Direct analysis of dried blood spots coupled with mass spectrometry: concepts and biomedical applications.

Because of the emergence of dried blood spots (DBS) as an attractive alternative to conventional venous plasma sampling in many pharmaceutical companies and clinical laboratories, different analytical approaches have been developed to enable automated handling of DBS samples without any pretreatment. Associated with selective and sensitive MS-MS detection, these procedures give good results in the rapid identification and quantification of drugs (generally less than 3 min total run time), which is desirable because of the high throughput requirements of analytical laboratories. The objective of this review is to describe the analytical concepts of current direct DBS techniques and to present their advantages and disadvantages, with particular focus on automation capacity and commercial availability. Finally, an overview of the different biomedical applications in which these concepts could be of major interest will be presented.

Cardenolides in nectar may be more than a consequence of allocation to other plant parts: a phylogenetic study of Asclepias

The primary function of secondary plant metabolites is thought to be defence against herbivores. The frequent occurrence of these same noxious compounds in floral nectar, which functions primarily to attract pollinators, has been seen as paradoxical. Although these compounds may have an adaptive purpose in nectar, they may also occur as a nonadaptive consequence of chemical defence in other plant parts. If nectar chemistry reflects physiological constraints or passive leakage from other tissues, we expect that the identity and relative concentration of nectar cardenolides to be correlated with those of other plant parts; in contrast, discordant distributions of compounds in nectar and other tissues may suggest adaptive roles in nectar. We compared the concentrations and identities of cardenolides in the nectar, leaves and flowers of 12 species from a monophyletic clade of Asclepias. To measure putative toxicity of nectar cardenolides, we then examined the effects of a standard cardenolide (digoxin) on the behaviour of bumblebees, a common generalist pollinator of Asclepias. We found that the average cardenolide concentrations in nectar, leaves and flowers of the 12 Asclepias species were positively correlated as predicted by nonadaptive hypotheses. However, significant differences in the identities and concentrations of individual cardenolides between nectar and leaves suggest that the production or allocation of cardenolides may be independently regulated at each plant part. In addition, cardenolide concentrations in leaves and nectar exhibited no phylogenetic signal. Surprisingly, bumblebees did not demonstrate an aversion to digoxin-rich nectar, which may indicate that nectar cardenolides have little effect on pollination. Although the idea that discordant patterns of secondary metabolites across tissue types may signal adaptive functions is attractive, there is evidence to suggest constraint contributes to nectar secondary chemistry. Further work testing the ecological impacts of such patterns will be critical in determining the functional significance of nectar cardenolides

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression.

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

Variation in intensity of a parasitic mite (Spinturnix myoti) in relation to the reproductive cycle and immunocompetence of its bat host (Myotis myotis)

Given the intimate association in host-parasite systems, parasites are expected to initiate their own reproduction when vulnerable hosts become abundant and/or when adult hosts are less resistant. In this study, we examined the variation in the intensities of a blood-sucking mite (Spinturnix myoti, Acarina) with respect to the reproductive cycle and immunocompetence of its host, the greater mouse-eared bat Myotis myotis. Reproductive, pregnant females were less immunocompetent and harboured more parasites than nonreproductive females, whilst, during lactation, immunocompetence was positively associated with female body mass. There was a dramatic increase in the T-cell response of gravid females with the advancement of gestation, which coincided with a diminution of individual parasite loads and a progressive switch of parasites from adults to juveniles. The latter not only harboured greater numbers of mites than adult female bats, but they also exhibited gravid parasites in higher proportions, indicating that juvenile hosts are more attractive for parasite reproduction than adult females.

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13.

Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA(Gly) genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

Identification of pharmaceutical tablets by Raman spectroscopy and chemometrics

Raman spectroscopy has become an attractive tool for the analysis of pharmaceutical solid dosage forms. In the present study it is used to ensure the identity of tablets. The two main applications of this method are release of final products in quality control and detection of counterfeits. Twenty-five product families of tablets have been included in the spectral library and a non-linear classification method, the Support Vector Machines (SVMs), has been employed. Two calibrations have been developed in cascade: the first one identifies the product family while the second one specifies the formulation. A product family comprises different formulations that have the same active pharmaceutical ingredient (API) but in a different amount. Once the tablets have been classified by the SVM model, API peaks detection and correlation are applied in order to have a specific method for the identification and allow in the future to discriminate counterfeits from genuine products. This calibration strategy enables the identification of 25 product families without error and in the absence of prior information about the sample. Raman spectroscopy coupled with chemometrics is therefore a fast and accurate tool for the identification of pharmaceutical tablets.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Molecular and physiological characterisation of lonotropic receptors in the sacculus in drosophila

