High miR-126-3p levels associated with cardiovascular events in a general population.

BACKGROUND: Endothelial dysfunction is a forerunner of atherosclerosis, leading to cardiovascular disease, and albuminuria is a marker of endothelial dysfunction. Circulating levels of microRNAs are emerging as potential biomarkers for cardiovascular disease. Here we estimate the predictive value of a plasma microRNAs signature associated with albuminuria in the incidence of cardiovascular events. METHODS: Plasma microRNAs quantified in hypertensive patients by next generation sequencing were validated in a cohort of patients and controls by real-time quantitative PCR. The microRNAs found to be associated with albuminuria were analysed for their prognostic value in predicting cardiovascular events incidence on a retrospective, population-based study (Hortega Study), using Cox proportional hazard models. RESULTS: A plasma microRNA profile was identified in the discovery cohort (n = 48) associated with albuminuria and three microRNAs (miR-126-3p, miR-1260b and miR-374a-5p) were confirmed in the validation cohort (n = 98). The microRNA signature discriminates urinary albumin excretion at baseline (n = 1025), and predicts the incidence of cardiovascular events and coronary heart disease and stroke in a general population retrospective study within a 14-year follow-up (n = 926). High miR-126-3p levels were associated with a shorter time free of both cardiovascular events (HR=1.48, (1.36-1.62), p < 0.0001), as well as coronary artery disease and stroke combined (HR=2.49, (2.19-2.83), p < 0.0001). CONCLUSIONS: An increased plasma microRNAs profile was identified in hypertensive patients with albuminuria. Increased miR-126-3p suggest it may serve as a prognostic marker for cardiovascular events in a long-term general population. Further studies will assess the potential role of miR-126-3p as a guide for the status of endothelial dysfunction.

Serum vascular endothelial growth factor b and metabolic syndrome incidence in the population based cohort Di@bet.es study.

BACKGROUND/OBJECTIVES: Although vascular endothelial growth factor b (VEGFb) might have an impact on the development of obesity, diabetes and related disorders, the possible relationship between VEGFb serum levels and the incidence of these metabolic complications in humans is still unknown. The aim of our study was to evaluate the association between VEGFb serum levels and the new-onset of metabolic syndrome (MS) and its components in the Spanish adult population after 7.5 years of follow-up. SUBJECTS/METHODS: A total of 908 subjects from the Di@bet.es cohort study without MS at cross-sectional stage according to International Diabetes Federation (IDF) or Adult Treatment Panel III (ATP-III) criteria were included. Additionally, five sub-populations were grouped according to the absence of each MS component at baseline. Socio-demographic, anthropometric and clinical data were recorded. The Short Form of International Physical Activity Questionnaire (SF-IPAQ) was used to estimate physical activity. A fasting blood extraction and an oral glucose tolerance test were performed. Serum determinations of glucose, lipids, hsCRP and insulin were made. VEGFb levels were determined and categorized according to the 75th percentile of the variable. New cases of MS and its components were defined according to ATPIII and IDF criteria. RESULTS: A total of 181 or 146 people developed MS defined by IDF or ATP-III criteria respectively. Serum triglyceride levels, hs-CRP and systolic blood pressure at the baseline study were significantly different according to the VEGFb categories. Adjusted logistic regression analysis showed that the likelihood of developing MS and abdominal obesity was statistically reduced in subjects included in the higher VEGFb category. CONCLUSION: Low serum levels of VEGFb may be considered as early indicators of incident MS and abdominal obesity in the Spanish adult population free of MS, independently of other important predictor variables.

Genetic variants in obesity-related genes and the risk of osteoporotic fracture. The Hortega Follow-up Study.

BACKGROUND: Osteoporosis and obesity are major public health problems that are closely correlated, as they share various features, including a genetic predisposition. A genetic correlation between obesity and osteoporosis due to the biological common pathways of bone and fat metabolism, which implies pleiotropic genes regulating has been described. The objective of our study was to analyse whether polymorphisms in obesity-related genes modify the risk of osteoporotic bone fracture. METHODS: We studied 575 subjects from the Hortega Study. The subjects were followed-up for 12-14 years. 202 subjects were overweight, 143 obese and 221 had bone fractures. The distribution of 39 genetic variants in 22 obesity-related genes were studied. RESULTS: The results showed a relationship between polymorphisms in the FTO and NEGR1 genes and the susceptibility to osteoporotic fracture. The variant genotype of the rs2568958 NEGR1 polymorphism and the rs6499649, rs3751812, and rs8044769 genetic variants in FTO were associated with susceptibility to bone fracture. In the best of our knowledge, this is the first time that these variants in NEGR1 and FTO genes have been associated with the susceptibility to osteoporotic bone fracture, supporting the hypothesis that the NEGR1 and FTO genes might be candidates for osteoporosis and bone fracture. CONCLUSIONS: In conclusion, this study associates obesity-related polymorphisms in the NEGR1 and FTO genes with osteoporotic bone fracture, reinforcing the hypothesis that obesity and bone metabolism are closely correlated genetically.

Association between genetic variants in oxidative stress-related genes and osteoporotic bone fracture. The Hortega follow-up study.

The most widely accepted etiopathogenesis hypothesis of the origin of osteoporosis and its complications is that they are a consequence of bone aging and other environmental factors, together with a genetic predisposition. Evidence suggests that oxidative stress is crucial in bone pathologies associated with aging. The aim of this study was to determine whether genetic variants in oxidative stress-related genes modified the risk of osteoporotic fracture. We analysed 221 patients and 354 controls from the HORTEGA sample after 12-14 years of follow up. We studied the genotypic and allelic distribution of 53 SNPs in 24 genes involved in oxidative stress. The results showed that being a carrier of the variant allele of the SNP rs4077561 within TXNRD1 was the principal genetic risk factor associated with osteoporotic fracture and that variant allele of the rs1805754 M6PR, rs4964779 TXNRD1, rs406113 GPX6, rs2281082 TXN2 and rs974334 GPX6 polymorphisms are important genetic risk factors for fracture. This study provides information on the genetic factors associated with oxidative stress which are involved in the risk of osteoporotic fracture and reinforces the hypothesis that genetic factors are crucial in the etiopathogenesis of osteoporosis and its complications.

Urinary- and Plasma-Derived Exosomes Reveal a Distinct MicroRNA Signature Associated With Albuminuria in Hypertension.

Urinary albumin excretion (UAE) is a marker of cardiovascular risk and renal damage in hypertension. MicroRNAs (miRNAs) packaged into exosomes function as paracrine effectors in cell communication and the kidney is not exempt. This study aimed to state an exosomal miRNA profile/signature associated to hypertension with increased UAE and the impact of profibrotic TGF-ß1 (transforming growth factor ß1) on exosomes miRNA release. Therefore, exosomes samples from patients with hypertension with/without UAE were isolated and characterized. Three individual and unique small RNA libraries from each subject were prepared (total plasma, urinary, and plasma-derived exosomes) for next-generation sequencing profiling. Differentially expressed miRNAs were over-represented in Kyoto Encyclopedia of Genes and Genomes pathways, and selected miRNAs were validated by real-time quantitative polymerase chain reaction in a confirmation cohort. Thus, a signature of 29 dysregulated circulating miRNAs was identified in UAE hypertensive subjects, regulating 21 pathways. Moreover, changes in the levels of 4 exosomes-miRNAs were validated in a confirmation cohort and found associated with albuminuria. In particular miR-26a, major regulator of TGF-ß signaling, was found downregulated in both type of exosomes when compared with healthy controls and to hypertension normoalbuminurics (P<0.01). Similarly, decreased miR-26a levels were found in podocyte-derived exosomes after TGF-ß stress. Our results revealed an exosomes miRNA signature associated to albuminuria in hypertension. In particular, exosomes miR-26a seemed to play a key role in the regulation of TGF-ß, a relevant effector in podocyte damage. These findings support the use of exosomes miRNAs as biomarkers of cardiovascular risk progression and therapeutic tools in early kidney damage.

