A fluorescent nanoprobe for single bacterium tracking: functionalization of silver nanoparticles with tryptophan to probe the nanoparticle accumulation with single cell resolution

The investigation of the interaction of silver nanoparticles and live bacteria cells is of particular importance for understanding and controlling their bactericidal properties. In this study, the process of internalization of silver nanoparticles in Escherichia coli cells was followed by means of synchrotron excitation deep ultraviolet (DUV) fluorescence imaging. Antimicrobial nanostructures that can absorb and emit light in the UV region were prepared by functionalization of silver nanoparticles with tryptophan amino acid and used as environmentally sensitive fluorescent probes. The nanostructures were characterized by morphological (TEM) and spectroscopic methods (UV-vis, FTIR, XPS, and photoluminescence). The TEM images and the analyses of the UV-vis spectra suggested that the addition of tryptophan led to the formation of hybrid nanostructures with pronounced eccentricity and larger sizes with respect to that of the initial silver nanoparticles. The DUV imaging showed that it was possible to distinguish the fluorescent signal pertaining to silver-tryptophan nanostructures from the autofluorescence of the bacteria. The spatial resolution of the fluorescence images was 154 nm which was sufficient to perform analyses of the accumulation of the nanostructures within a single bacterium. The DUV imaging results imply that the tryptophan-functionalized silver nanoparticles interact with cell membranes via insertion of the amino acid into the phospholipid bilayer and enter the cells

Somatostatin analogues for receptor targeted photodynamic therapy

Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2+ AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate

Anti-infectives in Drug Delivery-Overcoming the Gram-Negative Bacterial Cell Envelope.

Infectious diseases are becoming a major menace to the state of health worldwide, with difficulties in effective treatment especially of nosocomial infections caused by Gram-negative bacteria being increasingly reported. Inadequate permeation of anti-infectives into or across the Gram-negative bacterial cell envelope, due to its intrinsic barrier function as well as barrier enhancement mediated by resistance mechanisms, can be identified as one of the major reasons for insufficient therapeutic effects. Several in vitro, in silico, and in cellulo models are currently employed to increase the knowledge of anti-infective transport processes into or across the bacterial cell envelope; however, all such models exhibit drawbacks or have limitations with respect to the information they are able to provide. Thus, new approaches which allow for more comprehensive characterization of anti-infective permeation processes (and as such, would be usable as screening methods in early drug discovery and development) are desperately needed. Furthermore, delivery methods or technologies capable of enhancing anti-infective permeation into or across the bacterial cell envelope are required. In this respect, particle-based carrier systems have already been shown to provide the opportunity to overcome compound-related difficulties and allow for targeted delivery. In addition, formulations combining efflux pump inhibitors or antimicrobial peptides with anti-infectives show promise in the restoration of antibiotic activity in resistant bacterial strains. Despite considerable progress in this field however, the design of carriers to specifically enhance transport across the bacterial envelope or to target difficult-to-treat (e.g., intracellular) infections remains an urgently needed area of improvement. What follows is a summary and evaluation of the state of the art of both bacterial permeation models and advanced anti-infective formulation strategies, together with an outlook for future directions in these fields

Introduction to a culturally sensitive measure of well-being: Combining life satisfaction and interdependent happiness across 49 different cultures

How can one conclude that well-being is higher in country A than country B, when wellbeing is being measured according to the way people in country A think about wellbeing? We address this issue by proposing a new culturally sensitive method to comparing societal levels of well-being. We support our reasoning with data on life satisfaction and interdependent happiness focusing on individual and family, collected mostly from students, across forty-nine countries. We demonstrate that the relative idealization of the two types of wellbeing varies across cultural contexts and are associated with culturally different models of selfhood. Furthermore, we show that rankings of societal well-being based on life satisfaction tend to underestimate the contribution from interdependent happiness. We introduce a new culturally sensitive method for calculating societal well-being, and examine its construct validity by testing for associations with the experience of emotions and with individualism-collectivism. This new culturally sensitive approach represents a slight, yet important improvement in measuring well-being

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

In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra (hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed