Combined Computational-Experimental Analyses of CFTR Exon Strength Uncover Predictability of Exon-Skipping Level.

International audienceWith the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance

IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro

AbstractRecent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6–48 h causes a marked inhibition of the amiloride-sensitive Na+ channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca2+-dependent Cl− channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Rα. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the γ amiloride-sensitive Na+ channel mRNA, one of the subunits of the amiloride-sensitive Na+ channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies

Target enrichment sequencing coupled with GWAS identifies MdPRX10 as a candidate gene in the control of budbreak in apple

The timing of floral budbreak in apple has a significant effect on fruit production and quality. Budbreak occurs as a result of a complex molecular mechanism that relies on accurate integration of external environmental cues, principally temperature. In the pursuit of understanding this mechanism, especially with respect to aiding adaptation to climate change, a QTL at the top of linkage group (LG) 9 has been identified by many studies on budbreak, but the genes underlying it remain elusive. Here, together with a dessert apple core collection of 239 cultivars, we used a targeted capture sequencing approach to increase SNP resolution in apple orthologues of known or suspected A. thaliana flowering time-related genes, as well as approximately 200 genes within the LG9 QTL interval. This increased the 275 223 SNP Axiom® Apple 480 K array dataset by an additional 40 857 markers. Robust GWAS analyses identified MdPRX10, a peroxidase superfamily gene, as a strong candidate that demonstrated a dormancy-related expression pattern and down-regulation in response to chilling. In-silico analyses also predicted the residue change resulting from the SNP allele associated with late budbreak could alter protein conformation and likely function. Late budbreak cultivars homozygous for this SNP allele also showed significantly up-regulated expression of C-REPEAT BINDING FACTOR (CBF) genes, which are involved in cold tolerance and perception, compared to reference cultivars, such as Gala. Taken together, these results indicate a role for MdPRX10 in budbreak, potentially via redox-mediated signaling and CBF gene regulation. Moving forward, this provides a focus for developing our understanding of the effects of temperature on flowering time and how redox processes may influence integration of external cues in dormancy pathways

BIOCARBURANTS PSA Peugeot Citroën et les biocarburants

Les biocarburants représentent une filière essentielle pour PSA Peugeot Citroën dans le cadre de la protection de l’environnement, un axe stratégique majeur pour le groupe - de la production de ses véhicules au recyclage, de la qualité de l’air à l’effet de serre et au développement d’énergies renouvelables. Depuis plusieurs années, PSA a intégré la filière huiles végétales/biocarburants dans ses activités pour atteindre ses objectifs globaux d’innovation et de croissance. De nombreuses flottes de véhicules circulent en France avec du gazole contenant 30 % de biocarburant issu des oléagineux : une expérience réussie, qui dure depuis plus de dix ans. Le constructeur est favorable à l’utilisation de biocarburants en mélange dans les carburants pétroliers conventionnels, essence et gazole - une voie qui présente plusieurs avantages : * Un bilan CO2 global favorable (par absorption de CO2 lors de la photosynthèse de la plante-source), qui contribue à limiter « l’effet de serre ». * Une source complémentaire de carburant pour réduire les importations de pétrole (et de gazole). * Un soutien au secteur agricole (débouché supplémentaire, dans le cadre de la valorisation des terres en jachère). Issus de la transformation des céréales ou de la betterave à sucre, ou des plantes oléagineuses comme le colza, le tournesol ou le soja, les biocarburants sont par essence des énergies renouvelables. Outre leur bilan exceptionnellement favorable pour lutter contre l’effet de serre, ces carburants offrent de réelles performances environnementales, en particulier par la réduction des émissions de particules (cas des EMHV)

Modification of the spontaneous seat-to-stand transition in cycling with bodyweight and cadence variations

International audienceWhen a high power output is required in cycling, a spontaneous transition by the cyclist from a seated to a standing position generally occurs. In this study, by varying the cadence and cyclist bodyweight, we tested whether the transition is better explained by the greater power economy of a standing position or by the emergence of mechanical constraints that force cyclists to stand. Ten males participated in five experimental sessions corresponding to different bodyweights (80%, 100%, or 120%) and cadences (50 RPM, 70 RPM, or 90 RPM). In each session, we first determined the seat-to-stand transition power (SSTP) in an incremental test. The participants then cycled at 20%, 40%, 60%, 80%, 100%, or 120% of the SSTP in the seated and standing positions, for which we recorded the saddle forces and electromyogram (EMG) signals of eight lower limb muscles. We estimated the cycling cost using an EMG cost function (ECF) and the minimal saddle forces in the seated position as an indicator of the mechanical constraints.Our results show the SSTP to vary with respect to both cadence and bodyweight. The ECF was lower in the standing position above the SSTP value (i.e., at 120%) in all experimental sessions. The minimal saddle forces varied significantly with respect to both cadence and bodyweight.These results suggest that optimization of the muscular cost function, rather than mechanical constraints, explain the seat-to-stand transition in cycling

