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The item-level tracing and tracking requirements are growing more and more in the pharmaceutical sector, where the counterfeiting problem and the significant fragmentation of the market contribute significantly to complicate the scenario. The Radio Frequency Identification (RFID) technology holds the promise to eliminate many of the previous problems. Unfortunately, there are still some barriers limiting the largescale deployment of these innovative technologies. Currently, it is not easy finding an exhaustive analysis about potential effects of exposure to electromagnetic fields of the RFID systems on drugs. This work aimed to evaluate the effects of RFID systems on the molecular structure and potency of a biological drug. In particular, some samples of a commercial human insulin preparation (ActrapidTM) were exposed for different periods to electromagnetic fields generated by RFID devices in UHF band. In order to evaluate both possible alterations of the molecular structure and possible adverse effects on drug performance, the following techniques have been used: Reverse Phase-High Pressure Liquid Chromatography and in vitro cell proliferation assays. The experimental results have shown that the electromagnetic field generated by UHF RFID devices does not cause significant biological effects on ActrapidTM insulin.

Lipid and fatty acid compositions of Mytilus galloprovincialis cultured in the Mar Grande of Taranto (southern Italy): feeding strategies and trophic relationships. Zoological Studies 49(2): 211-219. Lipid and fatty acid (FA) compositions were determined in the mussel Mytilus galloprovincialis collected from June 2006 to May 2007 in the Mar Grande of Taranto, southern Italy. Total lipids significantly differed throughout the study period (ANOVA, p < 0.05), with higher values in summer (24.7% dry weight (DW)) and the lowest values in winter (3.5% DW). Triacylglycerols (TAGs) were the dominant lipid class in spring and summer accounting for 55.28% and 60.3% of total lipids, respectively, while in the autumn and winter phospholipids (PLs) were considerably greater than TAGs, comprising 55.16% and 47.5% of total lipids, respectively. Cholesterol did not show large variations over the seasons. Predominant FAs were saturated FAs (SAFAs) followed by monounsaturated FAs (MUFAs). The amount of polyunsaturated FAs (PUFAs) was low. The 14:0, 16:0, 18:0, and 22:0 SAFAs, together with 14:1, 16:1 omega 7, 18:1 omega 9, 18:1 omega 7, 20:1 omega 9, and 24:1 omega 9 MUFAs, and the PUFA, non-methylene interrupted dienoic (NMID), were the most abundant FAs. FA biomarkers are frequently used to identify trophic relationships among marine invertebrates. In order to obtain indications on food sources of M. galloprovincialis, a variety of FA ratios and the sum of some FAs were determined. The sum of 18:1 omega 7 + odd-branched FAs indicated a moderate bacterial contribution to the mussel diet. A high 18:1 omega 9/18:1 omega 7 ratio together with a high level of 20:1w9 indicated an animal dietary input. Trophic markers suggested lows contribution of diatoms and dinoflagellates to the diet of M. galloprovincialis.

Gynecological cancer management remains challenging and a better understanding of molecular mechanisms that lead to carcinogenesis and development of these diseases is needed to improve the therapeutic approaches. The Na+/H+ exchanger regulatory factor 1 (NHERF1) is a scaffold protein that contains modular protein-interaction domains able to interact with molecules with an impact on carcinogenesis and cancer progression. During recent years, its involvement in gynecological cancers has been explored, suggesting that NHERF1 could be a potential biomarker for the development of new targeted therapies suitable to the management of these tumors. This comprehensive review provides an update on the recent study on NHERF1 activity and its pathological role in cervical and ovarian cancer, as well as on its probable involvement in the therapeutic landscape of these cancer types.

Hydroxytyrosol (HT), a peculiar olive and olive oil phenolic antioxidant, plays a significant role in the endothelial and cardiovascular protection associated with olive oil consumption. However, studies examining the effects of HT on the whole-genome expression of endothelial cells, which are prominent targets for vasculo-protective effects of olive oil polyphenols, have been lacking. This study aims to comprehensively evaluate the genomic effects exerted by HT, at the transcriptional level, in endothelial cells under resting or proinflammatory conditions. Human umbilical vein endothelial cells (HUVECs) were treated with 10 µmol/L HT for 1 h and then stimulated with 5 ng/mL interleukin (IL)-1β for 3 h. Total RNA was extracted, and gene expression profile assessed with microarray analysis. Functional enrichment analysis and pathway analysis were performed by Ingenuity Pathways Analysis. Microarray data were validated by qRT-PCR. Fixing a significance threshold at 1.5-fold change, HT affected the expression of 708 and 599 genes, respectively, in HUVECs under resting and IL-1β-stimulated conditions, among these, 190 were common to both conditions. Unfolded protein response (UPR) and endoplasmic reticulum stress resulted from the two top canonical pathways common between HT and HT-IL-1β affected genes. IL-17F/A signaling was found in the top canonical pathways of HT modified genes under resting unstimulated conditions, whereas cardiac hypertrophy signaling was identified among the pathways affected by HT-IL-1β. The transcriptomic analysis allowed pinpointing immunological, inflammatory, proliferative, and metabolic-related pathways as the most affected by HT in endothelial cells. It also revealed previously unsuspected genes and related gene pathways affected by HT, thus broadening our knowledge of its biological properties. The unbiased identification of novel genes regulated by HT improves our understanding of mechanisms by which olive oil prevents or attenuates inflammatory diseases and identifies new genes to be enquired as potential contributors to the inter-individual variation in response to functional food consumption.

