Metrological traceability of Polycyclic Aromatic Hydrocarbons (PAHs) measurements in green tea and mate

The development of suitable analytical methods to obtain metrologically traceable results in the determination of toxicants in food matrices is an important issue, as food represents the main way of assumption of many contaminants, among which the Polycyclic Aromatic Hydrocarbons (PAHs). The present work deals with the set up and internal validation of an analytical method carried out at INRiM for the quantification by gascromatography coupled with mass spectrometry (GC–MS) of some priority PAHs in green tea (Camellia sinensis) and yerba mate (Ilex paraguariensis), in order to obtain metrologically traceable results. Two approaches for the quantification were applied: an external calibration, for determining the GC–MS calibration curves by means of standard reference solutions and an internal calibration by using perdeuterated standards. For the external calibration, Weighted (WLS) and Weighted Total (WTLS) Least Squares fitting procedures were applied. The measurement uncertainty evaluation was carried out by applying the Law of Propagation of Uncertainty

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations

Correlation of test results and influence of a mass balance constraint on risks in conformity assessment of a substance or material

When components of a substance or material are subject to a mass balance constraint, test results of the components’ contents are intrinsically correlated because of the constraint. This so-called ‘spurious’ correlation is observed in addition to possible metrologically-related correlation of test results, and natural and/or technological correlation of the components’ contents. Such correlations may influence understanding of test results and evaluation of risks of false decisions, due to measurement uncertainty, in conformity assessment of the substance or material. The objective of the present paper is the development of a technique for appropriate evaluation of the risks. A Bayesian multivariate approach to evaluate the conformance probability of materials or objects and relevant risks is discussed for different scenarios of the data modelling, taking into account all observed correlations. A Monte Carlo method, including the mass balance constraint, written in the R programming environment, is provided for the necessary calculations

Interplay between distribution of live cells and growth dynamics of solid tumours

Experiments show that simple diffusion of nutrients and waste molecules is not sufficient to explain the typical multilayered structure of solid tumours, where an outer rim of proliferating cells surrounds a layer of quiescent but viable cells and a central necrotic region. These experiments challenge models of tumour growth based exclusively on diffusion. Here we propose a model of tumour growth that incorporates the volume dynamics and the distribution of cells within the viable cell rim. The model is suggested by in silico experiments and is validated using in vitro data. The results correlate with in vivo data as well, and the model can be used to support experimental and clinical oncology

Generation of CO2 gas mixtures by dynamic dilution for the development of gaseous certified reference materials

The use of Certified Reference Materials (CRMs) is of utmost importance to achieve the comparability and traceability of data, which are essential features of measurement results in environmental and climate fields. The present paper focuses on the generation of gas mixtures at known composition of carbon dioxide at atmospheric amount-of-substance fraction in synthetic air by means of a dynamic dilution system, designed and implemented at the Istituto Nazionale di Ricerca Metrologica (INRiM). The validation of the dynamic system in terms of amount-of-substance fraction is presented. The system was also used to verify the carbon dioxide amount-ofsubstance fraction of a suite of gas mixtures gravimetrically prepared at INRiM in the framework of the EMPIR Joint Research Project 19ENV05 – STELLAR. Dynamic dilution proved to be an effective tool for the preparation and certification of CRMs for gaseous pollutants (i.e. carbon dioxide, nitrogen oxides) relevant for monitoring environmental pollution and climate changes

Attività biologica di metaboliti secondari da Passiflora palmeri su linee tumorali umane

