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In recent years, the scientific community has stressed the need to invest in the first 1,000 days of life - the time spanning between conception and the 2nd birthday - because it is during this period that the foundations of health are laid and whose effects will be present throughout the life and may influence the next generation. Taking this into account, in 2013 the National Centre for Disease Prevention and Control (CCM) of the Italian Ministry of Health promoted and financed a project to test a surveillance system of the main determinants of health concerning the child between the conception period and the 2nd years of life which are included in the National Programme "GenitoriPiù": folic acid before and during pregnancy, abstention from tobacco and alcohol during pregnancy and lactation, breastfeeding, infant sleep position, vaccination attitude, and early reading. The Project, started in January 2014 and ended in August 2016, has piloted the design, testing, and evaluation of the surveillance system with the view to national extension and the repeatability over time. The surveillance system has been designed to collect data through a questionnaire compiled by mothers in vaccination centres, in order to produce indicators which will enable territorial and intertemporal comparisons to be made. The project has shown the feasibility of this system, identifying favourable conditions and possible difficulties, and its ability to collect important information on children's health.

We report that in endothelial cells, the angiogenic effect of 17beta-estradiol (E2) is inhibited by the estrogen receptor (ER) antagonist ICI or the NO synthase (NOS) inhibitor 7-nitroindazole via downregulation of hTERT, the telomerase catalytic subunit, suggesting that E2 and NO are involved in controlling hTERT transcription. Quantitative Real-Time PCR and chromatin immunoprecipitations in E2-treated human umbilical vein endothelial cells, showed recruitment of ERs on the hTERT promoter and concomitant enrichment in histone 3 methylation at Lysine 79, a modification associated with transcription-competent chromatin. Confocal microscopy and re-chromatin immunoprecipitations revealed that on E2 induction, endothelial (e)NOS rapidly localized into the nucleus and associated with ERalpha on the hTERT promoter. Transfections of a constitutively active eNOS mutant (S1177D) strongly induced the hTERT promoter, indicating a direct role of the protein in hTERT transcriptional regulation. Mutation of the estrogen response element in the promoter abolished response to both ERs and active eNOS, demonstrating that the estrogen response element integrity is required for hTERT regulation by these factors. To investigate this novel regulation in a reduced NO environment, pulmonary endothelial cells were isolated from eNOS(-/-) mice and grown with/without E2. In wild-type cells, E2 significantly increased telomerase activity. In eNOS(-/-) cells, basal telomerase activity was rescued by exogenous eNOS or an NO donor, whereas responsiveness to E2 demanded the active protein. In conclusion, we document the novel findings of a combinatorial eNOS/ERalpha complex at the hTERT estrogen response element site and that active eNOS and ligand-activated ERs cooperate in regulating hTERT expression in the endothelium.

The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)- and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinase-dead form of AKT1 into breast cancer cells abrogated TMZ-induced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O-6-guanine-methylating agents.