Tpk3 and Snf1 protein kinases regulate Rgt1 association with Saccharomyces cerevisiae HXK2 promoter

Hexokinase 2 is an essential factor for signalling repression through the Saccharomyces cerevisiae high-glucose sensing pathway. The main regulatory mechanism that controls the HXK2 gene expression in yeast is mediated by the Rgt1 and Med8 transcription factors, which repress HXK2 expression in low-glucose containing media. In this study, we show that the repression activity of Rgt1 is regulated by Snf1 and Tpk3 protein kinases. Binding of Rgt1 to the HXK2 promoter requires Rgt1 phosphorylation by Snf1 or by an Snf1-dependent protein kinase. Conversely, Rgt1 hyperphosphorylation by the Tpk3 or by a Tpk3-dependent protein kinase dissociates Rgt1 from the repressor complex. Two-hybrid and chromatin immunoprecipitation experiments indicate that an Snf1-dependent interaction between Rgt1 and Med8 in the repressor complex is also essential for Rgt1 repression. The repression of HXK2 transcription by Rgt1 likely occurs through the formation of a DNA loop in the HXK2 locus, spanning the promoter and coding regions. These results suggest that a novel silent-chromatin loop is responsible for Rgt1-dependent transcriptional regulation of the HXK2 gene

Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level

[EN] Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force¿extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour- length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2011-29038 and BFU2010-15703) and the Comunidad de Madrid (S2009/MAT/1507). IRA.-G. acknowledges a Ramon y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2007-01765). Work in the F.M.-H. laboratory was supported by a Starting Grant from the European Research Council (no. 206117) and a grant from the Spanish Ministry of Science and Innovation (FIS2011-24638). We thank M. S. Dillingham for kindly providing the pSP73-JY0 plasmid, M. Menendez for access to a spectropolarimeter, A. Monserrate for polylysine-AFM control experiments, and B. Ibarra for fruitful discussions.Herrero-Galán, E.; Fuentes-Perez. M.E.; Carrasco, C.; Valpuesta, J.; Carrascosa, J.; Moreno-Herrero, F.; Arias-Gonzalez, JR. (2013). Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level. Journal of the American Chemical Society. 135(1):122-131. https://doi.org/10.1021/ja3054755S122131135

Determination of a lectin protein allergen in food by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An aptamer affinity sorbent was prepared for clean-up, preconcentration, separation and characterization of a food allergen protein by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). SPE microcartridges were packed with a sorbent based on magnetic bead particles modified with an aptamer against the target lectin protein concanavalin A (Con A). After optimization of several parameters of the SPE-CE-MS method, the sample (ca. 30 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while the retained protein was eluted with 100 mM NH4OH (pH 11.2) (ca. 100 nL). The developed method was linear between 0.5 and 20 mg·L-1 and the limit of detection (LOD) was 0.25 mg·L-1, which was 100 times lower than by CE-MS. The repeatability of the method was satisfactory, with relative standard deviations (RSD) for migration times and peak areas below 1.9 and 8.1%, respectively. In addition, the microcartridges could be reused more than 25 analyses without significant loss of extraction efficiency. Finally, the applicability and versatility of the developed method were demonstrated by analyzing low levels of Con A in different food matrices (i.e. white beans, as well as chickpea, lentils, and wheat flours), leading to satisfactory results, with recoveries between 87 and 115%

Accurate determination of the milk protein allergen β-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 µg·mL-1 and the limit of detection (LOD) was 0.05 µg·mL-1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (< 0.5 % and 2.4 %, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100 % cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control

