252 research outputs found

    Smuggler’s Paradise

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    Tertium Datur

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    Nueva estrategia para el diseño y fabricación de cabezales de fresadora de altas prestaciones

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    Los cabezales de transmisión mecánica de altas prestaciones son una de las partes más complejas, costosas y problemáticas de una maquina fresadora. Esto es debido a la gran cantidad de canalizaciones para la transmisión de fluidos que deben de contener y a las altas prestaciones mecánicas que se les exigen. En este trabajo se presenta una estrategia para optimizar el diseño y la fabricación de los cuerpos de estos cabezales basada en la incorporación de aleaciones de aluminio y la inclusión de insertos tubulares de acero en la propia fundición que conforma el cabezal. Los insertos tubulares sustituyen a los canales de refrigeración realizados actualmente por taladros de mecanizado y quedan integrados en la masa de aluminio. La nueva metodología ha permitido realizar un diseño que mantiene las prestaciones mecánicas, aumenta la fiabilidad y reduce la masa de estos elementos móviles de fresadoras como ha demostrado la validación experimental de un primer prototipo.High-performance mechanical-transmission heads are one of the most complex, costly and problematic parts of a milling machine, owing to the large amount of piping required for transporting fluids and to the high level of mechanical performance that is required from them. This study proposes a strategy for optimising the design and manufacture of head bodies by using aluminium alloys and by integrating tubular stainless steel inserts in the casting of the head. These tubular inserts that are integrated into the aluminium mass are an alternative to cooling conduits currently made by machine drilling. As demonstrated in the experimental validation of the first prototype, the new method has created a design that retains the same mechanical performance, increases its reliability and reduces the weight of the milling machine’s moving parts.Spanish Ministry of Science and Innovation in the PROFIT Project FIT-020500-2006-50 and by the Agencia de Desarrollo Economico of the Junta de Castilla-Leon (04/06/BU/0008)

    Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: A case study with the metastatic breast cancer cell line MDA-MB-231

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    Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (−50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism

    The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

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    Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris

    Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering

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    In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes
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