Innovative Approaches to Active and Healthy Ageing: Campania Experience to Improve the Adoption of Innovative Good Practices.

The demographic projections on the European population predict that people aged over 60 will increase by about two million/year in the next decades. Since 2012, the Campania Reference Site of the European Innovation Partnership on Active and Healthy Ageing supports the innovation of the Regional Health System, to face up demographic changes and sustainability. Campania Reference Site provides the opportunity to connect loco-regional stakeholders in social and health care services (universities, healthcare providers, social services, local communities and municipalities), with international organizations, in order to adopt and scale up innovative solutions and approaches. This paper describes the building process of Campania Reference Site and the main results achieved, that have been allowing it to become a hub for open innovation in the field of active and healthy aging at regional, national and international level.

Pilot study on microvascular anastomosis: performance and future educational prospects.

The introduction of microvascular free flaps has revolutionised modern reconstructive surgery. Unfortunately, access to training opportunities at standardised training courses is limited and expensive. We designed a pilot study on microvascular anastomoses with the aim of verifying if a short course, easily reproducible, could transmit microvascular skills to participants; if the chosen pre-test was predictive of final performance; and if age could influence the outcome. A total of 30 participants (10 students, 10 residents and 10 surgeons) without any previous microvascular experience were instructed and tested during a single 3 to 5 hour course. The two microanastomoses evaluated were the first ever performed by each participant. More than the half of the cohort was able to produce both patent microanastomoses in less than 2 hours; two-thirds of the attempted microanastomoses were patent. The pretest predicted decent scores from poor performances with a sensitivity of 61.5%, specificity of 100%, positive predictive value of 100% and negative predictive value of 40%. Students and residents obtained significantly higher scores than surgeons. Since our course model is short, cost-effective and highly reproducible, it could be introduced and implemented anywhere as an educational prospect for preselecting young residents showing talent and natural predisposition and having ambitions towards microvascular reconstructive surgery.

Head and neck reconstruction with pedicled flaps in the free flap era.

Nowadays, the transposition of microvascular free flaps is the most popular method for management of head and neck defects. However, not all patients are suitable candidates for free flap reconstruction. In addition, not every defect requires a free flap transfer to achieve good functional results. The aim of this study was to assess whether pedicled flap reconstruction of head and neck defects is inferior to microvascular free flap reconstruction in terms of complications, functionality and prognosis. The records of consecutive patients who underwent free flap or pedicled flap reconstruction after head and neck cancer ablation from 2006 to 2015, from a single surgeon, in the AOUC Hospital, Florence Italy were analysed. A total of 93 patients, the majority with oral cancer (n = 59), were included, of which 64 were pedicled flap reconstructions (69%). The results showed no significant differences in terms of functional outcome, flap necrosis and complications in each type of reconstruction. Multivariate regression analysis of flap necrosis and functional impairments showed no associated factors. Multivariate regression analysis of complicated flap healing showed that only comorbidities remained an explaining factor (p = 0.019). Survival analysis and proportional hazard regression analysis regarding cancer relapse or distant metastasis, showed no significant differences in prognosis of patients concerning both types of reconstruction. In this retrospective, non-randomised study cohort, pedicled flaps were not significantly inferior to free flaps for reconstruction of head and neck defects, considering functionality, complications and prognosis.

Outcome of lumbar intervertebral foraminal stenosis surgery and depression.

A total of 58 patients consecutively underwent surgical treatment for lumbar intervertebral foraminal stenosis. We performed a microsurgical combined transarticular lateral and medial procedure with partial facetectomy in all patients to decompress the affected nerve root. All patients underwent assessment of depressive symptoms by means of the Zung Self Depression Scale (SDS). Subjective pain was self-evaluated by the Visual Analogue Scale (VAS). Both the tools were administered preoperatively, at 3 and 12 months' follow-up 0. The difference between the three SDS scores was significant (Friedman ANOVA, χ(2) = 53.171, p < 0.00001). The Wilcoxon rank test showed significant difference between preoperative SDS scores as compared with three months follow-up (Z = -6.393, p < 0.0001) and the last, in turn, as compared with twelve months follow- up (Z = -3.720, p = 0.0002). The comparison between preoperative and 12 months' follow-up also reached significance (Z = -3.285, p = 0.001). About VAS, the difference between the three VAS scores was significant (Friedman ANOVA, χ(2) = 69.932, p < 0.00001). The Wilcoxon rank test showed significant difference between preoperative VAS scores as compared with 3 months' follow-up (Z = -6.567, p < 0.0001) and the last, in turn, as compared with 12 months' follow-up (Z = -3.153, p < 0.002). The comparison between preoperative and 12 months' follow-up was also significance (Z = -5.520, p < 0.0001). Our results would alert clinicians to accurately consider the real need to treat and to include a careful psychiatric and psychological evaluation of these patients in the diagnosis and follow-up 0.

Frontal Behavioural Inventory in the differential diagnosis of dementia.

OBJECTIVE: To evaluate diagnostic properties of the Frontal Behavioural Inventory (FBI) in patients suffering from different forms of dementia. METHODS: The FBI was administered with other psychometric tests investigating cognitive performances and behavioral scales to the caregivers of 35 patients with the frontal variant of frontotemporal dementia (fv-FTD), 22 patients with Alzheimer's disease (AD) and 15 with vascular dementia (VaD). All patients were comparable for degree of dementia severity and level of executive impairment. RESULTS: The FBI showed high concurrent validity, internal consistency and good inter-rater and test-retest reliability. The discriminant validity was also very high. A new FBI cut-off score of 23 gave 97% sensitivity and 95% specificity in distinguishing fv-FTD from non-FTD patients. Conversely, the Neuropsychiatic Inventory (NPI) score was unable to differentiate fv-FTD from AD. CONCLUSIONS: The FBI is a neurobehavioral tool suitable to distinguish fv-FTD from other forms of dementia also when data from cognitive testing or other behavioral scales fail to support the differential diagnosis.

Prognostic role of depression after lumbar disc surgery.

A total of 73 patients underwent microdiscectomy for lumbar disc herniation between September 2001 and May 2002 at the Department of Neurosurgery of the Second University of Naples. Preoperatively and 3 and 6 months after surgery, patients were assessed on the Zung Self-rating Depression Scale (SDS) and on a visual analogue scale (VAS) for the subjective perception of pain. At 3 and 12 months, we found that patients with lower SDS scores (n=41) had a better outcome regarding pain than patients with relevant depressive symptoms (n=32). In agreement with the literature, our results confirm the negative role of depression in outcome after lumbar disc surgery. We emphasize the consideration of psychological factors in the management of lumbar disc herniation.

Monoclonal antibody panels for acute leukemia and myelodysplastic syndrome diagnosis. Results of a co-operative quality control group.

The need for standardization criteria and result reproducibility in immunophenotyping hematological diseases has increased along with their clinical importance. Our group "Policentric Study Group on Immunological Markers", is composed of 40 laboratories. Its aim, over recent years, has been to find a standardized way of immunophenotypic analysis applicable to various hematological diseases. The objective of this study is to contribute to the debate concerning standardization of monoclonal antibody panels and immunophenotypic analysis procedures in acute leukemia (AL) and myelodysplastic syndrome (MDS), with the following targets: to improve interlaboratory reproducibility of the immunophenotyping data, and interpretative results; to study, with improved feasibility, correlation between immunophenotype and clinical or biological findings on a large number of AL and MDS cases; to verify the utility of the proposed monoclonal antibody panels for proper AL and MDS classification, and to detect minimal residual disease. In the field of AL and MDS our experience is based on about 1800 and 700 cases respectively analyzed over the last five years. Starting from these experiences and data of the literature we have elaborated the proposed panels of monoclonal antibodies and the methods of analysis. We have suggested a standardized immunophenotypic approach to study AL and MDS. In particular our work has focused on the gating strategy. This aims at drawing a gate of analysis having high purity and recovery, and on the choice of monoclonal antibody combinations for multiparametric analysis, particularly the normal antigen expression on each step of lineage differentiation or their clinically relevant aberrant expressions. A standardized criteria has become a necessary starting point in any kind of analytical process. In the field of acute leukemias and myelodysplastic syndromes the work of this polycentric group has focused on the pre-analytical and analytical steps to be taken in cytometric evaluation of hematological malignancies. The results obtained may contribute to reaching intra and inter-laboratory reproducibility.

Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells.

