Genetics of cadmium hyperaccumulation variance in Thlaspi caerulescens natural populations from Switzerland

SUMMARY Heavy metal presence in the environment is a serious concern since some of them can be toxic to plants, animals and humans once accumulated along the food chain. Cadmium (Cd) is one of the most toxic heavy metal. It is naturally present in soils at various levels and its concentration can be increased by human activities. Several plants however have naturally developed strategies allowing them to grow on heavy metal enriched soils. One of them consists in the accumulation and sequestration of heavy metals in the above-ground biomass. Some plants present in addition an extreme strategy by which they accumulate a limited number of heavy metals in their shoots in amounts 100 times superior to those expected for a non-accumulating plant in the same conditions. Understanding the genetic basis of the hyperaccumulation trait - particularly for Cd - remains an important challenge which may lead to biotechnological applications in the soil phytoremediation. In this thesis, Thlaspi caerulescens J. & C. Presl (Brassicaceae) was used as a model plant to study the Cd hyperaccumulation trait, owing to its physiological and genetic characteristics. Twenty-four wild populations were sampled in different regions of Switzerland. They were characterized for environmental and soil parameters as well as intrinsic characteristics of plants (i.e. metal concentrations in shoots). They were as well genetically characterized by AFLPs, plastid DNA polymorphism and genes markers (CAPS and microsatellites) mainly developed in this thesis. Some of the investigated genes were putatively linked to the Cd hyperaccumulation trait. Since the study of the Cd hyperaccumulation in the field is important as it allows the identification of patterns of selection, the present work offered a methodology to define the Cd hyperaccumulation capacity of populations from different habitats permitting thus their comparison in the field. We showed that Cd, Zn, Fe and Cu accumulations were linked and that populations with higher Cd hyperaccumulation capacity had higher shoot and reproductive fitness. Using our genetic data, statistical methods (Beaumont & Nichols's procedure, partial Mantel tests) were applied to identify genomic signatures of natural selection related to the Cd hyperaccumulation capacity. A significant genetic difference between populations related to their Cd hyperaccumulation capacity was revealed based on somè specific markers (AFLP and candidate genes). Polymorphism at the gene encoding IRTl (Iron-transporter also participating to the transport of Zn) was suggested as explaining part of the variation in Cd hyperaccumulation capacity of populations supporting previous physiological investigations. RÉSUMÉ La présence de métaux lourds dans l'environnement est un phénomène préoccupant. En effet, certains métaux lourds - comme le cadmium (Cd) -sont toxiques pour les plantes, les animaux et enfin, accumulés le long de la chaîne alimentaire, pour les hommes. Le Cd est naturellement présent dans le sol et sa concentration peut être accrue par différentes activités humaines. Certaines plantes ont cependant développé des stratégies leur permettant de pousser sur des sols contaminés en métaux lourds. Parmi elles, certaines accumulent et séquestrent les métaux lourds dans leurs parties aériennes. D`autres présentent une stratégie encore plus extrême. Elles accumulent un nombre limité de métaux lourds en quantités 100 fois supérieures à celles attendues pour des espèces non-accumulatrices sous de mêmes conditions. La compréhension des bases génétiques de l'hyperaccumulation -particulièrement celle du Cd - représente un défi important avec des applications concrètes en biotechnologies, tout particulièrement dans le but appliqué de la phytoremediation des sols contaminés. Dans cette thèse, Thlaspi caerulescens J. & C. Presl (Brassicaceae) a été utilisé comme modèle pour l'étude de l'hyperaccumulation du Cd de par ses caractéristiques physiologiques et génétiques. Vingt-quatre populations naturelles ont été échantillonnées en Suisse et pour chacune d'elles les paramètres environnementaux, pédologique et les caractéristiques intrinsèques aux plantes (concentrations en métaux lourds) ont été déterminés. Les populations ont été caractérisées génétiquement par des AFLP, des marqueurs chloroplastiques et des marqueurs de gènes spécifiques, particulièrement ceux potentiellement liés à l'hyperaccumulation du Cd (CAPS et microsatellites). La plupart ont été développés au cours de cette thèse. L'étude de l'hyperaccumulation du Cd en conditions naturelles est importante car elle permet d'identifier la marque, éventuelle de sélection naturelle. Ce travail offre ainsi une méthodologie pour définir et comparer la capacité des populations à hyperaccumuler le Cd dans différents habitats. Nous avons montré que les accumulations du Cd, Zn, Fe et Cu sont liées et que les populations ayant une grande capacité d'hyperaccumuler le Cd ont également une meilleure fitness végétative et reproductive. Des méthodes statistiques (l'approche de Beaumont & Nichols, tests de Martel partiels) ont été utilisées sur les données génétiques pour identifier la signature génomique de la sélection naturelle liée à la capacité d'hyperaccumuler le Cd. Une différenciation génétique des populations liée à leur capacité d'hyperaccumuler le Cd a été mise en évidence sur certains marqueurs spécifiques. En accord avec les études physiologiques connues, le polymorphisme au gène codant IRT1 (un transporteur de Fe impliqué dans le transport du Zn) pourrait expliquer une partie de la variance de la capacité des populations à hyperaccumuler le Cd.

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses.

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.

A methodology for the adaptation of a PMP at the determination of a PMF

This paper presents a new type of very fine grid hydrological model based on the spatiotemporal repartition of a PMP (Probable Maximum Precipitation) and on the topography. The goal is to estimate the influence of this rain on a PMF (Probable Maximum Flood) on a catchment area in Switzerland. The spatiotemporal distribution of the PMP was realized using six clouds modeled by the advection-diffusion equation. The equation shows the movement of the clouds over the terrain and also gives the evolution of the rain intensity in time. This hydrological modeling is followed by a hydraulic modeling of the surface and subterranean flow, done considering the factors that contribute to the hydrological cycle, such as the infiltration, the resurgence and the snowmelt. These added factors make the developed model closer to reality and also offer flexibility in the initial condition that is added to the factors concerning the PMP, such as the duration of the rain, the speed and direction of the wind. All these initial conditions taken together offer a complete image of the PMF.

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.

Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome.

Cell-free translation of total RNA isolated from vaccinia virus-infected cells late in infection results in a complex mixture of polypeptides. A monospecific antibody directed against one of the major structural proteins of the virus particle immunoprecipitated a single polypeptide with a molecular weight of 11,000 (11K) from this mixture. Immunoprecipitation was therefore used to identify the structural polypeptide among the in vitro translation products of RNA purified by hybridization selection to restriction fragments of the vaccinia virus genome. This allowed us to map the mRNA coding for the 11K polypeptide to the extreme left-hand end of the HindIII E fragment. Detailed transcriptional mapping of this region of the genome by nuclease S1 analysis revealed the presence of a late RNA transcribed from the rightward-reading strand. Its 5' end mapped at ca. 130 base pairs to the left of the HindIII site at the junction between the HindIII F and E fragments. The map position of this RNA coincided precisely with the map position of the late message coding for the 11K polypeptide.

Geochemical compositions of marine fossils as proxies for reconstructing ancient environmental conditions.

A brief summary is given here about some of the geochemical methodologies that are often used to obtain information from fossils and sediments about the past environment. Such methods are frequently applied in our project in which the formation of Paleogene phosphate sequences in North Africa is investigated. These layers were deposited in shallow marine seas during a period of extreme warm climate with a high CO(2) concentration in the atmosphere. Some of the characteristics of this greenhouse interval are similar to the modern anthropogenic situation hence it is intensively investigated from several aspects by many scientists. Here the geochemical compositions of fossils deposited during this time are discussed, focusing on how the data are obtained and how they could be evaluated in terms of palaeo-environmental conditions.

Extended bottom-up proteomics with secreted aspartic protease Sap9.

We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7kDa on average) peptides in bottom-up proteomics to about twice longer (~3.2kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, ~1h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. BIOLOGICAL SIGNIFICANCE: Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (<3kDa) peptides, which may exceed the analytical capabilities of liquid chromatography and mass spectrometry. Information on localization of protein modifications may also be affected by the small size of typically produced peptides. On the other hand, advances in high-resolution mass spectrometry and liquid chromatography have created an intriguing opportunity of improving proteome analysis by gradually increasing the size of enzymatically-derived peptides in MS-based bottom-up proteomics. Bioinformatics has already confirmed the envisioned advantages of such approach. The remaining bottle-neck is an enzyme that could produce longer peptides. Here, we report on the characterization of a possible candidate enzyme, Sap9, which may be considered for producing longer, e.g., 3-7kDa, peptides and lead to a development of extended bottom-up proteomics.

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane.

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca(2+) channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca(2+) and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca(2+)channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.

Energy requirements during reproduction and reproductive effort in shrews (Soricidae)

The energy requirements of reproducing and non-reproducing females of three species of Crocidura (C. russula, C. viaria, C. olivieri), and two species of Sorex (S. coronatus, S. minutus) were measured. Members of these two genera show different rates of metabolism and reproductive strategies (extreme altriciality and larger litter size in Sorex). During pregnancy, the daily energy intake (on either an absolute or a mass-specific basis) remained close to the non-reproductive value in all species. The absolute energy intake increased strongly after parturition and was influenced by the litter size. Peak energy intake of lactating females was extremely high, typically between 100% and 200% above the non-reproductive requirements in the Crocidura and about 300% above the non-reproductive intake in the Sorex. The mass-specific daily energy intake was reduced during lactation in the three smaller species but not in C. viaria and C. olivieri. This decrease probably involves the different thermoregulatory abilities and/or basal rate of metabolism of the pups. Average reproductive effort was about 50%, in the Crocidura species and above 150% in the Sorex species. The higher effort in the latter is partly due to a larger litter size. But in addition, extreme altriciality in the Sorex leads to an earlier increase in the energy requirements and thus is an energetically more expensive reproductive mode. The present results support the hypothesis that a higher basal rate of metabolism is associated with a higher reproductive effort in shrews.