Il genere Passiflora comprende diverse centinaia di specie originarie delle regioni tropicali e sub-tropicali dell’America ed è da lungo tempo conosciuto in medicina tradizionale per le sue proprietà terapeutiche. Questa caratteristica, assieme alle osservazioni dell’interazione della pianta con l’ambiente ed alle informazioni chemotassonomiche a disposizione, ne fanno un buon candidato per la ricerca di molecole naturali con potenziale attività farmacologica. Nonostante questo, il genere Passiflora è ancora poco studiato e fino ad oggi non sono stati pubblicati dati sull’attività biologica nei confronti di linee tumorali umane. La prima parte del lavoro di tesi è stata dedicata all’individuazione di specie con potenziale attività antitumorale ed ha compreso lo studio di alcuni meccanismi molecolari che stanno alla base di tale attività. Dallo screening iniziale condotto sulla linea tumorale MDA-MB-231, P. palmeri e P. foetida sono risultate le specie con la più alta attività citotossica. Inoltre, l’estratto metanolico da foglia di P. palmeri si è dimostrato in grado indurre la morte selettiva delle cellule tumorali testate rispetto ai fibroblasti primari umani, con un meccanismo che si è rivelato in parte di tipo apoptotico. Nella seconda parte del lavoro è stata condotta l’analisi dei metaboliti secondari degli estratti attivi, con lo scopo di fornirne una caratterizzazione e di identificare i composti responsabili dell’attività citotossica. A tal fine è stato affiancato al classico approccio ‘bioguidato’, un approccio di tipo ‘metabolomico’, che ha compreso un’analisi più ampia e dettagliata dei metaboliti presenti all’interno degli estratti. Ai dati ottenuti mediante le analisi LC-ESI-MS degli estratti attivi e non attivi è stata applicata l’analisi delle componenti principali (PCA), in base alla quale sono state individuate le molecole che contribuivano maggiormente alla separazione tra i due tipi di estratto. Le analisi hanno rivelato la presenza negli estratti attivi di numerosi derivati di apigenina e luteolina. Mediante confronto con degli standard commerciali è stato possibile confermare la presenza di luteolina, vitexina, luteolina 7-O-glucoside e apigenina 7-O-glucoside. I due approcci applicati (‘bioguidato’ e ‘metabolomico’) hanno dato in parte risultati sovrapponibili, ma in nessun caso è stato possibile trovare all’interno dell’estratto delle componenti che potessero spiegare in modo esaustivo la sua attività. E’ stato comunque possibile individuare alcune molecole (apigenina, luteolina e crisoeriolo) in grado di partecipare all’azione citotossica, le quali, in linea con i risultati ottenuti per l’estratto totale, hanno dimostrato di possedere una specificità di azione nei confronti delle cellule tumorali. Dal momento che uno degli obiettivi della moderna terapia contro il cancro è l’aumento di selettività dei farmaci nei confronti delle cellule tumorali, è possibile concludere che l’estratto metanolico da foglia di P. palmeri rappresenta una possibile fonte di composti per lo sviluppo di farmaci specifici contro questo tipo di cellula.The genus Passiflora, comprising several hundred species that are indigenous to the tropical and semi-tropical areas of America, is well known in traditional medicine for its therapeutics properties. This feature, together with the observations of the interaction of the plants with their environment and the available chemotaxonomic information, makes the genus Passiflora a potential source of natural molecules of pharmacological relevance. However, only a few species of this genus have been widely studied from a phytochemical point of view and the biological activity on human tumor cell lines has not been investigated yet. In the first part of this work we searched for species with potential antitumor activity, focusing on molecular mechanisms involved. Preliminary screening on MDA-MB-231 cell line showed that P. palmeri and P. foetida have the strongest cytotoxic activity. Moreover, methanolic extract from leaves of P. palmeri kills tumoral cells in a selective manner, with a weak effect on normal fibroblasts; the apoptotic pathway is involved in the observed cell death. In order to give a phytochemical characterization and to identify the compounds responsible for the cytotoxic activity, in the second part of this work we analyzed the composition of secondary metabolites from active extracts. For this purpose, we combined a classical ‘bioguided’ approach with a ‘metabolomic’ approach that included a wider analysis of metabolites from the methanolic extracts. By applying principal component analysis (PCA) to LC-ESI-MS data from active and non active extracts, we identified the molecules that significantly contribute to the separation of the two types of extracts. The analysis of the active extracts revealed the presence of several apigenin and luteolin derivatives. By comparison with available commercial standards we confirmed the presence of luteolin, vitexin, luteolin 7-O-glucoside and apigenin 7-O-glucoside. The two different approaches (‘bioguided’ and ‘metabolomic’) employed have produced comparable results, even if it was not possible to find in the extracts any components that could exhaustively explain its cytotoxic activity. However we identified some molecules (i.e. apigenin, luteolin and chrysoeriol) that can contribute to this activity by acting specifically against tumoral cells, in line with previous observation on total extract. Since one of the goals of modern cancer therapy is to increase the selectivity of antitumor drugs, we can conclude that methanolic extract from leaves of P. palmeri is a possible source of compounds for the development of selective anticancer drugs

Chemistry and Metrology: an Overview of European Activities

The interest of the metrology community towards chemistry started quite late, compared to “classical” metrology. In chemistry there were many metrological activities (for ex. determination of atomic weights, validation of analytical methods) carried out outside the metrological organizations. The mole, the unit of amount of substance, was introduced in the International System of Units only in 1971. The growing need to assure accuracy, stability, comparability in time and space of chemical measurements led to a better rationalisation and control of the metrological activities, leading to a convergence of the international collaborations inside the Metre Convention at the beginning of the 90s with the establishment of CCQM. At European level, in 1991 the Amount of Substance sector within EUROMET (now EURAMET, the European Association of National Metrology Institutes) was founded. In the following years the Technical Committee of Metrology in Chemistry (TC-MC) was established as a Joint EURAMET-Eurachem Technical Committee chaired by EURAMET. TC-MC is concerned with primary methods and reference materials for chemical measurements and research in metrology to support different sectors in chemistry. The activities undertaken in its four Technical Subcommittees range from organic and inorganic chemical analysis to electrochemistry and gas analysis. The work is carried out within projects in the framework of both the usual metrological approach (i.e. organisation of measurement comparisons to establish the mutual agreement among European National Metrology Institutes and Designated Institutes) and in the European programmes on metrology (EMRP and EMPIR), where a very successful participation of NMIs and DIs active in the chemical field has been recorded

Reference materials: preparation, homogeneity, stability and value assignment

Reference Materials (RMs) and Certified Reference Materials (CRMs) are widely used in all stages of measurement procedures and in interlaboratory comparisons. CRMs, in particular, play a key role in implementing the concept of metrological traceability of measurement results in chemistry, biology and physics among other sciences dealing with substances and materials and, in this context, laboratories use CRMs as readily accessible measurement standards. In the past decades, an extensive production of RMs and CRMs was carried out. Although some of them are intended for applications in physics, the great majority belongs to the amount of substance related fields. There are various normative references dealing with RMs and CRMs, and broad scientific literature. This work aims at presenting a general overview on the production and characterization of RMs and CRMs, with particular focus on gaseous CRMs. An example of application of gaseous CRMs to support climate change studies is also given