Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative lowabundance differential clones. Following cloning, a second reverseNorthern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very lowabundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input

ER5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61%sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression

Incorporation of chromaffin granule membranes into large-size vesicles suitable for patch-clamp recording

AbstractIncubation of chromaffin granules with excess liposomes at pH 6.0 resulted in the formation of cell-size structures, which were purified by centrifugation on sucrose gradients. Experiments with fluorescein-labeled granules indicated incorporation of granule membrane to these structures. The preparation contained various vesicular structures with a diameter up to 15 μm. The largest elements were studied by the ‘patch-clamp’ technique. ‘Cell-attached’ and ‘whole-cell’ recordings indicated the presence of currents corresponding to unitary conductances ranging from 100 to 500 pS

Addressing the problem of plastic waste: Development of an enzymatic process for PET recycling

Every day, media and NGOs describe the society\u27s disaffection for plastics accused of polluting the planet. All major brand-owners made commitments to solve this problem (e.g. Coca-Cola, Nestlé, Danone, PepsiCo, Suntory, Unilever, L’Oréal, Nike) and announced a future with less plastic waste by 2025. Nevertheless, only 6 years before the announced term, no effective solution is yet available to meet these goals. Indeed, existing technologies like thermo-mechanical recycling leads to loss in mechanical properties of the polymer and even if several chemical recycling processes are under development, they suffer from the disadvantages of using organic solvents, high reaction temperatures and the need of an intensive waste sorting. Consequently, enzymatic recycling appears as a pertinent solution notably because the enzyme selectivity avoids a drastic sorting of waste and enables the recycling of complex plastics (multi-layers construction in some bottles of sparkling water for instance), it is an eco-friendly reaction in water and because of savings in energy consumption due to a low temperature of reaction. Using a computer-aided engineering strategy, we drastically improved the depolymerizing performance of the best identified enzyme candidate. Utilizing site-directed mutagenesis targeted at the active site, combined with three-dimensional fold stabilization, we engineered an enzyme variant, demonstrating an astounding increase in thermostability combined with a high activity. This enzyme is able to depolymerize 90% of PET waste (200g/kg) into monomers, terephthalic acid and ethylene glycol, in less than 10 hours. The downstream processing was developed and optimized leading to the demonstration that this enzymatic technology could enable the use of an industrial plastic waste to produce again PET monomers and ultimately a bottle from this recycled PET. We hope to demonstrate the strong potential of the enzymatic technology jointly developed by CARBIOS and LISBP to provide a breakthrough solution to help solve society’s growing plastic waste problem

Pet recycling: From enzyme and process optimization to an industrial plant

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Learning Bloch Simulations for MR Fingerprinting by Invertible Neural Networks

Magnetic resonance fingerprinting (MRF) enables fast and multiparametric MR imaging. Despite fast acquisition, the state-of-the-art reconstruction of MRF based on dictionary matching is slow and lacks scalability. To overcome these limitations, neural network (NN) approaches estimating MR parameters from fingerprints have been proposed recently. Here, we revisit NN-based MRF reconstruction to jointly learn the forward process from MR parameters to fingerprints and the backward process from fingerprints to MR parameters by leveraging invertible neural networks (INNs). As a proof-of-concept, we perform various experiments showing the benefit of learning the forward process, i.e., the Bloch simulations, for improved MR parameter estimation. The benefit especially accentuates when MR parameter estimation is difficult due to MR physical restrictions. Therefore, INNs might be a feasible alternative to the current solely backward-based NNs for MRF reconstruction.Comment: Accepted at MICCAI MLMIR 202

Incomplete vesicular docking limits synaptic strength under high release probability conditions

Central mammalian synapses release synaptic vesicles in dedicated structures called docking/release sites. It has been assumed that when voltage-dependent calcium entry is sufficiently large, synaptic output attains a maximum value of one synaptic vesicle per action potential and per site. Here we use deconvolution to count synaptic vesicle output at single sites (mean site number per synapse: 3.6). When increasing calcium entry with tetraethylammonium in 1.5 mM external calcium concentration, we find that synaptic output saturates at 0.22 vesicle per site, not at 1 vesicle per site. Fitting the results with current models of calcium-dependent exocytosis indicates that the 0.22 vesicle limit reflects the probability of docking sites to be occupied by synaptic vesicles at rest, as only docked vesicles can be released. With 3 mM external calcium, the maximum output per site increases to 0.47, indicating an increase in docking site occupancy as a function of external calcium concentration

Influence of Gas Turbulence on the Instability of an Air-Water Mixing Layer

International audienceWe present the first evidence of the direct influence of gas turbulence on the shear instability of a planar air-water mixing layer. We show with two different experiments that increasing the level of velocity fluctuations in the gas phase continuously increases the frequency of the instability, up to a doubling of frequency for the largest turbulence intensity investigated. A modified spatiotemporal stability analysis taking turbulence into account via a simple Reynolds stress closure provides the right trend and magnitude for this effect

Asymmetric hysteresis of N\'eel caps in flux-closure magnetic dots

We investigated with XMCD-PEEM magnetic imaging the magnetization reversal processes of N\'eel caps inside Bloch walls in self-assembled, micron-sized Fe(110) dots with flux-closure magnetic state. In most cases the magnetic-dependent processes are symmetric in field, as expected. However, some dots show pronounced asymmetric behaviors. Micromagnetic simulations suggest that the geometrical features (and their asymmetry) of the dots strongly affect the switching mechanism of the N\'eel caps.Comment: Proceeding for MMM-Intermag 2010 (Washington