A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Il Piano della Performance nei Comuni medi. La proposta di una nuova configurazione per superare l���inadeguatezza delle prime applicazioni

Il D.Lgs. 150/09 ha inteso dar vita ad una ���riforma organica��� della PA italiana, improntandone il funzionamento a logiche di programmazione e controllo delle performance. Attorno a tale concetto la riforma ha costruito un Sistema teso a programmare, misurare, controllare, valutare e comunicare la performance degli enti. Il lavoro si focalizza sulla programmazione, e in particolare sullo strumento cardine introdotto dal D.Lgs. 150/09: il Piano della Performance (PdP). Il contributo, basato su una metodologia deduttivo-induttiva, si concentra sui comuni medi italiani, scelti in quanto statisticamente rappresentativi del livello medio di complessit�� degli enti locali. Sono stati oggetto di indagine i PdP pubblicati sui siti istituzionali degli enti considerati, al fine di verificarne sia il livello di aderenza alle Linee Guida (LG) emanate dalla Commissione Indipendente per la Valutazione, la Trasparenza e l���Integrit�� delle amministrazioni pubbliche (CIVIT) e dalla Associazione Nazionale Comuni Italiani (ANCI), sia il loro livello di adeguatezza economico-aziendale. Preliminarmente si indagher�� il tema della programmazione, sotto il profilo normativo-dottrinale, concentrandosi su soggetti, processi e strumenti. Poi si sposter�� il focus sul PdP: dopo aver definito obiettivi, quesiti e metodologia della ricerca, verranno esplicitate le configurazioni di PdP emergenti dalle LG CIVIT e ANCI. Verranno poi illustrati i risultati della ricerca empirica, mettendo in luce il livello di allineamento dei PdP dei comuni medi alle LG, nonch�� il livello di adeguatezza economico-aziendale degli stessi. Si tratteggeranno quindi alcune brevi conclusioni.

External validation of the 2008 Framingham cardiovascular risk equation for CHD and stroke events in a European population of middle-aged men. The PRIME study

To test the applicability of the sex-specific 2008 Framingham general cardiovascular risk equation for coronary heart disease (CHD) and stroke in European middle-aged men from Ireland and France.

Multiple Biomarkers for the prediction of ischemic stroke: the PRIME study

Objective: To simultaneously evaluate 14 biomarkers from distinct biological pathways for risk prediction of ischemic stroke, including biomarkers of hemostasis, inflammation, and endothelial activation as well as chemokines and adipocytokines.<br/>Methods and Results: The Prospective Epidemiological Study on Myocardial Infarction (PRIME) is a cohort of 9771 healthy men 50 to 59 years of age who were followed up over 10 years. In a nested case���control study, 95 ischemic stroke cases were matched with 190 controls. After multivariable adjustment for traditional risk factors, fibrinogen (odds ratio [OR], 1.53; 95% confidence interval [CI], 1.03���2.28), E-selectin (OR, 1.76; 95% CI, 1.06���2.93), interferon-��-inducible-protein-10 (OR, 1.72; 95% CI, 1.06���2.78), resistin (OR, 2.86; 95% CI, 1.30���6.27), and total adiponectin (OR, 1.82; 95% CI, 1.04���3.19) were significantly associated with ischemic stroke. Adding E-selectin and resistin to a traditional risk factor model significantly increased the area under the receiver-operating characteristic curve from 0.679 (95% CI, 0.612���0.745) to 0.785 and 0.788, respectively, and yielded a categorical net reclassification improvement of 29.9% (P=0.001) and 28.4% (P=0.002), respectively. Their simultaneous inclusion in the traditional risk factor model increased the area under the receiver-operating characteristic curve to 0.824 (95% CI, 0.770���0.877) and resulted in an net reclassification improvement of 41.4% (P&lt;0.001). Results were confirmed when using continuous net reclassification improvement.<br/>Conclusion: Among multiple biomarkers from distinct biological pathways, E-selectin and resistin provided incremental and additive value to traditional risk factors in predicting ischemic stroke.

All-Cause Mortality up to and After Coronary Heart Disease and Stroke Events in European Middle-Aged Men The PRIME Study

<p>Background and Purpose-The aim was to investigate prospectively the all-cause mortality risk up to and after coronary heart disease (CHD) and stroke events in European middle-aged men.</p><p>Methods-The study population comprised 10 424 men 50 to 59 years of age recruited between 1991 and 1994 in France (N=7855) and Northern Ireland (N=2747) within the Prospective Epidemiological Study of Myocardial Infarction. Incident CHD and stroke events and deaths from all causes were prospectively registered during the 10-year follow-up. In Cox's proportional hazards regression analysis, CHD and stroke events during follow-up were used as time-dependent covariates.</p><p>Results-A total of 769 CHD and 132 stroke events were adjudicated, and 569 deaths up to and 66 after CHD or stroke occurred during follow-up. After adjustment for study country and cardiovascular risk factors, the hazard ratios of all-cause mortality were 1.58 (95% confidence interval 1.18-2.12) after CHD and 3.13 (95% confidence interval 1.98-4.92) after stroke.</p><p>Conclusions-These findings support continuous efforts to promote both primary and secondary prevention of cardiovascular disease.</p>

Predictive Value of Depressive Symptoms for All-Cause Mortality: Findings From the PRIME Belfast Study Examining the Role of Inflammation and Cardiovascular Risk Markers

<p>OBJECTIVES: To improve understanding about the potential underlying biological mechanisms in the link between depression and all-cause mortality and to investigate the role that inflammatory and other cardiovascular risk factors may play in the relationship between depressive symptoms and mortality.</p><p>METHODS: Depression and blood-based biological markers were assessed in the Belfast PRIME prospective cohort study (N = 2389 men, aged 50-59 years) in which participants were followed up for 18 years. Depression was measured using the 10-item Welsh Pure Depression Inventory. Inflammation markers (C-reactive protein [CRP], neopterin, interleukin [IL]-1 receptor antagonist [IL-1Ra], and IL-18) and cardiovascular-specific risk factors (N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, C-terminal pro-endothelin-1 [CT-proET]) were obtained at baseline. We used Cox proportional hazards modeling to examine the association between depression and biological measures in relation to all-cause mortality and explore the mediating effects.</p><p>RESULTS: During follow-up, 418 participants died. Higher levels of depressive symptoms were associated with higher levels of CRP, IL-1Ra, and CT-proET. After adjustment for socioeconomic and life-style risk factors, depressive symptoms were significantly associated with all-cause mortality (hazard ratio = 1.10 per scale unit, 95% confidence interval = 1.04-1.16). This association was partly explained by CRP (7.3%) suggesting a minimal mediation effect. IL-1Ra, N-terminal pro-b-type natriuretic peptide, midregion pro-atrial natriuretic peptide, midregion pro-adrenomedullin, and CT-proET contributed marginally to the association between depression and subsequent mortality.</p><p>CONCLUSIONS: Inflammatory and cardiovascular risk markers are associated with depression and with increased mortality. However, depression and biological measures show additive effects rather than a pattern of meditation of biological factors in the association between depression and mortality.</p>

A Role for Behavior in the Relationships Between Depression and Hostility and Cardiovascular Disease Incidence, Mortality, and All-Cause Mortality: the Prime Study

<p>BACKGROUND: Behavioral factors are important in disease incidence and mortality and may explain associations between mortality and various psychological traits.</p><p>PURPOSE: These analyses investigated the impact of behavioral factors on the associations between depression, hostility and cardiovascular disease(CVD) incidence, CVD mortality, and all-cause mortality.</p><p>METHODS: Data from the PRIME Study (N���=���6953 men) were analyzed using Cox proportional hazards models, following adjustment for demographic and biological CVD risk factors, and other psychological traits, including social support.</p><p>RESULTS: Following initial adjustment, both depression and hostility were significantly associated with both mortality outcomes (smallest SHR���=���1.24, p���&lt;���0.001). Following adjustment for behavioral factors, all relationships were attenuated both when accounting for and not accounting for other psychological variables. Associations with all-cause mortality remained significant (smallest SHR���=���1.14, p���=���0.04). Of the behaviors included, the most significant contribution to outcomes was found for smoking, but a role was also found for fruit and vegetable intakes and high alcohol consumption.</p><p>CONCLUSIONS: These findings demonstrate well-known associations between depression, hostility, and mortality and suggest the potential importance of behaviors in explaining these relationships.</p>