EXTRACELLULAR SPHINGOSINE-1-PHOSPHATE: A NOVEL ACTOR IN HUMAN GLIOBLASTOMA STEM CELL SURVIVAL PROPERTIES

Glioblastoma multiforme (GBM) is the most frequent and aggressive intracranial tumour in humans. The prognosis of GBM patients remains unfavourable even after aggressive treatments based on multiple approaches, due to the high proliferation rate, migrating-invasive properties, and resistance to therapeutic intervention. The introduction of the alkylating agent TMZ in glioblastoma therapy has improved patient survival, but drug resistance mechanisms limit its benefits. The aim of this study was to provide a contribution to the understanding of the malignant and chemoresistance properties in GBM by focusing on the role of the bioactive sphingoid molecules ceramide and S1P, which act as antagonists in regulating cell properties and survival. Accumulating literature indicates that ceramide is a tumour suppressor sphingolipid, able to induce antiproliferative and apoptotic responses, and that it is able to act as a major player in the mechanism of action of many chemotherapeutic drugs. We demonstrated that the treatment of T98G human glioblastoma cells with cytotoxic TMZ concentrations results in a significant increase in intracellular ceramide, which in turn promotes cell death. On the other hand, TMZ is not able to induce ceramide accumulation in TMZ-resistant glioblastoma cells (TMZ-R). These data suggest a role of ceramide as a mediator of TMZ-induced toxicity. A large amount of evidence underlines the role of S1P as an important tumour-promoting sphingolipid, acting predominantly in the extracellular milieu after interaction with specific G protein-coupled receptors and exerting opposite effects on cell survival compared to ceramide. Parallel studies demonstrated that S1P secretion in TMZ-R cells is functional to inhibit the cytotoxic effect of ceramide and to confer TMZ-resistant properties to glioblastoma cells. Stimulated by these findings, we next evaluated the role of sphingolipid mediators in the malignant features of glioblastoma stem cells (GSCs), a cell subpopulation within the tumour mass involved in the aberrant expansion and therapy resistance properties of glioblastomas. To this purpose we used GSCs isolated from the human U87-MG glioblastoma cell line and GSCs isolated from a primary culture of human glioblastoma. We found that both GSC models efficiently form typical neurosphere structures in mitogen-defined medium and express high levels of recognized cancer stem cell markers. Moreover, GSCs exhibit resistance to TMZ at concentrations that are cytotoxic in U87-MG, despite not expressing the DNA repair protein MGMT, a major contributor to TMZ-resistance. Even though a large amount of evidence underlines that S1P is able to favor growth, invasion and chemotherapy resistance of glioblastoma cells, so far little is known on the possible role of S1P as a factor modulating GSCs malignant properties. Further experiments revealed that glioblastoma cells and GSCs are able to efficiently synthesize S1P and also to release it in the culture medium. Notably the intracellular S1P level was found much lower in GSC models than in the glioblastoma cell line; meanwhile the extracellular S1P level was significantly higher in GSC models than in U87-MG cells. These differences resulted in an extracellular S1P-intracellular S1P ratio at least 10 times higher in GSCs compared to U87-MG. Furthermore, this ratio is about 1:1 in both GSCs, thus suggesting that these cells are an efficient source of S1P in the extracellular microenvironment. Furthermore we found that ceramide-extracellular S1P ratio is at least 2-fold lower in GSCs than in U87-MG. Since S1P and ceramide exert opposing effects on cell survival, according to the \u201csphingolipid rheostat\u201d model, this different ratio could promote GSC survival observed after TMZ treatment. Interestingly, enzyme activity assays excluded the presence of sphingosine kinase (SK), the enzyme responsible for S1P byosinthesis, in GSC medium, implicating an efficient secretion of S1P in GSCs. The analyses of the expression of the ABC-transporters known to be involved in S1P export (ABCG2, ABCA1 and ABCC1), revealed that only ABCA1 is expressed in GSCs. Notwithstanding, after ABCA1 inhibition, no variations in S1P release was observed, suggesting that other mechanisms different from those known are involved. We also investigated the role of S1P in glioblastoma resistance to TMZ. A first interesting finding was that exogenously administered S1P protected U87-MG cells against TMZ cytotoxic effects. In addition, we found that, after co-treatment with TMZ and an inhibitor of S1P biosynthesis, GSCs became sensitive to the toxic effect of the drug. Of note, exogenous S1P administration was able to revert this effect. These data strongly support extracellular S1P as an important mediator in TMZ-resistance of GSCs. Furthermore, results obtained in GSCs isolated from two patients affected by glioblastoma with different aggressive phenotype, revealed that the extracellular release of S1P was significantly higher by cells isolated from the most aggressive tumour, suggesting that the release and thus the levels of extracellular S1P might be related to tumour aggressiveness and patient prognosis. In conclusion, our data implicate for the first time GSCs as an important source of S1P in the extracellular microenvironment, where, on its turn, S1P can act as an autocrine/paracrine messenger able to contribute to the GSC survival properties. A better understanding of S1P role in GSCs aggressive phenotype could represent a critical start point that sets the bases for the development of new compounds able to sensitize GSCs to chemotherapeutic treatments, thus improving survival rates in GBM patients

Cognitive Functions and White Matter Tract Damage in Amyotrophic Lateral Sclerosis: A Diffusion Tensor Tractography Study

