A doubly responsive probe for the detection of Cys4-tagged proteins

International audienceRecombinant proteins bearing a tag are crucial tools for assessing protein location or function. Small tags such as Cys4 tag (tetracysteine; Cys–Cys–X–X–Cys–Cys) are less likely disrupt protein function in the living cell than green fluorescent protein. Herein we report the first example of the design and synthesis of a dual fluorescence and hyperpolarized 129Xe NMR-based sensor of Cys4-tagged proteins. This sensor becomes fluorescent when bound to such Cys4-tagged peptides, and the 129Xe NMR spectrum exhibits a specific signal, characteristic of the biosensor-peptide association

Comparison of distribution and activity of nanoparticles with short interfering DNA (Dbait) in various living systems

Introducing small DNA molecules (Dbait) impairs the repair of damaged chromosomes and provides a new method for enhancing the efficiency of radiotherapy in radio-resistant tumors. The radiosensitizing activity is dependent upon the efficient delivery of Dbait molecules into the tumor cells. Different strategies have been compared, to improve this key step. We developed a pipeline of assays to select the most efficient nanoparticles and administration protocols before preclinical assays: (i) molecular analyses of complexes formed with Dbait molecules, (ii) cellular tests for Dbait uptake and activity, (iii) live zebrafish embryo confocal microscopy monitoring for in vivo distribution and biological activity of the nanoparticles and (iv) tumor growth and survival measurement on mice with xenografted tumors. Two classes of nanoparticles were compared, polycationic polymers with linear or branched polyethylenimine (PEI) and covalently attached cholesterol (coDbait). The most efficient Dbait transfection was observed with linear PEI complexes, in vitro and in vivo. Doses of coDbait ten-fold higher than PEI/Dbait nanoparticles, and pretreatment with chloroquine, were required to obtain the same antitumoral effect on xenografted melanoma. However, with a 22-fold lower ‘efficacy dose/toxicity dose' ratio as compared with Dbait/PEI, coDbait was selected for clinical trials

Xe NMR-based sensors: biological applications and recent methods

International audienceXenon is a first-rate sensor of biological events, due to its large polarizable electron cloud inducing significant modification of NMR parameters through slight changes in its local environment. The use of xenon as a sensor is of increasing interest for sensitive magnetic resonance imaging, since its signal can be enhanced by several orders of magnitude, mainly by spin-exchange optical pumping. Furthermore xenon can be vectorized toward targets of interest by using functionalized host systems, enabling their detection at subnanomolar concentrations. Associated with a new generation of detection methods this gives rise to a powerful molecular imaging approach, where xenon can be delivered on purpose several times after introduction of the functionalized host system

¹²⁹Xe ultra-fast Z spectroscopy enables micromolar detection of biosensors on a 1 T benchtop spectrometer

The availability of a benchtop nuclear magnetic resonance (NMR) spectrometer, of low cost and easily transportable, can allow detection of low quantities of biosensors, provided that hyperpolarized species are used. Here we show that the micromolar threshold can easily be reached by employing laser-polarized xenon and cage molecules reversibly hosting it. Indirect detection of caged xenon is made via chemical exchange, using ultra-fast Z spectroscopy based on spatio-temporal encoding. On this non-dedicated low-field spectrometer, several ideas are proposed to improve the signal.</p

A doubly responsive probe for the detection of Cys4-tagged proteins

International audienceIntroduction Full understanding of intracellular phenomena involves sensitive and non-invasive detection. A less disruptive method than labeling of fluorescent proteins uses binding between a tag of only six natural amine acids that can be genetically incorporated into the protein of interest and a small molecule ca lied FIAsH[1]. This molecule has the ability to fluoresce only when it binds to its 4Cys-tag target. Another technique based on 129Xe NMR has emerged. Xenon is hyperpolarized to enhance the NMR signal by orders of magnitude and its reversible encapsulation in functionalized host systems gives it a specifie spectral signature[2]. Capability of the noble gas to cross cell membranes without losing its polarization[3] enables in cellule investigations. Here we report the first design and study of a dual fluorescence-and 129Xe NMR-based sensor of Cys4-tagged proteins[4]

EPJ Web of Conferences, Vol 30: Experiment and Modelling in Structural NMR.

The school covers several topics in the field of structural NMR. In the first part, following an introduction on general principles, the basic knowledge in isotropic and anisotropic media is given. The second part deals with the structural studies of biological systems and materials while the third gives an overview of a some advanced concepts including Hyper-polarization, Fast NMR and Diffusion process

Vasoactive intestinal contractor (VIC) peptide structure in solution as determinated by proton NMR

The Vasoactive Intestinal Contractor peptide has been synthesized and studied by 1H NMR in 50/50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. Modeling was performed with constraints obtained by NOEs. Some distances used in modeling were calculated from build-up data. Kinetic exchange of the amide protons showed that the 9-15 helical segment is stabilized by at least three hydrogen bonds. The proposed structure is compared with those of Nle-7 endothelin and Sarafotoxin-S6b previously obtained in same conditions

High HEV seroprevalence in forestry workers and in wild boars in France.

HEV is a fecal orally-transmitted human pathogen of worldwide distribution. In industrial countries, HEV is observed in an increasing number of autochthonous cases and is considered to be an emerging pathogen. A growing body of evidence suggests that HEV is a zoonotic disease, and pig handlers and pig veterinarians have been reported to be high-risk groups for HEV infection. The aims of the present study were to establish the prevalence of anti-HEV in wild boar in France and to identify whether forestry workers are at a higher risk of HEV infection. Three different anti-HEV tests were used to compare their effectiveness in detecting anti-HEV in the general population. The most sensitive test was then used to investigate HEV seroprevalence in 593 forestry workers and 421 wild boars.Anti-HEV was detected in 31% of the forestry workers and 14% of the wild boar. Detection of anti-HEV in humans was correlated with age, geographical location and occupational activity, and in wild boar with geographical location. HEV infection is frequent in woodcutters in France, and it varies geographically. Further studies are needed to confirm these findings and to elucidate the transmission route and the exact virus reservoirs

A doubly responsive probe for the detection of Cys4-tagged proteins

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