Charging and discharging of graphene in ambient conditions studied with scanning probe microscopy

4 páginas, 3 figuras.-- Trabajo presentado como comunicación oral a la "European Conference on Surface Science ECOSS27" celbrado en Holanda en septiembre de 2010: http://www.ecoss27.eu/By means of scanning probe microscopy we are able to inject charges in isolated graphene sheets deposited on SiO2/Si wafers and characterize the discharge induced by water in controlled ambient conditions. Contact potential differences between the graphene surface and the probe tip, measured by Kelvin probe microscopy, show a linear relationship with the tip bias during charge injection. The discharge depends on relative humidity and decays exponentially with time constants of the order of tens of minutes. We propose that graphene discharges through the water film adsorbed on the SiO2 surface.This work was supported by the Ministerio de Educación y Ciencia (MEC), Spain, through Grant No. FIS2006-12117- C04-01, and by the EXPLORA program NAN2007-29375-E.Peer reviewe

Oligosaccharyltransferase Inhibition Induces Senescence in RTK-Driven Tumor Cells

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high throughput screen and lead compound optimization campaign that delivered a cell permeable inhibitor (NGI-1). NGI-1 targets the oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small cell lung cancer cells NGI-1 blocks cell surface localization and signaling of the EGFR glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or FGFR) for survival. In these cell lines OST inhibition causes cell cycle arrest accompanied by induction of p21, autofluorescence, and changes in cell morphology; all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor tyrosine kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells

Characterization and localization of intracellular signals emanating from an oncogenic mutant of the cytokine receptor Gp130

IL-6 is a major regulator of the acute phase response (APR) and signals via a hexameric complex comprising two molecules IL-6, IL-6Rα and gp130, respectively. Upon formation of the signaling complex cytoplasmic Tyr-residues of the receptor get phosphorylated by receptor associated Jaks leading to the initiation of two major signaling pathways: the Jak/STAT and the MAPK/Erk pathway. Within the Jak/STAT pathway STAT1 and STAT3 are recruited to the four most membrane-distal pTyr-residues, homo- and/or heterodimerize and translocate into the nucleus to induce the expression of target genes. Among the most important target genes is the feedback inhibitor SOCS3 that binds to the pTyr 759 and exerts its action primarily via direct inhibition of the Jaks. Furthermore, SOCS3 recruits an E3-ubiquitin ligase complex leading to the degradation of its signaling partners. pTyr 759 also serves as a docking site for SHP2 and is therefore important for initiation of the MAPK/Erk pathway. This cascade leads to nuclear translocation of Erk and target gene induction. In 2009 small in-frame somatic deletions were identified within the gene encoding gp130 in benign liver tumors designated as IHCAs. These mutations target the interaction site of gp130 with IL-6 and confer constitutive and ligand-independent activity to the receptor by means of STAT3-phosphorylation and induction of SOCS3. This work focuses on the most potent of the detected deletion mutants of gp130 that is designated as CAgp130.In the first part of this thesis we examined the processing-trafficking-signaling axis of CAgp130 in comparison to WTgp130. For this purpose WT and mutant receptor were tagged with fluorescent proteins and cell lines were generated that allowed expression of both receptors in a stable and inducible manner. Our results reveal that in contrast to WTgp130 that is prevalent in its high glycosylated form, CAgp130 is predominantly expressed in the lower glycosylated and therefore incompletely processed form. In line with these data CAgp130 accumulates in intracellular compartments that resemble the ER and Golgi and reaches the cell membrane to a much lower extent than WTgp130. We come to the conclusion that defects in processing and trafficking of CAgp130 cannot be attributed to constitutive receptor-phosphorylation, as described for several RTKs, as a receptor mutant where all Tyr-residues have been mutated does not differ in its intracellular distribution. Concerning the signaling properties, WTgp130 as well as CAgp130 lead to a full-fledged activation of the Jak/STAT pathway according to phosphorylation of the respective Tyr- and Ser-residues on STAT3 and STAT1. In contrast to WTgp130, that clearly activates the MAPK/Erk pathway, CAgp130 just leads to its partial activation as it phosphorylates SHP2 but not Erk. We conclude that partial activation of the MAPK/Erk cascade by CAgp130 is due to its reduced cell surface expression as this pathway reportedly is strictly limited to the plasma membrane. By the use of so-called add-back mutants of CAgp130 were only single cytoplasmic Tyr-residues are available for signaling we confirmed a differential contribution of the Tyr-residues for activation of the Jak/STAT and MAPK/Erk pathways that, however, is identical to the WT. In the second part of this thesis we investigated the spatial distribution of constitutive signaling. In order to analyze the signaling potential of CAgp130 on its way to the plasma membrane we initially tried to retain the receptor within the cell by the use of ER-retention sequences. As a second approach we utilized the trafficking-inhibitor brefeldin A and found that mutant receptor is able to activate STAT3 before reaching the cell membrane. Analysis of signaling emanating from CAgp130 located at the plasma membrane or upon its endocytosis was performed utilizing neutralizing gp130 Abs and dominant-negative dynamin, respectively. In both cases STAT3-activation did not show any perturbance leading us to the conclusion that neither cell surface CAgp130 nor CAgp130 at endosomal compartments significantly contributes to constitutive signaling. Downregulation of constitutive signaling was achieved by transfection of dominant-negative STAT3. In the third part of this thesis we examined the role of SOCS3 that is induced by CAgp130 for downregulation of constitutive signaling. Our results using the Y759F mutant of gp130 reveal that signaling emanating from CAgp130 is inhibited by SOCS3 to a certain extent. Further data indicate a possible role of SOCS3 in lysosomal degradation of the receptor mutant as CAgp130 but not its Y759F mutant gets stabilized in the presence of inhibitors of lysosomal degradation