International audience; Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in various cell models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms regulating oligodendrocyte differentiation after PKC activation are still unclear. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to provide a comprehensive analysis of the proteome expression changes in the MO3.13 oligodendroglial cell line after PKC activation. Our findings suggest that multiple networks that communicate and coordinate with each other may finally determine the fate of MO3.13 cells, thus identifying a modular and functional biological structure. In this work, we provide a detailed description of these networks and their participating components and interactions. Such assembly allows perturbing each module, thus describing its physiological significance in the differentiation program. We applied this approach by targeting the Rho-associated protein kinase (ROCK) in PKC-activated cells. Overall, our findings provide a resource for elucidating the PKC-mediated network modules that contribute to a more robust knowledge of the molecular dynamics leading to this cell fate transition.

Microbial communities and human cells, through a dynamic crosstalk, maintain a mutualistic relationship that contributes to the maintenance of cellular metabolism and of the immune and neuronal systems. This dialogue normally occurs through the production and regulation of hormonal intermediates, metabolites, secondary metabolites, proteins, and toxins. When the balance between host and microbiota is compromised, the dynamics of this relationship change, creating favorable conditions for the development of diseases, including cancers. Microbiome metabolites can be important modulators of the tumor microenvironment contributing to regulate inflammation, proliferation, and cell death, in either a positive or negative way. Recent studies also highlight the involvement of microbiota metabolites in inducing epithelial–mesenchymal transition, thus favoring the setup of the metastatic niche. An investigation of microbe-derived metabolites in “liquid” human samples, such as plasma, serum, and urine, provide further information to clarify the relationship between host and microbiota.

International audience; It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial-mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

International audience; Members of the carbonic anhydrase family are functionally involved in the regulation of intracellular and extracellular pH in physiological and pathological conditions. Their expression is finely regulated to maintain a strict control on cellular homeostasis, and it is dependent on the activation of extracellular and intracellular signaling pathways. Combining RNA sequencing (RNA-seq), NanoString, and bioinformatics data, we demonstrated that the expression of carbonic anhydrase 12 (CAXII) is significantly different in luminal and triple negative breast cancer (BC) models and patients, and is associated with the activation of an epithelial mesenchymal transition (EMT) program. In BC models, the phorbol ester 12-myristate 13-acetate (PMA)-mediated activation of protein kinase C (PKC) induced a down-regulation of CAXII with a concomitant modulation of other members of the transport metabolon, including CAIX and the sodium bicarbonate cotransporter 3 (NBCn1). This is associated with a remodeling of tumor glycolytic metabolism induced after PKC activation. Overall, this analysis highlights the dynamic nature of transport metabolom and identifies signaling pathways finely regulating this plasticity.

International audience; The epithelial mesenchymal transition (EMT) program is defined as a cellular transition from an epithelial to a mesenchymal state. This process occurs to provide the cell with new phenotypic assets and new skills to perform complex processes. EMT is regulated at multilayer levels, including transcriptional control of gene expression, regulation of RNA splicing, and translational/post-translational control. Although transcriptional regulation by EMT-inducing transcription factors (EMT-TFs), including Zeb, Snail and Slug members, is generally considered the master step in this process, emerging data indicate that all these regulatory networks may have a role in the control of EMT. There is a sort of parallelism between the biological and still unrevealed EMT complexity and the cosmological hypothesis that sustains the universe may exist as a multiverse. The presence of different EMT transition states together with the occurrence of multiple layers of regulation support the idea that EMT is just one on many out there. Is the activation of a single layer of regulation sufficient to initiate the whole EMT program? Can we postulate the activation of different EMT "dimensions"? If we think about these layers as multiple separate "universes", various scenarios can be revealed.

Microglia cells are the primary immune population of the central nervous system with a role in the regulation of several physiological and pathological conditions. Upon appropriate stimulation, microglia cells can be polarized in a pro-inflammatory M1-like or anti-inflammatory M2-like status. Biological processes and pathways engaged in microglia polarization are starting to be elucidated. To help clarify this, we used a liquid chromatography-mass spectrometry (LC-MS/MS) label free approach to characterize the proteomic profile of human microglia cell line (CHME-5) stimulated with gamma-interferon (IFN-&gamma, ) and interleukin-4 (IL-4) to induce a M1 or M2 phenotype, respectively. Outside the classical M1/M2 polarization markers, the M1 status appears to center around the activation of a classical inflammatory response and through the activation of multiple signaling pathways. M2 polarization resulted in a different pattern of protein modulation related to RNA and cellular metabolic processes. Together, our findings provide information regarding the protein changes specific to M1 and M2 activation states, and potentially link the polarization of microglia cells to the acquisition of a specific proteomic profile.

