The Life Sciences Mass Spectrometry Research Unit

The Life Sciences Mass Spectrometry (LSMS) research unit focuses on the development of novel analytical workflows based on innovative mass spectrometric and software tools for the analysis of low molecular weight compounds, peptides and proteins in complex biological matrices. The present article summarizes some of the recent work of the unit: i) the application of matrix-assisted laser desorption/ionization (MALDI) for mass spectrometry imaging (MSI) of drug of abuse in hair, ii) the use of high resolution mass spectrometry for simultaneous qualitative/quantitative analysis in drug metabolism and metabolomics, and iii) the absolute quantitation of proteins by mass spectrometry using the selected reaction monitoring mode

High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices

Quantitative and qualitative high-resolution (HR) dependent and independent acquisition schemes on a QqTOF MS (with resolving power 20,000-40,000) were investigated for the analysis of pharmaceutical compounds in biological fluids. High-resolution selected reaction monitoring (HR-SRM) was found to be linear over three orders of magnitude for quantitative analysis of paracetamol in human plasma, offering a real alternative to triple quadrupole LC-SRM/MS. Metabolic stability of talinolol in microsomes was characterized by use of three different acquisition schemes: (i) information-dependent acquisition (IDA) with a TOF MS experiment as survey scan and product-ion scan as dependent scan; (ii) MSALL by collecting TOF mass spectra with and without fragmentation by alternating the collision energy of the collision cell between a low (i.e., 10eV) and high setting (i.e., 40eV); and (iii) a novel independent acquisition mode referred to as "sequential window acquisition of all theoretical fragment-ion spectra” (SWATH) or "global precursor ions scan mode” (GPS) in which sequential precursor ions windows (typically 20 u) are used to collect the same spectrum precursor and fragment ions using a collision energy range. SWATH or GPS was found to be superior to IDA or MSALL in combination with UHPLC for qualitative analysis but requires a rapidly acquiring mass spectrometer. Finally, the GPS concept was used for QUAL/QUAN analysis (i.e. integration of qualitative and quantitative analysis) of bosentan and its metabolites in urine over a concentration range from 5 to 2,500ngmL−

Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry

The liquid chromatography-mass spectrometry (LC-MS) analysis of complex samples such as biological fluid extracts is widespread when searching for new biomarkers as in metabolomics. The success of this hyphenation resides in the orthogonality of both separation techniques. However, there are frequent cases where compounds are co-eluting and the resolving power of mass spectrometry (MS) is not sufficient (e.g., isobaric compounds and interfering isotopic clusters). Different strategies are discussed to solve these cases and a mixture of eight compounds (i.e., bromazepam, chlorprothixene, clonapzepam, fendiline, flusilazol, oxfendazole, oxycodone, and pamaquine) with identical nominal mass (i.e., m/z 316) is taken to illustrate them. Among the different approaches, high-resolution mass spectrometry or liquid chromatography (i.e., UHPLC) can easily separate these compounds. Another technique, mostly used with low resolving power MS analyzers, is differential ion mobility spectrometry (DMS), where analytes are gas-phase separated according to their size-to-charge ratio. Detailed investigations of the addition of different polar modifiers (i.e., methanol, ethanol, and isopropanol) into the transport gas (nitrogen) to enhance the peak capacity of the technique were carried out. Finally, a complex urine sample fortified with 36 compounds of various chemical properties was analyzed by real-time 2D separation LC×DMS-MS(/MS). The addition of this orthogonal gas-phase separation technique in the LC-MS(/MS) hyphenation greatly improved data quality by resolving composite MS/MS spectra, which is mandatory in metabolomics when performing database generation and searc

