Piero Del Boccio

Liquid chromatography-tandem mass spectrometry analysis of oleuropein and its metabolite hydroxytyrosol in rat plasma and urine after oral administration

We describe a liquid chromatography-electrospray ionisation tandem mass spectrometry method for the qualitative and quantitative determination of the secoiridoid oleuropein and its bioactive metabolite hydroxytyrosol in rat plasma and urine. Samples were prepared by liquid-liquid extraction using ethyl acetate with a recovery for both compounds of about 100% in plasma and about 60% in urine. The chromatographic separation was performed with a RP-ODS column using a water-acetonitrile linear gradient. The calibration curve was linear for both biophenols over the range 2.5-1000 ng/ml (LOD 1.25 ng/ml) for plasma and 5-1000 ng/ml (LOD 2.5 ng/ml) for urine. Plasma concentrations of oleuropein and hydroxytyrosol were measured after oral administration of a single dose (100 mg/kg) of oleuropein. Analysis of treated rat plasma showed the presence of unmodified oleuropein, reaching a peak value of 200 ng/ml within 2 h, with a small amount of hydroxytyrosol, whereas in urine, both compounds were mainly found as glucuronides.

A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines.

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.

A hyphenated microLC-Q-TOF-MS platform for exosomal lipidomics investigations: Application to RCC urinary exosomes

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC‐Q‐TOF‐MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed‐phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynxTM (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q‐TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.

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.

Integration of metabolomics and proteomics in multiple sclerosis: From biomarkers discovery to personalized medicine

Personalized medicine is the science of individualized prevention and therapy. In the last decade, advances in high‐throughput approaches allowed the development of proteomic and metabolomic studies in evaluating the association of genetic and phenotypic variability with disease sensitivity and analgesic response. These considerations have more value in case of multiple sclerosis (MuS), a multifactorial disease with high heterogeneity in clinical course and treatment response. In this review, we reported and updated about proteomic and metabolomic studies for the research of new candidate biomarkers in MuS, and difficulties in their clinical applications. We focused especially on the description of both “omics” approaches that, once integrated, may synergically describe pathophysiology conditions. To prove this assumption, we rebuilt interaction between proteins and metabolites described in the literature as potential biomarkers for MuS, and a pathway analysis of these molecules was performed. The result of such speculation demonstrated a strong convergence of proteomic and metabolomic results in this field, showing also a poorness of available tools for incorporating “omics” approaches. In conclusion, the integration of Metabolomics and Proteomics may allow a more complete characterization of such a heterogeneous disease, providing further insights into personalized healthcare.

Serum lipidomic study reveals potential early biomarkers for predicting response to chemoradiation therapy in advanced rectal cancer: A pilot study

Purpose Prospective detection of patients with advanced rectal cancer (LARC) who have a higher probability of responding to preoperative chemoradiotherapy (CRT) may provide individualized therapy. Lipidomics is an emerging science dedicated to the characterization of lipid fingerprint involved in different pato-physiological conditions. The purpose of this study is to highlight a typical lipid signature able to predict the tumor response to CRT. Experimental Design A prospective global analysis of lipids in 54 sera from 18 LARC patients treated with preoperative CRT was performed. Samples were collected at 3 time points: before (T0), at 14th day and at 28th day of CRT. An open LC-MS/MS analysis was performed to characterize lipid expression at T0. Differential lipids were validated by an independent approach and studied during treatment. Results From 65 differential lipids highlighted between responder (RP) vs not responder (NRP) patients, five lipids were validated to predict response at T0: SM(d18:2/18:1), LysoPC (16:0/0:0), LysoPC (15:1(9z)/0:0), Lyso PE (22:5/0:0) and m/z= 842.90 corresponding to a PC containing 2 fatty acids of 40 carbons totally. The levels of these lipids were lower in NRP before treatment. The ROC curve obtained by combining these five lipid signals showed an AUC of 0.95, evidence of good sensitivity and specificity in discriminating groups. Conclusion Our results are in agreement with previous evidences about the role of lipids in determining the tumor response to therapy and suggest that the study of serum lipid could represent a useful tool in prediction of CRT response and in personalizing treatment.

Enhanced release of acid sphingomyelinase-enriched exosomes generates a lipidomics signature in CSF of Multiple Sclerosis patients

Multiple Sclerosis (MuS) is a complex multifactorial neuropathology, resulting in heterogeneous clinical presentation. A very active MuS research field concerns the discovery of biomarkers helpful to make an early and definite diagnosis. The sphingomyelin pathway has emerged as a molecular mechanism involved in MuS, since high levels of ceramides in cerebrospinal fluid (CSF) were related to axonal damage and neuronal dysfunction. Ceramides are the hydrolysis products of sphingomyelins through a reaction catalyzed by a family of enzymes named sphingomyelinases, which were recently related to myelin repair in MuS. Here, using a lipidomic approach, we observed low levels of several sphingomyelins in CSF of MuS patients compared to other inflammatory and non-inflammatory, central or peripheral neurological diseases. Starting by this result, we investigated the sphingomyelinase activity in CSF, showing a significantly higher enzyme activity in MuS. In support of these results we found high number of total exosomes in CSF of MuS patients and a high number of acid sphingomyelinase-enriched exosomes correlated to enzymatic activity and to disease severity. These data are of diagnostic relevance and show, for the first time, high number of acid sphingomyelinase-enriched exosomes in MuS, opening a new window for therapeutic approaches/targets in the treatment of MuS.

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 (FDR<2.38) for the MVs fraction. To uncover the molecular events underlying these proteins profiles, we studied the Gene Ontology (GO) information in terms of biological process and molecular function by using PANTHER software and we observed that about 70% of the identified proteins resulted were involved in binding processes, while 40% of them were related to cell communication. Ingenuity Pathway Analysis (IPA) of the identified MVs proteins revealed that the top network associated to them are “Cellular Movement, Hematological Disease, Immunological Disease”, well matching with MS. Among the upstream regulators, the most significant one is PRDM with a p-value of 6.68E-07. The remaining upstream regulators, including APOE (the most relevant lipid carrier protein in the brain involved in brain development and repair), well related to neurological disease. These data underlined that MVs form neuronal and microcglial origin are detectable not only in the CSF, but also in tears from MuS patients. Of note, MVs of CSF origin carry relevant targets involved in immune responses in MuS patients, therefore they might be proposed as useful tools in MuS diagnosis and characterization.

Inside front cover: Integration of metabolomics and proteomics in multiple sclerosis: From biomarkers discovery to personalized medicine

DOI: 10.1002/prca.201500083 The image represents the idea that integration ofmetabolomic and proteomic results may generate detailed networks. The capsule, representative of personalized medicine, may be generated from the combination of these omic techniques. This strategy may be particularly useful in the characterization of complex disorders such as Multiple Sclerosis. Further details can be found in the article by Piero Del Boccio et al. on page 470.

Identification of an elongation factor 1Bγ protein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.