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1. Climate warming is causing changes in the physics of deep lakes, such as longer summer stratification, increased water column stability, reduced ice cover, and a shallower depth of winter overturns. An ultimate consequence of warming would be a transition to a different mixing regime. Here we investigate the role of physical, chemical, and biological feedback mechanisms that unfold during a shift in mixing regime, and whether these feedbacks could prompt and stabilise the new regime. Although climate, interannual temperature variation, and lake morphometry are the main determinants of a mixing regime, when climate change causes shifts in mixing regime, internal feedback mechanisms may gain in importance and modify lake ecosystem functioning. 2. We review the role of these feedbacks in three mixing regime shifts: from polymictic to seasonally stratified, from dimictic to monomictic, and from holomictic to oligomictic or meromictic. 3. Polymictic lakes of intermediate depth (c. 3–10 m mean depth) could experience seasonal stratification if a stratification event triggers phytoplankton blooms or dissolved organic matter release, reducing transparency and therefore further heating the surface layer. However, this feedback is only likely to have influence in small and clear lakes, it would be easily disturbed by weather conditions, and the resulting stratified state does not remain stable in the long term, as stratification is lost in winter. 4. The ice‐albedo feedback might cause an accelerated shift from ice‐covered (dimictic) to ice‐free (monomictic) winters in sufficiently deep (mean depth 50 m or more) lakes, where temperature memory is carried over from one winter to the next. Nevertheless, there is an ongoing debate into whether this process can persist during natural weather variations and overcome self‐stabilising mechanisms such as thermal insulation by snow. The majority of studies suggest that a gradual transition from dimictic to monomictic is more likely than an abrupt transition. 5. A shift from a holomictic to a meromictic regime can occur if anoxia is triggered by incomplete mixing and an increase in deep‐water density—through the accumulation of solutes—exceeds a density decrease by hypolimnetic warming. A shift to meromixis would strongly alter the biology of a lake and might be difficult to reverse. If solutes accumulate only minimally in the hypolimnion, an oligomictic regime is formed, in which years with complete and incomplete mixing alternate. 6. Understanding the importance of feedback mechanisms and the role of biogeochemistry when lakes shift in mixing regime could lead to a better understanding of how climate change affects lake ecosystems.

International audience; Campylobacter is among the most common causes of gastroenteritis worldwide. Campylobacter jejuni and Campylobacter coli are the most common species causing human-disease. DNA-sequence-based methods for strain characterization have focussed largely on C. jejuni, responsible for 80-90% of infections, meaning that C. coli epidemiology has lagged behind. Here we have analyzed the genome of 450 C. coli isolates to determine genetic markers that can discriminate isolates sampled from 3 major reservoir hosts (chickens, cattle and pigs). These markers were then applied to identify the source of infection of 147 C. coli from French clinical cases. Using STRUCTURE software, 259 potential host-segregating markers were revealed by probabilistic characterization of SNP frequency variation in strain collections from three different hosts. These SNPs were found in 41 genes or intergenic regions, mostly coding for proteins involved in motility and membrane functions. Source attribution of clinical isolates based on the differential presence of these markers confirmed chicken as the most common source of C. coli infection in France.IMPORTANCE Genome-wide and source attribution studies based on Campylobacter species have shown their importance for the understanding of foodborne infections. Although the use of MLST based on 7 genes from C. jejuni is a powerful method to structure populations, when applied to C. coli results have not clearly demonstrated their robustness. Therefore, we aim here to provide more accurate data based on the identification of single-nucleotide polymorphisms. Results from this study reveal an important number of host-segregating SNPs, found in proteins implied in motility, membrane functions or DNA repair systems. These findings offer new interesting opportunities for further study on C. coli adaptation to its environment. Additionally, the results demonstrate that poultry is potentially the main reservoir of C. coli in France.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable., Better and cheaper SARS-CoV-2 qRT-PCR tests are needed, but it is known that human and viral nucleic acid quantities in swab samples correlate, showing the importance of a human quality control probe. This study describes multiplex assays that perform equally well to commercial tests, but at ~10% of the cost.

