Université PSL



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Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells
G. Lia, B. Michel and J.-F. Allemand
Science - 335(6066) :328–31 - DOI:10.1126/science.1210400 - 2012
DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.
Single-molecule mechanical identification and sequencing
F. Ding, M. Manosas, M. M. Spiering, S. J. Benkovic, D. Bensimon, J.-F. Allemand and V. Croquette
Nat. Methods - 9(4) :367–72 - DOI:10.1038/nmeth.1925 - 2012
High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.
UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution
A. Diguet, M. Yanagisawa, Y. J. Liu, E. Brun, S. Abadie, S. Rudiuk and D. Baigl
JACS - 134(10) :4898-904 - DOI:10.1021/ja211664f - 2012
We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (Lo) phase and a DOPC-rich liquid-disordered (Ld) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Yburst), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Yburst decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Yburst increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of Lo phase (with increasing [Chol] = 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Yburst = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.
Carbamazepine removal from water by dielectric barrier discharge : comparison of ex situ and in situ discharge on water
Y. Liu, S. Mei, D. Iya-Sou, S. Cavadias, S. Ognier
Chemical Engineering And Processing - 56 :10-18 - DOI:10.1016/j.cep.2012.03.003 - 2012
Dielectric barrier discharges (DBD) were used for the degradation of carbamazepine (CBZ) in aqueous solution. The electric discharge was generated either ex situ or in situ directly on the water surface. To maintain the same ozone concentration of 40 ppm in both instances, the power injected was 0.7 W in the ex situ discharge and 12 W in the in situ discharge. The results showed 100% CBZ removal after 3 min of treatment with the ex situ discharge, while the in situ discharge only removed 81% of the CBZ after 60 min. According to measurements of UV absorbance at 285 nm and 254 nm, and of total organic carbon, the ex situ discharge system also proved to be more effective than the in situ system. The measurement of nitrogen oxides in both gaseous and liquid phases indicated that high energy in situ discharges produced a large amount of NOx. These species contributed to decreased pH and significantly slowed the CBZ oxidation rate, due to their competition with ozone. Production of NOx should be avoided when using the DBD technique for wastewater treatment.
Atmospheric Pressure Deposition of Thin Functional Coatings: Polymer Surface Patterning by DBD and Post-Discharge Polymerization of Liquid Vinyl Monomer from Surface Radicals
J.P. Borra, A. Valt, F. Arefi-Khonsari, M. Tatoulian
Plasma Process - 9(11-12) :1104-15 - DOI:10.1002/ppap.201100210 - 2012
We present a route for grafting polyacid and polyether coatings on polymers by post-discharge polymerization of liquid vinyl monomer. Surface modifications of polymer films by Micro-Discharges in air Dielectric Barrier Discharge (MD, DBD) are depicted with sub-micrometer craters homogeneously distributed. Both the energy per MD and the power density are critical to avoid thermal film deformation. Homogeneous surface composition is related to the DBD energy density. The polymerization mechanism is depicted from yields versus DBD energy density and time of exposure to air between DBD and monomer deposition. Both parameters control the surface density of radicals and peroxides, triggering the post-DBD polymerization with 80 and 73% of monomer functionality remaining in acid and ether coatings, respectively. The effect of deposition conditions on coatings properties is shown as well as the stability of coatings upon washing.
Fabrication Of Metallic Patterns On PDMS Using Transfer Technology: Application To MRI Microcoils
M. Couty, S. Nazeer, C. Jelita, E. Martincic, M. Woytasik, J.C. Ginefri, L. Darrasse, M. Tatoulian, E. Dufour-Gergam
MICRO & NANO LETTERS - 7(6) :519-22 - DOI:10.1049/mnl.2012.0271 - 2012
Despite the large use of this material in the microsystem field, fabrication of metallic patterns on polydimethylsiloxane (PDMS) still remains a challenge. In this Letter, we present a new process based on the transfer principle and report its application to MRI microcoils. These double-side structures are well aligned and the transfer yield is higher than 90%. The limit of the working range for these flexible coils is a bending radius of 2 mm, similar to the radius of the coil. The developed process opens a wide range of further applications for flexible devices.
