In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell‐specific quantification of protein abundance changes, we sought to apply an enzyme‐activated cellular fluorescence system in vivo by delivering ester‐masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES‐1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.     

Synthesis and in Vitro Characterisation of Ifenprodil‐Based Fluorescein Conjugates as GluN1/GluN2B N‐Methyl‐D‐aspartate Receptor Antagonists

GluN2B‐containing NMDA receptors are involved in many important physiological functions and play a pivotal role in mediating pain as well as in several neurodegenerative disorders. We aimed to develop fluorescent probes to target the GluN2B subunit selectively in order to allow better understanding of the relationships between receptor localisation and physiological importance. Ifenprodil, known as the GluNR2B antagonist of reference, was chosen as the template for the elaboration of probes. We had previously reported a fluorescein conjugate that was shown (by confocal microscopy imaging of DS‐red‐labelled cortical neurons) to bind specifically to GluN2B. To elaborate this probe, we explored the influence of both the nature and the attachment point of the spacer between the fluorophore and the parent compound, ifenprodil. We performed chemical modifications of ifenprodil at the benzylic position and on the phenol ring by introducing secondary amine or amide functions and evaluated alkyl chains from two to 20 bonds either including or not including secondary amide functions as spacers. The previously developed probe was found to display the greatest activity in the inhibition of NMDA‐induced Ca²⁺ influx by calcium imaging experiments on HEK293 cells transfected with the cDNA encoding for GluN1‐1A and GluN2B. Further investigations revealed that this probe had a neuroprotective effect equivalent to that of ifenprodil in a standard test for neurotoxicity. Despite effects of lesser amplitude with these probes relative to ifenprodil, we demonstrated that they displaced [³H]ifenprodil in mouse brain slices in a similar manner.     

Linear viscoelastic behavior of compatibilized PMMA/PP blends

In this work, the morphology and linear viscoelastic behavior of PMMA/PP blends to which a graft copolymer PP‐g‐PMMA has been added was studied. The copolymer concentration varied from 1 to 10 wt % relative to the dispersed phase concentration. The rheological data were used to infer the interfacial tension between the blended components. It was observed that PP‐g‐PMMA was effective as a compatibilizer for PMMA/PP blends. For PP‐g‐PMMA concentration added below the critical concentration of interface saturation, two rheological behaviors were observed depending on the blend concentration: for 70/30 blend, the storage modulus, at low frequencies, increased as compared to the one of the unmodified blend; for 90/10 blend, it decreased. For 90/10 blend, the relaxation spectrum presented an interfacial relaxation time related to the presence of the compatibilizer (τ_β). For PP‐g‐PMMA concentrations added above the critical concentration of interface saturation, the storage modulus of all blends increased as compared with the one of the unmodified blend. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Heterodera schachtii Nematodes Interfere with Aphid-Plant Relations on Brassica oleracea

Aboveground and belowground herbivore species modify plant defense responses differently. Simultaneous attack can lead to non-additive effects on primary and secondary metabolite composition in roots and shoots. We previously found that aphid (Brevicoryne brassicae) population growth on Brassica oleracea was reduced on plants that were infested with nematodes (Heterodera schachtii) prior (4 weeks) to aphid infestation. Here, we examined how infection with root-feeding nematodes affected primary and secondary metabolites in the host plant and whether this could explain the increase in aphid doubling time from 3.8 to 6.7 days. We hypothesized that the effects of herbivores on plant metabolites would depend on the presence of the other herbivore and that nematode-induced changes in primary metabolites would correlate with reduced aphid performance. Total glucosinolate concentration in the leaves was not affected by nematode presence, but the composition of glucosinolates shifted, as gluconapin concentrations were reduced, while gluconapoleiferin concentrations increased in plants exposed to nematodes. Aphid presence increased 4-methoxyglucobrassicin concentrations in leaves, which correlated positively with the number of aphids per plant. Nematodes decreased amino acid and sugar concentrations in the phloem. Aphid population doubling time correlated negatively with amino acids and glucosinolate levels in leaves, whereas these correlations were non-significant when nematodes were present. In conclusion, the effects of an herbivore on plant metabolites were independent of the presence of another herbivore. Nematode presence reduced aphid population growth and disturbed feeding relations between plants and aphids.     

Effect of GABA,a Bacterial Metabolite,on Pseudomonas fluorescens Surface Properties and Cytotoxicity

Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10⁻⁵ M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABA_A receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains.     

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.     

Conversion of Nuclear Waste to Molten Glass: Cold‐Cap Reactions in Crucible Tests

The feed‐to‐glass conversion, which comprises complex chemical reactions and phase transitions, occurs in the cold cap during nuclear waste vitrification. To investigate the conversion process, we analyzed heat‐treated samples of a simulated high‐level waste feed using X‐ray diffraction, electron probe microanalysis, leaching tests, and residual anion analysis. Feed dehydration, gas evolution, and borate phase formation occurred at temperatures below 700°C before the emerging glass‐forming melt was completely connected. Above 700°C, intermediate aluminosilicate phases and quartz particles gradually dissolved in the continuous borosilicate melt, which expanded with transient foam. Knowledge of the chemistry and physics of feed‐to‐glass conversion will help us control the conversion path by changing the melter feed makeup to maximize the glass production rate.     

Influence of granulometry and organic treatment of a Brazilian montmorillonite on the properties of poly(styrene‐co‐n‐butyl acrylate)/layered silicate nanocomposites prepared by miniemulsion polymerization

The influence of granulometry and organic treatment of a Brazilian montmorillonite (MMT) clay on the synthesis and properties of poly(styrene‐co‐n‐butyl acrylate)/layered silicate nanocomposites was studied. Hybrid latexes of poly(styrene‐co‐butyl acrylate)/MMT were synthesized via miniemulsion polymerization using either sodium or organically modified MMT. Five clay granulometries ranging from clay particles smaller than 75 μm to colloidal size were selected. The size of the clay particles was evaluated by specific surface area measurements (BET). Cetyl trimethyl ammonium chloride was used as an organic modifier to enhance the clay compatibility with the monomer phase before polymerization and to improve the clay distribution and dispersion within the polymeric matrix after polymerization. The sodium and organically modified natural clays as well as the composites were characterized by X‐ray diffraction analysis. The latexes were characterized by dynamic light scattering. The mechanical, thermal, and rheological properties of the composites obtained were characterized by dynamical‐mechanical analysis, thermogravimetry, and small amplitude oscillatory shear tests, respectively. The results showed that smaller the size of the organically modified MMT, the higher the degree of exfoliation of nanoplatelets. Hybrid latexes in presence of Na‐MMT resulted in materials with intercalated structures. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009