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.     

Yeast ecology in French cider and black olive natural fermentations

In this study, rDNA ITS restriction analysis was used to identify yeasts from two naturally fermented products: French ciders and black olives. In cider musts and bottled ciders, the PCR-RFLP method generated 15 different ITS/RFLP profiles for a total of 208 isolates. The predominant yeasts corresponded to Saccharomyces bayanus, Saccharomyces cerevisiae, Lachancea cidri and Dekkera anomala. Three identified species: Candida sake, Candida tropicalis and Kluyveromyces marxianus had never been described before in ciders. For the black olive fermentation, the method allowed for identification of 11 profiles for a total of 137 isolates. A sequential apparition of yeasts was observed with Pichia anomala, Candida boidinii and Debaryomyces etchellsii being the predominant species. Four isolates did not correspond to any known species based on the sequencing of the D1/D2 region of the 26S rRNA gene. By using the rDNA ITS method, valuable information on yeast population biodiversity and dynamics in the naturally fermented food products studied was obtained.     

Prion degradation in soil: possible role of microbial enzymes stimulated by the decomposition of buried carcasses

This study is part of a European project focused on understanding the biotic and abiotic mechanisms involved in the retention and dissemination of transmissible spongiform encephalopathies (TSE) infectivity in soil in order to propose practical recommendations to limit environmental contamination. A 1-year field experiment was conducted with lamb carcasses buried in a pasture soil at three depths (25, 45, and 105 cm). Microbial community response to carcasses was monitored through the potential proteolytic activity and substrate induced respiration (SIR). Soil above carcasses and control soil exhibited low proteolytic capacity, whatever the depth of burial. Contrastingly, in soil beneath the carcasses, proteolysis was stimulated. Decomposing carcasses also stimulated SIR, i.e., microbial biomass, suggesting that proteolytic populations specifically developed on lixiviates from animal tissues. Decomposition of soft tissues occurred within 2 months at subsurface while it lasted at least 1 year at deeper depth where proteolytic activities were season-dependent. The ability of soil proteases to degrade the beta form of prion protein was shown in vitro and conditions of burial relevant to minimize the risk of prion protein dissemination are discussed.     

The effect of the temporal structure of spoken words on paired-associate learning.

In a series of experiments, participants learned to associate black-and-white shapes with nonsense spoken labels (e.g., “joop”). When tested on their recognition memory, participants falsely recognized as correct a shape paired with a label that began with the same sounds as the shape’s original label (onset-overlapping lure; e.g., joob) more often than a shape paired with a label that overlapped with the original label at offset (offset-overlapping lure; e.g., choop). Furthermore, the false-alarm rate was modulated by the phonetic distance between the sounds that distinguished the original label and the lures. Greater false-alarm rates to onset-overlapping labels were not predicted by explicit similarity ratings or by consonant identification and were not dependent upon label familiarity. The asymmetry at erroneously recognizing onset- versus offset-overlapping lures remained unchanged as the presentation of the shape at test was delayed in time, suggesting that response anticipation based on the first sounds of the spoken label did not contribute much to the false recognition of onset-overlapping lures. Thus, learning 2 words whose names differ in their last sounds appears to pose greater difficulty than learning 2 words whose names differ in their first sounds because, we argue, people are biased to give more importance to the early sounds of a name than to its last sounds when learning a novel label–referent association. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Digital titration: Automated image acquisition and analysis of load and growth of Chlamydophila psittaci

