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Background: Infertility, defined as the failure to conceive after one year of regular, unprotected intercourse, affects 50–80 million people worldwide. A male factor is involved in approximately 20–30% of cases. In the etiology of male infertility, the association between poor semen quality and oxidative stress (OS) is well known. High levels of reactive oxygen species (ROS) allow the oxidation of DNA, proteins, and lipids of sperm cells, modifying their vitality, motility, and morphology. Methods: To evaluate the effects of antioxidants on sperm in infertile men, we queried the MEDLINE database (via the PubMed interface) for published studies in the last 10 years (2011–2021). The following keywords were used: “infertility” and -“inositol”, -“alpha-lipoic acid”, -“zinc”, -“folate”, -“coenzyme Q10”, -“selenium”, and -“vitamin”. Results: Inositol regulates OS levels in sperm cells thanks to its role in mitochondrial reactions and is involved in several processes favoring sperm–oocyte interactions. Alpha-lipoic acid (ALA) reduces ROS damage and improves semen parameters in terms of spermatozoa’s motility, morphology, and count. Poor zinc nutrition may be related to low quality of sperm. Supplementation of folate plus zinc has a positive effect on the sperm concentration and morphology. Supplementation with CoQ10 increases sperm concentration, total and progressive motility. Selenium (Se) supplementation improves the overall semen quality and is related to a higher ejaculated volume. Among vitamins, only vitamin B12 shows a positive effect on semen quality; it increases sperm count and motility and reduces sperm DNA damage. Conclusions: In men showing low-quality semen, diet supplementation with antioxidants may improve the sperm quality by alleviating OS-induced sperm damage and enhancing hormone synthesis and spermatozoa concentration, motility, and morphology. Future clinical trials should be focused on the possible association of several antioxidants to take advantage of combined mechanisms of action.

The epidemic spread of obesity is nowadays recognized as a global health and economic burden, arising great interest in the scientific community. The rate of adult obesity steadily increases concomitantly with the cancer incidence. As has been comprehensively reported, obesity is included among the multiple cancer risk factors and can progressively cause and/or exacerbate certain cancer types, as colorectal and breast cancers. The term adiponcosis was forged precisely to emphasize the interconnection between obesity and cancer onset and progression. The underlying mechanisms of adiponcosis have not been fully elucidated yet, may vary on cancer type, and depend on body fat distribution. It has been proposed that insulin resistance and related chronic hyperinsulinemia, increased insulin-like growth factors production, chronic inflammation or increased bioavailability of steroid hormones could be responsible of cancer hallmarks. Additionally, it has been suggested that adipose tissue-derived hormones, cytokines and adipokines, such as leptin, adiponectin and inflammatory markers, may reflect mechanisms linked to tumorigenesis. This review summarizes the current evidence on pathways, hormones, cytokines and low-chronic inflammation subtending adiponconsis, focusing on breast and colorectal cancers. In addition, we analyzed the lifestyle interventions that could attenuate the driving forces of obesity-related cancer incidence and progression. Moreover, current targets and drugs, their pros and cons, as well as new mechanisms and targets with promising therapeutic potential in cancer are discussed. Depicting this complex interconnection will provide insights for establishing new therapeutic approaches to halt the obesity impacts and thwart cancer onset and progression.

Transcriptional regulation is a critical biological process that allows the cell or an organism to respond to a variety of intra- and extracellular signals, to define cell identity during development, to maintain it throughout its lifetime, and to coordinate cellular activity [...]

Abstract: Breast cancer, even today, can cause death. Therefore, prevention and early detection are fundamental factors. The mechanisms that favour it are genetic and epigenetic, and seem to play a significant role; also, the microbiota can change estrogen levels and can induce chronic inflammation in the neoplastic site, alternating the balance between proliferation and cell death. Activated steroid hormone receptors induce transcription of genes that encode for proteins involved in cell proliferation and activate another transduction pathway, inducing cell cycle progression and cell migration. These important studies have allowed to develop therapies with selective modulators of estrogen receptors (SERMs), able to block their proliferative and pro-tumorigenic action. Of fundamental importance is also the role played by the microbiota in regulating the metabolism of estrogens and their levels in the blood. There are microbial populations that are able to promote the development of breast cancer, through the production of enzymes responsible for the deconjugation of estrogens, the increase of these in the intestine, subsequent circulation and migration to other locations, such as the udder. Other microbial populations are, instead, able to synthesize estrogen compounds or mimic estrogenic action, and interfere with the metabolism of drugs, affecting the outcome of therapies. The microbial composition of the intestine and hormonal metabolism depend largely on eating habits; the consumption of fats and proteins favours the increase of estrogen in the blood, unlike a diet rich in fiber. Therefore, in-depth knowledge of the microbiota present in the intestine-breast axis could, in the future, encourage the development of new diagnostic and therapeutic approaches to breast cancers.

