An integrated metabolomics approach for the research of new cerebrospinal fluid biomarkers of multiple sclerosis

(1) Lipid profiling in MuS and OND patients. (2) Search of alterations associated with MuS. (3) Characterization of differences

LIMPIC: a computational method for the separation of protein MALDI-TOF-MS signals from noise

BACKGROUND: Mass spectrometry protein profiling is a promising tool for biomarker discovery in clinical proteomics. However, the development of a reliable approach for the separation of protein signals from noise is required. In this paper, LIMPIC, a computational method for the detection of protein peaks from linear-mode MALDI-TOF data is proposed. LIMPIC is based on novel techniques for background noise reduction and baseline removal. Peak detection is performed considering the presence of a non-homogeneous noise level in the mass spectrum. A comparison of the peaks collected from multiple spectra is used to classify them on the basis of a detection rate parameter, and hence to separate the protein signals from other disturbances. RESULTS: LIMPIC preprocessing proves to be superior than other classical preprocessing techniques, allowing for a reliable decomposition of the background noise and the baseline drift from the MALDI-TOF mass spectra. It provides lower coefficient of variation associated with the peak intensity, improving the reliability of the information that can be extracted from single spectra. Our results show that LIMPIC peak-picking is effective even in low protein concentration regimes. The analytical comparison with commercial and freeware peak-picking algorithms demonstrates its superior performances in terms of sensitivity and specificity, both on in-vitro purified protein samples and human plasma samples. CONCLUSION: The quantitative information on the peak intensity extracted with LIMPIC could be used for the recognition of significant protein profiles by means of advanced statistic tools: LIMPIC might be valuable in the perspective of biomarker discovery

12 Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of notch signalling in a muine model

Abstract Aims Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms, in general, and sex-related mechanisms in particular. To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods Twenty-four-month-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a down-regulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and—to a lesser extent—by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity

Proteomic insights in extracellular microvesicles from multiple sclerosis patients

To date the most important biomarkers for Multiple Sclerosis (MuS) diagnosis are the oligoclonal bands (OCBs) in CSF and Link Index. CSF is the body fluid that might better provide information about the pathological processes occurring in the CNS, because of its proximity. Anyway, it is obtained through an invasive procedure, thus tears, may represent an useful alternative source of biomarkers. Emerging evidences showed that distinct types of brain cells release high number of Extracellular Vesicles (EVs), that play important roles in the CNS, and represent a relevant source of biomarkers, relative free from confounding factors. In the present study, we analysed EVs from MuS patients obtained from tears and CSF samples. In details, 50μl of CSF or 50 μl of tears/sample were processed by a common flow cytometry no-lyse and no-wash method, in order to identify EVs. Exosomes and microvesicles (MVs) were sorted (70 μm nozzle, FACSAria III cell sorter, BD) from pooled CSF samples on the basis of their positivity to specific tetraspainins (for exosomes) or markers identifying each MV subset. Fractions were analysed by electron microscopy and Dynamic Light Scattering. Purified MV fractions undergone to FASP tryptic digestion and nanoLC-ESI-QTOF-MS/MS based shotgun proteomic approach. Identified MVs proteins were processed by Ingenuity Pathway Analysis (IPA) and PANTHER - Gene List Analysis. Our data shows the presence of subpopulations of extracellular MVs of neuronal and microglia origins in tears , indicating a cross talk between the two compartment. We also identified 55 proteins (FD

Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing

Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry

Dry eye disease (DED) is a multifactorial disorder of the ocular surface unit resulting in eye discomfort, visual disturbance, and ocular surface damage; the risk of DED increases with age in both sexes, while its incidence is higher among females caused by an overall hormonal imbalance. The role of androgens has recently investigated and these hormones were considered to have a protective function on the ocular surface. In order to correlate DED to tear steroid levels, a robust, specific, and selective method for the simultaneous quantification of cortisol (CORT), corticosterone (CCONE), 11-deoxycortisol (11-DECOL), 4-androstene-3,17-dione (ADIONE), testosterone (TESTO), 17α-hydroxyprogesterone (17-OHP), and progesterone (PROG) was developed and applied for the analysis of tear samples. The method involves a simple extraction procedure of steroids from tears collected on Schirmer strips, followed by a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In total, tear samples from 14 DED female patients and 13 healthy female controls were analysed and, CORT, ADIONE, and 17-OHP response levels resulted significantly decreased in dry eye patients respect to controls. The receiver operating characteristic (ROC) curve obtained by the combination of these three steroids (AUC = 0.964) demonstrated the good diagnostic power of the differential tear steroids in identifying DED. In conclusion, the present method made it possible, for the first time, to study steroid profiling directly in tear fluid

In vitro and in vivo lipidomics as a tool for probiotics evaluation

The probiotic bacteria are helpful for nutritional and therapeutic purposes, and they are commercially available in various forms, such as capsules or powders. Increasing pieces of evidence indicate that different growth conditions and variability in manufacturing processes can determine the properties of probiotic products. In recent years, the lipidomic approach has become a useful tool to evaluate the impact that probiotics induce in host physiology. In this work, two probiotic formulations with identical species composition, produced in two different sites, the USA and Italy, were utilized to feed Caenorhabditis elegans, strains and alterations in lipid composition in the host and bacteria were investigated. Indeed, the multicellular organism C. elegans is considered a simple model to study the in vivo effects of probiotics. Nematodes fat metabolism was assessed by gene expression analysis and by mass spectrometry–based lipidomics. Lipid droplet analysis revealed a high accumulation of lipid droplets in worms fed US-made products, correlating with an increased expression of genes involved in the fatty acid synthesis. We also evaluated the lifespan of worms defective in genes involved in the insulin/IGF-1-mediated pathway and monitored the nuclear translocation of DAF-16. These data demonstrated the involvement of the signaling in C. elegans responses to the two diets. Lipidomics analysis of the two formulations was also conducted, and the results indicated differences in phosphatidylglycerol (PG) and phosphatidylcholine (PC) contents that, in turn, could influence nematode host physiology. Results demonstrated that different manufacturing processes could influence probiotics and host properties in terms of lipid composition

Advances in Lipidomics for Cancer Biomarkers Discovery

Lipids play critical functions in cellular survival, proliferation, interaction and death, since they are involved in chemical-energy storage, cellular signaling, cell membranes, and cell–cell interactions. These cellular processes are strongly related to carcinogenesis pathways, particularly to transformation, progression, and metastasis, suggesting the bioactive lipids are mediators of a number of oncogenic processes. The current review gives a synopsis of a lipidomic approach in tumor characterization; we provide an overview on potential lipid biomarkers in the oncology field and on the principal lipidomic methodologies applied. The novel lipidomic biomarkers are reviewed in an effort to underline their role in diagnosis, in prognostic characterization and in prediction of therapeutic outcomes. A lipidomic investigation through mass spectrometry highlights new insights on molecular mechanisms underlying cancer disease. This new understanding will promote clinical applications in drug discovery and personalized therapy

Molecular biomarkers in primary open-angle glaucoma: From noninvasive to invasive

Glaucoma, the first cause of irreversible blindness worldwide, is a neurodegenerative disease characterized by the progressive loss of retinal ganglion cells. There are different subtypes of glaucoma, all expression of a common optic neuropathy; primary open-angle glaucoma (POAG) is the most diffuse subtype in western countries. To date, unfortunately, several questions still remain unsolved in the glaucoma management, such as the availability of powerful methods for screening high-risk populations, early diagnosis, timely detection of damage progression, and prediction of response to therapy. Over the last years, biomarkers have gained immense scientific and clinical interest to solve these issues, with countless molecules that have been candidate as potential biomarkers. In the present review, we summarize the current knowledge about the most robust molecular biomarkers proposed in POAG, distinguishing noninvasive from minimally invasive, and invasive biomarkers, according to the procedure adopted to collect fluid samples

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS