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RNA editing is being recognized as an important post-transcriptional mechanism that may have crucial roles in introducing genetic variation and phenotypic diversity. Despite microRNA editing recurrence, defining its biological relevance is still under extended debate. To better understand microRNA editing function and regulation we performed an exhaustive characterization of the A-to-I site-specific patterns in mir-376a-1, a mammalian microRNA which RNA editing is involved in the regulation of development and in disease. Thorough an integrative approach based on high-throughput small RNA sequencing, Sanger sequencing and computer simulations we explored mir-376a-1 editing in samples from various individuals and primate species including human placenta and macaque, gorilla, chimpanzee and human brain cortex. We observed that mir-376a-1 editing is a common phenomenon in the mature and primary microRNA molecules and it is more frequently detected in brain than in placenta. Primary mir-376a-1 is edited at three positions, -1, +4 and +44. Editing frequency estimations and in silico simulations indicated that editing was not equally recurrent along the three mir-376a-1 sites, nevertheless no epistatic interactions among them were observed. Particularly, the +4 site, located in the seed region of the mature miR-376a-5p, reached the highest editing frequency in all samples. Secondary structure predictions revealed that the +4 position was the one that conferred the highest stability to the mir-376a-1 hairpin. We suggest that molecular stability might partially explain the editing recurrence observed in certain microRNAs and that editing events conferring new functional regulatory roles in particular tissues and species could have been conserved along evolution, as it might be the case of mir-376a-1 in primate brain cortex.

Objective: Clinical progression in lacunar strokes (LS) is an unpredictable and fearful complication. Endothelial dysfunction (ED) is believed to be the first step in the pathophysiology of LS therefore we aimed to analyze the association of three markers of ED: albuminuria, von Willebrand factor (vWF), and oxidized LDL cholesterol (ox-LDL) with LS progression. Methods: From December 2007 to December 2010, 127 LS patients admitted within 6 h of symptom onset were prospectively assessed. Progression was defined as initial NIHSS score worsening ≥4 points within the first 72 h. Analysis of vWF and ox-LDL was done at admission. Albuminuria was measured in the first morning spot urine. Association between 3 biomarkers and progression was tested using logistic regression analysis. Other clinical variables of interest were also studied. Discriminative power was analyzed with a receiver operator curve. Results: Twenty-two patients (17.3%) progressed. Progression was associated with worse outcome at 90 days. Albuminuria and ox-LDL were associated in univariate analysis; vWF was not. Adjusted OR were: ox-LDL [OR: 1.03; 95% CI: 1.01–1.07, p = 0.019], albuminuria [OR: 2.07; 95% CI: 1.04–4.13, p = 0.039]. Association was linear without a cut-off point. Clinical variables were not associated with progression. The model including albuminuria and ox-LDL had a good predictive value [AUC: 0.80 [0.70–0.89)]. Conclusions: Albuminuria and ox-LDL levels are independently associated with higher risk of progression in LS. The lack of reliable clinical predictors makes biomarker research a priority to improve progression detection in this subtype of ischemic strokes. © 2011 Elsevier Ireland Ltd. All rights reserved.

Background/Aims: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca 2+ ] i ) and cell contractility. Methods: Binding studies using [ 3 H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca 2+ ] i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. Results: Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1±1.4×10 4 sites/cell with a Kd of 2.1±0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca 2+ ] i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca 2+ ] i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. Conclusion: Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca 2+ ] i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.