Chemosensation is the detection of chemical signals in the environment that enable an animal to make informed decisions about food choice, mate preference or predator detection. Dissecting the molecular and neural mechanisms by which animals detect chemical cues is an important goal towards understanding how they interact with the environment. An attractive system to dissect the mechanisms of chemosensation is the olfactory system. One of the most-investigated olfactory systems is that of Drosophila melanogaster, a model organism that is amenable to a powerful combination of genetic and physiological analyses. Embedded within the antennal olfactory organ of Drosophila is an unusual sensory structure called the sacculus. The sacculus is comprised of three distinct chambers, each lined with several sensilla housing two to three neurons. Previous morphological, anatomical and surgical studies of sacculus neurons have implicated sacculus neurons in chemosensation, hygrosensation and/or thermosensation. While a subset of sacculus neurons have been physiologically characterised as temperature sensors, the role of this organ has remained largely mysterious, due to its inaccessibility to peripheral electrophysiological analysis. Recently a new family of olfactory receptors, the lonotropic Receptors (IRs), was identified. Five IRs are expressed in sacculus neurons providing the first selective molecular markers for these cells. In this thesis I describe the molecular, physiological and anatomical characterisation of these neurons. Genetic labelling of specific populations of sacculus neurons with anatomical (CD8:GFP) reporters has identified neurons in sacculus chambers I and II express IR40a+IR93a together with their co- receptor IR25a, while neurons in chamber III express IR64a with its co-receptor IR8a. Both these sets of neurons project to two distinct glomeruli in the antennal lobe; IR40a neurons project to the column and arm, IR64a neurons project to DC4 and DP1m. Through a live optical imaging screen I showed that these neurons are indeed olfactory and IR64a neurons recognise acidic ligands, while IR40a neurons recognise amine ligands. IR40a and IR64a neurons are in fact composed of anatomically and physiologically distinct subpopulations, strongly implying the existence of other factors that define their functional properties. My thesis identifies the sacculus as a specialised olfactory organ capable of detecting acids and bases, which are of widespread importance to insects. The data from my thesis along with data from other labs show the sacculus is composed of different populations of olfactory sensory neurons and thermosensory neurons. Comparative genomic analysis of sacculus IRs across insects reveals them to be among the most conserved of this receptor repertoire, suggesting that the sacculus represents an evolutionarily ancient insect olfactory acid-base sensor. - La détection des produits chimiques se trouvant dans l'environnement (perception chimiosensorielle) permet à un animal de choisir sa nourriture, son partenaire ou encore d'identifier ses prédateurs. Décortiquer les mécanismes moléculaires et neuronaux grâce auxquels les animaux détectent ces signaux chimiques permet de comprendre comment ces animaux interagissent avec leur environnement. Un système intéressant pour décortiquer ces mécanismes de perception chimiosensorielle est le système olfactif, de la drosophile (Drosophila melanogaster), aussi appelée mouche du vinaigre. C'est un animal modèle très utile grâce à la combinaison d'outils génétiques puissants et d'analyses physiologiques facilement réalisables. Dans l'antenne de la drosophile, qui est l'organe olfactif principal de cet animal, se trouve une structure appelée sacculus. Celui-ci est composé de trois chambres distinctes, chacune comprenant plusieurs sensilles à l'intérieur desquelles se trouvent deux à trois neurones. De précédentes études morphologiques et anatomiques des ces neurones ont déterminé qu'ils sont impliqués dans la perception des odeurs, de l'humidité et de la température. Malgré ceci, la fonction principale de cet organe reste largement inconnue, principalement car il est inaccessible aux analyses électrophysiologiques. Récemment, une nouvelle famille de soixante-six récepteurs olfactifs, nommés Récepteurs lonotropiques (IRs), a été découverte chez la drosophile. Cinq IRs sont exprimés dans les neurones du sacculus. Pour la première fois, une sélection de marqueurs moléculaires est disponible pour l'étude de ces cellules. Dans cette thèse, les caractéristiques moléculaires, physiologiques et anatomiques des neurones du sacculus sont décrites. Ces populations de neurones situés dans le sacculus ont été marquées avec des gènes rapporteurs (CD8:GFP). Ceci a montré que les récepteurs IR40a et IR93a sont exprimés ensemble avec le co-récepteur IR25a dans les chambres I et II, tandis que les neurones de la chambre III expriment IR64a avec son co-récepteur IR8a. Ces deux groupes de neurones projettent vers deux glomérules distincts du lobe antennaire : les neurones IR40a projettent vers la column et le