MicroRNAs (miRNAs) represent a family of small non-coding ribonucleic acids that post-transcriptionally inhibits the expression of their target messenger RNAs (mRNAs), thereby acting as general gene repressors. In this study we examined the relative quantity and stability of miRNA subjected to a long period of freezing; we compared the stability of eight miRNAs in the plasma of five human healthy controls before freezing and after six and 12 months of storage at −80 °C. In addition, we examined the plasma frozen for 14 years and the amount of miRNA still available. Using a Life Technologies protocol to amplify and quantify plasma miRNAs from EDTA (Ethylene Diamine Tetraacetic Acid)-treated blood, we analyzed the stability of eight miRNAs, (miR-125b-5p, miR-425-5p, miR-200b-5p, miR-200c-3p, miR-579-3p, miR-212-3p, miR-126-3p, and miR-21-5p). The miRNAs analyzed showed a high stability and long frozen half-life.

BackgroundThis study examined the effect of storage temperature on the protein profile of human plasma. Plasma samples were stored for 13 days at -80°C, -20°C, +4°C and room temperature (20-25°C) prior to proteomic analysis. The proteomic comparisons were based on the differences of mean intensity values of protein spots between fresh plasma samples (named “time zero”) and plasma samples stored at different temperatures. To better understand the thermally induced biochemical changes that may affect plasma proteins during storage we identified proteins with different expressions with respect to the time zero sample.ResultsUsing two-dimensional electrophoresis followed by MALDI-TOF MS and /or LC-MS/MS 20 protein spots representing 10 proteins were identified with significant differences in abundance when stored at different temperatures. Our results, in agreement with various authors, indicate that during storage for a short period (13 days) at four different temperatures plasma proteins were more affected by degradation processes at +4°C compared to the other temperatures analysed. However, we founded that numerous protein spots (vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein A-I, apolipoprotein E, haptoglobin and complement factor B) decrease in abundance with increasing temperature up to 4°C, but at room temperature their intensity mean values are similar to those of time zero and -80°C. We hypothesize that these proteins are labile at 4°C, but at the same time they are stable at room temperature (20-25°C). Furthermore we have grouped the proteins based on their different sensitivity to the storage temperature. Spots of serum albumin, fibrinogen gamma chain and haptoglobin are more resistant to the higher temperatures tested, as they have undergone changes in abundance only at room temperature; conversely, other spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as they have undergone changes in abundance at all temperatures except at -80°C.ConclusionsAlthough there are many studies concerning protein stability of clinical samples during storage these findings may help to provide a better understanding of the changes of proteins induced by storage temperature.

Inferring gene networks is a daunting task. We here describe several algorithms we used in the Dialogue for Reverse Engineering Assessments and Methods (DREAM2) Reverse Engineering Competition 2007: an algorithm based on first-order partial correlation for discovering BCL6 targets in Challenge 1 and an algorithm using nonlinear optimization with winning performance in Challenge 3. After the gold standards for the challenges were released, the performance of alternative variants of the algorithms could be evaluated. The DREAM competition taught us some strong lessons. Amazingly, simpler methods performed in general better than more advanced, theoretically motivated approaches. Also, the challenges strongly showed that inferring gene networks requires controlled experimentation using a well-defined experimental design. Analyzing data obtained through merging many unrelated datasets indeed resulted in weak performances of all algorithms, while algorithms that explicitly took the experimental design into account performed best.