Urinary exosomal miR-146a as a marker of albuminuria, activity changes and disease flares in lupus nephritis.

BACKGROUND: Urinary exosomes, especially microRNAs (miRNAs) packaged within, are ideal sources of renal damage markers. We investigated the association between exosomal miR-146a, (anti-inflammatory regulator) and disease activity, proteinuria and systemic lupus erythematosus (SLE) flares over a 36-month follow-up period. METHODS: We isolated urinary exosomes from 41 SLE patients, 27 with lupus nephritis (LN) and 20 healthy controls, and exosomal miR-146a, quantified by the real-time quantitative polymerase chain reaction (RT-qPCR), was correlated with histological features in 13 renal biopsies. We also analysed the association between the exosomal miR-146a and TRAF6 axis. RESULTS: Exosomal miR-146a showed an inverse association with circulating C3 and C4 complement components, proteinuria, and with histological features such as chronicity index. This marker was able to identify LN with an AUC of 0.82 (p = 0.001). Basal exosomal miR-146a was associated with disease activity and proteinuria changes and was an independent marker of 36-month follow-up flares (OR 7.08, p = 0.02). Pathway analysis identified IRAK1 and TRAF6 as miR-146a target genes. Finally, in vitro experiments suggested that miR-146a exerts a protective effect through negative regulation of inflammation by suppressing IRAK1 and TRAF6. CONCLUSIONS: Urinary exosomal miR-146a levels are correlated with lupus activity, proteinuria and histological features, discriminating patients with LN and being a good baseline marker of SLE flares. We have identified a relevant biological miR-146a-TRAF6 axis association in LN renal fibrosis progression.

The Rab-Rabphilin system in injured human podocytes stressed by glucose overload and angiotensin II.

Kidney injury in hypertension and diabetes entails, among in other structures, damage in a key cell of the glomerular filtration barrier, the podocyte. Podocytes are polarized and highly differentiated cells in which vesicular transport, partly driven by Rab GTPases, is a relevant process. The aim of the present study was to analyze Rab GTPases of the Rab-Rabphilin system in human immortalized podocytes and the impact of high glucose and angiotensin II. Furthermore, alterations of the system in urine cell pellets from patients with hypertension and diabetes were studied. Apoptosis was analyzed in podocytes, and mRNA level quantification, Western blot analysis, and immunofluorescence were developed to quantify podocyte-specific molecules and Rab-Rabphilin components (Rab3A, Rab27A, and Rabphilin3A). Quantitative RT-PCR was performed on urinary cell pellet from patients. The results showed that differentiated cells had reduced protein levels of the Rab-rabphillin system compared with undifferentiated cells. After glucose overload and angiotensin II treatment, apoptosis was increased and podocyte-specific proteins were reduced. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of podocyte markers and Rab27A under treatments. Finally, Rab3A and Rab27A were increased in patient urine pellets and showed a direct relationship with albuminuria. Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.

Polymorphisms in genes involved in inflammation, the NF-kB pathway and the renin-angiotensin-aldosterone system are associated with the risk of osteoporotic fracture. The Hortega Follow-up Study.

Osteoporosis is the most common bone disorder worldwide and is associated with a reduced quality of life with important clinical and economic consequences. The most widely accepted etiopathogenic hypothesis on the origin of osteoporosis and its complications is that they are a consequence of the synergic action of environmental and genetic factors. Bone is constantly being remodelled through anabolic and catabolic pathways in which inflammation, the NF-kB pathway and the renin-angiotensin-aldosterone system (RAAS) are crucial. The aim of our study was to determine whether polymorphisms in genes implicated in inflammation, the NF-kB pathway and RAAS modified the risk of osteoporotic fracture. We analysed 221 patients with osteoporotic fracture and 354 controls without fracture from the HORTEGA sample after 12-14 years of follow up. In addition, we studied the genotypic distribution of 230 single nucleotide polymorphisms (SNPs) in genes involved in inflammation, NF-kB pathway and RAAS. Our results showed that be carrier of the C allele of the rs2228145 IL6R polymorphism was the principal genetic risk factor associated with osteoporotic fracture. The results also showed that variant genotypes of the rs4762 AGT, rs4073 IL8, rs2070699 END1 and rs4291 ACE polymorphisms were important genetic risk factors for fracture. The study provides information about the genetic factors associated with inflammation, the NF-kB pathway and RAAS, which are involved in the risk of osteoporotic fracture and reinforces the hypothesis that genetic factors are crucial in the etiopathogenesis of osteoporosis and its complications.

Optimization of small RNA library preparation protocol from human urinary exosomes.

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.

Easy One-Step Amplification and Labeling Procedure for Copy Number Variation Detection.

BACKGROUND: The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS: We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS: The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS: EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.

Nearly Complete Genome Sequences of Human Norovirus Belonging to Several Genotypes from Valencia, Spain.

Human noroviruses are responsible for most nonbacterial acute gastroenteritis cases. The GII.2, GII.4, and GII.17 genotypes of human noroviruses have recently arisen as the most frequent genotypes found in humans worldwide. We report here seven nearly complete genomes of these genotypes from patients with acute gastroenteritis in Valencia, Spain.

Nearly Complete Genome Sequence of a Human Norovirus GII.P17-GII.17 Strain Isolated from Brazil in 2015.

Human noroviruses are the most common cause of nonbacterial acute gastroenteritis worldwide. We report here the nearly complete genome sequence (7,551 nucleotides) of a human norovirus GII.P17-GII.17 strain detected in July 2015 in the stool sample from an adult with acute gastroenteritis in Brazil.

Immune-unreactive urinary albumin as a predictor of cardiovascular events: the Hortega Study.

BACKGROUND: We aimed to determine if immune-unreactive albumin excretion (IURAE) is associated with cardiovascular (CV) events in a representative sample of a general population from Spain. METHODS: We included 1297 subjects (mean age ± standard error 48.0 ± 0.2 years, 48% females), who participated in the Hortega Follow-Up Study. The primary endpoint was incidence of fatal and non-fatal CV events. Urinary albumin excretion (UAE) was measured in spot voided urine, frozen at -80°C, by immunonephelometry [immune-reactive albumin excretion (IRAE)] and by high-performance liquid chromatography (HPLC) [total albumin excretion (AE)]. IURAE was calculated as the difference between HPLC measurements and IRAE. We estimated fully adjusted hazard ratios (HRs) of CV incidence by Cox regression for IRAE, IURAE and total AE. RESULTS: After an average at-risk follow-up of 13 years, we observed 172 CV events. urinary albumin to creatinine ratio (UACR) of ≥30 mg/g assessed by IRAE, IURAE or total AE concentrations was observed in 74, 273 and 417 participants, respectively. Among discordant pairs, there were 49 events in those classified as micro- and macroalbuminuric by IURAE, but normoalbuminuric by IRAE. Only the IRAE was a significant independent factor for the incidence of CV events [HR (95% confidence interval) 1.15 (1.04-1.27)]. The association of UAE with CV events was mainly driven by heart failure (HF) [HR 1.33 (1.15-1.55) for IRAE; HR 1.38 (1.06-1.79) for IURAE; HR 1.62 (1.22-2.13) for total AE]. Those subjects who were micro- and macroalbuminuric by both IRAE and IURAE had a significant increase in risk for any CV event, and especially for HF. CONCLUSIONS: IRAE, IURAE and AE were associated with an increased risk for CV events, but IRAE offered better prognostic assessment.

Urinary exosome miR-146a is a potential marker of albuminuria in essential hypertension.

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.

Urinary podocyte-associated molecules and albuminuria in hypertension.

