An Extensive Evaluation of Read Trimming Effects on Illumina NGS Data Analysis

Next Generation Sequencing is having an extremely strong impact in biological and medical research and diagnostics, with applications ranging from gene expression quantification to genotyping and genome reconstruction. Sequencing data is often provided as raw reads which are processed prior to analysis 1 of the most used preprocessing procedures is read trimming, which aims at removing low quality portions while preserving the longest high quality part of a NGS read. In the current work, we evaluate nine different trimming algorithms in four datasets and three common NGS-based applications (RNA-Seq, SNP calling and genome assembly). Trimming is shown to increase the quality and reliability of the analysis, with concurrent gains in terms of execution time and computational resources needed.     

An Immunomodulating Fatty Acid Analogue Targeting Mitochondria Exerts Anti-Atherosclerotic Effect beyond Plasma Cholesterol-Lowering Activity in apoE-/- Mice

Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator-activated receptors (PPARs) and proliferation of mitochondria. This study aimed to penetrate the effect of TTA on the development of atherosclerotic lesions in apolipoprotein (apo)-E^-/- mice fed a high-fat diet containing 0.3% TTA for 12 weeks. These mice displayed a significantly less atherosclerotic development vs control. Plasma cholesterol was increased by TTA administration and triacylglycerol (TAG) levels in plasma and liver were decreased by TTA supplementation, the latter, probably due to increased mitochondrial fatty acid oxidation and reduced lipogenesis. TTA administration also changed the fatty acid composition in the heart, and the amount of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was reduced and increased, respectively. The heart mRNA expression of inducible nitric oxidase (NOS)-2 was decreased in TTA-treated mice, whereas the mRNA level of catalase was increased. Finally, reduced plasma levels of inflammatory mediators as IL-1α, IL-6, IL-17, TNF-α and IFN-γ were detected in TTA-treated mice. These data show that TTA reduces atherosclerosis in apoE^-/- mice and modulates risk factors related to atherosclerotic disorders. TTA probably acts at both systemic and vascular levels in a manner independent of changes in plasma cholesterol, and triggers TAG catabolism through improved mitochondrial function.     

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.     

Diagnostic Potential of Plasmatic MicroRNA Signatures in Stable and Unstable Angina

Purpose

We examined circulating miRNA expression profiles in plasma of patients with coronary artery disease (CAD) vs. matched controls, with the aim of identifying novel discriminating biomarkers of Stable (SA) and Unstable (UA) angina.

Methods

An exploratory analysis of plasmatic expression profile of 367 miRNAs was conducted in a group of SA and UA patients and control donors, using TaqMan microRNA Arrays. Screening confirmation and expression analysis were performed by qRT-PCR: all miRNAs found dysregulated were examined in the plasma of troponin-negative UA (n=19) and SA (n=34) patients and control subjects (n=20), matched for sex, age, and cardiovascular risk factors. In addition, the expression of 14 known CAD-associated miRNAs was also investigated.

Results

Out of 178 miRNAs consistently detected in plasma samples, 3 showed positive modulation by CAD when compared to controls: miR-337-5p, miR-433, and miR-485-3p. Further, miR-1, -122, -126, -133a, -133b, and miR-199a were positively modulated in both UA and SA patients, while miR-337-5p and miR-145 showed a positive modulation only in SA or UA patients, respectively. ROC curve analyses showed a good diagnostic potential (AUC ≥ 0.85) for miR-1, -126, and -483-5p in SA and for miR-1, -126, and -133a in UA patients vs. controls, respectively. No discriminating AUC values were observed comparing SA vs. UA patients. Hierarchical cluster analysis showed that the combination of miR-1, -133a, and -126 in UA and of miR-1, -126, and -485-3p in SA correctly classified patients vs. controls with an efficiency ≥ 87%. No combination of miRNAs was able to reliably discriminate patients with UA from patients with SA.

Conclusions

This work showed that specific plasmatic miRNA signatures have the potential to accurately discriminate patients with angiographically documented CAD from matched controls. We failed to identify a plasmatic miRNA expression pattern capable to differentiate SA from UA patients.     

Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction

The human pulmonary vasculature constricts in response to hypercapnia and hypoxia, with important consequences for homeostasis and adaptation. One function of these responses is to direct blood flow away from poorly-ventilated regions of the lung. In humans it is not known whether the stimuli of hypercapnia and hypoxia constrict the pulmonary blood vessels independently of each other or whether they act synergistically, such that the combination of hypercapnia and hypoxia is more effective than the sum of the responses to each stimulus on its own. We independently controlled the alveolar partial pressures of carbon dioxide (Pa_{co 2}) and oxygen (Pa_{o 2}) to examine their possible interaction on human pulmonary vasoconstriction. Nine volunteers each experienced sixteen possible combinations of four levels of Pa_{co 2} (+6, +1, −4 and −9 mmHg, relative to baseline) with four levels of Pa_{o 2} (175, 100, 75 and 50 mmHg). During each of these sixteen protocols Doppler echocardiography was used to evaluate cardiac output and systolic tricuspid pressure gradient, an index of pulmonary vasoconstriction. The degree of constriction varied linearly with both Pa_{co 2} and the calculated haemoglobin oxygen desaturation (1-So ₂). Mixed effects modelling delivered coefficients defining the interdependence of cardiac output, systolic tricuspid pressure gradient, ventilation, Pa_{co 2} and So ₂. No interaction was observed in the effects on pulmonary vasoconstriction of carbon dioxide and oxygen (p>0.64). Direct effects of the alveolar gases on systolic tricuspid pressure gradient greatly exceeded indirect effects arising from concurrent changes in cardiac output.     