Traditionally, the amount of infective chlamydiae in a given sample is determined by inoculating dilution series into cell cultures and physically counting chlamydial inclusions. This approach is time consuming, tedious, and error prone, mainly when dealing with high titers. Therefore, this paper describes a largely automated technique that was developed to standardize the determination of chlamydial load in vitro. Cells are fixed at 36 h post-inoculation and bacteria visualized using standard immunological detection methods. Consequently, for 81 microscopic fields, an image is recorded at the interpolated focal plane. These images are then automatically processed using an ImageJ plugin and the obtained results are imported into Excel to determine the number of inclusion forming units per mL in the sample. The main advantage of this technique is that no or minimal sample dilution is required, thus minimizing dilution errors. In addition, this technique was employed during the early, middle and late growth stages of the chlamydial developmental cycle and results correlated well (P < 0.01) with 16S rRNA values from previous experiments, thereby proving its suitability to follow chlamydial growth in vitro. The method described is highly suitable for high throughput titration of cell culture inoculated samples and assessment of possible antichlamydial effects of novel compounds throughout the chlamydial growth cycle. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Paclitaxel-Loaded Microparticles for Intratumoral Administration via the TMT Technique: Preparation,Characterization, and Preliminary Antitumoral Evaluation

In our pursuit to develop suitable therapeutic particulate systems for intratumoral delivery by the targeted multi-therapy (TMT) technique, we describe the preparation of paclitaxel-loaded poly(d,l-lactic-co-glycolic) acid (PLGA) microparticles (MPs) (drug loading 35–38%, wt/wt; size 0.7–5 μm). Magnetite (15%, wt/wt) was also incorporated in some preparations for a future magnetic resonance imaging (MRI)-guided delivery. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) experiments showed that paclitaxel was not encapsulated in its initial crystalline form. The paclitaxel in vitro release pattern showed a biphasic tendency with a burst effect followed by a sustained release (28% released amount after 1 month), which was accompanied with MP erosion and degradation signs as confirmed by scanning electronic microscopy (SEM) micrographs. The paclitaxel-loaded MPs demonstrated a dose-dependent antitumor effect on human uterine cancer cells, with an IC₅₀ value relatively close to that of commercial Taxol®. This paclitaxel delivery system represents a potent antiprofilerative and radiosensitizer agent for intratumoral administration via the TMT technique.     

How to increase the safety and efficacy of compounds against neurodegeneration? A multifunctional approach

Successful drug design requires not only the detailed knowledge of the pharmacokinetic and pharmacodynamic profiles of the drug candidate portfolio but also a thorough documentation of the possible toxic effects on humans and the environment. Thus, experimental and computational strategies able to measure or predict specific profiles of designed compounds related to their potential toxicity are highly desired. Moreover, a strategy to avoid toxic effects thus enhancing the potential efficacy of drug candidates is of great interest. To fulfil this aim, the pharmacochemistry research unit at the EPGL has recently developed and improved methodologies that detect the potential human health and environmental hazards of compounds active against neurodegeneration at an early stage. A three-step strategy is presented herein. In particular, i) an alternative index to model the bioconcentration of chemicals in the environment was determined; ii) the antioxidant activity of chemical species against free radicals was evaluated. Moreover, since antioxidants play a key role in both toxicity prevention and neuroprotection, iii) the potential interaction of such compounds with enzymatic targets involved in the neurodegenerative cascade was investigated in silico.     

Impact of the Baking Duration on Bread Staling Kinetics

This paper presents a study on the impact of the duration of the baking plateau on staling kinetics in the case of bread crumb made of sourdough; it follows Le-Bail et al. Journal of Cereal Science 50:235–240, (2009)a previous study proposed by Le-Bail et al. Journal of Cereal Science 50:235–240, (2009) on the impact of heating rate during baking on staling parameters. Degassed bread dough was baked in a miniaturized baking system with baking plateau of 0, 4, and 8 min at 98 °C corresponding to a total baking time of 10, 14 and 18 min respectively (simulating from underbaked to fully baked bread). Results showed that longer baking time resulted in the higher Young’s modulus of the baked dough at the end of staling was. It was observed as in Le-Bail et al. Journal of Cereal Science 50:235–240, (2009) that the crystallization of amylopectin occurred a few days before the hardening of the baked crumb during staling. The amount of freezable water decreased during staling (over 10 days period), which was in agreement with the increase in amylopectin crystallites during staling which trap water. The amount of soluble amylose increased with increasing duration of the baking plateau at 98 °C, indicating that for prolonged baking, an increasing amount of amylose is leached outside of the starch granules. This was proposed as an explanation for the higher Young’s modulus of the crumb at the end of staling.