Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects

Strategies for Controlled Placement of Nanoscale Building Blocks

The capability of placing individual nanoscale building blocks on exact substrate locations in a controlled manner is one of the key requirements to realize future electronic, optical, and magnetic devices and sensors that are composed of such blocks. This article reviews some important advances in the strategies for controlled placement of nanoscale building blocks. In particular, we will overview template assisted placement that utilizes physical, molecular, or electrostatic templates, DNA-programmed assembly, placement using dielectrophoresis, approaches for non-close-packed assembly of spherical particles, and recent development of focused placement schemes including electrostatic funneling, focused placement via molecular gradient patterns, electrodynamic focusing of charged aerosols, and others

A new model for surface potential decay of corona-charged polymers

Surface potential measurement provides a useful tool to gauge the electrical properties of materials. It has been observed that the potential of a sample with an initial high surface potential decays faster than that with an initial lower surface potential, known as the cross-over phenomenon. The phenomenon was found a few decades ago and various theories and models have been proposed. A common feature of the existing models is based on single charge carrier injection from a corona-charged surface. With our recent space charge measurement results on corona-charged samples, double injection from both electrodes has been verified. Based on this new fact, a new model based on bipolar charge injection is proposed and initial numerical simulation reveals that the surface potential cross-over phenomenon can occur under bipolar charge injection

Determination of the evolution of the surface potential of a charging insulator by measuring the intensity of its X-ray characteristic peaks

The charge developed in an insulator by an electron beam has often been studied by following the evolution of the secondary electron emission of the target. However this latter proves to be insufficient when one attempts to correlate it with physical properties of the sample such as its density of traps and their spatial distribution. An important step would be to have access to the time evolution of the surface potential Vs(t). However, this latter quantity is not easy to measure. In this paper, we propose a new method to measure the evolution of the surface potential by relating it to the X-ray emission induced by the electronic bombardment. More exactly, we show that there is a relation between the intensity of the characteristic peaks and the potential existing under the beam impact at a given instant of the charge. After having explained the method, we check its validity both experimentally and also by comparison with Monte Carlo simulations. We then present initial results obtained for amorphous SiO2 targets

Monte Carlo simulation of the secondary electron yield of an insulating target bombarded by a defocused primary electron beam

International audienceWe study the charge of an insulating target irradiated by a broad electron beam of a few keV with our Monte Carlo simulation model. We are particularly interested in the dynamics which leads the system towards a stationary state. We examine successively the role of parameters such as the density of current in the primary beam, the density of traps, their activation energy. According to the situation considered, one observes that the regulation of the system can sometimes be stopped, either because the traps become saturated, or, in the case of thin targets, by the appearance of a leakage current towards the ground, due to carriers released from the traps

Interpretation method for mirror experiments based on a Monte Carlo charge implantation model

The scanning electron microscope mirror (SEMM) method is considered as a suitable approach for the characterisation of the charge trapping ability of insulators. However, the interpretation of the experimental results is far from being obvious. The aim of this paper is to give some new insights in this problem. Our description of the mirror plots is conventionally based on a multipole analysis of the charge distribution and on the radius of curvature approximation. This approach indicates that the trend of the plots is mainly controlled by the quadrupole contribution. This clearly illustrates the non- uniqueness of the interpretation since different trapped charge distributions, but with nearly equal quadrupole terms, can produce rather similar mirror plots. To make the problem unambiguous, a semi-ellipsoidal shell model is used to account for the implanted space charge. Within this model, we propose a simple method to get the mean density of trapped charge ρ0. Our Monte Carlo simulations of the charge implantation phase show that ρ0 can strongly differ from the density of traps NT. The reasons for these deviations are discussed for a significant set of values of NT and of the primary energy Ep

Influence on the secondary electron yield of the space charge induced in an insulating target by an electron beam

International audienceThe building up of the space charge induced by electron bombardment in an insulating target is due to the stabilization of self-trapped electrons and holes in polaronic traps. For the energies considered, the target charges positively and the secondary electrons emitted at low energies can be attracted back to the surface. This results in a self-regulation effect where the total secondary yield tends to unity and the surface potential stabilizes at a low positive value. This conclusion is checked for various experimental conditions. The electrons landing on the target form a ring of negative charges that progressively spread out on the surface of the sample

Etude des mouvements de parois de domaines sur BaSrTiO3 dans la gamme des frequencies microondes

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