Characterization of the erythropoietin/erythropoietin receptor axis in a rat model of liver damage and cholangiocarcinoma development

It has been recently shown that the biological effects of erythropoietin (EPO) are not limited to the hematopoietic compartment but, as pleiotropic glycoprotein, this hormone can exert pro-angiogenic and tissue-protective functions also in a wide range of non-hematopoietic organs. The role of EPO and the effective functionality of its receptor in solid tumors are still a controversial point of debate. In the present work we analyzed the gene expression of EPO and its cognate receptor (EpoR) in a rat model of thioacetamide-induced damage and tumor. An analysis of the EPO/EpoR axis was also performed on human cholangiocarcinoma (CC) cell lines. A progressive increase of EPO and EpoR mRNA can already be observed during the fibrotic–cirrhotic development with a peak of expression rising at tumor formation (24.7 ± 9.9-fold increase and 15.5 ± 1.1-fold increase, respectively, for the two genes). Co-localization studies by immunofluorescence revealed hepatocytes in the regenerative cirrhotic nodules (Hep Par-1⁺) and in the dysplastic bile duct cells (CK19⁺) as the major EPO producers in this specific condition. The same cell populations, together with endothelial cells, exhibited an increased expression of EpoR, although all the non-parenchymal cell populations in the liver exhibited modest basal mRNA levels. Challenging human CC cells, Mz-Cha-2, with a combination of EPO and SCF resulted in a synergistic effect on the gene expression of EPO, CyclinD1 and PCNA. This study suggests that the autocrine and paracrine release of endogenous EPO in the microenvironment may contribute to the development and maintenance of the CC possibly in cooperation with other signaling pathways.     

Maintenance of photosynthetic capacity in flooded tomato plants with reduced ethylene sensitivity

Ethylene is considered one of the most important plant hormones orchestrating plant responses to flooding stress. However, ethylene may induce deleterious effects on plants, especially when produced at high rates in response to stress. In this paper, we explored the effect of attenuated ethylene sensitivity in the Never ripe (Nr) mutant on leaf photosynthetic capacity of flooded tomato plants. We found out that reduced ethylene perception in Nr plants was associated with a more efficient photochemical and non-photochemical radiative energy dissipation capability in response to flooding. The data correlated with the retention of chlorophyll and carotenoids content in flooded Nr leaves. Moreover, leaf area and specific leaf area were higher in Nr, indicating that ethylene would exert a negative role in leaf growth and expansion under flooded conditions. Although stomatal conductance was hampered in flooded Nr plants, carboxylation activity was not affected by flooding in the mutant, suggesting that ethylene is responsible for inducing non-stomatal limitations to photosynthetic CO₂ uptake. Upregulation of several cysteine protease genes and high protease activity led to Rubisco protein loss in response to ethylene under flooding. Reduction of Rubisco content would, at least in part, account for the reduction of its carboxylation efficiency in response to ethylene in flooded plants. Therefore, besides its role as a trigger of many adaptive responses, perception of ethylene entails limitations in light and dark photosynthetic reactions by speeding up the senescence process that leads to a progressive disassembly of the photosynthetic machinery in leaves of flooded tomato plants.     

Assessing the genetic diversity in Argopecten nucleus (Bivalvia: Pectinidae), a functional hermaphrodite species with extremely low population density and self‐fertilization: Effect of null alleles

Argopecten nucleus is a functional hermaphroditic pectinid species that exhibits self‐fertilization, whose natural populations have usually very low densities. In the present study, the genetic diversity of a wild population from Neguanje Bay, Santa Marta (Colombia), was estimated using microsatellite markers, and the effect of the presence of null alleles on this estimation was assessed. A total of 8 microsatellite markers were developed, the first described for this species, and their amplification conditions were standardized. They were used to determine the genotype of 48 wild individuals from Naguanje Bay, and 1,010 individuals derived from the offspring of 38 directed crosses. For each locus, the frequencies of the identified alleles, including null alleles, were estimated using the statistical package Micro‐Checker, and the parental genotypes were confirmed using segregation analysis. Three to 8 alleles per locus with frequencies from 0.001 to 0.632 were detected. The frequencies of null alleles ranged from 0.10 to 0.45, with Ho from 0.0 to 0.79, and He from 0.53 to 0.80. All loci were in H‐W disequilibrium. The null allele frequencies values were high, with lower estimations using segregation analysis than estimated using Micro‐Checker. The present results show high levels of population genetic diversity and indicate that null alleles were not the only cause of deviation from H‐W equilibrium in all loci, suggesting that the wild population under study presents signs of inbreeding and Wahlund effect.     

The emergent role of sarcopenia: Preliminary Report of the Observatory of Sarcopenia of the Spanish Society of Geriatrics and Gerontology

Sarcopenia is a common and prominent geriatric syndrome, of major interest for daily clinical practice of professionals working with older people. The number of affected individuals and its relation with disability, frailty, many chronic diseases, lifestyle and adverse outcomes are extremely relevant for geriatric care. Moreover, biological changes that lead to the loss of muscle mass and strength are intrinsically related to the mechanisms of aging. It is not therefore surprising that research in this field is growing exponentially in recent years, and sarcopenia has been placed in recent years in the forefront of research in geriatric medicine and gerontology. The Spanish Society of Geriatrics and Gerontology has recently created an Observatory of Sarcopenia, which aims to promote educational and research activities in this field. The first activity of the Observatory has been to offer the Spanish speaking scientific community a review of the current status of sarcopenia, that may allow unifying concepts and fostering interest in this promising field of geriatrics.