Signaling in natural killer cells: SHIP, 2B4 and the Kinome

The NK cell is a large granular lymphocyte that plays a key role in protecting the body against numerous pathogens including parasites, intracellular bacteria, viral infections, as well as showing anti-tumor activity and playing a role in the rejection of allogeneic BM. Unlike other lymphocytic cell types, that utilize rearranging receptors, NK cells are regulated by a complex array of germ line encoded activating and inhibitory receptors. NK cells are often described as a front line or rapid defense given their response to stimuli can be immediate, although they also maintain functions that extend their role well into the adaptive immune system. Inhibitory receptors that recognize MHC class I molecules regulate NK cell responses and self-tolerance. Recent evidence indicates self-ligands not present in the MHC locus can also modulate NK function. We previously demonstrated that the NK receptor repertoire is disrupted by SHIP-deficiency. Here we show that an inhibitory receptor, 2B4, that recognizes an MHC-independent ligand is over expressed in NK cells of SHIP-/- mice at all stages of NK development and differentiation. Overexpression of 2B4 compromises key cytolytic NK functions, including killing of allogeneic, tumor and viral targets. These results demonstrate that in SHIP-/- NK cell 2B4 is the dominant inhibitory receptor. We then furthered this finding by examining the molecular basis of 2B4 dominance. We show that in SHIP-/- NK cells there is increased 2B4 expression as well as a strong bias towards the 2B4L isoform. We have also identified a greater than tenfold increase in SHP1 recruitment to 2B4. Consistent with this SHP1 over recruitment,both a broad and a selective SHP1 inhibitor restore SHIP-/- NK killing of complex targets.Through this study we have identified the molecular mechanism of 2B4 receptor dominance as SHP1 over-recruitment.In addition we have utilized protein array technology to explore NK signaling through the determination of the NK kinome. To this end we have been able to identify multiple pathways that may mark crucial differences between the mature and immature NK cell

Novel antiproliferative biphenyl nicotinamide: NMR metabolomic study of its effect on the MCF-7 cell in comparison with cisplatin and vinblastine

A 1H-NMR-based metabolomic study was performed on MCF-7 cell lines treated with a novel nicotinamide derivative (DT-8) in comparison with two drugs characterized by a well-established mechanism of action, namely the DNA-metalating drug cisplatin (cis-diamminedichloridoplatinum(II), CDDP) and the antimitotic drug vinblastine (vinblastine, VIN). The effects of the three compounds, each one at the concentration corresponding to the IC50 value, were investigated, with respect to the controls (K), by the 1H-NMR of cells lysates and multivariate analysis (MVA) of the spectroscopic data. Relevant differences were found in the metabolic profiles of the different treatments with respect to the controls. A large overlap of the metabolic profiles in DT-8 vs. K and VIN vs. K suggests a similar biological response and mechanism of action, significantly diverse with respect to CDDP. On the other hand, DT8 seems to act by disorganizing the mitotic spindle and ultimately blocking the cell division, through a mechanism implying methionine depletion and/or S-adenosylmethionine (SAM) limitation

CaCO3 as an environmentally friendly renewable material for drug delivery systems: Uptake of HSA-CaCO3 nanocrystals conjugates in cancer cell lines

Chemical and biochemical functionalization of nanoparticles (NPs) can lead to an active cellular uptake enhancing their efficacy thanks to the targeted localization in tumors. In the present study calcium carbonate nano-crystals (CCNs), stabilized by an alcohol dehydration method, were successfully modified by grafting human serum albumin (HSA) on the surface to obtain a pure protein corona. Two types of CCNs were used: naked CaCO3 and the (3-aminopropyl)triethoxysilane (APTES) modified CaCO3-NH2. The HSA conjugation with naked CCN and amino-functionalized CCN (CCN-NH2) was established through the investigation of modification in size, zeta potential, and morphology by Transmission Electron Microscopy (TEM). The amount of HSA coating on the CCNs surface was assessed by spectrophotometry. Thermogravimetric analysis (TGA) and Differential scanning calorimetry (DSC) confirmed the grafting of APTES to the surface and successive adsorption of HSA. Furthermore, to evaluate the effect of protein complexation of CCNs on cellular behavior, bioavailability, and biological responses, three human model cancer cell lines, breast cancer (MCF7), cervical cancer (HeLa), and colon carcinoma (Caco-2) were selected to characterize the internalization kinetics, localization, and bio-interaction of the protein-enclosed CCNs. To monitor internalization of the various conjugates, chemical modification with fluorescein-isothiocyanate (FITC) was performed, and their stability over time was measured. Confocal microscopy was used to probe the uptake and confirm localization in the perinuclear region of the cancer cells. Flow cytometry assays confirmed that the bio-functionalization influence cellular uptake and the CCNs behavior depends on both cell line and surface features

1H-NMR metabolite fingerprinting analysis reveals a disease biomarker and a field treatment response in xylella fastidiosa subsp. Pauca-infected olive trees

Xylella fastidiosa subsp. pauca is a xylem-limited bacterial phytopathogen currently found associated on many hectares with the “olive quick decline syndrome” in the Apulia region (Southern Italy), and the cultivars Ogliarola salentina and Cellina di Nardò result in being particularly sensitive to the disease. In order to find compounds showing the capability of reducing the population cell density of the pathogen within the leaves, we tested, in some olive orchards naturally-infected by the bacterium, a zinc-copper-citric acid biocomplex, namely Dentamet®, by spraying it to the crown, once per month, during spring and summer. The occurrence of the pathogen in the four olive orchards chosen for the trial was molecularly assessed. A 1H NMR metabolomic approach, in conjunction with a multivariate statistical analysis, was applied to investigate the metabolic pattern of both infected and treated adult olive cultivars, Ogliarola salentina and Cellina di Nardò trees, in two sampling periods, performed during the first year of the trial. For both cultivars and sampling periods, the orthogonal partial least squares discriminant analysis (OPLS-DA) gave good models of separation according to the treatment application. In both cultivars, some metabolites such as quinic acid, the aldehydic form of oleoeuropein, ligstroside and phenolic compounds, were consistently found as discriminative for the untreated olive trees in comparison with the Dentamet®-treated trees. Quinic acid, a precursor of lignin, was confirmed as a disease biomarker for the olive trees infected by X. fastidiosa subsp. pauca. When treated with Dentamet®, the two cultivars showed a distinct response. A consistent increase in malic acid was observed for the Ogliarola salentina trees, whereas in the Cellina di Nardò trees the treatments attenuate the metabolic response to the infection. To note that in Cellina di Nardò trees at the first sampling, an increase in γ-aminobutyric acid (GABA) was observed. This study highlights how the infection incited by X. fastidiosa subsp. pauca strongly modifies the overall metabolism of olive trees, and how a zinc-copper-citric acid biocomplex can induce an early re-programming of the metabolic pathways in the infected trees

Treated Unconventional Waters Combined with Different Irrigation Strategies Affect 1 H NMR Metabolic Profile of a Monovarietal Extra Virgin Olive Oil

none8noThe agricultural sector is facing a decrease in water supply and water quality at a global level and this is a problem that strictly affects all the Mediterranean olive growing areas. The aim of this work was to evaluate, for the first time, by NMR Spectroscopy and multivariate data analysis the metabolic profiling of the oils produced under different irrigation schemes. Arbosana olive oils were obtained from the use of saline reclaimed water (RW) and treated municipal wastewater (DW), combined with: full irrigation (FI) and regulated deficit irrigation (RDI). The results show a higher relative content of saturated fatty acids in EVOOs obtained from RDI strategy, regardless of the water source. Moreover, an increase in unsaturated fatty acids, a ω6/ω3 ratio content was observed in EVOOs obtained from RW when compared with DW water. Furthermore, the RW–RDI showed an increase in secoiridoid derivatives and hydroperoxides with respect to DW–RDI. A sustainable irrigation management, by combining a deficit irrigation strategy and saline reclaimed water source, could be crucial in order to overcome the problem of water scarcity and to guarantee the olive oil nutraceutical properties. The1 H NMR-based metabolomic approach proved a powerful and versatile tool for this specific investigation.openAngile F.; Vivaldi G.A.; Girelli C.R.; Del Coco L.; Caponio G.; Lopriore G.; Fanizzi F.P.; Camposeo S.Angile, F.; Vivaldi, G. A.; Girelli, C. R.; Del Coco, L.; Caponio, G.; Lopriore, G.; Fanizzi, F. P.; Camposeo, S

The response of the algae Fucus virsoides (Fucales, Ochrophyta) to Roundup® solution exposure: A metabolomics approach

8noGlyphosate, as a broad-spectrum herbicide, is frequently detected in water and several studies have investigated its effects on several freshwater aquatic organisms. Yet, only few investigations have been performed on marine macroalgae. Here, we studied both the metabolomics responses and the effect on primary production in the endemic brown algae Fucus virsoides exposed to different concentration (0, 0.5, 1.5 and 2.5 mg L−1) of a commercial glyphosate-based herbicide, namely Roundup®. Our results show that Roundup® significantly reduced quantum yield of photosynthesis (Fv/Fm) and caused alteration in the metabolomic profiles of exposed thalli compared to controls. Together with the decrease in the aromatic amino acids (phenylalanine and tyrosine), an increase in shikimate content was detected. The branched-amino acids differently varied according to levels of herbicide exposure, as well as observed for the content of choline, formate, glucose, malonate and fumarate. Our results suggest that marine primary producers could be largely affected by the agricultural land use, this asking for further studies addressing the ecosystem-level effects of glyphosate-based herbicides in coastal waters.partially_openopenFelline, Serena; Del Coco, Laura; Kaleb, Sara; Guarnieri, Giuseppe; Fraschetti, Simonetta; Terlizzi, Antonio; Fanizzi, Francesco Paolo; Falace, AnnalisaFelline, Serena; Del Coco, Laura; Kaleb, Sara; Guarnieri, Giuseppe; Fraschetti, Simonetta; Terlizzi, Antonio; Fanizzi, Francesco Paolo; Falace, Annalis

