Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations

Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides. In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome. Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding. We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications

Mice Doubly-Deficient in Lysosomal Hexosaminidase A and Neuraminidase 4 Show Epileptic Crises and Rapid Neuronal Loss

Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts GM2 to GM3 ganglioside. Hexa−/− mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise GM2 ganglioside via a lysosomal sialidase into glycolipid GA2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4−/−;Hexa−/−) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa−/− or Neu4−/− siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating GM2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa−/− mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa−/− mice

Rôle de l'efflux dans la résistance de Clostridium difficile aux fluoroquinolones et aux composés toxiques

Clostridium difficile est un bacille à Gram positif anaérobie strict, sporulé et producteur des toxines A et B. Il représente la principale étiologie des diarrhées nosocomiales de l'adulte. Le taux de résistance acquise aux quinolones en France était de 7 % entre 1991 et 1997. Cette résistance était toujours associée à la présence d'une mutation dans les gènes de l'ADN gyrase. C. difficile ne possède qu'une seule cible des quinolones, l'ADN gyrase, et est dénué d'ADN topoisomérase IV. Nous avons cloné une protéine d'efflux appelée CdeA appartenant au cluster 3 de la famille MATE. Elle est capable de conférer une résistance aux fluoroquinolones lorsqu'elle est surexprimée chez E. coli. L'exposition au bromure d'éthidium augmente la transcription du gène cdeA. Nous avons réussi à introduire par conjugaison un nouveau plasmide réplicatif exprimant un ARN antisens chez C. difficile. Cependant, le rôle de CdeA dans la résistance aux fluoroquinolones et aux toxiques n'a pu être démontré.C. difficile represents the main cause of nosocomial diarrhea in adults. The prevalence of acquired resistance to quinolones in France was 7% and identical for the years 1991 and 1997. All isolates with decreased susceptibility carried a mutation in the DNA gyrase gyrA or gyrB genes. We found that C. difficile lacks the genes coding for DNA topoisomerase IV. CdeA is the first multidrug efflux transporter identified in C. difficile. CdeA belongs to the cluster 3 of the MATE family. It was responsible for quinolone resistance in E. coli when overexpressed. The presence of subinhibitory concentration of ethidium bromide significantly increased the transcription of cdeA. We have successfully introduced by conjugation between E. coli and C. difficile a new replicative vector which was used to introduce antisens RNA for cdeA. However, there was no significant difference in the susceptibility to quinolones or toxics between the recombinant and the parental strain.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

On the relationship between the formation factor and diffusion coefficients of Portland cement mortars

International audienceConcrete durability continues to be a subject of considerable interest, especially with the use of cement based materials on structures requiring improved sustainability and resistance to aggressive ions penetration or radionuclide release. Diffusion is considered as one of the main transport phenomena that causes migration of aggressive solutes and radionuclide in a porous media according to most studies.In this work, two different tracers (an ion, and a radionuclide) were tested on the same formulations of mortars (water / cement = 0.4 and sand volume fractions from 0 to 60%) by the through-out diffusion, in order to determine the effective diffusion coefficients of each tracer and each formulation. The obtained results have proven the validity of the formation factor equation relating the effective diffusivity of a tracer to its diffusion coefficient in pure water

The validity of the formation factor concept from through-out diffusion tests on Portland cement mortars

International audienceThe diffusion coefficients of ions and radionuclides in cementitious materials are the basic parameters to evaluate the state of the degradation of structures. In this article, three different tracers (two ions, and a radionuclide) were tested on the same formulations of mortars (sand volume fractions from 0 to 60 per cent) in terms of the through-out diffusion, to determine the effective diffusion coefficients of each tracer and each formulation. The aim of this study is to prove the validity of the formation factor equation relating the effective diffusivity of a tracer in cementitious material to its diffusion coefficient in pure water. This result is extremely interesting because once the geometric formation factor of a material is known, it is possible to determine the values of the effective diffusion coefficients of any other diffusing species in this material

Link between microstructure and tritiated water diffusivity in mortars

Ions and radionuclide diffusivity in concrete is one of the most important factors that determine service life and safety assessment of cement based structures in nuclear power plants and radioactive-waste repositories. Apart from the influence of cement paste microstructure, the presence of aggregates may have an impact on transport properties of the material. The well-known interfacial transition zone, denoted by ITZ, is created near the aggregates and characterized by a greater porosity. The goal of this study is to investigate the competition between the more diffusing ITZ zone and the less diffusing aggregates. To this end, several series of tritiated water diffusion tests are conducted on mortars characterized by different water-to-cement ratios and sand volume fractions. In parallel, microstructure of these materials is explored by mercury and water porosimetry. It was observed that at low sand content (0% – 50%), diffusion properties of mortars are dominated by aggregates dilution effect. At 60% sand, diffusion increases significantly suggesting that percolation’s pores threshold has been reached. Results indicate also that sand particle size distribution has a great impact on the diffusivity of mortars