Phosphatidylinositol 3-Kinase/AKT pathway regulates the endoplasmic reticulum to Golgi traffic of ceramide in glioma cells : a link between lipid signaling pathways involved in the control of cell survival

Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER

Sphingolipids : key regulators of apoptosis and pivotal players in cancer drug resistance

Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (\u3b1Neu5Ac(2-8)\u3b1Neu5Ac(2-3)\u3b2Gal(1-4)\u3b2Glc(1-1)Cer) or N-glycolyl GM3 (\u3b1Neu5Ac (2-3)\u3b2Gal(1-4)\u3b2Glc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)\u3b2Gal(1-4)\u3b2Glc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy

Aluminum alloy production for the reinforcement of the CMS conductor

The Compact Muon Solenoid (CMS) is one of the general-purpose detectors to be provided for the Large Hadron Collider (LHC) project at CERN. The design field of the CMS superconducting magnet is 4 T, the magnetic length is 12.5 m and the free bore is 6 m. To reinforce the high-purity (99.998%) Al-stabilized conductor of the magnet against the magnetic loadings experienced during operation at 4.2 K, two continuous sections of Al-alloy (AA) reinforcement are Electron Beam (EB) welded to it. The reinforcements have a section of 24*18 mm and are produced in continuous 2.55 km lengths. The alloy EN AW-6082 has been selected for the reinforcement due to its excellent extrudability, high strength in the precipitation hardened states, high toughness and strength at cryogenic temperature and good EB weldability. Each of the continuous lengths of the reinforcement is extruded billet on billet and press quenched on-line from the extrusion temperature in an industrial extrusion plant. In order to insure the ready EB weldability of the reinforcement onto the pure aluminum of the insert, tight dimensional tolerances and proper surface finish of the reinforcement are required in the as-extruded state. As well, in order to facilitate the winding operation of the conductor, the uniformity of the mechanical properties of the extruded reinforcement, especially at the billet on billet joints, is critical. To achieve these requirements in an industrial environment, substantial effort was made to refine existing production techniques and to monitor critical extrusion parameters during production. This paper summarizes the main results obtained during the establishment of the extrusion line and of the production phase of the reinforcement. (10 refs)

The effect of calving in the summer on the hepatic transcriptome of Holstein cows during the peripartal period

The liver is the main metabolic organ coordinating the adaptations that take place during the peripartal period of dairy cows. A successful transition into lactation, rather than management practices alone, depends on environmental factors such as temperature, season of parturition, and photoperiod. Therefore, we analyzed the effect of calving season on the hepatic transcriptome of dairy cows during the transition period. A total of 12 Holstein dairy cows were assigned into 2 groups based on calving season (6 cows March-April, spring; 6 cows June-July, summer, SU). The RNA was extracted from liver samples obtained at -30, 3, and 35 DIM via percutaneous biopsy and hybridized to the Agilent 44K Bovine (V2) Gene Expression Microarray (Agilent Technologies Inc., Santa Clara, CA). A quantitative PCR on 22 target genes was performed to verify and expand the analyses. A total of 4,307 differentially expressed genes were detected (false discovery rate ≤0.05) in SU compared with spring. Furthermore, 73 unique differentially expressed genes were detected in SU compared with spring cows after applying a fold-change threshold ≥3 and ≤-3. For Kyoto Encyclopedia of Genes and Genomes pathways analysis of differentially expressed genes, we used the dynamic impact approach. Ingenuity Pathway Analysis software was used to analyze upstream transcription regulators and perform gene network analysis. Among metabolic pathways, energy metabolism from lipids, carbohydrates, and amino acids was strongly affected by calving in SU, with a reduced level of fatty acid synthesis, oxidation, re-esterification, and synthesis of lipoproteins, leading to hepatic lipidosis. Glycan-synthesis was downregulated in SU cows probably as a mechanism to counteract the progression of this lipidosis. In contrast, calving in the SU resulted in upregulation of gluconeogenesis but also greater use of glucose as an energy source. Among nonmetabolic pathways, the heat-shock response was obviously activated in SU cows but was also associated with inflammatory and intracellular stress response. Furthermore, data support a recent finding that cows experience endoplasmic reticulum stress around parturition. Transcription regulator analysis revealed how metabolic changes are related to important regulatory mechanisms, including epigenetic modification. The holistic analyses of the liver transcriptome response to calving in the summer at high environmental temperatures underscore how transition cows should be carefully managed during this period, as they experience alterations in liver energy metabolism and inflammatory state increasing susceptibility to health disorders in early postpartum