Absentismo laboral en trabajadores públicos de Mallorca

Objetivo. Valorar los procesos de absentismo en una empresa pública en el periodo 1991-2008 y la influencia de algunas variables socio demográficas. Material y método. Estudio descriptivo transversal un total de 10154 procesos de absentismo (incapacidad temporal, accidente de trabajo e indisposición) en trabajadores públicos. Se analiza la influencia de la edad y el sexo. Resultados. Se estudian un total de 6542 episodios de incapacidad temporal, 2297 indisposiciones y 615 accidentes laborales. El número total de días perdidos es de 302147. Las mujeres y los trabajadores mayores de 45 años son los grupos que presentan mayor número de procesos de absentismo y los que pierden mayor número de días. Conclusiones. El presente trabajo es uno de los primeros que presenta datos de indisposiciones, un aspecto del absentismo que muy pocas veces se ha analizado.Objective. Assess the processes of absenteeism in a public company between 1991-2008 and the influence of some socio demographic variables. Methods. Cross-sectional study of 10154 absences processes (temporary disability, industrial accident and mild ailments) in public workers. The influence of age and sex is analyzed. Results. 6542 episodes of temporary disability, 2297 mild ailments and 615 industrial accidents were studied. The total number of “lost days” is 302147. Women and workers over 45 are the groups with the highest number of absenteeism processes and more “lost days”. Conclusions. This work is one of the first to present data of mild ailments, one aspect of absenteeism rarely been analyzed

Effect of soiling in CPV systems

The effect of soiling in flat PV modules has been already studied, causing a reduction of the electrical output of 4% on average. For CPV's, as far as soiling produces light scattering at the optical collector surface, the scattered rays should be definitively lost because they cannot be focused onto the receivers again. While the theoretical study becomes difficult because soiling is variable at different sites, it becomes easier to begin the monitoring of the real field performance of concentrators and then raise the following question: how much does the soiling affect to PV concentrators in comparison with flat panels?? The answers allow to predict the PV concentrator electrical performance and to establish a pattern of cleaning frequency. Some experiments have been conducted at the IES-UPM and CSES-ANU sites, consisting in linear reflective concentration systems, a point focus refractive concentrator and a flat module. All the systems have been measured when soiled and then after cleaning, achieving different increases of ISC. In general, results show that CPV systems are more sensitive to soiling than flat panels, accumulating losses in ISC of about 14% on average in three different tests conducted at IESUPM and CSES-ANU test sites in Madrid (Spain) and Canberra (Australia). Some concentrators can reach losses up to 26% when the system is soiled for 4 months of exposure

Proses Pengolahan dan Kualitas Air Minum di Kompleks Perumahan Karyawan PT. Chevron Pacific Indonesia (PT. CPI) Duri-Riau Tahun 2014

Water is one of the important matter after the air, nobody can life without drinking until 4-5 days. Drinking water which is not qualified, can resulting disease or even death. Water treatment can prevents disease and resulting qualified drinking water. PT. Chevron Pacific Indonesia (PT. CPI) is one of the companies which has a water treating plant to makes drinking water from raw water The purpose of this research is to know the process of water treatment, the quality of raw water after treatment and quality of distributed drinking water to employees housing PT. Chevron Pacific Indonesia Duri. This research was descriptive survey using simple random sampling technique with 1102 populations and 30 samples. Data were analyzed descriptively in tabulation and narration. The result of the research showed that the water treatment was using coagulation, floculation, sedimentation and desinfectan process. Based on the analysis of obtaained data, the quality of raw water was qualified, whereas the quality of water which was finish the treatment and the quality of distributed drinking water to employees housing on Maret 2014 were not qualified in one parameter. Most of the respondents have been using water as drinking water. The conclusion of this research is the water treatmen process in PT. Chevron Pacific Indonesia was conventional water treatment (complete water treatment). Overall water quality is good, it is recommended to improve adsorption using activated carbon, renew and re-examine the tools used and provision of information about water quality to water users

Force determination in lateral magnetic tweezers combined with TIRF microscopy

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA:protein interactions. In particular, combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of the flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.We thank the financial support from the Spanish MINECO (FIS2014-58328-P) and from (ERC) under the European Union’s Horizon2020 Research and Innovation programme (grant agreement No 681299). J. M. M. acknowledges a Predoctoral PhD fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174). ). G. L. M. F was supported by the Biotechnology and Biological Sciences Research Council (1363883). M. S. D was supported by the Wellcome Trust (100401 and 077368).Peer reviewe