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Murviel-lès-Montpellier, France

Lau A.A.,Lysosomal Diseases Research Unit | Lau A.A.,University of Adelaide | Hopwood J.J.,Lysosomal Diseases Research Unit | Hopwood J.J.,University of Adelaide | And 4 more authors.
Molecular Genetics and Metabolism | Year: 2010

Many viral backbones have been used as gene transfer vectors. However, the efficacy of therapy based on human-derived vectors may be limited by the high incidence of pre-existing humoral and cellular memory immunity. To circumvent some of the clinical disadvantages of vectors derived from common human pathogens, we have used an E1-deleted vector derived from a xenogenic adenovirus, canine adenovirus serotype 2 (CAV-2) to ameliorate neuropathological changes associated with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). This presently untreatable condition is caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency and is characterized by heparan sulfate accumulation and progressive neurodegeneration. Injection of CAV-SGSH-GFP into the thalamus of adult MPS IIIA mouse brain resulted in short-term gene expression. In contrast, intra-ventricular injection of newborn mice yielded dose-dependent transgene expression which persisted for at least 20-weeks and improved neuropathology. Together, these studies suggest that this E1-deleted CAV-2 vector is capable of mediating regional medium-term gene expression and facilitating improvements in neuropathology in MPS IIIA mice. © 2010 Elsevier Inc. All rights reserved.

Baronian G.,French National Center for Scientific Research | Ginda K.,Wroclaw University | Berry L.,French National Center for Scientific Research | Cohen-Gonsaud M.,Universites Montpellier I et | And 3 more authors.
PLoS ONE | Year: 2015

Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro . ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis. © 2015 Baronian et al.

Cattoni D.I.,Institute Nationale Of La Sante Et La Recherche Medicale | Cattoni D.I.,Universites Montpellier I et | Fiche J.-B.,Institute Nationale Of La Sante Et La Recherche Medicale | Fiche J.-B.,Universites Montpellier I et | And 5 more authors.
PLoS ONE | Year: 2013

Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level. © 2013 Cattoni et al.

Olofsson L.,French National Center for Scientific Research | Olofsson L.,French Institute of Health and Medical Research | Olofsson L.,Universites Montpellier I et | Margeat E.,French National Center for Scientific Research | And 2 more authors.
Optics Express | Year: 2013

Pulsed Interleaved Excitation (PIE) improves fluorescence crosscorrelation spectroscopy (FCCS) and single pair Förster Resonance Energy Transfer (spFRET) measurements, by correlating each detected photon to the excitation source that generated it. It relies on the interleaving of two picosecond laser sources and time correlated single photon counting (TCSPC) detection. Here, we present an optical configuration based on a commercial supercontinuum laser, which generates multicoulour interleaved picosecond pulses with arbitrary spacing and wavelengths within the visible spectrum. This simple, yet robust configuration can be used as a versatile source for PIE experiments, as an alternative to an array of picosecond lasers and drivers. © 2012 Optical Society of America.

Olofsson L.,French National Center for Scientific Research | Olofsson L.,French Institute of Health and Medical Research | Olofsson L.,Universites Montpellier I et | Felekyan S.,Heinrich Heine University Dusseldorf | And 20 more authors.
Nature Communications | Year: 2014

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor. We demonstrate that most receptors oscillate between a resting- and an active-conformation on a sub-millisecond timescale. Interestingly, we demonstrate that differences in agonist efficacies stem from differing abilities to shift the conformational equilibrium towards the fully active state, rather than from the stabilization of alternative static conformations, which further highlights the dynamic nature of mGluRs and revises our understanding of receptor activation and allosteric modulation. © 2014 Macmillan Publishers Limited. All rights reserved.

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