Mass Spectrometric QUAL/QUAN Approaches for Drug Metabolism and Metabolomics

A liquid chromatography–high-resolution mass spectrometry platform was used for simultaneous qualitative and quantitative (QUAL/QUAN) acquisition, enabling drug metabolism and metabolomics investi- gations. Plasma study samples were monitored for three different groups of patients at a single time-point (1 h after drug administration): one group received acetaminophen (APAP), one group received both APAP and ketorolac and one group was a control group. The quantification of APAP and two of its metabolites (APAP-glucuronide and APAP-cysteine) was performed on a fast acquisition quadrupole-Time-Of-Flight (50–100 ms duty cycle, resolving power of 30,000) compatible with UHPLC time constraints. High-resolution Selected Reaction Monitoring was used for quantification of APAP and its metabolites from 50–10,000 ng/mL using a 50 ?L plasma aliquot. Average measured concentrations were for APAP 6,650 ng/mL vs 6,160 ng/mL, APAP-CYS concentrations were 154.2 ng/mL vs 140.6 ng/mL and APAP-GLU concentrations 8,750 ng/mL vs 8,430 ng/mL between the group that received only APAP (n = 11) and the group that received APAP in combination with ketorolac (n = 11). No major differences were observed between the two groups of patients, as it would be expected due to the differing metabolism pathway for both substances. For the qualitative aspect, a metabolomics data processing platform with biological QC samples was applied to the study samples to search for unanticipated metabolites and biomarkers related to APAP and ketorolac metabolism. Multivariate analysis (i.e. Principle Component Analysis), variables grouping tools (i.e. PCVG) and high-resolution MS(/MS) spectra from the MSALL acquisition strategy enabled the profiling and characterization of circulating metabolites of APAP in plasma such as APAP-sulfate, APAP-mercapturate as well as ketorolac

Informal recycling of Congolese refugee youth in Kampala

This article explores the informal recycling of young Congolese refugees based in threeneighborhoods of Kampala: Kamwokya, Nakulabye, and Lungujja. Livelihood experiences ofCongolese youth related to the waste picking is identified as an important asset to navigate theeveryday exclusion of the city. If the right-allocating mechanism positions refugees in stratifiedmembership around certain rights, in Uganda those who chose to settle in cities independently are noteligible of humanitarian aid and, therefore, most of them remain in precarious conditions. Congoleseyouth’ urban hustle highlights the importance of strengthening local and national knowledge aboutforced migration in urban settings. Questo articolo esplora il riciclo informale dei giovani rifugiati congolesi che vivono in tre quartieridi Kampala: Kamwokya, Nakulabye e Lungujja. Le esperienze dei giovani congolesi legate allaraccolta dei rifiuti sono identificate come una risorsa importante per navigare l'esclusione quotidianadella città. Se il meccanismo di assegnazione dei diritti posiziona i rifugiati in un'appartenenzastratificata intorno a certi diritti, in Uganda coloro che hanno scelto di stabilirsi in città in modoindipendente non hanno diritto agli aiuti umanitari e, quindi, la maggior parte di loro rimane incondizioni precarie. L’economia informale dei giovani congolesi evidenzia l'importanza di rafforzarele conoscenze locali e nazionali sulla migrazione forzata in ambiente urbano

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion.

The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression. To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and three-dimensional cultures of MCF-10░A cells. We show that upon Dbl expression MCF-10░A cells undergo EMT. In addition, we found that Dbl overexpression sustain

Paper Session II-A - Iris-LAGEOS 2 Mission

The developing and launching of LAGEOS 2 must be considered an important step in the evolution of Italian space and scientific activities. This paper will describe the scientific objectives of the IRIS-LAGEOS 2 mission, giving full details, characteristics and performances of the LAGEOS system and its launcher, IRIS: LAGEOS 2 is a scientific satellite to be utilized for geodesy research being developed in Italy under a joint agreement between the Italian (ASI) and U.S. (NASA) Space Agencies. IRIS (Italian Research Interim Stage), funded and managed by the Italian Space Agency, is a spinning, solid propellant upper stage system to be used in conjunction with the NASA Space Transportation System. An overview of pre-launch ground activities and flight operations will be provided. The program status will be summarized. The industrial organization in charge of developing both systems will be, briefly, indicated

DBNet, a tool to convert Dynamic Fault Trees into Dynamic Bayesian Networks

The unreliability evaluation of a system including dependencies involving the state of components or the failure events, can be performed by modelling the system as a Dynamic Fault Tree (DFT). The combinatorial technique used to solve standard Fault Trees is not suitable for the analysis of a DFT. The conversion into a Dynamic Bayesian Network (DBN) is a way to analyze a DFT. This paper presents a software tool allowing the automatic analysis of a DFTexploiting its conversion to a DBN. First, the architecture of the tool is described, together with the rules implemented in the tool, to convert dynamic gates in DBNs. Then, the tool is tested on a case of system: its DFT model and the corresponding DBN are provided and analyzed by means of the tool. The obtained unreliability results are compared with those returned by other tools, in order to verify their correctness. Moreover, the use of DBNs allows to compute further results on the model, such as diagnostic and sensitivity indices

Decreta sacrae Congreg. Concilii J. D.N. Vrbani Papae VIII ius edita de Regularibus Apostatis, [et] Eiectis.

Port. con esc. xil. de los franciscanos