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

Harvest year effects on Apulian EVOOs evaluated by (1)H NMR based metabolomics

none5noNine hundred extra virgin olive oils (EVOO) were extracted from individual olive trees of four olive cultivars (Coratina, Cima di Mola, Ogliarola, Peranzana), originating from the provinces of Bari and Foggia (Apulia region, Southern Italy) and collected during two consecutive harvesting seasons (2013/14 and 2014/15). Following genetic identification of individual olive trees, a detailed Apulian EVOO NMR database was built using 900 oils samples obtained from 900 cultivar certified single trees. A study on the olive oil lipid profile was carried out by statistical multivariate analysis (Principal Component Analysis, PCA, Partial Least-Squares Discriminant Analysis, PLS-DA, Orthogonal Partial Least-Squares Discriminant Analysis, OPLS-DA). Influence of cultivar and weather conditions, such as the summer rainfall, on the oil metabolic profile have been evaluated. Mahalanobis distances and J2 criterion have been measured to assess the quality of resulting scores clusters for each cultivar in the two harvesting campaigns. The four studied cultivars showed non homogeneous behavior. Notwithstanding the geographical spread and the wide number of samples, Coratina showed a consistent behavior of its metabolic profile in the two considered harvests. Among the other three Peranzana showed the second more consistent behavior, while Cima di Mola and Ogliarola having the biggest change over the two years.Girelli, Chiara R; Del Coco, Laura; Papadia, Paride; De Pascali, Sandra A; Fanizzi, Francesco PGirelli, CHIARA ROBERTA; DEL COCO, Laura; Papadia, Paride; DE PASCALI, SANDRA ANGELICA; Fanizzi, Francesco Paol

1H NMR Spectroscopy and MVA Analysis of Diplodus sargus Eating the Exotic Pest Caulerpa cylindracea

6noThe green alga Caulerpa cylindracea is a non-autochthonous and invasive species that is severely affecting the native communities in the Mediterranean Sea. Recent researches show that the native edible fish Diplodus sargus actively feeds on this alga and cellular and physiological alterations have been related to the novel alimentary habits. The complex effects of such a trophic exposure to the invasive pest are still poorly understood. Here we report on the metabolic profiles of plasma from D. sargus individuals exposed to C. cylindracea along the southern Italian coast, using 1H NMR spectroscopy and multivariate analysis (Principal Component Analysis, PCA, Orthogonal Partial Least Square, PLS, and Orthogonal Partial Least Square Discriminant Analysis, OPLS-DA). Fish were sampled in two seasonal periods from three different locations, each characterized by a different degree of algal abundance. The levels of the algal bisindole alkaloid caulerpin, which is accumulated in the fish tissues, was used as an indicator of the trophic exposure to the seaweed and related to the plasma metabolic profiles. The profiles appeared clearly influenced by the sampling period beside the content of caulerpin, while the analyses also supported a moderate alteration of lipid and choline metabolism related to the Caulerpa-based diet.openopenS. A. De Pascali; L. Del Coco; S. Felline; E. Mollo; A. Terlizzi; F. P. FanizziS. A., De Pascali; L., Del Coco; S., Felline; E., Mollo; Terlizzi, Antonio; F. P., Fanizz

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters