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Non-invasive continuous glucose monitoring (NI-CGM) sensors are still at an early stage of development, but, in the near future, they could become particularly appealing in diabetes management. Solianis Monitoring AG (Zurich, Switzerland) has proposed an approach for NI-CGM based on a multi-sensor concept, embedding primarily dielectric spectroscopy and optical sensors. This concept requires a mathematical model able to estimate glucose levels from the 150 channels directly measured through the Multisensor. A static multivariate linear regression model (with order and parameters common to the entire population of subjects) was proposed for such a scope (Caduff et al., Biosens Bioelectron 26:3794-3800, 2011). The aim of this work is to evaluate the accuracy in the estimation of glucose levels and trends that the NI-CGM Multisensor platform can achieve by exploiting different techniques for model identification, namely, ordinary least squares, subset variable selection, partial least squares and least absolute shrinkage and selection operator (LASSO). Data collected in human beings monitored for a total of 45 study days were used for model identification and model test. Several metrics of standard use in the diabetes scientific community to measure point and clinical accuracy of glucose sensors were used to assess the models. Results indicate that the LASSO technique is superior to the others shrinking many channel weights to zero thus leading to smoother glucose profiles and resulting in a more robust model to possible artifacts in the Multisensor data. Although, as expected, the performance of the NI-CGM system with the LASSO model is not yet comparable with that of enzyme-based needle glucose sensors, glucose trends are satisfactorily estimated. Considering the non-invasive nature of the multi-sensor platform, this result can have an immediate impact in the current clinical practice, e.g., to integrate sparse self-monitoring of blood glucose data with an indication of the glucose trend to aid the diabetic patient in dealing with, or even preventing in the short time scale, the threats of critical events such as hypoglycaemia.

To better define the neural networks related to preparation of reaching, we applied transcranial magnetic stimulation (TMS) to the lateral parietal and frontal cortex. TMS did not evoke effects closely related to preparation of reaching, suggesting that neural networks already identified by our group are not larger than previously thought. We also replicated previous TMS/EEG data by applying TMS to the parietal cortex: new analyses were performed to better support reliability of already reported findings (Zanon et al., 2010; Brain Topography 22, 307-317). We showed the existence of neural circuits ranging from posterior to frontal regions of the brain after the stimulation of parietal cortex, supporting the idea of strong connections among these areas and suggesting their possible temporal dynamic. Connection with ventral stream was confirmed. The present work helps to define those areas which are involved in preparation of natural reaching in humans. They correspond to parieto-occipital, parietal and premotor medial regions of the left hemisphere, i.e., the contralateral one with respect to the moving hand, as suggested by previous studies. Behavioral data support the existence of a discrete stream involved in reaching. Besides the serial flow of activation from posterior to anterior direction, a parallel elaboration of information among parietal and premotor areas seems also to exist. Present cortico-cortical interactions (TMS/EEG experiment) show propagation of activity to frontal, temporal, parietal and more posterior regions, exhibiting distributed communication among various areas in the brain. The neural system highlighted by TMS/EEG experiments is wider with respect to the one disclosed by the TMS behavioral approach. Further studies are needed to unravel this paucity of overlap. Moreover, the understanding of these mechanisms is crucial for the comprehension of response inhibition and changes in prepared actions, which are common behaviors in everyday life.

Purpose: To assess the role of Apollon in melanoma resistance to intrinsic and extrinsic pathways of apoptosis and to identify strategies to reduce its expression., Experimental Design: Apollon expression was assessed in melanoma cells in vitro and in vivo. Apollon modulation and melanoma apoptosis were evaluated by Western blot and/or flow cytometry in response to cytotoxic drugs, mitogen-activated protein/extracellular signal-regulated kinase (MEK)-, BRAF(V600E)-, and mTOR-specific inhibitors, TRAIL and anti-HLA class II monoclonal antibodies (mAb). Mitochondrial depolarization, caspase activation, apoptosis assays, and gene expression profiling were used to test effects of Apollon silencing, by siRNA, on melanoma response to antitumor agents., Results: Apollon was constitutively expressed by melanoma cells, in vitro and in vivo, and at higher levels than in benign melanocytic lesions. Melanoma apoptosis correlated significantly with Apollon protein downmodulation in response to cytotoxic drugs, MEK, or BRAF(V600E)-specific inhibitors. Combinatorial treatment with MEK and mTOR inhibitors and HLA class II ligation, by a specific mAb, promoted Apollon downmodulation and enhanced melanoma apoptosis. Apollon downmodulation induced by antitumor agents was caspase independent, but proteasome dependent. Knockdown of Apollon, by siRNA, triggered apoptosis and/or significantly enhanced melanoma cell death in response to cytotoxic drugs, MEK- and BRAF(V600E)-specific inhibitors, and soluble or membrane-bound TRAIL. Apollon silencing promoted mitochondrial depolarization and caspase-2, caspase-8, caspase-9, and caspase-3 activation in response to different antitumor agents and altered the profile of genes modulated by MEK or BRAF(V600E)-specific inhibitors., Conclusions: Targeting of Apollon may significantly improve melanoma cell death in response to antitumor agents that trigger the intrinsic or the extrinsic apoptosis pathways., (©2012 AACR.)

Fusarium equiseti and Fusarium semitectum represent the most abundant species in the Fusarium complex isolated from flowers, soybean pods and seeds in Argentina. The aim of the present study was to assess the production of major type A and type B trichothecenes (diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin, nivalenol, deoxynivalenol) and zearalenone by 40 F. equiseti and 22 F. semitectum isolates on rice culture. Mycotoxins were determined by HPLC with fluorescence detection after derivatisation with 1-anthronylnitrile for type A trichothecenes (i.e. diacetoxyscirpenol, neosolaniol, T-2 toxin and HT-2 toxin), by HPLC with UV detection for type B trichothecenes (i.e. nivalenol and deoxynivalenol), and by TLC for zearalenone. A total of 22 of 40 F. equiseti isolates produced diacetoxyscirpenol, nivalenol and ZEA alone or in combination, whereas only two of 20 F. semitectum isolates were nivalenol and ZEA producers. Both Fusarium species did not produce any deoxynivalenol, neosolaniol, T-2 toxin and HT-2 toxin. The variable retention in toxigenicity displayed by both fungal species suggests that these species have a saprophytic lifestyle in the soybean agroecosystem in Argentina.

Soybean (Glycine max L.), the main source of protein throughout the world, is used both as a food and a feedstuff. Currently, limited information about the occurrence of Fusarium species and mycotoxins in soybean grain and by-products is available. The aims of the present study were: (1) to identify toxigenic Fusarium species associated with soybean during crop reproductive stages; (2) to determine the occurrence of deoxynivalenol (DON) and nivalenol (NIV) in soybean seeds; (3) to determine the genotype and chemotype of selected Fg complex strains using molecular and chemical analysis, respectively; and (4) to characterize the strains using AFLP(s) markers. One soybean field located at Córdoba Province, Argentina, was monitored and samples of soybean tissue were harvested at three reproductive stages: flowering (R2), full seed (R6) and full maturity (R8). A total of 389 Fusarium strains F. equiseti (40%) was the most frequently species recovered followed by F. semitectum (27%) and F. graminearum (Fg) (11%). From the 40 soybean samples analysed, only two presented detectable DON levels. Based on DON occurrence on soybean seeds at ripening stages, the toxigenic ability of Fg complex strains isolated from soybean seeds, pods and flowers were analysed. The trichothecene genotype was determined by a multiplex PCR using primers based on Tri3, Tri5 and Tri7 toxin genes and then the chemotype was verified by chemical analysis. Most Fg complex strains showed 15-ADON genotype and five strains presented a DON/NIV; these also produced both toxins under in vitro culture. Neither the NIV nor the 3-ADON genotypes were detected among the members of the population evaluated. All the 15-ADON genotype strains were characterized as F. graminearum sensu stricto (lineage 7), while the strains presented a DON/NIV genotype were characterized as F. meridionale (lineage 2). The present study contributes new information on the occurrence of Fusarium species and trichothecenes toxins on soybean at the pre-harvest stages. Also, this is the first report on the chemotype, genotype and lineages among Fg complex isolated from soybean.

New scenarios in diabetes treatment have been opened in the last ten years by continuous glucose monitoring (CGM) sensors. In particular, Non-Invasive CGM sensors are particularly appealing, even though they are still at an early stage of development. Solianis Monitoring AG (Zürich, Switzerland) has proposed an approach based on a multisensor concept, embedding primarily dielectric spectroscopy and optical sensors. This concept requires a mathematical model able to reconstruct the glucose concentration from the 150 channels measured with the device. Assuming a multivariate linear regression model (valid and usable for different individuals), the aim of this paper is the assessment of some techniques usable for determining such a model, namely Ordinary Least Squares (OLS), Partial Least Squares (PLS) and Least Absolute Shrinkage and Selection Operator (LASSO). Once the model is identified on a training set, the accuracy of prospective glucose profiles estimated from "unseen" multisensor data is assessed. Preliminary results obtained from 18 in-clinic study days show that sufficiently accurate reconstruction of glucose levels can be achieved if suitable model identification techniques, such as LASSO, are considered.

Parietal cortex subserves various cognitive tasks, ranging from attention to visuo-motor skills. It is part of a parieto-frontal network involved in attention, and part of the visual dorsal stream, opposed to the visual ventral stream, although increasing evidence suggests interchange of information between them. In this study, co-registration of Transcranial Magnetic Stimulation (TMS) and Electroencephalographic activity (EEG) has been used to investigate the spreading of cortical connections from the parietal cortex in healthy volunteers. TMS on the left parietal cortex activated a network of prefrontal regions in the contra-lateral hemisphere in a time range of 102-167 ms after the stimulus. Moreover, activation in the ipsi-lateral middle temporal and fusiform gyri was observed at 171-177 ms after delivery of TMS. Findings suggest the existence of late driven connections between parietal and prefrontal regions that could partially represent the neural pathway related to attention, even if, in this experiment, no attentional processing was requested. Late connections between dorsal and ventral streams were also evident, confirming previous evidence about interchange of information between them. Conclusively, the present investigation confirms that a great amount of information spreads from parietal cortex to different regions in the brain, supporting the idea that connections are more complex and articulated than those proposed. Present findings also suggest that the simultaneous recording of EEG during the application of TMS is a promising tool for the study of connections in the brain.

Objective: The aim of this study was to evaluate the prevalence of subclinical atherosclerosis in patients with psoriatic arthritis (PsA), correlated with some traditional risk factors of atherosclerosis and with PsA-related disease factors., Methods: Forty-one patients and 41 healthy subjects were evaluated for intima-media thickness (IMT) and flow-mediated dilation (FMD), using carotid duplex scanning. IMT values were expressed like IMT mean (cumulative mean of all the IMT mean) and M-MAX (cumulative mean of all the higher IMT). Subclinical atherosclerosis markers were correlated with age, body mass index (BMI) and blood pressure in both groups, with duration of arthritis, duration of psoriasis, tender and swollen joints, BASDAI (Bath Ankylosing Spondylitis Disease Activity Index), BASFI (Bath Ankylosing Spondylitis Functional Index), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in patients., Results: IMT mean and M-MAX were both higher in PsA patients compared with controls (0.7+/-0.15 vs 0.62+/-0.09 mm; p<0.01 and 0.86+/-0.21 vs. 0.74+/-0.13 mm; p<0.01 respectively). FMD was smaller in patients than in controls (5.9+/-2 vs 7.5+/-2.8%; p<0.01). Univariate analysis showed a correlation between IMT mean and SBP (r=0.217; p=0.05) and a correlation between M-MAX and age (r=0.392; p<0.001), BMI (r=0.252; p<0.05), SBP (r=0.446; p<0.001) in both groups. In PsA patients M-MAX resulted correlated with ESR (r=0.338; p<0.05) and BASDAI (r=0.322; p<0.05)., Conclusions: PsA patients exhibited endothelial dysfunctions which is an early marker of subclinical atherosclerosis, as well as an higher IMT. An interesting correlation between M-MAX and PsA activity index (ESR and BASDAI) was found.

Acquired resistance to mechanisms of programmed cell death is one of the hallmarks of cancer. Human melanoma, in advanced stage, is hardly curable, due to development of several strategies that impair apoptosis induced by the death receptor and the mitochondrial pathways of apoptosis. Among these apoptosis escape strategies, one is based on inactivation of pro-apoptotic factors such as Apoptotic Protease Activating Factor-1 (APAF-1). APAF-1 couples cytochrome c release from the mitochondria to caspase-9 activation and has been considered a central adaptor in the intrinsic pathway of programmed cell death. Inactivation of APAF-1 in human melanoma may impair the mitochondrial pathway of apoptosis induced by chemotherapeutic drugs that activate the p53 pathway, thus contributing to the development of chemoresistance. In-vivo, loss of expression of APAF-1 is associated with tumor progression, suggesting that APAF-1 inactivation may provide a selective survival advantage to neoplastic cells. However, recent results have indicated the existence of APAF-1-independent pathways of caspase activation and apoptosis in normal and neoplastic cells. Moreover, it has been found that expression of APAF-1 is not necessary for the apoptotic response of melanoma cells to different pro-apoptotic drugs. The emerging picture from results obtained in melanoma and other human tumors is that the relevance of the APAF-1 pathway in programmed cell death is cell-context-dependent and related to the specificity of the pro-apoptotic-stimuli.

