AUTHOR]

Myopericytoma (MPC) is a rare tumor with perivascular proliferation of pluripotent stem-cell–like pericytes. Although indolent, MPC may be locally aggressive with recurrent disease. The pathogenesis and diagnostic biomarkers of MPC are poorly understood. We discovered that 15% of benign MPCs (thyroid, skin; 3 of 20 samples) harbored BRAFWT/V600E; 33.3% (1 of 3 samples) of BRAFWT/V600E-MPCs were multifocal/infiltrative/recurrent. Patient-MPC and primary MPC cells harbored BRAFWT/V600E, were clonal and expressed pericytic-differentiation biomarkers crucial for its microenvironment. BRAFWT/V600E-positive thyroid MPC primary cells triggered in vitro (8.8-fold increase) and in vivo (3.6-fold increase) angiogenesis. Anti-BRAFV600E therapy with vemurafenib disrupted angiogenic and metabolic properties (~3-fold decrease) with down-regulation (~2.2-fold decrease) of some extracellular-matrix (ECM) factors and ECM-associated long non-coding RNA (LincRNA) expression, with no effects in BRAFWT-pericytes. Vemurafenib also inhibited (~3-fold decrease) cell viability in vitro and in BRAFWT/V600E-positive thyroid MPC patient-derived xenograft (PDX) mice (n = 5 mice per group). We established the first BRAFWT/V600E-dependent thyroid MPC cell culture. Our findings identify BRAFWT/V600E as a novel genetic aberration in MPC pathogenesis and MPC-associated biomarkers and imply that anti-BRAFV600E agents may be useful adjuvant therapy in BRAFWT/V600E-MPC patients. Patients with BRAFWT/V600E-MPC should be closely followed because of the risk for multifocality/recurrence. [ABSTRACT FROM PUBLISHER]