The Study of a 231 French Patient Cohort Significantly Extends the Mutational Spectrum of the Two Major Usher Genes MYO7A and USH2A

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing loss combined with retinitis pigmentosa, and in some cases, vestibular areflexia. Three clinical subtypes are distinguished, and MYO7A and USH2A represent the two major causal genes involved in Usher type I, the most severe form, and type II, the most frequent form, respectively. Massively parallel sequencing was performed on a cohort of patients in the context of a molecular diagnosis to confirm clinical suspicion of Usher syndrome. We report here 231 pathogenic MYO7A and USH2A genotypes identified in 73 Usher type I and 158 Usher type II patients. Furthermore, we present the ACMG classification of the variants, which comprise all types. Among them, 68 have not been previously reported in the literature, including 12 missense and 16 splice variants. We also report a new deep intronic variant in USH2A. Despite the important number of molecular studies published on these two genes, we show that during the course of routine genetic diagnosis, undescribed variants continue to be identified at a high rate. This is particularly pertinent in the current era, where therapeutic strategies based on DNA or RNA technologies are being developed.     

Salt-Sensitive Hypertension in GR+/− Rats Is Accompanied with Dysregulation in Adrenal Soluble Epoxide Hydrolase and Polyunsaturated Fatty Acid Pathways

Mutations within the glucocorticoid receptor (GR) gene locus lead to glucocorticoid resistance which is characterized by several clinical symptoms such as adrenal gland hyperplasia and salt-sensitive hypertension, although the underlying mechanisms are still unknown. We studied GR haploinsufficient (GR^+/−) Sprague Dawley rats which, on a standard diet, showed significantly increased plasma aldosterone and corticosterone levels and an adrenocortex hyperplasia accompanied by a normal systolic blood pressure. Following a high salt diet, these rats developed salt-sensitive hypertension and maintained elevated enzyme-soluble epoxide hydrolase (sEH) in adrenal glands, while sEH was significantly decreased in wild-type rats. Furthermore, GR^+/− rats showed dysregulation of the equilibrated linoleic and arachidonic acid pathways, with a significant increase of less active metabolites such as 8,9-DiHETrE. In Sprague Dawley rats, GR haploinsufficiency induced steroid disturbances, which provoked hypertension only in combination with high salt intake, which was accompanied by disturbances in sEH and fatty acid metabolism. Our results suggest that sEH inhibition could be a potential target to treat hypertension in patients with GR haploinsufficiency.     

Discovery of novel non-cyclam polynitrogenated CXCR4 coreceptor inhibitors

HIV cell fusion and entry have been validated as targets for therapeutic intervention against infection. Bicyclams were the first low-molecular-weight compounds to show specific interaction with CXCR4. The most potent bicyclam was AMD3100, in which the two cyclam moieties are tethered by a 1,4-phenylenebis(methylene) bridge. It was withdrawn from clinical trials owing to its lack of oral bioavailability and cardiotoxicity. We have designed a combinatorial library of non-cyclam polynitrogenated compounds by preserving the main features of AMD3100. At least two nitrogen atoms on each side of the p-phenylene moiety, one in the benzylic position and the other(s) in the heterocyclic system were maintained, and the distances between them were similar to the nitrogen atom distances in cyclam. A selection of diverse compounds from this library were prepared, and their in vitro activity was tested in cell cultures against HIV strains. This led to the identification of novel potent CXCR4 coreceptor inhibitors without cytotoxicity at the tested concentrations.     

Influence of the co-firing on the leaching of trace pollutants from coal fly ash

The (co)-firing of low-cost alternative fuels is expected to increase in the forthcoming years in the EU because of the economic and environmental benefits provided by this technology. This study deals with the impact of the different coal/waste fuel ratio of the feed blend on the mineralogy, the chemical composition and especially on the leaching properties of fly ash. Different blends of coal, petroleum coke, sewage sludge, wood pellets, coal tailings and other minor biomass fuels were tested in PCC (pulverised coal combustion) and FBC (fluidized bed combustion) power plants. The co-firing of the studied blends did not drastically modify the mineralogy, bulk composition or the overall leaching of the fly ash obtained. This suggests that the co-firing process using the alternative fuels studied does not entail significant limitations in the re-use or management strategies of fly ash.     

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and itscomplexes with various species-variant and mutant lysozymeshave been the subject of considerable experimental and theoreticalinvestigation. The affinity of HyHEL-5 for bobwhite quail lysozyme(BWQL) is over 1000-fold lower than its affinity for the originalantigen, hen egg lysozyme (HEL). This difference is believedto arise almost entirely from the replacement in BWQL of thestructural and energetic epitope residue Arg68 by lysine. Inthis study, the association and dissociation kinetics of BWQLwith HyHEL-5 were investigated under a variety of conditionsand compared with previous results for HEL. HyHEL-5–BWQLassociation follows a bimolecular mechanism and the dissociationof the antibody–antigen complex is a first-order process.Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0to 10.0) produced about a 2-fold change in the association anddissociation rates. The effect of viscosity modifiers on theassociation reaction was also studied. The large differencein the HEL and BWQL affinities for HyHEL-5 is essentially dueto differences in the dissociation rate constant.     

