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Additional file 2.

Figure S1. Representative images of in vitro cell migration and invasion in scramble control (si-Ctrl) or Sema5A silenced (si-Sema5A) and plasmid control (Ctrl) or ECD-Sema5A overexpressing (ECD-Sema5A) M14 cells. Figure S2. Representative images of in vitro cell migration of plasmid control (Ctrl) or ECD-Sema5A overexpressing (ECD-Sema5A) M14 cells treated with 10 nM Trametinib or drug vehicle (untreated) for 6 h, prior performing migration assay. Figure S3. Western blotting analysis of Sema5A expression in M14 melanoma cells stably overexpressing the full-length Sema5A protein (FL-Sema5A/1 and Sema5A/6). HSP70/72 expression was evaluated to confirm equivalent transfer and loading. Representative images of two independent experiments are reported. Figure S4. Western blotting analysis of c-Myb protein expression in melanoma parental cell lines, cultured as previously reported [22, 26, 27]. Reported images are representative of two independent experiments with similar results. β-actin expression was evaluated to confirm equivalent transfer and loading. Representative images of two independent experiments are reported. (PDF 630 kb)

Table S1. Primer sequences used in ChIP analysis. (PDF 224 kb)

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly. published

The aim of the present study was to investigate the proportion of physicians and nurses who agree with the administration of antibiotic therapy (AT), artificial hydration (AH), and artificial nutrition (AN) in patients with advanced dementia and different life expectancies. Furthermore, we aimed at analyzing the correlates of the opinion according to which medical treatments should no longer be given to advanced dementia patients once their life expectancy falls.End-of-life decisions and opinions were measured with a questionnaire that was sent to geriatric units, hospices and nursing homes in three different regions of Italy. Multivariate logistic regressions were carried out to ascertain the correlates of the agreement with the administration of AH, AT or AN.When the patient's life expectancy was 1-6 months, 83% of respondents agreed with AH, 79% with AT and 71% with AN. When the life expectancy was less than 1 month, a large proportion of respondents still agreed with AH and AT (73% and 61%), whereas less than one in two respondents (48%) agreed with AN.The findings of the present study showed that AN creates more ethical dilemmas in the clinical management of end of life than other treatments, such as AH or AT. Opinions on whether or not these practices are appropriate at the end of life were related with feelings, thoughts and ethical issues that played a different part for physicians and nurses. Geriatr Gerontol Int 2017; 17: 487-493.