Continuous EB welding of the reinforcement of the CMS conductor

The Compact Muon Solenoid (CMS) is one of the general-purpose detectors to be provided for the LHC project at CERN. The design field of the CMS superconducting magnet is 4 T, the magnetic length is 12.5 m and the free bore is 6 m. In order to withstand the electro-mechanical forces during the operation of the CMS magnet, the superconducting cable embedded in a 99.998% pure aluminum matrix is reinforced with two sections of aluminum alloy EN AW-6082 assembled by continuous Electron Beam Welding (EBW). A dedicated production line has been designed by Techmeta, a leading company in the field of EBW. The production line has a total length of 70 m. Non-stop welding of each of the 20 lengths of 2.5 km, required to build the coil, will last 22 hours. EBW is the most critical process involved in the production line. The main advantage of the EBW process is to minimize the Heat Affected Zone; this is particularly important for avoiding damage to the superconducting cable located only 4.7 mm from the welded joints. Two welding guns of 20 kW each operate in parallel in a vacuum chamber fitted with dynamic airlocks. After welding, the conductor is continuously machined on the four faces and on each corner to obtain the required dimensions and surface finish. Special emphasis has been put on quality monitoring. All significant production parameters are recorded during operation and relevant samples are taken from each produced length for destructive testing purposes. In addition, a continuous phased array ultrasonic checking device is located immediately after the welding unit for the continuous welding quality control, along with a dimension laser measurement unit following the machining. (8 refs)

The CMS conductor

The Compact Muon Solenoid (CMS) is one of the experiments, which are being designed in the framework of the Large Hadron Collider (LHC) project at CERN, the design field of the CMS magnet is 4 T, the magnetic length is 13 m and the aperture is 6 m. This high magnetic field is achieved by means of a 4 layer, 5 modules superconducting coil. The coil is wound from an Al-stabilized Rutherford type conductor. The nominal current of the magnet is 20 kA at 4.5 K. In the CMS coil the structural function is ensured, unlike in other existing Al-stabilized thin solenoids, both by the Al-alloy reinforced conductor and the external former. In this paper the retained manufacturing process of the 50-km long reinforced conductor is described. In general the Rutherford type cable is surrounded by high purity aluminium in a continuous co-extrusion process to produce the Insert. Thereafter the reinforcement is joined by Electron Beam Welding to the pure Al of the insert, before being machined to the final dimensions. During the manufacture the bond quality between the Rutherford cable and the high purity aluminium as well as the quality of the EB welding are continuously controlled by a novel ultrasonic phased array system. The dimensions of the insert and the final conductor are measured by laser micrometer. (8 refs)

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

The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells

Plants have evolved different strategies to resist drought, of which the best understood is the abscisic acid (ABA)-induced closure of stomatal pores to reduce water loss by transpiration. The availability of useful promoters that allow for precise spatial and temporal control of gene expression in stomata is essential both for investigating stomatal regulation in model systems and for biotechnological applications in field crops. Previous work indicated that the regulatory region of the transcription factor AtMYB60 specifically drives gene expression in guard cells of Arabidopsis, although its activity is rapidly down-regulated by ABA. Here, the activity of the full-length and minimal AtMYB60 promoters is reported in rice (Oryza sativa), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum), using a reporter gene approach. In rice, the activity of both promoters was completely abolished, whereas it was spatially restricted to guard cells in tobacco and tomato. To overcome the negative effect of ABA on the AtMYB60 promoter, a chimeric inducible system was developed, which combined the cellular specificity of the AtMYB60 minimal promoter with the positive responsiveness to dehydration and ABA of the rd29A promoter. Remarkably, the synthetic module specifically up-regulated gene expression in guard cells of Arabidopsis, tobacco, and tomato in response to dehydration or ABA. The comparative analysis of different native and synthetic regulatory modules derived from the AtMYB60 promoter offers new insights into the functional conservation of the cis-mechanisms that mediate gene expression in guard cells in distantly related dicotyledonous species and provides novel tools for modulating stomatal activity in plants.Fabio Rusconi, Fabio Simeoni, Priscilla Francia, Eleonora Cominelli, Lucio Conti, Matteo Riboni, Laura Simoni, Cathie R. Martin, Chiara Tonelli, and Massimo Galbiat

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G\u2009>\u2009C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3'-phosphate, 5'-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here

Prognostic value of preoperative von Willebrand factor plasma levels in patients with Glioblastoma

Circulating biomarker for malignant gliomas could improve both differential diagnosis and clinical management of brain tumor patients. Among all gliomas, glioblastoma (GBM) is considered the most hypervascularized tumor with activation of multiple proangiogenic signaling pathways that enhance tumor growth. To investigate whether preoperative antigen plasma level of von Willebrand Factor (VWF:Ag) might be possible marker for GBM onset, progression, and prognosis, we retrospectively examined 57 patients with histological diagnosis for GBM and 23 meningiomas (MNGs), benign intracranial expansive lesions, enrolled as controls. Blood samples were collected from all the patients before tumor resection. Plasma von Willebrand Factor (VWF):Ag levels were determined by using a latex particle-enhanced immunoturbidimetric assay. The median levels of vWF:Ag were significantly higher in GBMs than in meningiomas (MNGs) (183 vs. 133IU/dL, P=0.01). The cumulative 1-year survival was significantly shorter in patients with VWF:Ag levels 200IU/dL than in those with levels <200IU/dL and increased VWF levels were associated with a threefold higher risk of death in GBM patients. Our data suggest that VWF:Ag could be a circulating biomarker of disease malignancy, that could be considered, in association with other genetic and epigenetic factors, currently available in the GBM management. Future studies should investigate whether plasma VWF:Ag levels could also be used to monitor therapeutic effects and whether it may have a prognostic value