The 3Rs principles (Replacement, Reduction and Refinement) are focused on finding alternatives to the use of animals in research. In this regard, cell lines are popular and useful tools for the replacement of primary cells in in vitro studies. However, around 15–30% of cell lines used in research have been misidentified or cross-contaminated generating concerns about the results obtained from experiments that use them. Here we described how old aliquots of an equine macrophage cell line (e-CAS) stored at the Animal Health Trust did not contain equine cells but macrophages of murine origin (m-CAS).

Background Helicobacter pylori are stomach-dwelling bacteria that are present in about 50% of the global population. Infection is asymptomatic in most cases, but it has been associated with gastritis, gastric ulcers and gastric cancer. Epidemiological evidence shows that progression to cancer depends upon the host and pathogen factors, but questions remain about why cancer phenotypes develop in a minority of infected people. Here, we use comparative genomics approaches to understand how genetic variation amongst bacterial strains influences disease progression. Results We performed a genome-wide association study (GWAS) on 173 H. pylori isolates from the European population (hpEurope) with known disease aetiology, including 49 from individuals with gastric cancer. We identified SNPs and genes that differed in frequency between isolates from patients with gastric cancer and those with gastritis. The gastric cancer phenotype was associated with the presence of babA and genes in the cag pathogenicity island, one of the major virulence determinants of H. pylori, as well as non-synonymous variations in several less well-studied genes. We devised a simple risk score based on the risk level of associated elements present, which has the potential to identify strains that are likely to cause cancer but will require refinement and validation. Conclusion There are a number of challenges to applying GWAS to bacterial infections, including the difficulty of obtaining matched controls, multiple strain colonization and the possibility that causative strains may not be present when disease is detected. Our results demonstrate that bacterial factors have a sufficiently strong influence on disease progression that even a small-scale GWAS can identify them. Therefore, H. pylori GWAS can elucidate mechanistic pathways to disease and guide clinical treatment options, including for asymptomatic carriers. Electronic supplementary material The online version of this article (10.1186/s12915-018-0550-3) contains supplementary material, which is available to authorized users.

International audience; Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of $Pseudomonas\ syringae\ sensu\ lato$, LINs were compared with a $P.\ syringae$ genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of $P.\ syringae$ pathovar $actinidiae$, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

The behavioural rhythms of organisms are thought to be under strong selection, influenced by the rhythmicity of the environment1,2,3,4. Such behavioural rhythms are well studied in isolated individuals under laboratory conditions1,5, but free-living individuals have to temporally synchronize their activities with those of others, including potential mates, competitors, prey and predators6,7,8,9,10. Individuals can temporally segregate their daily activities (for example, prey avoiding predators, subordinates avoiding dominants) or synchronize their activities (for example, group foraging, communal defence, pairs reproducing or caring for offspring)6,7,8,9,11. The behavioural rhythms that emerge from such social synchronization and the underlying evolutionary and ecological drivers that shape them remain poorly understood5,6,7,9. Here we investigate these rhythms in the context of biparental care, a particularly sensitive phase of social synchronization12 where pair members potentially compromise their individual rhythms. Using data from 729 nests of 91 populations of 32 biparentally incubating shorebird species, where parents synchronize to achieve continuous coverage of developing eggs, we report remarkable within- and between-species diversity in incubation rhythms. Between species, the median length of one parent’s incubation bout varied from 1–19 h, whereas period length—the time in which a parent’s probability to incubate cycles once between its highest and lowest value—varied from 6–43 h. The length of incubation bouts was unrelated to variables reflecting energetic demands, but species relying on crypsis (the ability to avoid detection by other animals) had longer incubation bouts than those that are readily visible or who actively protect their nest against predators. Rhythms entrainable to the 24-h light–dark cycle were less prevalent at high latitudes and absent in 18 species. Our results indicate that even under similar environmental conditions and despite 24-h environmental cues, social synchronization can generate far more diverse behavioural rhythms than expected from studies of individuals in captivity5,6,7,9. The risk of predation, not the risk of starvation, may be a key factor underlying the diversity in these rhythms., The study was supported by the Max Planck Society (to B.K.). M.B. is a PhD student in the International Max Planck Research School for Organismal Biology.

15 pages; International audience; We devised a simple model for assessing the role of development in shaping the evolution of morphological disparity. Disparity of a clade at any given time is expressed in terms of the developmental dynamics that lead to the variety of adult morphotypes observed. We use assumed phenotypic manifestations of developmental processes, as they could be detected from allometric characterizations, to distinguish a few, nonexclusive types of evolutionary changes in ontogeny. On the basis of this formalization, we describe the diversification of hypothetical clades, using the standard curve of adult morphological disparity, the curve of juvenile disparity, and the curve of allometric disparity, the latter quantifying the diversification of clades in allometric space. Contrasts of these curves reflect the underlying developmental scheme that drives temporal changes in disparity. We then vary the parameters of the model to assess the expected signature of each metric under specific conditions: changes in the relative frequencies of the types of evolutionary developmental changes, changes in the transition magnitude attached to each of them, and effects of temporal variation in average adult size on disparity curves and patterns of morphospace occupation. Results emphasize the potential contribution of these proxies for developmental dynamics—juvenile morphological disparity, allometric disparity, and average adult size—in enriching the interpretation of standard disparity curves and the description of clade histories, with possible process-oriented inferences.

Objective— Bexarotene (Targretin) is a clinically used antitumoral agent which exerts its action through binding to and activation of the retinoid-X-receptor (RXR). The most frequent side-effect of bexarotene administration is an increase in plasma triglycerides, an independent risk factor of cardiovascular disease. The molecular mechanism behind this hypertriglyceridemia remains poorly understood. Methods and Results— Using wild-type and LXRα/β-deficient mice, we show here that bexarotene induces hypertriglyceridemia and activates hepatic LXR-target genes of lipogenesis in an LXR-dependent manner, hence exerting a permissive effect on RXR/LXR heterodimers. Interestingly, RNA analysis and Chromatin Immunoprecipitation assays performed in the liver reveal that the in vivo permissive effect of bexarotene on the RXR/LXR heterodimer is restricted to lipogenic genes without modulation of genes controlling cholesterol homeostasis. Conclusion— These findings demonstrate that the hypertriglyceridemic action of bexarotene occurs via the RXR/LXR heterodimer and show that RXR heterodimers can act with a selective permissivity on target genes of specific metabolic pathways in the liver.

International audience; : Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.

International audience; We investigated the relationship between genomic and phenotypic evolution among replicate populations of Escherichia coli evolved for 1000 generations in four different environments. By resequencing evolved genomes, we identified parallel changes in genes encoding transcription regulators within and between environments. Depending on both the environment and the altered gene, genetic parallelism at the gene level involved mutations that affected identical codons, protein domains or were widely distributed across the gene. Evolved clones were characterized by parallel phenotypic changes in their respective evolution environments but also in the three alternative environments. Phenotypic parallelism was high for clones that evolved in the same environment, even in the absence of genetic parallelism. By contrast, clones that evolved in different environments revealed a higher parallelism in correlated responses when they shared mutated genes. Altogether, this work shows that after an environmental change or the colonization of a new habitat, similar ecological performance might be expected from individuals that share mutated genes or that experienced similar past selective pressures.

15 pages; International audience; Aim In this study, we analyse microevolutionary processes in common voles (Microtus arvalis) through the investigation of tooth morphological structure, in order to assess the relative impact of climate and phylogeographical history. Microevolutionary studies have shown that climate change may play a role in both population phylogeography and phenotypic differentiation. However, relatively little is known about the precise relationship between phylogeography and phenotypic variability and about how organisms respond to climate change. Location France, from sea level to the Alps (5 to > 2300 m a.s.l.). Methods This morphological analysis is based on first lower molar measurements from 16 geographically distinct common vole populations. Size and shape components are assessed separately. Population structure patterns are characterized using canonical variate analysis. We use phylogenetic analyses of two regions of mitochondrial DNA (the control region and the cytochrome b gene) to infer genetic structure. We calculate climate parameters from temperature and precipitation data. We investigate the influence of climate, geography and phylogeographical history on the phenotype using (1) multiple regression tests, (2) pairwise comparison of observation-by-variable matrices, and (3) a correlation method designed to compare three matrices. Results All populations were clearly structured, whatever the dataset. Neither size nor shape variation was correlated with climate parameters, but tooth shape was strongly correlated with both genetic structure and geographical distance. Main conclusions In French Microtus arvalis populations, molar shape differentiation is clearly associated with both phylogeographical history and geographical distance. Population phylogeographical history has a greater relevance than climate in accounting for variation in tooth morphology.

4 páginas, 1 tabla.-- Uwe Strähle ... et al., Small fresh water fishes such as the zebrafish (Danio rerio) have become important model organisms for biomedical research. They currently represent the best vertebrate embryo models in which it is possible to derive quantitative data on gene expression, signaling events, and cell behavior in real time in the living animal. Relevant phenotypes in fish mutants are similar to those of other vertebrate models and human diseases. They can be analyzed in great detail and much faster than in mammals. In recent years, approximately 2500 genetically distinct fish lines have been generated by European research groups alone. Their potential, including their possible use by industry, is far from being exploited. To promote zebrafish research in Europe, EuFishBioMed was founded and won support by the EU COST programme (http://www.cost.esf.org/). The main objective of EuFishBioMed is to establish a platform of knowledge exchange for research on small fish models with a strong focus on widening its biomedical applications and an integration of European research efforts and resources. EuFishBioMed currently lists more than 300 member laboratories in Europe, offers funding for short-term laboratory visits, organizes and co-sponsors meetings and workshops, and has successfully lobbied for the establishment of a European Zebrafish Resource Centre. To maintain this network in the future, beyond the funding period of the COST Action, we are currently establishing the European Society for Fish Models in Biology and Medicine, Our network is financed by the EU COST Action Eu- FishBioMed (BM 0804) from 2009 to 2013. The European Zebrafish Resource Center is partially funded by the Klaus Tschira Foundation (Project No. 00.170.2010) and KIT. US, RG, and LB-C are supported by the European Union’s 7th Framework Programme (EC Grant Agreement HEALTH-F4- 2010-242048, ZF-HEALTH).

PubMedID: 22504343 One of the fundamental principles of the life-history theory is that parents need to balance their resources between current and future offspring. Deserting the dependent young is a radical life-history decision that saves resources for future reproduction but that may cause the current brood to fail. Despite the importance of desertion for reproductive success, and thus fitness, the neuroendocrine mechanisms of brood desertion are largely unknown. We investigated two candidate hormones that may influence brood desertion in the Kentish plover Charadrius alexandrinus: prolactin ('parental hormone') and corticosterone ('stress hormone'). Kentish plovers exhibit an unusually diverse mating and parental care system: brood desertion occurs naturally since either parent (the male or the female) may desert the brood after the chicks hatch and mate with a new partner shortly after. We measured the hormone levels of parents at hatching using the standard capture and restraint protocol. We subsequently followed the broods to determine whether a parent deserted the chicks. We found no evidence that either baseline or stress-induced prolactin levels of male or female parents predicted brood desertion. Although stress-induced corticosterone levels were generally higher in females compared with males, individual corticosterone levels did not explain the probability of brood desertion. We suggest that, in this species, low prolactin levels do not trigger brood desertion. In general, we propose that the prolactin stress response does not reflect overall parental investment in a species where different parts of the breeding cycle are characterized by contrasting individual investment strategies. © 2012 Elsevier Inc.. Deutscher Akademischer Austauschdienst OMFB-00365/2010, 22895WE Hungarian Scientific Research Fund Hungarian Scientific Research Fund: K81953, PD76862 TUBITAK-108Y329, OMFB-00581/2009 The study was supported by the Hungarian Scientific Research Fund (OTKA, K81953 , PD76862 ) and the Hungarian-French Intergovernmental S&T Cooperation Programme (OMFB-00365/2010, Egide 22895WE). Fieldwork was supported by a grant from the Hungarian–Turkish Intergovernmental S&T Cooperation Programme ( OMFB-00581/2009 , TUBITAK-108Y329 ). CK was supported by a DAAD fellowship. At the CEBC, we thank Colette Trouvé for her technical assistance in performing the prolactin and corticosterone assays. We are grateful to Peter Sharp and Alexander Badyaev for discussion and useful advice on the methods. We are grateful for two anonymous referees for their excellent comments that improved the manuscript.

International audience; Epistatic interactions between mutations play a prominent role in evolutionary theories. Many studies have found that epistasis is widespread, but they have rarely considered beneficial mutations. We analyzed the effects of epistasis on fitness for the first five mutations to fix in an experimental population of Escherichia coli. Epistasis depended on the effects of the combined mutations--the larger the expected benefit, the more negative the epistatic effect. Epistasis thus tended to produce diminishing returns with genotype fitness, although interactions involving one particular mutation had the opposite effect. These data support models in which negative epistasis contributes to declining rates of adaptation over time. Sign epistasis was rare in this genome-wide study, in contrast to its prevalence in an earlier study of mutations in a single gene.