Background. In developed societies, the main causes of death are represented by degenerative illnesses, and an increasing number of patients in intensive care yards remain in a coma or in a terminal phase for long time. In such a context, clinical decisions at the end of life are becoming more and more complex, and involve medical, psychological and ethical aspects. A multidisciplinary reflection on these problems increased the interest toward end-of-life issues in the national and international context, highlighting important changes in shared thoughts and different opinions on these topics by Italian professionals compared to European or American colleagues. Topics such as end-of-life decisions, cures and treatments in the terminal phase, respect for living will are increasingly present in the literature. However, only a few studies investigated these topics in the geriatric field, particularly in Italy. Aims. The E.L.D.Y. study (End of Life Decision studY) aims at: investigate the type and frequency of decisions taken, in the clinical practice during the terminal phase of patients’ life, by physicians and professional nurses, especially in the geriatric field in the Italian Regions Veneto, Trentino Alto-Adige and Marche; assess the opinions concerning end-of-life decisions by professionals involved in the assistance of elderly patients; analyze the possible associations between end-of-life opinions and individual characteristics; compare the obtained findings with those of similar studies previously conducted both in the Italian and in the European context. Materials and Methods. Through an articulated multidisciplinary work, a new questionnaire has been constructed, taking into account the critiques formulated to the previous national and international studies. The instrument was send to approximately 6000 physicians, nurses, psychologists and healthcare workers in elderly patients assistance units in Veneto, Trentino Alto-Adige and Marche Regions. The questionnaire was composed by three parts: the first, to be compiled by physicians and nurses only, concerned the decisions taken about the last death patient they assisted; the second, regarding health workers in general, aimed at investigating end-of-life related opinions; the third assessed some individual characteristics of the professionals. Results. Respondents were 1545 social and health workers: 301 physicians (MD), 788 registered nurses (RN), 231 healthcare workers, 108 psychologists; 117 were other workers in the structures. The questionnaires that met the selection criteria for the statistical analyses were 680 for end-of-life decisions and 1425 for the investigated opinions. A total of 161 decisions to end life (DEL) emerged (24% of deaths that did not occur suddenly and that professionals had the opportunity to follow). They were divided into 141 non-treatment decisions and 20 cases of medically assisted death. Among the latter, 2 were cases of euthanasia (MD: 1 and RN: 1), while 16 were cases of death suppression without explicit request by the patient (MD: 3 and RN: 13). Overall, 1.4% of physicians stated that they intervened directly and intentionally to anticipate the end of life. Fifty percent of physicians reported that they had started or not stopped a treatment taking into account the possibility that this action could have prolonged the patient’s life; the 31%, instead, reported that they did not start or that they stopped a treatment taking into account the possibility that this action could have hastened the death of a terminal ill patient. Concerning opinions, only a minority (26%) of physicians and nurses agree with the principle according to which there is not a right to die, while the majority (76%) agree with the right of a patient to decide on the possibility to hasten the end of life. About half of our respondents (52%) think that it is acceptable to use lethal drugs if requested by a terminally ill patient with untreatable suffering. About 60% of participants think that living will should always be respected, even if this could hasten death. Finally, the separation of the items concerning artificial nutrition and artificial hydration allowed us to observe that the latter is administered more frequently than the former, suggesting that artificial hydration only is considered as a basic form of care. Concerning communication, results indicate that physicians tend to discuss the proposed topics more with relatives than with patients, differently from what emerged in other European countries. Regarding differences between professionals, the main ones concern the answers provided by psychologists. The psychologists in our study, almost all from Triveneto, consistently with their professional role, tend to attribute particular importance to topics such as communication with patients, respect of their indications and will, and the act of listening in the relations with relatives. The main differences related to regional origin concern the administration of treatments such as artificial nutrition, dialysis and ventilation, that is considered as more necessary in Marche than in Triveneto. Conclusions. The conducted study presents some limitations: data concern three Italian Regions, two from the North and one in Central Italy and, therefore, in the future it would be useful to extend the investigation to other Italian Regions and possibly to other nations; many of our variables regard the opinions of respondents and not only their actual behaviours. Despite these limitations, this work is certainly useful to provide indications concerning fundamental problems of a clinical, psychological and ethical nature that health professionals encounter when facing end-of-life issues. Moreover, given the high percentages of respondents who reported interest toward the questionnaire, so as the absence of substantial criticisms, we can conclude that the aim of creating a new instrument, useful to effectively assess practices and opinions related to end-of-life in Italy, has been reached.

// Fabio Ciccarone 1,2 , Elisabetta Valentini 1,2 , Maria Giulia Bacalini 3 , Michele Zampieri 1,2 , Roberta Calabrese 1,2 , Tiziana Guastafierro 1,2 , Germano Mariano 1,2 , Anna Reale 1,2 , Claudio Franceschi 3 and Paola Caiafa 1,2 1 Department of Cellular Biotechnologies and Hematology, “Sapienza” University of Rome, Rome, Italy 2 Pasteur Institute-Fondazione Cenci Bolognetti, Rome, Italy 3 Department of Experimental Pathology, Alma Mater Studiorum, University of Bologna, Bologna, Italy Correspondence: Fabio Ciccarone, email: // Paola Caiafa, email: // Keywords : poly(ADP-ribosyl)ation, TET1, DNA methylation, 5-hydroxymethylcytosine Received : February 07, 2014 Accepted : April 16, 2014 Published : April 17, 2014 Abstract 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 expressionmaintaining 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 down-regulation 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.