OBJECTIVE: Hypertension-induced podocyte damage and the relationship with UAE is analyzed in diabetic and nondiabetic participants. PATIENTS AND METHODS: Sixty-four hypertensive patients, 30 diabetics, with glomerular filtration rate (eGFR) greater than 60 ml/min per 1.73 m were included. Urinary albumin excretion was measured in morning urine using a nephelometric immunoassay and expressed as albumin/creatinine ratio. Urinary pellets were obtained from fresh urine and mRNA was assessed by real-time quantitative PCR. Likewise, protein podocyte-specific molecules were measured by western blot using specific antibodies. RESULTS: Fourteen nondiabetics and 20 diabetics had increased UAE greater than 30 mg/g. In individuals with increased EUA, the mRNA expression of nephrin and CD2AP was low in diabetics, whereas only nephrin mRNA in nondiabetics. No differences were observed in podocalyxin and aquaporin-1 mRNA levels. Concerning the protein values, in both nondiabetic and diabetic patients, nephrin, CD2AP and podocalyxin were increased in patients with increased UAE, with no differences in aquaporin-1. A significant positive relationship was observed between log UAE and nephrin protein values, and an inverse association observed with mRNA. CONCLUSION: Hypertensive patients who had elevated UAE showed increased urinary excretion of podocyte-specific proteins coupled with a phenotype of decreased mRNA expression. The phenotype of podocyte-specific mRNA and the increment of nephrin can be used as a valuable marker of early glomerular injury.

Iron deficiency is associated with Hypothyroxinemia and Hypotriiodothyroninemia in the Spanish general adult population: Di@bet.es study.

Previous studies have suggested that iron deficiency (ID) may impair thyroid hormone metabolism, however replication in wide samples of the general adult population has not been performed. We studied 3846 individuals free of thyroid disease, participants in a national, cross sectional, population based study representative of the Spanish adult population. Thyroid stimulating hormone (TSH), free thyroxin (FT4) and free triiodothyronine (FT3) were analyzed by electrochemiluminescence (E170, Roche Diagnostics). Serum ferritin was analyzed by immunochemiluminescence (Architect I2000, Abbott Laboratories). As ferritin levels decreased (>100, 30-100, 15-30, <15 µg/L) the adjusted mean concentrations of FT4 (p < 0.001) and FT3 (p < 0.001) descended, whereas TSH levels remained unchanged (p = 0.451). In multivariate logistic regression models adjusted for age, sex, UI, BMI and smoking status, subjects with ferritin levels <30 µg/L were more likely to present hypothyroxinemia (FT4 < 12.0 pmol/L p5): OR 1.5 [1.1-2.2] p = 0.024, and hypotriiodothyroninemia (FT3 < 3.9 pmol/L p5): OR 1.8 [1.3-2.6] p = 0.001 than the reference category with ferritin ≥30 µg/L. There was no significant heterogeneity of the results between men, pre-menopausal and post-menopausal women or according to the iodine nutrition status. Our results confirm an association between ID and hypothyroxinemia and hypotriiodothyroninemia in the general adult population without changes in TSH.

Urinary levels of sirtuin-1 associated with disease activity in lupus nephritis.

Identifying new markers of disease flares in lupus nephritis (LN) that facilitate patient stratification and prognosis is important. Therefore, the aim of the present study was to analyze whether urinary SIRT1 expression was altered in LN and whether SIRT1 values in urine could be valuable biomarker of disease activity. In a cohort study, urinary pellets from 40 patients diagnosed with systemic lupus erythematosus (SLE) were analyzed. Clinical measures of lupus activity were assessed. The expression of SIRT1 was quantified by quantitative PCR (qRT-PCR) and immunoblot, then compared between patients with active lupus nephritis, in remission and healthy controls. Association with lupus activity and renal histological features was also analyzed. A significant increase in SIRT1 mRNA levels in patients with active LN was observed compared with those in remission (P=0.02) or healthy controls (P=0.009). In addition, SIRT-1 protein levels were also augmented in LN group than remission (P=0.029) and controls (P=0.001). A strong association was found between SIRT1 expression with anti-dsDNA in SLE and in patients with LN. In addition, histological features in LN biopsies were related with SIRT1, increasing its expression in proliferative forms. Finally, SIRT1 expression values showed a strong discriminatory power of renal injury in SLE. Our study demonstrated an altered urinary expression of SIRT1 and a strong association with disease activity in LN patients, being a valuable marker of renal injury. These results showed the role of the SIRT1 pathway in the SLE pathogenesis.