Pédiatrie [Current progress in pediatrics: obesity and diagnosis approach to the febrile child]

The literature shows that obesity is a public health problem concerning especially the general paediatrician. While prevention has probably more chances of success than treatment, drugs or, in case of failure, the surgical approach are reserved for extreme cases. In the domain of infectiology the different laboratory tests allow only partially to diagnose severe infections. But in the context of a potential influenza pandemic rapid virologic tests become more and more important. They allow a more precise diagnosis and a reduction of hospitalisations and of antibiotic prescriptions. A review of the north American experience with the pneumococal vaccine shows that the heptavalent vaccine will change our approach in infants with fever in whom we suspect a severe infection.

A systematic review and meta-analysis of perineural dexamethasone for peripheral nerve blocks.

We systematically reviewed the safety and efficacy of perineural dexamethasone as an adjunct for peripheral nerve blockade in 29 controlled trials of 1695 participants. We grouped trials by the duration of local anaesthetic action (short- or medium- vs long-term). Dexamethasone increased the mean (95% CI) duration of analgesia by 233 (172-295) min when injected with short- or medium-term action local anaesthetics and by 488 (419-557) min when injected with long-term action local anaesthetics, p < 0.00001 for both. However, these results should be interpreted with caution due to the extreme heterogeneity of results, with I2 exceeding 90% for both analyses. Meta-regression did not show an interaction between dose of perineural dexamethasone (4-10 mg) and duration of analgesia (r2 = 0.02, p = 0.54). There were no differences between 4 and 8 mg dexamethasone on subgroup analysis.

The political economy of childcare in OECD countries : Explaining cross-national variation in spending and coverage rates

If childcare policy has become topical in most OECD countries over the last ten years or so, actual developments display huge cross-national variations. Countries like Sweden and Denmark spend around 2 per cent of GDP on this service, and provide affordable childcare places to most children below school age. At the other extreme, in Southern Europe, only around 10 per cent of this age group has access to formal daycare. Against this background, this article aims to account for cross-national variations in childcare services. It distinguishes two dependent variables: the coverage rate and the proportion of GDP spent subsidising childcare services. Using a mix of cross-sectional and pooled times-series methods, it tests a series of hypotheses concerning the determinants of the development of this policy. Its main conclusion for the coverage rate is that key factors are public spending and wage dispersion (both positive). For spending, key factors are the proportion of women in parliaments (positive) and spending on age-related policies (negative).

Error and attack tolerance of layered complex networks.

Many complex systems may be described by not one but a number of complex networks mapped on each other in a multi-layer structure. Because of the interactions and dependencies between these layers, the state of a single layer does not necessarily reflect well the state of the entire system. In this paper we study the robustness of five examples of two-layer complex systems: three real-life data sets in the fields of communication (the Internet), transportation (the European railway system), and biology (the human brain), and two models based on random graphs. In order to cover the whole range of features specific to these systems, we focus on two extreme policies of system's response to failures, no rerouting and full rerouting. Our main finding is that multi-layer systems are much more vulnerable to errors and intentional attacks than they appear from a single layer perspective.

Bioclimatic constraints to Andean cat distribution: a modelling application for rare species

AimTo identify the bioclimatic niche of the endangered Andean cat (Leopardus jacobita), one of the rarest and least known felids in the world, by developing a species distribution model.LocationSouth America, High Andes and Patagonian steppe. Peru, Bolivia, Chile, Argentina.MethodsWe used 108 Andean cat records to build the models, and 27 to test them, applying the Maxent algorithm to sets of uncorrelated bioclimatic variables from global databases, including elevation. We based our biogeographical interpretations on the examination of the predicted geographic range, the modelled response curves and latitudinal variations in climatic variables associated with the locality data.ResultsSimple bioclimatic models for Andean cats were highly predictive with only 3-4 explanatory variables. The climatic niche of the species was defined by extreme diurnal variations in temperature, cold minimum and moderate maximum temperatures, and aridity, characteristic not only of the Andean highlands but also of the Patagonian steppe. Argentina had the highest representation of suitable climates, and Chile the lowest. The most favourable conditions were centrally located and spanned across international boundaries. Discontinuities in suitable climatic conditions coincided with three biogeographical barriers associated with climatic or topographic transitions.Main conclusionsSimple bioclimatic models can produce useful predictions of suitable climatic conditions for rare species, including major biogeographical constraints. In our study case, these constraints are also known to affect the distribution of other Andean species and the genetic structure of Andean cat populations. We recommend surveys of areas with suitable climates and no Andean cat records, including the corridor connecting two core populations. The inclusion of landscape variables at finer scales, crucially the distribution of Andean cat prey, would contribute to refine our predictions for conservation applications.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.