The role of Clusterin in attenuation of inflammation and reverse cholesterol transfer makes this molecule a potential candidate as a marker for cancer, cardiovascular disease, diabetes mellitus, and metabolic syndrome. In elderly subjects cardiovascular diseases represent the primary cause of death and different clinical studies have shown a positive correlation of these diseases with changes in the lipid pattern. This work aimed at evaluating the relationship between circulating clusterin and the biochemical parameters that characterize the lipid profile of a Sardinian population divided into five age groups including centenarians; the high frequency in Sardinia of these long-lived individuals gave us the opportunity to extend the range of the age groups to be analyzed to older ages and to better evaluate the changes in the lipid balance during ageing and its relationship with clusterin concentration in plasma. Our results showed that Clusterin concentration values of the youngest group were more similar with the centenarian’s group compared to the other age groups, and a positive correlation arises with LDL. Furthermore given the high prevalence of cardiovascular diseases in the population examined and the association of Clusterin with these pathologies we evaluated Clusterin concentration variation in two groups with or without cardiovascular diseases. In presence of cardiovascular disease, Clusterin is significantly related to the most atherogenic components of lipid profile (total cholesterol and LDL), especially in women, suggesting its potential role in modulating cardiovascular metabolic risk factors.

These results indicate that specific proteomic signatures can be detected and useful in terms of treatment selection and in early COPD patient monitoring.

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1Background and Aims: Chronic kidney disease (CKD) is characterized by increased oxidative 2 stress (OS). In consideration of the well-known link between OS and DNA methylation we assessed 3 DNA methylcytosine (mCyt) concentrations in CKD patients at baseline and during cholesterol 4 lowering treatment. 5 Methods and Results: DNA methylation and OS indices (malonyldialdehyde, MDA; 6allantoine/uric acid ratio, All/UA) were measured in 30 CKD patients randomized to three 7 cholesterol lowering regimens for 12 months (simvastatin 40 mg/day, ezetimibe/simvastatin 10/20 8 mg/day, or ezetimibe/simvastatin 10/40 mg/day) and 30 age-and sex-matched healthy controls. 9 DNA methylation was significantly lower in CKD patients vs. controls (4.06±0.20 % vs. 4.27±0.17 10 % mCyt, p=0.0001). Treatment significantly increased mCyt DNA concentrations in all patients 11 (4.06±0.04 % at baseline; 4.12±0.03% at 4 months; 4.17± 0.03% at 8 months; and 4.20± 0.02% at 12 12 months, p=0.0001 for trend). A trend for a greater effect on DNA methylation was observed with 13 combined treatment ezetimibe/simvastatin 10/40 mg/day (+5.2% after one year treatment). The 14 treatment-associated mCyt increase was significantly correlated with the concomitant reduction in 15 MDA concentrations and All/AU ratios. 16Conclusion: Our results demonstrate that CKD patients have a lower degree of DNA methylation 17 and that cholesterol lowering treatment restores mCyt DNA concentrations to levels similar to 18 healthy controls. The treatment-associated increase in DNA methylation is correlated with a 19 concomitant reduction in OS markers. 20 21 1.INTRODUCTION 1 There is unequivocal evidence that patients with chronic kidney disease (CKD) develop 2 accelerated atherosclerosis, with a consequent increase in cardiovascular morbidity and mortality 3 [1-3]. The main mechanisms underlying the increased cardiovascular disease (CVD) risk in this 4 population are related to the relatively high prevalence of hypertension, diabetes and obesity [4-5]. 5 Moreover, patients with CKD often have alterations in lipoprotein metabolism, which might result 6 in severe dyslipidemia [6]. Therefore, in view of the cardiovascular risk reduction reached in 7 hypercholesterolaemic patients, the pharmacological management of hypercholesterolaemia 8 represents a promising target to reduce CVD risk in CKD patients [7]. Several clinical studies show 9 that statins, aside from decreasing plasma cholesterol concentrations, may have specific 10 renoprotective properties and, when combined with renin-angiotensin system (RAS) inhibitors, may 11 have additional antiproteinuric effects [8]. The combination of statins with ezetimibe (EZE), a 12 cholesterol absorption inhibitor, exerts additional lipid lowering effects vs statins alone [9]. We 13 have recently reported that combined simvastatin/ezetimibe therapy reduces inflammatory status 14 and decreases plasma markers of endothelial dysfunction through a reduction in oxidative stress 15 (OS) in stage III-IV CKD patients [10-11]. OS occurs ...