Retinoic acid (RA) induces cell cycle arrest and differentiation of human neuroblastoma (NB) cells. Typically, NB cells differentiate along the neuronal lineage, but quiescent, "flat" cell types frequently have been described after treatment with differentiating agents. Two indistinguishable subclones of the cell line SK-N-SH, SK-N-SH-N (SH-N) and SK-N-SH-F (SH-F), display dramatically different responses to RA. In SH-N, RA induces neuronal differentiation, but in SH-F it transforms the small neuroblastic cells into large, flattened, epithelium-like cells. Here we analyze the mechanistic basis for the different effects of RA in the two NB subclones. First, we show that the flattened RA-treated SH-F expresses markers of cells undergoing replicative senescence. Inhibition of DNA synthesis by RA is significantly more rapid in SH-F than in SH-N. SH-F, which expresses basal amounts of p16(INK4A), responds to RA with elevation of p18(INK4C), marked down-regulation of cyclin D1, and swift inhibition of cyclin D-dependent kinases (cdks). Conversely, after addition of RA, SH-N retains cell cycling due to high expression of cyclin D1, the absence of Ink4 inhibitors, and accumulation of p21(Cip1). These changes result in sustained cdk activity. Accordingly, overexpression of p21(Cip1) but not p16(INK4A) induces neuronal differentiation of untreated NB cells. We propose that rapid inhibition of cdks by RA in NB leads to early cell cycle arrest, prevents neuronal differentiation, and results in a senescence-like state.

Supercharged protein and peptide ions formed by electrospray ionization.

The multiple charging of large molecules in electrospray ionization provides key advantages for obtaining accurate molecular weights by mass spectrometry and for obtaining structural information by tandem mass spectrometry and MS(n) experiments. Addition of glycerol or m-nitrobenzyl alcohol into the electrospray solutions dramatically increases both the maximum observed charge state and the abundances of the high charge states of protein and peptide ions. Adding glycerol to acidified aqueous solutions of cytochrome c shifts the most abundant charge state from 17+ to 21+, shifts the maximum charge state from 20+ to 23+, and shifts the average charge state from 16.6+ to 20.9+. Much less m-nitrobenzyl alcohol (<1%) is required to produce similar results. With just 0.7% m-nitrobenzyl alcohol, even the 24+ charge state of cytochrome c is readily observed. Similar results are obtained with myoglobin and (Lys)4. For the latter molecule, the 5+ charge state is observed in the electrospray mass spectrum obtained from solutions containing 6.7% m-nitrobenzyl alcohol. This charge state corresponds to protonation of all basic sites in this peptide. Although the mechanism for enhanced charging is unclear, it does not appear to be a consequence of conformational changes of the analyte molecules. This method of producing highly charged protein ions should be useful for improving the performance of mass measurements on mass spectrometers with performances that decrease with increasing m/z. This should also be particularly useful for tandem mass spectrometry experiments, such as electron capture dissociation, for which highly charged ions are desired.

Evidence that Myc isoforms transcriptionally repress caveolin-1 gene expression via an INR-dependent mechanism.

The c-Myc oncoprotein contributes to oncogenesis by activating and repressing a repertoire of genes involved in cellular proliferation, metabolism, and apoptosis. Increasing evidence suggests that the repressor function of c-Myc is critical for transformation. Therefore, identifying and characterizing Myc-repressed genes is imperative to understanding the mechanisms of Myc-induced tumorigenesis. Here, we employ NIH 3T3 cell lines harboring c-Myc-ER or N-Myc-ER to dissect the relationship between Myc activation and caveolin-1 expression. In this well-established inducible system, treatment with estrogen like molecules, such as tamoxifen, leads to activation of Myc, but in a tightly controlled fashion. Using this approach, we show that Myc activation induces the repression of caveolin-1 expression at the transcriptional level. We also provide two independent lines of evidence suggesting that caveolin-1 is a direct target of Myc: (i) the effect of Myc activation on caveolin-1 expression is independent of new protein synthesis, as revealed through the use of cycloheximide; and (ii) Myc-mediated repression of the caveolin-1 promoter is dependent on an intact INR sequence. Moreover, we show that expression of caveolin-1, via an adenoviral vector approach, can suppress cell transformation that is mediated by Myc activation. In support of these observations, treatment with an adenoviral vector harboring anti-sense caveolin-1 specifically potentiates transformation induced by Myc activation. Taken together, our results indicate that caveolin-1 is a direct target of Myc repression, and they also provide evidence for an additional mechanism by which Myc repression can elicit a malignant phenotype.

Id proteins at the cross-road of development and cancer.

