DaMiRseq—an R/Bioconductor package for data mining of RNA-Seq data: normalization, feature selection and classification

Abstract Summary RNA-Seq is becoming the technique of choice for high-throughput transcriptome profiling, which, besides class comparison for differential expression, promises to be an effective and powerful tool for biomarker discovery. However, a systematic analysis of high-dimensional genomic data is a demanding task for such a purpose. DaMiRseq offers an organized, flexible and convenient framework to remove noise and bias, select the most informative features and perform accurate classification. Availability and implementation DaMiRseq is developed for the R environment (R ≥ 3.4) and is released under GPL (≥2) License. The package runs on Windows, Linux and Macintosh operating systems and is freely available to non-commercial users at the Bioconductor open-source, open-development software project repository (https://bioconductor.org/packages/DaMiRseq/). In compliance with Bioconductor standards, the authors ensure stable package maintenance through software and documentation updates. Supplementary information Supplementary data are available at Bioinformatics online

Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration.

Background/aims Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. Methods We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²⁺ mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. Results We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²⁺ signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. Conclusion Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries

Untargeted metabolomics to go beyond the canonical effect of acetylsalicylic acid

15openInternationalItalian coauthor/editorGiven to its ability to irreversibly acetylate the platelet cyclooxygenase-1 enzyme, acetylsalicylic acid (ASA) is successfully employed for the prevention of cardiovascular disease. Recently, an antitumoral effect of ASA in colorectal cancer has been increasingly documented. However, the molecular and metabolic mechanisms by which ASA exerts such effect is largely unknown. Using a new, untargeted liquid chromatography–mass spectrometry approach, we have analyzed urine samples from seven healthy participants that each ingested 100 mg of ASA once daily for 1 week. Of the 2007 features detected, 25 metabolites differing after ASA ingestion (nominal p 1) were identified, and pathway analysis revealed low levels of glutamine and of metabolites involved in histidine and purine metabolisms. Likewise, consistent with an altered fatty acid β-oxidation process, a decrease in several short- and medium-chain acyl-carnitines was observed. An abnormal β-oxidation and a lower than normal glutamine availability suggests reduced synthesis of acetyl-Co-A, as they are events linked to one another and experimentally related to ASA antiproliferative effects. While giving an example of how untargeted metabolomics allows us to explore new clinical applications of drugs, the present data provide a direction to be pursued to test the therapeutic effects of ASA—e.g., the antitumoral effect—beyond cardiovascular protectionopenDi Minno, Alessandro; Porro, Benedetta; Turnu, Linda; Manega, Chiara Maria; Eligini, Sonia; Barbieri, Simone; Chiesa, Mattia; Poggio, Paolo; Squellerio, Isabella; Anesi, Andrea; Fiorelli, Susanna; Caruso, Donatella; Veglia, Fabrizio; Cavalca, Viviana; Tremoli, ElenaDi Minno, A.; Porro, B.; Turnu, L.; Manega, C.M.; Eligini, S.; Barbieri, S.; Chiesa, M.; Poggio, P.; Squellerio, I.; Anesi, A.; Fiorelli, S.; Caruso, D.; Veglia, F.; Cavalca, V.; Tremoli, E

Treatment with PCSK9 inhibitors in patients with familial hypercholesterolemia lowers plasma levels of platelet-activating factor and its precursors: a combined metabolomic and lipidomic approach

13openInternationalItalian coauthor/editorIntroduction: Familial hypercholesterolemia (FH) is characterized by extremely high levels of circulating low-density lipoprotein cholesterol (LDL-C) and is caused by mutations of genes involved in LDL-C metabolism, including LDL receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/Kexin type 9 (PCSK9). Accordingly, PCSK9 inhibitors (PCSK9i) are effective in LDL-C reduction. However, no data are available on the pleiotropic effect of PCSK9i. To this end, we performed an untargeted metabolomics approach to gather a global view on changes in metabolic pathways in patients receiving treatment with PCSK9i. Methods: Twenty-five FH patients starting treatment with PCSK-9i were evaluated by an untargeted metabolomics approach at baseline (before PCSK9i treatment) and after 12 weeks of treatment. Results: All the 25 FH subjects enrolled were on maximal tolerated lipid-lowering therapy prior to study entry. After a 12 week treatment with PCSK9i, we observed an expected significant reduction in LDL-cholesterol levels (from 201.0 ± 69.5 mg/dL to 103.0 ± 58.0 mg/dL, p < 0.001). The LDL-C target was achieved in 36% of patients. After peak validation and correction, after 12 weeks of PCSK9i treatment as compared to baseline, we observed increments in creatine (p-value = 0.041), indole (p-value = 0.045), and indoleacrylic acid (p-value= 0.045) concentrations. Conversely, significant decreases in choline (p-value = 0.045) and phosphatidylcholine (p-value < 0.01) together with a reduction in platelet activating factor (p-value = 0.041) were observed. Conclusions: Taking advantage of untargeted metabolomics, we first provided evidence of concomitant reductions in inflammation and platelet activation metabolites in FH patients receiving a 12 week treatment with PCSK9iopenDi Minno, Alessandro; Orsini, Roberta Clara; Chiesa, Mattia; Cavalca, Viviana; Calcaterra, Ilenia; Tripaldella, Maria; Anesi, Andrea; Fiorelli, Susanna; Eligini, Sonia; Colombo, Gualtiero I; Tremoli, Elena; Porro, Benedetta; Di Minno, Matteo Nicola DarioDi Minno, A.; Orsini, R.C.; Chiesa, M.; Cavalca, V.; Calcaterra, I.; Tripaldella, M.; Anesi, A.; Fiorelli, S.; Eligini, S.; Colombo, G.I.; Tremoli, E.; Porro, B.; Di Minno, M.N.D

Inhibition of IL-1β signaling normalizes NMDA-dependent neurotransmission and reduces seizure susceptibility in a mouse model of Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a neurodegenerative disorder caused by prion protein (PrP) misfolding, clinically recognized by cognitive and motor deficits, electroencephalographic abnormalities, and seizures. Its neurophysiological bases are not known. To assess the potential involvement of NMDA receptor (NMDAR) dysfunction, we analyzed NMDA-dependent synaptic plasticity in hippocampal slices from Tg(CJD) mice, which model a genetic form of CJD. Because PrP depletion may result in functional upregulation of NMDARs, we also analyzed PrP knock-out (KO) mice. Long-term potentiation (LTP) at the Schaffer collateral-commissural synapses in the CA1 area of ∼100-d-old Tg(CJD) mice was comparable to that of wild-type (WT) controls, but there was an inversion of metaplasticity, with increased GluN2B phosphorylation, which is indicative of enhanced NMDAR activation. Similar but less marked changes were seen in PrP KO mice. At ∼300 d of age, the magnitude of LTP increased in Tg(CJD) mice but decreased in PrP KO mice, indicating divergent changes in hippocampal synaptic responsiveness. Tg(CJD) but not PrP KO mice were intrinsically more susceptible than WT controls to focal hippocampal seizures induced by kainic acid. IL-1β-positive astrocytes increased in the Tg(CJD) hippocampus, and blocking IL-1 receptor signaling restored normal synaptic responses and reduced seizure susceptibility. These results indicate that alterations in NMDA-dependent glutamatergic transmission in Tg(CJD) mice do not depend solely on PrP functional loss. Moreover, astrocytic IL-1β plays a role in the enhanced synaptic responsiveness and seizure susceptibility, suggesting that targeting IL-1β signaling may offer a novel symptomatic treatment for CJD.SIGNIFICANCE STATEMENT Dementia and myoclonic jerks develop in individuals with Creutzfeldt-Jakob disease (CJD), an incurable brain disorder caused by alterations in prion protein structure. These individuals are prone to seizures and have high brain levels of the inflammatory cytokine IL-1β. Here we show that blocking IL-1β receptors with anakinra, the human recombinant form of the endogenous IL-1 receptor antagonist used to treat rheumatoid arthritis, normalizes hippocampal neurotransmission and reduces seizure susceptibility in a CJD mouse model. These results link neuroinflammation to defective neurotransmission and the enhanced susceptibility to seizures in CJD and raise the possibility that targeting IL-1β with clinically available drugs may be beneficial for symptomatic treatment of the disease

Laser versus LED polymerization: Comparison of in vitro preventive sealing procedures

