Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.     

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeF_x) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeF_x is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, k_obs, for the formation of E2P decreases with [Pi] whereas that of E2BeF_x increases with [BeF_x]. This might wrongly be taken as evidence of a mechanism where the binding of BeF_x induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeF_x follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na⁺. Although E2P and E2BeF_x are able to form states with 2 occluded Rb⁺, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb⁺ is much lower in E2BeF_x than in E2P. The higher rates of Rb⁺ occlusion and deocclusion observed for E2BeF_x, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeF_x mimics the E2P ground state.     

Life cycle assessment of electricity transmission and distribution—part 1: power lines and cables

Purpose  

The purpose of this study is to provide life cycle inventory data and results for components of electrical grids to the larger community of life cycle assessment practitioners. This article is the first in a series of two, each focusing on different components of power grids. In part 1, the objects under scope are power lines and cables. Systems for overhead, underground, and subsea transmission are modeled here, including HVDC systems used in long-distance transmission.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Synthesis and potent antitumor activity of new arylamino derivatives of nor-beta-lapachone and nor-alpha-lapachone

Several arylamino derivatives of nor-beta-lapachone were synthesized in moderate to high yields and found to show very potent cytotoxicity against six neoplastic cancer cells: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (breast), HL-60 (leukaemia), PC-3 (prostate), and B-16 (murine melanoma), with IC(50) below 1 microg/mL. Their cytotoxicities were compared to doxorubicin and with their synthetic precursors, beta-lapachone and nor-beta-lapachone. The activity against a normal murine fibroblast L-929 showed that some of the compounds were selective against cancer cells. The absence of hemolytic activity (EC(50)>200 microg/mL), performed with erythrocyte suspensions, suggests that the cytotoxicity of the compounds was not related to membrane damage of mouse erythrocytes. For comparison purposes, one isomeric compound based on nor-alpha-lapachone was also synthesized and showed lower activity than the related ortho-derivative. The modified arylamino quinones appear as interesting new lead compounds in anti-cancer drug development.     

Ambient Air Pollution and the Progression of Atherosclerosis in Adults

Background

Cross-sectional studies suggest an association between exposure to ambient air pollution and atherosclerosis. We investigated the association between outdoor air quality and progression of subclinical atherosclerosis (common carotid artery intima-media thickness, CIMT).

Methodology/Principal Findings

We examined data from five double-blind randomized trials that assessed effects of various treatments on the change in CIMT. The trials were conducted in the Los Angeles area. Spatial models and land-use data were used to estimate the home outdoor mean concentration of particulate matter up to 2.5 micrometer in diameter (PM2.5), and to classify residence by proximity to traffic-related pollution (within 100 m of highways). PM2.5 and traffic proximity were positively associated with CIMT progression. Adjusted coefficients were larger than crude associations, not sensitive to modelling specifications, and statistically significant for highway proximity while of borderline significance for PM2.5 (P = 0.08). Annual CIMT progression among those living within 100 m of a highway was accelerated (5.5 micrometers/yr [95%CI: 0.13–10.79; p = 0.04]) or more than twice the population mean progression. For PM2.5, coefficients were positive as well, reaching statistical significance in the socially disadvantaged; in subjects reporting lipid lowering treatment at baseline; among participants receiving on-trial treatments; and among the pool of four out of the five trials.

Conclusion

Consistent with cross-sectional findings and animal studies, this is the first study to report an association between exposure to air pollution and the progression of atherosclerosis – indicated with CIMT change – in humans. Ostensibly, our results suggest that air pollution may contribute to the acceleration of cardiovascular disease development – the main causes of morbidity and mortality in many countries. However, the heterogeneity of the volunteering populations across the five trials, the limited sample size within trials and other relevant subgroups, and the fact that some key findings reached statistical significance in subgroups rather than the sample precludes generalizations to the general population.     

Impaired Innate Immunity in Tlr4 ?/? Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8⁺ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8⁺ T cells specific for H-2K^b-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2^−/−, Tlr4^−/−, Tlr9^{−/ −} or Myd88^−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8⁺ T cells at levels comparable to WT mice, although the frequency of IFN-γ⁺CD4⁺ cells was diminished in infected Myd88^−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4^−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.     

Evolutionary history of the European free-tailed bat,a tropical affinity species spanning across the Mediterranean Basin

The Mediterranean Basin is a global biodiversity hotspot, hosting a number of native species belonging to families that are found almost exclusively in tropical climates. Yet, whether or not these taxa were able to survive in the Mediterranean region during the Quaternary climatic oscillations remains unknown. Focusing on the European free-tailed bat (Tadarida teniotis), we aimed to (a) identify potential ancient populations and glacial refugia; (b) determine the post-glacial colonization routes across the Mediterranean; and (c) evaluate current population structure and demography. Mitochondrial and nuclear markers were used to understand T. teniotis evolutionary and demographic history. We show that T. teniotis is likely restricted to the Western Palearctic, with mitochondrial phylogeny suggesting a split between an Anatolian/Middle East clade and a European clade. Nuclear data pointed to three genetic populations, one of which is an isolated and highly differentiated group in the Canary Islands, another distributed across Iberia, Morocco, and France, and a third stretching from Italy to the east, with admixture following a pattern of isolation by distance. Evolutionary and demographic reconstruction supports a pre-Last Glacial Maximum (LGM) colonization of Italy and the Anatolian/Middle East, while the remaining populations were colonized from Italy after the Younger Dryas. We also found support for demographic expansion following the Iberian colonization. The results show that during the LGM T. teniotis persisted in Mediterranean refugia and has subsequently expanded to its current circum-Mediterranean range. Our findings raise questions regarding the physiological and ecological traits that enabled species with tropical affinities to survive in colder climates.     

Oxysterol-induced toxicity in R28 and ARPE-19 cells

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2,7-dichlorodihydrofluorescein diacetate [H₂DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.