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Background: Sequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomarkers, but exosomes have low amount of RNA, hindering adequate analysis and quantification. Here, we have assessed several steps in developing an optimized small RNA (sRNA) library preparation protocol for next-generation sequencing (NGS) miRNA analysis from urinary exosomes.Methods: A total of 24 urinary exosome samples from donors were included in this study. RNA was extracted by column-based methods. The quality of extracted RNA was assessed by spectrophotometric quantification and Bioanalyzer software analysis. All libraries were prepared using the CleanTag small RNA library preparation protocol and the effect of our additional modifications on adapter-dimer presence, sequencing data and tagged small RNA library population was also analyzed.Results: Our results show that good quality sequencing libraries can be prepared following our optimized small RNA library preparation protocol from urinary exosomes. When the size selection by gel purification step was included within the workflow, adapter-dimer was totally removed from cDNA libraries. Furthermore, the inclusion of this modification step within small RNA library protocol augmented the small RNA mapped reads, with an especially significant 37% increase in miRNA reads, and the gel purification step made no difference to the tagged miRNA population.Conclusions: This study provides researchers with an optimized small RNA library preparation workflow for next generation sequencing based exosome-associated miRNA analysis that yields a high amount of miRNA mapped reads without skewing the tagged miRNA population significantly. [ABSTRACT FROM AUTHOR]

Background: The possibility of integrating viral vectors to become a persistent part of the host genome makes them a crucial element of clinical gene therapy. However, viral integration has associated risks, such as the unintentional activation of oncogenes that can result in cancer. Therefore, the analysis of integration sites of retroviral vectors is a crucial step in developing safer vectors for therapeutic use. Results: Here we present VISMapper, a vector integration site analysis web server, to analyze next-generation sequencing data for retroviral vector integration sites. VISMapper can be found at: http://vismapper.babelomics.org. Conclusions: Because it uses novel mapping algorithms VISMapper is remarkably faster than previous available programs. It also provides a useful graphical interface to analyze the integration sites found in the genomic context. [ABSTRACT FROM AUTHOR]

Background Alterations in the redox balance are involved in the origin, promotion and progression of cancer. Inter-individual differences in the oxidative stress regulation can explain a part of the variability in cancer susceptibility. Purpose: The aim of this study was to evaluate if polymorphisms in genes codifying for the different systems involved in oxidative stress levels can have a role in susceptibility to breast cancer. Methods We have analyzed 76 single base polymorphisms located in 27 genes involved in oxidative stress regulation by SNPlex technology. First, we have tested all the selected SNPs in 493 breast cancer patients and 683 controls and we have replicated the significant results in a second independent set of samples (430 patients and 803 controls). Gene-gene interactions were performed by the multifactor dimensionality reduction approach. Results Six polymorphisms rs1052133 (OGG1), rs406113 and rs974334 (GPX6), rs2284659 (SOD3), rs4135225 (TXN) and rs207454 (XDH) were significant in the global analysis. The gene-gene interactions demonstrated a significant four-variant interaction among rs406113 (GPX6), rs974334 (GPX6), rs105213 (OGG1) and rs2284659 (SOD3) (p-value = 0.0008) with high-risk genotype combination showing increased risk for breast cancer (OR = 1.75 [95% CI; 1.26-2.44]). Conclusions The results of this study indicate that different genotypes in genes of the oxidant/antioxidant pathway could affect the susceptibility to breast cancer. Furthermore, our study highlighted the importance of the analysis of the epistatic interactions to define with more accuracy the influence of genetic variants in susceptibility to breast cancer. [ABSTRACT FROM AUTHOR]

