Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Parent-child agreement and prevalence estimates of diagnoses in childhood: direct interview versus family history method.

Diagnostic information on children is typically elicited from both children and their parents. The aims of the present paper were to: (1) compare prevalence estimates according to maternal reports, paternal reports and direct interviews of children [major depressive disorder (MDD), anxiety and attention-deficit and disruptive behavioural disorders]; (2) assess mother-child, father-child and inter-parental agreement for these disorders; (3) determine the association between several child, parent and familial characteristics and the degree of diagnostic agreement or the likelihood of parental reporting; (4) determine the predictive validity of diagnostic information provided by parents and children. Analyses were based on 235 mother-offspring, 189 father-offspring and 128 mother-father pairs. Diagnostic assessment included the Kiddie-schedule for Affective Disorders and Schizophrenia (K-SADS) (offspring) and the Diagnostic Interview for Genetic Studies (DIGS) (parents and offspring at follow-up) interviews. Parental reports were collected using the Family History - Research Diagnostic Criteria (FH-RDC). Analyses revealed: (1) prevalence estimates for internalizing disorders were generally lower according to parental information than according to the K-SADS; (2) mother-child and father-child agreement was poor and within similar ranges; (3) parents with a history of MDD or attention deficit hyperactivity disorder (ADHD) reported these disorders in their children more frequently; (4) in a sub-sample followed-up into adulthood, diagnoses of MDD, separation anxiety and conduct disorder at baseline concurred with the corresponding lifetime diagnosis at age 19 according to the child rather than according to the parents. In conclusion, our findings support large discrepancies of diagnostic information provided by parents and children with generally lower reporting of internalizing disorders by parents, and differential reporting of depression and ADHD by parental disease status. Follow-up data also supports the validity of information provided by adolescent offspring.

Giacomo Becattini and the Marshall's method. A Schumpeterian approach

The studies of Giacomo Becattini concerning the notion of the "Marshallian industrial district" have led a revolution in the field of economic development around the world. The paper offers an interpretation of the methodology adopted by Becattini. The roots are clearly Marshallian. Becattini proposes a return to the economy as a complex social science that operates in historical time. We adopt a Schumpeterian approach to the method in economic analysis in order to highlight the similarities between the Marshall and Becattini's approach. Finally the paper uses the distinction between logical time, real time and historical time which enable us to study the "localized" economic process in a Becattinian way.

A new method for geoelectrical investigations underwater

A new electrical method is proposed for determining the apparent resistivity of multi-earth layers located underwater. The method is based on direct current geoelectric sounding principles. A layered earth model is used to simulate the stratigraphic target. The measurement array is of pole-pole type; it is located underwater and is orientated vertically. This particular electrode configuration is very useful when conventional electrical methods cannot be used, especially if the water depth becomes very important. The calculated apparent resistivity shows a substantial quality increase in the measured signal caused by the underwater targets, from which little or no response is measured using conventional surface electrode methods. In practice, however, different factors such as water stratification, underwater streams or meteorological conditions complicate the interpretation of the field results. A case study is presented, where field surveys carried out on Lake Geneva were interpreted using the calculated apparent resistivity master-curves.

Application of the combined integral method to Stefan problems

In this paper we present a new, accurate form of the heat balance integral method, termed the Combined Integral Method (or CIM). The application of this method to Stefan problems is discussed. For simple test cases the results are compared with exact and asymptotic limits. In particular, it is shown that the CIM is more accurate than the second order, large Stefan number, perturbation solution for a wide range of Stefan numbers. In the initial examples it is shown that the CIM reduces the standard problem, consisting of a PDE defined over a domain specified by an ODE, to the solution of one or two algebraic equations. The latter examples, where the boundary temperature varies with time, reduce to a set of three first order ODEs.

