Differential Effects of STCH and Stress-Inducible Hsp70 on the Stability and Maturation of NKCC2

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.     

Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.     

The invasion of a large lake by the Eurasian genotype of common reed: The influence of roads and residential construction

The Eurasian genotype of common reed (Phragmites australis) is one of the most aggressive plant invading North American wetlands. There is, however, little published evidence on establishment patterns of populations along lakes of the St. Lawrence River–Great Lakes watershed. We tested the hypothesis that the recent invasion of Great Lake Saint-François (Québec, Canada) by common reed was facilitated by a dense road system and by an intense residence construction activity along lakeshores. A total of 345 and 2914 reed stands were mapped along lakeshores, and along the road system of the study area, respectively. The probability of finding a reed stand on a lakeshore increases with the proximity of the lake's outlet, and of a paved road, but decreases with the proximity of a residence built since 1990. It is likely that common reed first spread along the road system, and that wind dispersal of seeds then favored the establishment of populations on lakeshores. Our model does not support the hypothesis that residential construction facilitated the establishment of reed stands, probably because the recent residential construction boom occurred essentially in the southern part of the lake, where the number of roadside reed populations is much lower than in the northern part (lower seed rain). The invasion of Great Lake Saint-François shows that the spread of the plant is not restricted to major river or road systems. Large or small lakes, if submitted to intense diaspore pressure, can also be at risk.     

Constructal design of distributed cooling on the landscape

Here we constructal design to determine the area size (or number of users N) to be allocated to a central refrigeration plant, and to configure the distribution network for the distributed cooling system. The main objective is to maximize the net cooling capacity delivered to every user, and to minimize the pumping power required for transporting the chilled water. Two types of distribution networks are investigated: radial and tree‐shaped networks. First, when ducts with a single diameter are used, the net cooling capacity delivered to every user is almost the same, while the pumping power requirement for the tree network is greater than that for the radial network. Multi‐diameter tree networks are investigated next. The pumping power requirement for multi‐diameter tree‐shaped network is less than for radial network when N is greater than 19 if the flow is laminar, and when N is greater than 80 if the flow is turbulent. The global performance of the area‐based cooling system can be improved by increasing the freedom to morph the configuration. Copyright © 2010 John Wiley & Sons, Ltd.     

The fission yeast spindle orientation checkpoint: a model that generates tension?

In all eukaryotes, the alignment of the mitotic spindle with the axis of cell polarity is essential for accurate chromosome segregation as well as for the establishment of cell fate, and thus morphogenesis, during development. Studies in invertebrates, higher eukaryotes and yeast suggest that astral microtubules interact with the cell cortex to position the spindle. These microtubules are thought to impose pushing or pulling forces on the spindle poles to affect the rotation or movement of the spindle. In the fission yeast model, where cell division is symmetrical, spindle rotation is dependent on the interaction of astral microtubules with the cortical actin cytoskeleton. In these cells, a bub1-dependent mitotic checkpoint, the spindle orientation checkpoint (SOC), is activated when the spindles fail to align with the cell polarity axis. In this paper we review the mechanism that orientates the spindle during mitosis in fission yeast, and discuss the consequences of misorientation on metaphase progression.     

Electrochemical oxidation of hydrophobic compounds in aqueous micellar solutions and oil-in-water emulsions

The electrochemical oxidation of ferrocene (Fc) and tetrathiofulvalene (TTF) has been investigated in micelles and dodecane-in-water emulsions in order to compare the diffusional process involved in these systems. It is found that the diffusion coefficients of Fc and TTF, determined from the observed limiting currents at the rotating disc electrode, are governed by the solubilization equilibrium of the neutral substrates which are predominantly solubilized in the micelle or oil-droplet phase. In the emulsion, the cyclic voltammograms show that the current is controlled also by the rate constants of the substrate partition, which depend on the size of the emulsion droplets. The determination of the diffusion coefficients of Fc⁺ and TTF⁺, produced in situ by controlled potential electrolysis, showed that, except in SDS micellar solutions, the monocation radicals are solubilized mainly in the aqueous phase. As a result, the diffusion coefficients of the species involved in the redox system are different. In the case of TTF, the above results have a great influence upon the second oxidation step TTF⁺ → TTF²⁺. Indeed, the homogeneous solution reaction between TTF and the electrogenerated dication TTF²⁺ leads to the monocation TTF⁺ which diffuses more rapidly than its parent molecule TTF. The ratios of the two waves in cyclic and rotating disc electrode voltammetry show that the enhancement of the second wave depends mainly on the difference between the diffusion coefficients of TTF and TTF⁺ and that the nature of the solution or the size of the disperse phase has little influence upon the reaction rate of the homogeneous solution reaction.     

Priority organic pollutants in the urban water cycle (Toulouse, France)

Application of the European Water Framework Directive requires Member States to have better understanding of the quality of surface waters in order to improve knowledge of priority pollutants. Xenobiotics in urban receiving waters are an emerging concern. This study proposes a screening campaign of nine molecular species of xenobiotics in a separated sewer system. Five sites were investigated over one year in Toulouse (France) using quantitative monitoring. For each sample, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, nonylphenols, diethelhexylphthalate, linear alkylbenzene sulphonates, methyl tert-butylether, total hydrocarbons, estradiol and ethinylestradiol were analysed. Ground, rain and roof collected water concentrations are similar to treated wastewater levels. Run-off water was the most polluted of the five types investigated, discharged into the aquatic environment. The wastewater treatment plant reduced xenobiotic concentrations by 66% before discharge into the environment. Regarding environmental quality standards, observed concentrations in waters were in compliance with standards. The results show that xenobiotic concentrations are variable over time and space in all urban water compartments.     

Improving the transfer of near infrared prediction models by orthogonal methods

Eight calibration transfer methods based on the removal of orthogonal signal were compared for the standardization of whole soybean protein and oil models. Dynamic orthogonal projection (DOP), transfer by orthogonal projection (TOP), error removal by orthogonal subtraction (EROS), orthogonal signal correction (OSC), and orthogonal projections to latent structures (O-PLS) as well as the modification and extension of some of these methods were compared in the transfer of models in intra and inter-brand situations using two Foss Infratecs and two Bruins OmegAnalyzerGs. For each brand, a master was designated and its models transferred onto the second unit of its network and the two units of the second brand. Calibration models were transferable from brand to brand with similar or better precision than when each instrument was calibrated on its own calibration set (for Infratec 1229, the relative predictive determinant (RPD) increased in intra and inter-brand calibration transfer situations from 10.42 to 11.45 and 10.57, with DOP and EROS respectively). Performance of each method was different across parameters, instruments, and validation sets. DOP modifications on the determination of the difference matrix showed promising results while TOP and EROS extensions to include variability specifically present in certain crop years did not bring any beneficial effects.