Stability and robustness of blood variables in an antidoping context.

Introduction:  With the setting up of the newly Athlete's Biological Passport antidoping programme, novel guidelines have been introduced to guarantee results beyond reproach. We investigated in this context, the effect of storage time on the variables commonly measured for the haematological passport. We also wanted to assess for these variables, the within and between analyzer variations. Methods:  Blood samples were obtained from top level male professional cyclists (27 samples for the first part of the study and 102 for the second part) taking part to major stage races. After collection, they were transported under refrigerated conditions (2 °C < T < 12 °C), delivered to the antidoping laboratory, analysed and then stored at approximately 4 °C to conduct analysis at different time points up to 72 h after delivery. A mixed-model procedure was used to determine the stability of the different variables. Results:  As expected haemoglobin concentration was not affected by storage and showed stability for at least 72 h. Under the conditions of our investigation, the reticulocytes percentage showed a much better stability than previous published data (> 48 h) and the technical comparison of the haematology analyzer demonstrated excellent results. Conclusion:  In conclusion, our data clearly demonstrate that as long as the World Anti-Doping Agency's guidelines are followed rigorously, all blood results reach the quality level required in the antidoping context.

Lon protease negatively affects GacA protein stability and expression of the Gac/Rsm signal transduction pathway in Pseudomonas protegens.

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP-dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady-state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild-type cells. In starved cells, the loss of Lon function prolonged the half-life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.

Career decision making, stability and actualization of career intentions : the case of entrepreneurial intentions

Career counselors are often concerned with stability and likelihood of implementation of clients' career intentions. It is often assumed that the status in career decision making (CDM) is one likely indicator; yet, empirical support for this assumption is sparse. The present study focused on entrepreneurial career intentions (EI) and showed that German university students (N = 1,221), with high EI can be found in very different empirically derived CDM statuses that range from preconcern to mature decidedness. Longitudinal analyses (n = 561) showed that career choice foreclosure (high decidedness/low exploration) related to more EI stability and that mature decidedness (high decidedness/high exploration) amplified effects of EI on opportunity identification, a form of EI actualization. The results imply that CDM statuses are useful to estimate stability and actualization of career intentions.

Stability and change in party preferences.

This article analyses stability and volatility of party preferences using data from the Swiss Household-Panel (SHP), which, for the first time, allow studying transitions and stability of voters over several years in Switzerland. Analyses cover the years 1999- 2007 and systematically distinguish changes between party blocks and changes within party blocks. The first part looks at different patterns of change, which show relatively high volatility. The second part tests several theories on causes of such changes applying a multinomial random-effects model. Results show that party preferences stabilise with their duration and with age and that the electoral cycle, political sophistication, socio-structural predispositions, the household-context as well as party size and the number of parties each explain part of electoral volatility. Different results for withinand between party-block changes underlie the importance of that differentiation.

Thyroid hormones stimulate expression and modification of cytoskeletal protein during rat sciatic nerve regeneration.

Peripheral neurons can regenerate after axotomy; in this process, the role of cytoskeletal proteins is important because they contribute to formation and reorganization, growth, transport, stability and plasticity of axons. In the present study, we examined the effects of thyroid hormones (T3) on the expression of major cytoskeletal proteins during sciatic nerve regeneration. At various times after sciatic nerve transection and T3 local administration, segments of operated nerves from T3-treated rats and control rats were examined by Western blotting for the presence of neurofilament, tubulin and vimentin. Our results revealed that, during the first week after surgery, T3 treatment did not significantly alter the level of NF subunits and tubulin in the different segments of operated nerves compared to control nerves. Two or 4 weeks after operation, the concentration of NF-H and NF-M isoforms was clearly increased by T3 treatment. Moreover, under T3-treatment, NF proteins appeared more rapidly in the distal segment of operated nerves. Likewise, the levels of betaIII, and of acetylated and tyrosinated tubulin isotypes, were also up-regulated by T3-treatment during regeneration. However, only the tyrosinated tubulin form appeared earlier in the distal nerve segments. At this stage of regeneration, T3 had no effect on the level of vimentin expression. In conclusion, thyroid hormone improves and accelerates peripheral nerve regeneration and exerts a positive effect on cytoskeletal protein expression and transport involved in axonal regeneration. These results help us to understand partially the mechanism by which thyroid hormones enhance peripheral nerve regeneration. The stimulating effect of T3 on peripheral nerve regeneration may have considerable therapeutic potential.

Stochastic stability and the evolution of coordination in spatially structured populations.

Animals can often coordinate their actions to achieve mutually beneficial outcomes. However, this can result in a social dilemma when uncertainty about the behavior of partners creates multiple fitness peaks. Strategies that minimize risk ("risk dominant") instead of maximizing reward ("payoff dominant") are favored in economic models when individuals learn behaviors that increase their payoffs. Specifically, such strategies are shown to be "stochastically stable" (a refinement of evolutionary stability). Here, we extend the notion of stochastic stability to biological models of continuous phenotypes at a mutation-selection-drift balance. This allows us to make a unique prediction for long-term evolution in games with multiple equilibria. We show how genetic relatedness due to limited dispersal and scaled to account for local competition can crucially affect the stochastically-stable outcome of coordination games. We find that positive relatedness (weak local competition) increases the chance the payoff dominant strategy is stochastically stable, even when it is not risk dominant. Conversely, negative relatedness (strong local competition) increases the chance that strategies evolve that are neither payoff nor risk dominant. Extending our results to large multiplayer coordination games we find that negative relatedness can create competition so extreme that the game effectively changes to a hawk-dove game and a stochastically stable polymorphism between the alternative strategies evolves. These results demonstrate the usefulness of stochastic stability in characterizing long-term evolution of continuous phenotypes: the outcomes of multiplayer games can be reduced to the generic equilibria of two-player games and the effect of spatial structure can be analyzed readily.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Fanconi anemia and genome stability : the role of FANCM