First evidence for N7-Platinated Guanosine derivatives cell uptake mediated by plasma membrane transport processes

Nucleos(t)ide analogues (NA) belong to a family of compounds widely used in anticancer/antiviral treatments. They generally exhibit a cell toxicity limited by cellular uptake levels and the resulting nucleos(t)ides metabolism modifications, interfering with the cell machinery for nucleic acids synthesis. We previously synthesized purine nucleos(t)ide analogues N7-coordinated to a platinum centre with unaltered sugar moieties of the type: [Pt(dien)(N7-dGuo)]2+ (1; dien = diethylenetriamine; dGuo = 2′-deoxy-guanosine), [Pt(dien)(N7-dGMP)] (2; dGMP = 5′-(2′-deoxy)-guanosine monophosphate), and [Pt(dien)(N7-dGTP)]2− (3; dGTP = 5′-(2′-deoxy)-guanosine triphosphate), where the indicated electric charge is calculated at physiological pH (7.4). In this work, we specifically investigated the uptake of these complexes (1–3) at the plasma membrane level. Specific experiments on HeLa cervical cancer cells indicated a relevant cellular uptake of the model platinated deoxynucleos(t)ide 1 and 3 while complex 2 appeared unable to cross the cell plasma membrane. Obtained data buttress an uptake mechanism involving Na+-dependent concentrative transporters localized at the plasma membrane level. Consistently, 1 and 3 showed higher cytotoxicity with respect to complex 2 also suggesting selective possible applications as antiviral/antitumor drugs among the used model compounds

Uranium and thorium in lamprophyres of the Altai-Sayan folded region

The work is devoted to the study of lamprophyres of the Altai-Sayan region. According to X-ray phase analysis, these rocks have a complex mineral composition. According to the TAS diagram, the petrochemical composition varies from alkaline picrites to trachytes. As the INAA shows, the studied samples are significantly enriched with thorium and uranium. Combining the method of electron microscopy and fission radiography (f-radiography), the occurrence forms of thorium and uranium were studied, therefore concentrating minerals were identified

1H-NMR based serum metabolomics highlights different specific biomarkers between early and advanced hepatocellular carcinoma stages

The application of non-targeted serum metabolomics profiling represents a noninvasive tool to identify new clinical biomarkers and to provide early diagnostic differentiation, and insight into the pathological mechanisms underlying hepatocellular carcinoma (HCC) progression. In this study, we used proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy and multivariate data analysis to profile the serum metabolome of 64 HCC patients, in early (n = 28) and advanced (n = 36) disease stages. We found that1H-NMR metabolomics profiling could discriminate early from advanced HCC patients with a cross-validated accuracy close to 100%. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed significant changes in serum glucose, lactate, lipids and some amino acids, such as alanine, glutamine, 1-methylhistidine, lysine and valine levels between advanced and early HCC patients. Moreover, in early HCC patients, Kaplan\u2013Meier analysis highlighted the serum tyrosine level as a predictor for overall survival (OS). Overall, our analysis identified a set of metabolites with possible clinical and biological implication in HCC pathophysiology

Developing Central Nervous System and Vulnerability to Platinum Compounds

Comparative studies on the effects of the platinum complexes in use or in clinical trials are carried out in order to discover differences in the neurotoxic potential and the reversibility of neurotoxicity. In this paper, we summarized the current literature on neurotoxicity and chemoresistance of cisplatin (cisPt) and discussed our recent efforts on the interference of cisPt and a new platinum compound [Pt(O,O′-acac)(γ-acac)(DMS)] (PtAcacDMS), with high specific reactivity with sulphur ligands instead of nucleobases as cisPt, on some crucial events of rat postnatal cerebellum development. The acute effects of drug treatments on cell proliferation and death in the external granular layer and granule cell migration and the late effects on the dendrite growth of Purkinje cells were evaluated. Together with the demonstrated antineoplastic effectiveness in vitro, compared with cisPt, data suggest a lower neurotoxicity of PtAcacDMS, in spite of its presence in the brain that involves considerations on the blood brain barrier permeability