Positive Regulatory Domain (PRDM) gene family members commonly express two main molecular variants, the PR-plus isoform usually acting as tumor suppressor and the PR-minus one functioning as oncogene. Accordingly, PRDM2/RIZ encodes for RIZ1 (PR-plus) and RIZ2 (PR-minus). In human cancers, genetic or epigenetic modifications induce RIZ1 silencing with an expression level imbalance in favor of RIZ2 that could be relevant for tumorigenesis. Additionally, in estrogen target cells and tissues, estradiol increases RIZ2 expression level with concurrent increase of cell proliferation and survival. Several attempts to study RIZ2 function in HeLa or MCF-7 cells by its over-expression were unsuccessful. Thus, we over-expressed RIZ2 in HEK-293 cells, which are both RIZ1 and RIZ2 positive but unresponsive to estrogens. The forced RIZ2 expression increased cell viability and growth, prompted the G2-to-M phase transition and organoids formation. Accordingly, microarray analysis revealed that RIZ2 regulates several genes involved in mitosis. Consistently, RIZ silencing in both estrogen-responsive MCF-7 and -unresponsive MDA-MB-231 cells induced a reduction of cell proliferation and an increase of apoptosis rate. Our findings add novel insights on the putative RIZ2 tumor-promoting functions, although additional attempts are warranted to depict the underlying molecular mechanism.

PRDM12 is a member of the PRDI-BF1 (positive regulatory domain I-binding factor 1) homologous domain (PRDM)-containing protein family, a subfamily of Kruppel-like zinc finger proteins, controlling key processes in the development of cancer. PRDM12 is expressed in a spatio-temporal manner in neuronal systems where it exerts multiple functions. PRDM12 is essential for the neurogenesis initiation and activation of a cascade of downstream pro-neuronal transcription factors in the nociceptive lineage. PRDM12 inactivation, indeed, results in a complete absence of the nociceptive lineage, which is essential for pain perception. Additionally, PRDM12 contributes to the early establishment of anorexigenic neuron identity and the maintenance of high expression levels of pro-opiomelanocortin, which impacts on the program bodyweight homeostasis. PRDMs are commonly involved in cancer, where they act as oncogenes/tumor suppressors in a “Yin and Yang” manner. PRDM12 is not usually expressed in adult normal tissues but its expression is re-activated in several cancer types. However, little information is currently available on PRDM12 expression in cancers and its mechanism of action has not been thoroughly described. In this review, we summarize the recent findings regarding PRDM12 by focusing on four main biological processes: neurogenesis, pain perception, oncogenesis and cell metabolism. Moreover, we wish to highlight the importance of future studies focusing on the PRDM12 signaling pathway(s) and its role in cancer onset and progression.

Obesity is related to metabolic defects that may promote not only cancer initiation, but also its progression. The molecular basis for the association between obesity and cancer is not fully understood; however, many pathways are being investigated including hyperinsulinemia/insulin resistance (IR) and abnormalities of the insulin-like growth factor-1 (IGF-1) signaling, sex hormones biosynthesis and pathway, alterations in adipokines pathophysiology, and subclinical chronic low-grade inflammation. In this chapter, we analyze the current knowledge on the proposed biological mechanisms, especially focusing on the role of adiponectin (APN).

The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention.

Introduction: Telemedicine is the use of Information and Communication Technologies (ICT) to improve patient outcomes by increasing access to care, medical information and services. The aim of this pilot study was to evaluate and support the implementation of screening and early detection programs in the prevention of breast cancer and cardiovascular diseases with the establishment of a remote diagnosis through the use of ICT in mobile units. Materials and Methods: A total of 430 individuals were recruited in an area of Southern Italy. Particularly, 321 women were recruited to undergo breast cancer screening in accordance with Italian guidelines. Likewise, cardiovascular screening interested 109 subjects. A self-contained mobile unit with connectivity was provided to offer breast and cardiovascular screenings. To maximize the benefit, we have evaluated the return of investment. Results: The telemedicine screening program allowed the detection of early pathologies. In breast cancer screening, 40.8% of cases were negative to lesions, 34.9% were positive to benign lesions, and 3.1% presented suspicious malignant lesions; these lesions were further checked by histological analyses, which showed a positive response in 70% of cases. The cardiovascular screening concerned 109 participants based on age and other risk factors. We observed a significant difference among risk factors in patients with cardiac disease (p < 0.001); particularly, hypertension was significantly the most present risk factor (51.4%, p < 0.05), followed by smoking (28.4%, p < 0.05). A cardiovascular pathology was detected in 40.4% of enrolled subjects. A 3.3:1 return on investment was calculated. Conclusion: Our findings demonstrate that telemedicine may represent a promising approach to deliver several health services, such as screening programs, with users who cannot utilize services in their locations. The use of telemedicine on diagnostic campers greatly reduces the costs of screening for breast cancer and major cardiovascular diseases within the Southern Italian Health Service. We believe that public investment can have a further significant return on investment by implementing the principles of precision medicine.

The PR/SET domain gene family (PRDM) encodes 19 different transcription factors that share a subtype of the SET domain [Su(var)3-9, enhancer-of-zeste and trithorax] known as the PRDF1-RIZ (PR) homology domain. This domain, with its potential methyltransferase activity, is followed by a variable number of zinc-finger motifs, which likely mediate protein&ndash, protein, protein&ndash, RNA, or protein&ndash, DNA interactions. Intriguingly, almost all PRDM family members express different isoforms, which likely play opposite roles in oncogenesis. Remarkably, several studies have described alterations in most of the family members in malignancies. Here, to obtain a pan-cancer overview of the genomic and transcriptomic alterations of PRDM genes, we reanalyzed the Exome- and RNA-Seq public datasets available at The Cancer Genome Atlas portal. Overall, PRDM2, PRDM3/MECOM, PRDM9, PRDM16 and ZFPM2/FOG2 were the most mutated genes with pan-cancer frequencies of protein-affecting mutations higher than 1%. Moreover, we observed heterogeneity in the mutation frequencies of these genes across tumors, with cancer types also reaching a value of about 20% of mutated samples for a specific PRDM gene. Of note, ZFPM1/FOG1 mutations occurred in 50% of adrenocortical carcinoma patients and were localized in a hotspot region. These findings, together with OncodriveCLUST results, suggest it could be putatively considered a cancer driver gene in this malignancy. Finally, transcriptome analysis from RNA-Seq data of paired samples revealed that transcription of PRDMs was significantly altered in several tumors. Specifically, PRDM12 and PRDM13 were largely overexpressed in many cancers whereas PRDM16 and ZFPM2/FOG2 were often downregulated. Some of these findings were also confirmed by real-time-PCR on primary tumors.

PRDM2/RIZ is a member of a superfamily of histone/protein methyltransferases (PRDMs), which are characterized by the conserved N-terminal PR domain, with methyltransferase activity and zinc finger arrays at the C-terminus. Similar to other family members, two main protein types, known as RIZ1 and RIZ2, are produced from the PRDM2 locus differing by the presence or absence of the PR domain. The imbalance in their respective amounts may be an important cause of malignancy, with the PR-positive isoform commonly lost or downregulated and the PR-negative isoform always being present at higher levels in cancer cells. Interestingly, the RIZ1 isoform also represents an important target of estradiol action downstream of the interaction with hormone receptor. Furthermore, the imbalance between the two products could also be a molecular basis for other human diseases. Thus, understanding the molecular mechanisms underlying PRDM2 function could be useful in the pathophysiological context, with a potential to exploit this information in clinical practice. PRDM2/RIZ is a member of a superfamily of histone/protein methyltransferases (PRDMs), which are characterized by the conserved N-terminal PR domain, with methyltransferase activity and zinc finger arrays at the C-terminus. Similar to other family members, two main protein types, known as RIZ1 and RIZ2, are produced from the PRDM2 locus differing by the presence or absence of the PR domain. The imbalance in their respective amounts may be an important cause of malignancy, with the PR-positive isoform commonly lost or downregulated and the PR-negative isoform always being present at higher levels in cancer cells. Interestingly, the RIZ1 isoform also represents an important target of estradiol action downstream of the interaction with hormone receptor. Furthermore, the imbalance between the two products could also be a molecular basis for other human diseases. Thus, understanding the molecular mechanisms underlying PRDM2 function could be useful in the pathophysiological context, with a potential to exploit this information in clinical practice.

In the last decades, transcriptome profiling has been one of the most utilized approaches to investigate human diseases at the molecular level. Through expression studies, many molecular biomarkers and therapeutic targets have been found for several human pathologies. This number is continuously increasing thanks to total RNA sequencing. Indeed, this new technology has completely revolutionized transcriptome analysis allowing the quantification of gene expression levels and allele-specific expression in a single experiment, as well as to identify novel genes, splice isoforms, fusion transcripts, and to investigate the world of non-coding RNA at an unprecedented level. RNA sequencing has also been employed in important projects, like ENCODE (Encyclopedia of the regulatory elements) and TCGA (The Cancer Genome Atlas), to provide a snapshot of the transcriptome of dozens of cell lines and thousands of primary tumor specimens. Moreover, these studies have also paved the way to the development of data integration approaches in order to facilitate management and analysis of data and to identify novel disease markers and molecular targets to use in the clinics. In this scenario, several ongoing clinical trials utilize transcriptome profiling through RNA sequencing strategies as an important instrument in the diagnosis of numerous human pathologies.

Despite the use of systems biology is still at the beginning in the study of complex diseases, the '-omics'strategies are already contributing to novel insights into atherosclerosis and CVDs. The promise of this approach includes disease risk prediction and prevention as well as the possibility of personalized therapy. In this concern, research interest has been recently focused on evaluating the role of endothelial progenitor cells in tissue vascularization and regeneration. This circulating cell population has also been proposed as potential clinical marker for several diseases including cardiovascular ones, where they have begun to be used as a new therapeutic option [23]. Recent in vivo proteomic data suggest a potential effect of acute coronary syndrome in the protein profiles of these cells [24]. These findings suggest that integrated 'omics technologies, including metabolomics, may help to better characterize these cells and understand their biology as well as their pathophysiological role in CVD and tissue regeneration.

In the last decade, a novel molecular complex has been added to the RNA polymerase II-mediated transcription machinery as one of the major components. This multiprotein complex, named Integrator, plays a pivotal role in the regulation of most RNA Polymerase II-dependent genes. This complex consists of at least 14 different subunits. However, studies investigating its structure and composition are still lacking. Although it was originally discovered as a complex implicated in the 3'-end formation of noncoding small nuclear RNAs, recent studies indicate additional roles for Integrator in transcription regulation, for example during transcription pause release and elongation of polymerase, in the biogenesis of transcripts derived from enhancers, as well as in DNA and RNA metabolism for some of its components. Noteworthy, several subunits have been emerging to play roles during development and differentiation; more importantly, their alterations are likely to be involved in several human pathologies, including cancer and lung diseases. (C) 2016 Elsevier B.V. All rights reserved.

Mediator of RNA polymerase II transcription subunits (MEDs) serve to promote the assembly, activation, and regeneration of transcription complexes on core promoters during the initiation and re-initiation phases of transcription. There are no studies on the Mediator complex during development of endothelial progenitors (EPCs). Here, we have analysed all known MEDs during the differentiation of EPCs, by expression profile studies at RNA level and, for a limited subset of MED subunits, also at protein level. Since beneficial effects of l-arginine on EPCs have been described, we have also examined its effect on the expression of Mediator subunit coding genes. Through RT-PCR we have found increased expression for MED12 and decreased levels for MED30 after l-arginine treatment; Western blot analysis do not agree entirely with the RNA data in the identification of a putative protein product. Furthermore, we have analysed the three-dimensional nuclear positions of MED12 and MED30 genes in the presence of l-arginine treatment. Our major finding is the identification of a novel transcript of MED30, termed MED30 short (MED30s) generating by alternative splicing. Our results showed that the mRNA of this novel isoform is present only in circulating cells, but it is not expressed in cultured adherent cells. These findings are broadly relevant and will contribute to our understanding of the role of Mediator in eukaryotic gene expression. Despite the need to confirm the in vivo presence of the protein of this novel isoform, the presence of this novel RNA raises the possibility of regulating pathophysiological mechanism in progenitors. © 2010 Elsevier B.V. All rights reserved.

The GJB2 gene located on chromosome 13q12 and encoding the connexin 26 (Cx26) protein, a transmembrane protein involved in cell-cell attachment of almost all tissues, including the skin, causes autosomal recessive and sometimes dominant nonsyndromic sensorineural hearing loss. GJB2 mutations have also been identified in syndromic disorders exhibiting hearing loss associated with skin problems. Recently, a new mutation, p.G130V in the GJB2 gene has been reported as causative for Vohwinkel syndrome. In this case the p.G130V mutation was found in two patients (son and father) with palmoplantar keratoderma. The father also showed also skin constrictions of the 2nd and 3rd toes of the right foot. Here, we report on another family with palmoplantar keratoderma associated with a dominant form of hearing loss confirming the genotype-phenotype correlation between the mutation p.G130V and the skin abnormalities observed in syndromic disorders with hearing loss as described by [Snoeckx et al. (2005) Hum Mutat 26:60-65].

Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphorybosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism. © 2008 Elsevier B.V. All rights reserved.

To date, there is no report on the effect of antioxidants on endothelial progenitor cells (EPCs). This study shows that in vitro incubation of EPCs with vitamin C and E reverted the already well documented lowering effect of TNF-alpha on EPC number and increased p-p38 expression levels. In order to document major changes of gene expression levels and gain insight into signalling pathways, microarray analysis was performed and a significant variation of the expression of 5389 genes in EPCs following antioxidant treatment was detected. Also in vivo evidence is provided about the positive effect of antioxidant vitamins on EPCs, since vitamin C and E supplementation potentiated the physical training-induced increase of EPC number and VEGF levels. Together, these data indicate that antioxidant treatment ameliorates EPC number and causes major changes of gene expression within these cells in vitro. Furthermore, concomitant antioxidant supplementation and physical training in vivo raised the levels of circulating EPCs and serum VEGF more than physical training alone.

Background: Emerging infections are simply defined as "those whose incidence has increased within the past two decades or threatens to increase in the near future". Migration flows have greatly increased in recent years and are certainly one of the factors that have contributed to the rise of new or re-emerging pathogens, potentially dangerous for human and animals. Re-merging infections, such as malaria and tuberculosis, and new or emerging infectious diseases such as West Nile fever and Chikungunya fever, through blood transfusion is an important public health problem in endemic and not endemic areas. The increasing number of immigrants that request to become regular donors requires to make an appropriate assessment of the problems related to endemic diseases of the native regions. Aims: The project has been designed to implement a plan that monitors the organisms (Plasmodium falciparum, Mycobacterium tuberculosis, West Nile virus, Chikungunya virus) responsible of infectious diseases and to develop an appropriate prevention and surveillance program. This study can led to record the actual geographical distribution of pathogens and to assess the actual risks associated with transfusion therapy. Methods: we have collected 2000 foreign donors coming from Africa and Asia continents and resident in the Campania Region (Italy). They have been subjected to routine laboratories tests. To confirm the positivity of pathogen detection, we have used blood samples in real-time PCR assays that discriminate between microorganisms based on a signal from specific nucleic acid sequences.