A large body of evidence has been accumulated that demonstrates dominant effects of Id proteins on different aspects of cellular growth. Generally, constitutive expression of Id not only blocks cell differentiation but also drives proliferation. In some settings, it is sufficient to render cells immortal or induce oncogenic transformation. The participation of Id proteins in advanced human malignancy, where they are frequently deregulated, has been dramatically bolstered by the recent discovery that Id exert pivotal contributions to many of the essential alterations that collectively dictate malignant growth. Relentless proliferation associated with self-sufficiency in growth signals and insensitivity to growth inhibitory signals, sustained neoangiogenesis, tissue invasiveness and migration capabilities of tumor cells all share dependency on the unlimited availability of Id proteins. It is remarkable that many of these features recapitulate those physiologically propelled by Id proteins to support normal development. We propose that the participation of Id in multiple fundamental traits of cancer may be the basis for unprecedented therapeutic opportunities.

Effects of solvent on the maximum charge state and charge state distribution of protein ions produced by electrospray ionization.

The effects of solvent composition on both the maximum charge states and charge state distributions of analyte ions formed by electrospray ionization were investigated using a quadrupole mass spectrometer. The charge state distributions of cytochrome c and myoglobin, formed from 47%/50%/3% water/solvent/acetic acid solutions, shift to lower charge (higher m/z) when the 50% solvent fraction is changed from water to methanol, to acetonitrile, to isopropanol. This is also the order of increasing gas-phase basicities of these solvents, although other physical properties of these solvents may also play a role. The effect is relatively small for these solvents, possibly due to their limited concentration inside the electrospray interface. In contrast, the addition of even small amounts of diethylamine (<0.4%) results in dramatic shifts to lower charge, presumably due to preferential proton transfer from the higher charge state ions to diethylamine. These results clearly show that the maximum charge states and charge state distributions of ions formed by electrospray ionization are influenced by solvents that are more volatile than water. Addition of even small amounts of two solvents that are less volatile than water, ethylene glycol and 2-methoxyethanol, also results in preferential deprotonation of higher charge state ions of small peptides, but these solvents actually produce an enhancement in the higher charge state ions for both cytochrome c and myoglobin. For instruments that have capabilities that improve with lower m/z, this effect could be taken advantage of to improve the performance of an analysis.

Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins.

In mammalian cells, Id proteins coordinate proliferation and differentiation. Id2 is a dominant-negative antagonist of basic helix-loop-helix transcription factors and proteins of the retinoblastoma (Rb) family. Here we show that Id2-Rb double knockout embryos survive to term with minimal or no defects in neurogenesis and haematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma, an embryonal tumour derived from the neural crest, Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumour-suppressor function of Rb.

Transforming growth factor-beta1 recruits histone deacetylase 1 to a p130 repressor complex in transgenic mice in vivo.

Transforming growth factor (TGF)-beta1 functions as a tumor suppressor in vivo. Using transgenic mice, we show that hepatic TGF-beta1 overexpression inhibits abundance of the cyclin-dependent kinase activating tyrosine phosphatase cdc25A protein. The reduction in cdc25A protein levels was associated with increased binding of histone deacetylase 1 to p130 in the hepatic extracts. In cultured cells, HDAC1/p130 overexpression inhibited activity of the cdc25A promoter through an E2F site. TGF-beta1 treatment enhanced p130 binding to the cdc25A promoter E2F site assessed in chromatin immunoprecipitation assays. Hepatic proliferation induced by partial hepatectomy was associated with a decrease in the amount of HDAC1 bound to p130, without a significant decrease in p130 abundance, suggesting that HDAC1 binding to p130 may be regulated by proliferative stimuli. The induction of cdc25A abundance induced by partial hepatectomy correlated with the induction of DNA synthesis. These studies suggest that TGF-beta1 may enhance HDAC1 binding to p130 in vivo, thereby inhibiting cdc25A gene expression. TGF-beta1 regulation of HDAC1/pocket protein associations may provide a link between chromatin remodeling proteins and cdk inhibition through induction of cdc25A in vivo.

Induced differentiation of U937 cells by 1,25-dihydroxyvitamin D3 involves cell cycle arrest in G1 that is preceded by a transient proliferative burst and an increase in cyclin expression.