arm, alors que les neurones IR64a projettent vers DC4 et DP1m. Un screen d'imagerie optique a démontré que ces neurones sont en effet des neurones olfactifs, et que les neurones IR64a reconnaissent des ligands acides, tandis que les neurones IR40a reconnaissent des ligands aminés. De plus, les neurones IR40a et IR64a sont séparés en sous-populations distinctes anatomiquement et physiologiquement, et d'autres facteurs permettant de définir leurs propriétés fonctionnelles sont probablement impliqués. Cette thèse identifie ainsi le sacculus comme un organe olfactif spécialisé capable de détecter des acides et amines, lesquels sont très importants pour les insectes. Toutes les données collectées durant cette thèse, combinées aux données d'autres laboratoires, montrent que le sacculus est composé de différentes populations de neurones olfactifs et thermosenseurs. Ces IRs sont très conservés parmi les insectes, suggérant que le sacculus représente révolution d'un ancien détecteur olfactif d'acides et de bases chez l'insecte. - Tous les animaux sont capables de percevoir les signaux chimiques dans leur environnement, comme les odeurs ou le goût, via différents organes. L'odorat est le sens qui permet de percevoir les odeurs, et il est implique des neurones olfactifs qui se trouvent dans le nez des mammifères ou les antennes des insectes. La capacité d'un neurone olfactif à détecter une molécule odorante dépend des types de récepteurs olfactifs qu'il exprime. Il existe deux grandes familles de récepteurs qui perçoivent les odeurs : les Récepteurs Olfactifs, ORs, et Récepteurs lonotropiques IRs, qui détectent différents types d'odeurs avec différents mécanismes. Lorsqu'un récepteur reconnaît une molécule odorante, il convertit ce signal en un signal électrique qui est ensuite transmis au centre olfactif dans le cerveau. La drosophile (Drosophila melanogaster), aussi appelée mouche du vinaigre, est utilisée comme animal modèle pour étudier l'odorat, parce que son génome entier a été séquencé et que ses gènes sont facilement manipulables. De plus, l'anatomie du système olfactif de la mouche est similaire à celui des mammifères, malgré qu'il possède moins de neurones, ce qui le rend moins complexe. Ma thèse a pour objectif d'étudier les Récepteurs lonotropiques dans un organe spécifique, appelé le sacculus, situé dans les antennes. Les neurones du sacculus exprimant des IRs envoient leurs projections au centre olfactif du cerveau, suggérant que ces neurones perçoivent les odeurs. Une technique d'imagerie optique a été utilisée sur le cerveau de mouches vivantes afin de mesurer la réponse des neurones du le sacculus à différentes odeurs. J'ai démontré que ces récepteurs détectent des acides et des amines, qui sont très importants pour les insectes. Par exemple, les acides se retrouvent dans les fruits mûrs sur lesquels les mouches vont se nourrir, s'accoupler et poser leurs oeufs, et les amines sont souvent produites par des bactéries pouvant être nuisible pour la mouche. La principale découverte de ma thèse est donc l'identification du sacculus comme un organe capable de détecter deux des principales odeurs importantes pour la mouche. Ces récepteurs sont aussi présents dans d'autres insectes où ils jouent peut-être des rôles différents. Les acides et les amines se retrouvent aussi dans les excrétions (comme la sueur ou l'urine) de beaucoup de mammifères, qui pourraient potentiellement être dangereux pour la mouche, mais qui attirent les moustiques se nourrissant de leur sang.

Harmonic active contours.

We propose a segmentation method based on the geometric representation of images as 2-D manifolds embedded in a higher dimensional space. The segmentation is formulated as a minimization problem, where the contours are described by a level set function and the objective functional corresponds to the surface of the image manifold. In this geometric framework, both data-fidelity and regularity terms of the segmentation are represented by a single functional that intrinsically aligns the gradients of the level set function with the gradients of the image and results in a segmentation criterion that exploits the directional information of image gradients to overcome image inhomogeneities and fragmented contours. The proposed formulation combines this robust alignment of gradients with attractive properties of previous methods developed in the same geometric framework: 1) the natural coupling of image channels proposed for anisotropic diffusion and 2) the ability of subjective surfaces to detect weak edges and close fragmented boundaries. The potential of such a geometric approach lies in the general definition of Riemannian manifolds, which naturally generalizes existing segmentation methods (the geodesic active contours, the active contours without edges, and the robust edge integrator) to higher dimensional spaces, non-flat images, and feature spaces. Our experiments show that the proposed technique improves the segmentation of multi-channel images, images subject to inhomogeneities, and images characterized by geometric structures like ridges or valleys.