Mediator (MED) complex is a multiprotein playing a key role in the eukaryotic transcription. Alteration of MED function may have enormous pathophysiological consequences and several MED genes have been implicated in human diseases. Here, we have combined computational and experimental approaches to identify and characterize, new transcripts generated by alternative splicing (AS) for all MED genes, through the analysis of our recently published RNA-Sequencing datasets of endothelial progenitor cells (EPCs). This combined strategy allowed us to identify novel transcripts for MED4, MED9, MED11, MED14, MED27 and CDK8 most of them generated by AS. All the newly identified transcripts, except MED11, are predicted to encode novel protein isoforms. The identification of novel MED variants could lead to the finding of other MED complexes with different functions depending on their subunit composition. Finally, the expression profile of all MED genes, together with an extensive gene expression analysis, may be useful to better classify the diverse subsets of cell populations that contribute to neovascularization. © 2014. Mediator (MED) complex is a multiprotein playing a key role in the eukaryotic transcription. Alteration of MED function may have enormous pathophysiological consequences and several MED genes have been implicated in human diseases. Here, we have combined computational and experimental approaches to identify and characterize, new transcripts generated by alternative splicing (AS) for all MED genes, through the analysis of our recently published RNA-Sequencing datasets of endothelial progenitor cells (EPCs). This combined strategy allowed us to identify novel transcripts for MED4, MED9, MED11, MED14, MED27 and CDK8 most of them generated by AS. All the newly identified transcripts, except MED11, are predicted to encode novel protein isoforms. The identification of novel MED variants could lead to the finding of other MED complexes with different functions depending on their subunit composition. Finally, the expression profile of all MED genes, together with an extensive gene expression analysis, may be useful to better classify the diverse subsets of cell populations that contribute to neovascularization. © 2014.

Despite recent treatment advances, an increase in cardiovascular diseases (CVD) mortality is expected for the next years. Mediator (MED) complex plays key roles in eukaryotic gene transcription. Currently, while numerous studies have correlated MED alterations with several diseases, like cancer or neurological disorders, fewer studies have investigated MED role in CVD initiation and progression. The first finding of MED involvement in these pathologies was the correlation of missense mutations in MED13L gene with transposition of the great arteries. Nowadays, also MED13 and MED15 have been associated with human congenital heart diseases and others could be added, like MED12 that is involved in early mouse development and heart formation. Interestingly, a missense mutation in MED30 gene causes a progressive cardiomyopathy in homozygous mice suggesting a potential role for this subunit also in human CVDs. Moreover, several subunits like MED1, MED13, MED14, MED15, MED23, MED25 and CDK8 exert important roles in glucose and lipid metabolism. Although these evidences derive from in vitro and animal model studies, they indicate that their deregulation may have a significant role in human CVD-related metabolic disorders. Finally, alternative transcripts of MED12, MED19 and MED30 are differently expressed in circulating endothelial progenitor cells thus suggesting they can play a role in the field of regenerative medicine. Overall, further functional studies exploring MED role in human CVD are warranted. The results could allow identifying novel biomarkers to use in combination with imaging techniques for early diagnosis; otherwise, they could be useful to develop targets for novel therapeutic approaches. © 2014 Elsevier B.V.

Mediator complex (MED) is an evolutionarily conserved multiprotein, fundamental for growth and survival of all cells. In eukaryotes, the mRNA transcription is dependent on RNA polymerase II that is associated to various molecules like general transcription factors, MED subunits and chromatin regulators. To date, transcriptional machinery dysfunction has been shown to elicit broad effects on cell proliferation, development, differentiation, and pathologic disease induction, including cancer. Indeed, in malignant cells, the improper activation of specific genes is usually ascribed to aberrant transcription machinery. Here, we focus our attention on the correlation of MED subunits with carcinogenesis. To date, many subunits are mutated or display altered expression in human cancers. Particularly, the role of MED1, MED28, MED12, CDK8 and Cyclin C in cancer is well documented, although several studies have recently reported a possible association of other subunits with malignancy. Definitely, a major comprehension of the involvement of the whole complex in cancer may lead to the identification of MED subunits as novel diagnostic/prognostic tumour markers to be used in combination with imaging technique in clinical oncology, and to develop novel anti-cancer targets for molecular-targeted therapy. © 2013.

Introduction: Real time RT-PCR is a widely used technique to evaluate and confirm gene expression data obtained in different cell systems and experimental conditions. However, there are many conflicting reports about the same gene or sets of gene expression. A common method is to report the interest gene expression relative to an internal control, usually a housekeeping gene (HKG), which should be constant in cells independently of experimental conditions. Materials and Methods: In this study, the expression stability of ten HKGs was considered in parallel in two cell systems (endothelial and osteosarcoma cells): beta actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Cyclophilin A (PPIA), beta-2-microglobulin (B2M), glucuronidase beta (GUSB), eukaryotic translation elongation factor 1 alpha1 (EEF1A1), transferrin receptor (TFRC), ribosomal protein S18 (RPS18). In order to study the stability of candidate reference genes, data have been also analyzed by several algorithms (geNorm, NormFinder, BestKeeper and delta-Ct method). Results and Conclusions: The overall analysis obtained by the comprehensive ranking showed that RPS18 and PPIA are appropriate internal reference genes for tumor neovascularization studies where it is necessary to analyze both systems at the same time. © 2012 Federación de Sociedades Españolas de Oncología (FESEO).

Oxidation and glycation enhance foam cell formation via MAPK/JNK in euglycemic and diabetic subjects. Here, we investigated the effects of glycated and oxidized LDL (glc-oxLDL) on MAPK-ERK and JNK signaling pathways using human coronary smooth muscle cells. Glc-oxLDL induced a broad cascade of MAPK/JNK-dependent signaling transduction pathways and the AP-1 complex. In glc-oxLDL treated coronary arterioles, tumor necrosis factor (TNF) a increased JNK phosphorylation, whereas protein kinase inhibitor dimethylaminopurine (DMAP) prevented the TNF-induced increase in JNK phosphorylation. The role of MKK4 and JNK were then investigated in vivo, using apolipoprotein E knockout (ApoE-/-) mice. Peritoneal macrophages, isolated from spontaneously hyperlipidemic but euglycemic mice showed increases in both proteins and phosphorylated proteins. Compared to streptozotocin-treated diabetic C57BL6 and nondiabetic C57BL6 Wt mice, in streptozotocin-diabetic ApoE-/- mice, the increment of foam cell formation corresponded to an increment of phosphorylation of JNK1, JNK2, and MMK4. Thus, we provide a first line of evidence that MAPK-ERK/JNK pathways are involved in vascular damage induced by glycoxidation. J. Cell. Physiol. 227: 36393647, 2012. (C) 2012 Wiley Periodicals, Inc.

Mediator (MED) is a fundamental component of the RNA polymerase II-mediated transcription machinery playing a pivotal role in the regulation of eukaryotic mRNA synthesis. Human MED complexes contain at least 30 distinct MED subunits. Our previous study, aimed to analyse MED complex during the pattern of endothelial progenitor cells (EPCs) differentiation, found an alternative transcript of MED30 subunit expressed only in circulating immature progenitor cells. Here, we report two novel transcripts of MED12 and MED19 subunits both generated by alternative splicing and displaying similar expression patterns, thereby indicating their involvement during endothelial cell differentiation. © 2012 Elsevier Masson SAS. All rights reserved.

Background Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome. Methods Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. Results We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. Conclusions Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.

BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and Objective: Peripheral arterial disease (PAD) is a major health problem especially when associated to concomitant diabetes and hypercholesterolemia. Hyperglycemia with an overwhelming generation of oxygen radicals and formation of glycation end-products exacerbates oxidation-sensitive mechanisms activated by tissue ischemia. Administration of autologous bone marrow cells (BMC) is an increasing notable intervention to induce therapeutic angiogenesis, ameliorated by metabolic intervention (MT). Recently, hemangioblasts (HS) with functional properties were isolated. Methods: The effects of integrate regimen with intravenous BMC, HS, and MT (1.0% vitamin E, 0.05% vitamin C, and 6% l-arginine) were examined in the ischemic hindlimb of ApoE-/- diabetic and non-diabetic. Blood flow ratio was monitored by use of a laser Doppler blood flowmeter. Capillary density was determined in sections of the adductor and semimembranous muscles with antibody against CD31. Results: BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and, more consistently, in combination with MT ameliorated functional activity via decreased cellular senescence and increased telomerase and chemokine CXCR4 activities. Telomerase activity was also increased by HS alone or HS. +. MT and, more consistently, by BMC. +. HS alone or in combination with MT. Conclusions/interpretation: Intravenous autologous BMC and HS intervention together with MT increased therapeutic angiogenesis in the ApoE-/- diabetic mouse hindlimb. © 2009 Elsevier Ireland Ltd.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae . Nitric oxide (NO) and its precursor l -arginine ( l -arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l -arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l -arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella -infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l -arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Nebivolol is a cardioselective beta-blocker (BB) currently used for the treatment of hypertension. It has mild vasodilating properties attributed to its interaction with the L-arginine/nitric oxide pathway, a property not shared by other BBs. Carvedilol is a nonselective ß-adrenergic receptor antagonist that also blocks α1-adrenergic receptors and is a potent antioxidant. Anthracyclines (ANTs), daunorubicin and doxorubicin, are commonly used in the treatment of several tumours, but their cardiac toxicity prevents their use at maximum myelotoxic doses, representing an important problem. In this study, we have evaluated the role of these BBs administered in combination with ANTs (daunorubicin and doxorubicin) on a reduction in cardiac toxicity. The combination of BB and ANTs has reduced the release of GSSG and GSH; in particular, co-treatment with nebivolol to ANTs has shown a significant reduction. The total integrated creatine kinase and troponin T activities were improved by BB and ANTs co-treatment. A significant reduction of their release was observed when hearts were treated with nebivolol. Cardiac tissue activity of gluthatione reductase was not significant and similar among experimental groups. In contrast, gluthatione peroxidise, Mn-superoxide dismutase and nitrite/nitrate release were increased after co-treatment with nebivolol. Finally, three parameters have been used to evaluate the cardiac toxicity of ANTs: the left ventricular pressure developed under a constant perfusion pressure (LVDP), the rate of variation of this parameter during systole (contractility) (LV/dt)max and during diastole (relaxation) (LV(dP/dt)min. Combination with BB has shown a reduction in cardiac toxicity; in particular, nebivolol has exerted the most significant cardioprotective effect. © 2007 Elsevier Ltd. All rights reserved.