Identification of Mycobacteria by Thin Layer Chromatographic Analysis of Mycolic Acids and Conventional Biochemical Method: Four Years of Experience

Mycolic acids analysis by thin-layer chromatography (TLC) has been employed by several laboratories worldwide as a method for fast identification of mycobacteria. This method was introduced in Brazil by our laboratory in 1992 as a routine identification technique. Up to the present, 861 strains isolated were identified by mycolic acids TLC and by standard biochemical tests; 61% out of these strains came as clinical samples, 4% isolated from frogs and 35% as environmental samples. Mycobacterium tuberculosis strains identified by classical methods were confirmed by their mycolic acids contents (I, III and IV). The method allowed earlier differentiation of M. avium complex - MAC (mycolic acids I, IV and VI) from M. simiae (acids I, II and IV), both with similar biochemical properties. The method also permitted to distinguish M. fortuitum (acids I and V) from M. chelonae (acids I and II) , and to detect mixed mycobacterial infections cases as M. tuberculosis with MAC and M. fortuitum with MAC. Concluding, four years experience shows that mycolic acids TLC is an easy, reliable, fast and inexpensive method, an important tool to put together conventional mycobacteria identification methods.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

An approximate solution method for boundary layer flow of a power law fluid over a flat plate

The work in this paper deals with the development of momentum and thermal boundary layers when a power law fluid flows over a flat plate. At the plate we impose either constant temperature, constant flux or a Newton cooling condition. The problem is analysed using similarity solutions, integral momentum and energy equations and an approximation technique which is a form of the Heat Balance Integral Method. The fluid properties are assumed to be independent of temperature, hence the momentum equation uncouples from the thermal problem. We first derive the similarity equations for the velocity and present exact solutions for the case where the power law index n = 2. The similarity solutions are used to validate the new approximation method. This new technique is then applied to the thermal boundary layer, where a similarity solution can only be obtained for the case n = 1.

Conservation of a threatened European tree frog (Hyla arborea) metapopulation

Summary: Amphibians are among the most vulnerable animals of the world. One third of all species are currently threatened with extinction. Habitat loss is the major menace to pond- and stream-breeding species in the old world. In highly urbanized landscape like the Swiss Plateau, most species suffer from habitat reduction and fragmentation. Among all indigenous species, the European tree frog (Hyla arborea L., 1758) is one of the most endangered. It experienced an alarming decline during the last century and its regional long-term persistence is not guaranteed. We developed a monitoring framework based on calling male counts which included multiple visits to each wetland during the reproduction period in order to precisely determine its distribution on the Lemanic coast. Our results indicate that visiting populations 3 limes under suitable climatic conditions (temperature >20°C) provides reliable presence/absence data. Based on our monitoring data, we analyzed the species requirements regarding its breeding habitat. It appeared that anthropogenic activities had paradoxical effects on the species. On one hand, urbanization, traffic and intensive agriculture had a strong detrimental effect on tree frog distribution. On the other hand, large tree frog populations were frequently associated with gravel pits and military training grounds. Our results allowed us to create a habitat suitability map taking into account detrimental landscape elements around ponds (>1100m away from urban areas and >500m away from first class roads). In parallel, we developed a metapopulation model of the European tree frog in order to identify the critical threats to the long term persistence of the species. Our results indicated that suitable pond density is at the low end of the species requirements. Pond creation must therefore be considered an essential complementary approach to pond conservation and restoration. Our model also provided a mapping solution permitting the location of the must suitable area for pond creation from a metapopulation perspective. As many other amphibians, the European tree frog is not only exposed to an aquatic habitat (breeding and larval period), but also to a terrestrial stage (summer and overwintering habitats). Unfortunately, animals in their terrestrial phase are less conspicuous and, as a consequence, their terrestrial needs are relatively unknown. Using a recent tracking method (the Harmonic Direction Finder), we followed post-breeding frogs and identified favored terrestrial habitats, thus providing another practical conservation tool. We conclude that only the combination of multiple spatially explicit approaches (landscape-scale habitat suitability, metapopulation dynamics and terrestrial needs) is likely to provide wildlife managers with effective tools for the conservation of highly endangered amphibians. Résumé: Les amphibiens font partie des animaux les plus vulnérables du monde. Un tiers des espèces est actuellement menacé d'extinction. Dans l'ancien monde, la disparition des habitats constitue la principale menace pour les grenouilles, crapauds, tritons et salamandres. Dans les paysages fortement urbanisés comme le Plateau Suisse, la plupart des espèces souffrent d'une réduction et d'une fragmentation de leurs habitats. Parmi toutes les espèces indigènes, la rainette verte (Hyla arborea L., 1758) est l'une des plus menacée. Sa distribution a régressé de manière alarmante durant le siècle passé et sa survie régionale à long terme n'est pas assurée. Nous avons développé une méthode de suivi des populations se basant sur le comptage des mâles chanteurs durant la période de reproduction. Cette méthode requiert plusieurs visites à chaque plan d'eau de manière à déterminer précisément la distribution de l'espèce. Nos résultats démontrent que 3 visites par population dans des conditions climatiques favorable (température >20°C) permettent d'obtenir des données de présence/ absence valables. Sur la base de nos comptages sur la Côte lémanique, nous avons analysé les exigences de l'espèce concernant ses sites de reproduction. Il est apparu que les activités humaines avaient un effet paradoxal sur l'espèce. D'une part, l'urbanisation, le trafic routier et l'intensification de l'agriculture ont un effet fortement préjudiciable, tandis que d'autre part les plus grandes populations sont souvent associées à des gravières et autres places d'armes. Nos résultats ont permis de créer une carte de qualité d'habitat prenant en compte les éléments paysagers préjudiciables à la rainette (situé à plus de 1100m de zones urbaines et à plus de 500m de routes de première classe). En parallèle, nous avons développé un modèle métapopulationnel (incluant l'ensemble des populations) de manière à identifier les menaces prépondérantes sur la survie à long terme de l'espèce. Nos résultats ont permis de déterminer que la densité actuelle de plans d'eau adéquats est à la limite inférieure des exigences de l'espèce. La création d'étangs doit donc être considérée comme une approche indispensable et complémentaire à la protection et à la restauration des sites existants. Notre modèle a également fourni des résultats cartographiables permettant l'identification des sites les plus appropriés dans une perspective métapopulationnelle. Comme de nombreux autres amphibiens, la rainette verte est exposée à un habitat aquatique (reproduction et développement larvaire) ainsi qu'à un habitat terrestre (été et hiver). Les animaux étant particulièrement cryptiques dans cette seconde phase, leurs besoins terrestres sont relativement mal connus. Nous avons donc développé une nouvelle méthode de télémétrie basée sur le goniomètre harmonique. Cette méthode nous a permis de suivre des rainettes dans leurs migrations jusqu'à leurs habitats d'été et d'établir ainsi des recommandations pratiques pour la conservation de la rainette. Nous concluons que la combinaison de multiples approches spatialement explicites (qualité d'habitat, dynamique de métapopulation et habitats terrestres) est seule à même de produire des outils efficaces pour la conservation des espèces menacées d'amphibiens.

Development of a novel headspace sorptive extraction method to study the aging of volatile compounds in spent handgun cartridges

Estimating the time since the last discharge of firearms and/or spent cartridges may be a useful piece of information in forensic firearm-related cases. The current approach consists of studying the diffusion of selected volatile organic compounds (such as naphthalene) released during the shooting using solid phase micro-extraction (SPME). However, this technique works poorly on handgun car-tridges because the extracted quantities quickly fall below the limit of detection. In order to find more effective solutions and further investigate the aging of organic gunshot residue after the discharge of handgun cartridges, an extensive study was carried out in this work using a novel approach based on high capacity headspace sorptive extraction (HSSE). By adopting this technique, for the first time 51 gunshot residue (GSR) volatile organic compounds could be simultaneously detected from fired handgun cartridge cases. Application to aged specimens showed that many of those compounds presented significant and complementary aging profiles. Compound-to-compound ratios were also tested and proved to be beneficial both in reducing the variability of the aging curves and in enlarging the time window useful in a forensic casework perspective. The obtained results were thus particularly promising for the development of a new complete forensic dating methodology.