International audience; The dissection of natural selection and neutral processes remains a core problem for molecular evolutionary biologists. One of the longest-standing controversies con- cerns the causes of genome base compo- sition, notably the variation in the sum of G and C content (GC) between 17% and 75% in bacteria. Sueoka argued very early that GC content variation is driven by mutational biases and, as this bias affects non-synonymous sites, protein evolution might also be largely driven by neutral forces [1]. Later, Muto and Osawa showed that 4-fold degenerate positions in codons exhibit the largest range of GC content (GC4), whereas the non-degener- ate second codon positions (GC2) exhibit the narrowest (Figure 1) [2]. As the footprint of genomic GC variation is most evident in those sites under the least selective constraint for amino acid com- position, it has become accepted that GC content variation is primarily driven by neutral mutational effects and has little adaptive relevance [2].

12 pages; Les ammonoïdes qui perdurent 335 Ma constituent un modèle de choix dans l'analyse du fait évolutif. Cet article de synthèse se propose d'aborder (1) : leur place phylogénétique au sein des céphalopodes et le choix d'un modèle actuel de référence ; (2) : la construction d'espaces phénotypiques qui offrent un angle d'étude pertinent de l'évolution biologique ; (3) : les concordances et discordances entre reconstruction phylogénétique et registre fossile et (4) : les reconquêtes postcrises, modèles d'étude de l'évolution à grande échelle. Il ressort que les ammonoïdes peuvent servir d'appui à de nombreuses thématiques paléontologiques (dynamique de la biodiversité, analyse phylogénétique, analyse du registre fossile) qui permettent et qui permettront encore de mieux cerner l'évolution biologique, notamment dans un contexte d'étude à grande échelle.

The extent and nature of epistatic interactions between mutations are issues of fundamental importance in evolutionary biology. However, they are difficult to study and their influence on adaptation remains poorly understood. Here, we use a systems-level approach to examine epistatic interactions that arose during the evolution of Escherichia coli in a defined environment. We used expression arrays to compare the effect on global patterns of gene expression of deleting a central regulatory gene, crp. Effects were measured in two lineages that had independently evolved for 20,000 generations and in their common ancestor. We found that deleting crp had a much more dramatic effect on the expression profile of the two evolved lines than on the ancestor. Because the sequence of the crp gene was unchanged during evolution, these differences indicate epistatic interactions between crp and mutations at other loci that accumulated during evolution. Moreover, a striking degree of parallelism was observed between the two independently evolved lines; 115 genes that were not crp-dependent in the ancestor became dependent on crp in both evolved lines. An analysis of changes in crp dependence of well-characterized regulons identified a number of regulatory genes as candidates for harboring beneficial mutations that could account for these parallel expression changes. Mutations within three of these genes have previously been found and shown to contribute to fitness. Overall, these findings indicate that epistasis has been important in the adaptive evolution of these lines, and they provide new insight into the types of genetic changes through which epistasis can evolve. More generally, we demonstrate that expression profiles can be profitably used to investigate epistatic interactions., Author Summary The effect of a genetic mutation can depend on the genotype of the organism in which it occurs. For example, a mutation that is beneficial in one genetic background might be neutral or even deleterious in another. The interactions between genes that cause this dependence—known as epistasis—play an important role in many evolutionary theories. However, they are difficult to study and remain poorly understood. We used a novel approach to examine the evolution of interactions arising between a key regulatory gene, crp, and mutations that occurred during the adaptation of a bacterium, Escherichia coli, to a laboratory environment. To do this, we measured the effect of deleting crp on the expression of all genes in the organism, providing a sensitive measure to identify new interactions involving this gene. We found that deleting crp had a dramatic and parallel effect on gene expression in two independently evolved populations, but much less effect in their ancestor. An analysis of these changes identified a number of regulatory genes as candidates for harboring beneficial mutations that could account for the parallel changes. These findings indicate that epistasis has played an important role in the evolution of these populations, and they provide insight into the types of genetic changes through which epistasis can evolve.