Neoplastic cells are thought to have defective expression of costimulatory molecules. However, in this study, we show that human melanoma cells express LIGHT/TNFSF14, a ligand of herpesvirus entry mediator on T cells and of lymphotoxin beta receptor on stromal cells. In vitro, melanoma cells stained for LIGHT in the intracellular compartment, with weak or negative cell surface expression. However, LIGHT was expressed on tumor-derived microvesicles released from melanoma cells. In vivo, LIGHT was found in metastatic lesions, and the extent of lymphotoxin beta receptor expression on the stromal cells was significantly associated with a "brisk" T-cell infiltrate in the neoplastic tissue. In the lesions with a brisk T-cell infiltrate, stromal cells surrounding the tumor also stained for the T-cell attractant chemokine CCL21. The intratumoral T lymphocytes frequently expressed herpesvirus entry mediator and were characterized by a differentiated phenotype. Coculture of lymphocytes with LIGHT(+) melanoma-derived microvesicles or even with LIGHT(+) melanoma cells in the presence of interleukin-2 costimulated LIGHT-dependent CD3(+)CD8(+) T-cell proliferation. However, lymphocyte coculture with LIGHT(+) microvesicles in the presence of interleukin-2 was also associated with an apoptotic response as documented by increased binding of Annexin V by CD3(+)CD8(+) T cells. These data suggest that LIGHT constitutively expressed in human melanoma cells and microvesicles may contribute to regulate T-cell responses to tumor cells.

Human melanomas can express unique tumor antigens, resulting from mutated proteins, and shared epitopes encoded for by normal genes, but these two classes of antigens have not been previously compared for immunogenicity and retention in metastatic cells. Here, we identified a new unique antigen generated by a point mutation in the peroxiredoxin 5 (Prdx5) gene in an HLA-A*0201(+) human metastatic melanoma lacking the wild-type allele. An antioxidant assay, with recombinant Prdx5 proteins, and evaluation of peroxide accumulation in transiently transfected cells, indicated that the mutant protein retained its enzymatic activity. The mutation in the Prdx5 protein did not generate a new HLA agretope but yielded an HLA-A*0201-restricted T cell epitope (Prdx5(110-119)). By HLA-tetramer analysis, in a tumor-invaded lymph node, >50% of mutant Prdx5-specific CD8(+) T cells (frequency 0.37%/CD8(+)) showed a CCR7(+/-) CD45RA(-) "T(CM)" or "T(EM)" phenotype, as found in Melan-A/MART-1-specific T cells (frequency 0.68%/CD8(+)) in the same tissue. In agreement with their memory phenotype, the Prdx5-specific T cells readily expanded in vitro in mixed lymphocyte-tumor culture, as did the Melan-/MART-1-specific T cells. By immunohistochemistry of the invaded lymph node, the mutant Prdx5 protein was expressed in all neoplastic cells, in contrast with the heterogeneous expression of shared antigens as Melan-A/MART-1, gp100 and tyrosinase. Thus, a unique tumor antigen can be as immunogenic as the melanoma differentiation antigens but, in contrast to the latter, may be retained in all metastatic cells possibly as result of the relevant cellular function exerted by the mutated protein.

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1-dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1(-) cells) nor up-regulated (in APAF-1(+) cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1(+) and APAF-1(-) tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 -specific inhibitors in both APAF-1(+) and APAF-1(-) tumor cells. In response to 1 to 100 micromol/L of cisplatin, camptothecin, or celecoxib, APAF-1(+) melanomas (n = 12) did not show significantly increased levels of apoptosis compared with APAF-1(-) tumors (n = 7), with the exception of enhanced apoptosis in response to a very high dose (100 micromol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.