Endogenous and artificial miRNAs explore a rich variety of conformations: a potential relationship between secondary structure and biological functionality.

Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.

Stabilization vs. destabilization of G-quadruplex superstructures: the role of the porphyrin derivative having spermine arms.

The interaction of the porphyrin derivative H2TCPPSpm4, having spermine pendants in the four meso positions, with the G-quadruplex (GQ) structure formed by the DNA aptamer TGGGAG has been investigated by means of UV, electronic circular dichroism and PAGE studies. The results reported here demonstrate that the porphyrin derivative is capable of stabilizing or destabilizing the higher-ordered structures of parallel GQs, depending on the method used to reach their relative stoichiometry (titration vs. single addition). Noteworthily, when two equivalents of H2TCPPSpm4 were mixed directly with one equivalent of the (TGGGAG)4 GQ to reach a 2 : 1 H2TCPPSpm4 : GQ ratio T1/2 higher than 80 °C was also observed confirming the presence of higher-ordered GQ structures.

Association of the COMT synonymous polymorphism Leu136Leu and missense variant Val158Met with mood disorders.

BACKGROUND: Major depressive disorder (MDD) and bipolar disorder (BD) are the two most common mood disorders. Given the recognized involvement of catecholamines in depression, genetic research focused on the evaluation of polymorphisms in genes coding for proteins that regulate neurotransmitter release, transport and degradation. Here we aimed at evaluating the distribution of two genetic variants of catechol-O-methyltransferase (COMT), namely the well characterized missense polymorphism G1947A (Val158Met) and the recently reported synonymous polymorphism C1886G (Leu136Leu), in MDD and BD patients compared with healthy subjects. METHODS: Genotyping for COMT polymorphisms was carried out by DNA direct sequencing in 112 patients (54 MDD and 58 BD) and 58 healthy subjects. RESULTS: We did not find significant differences in the Val158Met variant distribution between patients and controls. Instead, we found that the C1886 major allele and the CC1886 wild-type genotype frequencies were significantly higher in controls than in both groups of patients. On the contrary, the G1886 minor allele and the heterozygous CG1886 genotype were significantly more present in both MDD and BD patients than in healthy subjects. When looking at combined polymorphisms, we found a significantly higher frequency of the double heterozygous diplotype CG/GAVal/Met158 in both MDD and BD patients than in controls. Instead, the diplotype CC/GAVal/Met158 showed a significantly higher frequency in controls than in BD patients. LIMITATIONS: The small size of our study cohort may limit the generalizability of the present findings. CONCLUSIONS: This work first showed the association of combined Leu136Leu and Val158Met variants of COMT gene with MDD and BD.

Transglutaminase 2 is involved in homocysteine-induced activation of human THP-1 monocytes.

Aberrant transglutaminase 2 (TG2) expression and protein cross-linking activity have been associated with several chronic neurodegenerative disorders in which inflammatory processes triggered by activated microglia and monocytes play a key role, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. Interestingly, mild-to-moderate hyperhomocysteinemia (HHcy), corresponding to increased plasma homocysteine (Hcy) concentrations in the range 16-60 µM, have recently been associated with the above-cited diseases. Using THP-1 monocytes, here we investigated the role of TG2 in cell response to mildly elevated Hcy concentrations. A five-day incubation with Hcy (∼25 µM) increased reactive oxygen species, peroxide lipids, as well as 8-hydroxyguanosine levels by twofold, and decreased the endogenous cell antioxidant defenses, that is reduced glutathione, by 50% in Hcy-exposed cultures compared with controls (p < 0.01). Hcy-induced oxidative stress was associated with increases in TG2 expression and activity, as well as nuclear factor kappa B activation. Notably, the latter was reduced in the presence of the TG-specific inhibitor R283. Hcy exposure also significantly increased the mRNA levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-1ß, as well as the level of Hcy-inducible endoplasmic reticulum (ER) stress protein, a marker of ER stress, in Hcy-exposed cultures compared with controls. Notably, these effects were dramatically reduced by R283. These preliminary findings indicate that TG2 plays a key role in Hcy-induced activation of THP-1 monocytes, involving oxidative as well as ER stress and inflammation. This underlines the potential of TG2 inhibition in the therapeutic management of inflammatory processes contributing to neurodegenerative disorders associated with mild HHcy.

Toxic effects of mildly elevated homocysteine concentrations in neuronal-like cells.