International audience; In a period of agricultural decline in the uplands of Europe, agriculture is ceasing to provide the primary rationale for the organisation, utilisation and functioning of rural space. Policy reform, market trends and changes to the way citizens and consumers think about the countryside all suggest a need for thinking strategically about the future development of these areas. However, without a broad involvement of stakeholders, land use conflicts, and social and cultural conflicts in general, may increase. Involving stakeholders in upland areas can be facilitated by using scenario technique and by discussing alternative futures in local stakeholder panels. In this paper we present four scenarios of land-use change for the year 2030, and their assessments by stakeholder panels in Scotland, France, Norway, Switzerland, Slovakia and Greece. The aim of the paper is to explore the ways in which stakeholders in these locations advocate and assess these scenarios. We also explore how stakeholders in different countries weight the visual landscape impacts, the livelihood and biodiversity aspects of the scenarios in their assessment, and the reasons for their prioritising. The cross-country analysis shows that stakeholders across the study areas are united in their overall rejection of agricultural liberalisation, advocating a production-oriented, but multifunctional and environmentally-friendly agriculture that maintains landscapes and biodiversity.

International audience; Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.

International audience; Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.

Nicotine, the addictive compound of tobacco products, exerts its effects in the brain by binding to neuronal acetylcholine nicotinic receptors (nAChRs). The aim of the present study was to increase the knowledge of nicotine s complex effects, the focus being on homomeric alpha7-nAChRs that are widely expressed in the brain. Nicotinic regulation of differential signalling molecules including transcriptional regulators was also studied. We found that the number of alpha7-nAChRs is increased in specific brain regions in mice, in a time-dependent manner after chronic oral nicotine administration. Our results suggest that in addition to alpha4beta2-nAChRs, the other major nAChR subtype expressed in the brain, the number of alpha7-nAChRs is affected by chronic presence of nicotine. We suggest that when studying the long-term effects of nicotine, the duration on administration is of great importance. Next, we observed that nicotine exposure induces accumulation of cAMP in cell cultures expressing nAChRs. Furthermore, nicotine-induced alpha7-nAChR upregulation was potentiated by treatments enhancing cAMP-signalling, suggesting a role for cAMP in the upregulation process. Protein kinase C (PKC) was found essential for the basal regulation of alpha7-nAChR number. The nicotine-evoked alpha7-nAChR upregulation could be further increased by PKC overexpression. Thirdly, the effects of nicotine on dopamine and cAMP regulated phosphoprotein (DARPP-32) were characterised in rat brain. The results show that DARPP-32 is regulated by both acute and long-term nicotine treatment in the striatal subdivisions. The effect of acute nicotine is dose-dependent and the three striatal regions display differential sensitivities to nicotine. Chronic nicotine is also able to regulate DARPP-32 signalling with prominent effect seen in the nucleus accumbens (NAc), suggesting a role for DARPP-32 in the mediation of long-term effects of nicotine. Finally, the regulation of transcription factors Elk-1 and FosB/deltaFosB by nicotine was investigated. We found that Elk-1 is activated by acute nicotine selectively in the NAc core and hippocampal area CA1, whereas acute nicotine does not affect FosB/deltaFosB. Long-term intermittent or continuous nicotine increases the level of total Elk-1 in the same brain regions as acute nicotine. FosB/deltaFosB is also affected by chronic nicotine. Thus, similarly to other drugs of abuse, nicotine regulates transcriptional regulators Elk-1 and FosB/deltaFosB. These results bring further support for a common mechanism underlying the development of addiction. Nicotine s positive effects on learning and memory might involve the transcription factor Elk-1 based on the changes seen in the hippocampus, the key area in cognitive functions. Nikotiini on tupakan riippuvuutta aiheuttava aine, jonka vaikutukset aivoissa välittyvät hermosolujen nikotiinireseptorien kautta. Väitöskirjatyön tarkoituksena oli lisätä tietämystä nikotiinin monimuotoisista vaikutuksista aivoissa. Työssä keskityttiin tutkimaan alfa7-reseptorialatyyppiä sekä selvitettiin nikotiinin vaikutuksia erilaisiin viestinvälittäjämolekyyleihin, kuten geeninsäätelytekijöihin. Havaitsimme, että pitkäaikainen nikotiinialtistus hiirille juomaveden mukana lisää alfa7-reseptorien määrää tietyillä aivoalueilla. Tulokset antavat viitteitä siitä, että aivojen toisen nikotiinireseptoripäätyypin, alfa4beeta2-reseptorien lisäksi, myös alfa7-reseptorien määrä muuttuu altistuttaessa nikotiinille. Uskomme, että nikotiinin annostelujakson pituudella on suuri merkitys selvitettäessä nikotiinin pitkäaikaisvaikutuksia. Tutkimuksen toisen osan päähavainto on, että solunsisäiset viestimolekyylit, syklinen AMP ja proteiinikinaasi C (PKC) säätelevät alfa7-reseptorimäärää. Havaitsimme muun muassa, että cAMP:n määrää lisäävät yhdisteet, samoin kuin PKC:n yli-ilmentäminen soluissa geenimanipulaation avulla, saavat nikotiinin aiheuttaman alfa7-reseptorimäärän kasvun lisääntymään entisestään. Selvitimme lisäksi nikotiinin vaikutuksia dopamiinin ja cAMP:n säätelemään fosfoproteiini DARPP-32:een rotan aivoissa. Havaitsimme, että nikotiini vaikuttaa monimuotoisesti tähän hermovälittäjäaine dopamiinin vaikutuksia välittävään viestimolekyyliin. Lyhykestoisen nikotiiniannon aikaansaamat muutokset DARPP-32:ssa ovat annosriippuvaisia ja eri aivoalueet poikkeavat toisistaan herkkyydessään näille vaikutuksille. DARPP-32:lla näyttäisi olevan merkitystä myös nikotiinin pitkäaikaisvaikutusten välittymisessä. Myös pitkäkestoisella nikotiiniannolla havaittiin selkeitä vaikutuksia DARPP-32:een. Lopuksi selvitimme nikotiinin vaikutuksia Elk-1 ja FosB/deltaFosB geeninsäätelytekijöihin. Lyhytkestoinen nikotiinianto sai aikaan Elk-1-aktivaation selektiivisesti tietyillä alueilla rotan aivoissa, mutta FosB/deltaFosB-säätelyyn akuutti nikotiini ei vaikuttanut. Pitkäaikaisen nikotiiniannon havaittiin vaikuttavan sekä Elk-1 että FosB/deltaFosB -säätelyyn. Tulokset antavat viitteitä siitä, että nikotiinilla on muiden riippuvuutta aiheuttavien aineiden tapaan vaikutuksia Elk-1 ja FosB/deltaFosB geeninsäätelytekijöihin. Väitöskirjatyön tulokset vahvistavat käsitystä, jonka mukaan riippuvuuden taustalla ovat samat syntymekanismit huolimatta käytetystä aineesta. Aivojen muistikeskus hippokampuksen alueella havaitut muutokset Elk-1-säätelyssä saattavat liittyä nikotiinin muistia ja oppimista parantavien vaikutusten syntyyn.

International audience; Genetic and experimental evidence points to a critical involvement of the atypical mammalian orphan receptor DAX-1 in reproductive development and steroidogenesis. Unlike conventional nuclear receptors, DAX-1 appears not to function as a DNA-bound transcription factor. Instead, it has acquired the capability to act as a transcriptional corepressor of steroidogenic factor 1 (SF-1). The interplay of DAX-1 and SF-1 is considered a central, presumably ligand-independent element of adrenogonadal development and function that requires tight regulation. This raises a substantial interest in identifying its modulators and the regulatory signals involved. Here, we uncover molecular mechanisms that link DAX-1 to the ubiquitin modification system via functional interaction with the E3 ubiquitin ligase RNF31. We demonstrate that RNF31 is coexpressed with DAX-1 in steroidogenic tissues and participates in repressing steroidogenic gene expression. We provide evidence for the in vivo existence of a corepressor complex containing RNF31 and DAX-1 at the promoters of the StAR and CYP19 genes. Our data suggest that RNF31 functions to stabilize DAX-1, which might be linked to DAX-1 monoubiquitination. In conclusion, RNF31 appears to be required for DAX-1 to repress transcription, provides means to regulate DAX-1 in ligand-independent ways, and emerges as a relevant coregulator of steroidogenic pathways governing physiology and disease.