VISMapper: ultra-fast exhaustive cartography of viral insertion sites for gene therapy.

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.

Coexistence of EGFR, KRAS, BRAF, and PIK3CA Mutations and ALK Rearrangement in a Comprehensive Cohort of 326 Consecutive Spanish Nonsquamous NSCLC Patients.

INTRODUCTION: Molecular screening is crucial for the care of nonsquamous non-small-cell lung cancer (NSCLC) patients. The coexistence of mutations could have important consequences regarding treatment. We described the mutational patterns and coexistence among patients and their outcomes after targeted treatment. MATERIALS AND METHODS: Data from consecutive patients with newly diagnosed nonsquamous NSCLC were prospectively collected. Next-generation sequencing analysis of mutational hotspots in the EGFR, KRAS, PIK3CA, and BRAF genes and analysis of anaplastic lymphoma kinase (ALK) rearrangement were performed. RESULTS: A total of 326 patients with nonsquamous NSCLC were identified. Of the 326 patients, 240 (73.6%) had EGFR, 141 (43.3%) KRAS, 137 (42.0%) BRAF, 130 (39.9%) PIK3CA mutation and 148 (45.4%) ALK rearrangement determined. Of the 240 with EGFR determination, 24.1% harbored EGFR mutations. Of these, 16.3% were activating mutations (43.6%, exon 19 deletion; 46.1%, exon 21; and 10.3%, exon 18) and 7.9% were nonsensitizing EGFR mutations. Furthermore, 39.0% had KRAS mutations, 2.9% BRAF mutations, 10.0% PIK3CA mutations, and 8.8% ALK rearrangements. Of the 154 stage IV patients with ≥ 1 mutations, analysis showed 19 coexisting cases (12.3%). Of 8 patients receiving targeted treatment, 6 had no response. Both responders to targeted treatment had coexistent PIK3CA mutations. CONCLUSION: Driver mutations can coexist in nonsquamous NSCLC. In our cohort, 12.3% of cases with stage IV disease had multiple mutations. Targeted treatment might not be as effective in patients with coexisting mutations; however, coexistence with PIK3CA might not preclude a response.

One-year follow-up of clinical, metabolic and oxidative stress profile of morbid obese patients after laparoscopic sleeve gastrectomy. 8-oxo-dG as a clinical marker.

Obesity has grown worldwide over the last few decades. In its different degrees, obesity is accompanied by many clinical and biochemical alterations reflecting the pathological condition of various body tissues. Among the mechanisms underlying the pathogenesis of obesity and associated complications, oxidative stress (OS) may be playing an important role. In the present study, we have characterized at systemic level the degree of OS status in a group of morbid obese patients (BMI>40kg/m2) at basal sate and its modulation during one year after bariatric surgery using the laparoscopic sleeve gastrectomy (LSG) technique. As compared with normal weight subjects matched in age, peripheral blood mononuclear cells (PBMc) of obese patients present a significant reduction of the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as well as a significant increase of the oxidized/reduced glutathione ratio (GSSG/GSH) in these cells. Lipid peroxidation is significantly increased in the patient group as shown by the increased levels of malondialdehyde (MDA) in PBMc and the amount of F2-Isoprostanes (F2-IsoPs) released in urine. In addition, the DNA damage product 8-oxo-7,8-2'-deoxyguanosine (8-oxo-dG) was also observed to be increased in serum and urine of morbid obese patients as compared with the control group. After LSG, an improvement of their ponderal and metabolic profile was accompanied by a progressive recovery of antioxidant enzyme activities and the decline of oxidative byproducts both in PBMc and biological fluids. The observed changes of urinary 8-oxo-dG levels correlate positively with its serum concentration, the lipid peroxidation products MDA and F2-IsoPs, triglycerides, glucose, insulin, HOMA index and body weight and negatively with the percentage of weight and BMI loss and antioxidant activities. We conclude that the analysis of urinary 8-oxo-dG could be validated as a useful marker for the monitoring of ponderal and metabolic status of morbid obese patients.