Background: Alterations in the redox balance are involved in the origin, promotion and progression of cancer. Inter-individual differences in the oxidative stress regulation can explain a part of the variability in cancer susceptibility. The aim of this study was to evaluate if polymorphisms in genes codifying for the different systems involved in oxidative stress levels can have a role in susceptibility to breast cancer. Methods: We have analyzed 76 single base polymorphisms located in 27 genes involved in oxidative stress regulation by SNPlex technology. First, we have tested all the selected SNPs in 493 breast cancer patients and 683 controls and we have replicated the significant results in a second independent set of samples (430 patients and 803 controls). Gene-gene interactions were performed by the multifactor dimensionality reduction approach. Results: Six polymorphisms rs1052133 (OGG1), rs406113 and rs974334 (GPX6), rs2284659 (SOD3), rs4135225 (TXN) and rs207454 (XDH) were significant in the global analysis. The gene-gene interactions demonstrated a significant four-variant interaction among rs406113 (GPX6), rs974334 (GPX6), rs105213 (OGG1) and rs2284659 (SOD3) (p-value = 0.0008) with high-risk genotype combination showing increased risk for breast cancer (OR = 1.75 [95% CI; 1.26-2.44]). Conclusions: The results of this study indicate that different genotypes in genes of the oxidant/antioxidant pathway could affect the susceptibility to breast cancer. Furthermore, our study highlighted the importance of the analysis of the epistatic interactions to define with more accuracy the influence of genetic variants in susceptibility to breast cancer. [ABSTRACT FROM AUTHOR]

Background The objective was to investigate the association between BMI and single nucleotide polymorphisms previously identified of obesity-related genes in two Spanish populations. Forty SNPs in 23 obesity-related genes were evaluated in a rural population characterized by a high prevalence of obesity (869 subjects, mean age 46yr, 62% women, 36% obese) and in an urban population (1425 subjects, mean age 54yr, 50% women, 19% obese). Genotyping was assessed by using SNPlex and PLINK for the association analysis. Results Polymorphisms of the FTO were significantly associated with BMI, in the rural population (beta 0.87, p-value <0.001). None of the other SNPs showed significant association after Bonferroni correction in the two populations or in the pooled analysis. A weighted genetic risk score (wGRS) was constructed using the risk alleles of the Tag-SNPs with a positive Beta parameter in both populations. From the first to the fifth quintile of the score, the BMI increased 0.45 kg/m2 in Hortega and 2.0 kg/m2 in Pizarra. Overall, the obesity predictive value was low (less than 1%). Conclusion The risk associated with polymorphisms is low and the overall effect on BMI or obesity prediction is minimal. A weighted genetic risk score based on genes mainly acting through central nervous system mechanisms was associated with BMI but it yields minimal clinical prediction for the obesity risk in the general population. [ABSTRACT FROM AUTHOR]

Background: There is increasing interest in using extracellular vesicle-derived microRNAs (miRNAs) as biomarkers in renal dysfunction and injury. Preliminary evidence indicates that miRNAs regulate the progression of glomerular disease. Indeed, exosomes from the renal system have provided novel evidence in the clinical setting of albuminuria. Thus, the aim of this study was to quantify the urinary miRNAs present in exosome and microvesicles (MVs), and to assess their association with the presence of increased urinary albumin excretion in essential hypertension.Methods: Exosomes were collected from urine specimens from a cohort of hypertensive patients with (n = 24) or without albuminuria (n = 28), and from 20 healthy volunteers as a control group. Urinary exosomes were phenotyped by Western blot, tunable resistive pulse sensing, and electronic microscopy. Expression of miR-146a and miR-335* was analysed by qRT-PCR and any associations between albuminuria and exosomal miRNAs were analysed.Results: Urinary miRNAs are highly enriched in exosome subpopulations compared to MVs, both in patients with or without increased albuminuria (p < 0.001), but not in the control group. High albuminuria was associated with 2.5-fold less miR-146a in exosomes (p = 0.017), whereas miR-146a levels in MV did not change. In addition, exosome miR-146a levels were inversely associated with albuminuria (r = 0.65, p < 0.0001), and discriminated the presence of urinary albumin excretion presence [area under the curve = 0.80, 95% confidence interval: 0.66-0.95; p = 0.0013].Conclusions: Our results indicate that miRNAs were enriched in the urinary exosome subpopulation in hypertensive patients and that low miR-146a expression in exosomes was associated with the presence of albuminuria. Thus, urinary exosome miR-146a may be a potentially useful tool for studying early renal injury in hypertension. [ABSTRACT FROM AUTHOR]