Characterization of forced oxidation of sardine oil: Physicochemical data and mathematical modeling

It is of major interest to the food industry to understand the mechanisms and kinetics underlying spontaneous oxidation of marine oils because these polyunsaturated fatty acid (PUFA)-rich oils, the object of several health claims, have been repeatedly recommended for dietary intake. The present study attempts to characterize forced oxidation and hydrolytic breakdown of glycerides and fatty acids in sardine oil. A simple, first-order mathematical model was postulated and successfully fitted to the experimental data. This model confirmed that the rate of decrease in concentration of intact fatty acid moieties is almost directly proportional to the number of double bonds present. Therefore, as expected, the rate of oxidative decay was virtually independent of chain length, with an overall activation energy of ca. 22 kJ mol⁻¹. Additionally, the rate of hydrolysis was correlated with the rate of oxidative decay. With the exception of fatty acids possessing more than four double bonds, PUFA proved to be relatively stable to oxidation for up to 10 h at 50–70°C, and the qualitatively richest pattern of volatiles was obtained when the reaction was performed at the highest temperature (80°C).     

Electron‐beam processing of destructurized allylurea–starch blends: Immobilization of plasticizer by grafting

Allylurea (AU) was used as a reactive additive with poor aptitude to homopolymerization for obtaining grafted plasticized starch films with stabilized physical properties. Potato starch was mixed with AU (30–50 parts per hundred/pph) in a mixer operating at 125°C. Upon storage in well‐defined hygrothermal conditions, the resulting thermoplastic material shows strong plasticizer migration revealed by AU crystals blooming at the samples surface and exhibits strong opacity assigned to phase separation of the organic additive inside the material. Freshly prepared thermoplastic films of appropriate thickness were exposed to a 175‐kV electron beam (EB) radiation for inducing covalent grafting of AU by a free radical process. FTIR monitoring of the resulting chemical changes in thin films of AU–starch blends indicates unambiguously the transformation of AU allylic bond. High irradiation doses are required for achieving complete conversion of AU in the blend. However, no detectable AU migration was observed for intermediate AU conversion, probably as a consequence of higher plasticizer solubility in the grafted polysaccharide. Examination of the viscoelastic properties by dynamic mechanical thermal analysis shows that artificial aging by placing the films alternatively in high and low relative humidity (RH) atmosphere does not significantly alter the thermomechanical spectrum of the material reconditioned in a cell at 58% RH. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 409–417, 1999     

Microwave analysis of PACVD diamond deposition reactor based on electromagnetic modelling

The production of high quality diamond films by microwave plasma assisted CVD, with rapid growth rates and good uniformity over large surfaces, requires perfectly optimized reactors from the microwave design point of view. Most MW plasma assisted CVD reactors used for diamond film deposition work on the resonant cavity principle. The design of such reactors relies on 3 choices: i) choice of a suitable resonant mode (i.e. with an electric field structure conducive to plasma ignition), ii) choice of a MW coupling system to excite the cavity, and iii) choice of a quartz window to delimit a reduced pressure zone inside the cavity, so as to obtain the plasma in front of the substrate [1].In this paper, we present an analysis method for MW plasma reactors relying on EM modelling, which allows for the identification of a resonant mode responsible for plasma ignition, applied to an existing reactor exhibiting plasma instabilities and requiring constant supervision.This analysis method, which can be generalised to any resonant cavity reactor, can describe the device behaviour (shape and location of the plasma, occurrence of instabilities) as a function of the various cavity geometrical configurations and to get a first estimate of the process performance.On the basis of such an analysis, it was possible to propose modifications to the reactor considered in order to improve process stability, and obtain higher growth rates. First growth tests done on diamond mono- and nano-crystalline films show excellent material quality and an increase in growth rate by more than an order of magnitude.     

Neutral lipids in non‐starch lipid and starch lipid extracts from Portuguese sourdough bread

This research effort was aimed at assessing the changes in extractable neutral lipids (NL) throughout the baking process of Broa, a Portuguese traditional sourdough bread. NL were accordingly isolated, purified and quantitated – starting from non‐starch lipid (NSL) and starch lipid (SL) extracts of maize and rye flours, as well as fermented dough and bread. NSL accounted for the major fraction of extracted lipids; furthermore, the NSL/SL ratio evolved throughout processing in agreement with the phenomena prevailing during dough preparation, fermentation, and baking. An analytical method based on resolution by normal‐phase HPLC coupled with detection by evaporative light scattering was accordingly developed for quantitation of the aforementioned NL classes. Distinct NL classes correlated well with the stage of bread making. The main NL in NSL were triacylglycerols (ca. 75% of the total), but relatively high concentrations of sterol esters and diacylglycerols were also found. Conversely, free fatty acids were the dominant component of SL, whereas monoacylglycerols and free sterols were comparable to those in NSL.     

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K_D=42 and 84 μM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K_D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.