BACKGROUND AND PURPOSE: ALS is predominantly a disease of the motor system, but cognitive and behavioral symptoms also are observed. DT MR imaging is sensitive to microstructural changes occurring in WM tracts of patients with ALS. In this study, we investigated the association between cognitive functions and extramotor WM tract abnormalities in ALS patients. MATERIALS AND METHODS: DT MR imaging was obtained from 16 nondemented patients with ALS and 15 healthy controls. Patients with ALS underwent a neuropsychologic and behavioral evaluation. DT tractography was used to asses the integrity of the CST, corpus callosum, and the major long-range association tracts. The relationship between DT MR imaging metrics and cognitive functions was tested by using linear model analyses, adjusting for age and clinical disability. RESULTS: Eleven patients (69%) scored below the fifth percentile in at least 1 cognitive test, and 2 of them had a mild executive impairment. Performances at tests assessing attention and executive functions correlated with DT MR imaging metrics of the corpus callosum, CST, and long association WM tracts bilaterally, including the cingulum, inferior longitudinal, inferior fronto-occipital, and uncinate fasciculi. Verbal learning and memory test scores were associated with fornix DT MR imaging values, whereas visual-spatial abilities correlated with left uncinate fractional anisotropy. CONCLUSIONS: WM tract degeneration is associated with neuropsychologic deficits in patients with ALS. DT tractography holds promise to gain insight into the role of the brain WM network abnormalities in the development of cognitive impairment in patients with ALS

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed "sand in the gearbox". Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptideinduced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer

Management of oxygen saturation monitoring in preterm newborns in the NICU: the Italian picture

Background: Although many studies emphasize the importance of using oxygen saturation (SpO2) targets in the NICUs, there is a wide variability in used saturation ranges among centers. Primary aim was to draw a representative picture on how the management of oxygen monitoring is performed in the Italian NICUs. Second aim was to identify healthcare-professionals related factors associated with oxygen targeting in the preterm population. Methods: Cross-sectional study with data collection via an electronic survey form. A questionnaire containing pre-piloted and open questions on monitoring and management of the SpO2 was administered to neonatologists across the network of the Italian Society of Neonatology. The questions focused on: the infrastructure, specific training, healthcare professionals and patients-related factors. The results of the survey were anonymously collected, summarized and analyzed. Results: Out of 378 questionnaires, 93 were correctly filled. Thirty-six different SpO2 ranges were observed. Centers using written standard operating procedures on oxygen management and SpO2 monitoring maintained a correct average range of SpO2 90–95%, avoided hyperoxia and reconsidered saturation targets in relation to comorbidities. 39.8% of responders disabled alarms during neonatal care. One center used biomarkers for complete monitoring of neonatal oxygenation status. Conclusions: There is considerable variation in SpO2 targets for preterm infants in the Italian NICUs. Standard operating procedures and specific training for health care personnel are the main factors playing a role for the correct maintenance of the recommended oxygen targets in preterms

Extracellular sphingosine-1-phosphate : a novel actor in human glioblastoma stem cell survival

Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs), a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P) a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ), and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source of extracellular S1P, which might act as an autocrine/paracrine signal contributing to their malignant properties

Plasma Dynamics

Contains table of contents for Section 2 and reports on two research projects.Princeton University/National Spherical Torus Experiment Grant S04020G PPPLU.S. Department of Energy Grant DE-FGO2-91-ER-54109National Science Foundation Grant ECS 94-24282Los Alamos National Laboratory Grant No. E29060017

Plasma Dynamics

Contains table of contents for Section 2 and reports on three research projects.U.S. Navy - Office of Naval Research Grant N00014-90-J-4130Princeton University/Tokamak Physics Experiment Grant S-03688-GU.S. Department of Energy Grant DE-FG02-91-ER-54109National Science Foundation Grant ATM 94-2428

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Baseline characteristics of patients in the reduction of events with darbepoetin alfa in heart failure trial (RED-HF)

<p>Aims: This report describes the baseline characteristics of patients in the Reduction of Events with Darbepoetin alfa in Heart Failure trial (RED-HF) which is testing the hypothesis that anaemia correction with darbepoetin alfa will reduce the composite endpoint of death from any cause or hospital admission for worsening heart failure, and improve other outcomes.</p> <p>Methods and results: Key demographic, clinical, and laboratory findings, along with baseline treatment, are reported and compared with those of patients in other recent clinical trials in heart failure. Compared with other recent trials, RED-HF enrolled more elderly [mean age 70 (SD 11.4) years], female (41%), and black (9%) patients. RED-HF patients more often had diabetes (46%) and renal impairment (72% had an estimated glomerular filtration rate <60 mL/min/1.73 m2). Patients in RED-HF had heart failure of longer duration [5.3 (5.4) years], worse NYHA class (35% II, 63% III, and 2% IV), and more signs of congestion. Mean EF was 30% (6.8%). RED-HF patients were well treated at randomization, and pharmacological therapy at baseline was broadly similar to that of other recent trials, taking account of study-specific inclusion/exclusion criteria. Median (interquartile range) haemoglobin at baseline was 112 (106–117) g/L.</p> <p>Conclusion: The anaemic patients enrolled in RED-HF were older, moderately to markedly symptomatic, and had extensive co-morbidity.</p&gt

Clinical correlates of grey matter pathology in multiple sclerosis

Traditionally, multiple sclerosis has been viewed as a disease predominantly affecting white matter. However, this view has lately been subject to numerous changes, as new evidence of anatomical and histological changes as well as of molecular targets within the grey matter has arisen. This advance was driven mainly by novel imaging techniques, however, these have not yet been implemented in routine clinical practice. The changes in the grey matter are related to physical and cognitive disability seen in individuals with multiple sclerosis. Furthermore, damage to several grey matter structures can be associated with impairment of specific functions. Therefore, we conclude that grey matter damage - global and regional - has the potential to become a marker of disease activity, complementary to the currently used magnetic resonance markers (global brain atrophy and T2 hyperintense lesions). Furthermore, it may improve the prediction of the future disease course and response to therapy in individual patients and may also become a reliable additional surrogate marker of treatment effect