RÉSUMÉ: Le génome de toute cellule est susceptible d'être attaqué par des agents endogènes et exogènes. Afin de préserver l'intégrité génomique, les cellules ont développé des multitudes de mécanismes. La réplication de l'ADN, une étape importante durant le cycle cellulaire, constitue un stress et présente un danger important pour l'intégrité du génome. L'anémie de Fanconi est une maladie héréditaire rare dont les protéines impliquées semblent jouer un rôle crucial dans la réponse au stress réplicatif. La maladie est associée à une instabilité chromosomique ainsi qu'à une forte probabilité de développer des cancers. Les cellules des patients souffrant de l'anémie de Fanconi sont sensibles à des agents interférant avec la réplication de l'ADN, et plus particulièrement àdes agents qui fient les deux brins d'ADN d'une manière covalente. L'anémie de Fanconi est une maladie génétiquement hétérogène. Treize protéines ont pu être identifiées. Elles semblent figurer dans une même voie de signalisation qui est aussi connue sous le nom de « FA/BRCA pathway », car un des gènes est identique au gène BRCA2 (breast cancer susceptibility gene 2). Huit protéines forment un complexe nucléaire dont l'intégrité est nécessaire à la monoubiquitination de deux autres protéines, FANCD2 et FANCI, en réponse à un stress réplicatif. A ce jour, la fonction moléculaire des protéines du « FA/BRCA pathway »reste encore mal décrite. Au début de mon travail de thèse, nous avons donc décidé de purifier les protéines du complexe nucléaire et d'étudier leurs propriétés biochimiques. Nous avons tout d'abord étudié les cinq protéines connues à l'époque qui sont FANCA, FANCC, FANCE, FANCF et FANCG. Par la suite, nous avons étendu notre étude à des protéines découvertes plus récemment, FANCL, FANCM et FAAP24, en concentrant finalement notre travail sur la caractérisation de FANCM. FANCM, contrairement aux autres protéines du complexe, est constituée de deux domaines conservés suggérant un rôle important dans le métabolisme de l'ADN. Il s'agit d'un domaine « DEAH box hélicase »situé dans la partie N-terminale et d'un domaine « ERCC4 nuclease »situé dans la partie C-terminale de la protéine. Dans cette étude, nous avons purifié avec succès la protéine FANCM entière à partir d'un système hétérologue. Nous montrons que FANCM s'attache de manière spécifique à des jonctions de Holliday et des fourches de réplication. De plus, nous démontrons que FANCM peut déplacer le point de jonction de ces structures via son domaine hélicase de manière dépendante de l'ATP. FANCM est aussi capable de dissocier de grands intermédiaires de la recombinaison, via la migration de jonctions de Holliday à travers une région d'homologie de 2.6 kb. Tous ces résultats suggèrent que FANCM peut s'attacher spécifiquement à des fourches de réplication et à des jonctions de Holliday in vitro et que son domaine hélicase est associé à une activité migratoire efficace. Nous pensons que FANCM peut avoir un rôle direct sur les intermédiaires de réplication. Ceci est en accord avec l'idée que les protéines de l'anémie de Fanconi coordonnent la réparation de l'ADN au niveau des fourches de réplication arrêtées. Nos résultats donnent une première indication quant au rôle de FANCM dans la cellule et peuvent contribuer à élucider la fonction de cette voie de signalisation peu comprise jusqu'à présent. SUMMARY: The genome of every cell is subject to a constant offence by endogenous and exogenous agents. Not surprisingly; cells have evolved a multitude of mechanisms which aim at preserving genomic integrity. A key step during the life cycle of a cell, DNA replication itself, constitutes a special danger to the integrity of the genome. The proteins defective in the rare hereditary disease Fanconi anemia (FA) are suspected to play a crucial role in the cellular response to DNA replication stress. The disease is associated with chromosomal instability and pronounced cancer susceptibility. Cells from Fanconi anemia patients are sensitive to a variety of agents which interfere with DNA replication, DNA interstrand cross-linking agents being particularly threatening to their survival. Fanconi anemia is a genetically heterogeneous disease with 13 different proteins identified, which seem to work together in a common pathway. Since one of the FA genes is identical to the breast cancer susceptibility gene BRCA2, it is also referred to as the FA/BRCA pathway. Eight proteins form a nuclear complex, whose integriry is required for the monoubiquitination of two other FA proteins, FANCD2 and FANCI, in response to DNA replication stress. Despite intensive research, the function of the FA/BRCA pathway at a molecular level has remained largely elusive so far. At the beginning of my thesis, we therefore decided to purify the proteins of the FA core complex and to investigate their biochemical properties. We started with the five proteins which were known at that time, FANCA, FANCC, FANCE, FANCF, and FACG. Later on, we extended our studies to the newly discovered proteins FANCL, FANCM, and FAAP24, and eventually focused our work on the characterisation of FANCM. In contrast to the other core complex proteins, FANCM contains two conserved domains, which point to a role in DNA metabolism: an N-terminal DEAH box helicase domain and a C-terminal ERCC4 nuclease domain. In this study, we have successfully purified full-length FANCM from a recombinant source. We show that purified FANCM binds to branched DNA molecules, such as Holliday junctions and replication forks, with high specificity and affinity. In addition, we demonstrate that FANCM can translocate the junction point of branched DNA molecules due to its helicase domain in an ATPase-dependent manner. FANCM can even dissociate large recombination intermediates, via branch migration of Holliday junctions through a 2.6 kb region of homology. Taken together, our data suggest that FANCM can specifically bind to replication forks and Holliday junctions in vitro, and that its DEAH box helicase domain is associated with a potent branch migration activity. We propose that FANCM might have a direct role in the processing of DNA replication intermediates. This is consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks. Our findings provide a first hint as to the context in which FANCM might play a role in the cell. We are optimistic that they might be key to further elucidate the function of a pathway which is far from being understood.

MRE11-RAD50-NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair.

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Design and evaluation of a solid sampler for the monitoring of airborne 1,6-hexamethylene diisocyanate (HDI) and its prepolymers in 2-component spray painting

An active, solvent-free solid sampler was developed for the collection of 1,6-hexamethylene diisocyanate (HDI) aerosol and prepolymers. The sampler was made of a filter impregnated with 1-(2-methoxyphenyl)piperazine contained in a filter holder. Interferences with HDI were observed when a set of cellulose acetate filters and a polystyrene filter holder were used; a glass fiber filter and polypropylene filter cassette gave better results. The applicability of the sampling and analytical procedure was validated with a test chamber, constructed for the dynamic generation of HDI aerosol and prepolymers in commercial two-component spray paints (Desmodur(R) N75) used in car refinishing. The particle size distribution, temporal stability, and spatial uniformity of the simulated aerosol were established in order to test the sample. The monitoring of aerosol concentrations was conducted with the solid sampler paired to the reference impinger technique (impinger flasks contained 10 mL of 0.5 mg/mL 1-(2-methoxyphenyl)piperazine in toluene) under a controlled atmosphere in the test chamber. Analyses of derivatized HDI and prepolymers were carried out by using high-performance liquid chromatography and ultraviolet detection. The correlation between the solvent-free and the impinger techniques appeared fairly good (Y = 0.979X - 0.161; R = 0.978), when the tests were conducted in the range of 0.1 to 10 times the threshold limit value (TLV) for HDI monomer and up to 60-mu-g/m3 (3 U.K. TLVs) for total -N = C = O groups.

Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL.

Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.

The longitudinal association between social functioning and theory of mind in first episode psychosis

Introduction. There is some cross-sectional evidence that theory of mind ability is associated with social functioning in those with psychosis but the direction of this relationship is unknown. This study investigates the longitudinal association between both theory of mind and psychotic symptoms and social functioning outcome in first-episode psychosis. Methods. Fifty-four people with first-episode psychosis were followed up at 6 and 12 months. Random effects regression models were used to estimate the stability of theory of mind over time and the association between baseline theory of mind and psychotic symptoms and social functioning outcome. Results. Neither baseline theory of mind ability (regression coefficients: Hinting test 1.07 95% CI 0.74, 2.88; Visual Cartoon test 2.91 95% CI 7.32, 1.51) nor baseline symptoms (regression coefficients: positive symptoms 0.04 95% CI 1.24, 1.16; selected negative symptoms 0.15 95% CI 2.63, 2.32) were associated with social functioning outcome. There was evidence that theory of mind ability was stable over time, (regression coefficients: Hinting test 5.92 95% CI 6.66, 8.92; Visual Cartoon test score 0.13 95% CI 0.17, 0.44). Conclusions. Neither baseline theory of mind ability nor psychotic symptoms are associated with social functioning outcome. Further longitudinal work is needed to understand the origin of social functioning deficits in psychosis.

Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform.

BACKGROUND: Hepcidin, a 25 amino acid peptide, plays an important role in iron homeostasis. Some hepcidin truncated peptides have antibiotic effects. RESULTS: A new analytical method for hepcidin determination in human plasma using LC-HRMS operating in full-scan acquisition mode has been validated. The extraction consists of protein precipitation and a drying reconstitution step; a 2.1 x 50 mm (idxL) C18 analytical column was used. Detection specificity, stability, accuracy, precision and recoveries were determined. The LOQ/LOD were 0.25/0.1 nM, respectively. More than 600 injections of plasma extracts were performed, allowing evaluation of the assay robustness. Hepcidin-20, hepcidin-22 and a new isoform, hepcidin-24, were detected in patients. CONCLUSION: The data underscore the usefulness of LC-HRMS for in-depth investigations related to hepcidin levels and pathways.