Electronic Circular Dichroism Spectroscopy of Proteins

Circular dichroism (CD) is an important spectroscopic technique that enables the characterization of protein secondary and tertiary structure. Proteins can undergo changes in their structure when they participate in processes, for example, ligand binding. CD, therefore, can be used to monitor secondary and tertiary structural changes when a protein (receptor) binds to a drug molecule (ligand).This review describes experimental studies of protein CD and theoretical and computational methods that compute spectra from structure or structure from spectra. CD is a technique that can be used to complement X-ray, NMR, and ultraviolet-visible (UV-vis) experiments on biomolecules and proteins, all of which can be assisted by molecular modeling, which has the capability of computing CD from first principles. A combination of experimental CD and molecular modeling has the capacity to greatly enhance future multi-disciplinary research to expand our knowledge of the structure, function, and dynamics of proteins

RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure

Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3' end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3'-DNA/ds siRNA and includes a 3'-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3'-DNA/ds siRNA, (II) passenger-3'-DNA/ds siRNA, (III) guide-3'-DNA/ss siRNA, and (IV) passenger-3'-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I-III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3'-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3'-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3'-DNA/siRNA constructs are discussed. Molecular Therapy-Nucleic Acids (2014) 2, e141; doi:10.1038/mtna.2013.68; published online 7 January 2014

A rhenium tris-carbonyl derivative as a single core multimodal probe for imaging (SCoMPI) combining infrared and luminescent properties.

International audienceA rhenium tris-carbonyl derivative has been designed to couple infrared and luminescent detection in cells. Both spectroscopies are consistent with one another; they point out the reliability of the present SCoMPI (for Single Core Multimodal Probe for Imaging) for bimodal imaging and unambiguously indicate a localization at the Golgi apparatus in MDA-MB-231 breast cancer cells

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack

Tomographie de fluorescence DUV sur des systèmes d’intérêt biologique

L’utilisation de la lumière synchrotron en ultraviolet lointain (DUV, de longueur d’onde comprise entre 120 et 300 nm) pour exciter la fluorescence des molécules en microscopie, permet de s’affranchir de marqueurs de contrastes. Un microscope inversé DUV est installé à SOLEIL sur la ligne de lumière DISCO. La motorisation axiale de l’échantillon et le traitement du signal par déconvolution rendent possible l’obtention de tomogrammes avec des résolutions de l’ordre de 100 nm. Deux applications biologiques sont présentées ici : l’activité d’une enzyme, l’amylase, sur un grain d’amidon; et le suivi de l’entrée de nanoparticules dans des cellules cancéreuses

Ultraviolets for better diagnosis

International audienceBackgroundThe invention of the UV lamp by Dr Wood triggered the observation of fluorescence induced by UV from tissues and biological samples at the beginning of the last century with notably, the classification of the observable color from dissected tissues under UV excitation1. In the following years, it was widely used for tumors observation2. We would like to demonstrate that one century after, this method may be rejuvenated.Materials and methodsDISCO Beamline3 is a bending magnet beamline at synchrotron SOLEIL covering the unusual 1-21 eV energy range. One of its branches is dedicated to UV microscopic imaging of biological samples4.ResultsUse of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because, it does not need external specific probes or labeling, but instead takes profit of the intrinsic fluorescence that arise from many biomolecules under deep ultraviolet excitation. Indeed, observation of label free biomolecules5 or active drugs6 ensures that the label will not modify the biolocalisation or any of its properties. UV monophotonic excitation does present real spectral excitation, leading the way to excitation imaging and a better selectivity of the chromophores. DUV excitation may also be used to track exogenous drugs or toxic compounds that present different spectral behaviour. Moreover, due to diffraction limit the lateral resolution is always increased when looking in the UV range allowing nanometric spatial resolution4. Examples of UV for diagnosis, in drug pharmacokinetic, in liver grafts quality and microcalcifications formations will be presented

Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria

International audienceResearchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are char- acterized by low permeability and often contain drug e ux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or e ux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identi ed clear correlations between e ux activ- ity and drug di usion and accumulation in both susceptible and resistant strains, and have also clari ed how certain chemical structures can a ect drug entry and residence time within the cell. In this Perspective, we discuss the biological underpinnings of drug permeability and export using several prototypical in ux and e ux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid e orts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics

Synchrotron UV/visible imaging discriminates historical pigments

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