In vitro analyses of Merkel cell polyomavirus cell biology

The work described in this dissertation was initiated in 2008, shortly after the discovery of Merkel cell polyomavirus (MCV). MCV was discovered as clonally integrated in 80% of Merkel cell carcinoma (MCC) and tumor-derived viral genomes were found to harbor individual large T antigen (LT) truncating deletions. The work presented in this thesis focuses on two main features associated with MCV cell biology: (1) Viral replication requirements that promote viral origin replication in the context of full length MCV LT (chapter 3) and (2) cellular targets that enhance human fibroblast proliferation in the presence of tumor-derived full length LT (chapter 4). As part of these studies, the novel MCV-positive MCC cell line MS-1 was generated, and its cellular and viral features are presented here (chapter 2). MCC cell lines are useful tools that can be used to study MCV biology, and test therapeutic compounds in vitro, as well as in a xenograft setting in vivo as described in the appendix of this thesis. Within the past 6 years, 8 new human polyomaviruses have been identified through basic biological approaches, highlighting the possibility that other human malignancies may be associated with polyomaviral infections. The identification of MCV in association with an aggressive human cancer also underscores the relevance of basic research in understanding human disease

Poly(ADP-ribosyl)ation is involved in the epigenetic control of TET1 gene transcription

TET enzymes are the epigenetic factors involved in the formation of the Sixth DNA base 5-hydroxymethylcytosine, whose deregulation has been associated with tumorigenesis. In particular, TET1 acts as tumor suppressor preventing cell proliferation and tumor metastasis and it has frequently been found down-regulated in cancer. Thus, considering the importance of a tight control of TET1 expression, the epigenetic mechanisms involved in the transcriptional regulation of TET1 gene are here investigated. The involvement of poly(ADP-ribosyl)ation in the control of DNA and histone methylation on TET1 gene was examined. PARP activity is able to positively regulate TET1 expression maintaining a permissive chromatin state characterized by DNA hypomethylation of TET1 CpG island as well as high levels of H3K4 trimethylation. These epigenetic modifications were affected by PAR depletion causing TET1 downregulation and in turn reduced recruitment of TET1 protein on HOXA9 target gene. In conclusion, this work shows that PARP activity is a transcriptional regulator of TET1 gene through the control of epigenetic events and it suggests that deregulation of these mechanisms could account for TET1 repression in cancer

Methylation-dependent PAD2 upregulation in multiple sclerosis peripheral blood

Background: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. Objectives: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. Methods: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. Results: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. Conclusion: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS

Validation of suitable internal control genes for expression studies in aging.

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors

PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression

To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosi

Ligaclip for Preauricular Skin Tags in the Newborn

Introduction Skin tags are benign lesions, that often represent only an aesthetic problem; if subjected to trauma, they can occasionally bleed with possible infection and pain. When they occur in the preauricular region, attention should be paid to the diagnosis and approach; in fact, controversy exists in the differential diagnosis between hair follicle nevi, accessory tragus, and skin tag. Misdiagnosis and failure of treatment can lead to serious consequences, such as chondritis. Materials and Methods In our retrospective study, we evaluated 19 newborns affected by single, unilateral skin tag in the preauricular region. Each patient underwent a careful clinical examination; lesions without a pilosebaceous unit and with a thin, soft pedicle were treated in the nursery with Ligaclip (Johnson & Johnson). Results Skin tag falls between day 7 and 10. We had no cases of edema, cellulitis, clip loss, or bleeding. Scarring results were extremely satisfactory at 3-month follow-up. Conclusion We believe that after a careful clinical examination, cases of skin tags in the preauricular area can be selected and treated with Ligaclip. This procedure can be considered rapid, safe, economical, and simple in the newborn patients

DNA hydroxymethylation levels are altered in blood cells from Down syndrome persons enrolled in the MARK-AGE project

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the ageing process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work we investigated the levels of 5-hydroxymethylcytosine (5hmC) and of the TET dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in ageing. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease of 5hmC, TET1 and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS

Global burden of headache disorders in children and adolescents 2007–2017

Headache disorders are prevalent and disabling conditions impacting on people of all ages, including children and adolescents with substantial impact on their school activities and leisure time. Our study aims to report specific information on headaches in children and adolescents based on the Global Burden of Disease (GBD) study, that provides estimates for incidence, prevalence, fatal and non-fatal outcomes. We relied on 2007 and 2017 GBD estimates for prevalence and Years Lived with Disability (YLDs) at the global level and in WHO regions. The results show that, migraine and tension-type headache (TTH) together account for 37.5% of all-cause prevalence and for 7% of all-cause YLDs. Over the past decade, prevalence rates showed a mild increase of TTH in all ages and of migraine alone for adolescents. The YLDs increased among females of all ages with some regional differences that might be connected to the unequal availability of effective acute and prophylactic treatments across world regions. GBD data support the need to promote public health policies and strategies including diagnosis, pharmacological and non-pharmacological treatments that are expected to help reduce the disability and burden associated to migraine and TTH among children and adolescents

Non-pharmacological approaches to headaches: Non-invasive neuromodulation, nutraceuticals, and behavioral approaches

Significant side effects or drug interactions can make pharmacological management of headache disorders very difficult. Non-conventional and non-pharmacological treatments are be-coming increasingly used to overcome these issues. In particular, non-invasive neuromodulation, nutraceuticals, and behavioral approaches are well tolerated and indicated for specific patient categories such as adolescents and pregnant women. This paper aims to present the main approaches reported in the literature in the management of headache disorders. We therefore reviewed the available literature published between 2010 and 2020 and performed a narrative presentation for each of the three categories (non-invasive neuromodulation, nutraceuticals, and behavioral therapies). Regarding non-invasive neuromodulation, we selected transcranial magnetic stimulation, supraor-bital nerve stimulation, transcranial direct current stimulation, non-invasive vagal nerve stimulation, and caloric vestibular stimulation. For nutraceuticals, we selected Feverfew, Butterbur, Riboflavin, Magnesium, and Coenzyme Q10. Finally, for behavioral approaches, we selected biofeedback, cognitive behavioral therapy, relaxation techniques, mindfulness-based therapy, and acceptance and commitment therapy. These approaches are increasingly seen as a valid treatment option in headache management, especially for patients with medication overuse or contraindications to drug treatment. However, further investigations are needed to consider the effectiveness of these approaches also with respect to the long-term effects

Circumferential dissection of deep fascia as ancillary technique in circumcision: is it possible to correct phimosis increasing penis size?

Background: Phimosis is the inability to retract the preputium downward over the glans penis. Despite the various techniques of preputial plasty described in literature, the most performed surgical treatment is still the conventional circumcision.Methods: In this paper we retrospectively reviewed data of a homogeneous population of 36 consecutive adult patients who underwent phimosis correction by circumcsion with dissection of the Deep Fascia. Patients were followed up by one independent plastic surgeon that measured penis length and circumference in nonerected state preoperatively and at 6 month time postoperatively.Results: The Wilcoxon Signed Rank Test showed a significant (p < 0.0001) difference between the two groups both in terms of length and circumference.Conclusions: In conclusion, the ancillary technique we described leads to an increase of penis size, is safe and easy to perform and does not increase significantly operative time nor complication rate to the conventional procedure