International audience; Several studies have identified a specific metabolic program that is associated with the process of epithelial-mesenchymal transition (EMT). Whereas much is known about the association between glucose metabolism and EMT, the contribution of lipid metabolism is not still completely understood. Here, we studied epithelial and mesenchymal breast cancer cells by proteomic and lipidomic approaches and identified significant differences that characterised these models concerning specific metabolic enzymes and metabolites including fatty acids and phospholipids. Higher levels of monounsaturated fatty acids together with increased expression of enzymes of de novo fatty acid synthesis is the distinct signature of epithelial with respect to mesenchymal cells that, on the contrary, show reduced lipogenesis, higher polyunsaturated fatty acids level and increased expression of genes involved in the triacylglycerol (TAG) synthesis and lipid droplets formation. In the mesenchymal model, the diacylglycerol acyltransferase (DGAT)-1 appears to be the major enzyme involved in TAG synthesis and inhibition of DGAT1, but not DGAT2, drastically reduces the incorporation of labeled palmitate into TAG. Moreover, knockdown of β-catenin demonstrated that this metabolic phenotype in under the control of a network of transcriptional factors and that β-catenin has a specific role in the regulation of lipid metabolism in mesenchymal cells.

With the advent of novel molecular platforms for high-throughput/next-generation sequencing, the communities of commensal and pathogenic microorganisms that inhabit the human body have been defined in depth. In the last decade, the role of microbiota-host interactions in driving human cancer plasticity and malignant progression has been well documented. Germ-free preclinical models provided an invaluable tool to demonstrate that the human microbiota can confer susceptibility to various types of cancer and can also modulate the host response to therapeutic treatments. Of interest, besides the detrimental effects of dysbiosis on cancer etiopathogenesis, specific microorganisms have been shown to exert protective activities against cancer growth. This has strong clinical implications, as restoration of the physiologic microbiota is being rapidly implemented as a novel anticancer therapeutic strategy. Here, we reviewed past and recent literature depicting the role of microbiota-host interactions in modulating key molecular mechanisms that drive human cancer plasticity and lead to malignant progression. We analyzed microbiota-host interactions occurring in the gut as well as in other anatomic sites, such as oral and nasal cavities, lungs, breast, esophagus, stomach, reproductive tract, and skin. We revealed a common ground of biological alterations and pathways modulated by a dysbiotic microbiota and potentially involved in the control of cancer progression. The molecular mechanisms most frequently affected by the pathogenic microorganisms to induce malignant progression involve epithelial-mesenchymal transition- (EMT-) dependent barrier alterations and tumor-promoting inflammation. This evidence may pave the way to better stratify high-risk cancer patients based on unique microenvironmental/microbial signatures and to develop novel, personalized, biological therapies.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia associated with alterations in carbohydrate, lipid, and protein metabolism. The prognosis of T2DM patients is highly dependent on the development of complications, and therefore the identification of biomarkers of T2DM progression, with minimally invasive techniques, is a huge need. In the present study, we applied a 1H-Nuclear Magnetic Resonance (1H-NMR)-based metabolomic approach coupled with multivariate data analysis to identify serum metabolite profiles associated with T2DM development and progression. To perform this, we compared the serum metabolome of non-diabetic subjects, treatment-na&iuml, ve non-complicated T2DM patients, and T2DM patients with complications in insulin monotherapy. Our analysis revealed a significant reduction of alanine, glutamine, glutamate, leucine, lysine, methionine, tyrosine, and phenylalanine in T2DM patients with respect to non-diabetic subjects. Moreover, isoleucine, leucine, lysine, tyrosine, and valine levels distinguished complicated patients from patients without complications. Overall, the metabolic pathway analysis suggested that branched-chain amino acid (BCAA) metabolism is significantly compromised in T2DM patients with complications, while perturbation in the metabolism of gluconeogenic amino acids other than BCAAs characterizes both early and advanced T2DM stages. In conclusion, we identified a metabolic serum signature associated with T2DM stages. These data could be integrated with clinical characteristics to build a composite T2DM/complications risk score to be validated in a prospective cohort.

Mitochondrial dysfunction is a key feature of cancer and is frequently associated with increased aggressiveness and metastatic potential. Recent evidence has brought to light a metabolic rewiring that takes place during the epithelial-to-mesenchymal transition (EMT), a process that drives the invasive capability of malignant tumors, and highlights a mechanistic link between mitochondrial dysfunction and EMT that has been only partially investigated. In this study, we characterized mitochondrial function and bioenergetic status of cultured human breast cancer cell lines, including luminal-like and basal-like subtypes. Through a combination of biochemical and functional studies, we demonstrated that basal-like cell lines exhibit impaired, but not completely inactive, mitochondrial function, and rely on a consequent metabolic switch to glycolysis to support their ATP demand. These altered metabolic activities are linked to modifications of key electron transport chain proteins and a significant increase in levels of reactive oxygen species compared to luminal cells. Furthermore, we observed that the stable knockdown of EMT markers caused functional changes in mitochondria that result in acquisition of a hybrid glycolysis/OXPHOS phenotype in cancer cells as a means to sustain their metabolic demand.