Previous studies showed that p66(Shc-/-) mice on a very-high-fat diet (HFD) had reduced oxidative stress, foam cell, and early atherosclerotic lesion formation. Here, the authors have used hypercholesterolemic apolipoprotein E (ApoE(-/-)) mice to investigate the role of p66Shc deletion in advanced atheroma. The authors generated mice deficient of both ApoE and p66Shc genes (ApoE(-/-) /p66(Shc-/-)). They used microsatellite polymerase chain reaction (PCR) analysis to analyze the genetic background and considered only animals with a constant percentages of C57B6L and 129SV background strands (it was obtained the 50.3% +/- 6.4% of C57B6L background). Computer-assisted analysis revealed that advanced atherosclerotic lesions in ApoE(-/-)/p66(Shc+/+) were significantly larger than those observed in ApoE(-/-)/p66(Shc-/-). Accordingly, the lipid-laden macrophage foam cells and oxidation-specific epitopes in ApoE(-/-)/p66(shc+/+) HFD-treated groups were higher than those observed in normal diet (ND)-treated groups. Thus, p66(Shc-/-) plays an important protective role also against advanced atherosclerotic lesion formation. Finally, the authors have used microarray to investigate major changes in gene expression in aortas of mice with ApoE(-/-)/p66(Shc-/-) background treated with a very HFD in comparison to ApoE(-/-)/p66(Shc+/+) (these data have been confirmed by by real-time PCR and immunohistochemistry). DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis revealed that CD36 antigen (CD36), tissue inhibitor of metalloproteinase 2 (TIMP2), apolipoprotein E (ApoE), acetyl-coenzyme A acetyltransferase 1 (ACAT1), and thrombospondin 1 (THBS1) can be involved in p66 deletion-dependent vascular protection through the adipocytokine/lipid signaling pathway.

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Despite advances in imaging, understanding the underlying pathways, and clinical translation of animal models of disease there remains an urgent need for therapies that reduce brain damage after stroke and promote functional recovery in patients. Blocking oxidant radicals, reducing matrix metalloproteinase-induced neuronal damage, and use of stem cell therapy have been proposed and tested individually in prior studies. Here we provide a comprehensive integrative management approach to reducing damage and promoting recovery by combining biological therapies targeting these areas. In a rat model of transient cerebral ischemia (middle cerebral artery occlusion) gene delivery vectors were used to overexpress tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP1 and TIMP2) 3 days before ischemia. After occlusion, autologous bone marrow cells alone or in combination with agents to improve NO bioavailability were administered intraarterially. When infarct size, BrdU incorporation, and motor function recovery were determined in the treatment groups the largest beneficial effect was seen in rats receiving the triple combined therapy, surpassing effects of single or double therapies. Our study highlights the utility of combined drug, gene, and cell therapy in the treatment of stroke. © 2007 by The National Academy of Sciences of the USA.

Cyclooxygenases (COXs) are enzymes catalysing prostaglandin synthesis and are implicated in the carcinogenesis of some cancer types. In addition, an important role of these enzymes in herpesvirus infections was demonstrated and it has recently been proposed that COX-2 may participate in herpesvirus-induced neoplasia such as Kaposi's sarcoma (KS). To date no immunohistochemical study has been performed to determine the identification of COX-1 and COX-2 in KS. We have investigated 35 cases of classic KS and 27 cases of epidemic KS form in order to study the distribution and localisation of COXs. We have examined by immunohistochemistry the expression of COX-1 and COX-2 in classic and epidemic forms of KS also in relationship to the characteristic morphological phases (patch, plaque and nodular stage) of KS and cell localisation by double immunostaining. Moreover, we have obtained COX-1 and COX-2 expression by Western blot analysis. Our results establish that (a) COX-1 and COX-2 are overexpressed significantly in classic and epidemic KS compared with control skin tissues (P < 0.01 and P > 0.03, respectively, for COX-1; P < 0.01 and P > 0.03, respectively, for COX-2); (b) the extent and intensity staining for both COXs were higher in classic than in epidemic form of KS. Our data support the hypothesis that both COXs may be involved in the pathogenesis of KS. © 2007 Elsevier Ltd. All rights reserved.