Epidemiological and experimental evidence indicated that hyperhomocysteinemia is associated with neurodegeneration. However, homocysteine neurotoxic effects have been so far investigated mostly by employing homocysteine concentrations (≥100 µM) much higher than homocysteine mean plasma levels (20 µM) observed in patients with neurodegenerative disorders. While evaluating the effects of a prolonged exposure to ~20 µM homocysteine in neuronal-like differentiated SH-SY5Y cells, we observed a 35% loss of cell viability and a four-fold increase in reactive oxygen species levels in cells incubated with homocysteine for five days compared with controls. Moreover, homocysteine increased by 30% and around two-fold, respectively, the Comet-positive cell number and DNA damage indexes (tail length, T-DNA, olive tail moment) compared with controls. Cell response to homocysteine-induced DNA damage involved the up-regulation of Bax and, at a greater extent, Bcl-2, but not caspase-3, in association with a p53-independent increase of p21 levels; concomitantly, also p16 levels were increased. When looking at time-dependent changes in cyclin expression, we found that a significant up-regulation of cyclins D1, A1, E1, but not B1, concomitant with p21 down-regulation, occurred in cells incubated with homocysteine for three days. However, in line with the observed increase of p21 and p16 levels, a five days incubation with homocysteine induced cyclin down-regulation accompanied by a strong reduction of phosphorylated pRB amounts. These results suggest that, when prolonged, the exposure of neuronal-like cells to mildly elevated homocysteine concentrations triggers oxidative and genotoxic stress involving an early induction of cyclins, that is late repressed by G1-S check-point regulators.

Relationship between major adverse cardiac events and angiographic findings in dialysis patients.

BACKGROUND: In dialysis patients, coronary angiography (CA) predicts major adverse coronary events (MACE) better than non-invasive tests. The aim of this study was to investigate in such patients the relationship between coronary atherosclerotic damage shown by angiography and MACE, during an average follow-up period of more than 5 years. PATIENTS AND METHODS: Coronary angiography was performed in 63 dialysis patients (mean age 56 ± 12 years, 49 men); 37 subjects awaiting kidney transplantation had no history of cardiac disease, whereas the remaining 26 patients had clinical evidence of coronary artery disease (CAD). During a follow-up period of 62 ± 20 months (range 12-109), all the MACE were recorded. Statistical analysis was carried out by dividing the patients into two groups, those who had MACE (MACE group) and those who were free of cardiac events (FCE group). Severe CAD on CA was defined as luminal stenosis ≥ 75% in at least one vessel. Logistic regression analysis and Cox regression analysis were carried out in order to evaluate which variable was associated with MACE. RESULTS: At the end of follow-up, 17 subjects had MACE and severe CAD was shown in the epicardial arteries of 31 patients (49%). Compared to the FCE group, the MACE group had older age (65 ± 10 vs 53 ± 11 years, P = 0.002), lower diastolic blood pressure (79 ± 7 vs 85 ± 7 mmHg, P = 0.0037), higher prevalence of CAD (82 vs 30%, P = 0.0002) and cerebrovascular disease (41 vs 15%, P = 0.0278). Coronary artery damage was higher in the MACE group than in the FCE group. Logistic and Cox regression analyses showed that age was the only variable independently associated with MACE (OR 1.109 95% CI 1.022-1.204, P = 0.0133, hazard ratio 1.066 95% CI 1.010-1.125, P = 0.02, respectively). After removal of age from the model, MACE were independently associated with haemodynamic stenosis of coronary arteries (OR 7.429 95% CI 1.829-30.173, P = 0.005, hazard ratio 5.992 95% CI 1.655-21.698, P = 0.006, respectively). Event-free survival was much better in the 37 renal transplant candidates with no history of CAD than in the 26 patients who had clinical evidence of CAD. CONCLUSIONS: This observational study confirms that in dialysis patients coronary atherosclerotic damage shown by angiography is strongly related to MACE and that age and severe CAD are major risk factors for MACE.

[Apropos of 1 case of anisakiasis contracted in Sicily]. / A propos d'un cas d'anisakiase contractée en sicile.

The authors describe a case of anisakiasis in Sicily. The diagnosis was based on the knowledge that a contaminated fish, Lepidopus caudatus, had probably been absorbed, as well as on clinical intestinal symptoms, intestinal lesions observed by endoscopy and O.G.D.S, duodenal infiltration by eosinophilic polymorphonuclear, positive ELISA anisakis serology and successful treatment by albendazole.