Elastic energy in locomotion: Spring-mass vs. poly-articulated models

International audienceThe human is often modeled as a Poly-Articulated Model (PAM) with rigid segments while some authors use a Spring Mass Model (SMM) for modeling locomotion. These two models are considered independent, and the objective of this study was to link them in order to enlighten the origin of the elasticity in locomotion.Using the characteristics of the two models, a theoretical relationship demonstrates that the variation of elastic energy of the SMM equals the variation of the internal kinetic energy minus internal forces work of the PAM. This theoretical relationship was experimentally investigated among 19 healthy participants walking and running on a treadmill.The results showed that the equality is verified except during the double support phase at 0.56 m s−1, at high walking speeds (1.67 and 2.22 m s−1) or during the aerial phase of running.The formal relationship showed that the global stiffness of the SMM is directly related to the work of the internal forces of the PAM, and thus, to the characteristics of the musculoskeletal system. It also showed the relevance of taking into account the participation of each joint in the global stiffness. Finally, the coordination of internal forces work to produce a global stiffness may be considered as a new criterion of movement optimization for clinical purposes or motion planning for humanoid robot

Transferability between Isolated Joint Torques and a Maximum Polyarticular Task: A Preliminary Study

International audienceThe aims of this study were to determine if isolated maximum joint torques and joint torques during a maximum polyarticular task (i.e. cycling at maximum power) are correlated despite joint angle and velocity discrepancies, and to assess if an isolated joint-specific torque production capability at slow angular velocity is related to cycling power. Nine cyclists completed two different evaluations of their lower limb maximum joint torques. Maximum Isolated Torques were assessed on isolated joint movements using an isokinetic ergometer and Maximum Pedalling Torques were calculated at the ankle, knee and hip for flexion and extension by inverse dynamics during cycling at maximum power. A correlation analysis was made between Maximum Isolated Torques and respective Maximum Pedalling Torques [3 joints x (flexion + extension)], showing no significant relationship. Only one significant relationship was found between cycling maximum power and knee extension Maximum Isolated Torque (r=0.68, p<0.05). Lack of correlations between isolated joint torques measured at slow angular velocity and the same joint torques involved in a polyarticular task shows that transfers between both are not direct due to differences in joint angular velocities and in mono-articular versus poly articular joint torque production capabilities. However, this study confirms that maximum power in cycling is correlated with slow angular velocity mono-articular maximum knee extension torque

An algorithm to decompose ground reaction forces and moments from a single force platform in walking gait

International audienceIn walking experimental conditions, subjects are sometimes unable to perform two steps on two different forceplates. This leads the authors to develop methods for discerning right and left ground reaction data while they are summed during the double support in walking. The aim of this study is to propose an adaptive transition function that considers the walking speed and ground reaction forces (GRF). A transition function is used to estimate left and right side GRF signals in double support. It includes a shape coefficient adjusted using single support GRF parameters. This shape coefficient is optimized by a non-linear least-square curve-fitting procedure to match the estimated signals with real GRF. A multiple regression is then performed to identify GRF parameters of major importance selected to compute the right and left GRF of the double support. Relative RMSE (RMSER), maximum GRF differences normalized to body mass and differences of center of pressure (CoP) are computed between real and decomposed signals. During double support, RMSER are 6%, 18%, 3.8%, 4.3%, 3%, and 12.3% for anterior force, lateral force, vertical force, frontal moment, sagittal moment and transverse moment, respectively. Maximum GRF differences normalized to body mass are lower than 1 N/kg and mean CoP difference is 0.0135 m, when comparing real to decomposed signals during double support. This work shows the accuracy of an adaptive transition function to decompose GRF and moment of right and left sides. This method is especially useful to accurately discern right and left GRF data in single force platform configurations

A preliminary study suggests that walk-to-run transition is consistent with mechanical optimization

The aim of this study was to compute moment cost function (MCF) at different walking and running speeds. We hypothesized that MCF would be optimal at low speeds during walking and at high speeds during running