Introduction: The technique of sealing is a widely accepted procedure for prevention of caries. The aim of our in vitro study was to compare the effect of two different curing units (traditional LED source and innovative laser diode lamp) on the integrity of fissure sealant material and its interface with tooth enamel. Materials and methods: Sixty healthy third molars were randomly assigned to two groups. In group A were teeth intended for polymerization by LED B lamp, and group B comprised teeth to be polymerized by an innovative laser diode. Both groups were treated with the traditional sealing technique, subjected to a metallization process, and analyzed by scanning electron microscope. Results: Micro-gaps between the sealant and the enamel were found in specimens in both A (43%) and B (40%) groups (p=0.793), and sealant shrinkage was seen. Significant differences between the groups emerged in the percentage of perimetric micro-erosion sites (80% vs. 100%, p=0.010) and the presence of holes and micro-bubbles on the sealant surface (21% vs. 63%, p=0.001). Conclusions: Although macroscopic clinical polymerization occurred with both instruments, the microscopic evaluation showed significant differences between the studied groups in terms of perimetric micro-erosion sites and micro-bubbles, which were higher in laser-cured samples

Application of AI in cardiovascular multimodality imaging

Technical advances in artificial intelligence (AI) in cardiac imaging are rapidly improving the reproducibility of this approach and the possibility to reduce time necessary to generate a report. In cardiac computed tomography angiography (CCTA) the main application of AI in clinical practice is focused on detection of stenosis, characterization of coronary plaques, and detection of myocardial ischemia. In cardiac magnetic resonance (CMR) the application of AI is focused on post-processing and particularly on the segmentation of cardiac chambers during late gadolinium enhancement. In echocardiography, the application of AI is focused on segmentation of cardiac chambers and is helpful for valvular function and wall motion abnormalities. The common thread represented by all of these techniques aims to shorten the time of interpretation without loss of information compared to the standard approach. In this review we provide an overview of AI applications in multimodality cardiac imaging

Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds

MiR-320a as a Potential Novel Circulating Biomarker of Arrhythmogenic CardioMyopathy

Diagnosis of Arrhythmogenic CardioMyopathy (ACM) is challenging and often late after disease onset. No circulating biomarkers are available to date. Given their involvement in several cardiovascular diseases, plasma microRNAs warranted investigation as potential non-invasive diagnostic tools in ACM. We sought to identify circulating microRNAs differentially expressed in ACM with respect to Healthy Controls (HC) and Idiopathic Ventricular Tachycardia patients (IVT), often in differential diagnosis. ACM and HC subjects were screened for plasmatic expression of 377 microRNAs and validation was performed in 36 ACM, 53 HC, 21 IVT. Variable importance in data partition was estimated through Random Forest analysis and accuracy by Receiver Operating Curves. Plasmatic miR-320a showed 0.53\u2009\ub1\u20090.04 fold expression difference in ACM vs. HC (p\u2009<\u20090.01). A similar trend was observed when comparing ACM (n\u2009=\u200913) and HC (n\u2009=\u200917) with athletic lifestyle, a ACM precipitating factor. Importantly, ACM patients miR-320a showed 0.78\u2009\ub1\u20090.05 fold expression change vs. IVT (p\u2009=\u20090.03). When compared to non-invasive ACM diagnostic parameters, miR-320a ranked highly in discriminating ACM vs. IVT and it increased their accuracy. Finally, miR-320a expression did not correlate with ACM severity. Our data suggest that miR-320a may be considered a novel potential biomarker of ACM, specifically useful in ACM vs. IVT differentiation

Measurements of the W production cross sections in association with jets with the ATLAS detector

This paper presents cross sections for the production of a W boson in association with jets, measured in proton–proton collisions at √s=7 TeV with the ATLAS experiment at the large hadron collider. With an integrated luminosity of 4.6 fb−1, this data set allows for an exploration of a large kinematic range, including jet production up to a transverse momentum of 1 TeV and multiplicities up to seven associated jets. The production cross sections for W bosons are measured in both the electron and muon decay channels. Differential cross sections for many observables are also presented including measurements of the jet observables such as the rapidities and the transverse momenta as well as measurements of event observables such as the scalar sums of the transverse momenta of the jets. The measurements are compared to numerous QCD predictions including next-to-leading-order perturbative calculations, resummation calculations and Monte Carlo generators