Stable modification of PDMS surface properties by plasma polymerization: Innovative process of allylamine PECVD deposition and microfluidic devices sealing
S. Massey, A. Duboin, D. Mantovani, P. Tabeling, M. Tatoulian
Surface & Coatings Technology - 206(19-20) :4303-9 - DOI:10.1016/j.surfcoat.2012.04.047 - 2012
This paper presents a new and innovative process of modification of wetting of open micro-channels involving a method to seal the microfluidic devices. Allylamine was polymerized on poly(dimethylsiloxane) (PDMS) by plasma-enhanced chemical vapour deposition (PECVD) to modify the wetting properties of open micro-channels. The sealing of the devices was done by thermal pressing. All the steps of the process were characterized by different analysis techniques to understand the mechanisms of the process and to assess the performance of the technique. Physicochemical analysis of the polymerized allylamine coatings (X-ray photoelectron spectroscopy and static water contact angle) showed that the coatings were resistant to the thermal pressing and were stable in ambient air and underwater up to 14 days of ageing, even if the water contact angle decreased during the underwater ageing. Parallel tests were undergone in microfluidic devices and the stability of ageing was tested by the production of the simple oil-in-water emulsions. All the experiments showed that this new PECVD/thermal press process is an effective way to modify the wetting properties of an open microfluidic device and includes a technique to seal effectively the system afterwards.
Tau Pathology modulates Pin1 post-translational modifications and may be relevant as biomarker
Ando K, Dourlen P, Sambo AV, Bretteville A, Bélarbi K, Vingtdeux V, Eddarkaoui S, Drobecq H, Ghestem A, Bégard S, Demey-Thomas E, Melnyk P, Smet C, Lippens G, Maurage CA, Caillet-Boudin ML, Verdier Y, Vinh J, Landrieu I, Galas MC, Blum D, Hamdane M, Serg
Neurobiol Aging - 34(3) :757-69 - DOI:10.1016/j.neurobiolaging.2012.08.004 - 2012
A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.
On-bead tryptic proteolysis: An attractive procedure for LC-MS/MS analysis of the Drosophila caspase 8 protein complex during immune response against bacteria
Fukuyama H & Ndiaye S, Hoffmann J, Rossier J, Liuu S, Vinh J, Verdier Y.
J Proteomics - 75(15) :4610-9 - DOI:10.1016/j.jprot.2012.03.003 - 2012
This study aims to characterize the immune response against bacteria in Drosophila melanogaster. Obtaining a description of the in vivo state of protein complexes requires their isolation as a snapshot of physiological conditions before their identification. Affinity purification with streptavidin-biotin system is widely used to address this issue. However, because of the extraordinary stability of the interaction between streptavidin and biotin, the release of biotin-labeled bait remains a challenge. We transfected Drosophila cells with a DNA construct encoding a biotin-tagged Dredd protein (ortholog of caspase 8). After affinity purification, different strategies were evaluated, and proteins analyzed by LC-MS/MS mass spectrometry. The on-bead digestion allowed the identification of more proteins associated to the Dredd complex than different protocols using competitive or acid elution. A functional assay showed that a large part of the proteins specifically identified in the Dredd sample are functionally involved in the activation of the Imd pathway. These proteins are immune response proteins (BG4, Q9VP57), stress response proteins (HSP7C, Q9VXQ5), structural proteins (TBB1, CP190), a protein biosynthesis protein (Q9W1B9) and an antioxidant system protein (SODC). Our results clearly show that on-bead digestion of proteins is an attractive procedure for the study of protein complexes by mass spectrometry. This article is part of a Special Issue entitled: Translational Proteomics.
Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis
Shahali Y, Sutra JP, Haddad I, Vinh J, Guilloux L, Peltre G, Sénéchal H, Poncet P.
Electrophoresis - 33(3) :462-9 - DOI:10.1002/elps.20110032 - 2012
Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one-dimensional gel electrophoresis (D1-DE) as an alternative to the 2-DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one-dimensional combination of IEF and SDS-PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43?kDa. D1-DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one-dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin-based protein-protein interactions. Therefore, D1-DE could be used in routine work as a convenient alternative to 2-DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.

405 publications.