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3.3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46.5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le résultat d'une lésion ou d'un dysfonctionnement du système nerveux. Les symptômes qui suivent la douleur neuropathique sont sévères et leur traitement inefficace. Une meilleure approche thérapeutique peut être proposée en se basant sur les mécanismes pathologiques de la douleur neuropathique. Lors d'une lésion périphérique une douleur neuropathique peut se développer et affecter le territoire des nerfs lésés mais aussi les territoires adjacents des nerfs non-lésés. Une hyperexcitabilité des neurones apparaît au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle épinière. Le but de ce travail consiste à mettre en évidence les modifications moléculaires associées aux nocicepteurs lésés et non-lésés au niveau des DRG et des laminae I et II de la corne dorsale, là où l'information nociceptive est intégrée. Pour étudier les changements moléculaires liés à la douleur neuropathique nous utilisons le modèle animal d'épargne du nerf sural (spared nerve injury model, SNI) une semaine après la lésion. Pour la sélection du tissu d'intérêt nous avons employé la technique de la microdissection au laser, afin de sélectionner une sous-population spécifique de cellules (notamment les nocicepteurs lésés ou non-lésés) mais également de prélever le tissu correspondant dans les laminae superficielles. Ce travail est couplé à l'analyse à large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons étudié les courants électriques et les propriétés biophysiques des canaux sodiques (Na,,ls) dans les neurones lésés et non-lésés des DRG. Aussi bien dans le système nerveux périphérique, entre les neurones lésés et non-lésés, qu'au niveau central avec les aires recevant les projections des nocicepteurs lésés ou non-lésés, l'analyse du transcriptome montre des différences de profil d'expression. En effet, nous avons constaté des changements transcriptionnels importants dans les nocicepteurs lésés (1561 gènes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 gènes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-lésés (60 gènes), mais important dans leurs laminae de projection (459 gènes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observé que plusieurs processus biologiques sont modifiés. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les mécanismes de réponse au stress sont affectés. Par contre dans les neurones non-lésés seuls les processus biologiques liés au métabolisme et au développement sont modifiés. Au niveau de la corne dorsale de la moelle, nous avons observé des modifications importantes des processus immuno-inflammatoires dans l'aire affectée par les nerfs lésés et des changements associés à l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-lésés. L'analyse approfondie des canaux sodiques a démontré plusieurs changements d'expression, principalement dans les neurones lésés. Les analyses fonctionnelles n'indiquent aucune différence entre les densités de courant tétrodotoxine-sensible (TTX-S) dans les neurones lésés et non-lésés même si les niveaux d'expression des ARNm des sous-unités TTX-S sont modifiés dans les neurones lésés. L'inactivation basale dépendante du voltage des canaux tétrodotoxine-insensible (TTX-R) est déplacée vers des potentiels positifs dans les cellules lésées et non-lésées. En revanche la vitesse de récupération des courants TTX-S et TTX-R après inactivation est accélérée dans les neurones lésés. Ces changements pourraient être à l'origine de l'altération de l'activité électrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En résumé, ces résultats suggèrent l'existence de mécanismes différenciés affectant les neurones lésés et les neurones adjacents non-lésés lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle épinière qui surviennent après lésion sont probablement intégrés différemment selon la perception de signaux des neurones périphériques lésés ou non-lésés. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqués dans la genèse et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied functionál properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Standartization of broth microdilution method for Mycobacterium tuberculosis

Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100% for all drugs and specifities of 91, 100, 96, 98 and 85% were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days) than the proportional method (20-28 days). The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.