A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity

The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses

Transport assay of the hSLCO5A1 protein with Tritium-labelled substrates in <i>X. laevis</i> oocytes.

<p><i>X. laevis</i> oocytes (8–12 oocytes) were injected with the cRNA of the WT SLCO5A1 or its L³³F mutant, or with the control (Tris-HCl). Oocytes were incubated with 1 µCi/ml Tritium-labelled substrate and 0.04 µCi/ml [¹⁴C]sucrose at room temperature for 30 minutes. [¹⁴C]sucrose served as internal leakage control. Radioactivity was measured using a Beckman scintillation counter. Mean CPM (counts per minute) values with standard deviation are displayed.</p

Analysis of gene expression using exon expression array and qRT-PCR.

<p>A) Gene expression profiling using exon expression arrays. RNA samples of mock-transfected HeLa cells respectively HeLa cells expressing the WT SLCO5A1 both treated with 1 µg/ml tetracycline for 24 h were collected and analyzed on Affymetrix Exon Arrays. Results of the WT SLCO5A1 sample were compared to the mock sample and expression values of genes with a fold-change of at least 2.0 were analyzed using the GeneSpring® GX 12.0 software. Selected genes were clustered according to their biological function using the GeneSpring Gene Ontology (GO) analysis tool (for complete results see supplemental information – <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083257#pone.0083257.s003" target="_blank">Table S1</a>). A fold-change expression of 30.2-fold was observed for SLCO5A1 (control) (not shown). B) Analysis of GeneChip Human Exon 1.0 ST microarray data by quantitative <i>real-time</i> PCR. The expression of the indicated genes was analyzed after application of mock-transfected HeLa cells and HeLa cells expressing the WT SLCO5A1 with tetracycline for 24 h. The relative expression levels of the WT SLCO5A1 sample were compared to the mock sample ( = 1) and normalized to GUSB (glucuronidase, beta) expression. Mean values with standard deviation of 3 biological replicates are displayed.</p