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1, 25(OH)2D3], is a potent inhibitor of cellular proliferation as well as an inducer of differentiation of myeloid leukemic cells to macrophages. We have previously reported that a number of genes are upregulated by 1,25(OH)2D3 during myeloid differentiation, including the cyclin-dependent kinase (CDK) inhibitors p21, p27, 15, and p18, suggesting that cell cycle arrest and differentiation are tightly linked processes. We further explore here the relationship between growth inhibition and differentiation. We report that, upon 1, 25(OH)2D3 treatment, U937 cells exhibited an early proliferative burst followed by growth inhibition and subsequent differentiation. Although CDK levels remain constant throughout, this transient increase in proliferation was accompanied by increases in cyclin A, D1, and E protein levels. p21 and p27 levels were also elevated during both the proliferative burst and subsequent inhibition of cell growth. Ectopic overexpression of p21 and/or p27 in U937 cells, in the absence of hormone, resulted in an induction of the expression of monocyte/macrophage-specific markers, whereas overexpression of p15 and p18 had no effect, suggesting that a subset of CDK inhibitors are important for both growth arrest and differentiation and that an early increase in proliferation is somehow a prerequisite for subsequent differentiation. However, no such biphasic behavior was detected in cells that are growth inhibited by 1,25(OH)2D3 but do not differentiate, such as MCF-7 cells. Taken together, these results indicate that both growth stimulation and subsequent inhibition precede differentiation and involve induction of both cyclins and p21 and p27, whereas cell cycle arrest of differentiated cells can be achieved simply by elevations in CDK inhibitors.

Dyscalculia in the early stages of Alzheimer's disease.

OBJECTIVE: To study mathematical deficits in the early stages of Alzheimer's disease (AD). METHODS: Sixty-eight patients with mild AD and 242 normal controls (NC) received a standardized battery (EC 301-R) assessing number processing and calculation abilities. AD patients also received testing for language, memory, visuo-spatial and executive-attentional domains. RESULTS: Sixty-four AD patients (94.1%) showed impaired performances on the EC 301-R. Mathematical deficits were evident both on calculation and number processing skills. Performance on the single tasks was related to attentional-executive resources and to impaired number representations. Heterogeneous patterns of preserved/impaired mathematical abilities were also observed in single cases. CONCLUSION: Dyscalculia is an early sign of AD. It should be included among the reliable clinical hallmarks for the diagnosis of AD. Identification of dyscalculic symptoms in these patients requires composite assessment procedure.

E2F and histone deacetylase mediate transforming growth factor beta repression of cdc25A during keratinocyte cell cycle arrest.

cdc25A is a tyrosine phosphatase that activates G1 cyclin-dependent kinases (Cdk's). In human keratinocytes, cdc25A expression is down-regulated after the initial drop in Cdk activity caused by cell exposure to the antimitogenic cytokine transforming growth factor beta (TGF-beta) or removal of serum factors. Here we show that the TGF-beta-inhibitory-response element in the cdc25A promoter maps to an E2F site at nucleotides -62 to -55 from the transcription start site. This site is not required for basal transcription in keratinocytes. We provide evidence that the cell cycle arrest program activated by TGF-beta in human keratinocytes includes the generation of E2F4-p130 complexes that in association with histone deacetylase HDAC1 inhibit the activity of the cdc25A promoter from this repressor E2F site. This mechanism is part of a program that places keratinocytes in the quiescent state following the initial drop in Cdk activity caused by cell exposure to TGF-beta.

Regulation of the cdk inhibitor p21 gene during cell cycle progression is under the control of the transcription factor E2F.

The control of cell cycle progression is orchestrated by an extraordinary diverse and dynamic in function group of proteins. Critical in the progression are the actions of the E2F family of transcription factors which regulate the expression of genes necessary for the G1/S transition and the WAF/CIP/KIP family of cdk inhibitors which can inhibit cell cycle progression. In this report, we have identified E2F binding sites in both the human and mouse p21 promoters that bind E2F protein complexes from nuclear extracts in a cell cycle-dependent manner. In ectopic expression experiments we determined that E2F1, but not E2F4, can strongly transactivate the human p21 gene through these E2F binding sites which are located in the -215/+1 region of the p21 gene. The transactivation of the p21 gene through regulatory elements within the -215/+1 region of the promoter was correlated with increased levels of endogenous E2F1 and p21 proteins at the G1/S boundary. The significance of transactivation of the p21 gene by E2F is that p21 function is important in cell cycle progression as well as for cell cycle arrest. Indeed, E2F-induced levels of p21 protein during the G1/ S transition is consistent with the recent findings demonstrating that p21 acts as an assembly factor for kinase active cyclin/cdk/p21 complexes.