Objective Maternal hypercholesterolaemia during pregnancy increases lipid peroxidation in mothers and fetuses and programs increased susceptibility to atherosclerosis later in life. The objective of this study was to elucidate the role of the placenta in mediating oxidative stress from mother to offspring. Design Comparison between normo- and hypercholesterolaemic mothers (n = 36 each) and their children. Setting Obstetric wards, hospitals of the University of Naples and Regione Campania. Population Healthy primiparas delivering by caesarean section. Methods Biochemical measurements of oxidative stress and serum leptin in cord plasma and placenta, immunochemistry of placenta microvessels, and vasoreactivity studies were performed. Main outcome measures Oxidative status (i.e. lipid composition and content of oxidised fatty acids, activity of pro- and antioxidant enzymes, immunohistochemical presence of oxidation-specific epitopes) in maternal and cord blood and in placental tissue, as well as vascular reactivity in omental arteries. Results Hypercholesterolaemia during pregnancy was associated with extensive changes in fatty acid composition of both maternal and cord blood lipids, sufficient to alter vasoreactivity of omental vessels. Results also indicated that the placenta is not only subject to substantial oxidative stress, but that it may further increase fetal oxidative stress through changes of pro- and antioxidant enzyme activities. Conclusions The placenta plays an important role in both transmitting and enhancing pathogenic effects of gestational hypercholesterolaemia.

We have identified and characterized a new pair of LINE and SINE elements, called Lucy-1 CR1-like LINE and P.s.1/SINE, respectively, in Podarcis sicula genome. The 3′-tail region in the 3′ untranslated region (UTR) of Lucy-1 element is almost identical to the of P.s.1/SINE element. This identity suggests that the P.s.1/SINE element, during evolution, has gained the 3′-end sequence of the Lucy-1 element and has exclusively recruited the enzymatic machinery of its partner CR1 LINE for retroposition. Moreover, the complex molecular organization around Lucy-1 insertion site is discussed and we found that Lucy-1 insertion is associated with the calcium binding transporter gene. Our results confirm that the retrotransposons can be an additional source of genomic diversification and the evolution of the retrotransposable elements can be a vector force shaping genomes by reassorting DNA domains thus forming a new DNA arrangement.

Aim Heart transplantation has evolved as the “gold standard” therapy for patients with end-stage heart failure (HF). Although mechanical circulatory support technology is improving, heart transplantation remains the preferred treatment. The mean risk factors for HF include environmental and genetic components. The intricate patterns of gene expression, during normal and disease states, are governed by elaborate signaling pathways that converge on the transcriptional mechanism. The general transcriptional mechanism can be recruited to promoters by Mediator complex (MED). MED is a large multi-subunit complex functioning to integrate diverse cellular signals by multiple mechanisms including recruitment of RNA polymerase II, chromatin modifying proteins and non-coding RNAs to promoters in a context dependent manner. Several MED subunits are involved in many human pathologies, including heart diseases. Particularly, some subunits like MED13 and MED13L are altered in congenital heart diseases. Similarly, MED30 mutation can lead to lethal cardiomyopathy in a mouse model. Methods To gain further insights, we have collected 12 human left atrium samples, which were acquired from hearts of 6 donors and 6 recipients during transplant intervention. Patients were affected by hypertrophic or dilated idiopathic cardiomyopathy. From these samples we have isolated RNA and started to analyze the expression patterns of MED subunits by real-time RT-PCR with specific oligonucleotides. Possible interactions between human leukocyte antigen (HLA) profile and MEDs are under investigation. Furthermore, RNA-seq analysis of samples from both donors and recipients is ongoing in our laboratory and whole transcriptome data will be also shown. Results Our preliminary data confirm that some MED subunits are altered in diseased hearts compared to healthy donor biopsies. Conclusions The identification of such alterations can lead to novel therapeutic targets regulating MED subunits, and HLA in advanced HF.

Aim Mediator (MED) complex functions as a pivotal adaptor between transcription factors bounded at gene regulatory elements, RNA polymerase II and general transcription factors. Different ancestral humanMED complexes including at least 30 distinct MED subunits (MEDs) have been isolated. Because of the importance of ancestral MED role in the transcription of the eukaryotic genes, disruption of MED function may have relevant pathophysiological consequence also in the cardiovascular system and transplantation. Here, we have analyzed the expression data relative to MEDs in human endothelial progenitor cells (EPCs) obtained by RNA-Seq on a next generation sequencing platform. The introduction of next generation sequencing (NGS) technologies has revealed the complexity of mammalian transcriptomes, enabling to effectively explore, with an unprecedented throughput capacity, simple and complex genomes and even their differences in health and disease conditions. Methods RNA was isolated from early human EPCs and after ribodepletion it was used for the RNA-Seq through SOLID System. Results By analysis of RNA-Seq data and RT-PCR validation we have identified novel transcripts in several MED genes (including MED1, MED 15, MED 17 and MED23). Some of these transcripts are different in their 5’ and 3’ UTRs. Other transcripts are differently spliced thus excluding or including known/new exons. Conclusions This findings contribute to the characterization of novel MED transcripts in EPCs that could participate to the regulation of genes involved in different cell states. Our findings could have relevant implications in the regenerative action of EPCs in the cardiovascular system immune response after cardiac transplantation.

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenylated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes--possibly novel miRNA targets or regulatory sites for gene transcription--were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders.