Ecotoxicological approaches to assess the long-term effects of four anticancer drugs and metabolites on Daphnia pulex

The aquatic environment is exposed continuously and increasingly to chemical substances such as pharmaceuticals. These medical compounds are released into the environment after having being consumed and body-excreted by patients. Pharmaceutical residues are synthetic molecules that are not always removed by traditional sewage treatment processes and thus escape degradation. Among pharmaceuticals that escape sewage treatment plants (STPs), the anticancer drugs were measured in STP effluents and natural waters. In the aquatic environment, their long-term effects at low concentrations are sparsely known on non-target species. Tamoxifen is an anticancer drug that is widely prescribed worldwide for the prevention and treatment of hormone receptor-positive breast cancers. Two of its metabolites, i.e., endoxifen and 4-hydroxy- tamoxifen (4OHTam), have high pharmacological potency in vivo and such as tamoxifen, they are excreted via faeces by patients. Tamoxifen was measured in STP effluents and natural waters but, to the best of our knowledge, its metabolites concentrations in waters have never been reported. Imatinib is another and recent anticancer compound that targets specific tumour cells. This pharmaceutical is also body excreted and because of its increasing use in cancer treatment, imatinib may reach the natural water. The effects of tamoxifen and imatinib are unknown upon more than one generation of aquatic species. And the effects of 4OHTam, endoxifen have never been studied in ecotoxicology so far. The aims of this thesis were threefold. First, the sensitivity of D. pulex exposed to tamoxifen, 4OHTam, endoxifen or imatinib was assessed using ecotoxicological experiments. Ecotoxicology is the science that considers the toxic effects of natural or synthetic substances, such as pharmaceuticals, on organisms, populations, community and ecosystem. Acute and multigenerational (2-4 generations) tests were performed on daphnids considering several studied endpoints, such as immobilisation, size, reproduction, viability and intrinsic rate of natural increase. Additional prospective assays were designed to evaluate whether 1) low concentrations of tamoxifen and 4OHTam were able to induce toxic effects when used in combination, and 2) daphnids were able to recover when offspring were withdrawn from solutions carrying the pharmaceutical. Second, the stability of tamoxifen, 4OHTam and endoxifen in incubation medium was evaluated in solution exempted from daphnids. Because the nominal concentrations of tamoxifen, 4OHTam and endoxifen did not correspond to the measured, we provide a predictive method to estimate the concentrations of these chemicals during long-term ecotoxicological tests. Finally, changes in protein expressions were analysed in D. pulex exposed 2 or 7 seven days to tamoxifen using ecotoxicoproteomic experiments with a shot-gun approach inducing a peptide fractionation step. Our results show that tamoxifen, 4OHTam and endoxifen induced adverse effects in D. pulex at environmentally relevant concentrations. At very low concentrations, these molecules displayed unusual and teratogenic effects because morphological abnormalities were observed in offspring, such as thick and short antennas, curved spines, premature neonates and aborted eggs. Tamoxifen was the most toxic compound among the test chemicals, followed by 4OHTam, endoxifen and imatinib. Tamoxifen no-observed effect concentrations (NOECs) that were calculated for size, reproduction and intrinsic rate were below or in the range of the concentrations measured in natural waters, i.e., between 0.12 µg/L and 0.67 µg/L. For instance, the tamoxifen NOECs that were calculated for reproduction were between 0.67 and 0.72 µg/L, whereas the NOEC was < 0.15 µg/L when based on morphological abnormalities. The NOECs of 4OHTam were higher but still in the same order of magnitude as tamoxifen environmental concentrations, with a value of 1.48 µg/L. Endoxifen NOEC for the intrinsic rate of natural increase (r) and the reproduction were 0.4 and 4.3 µg/L, respectively. Daphnids that were withdrawn from tamoxifen and 4OHTam were not able to recover. Also, the reproduction of D. pulex was reduced when the treated animals were exposed to the combination of tamoxifen and 4OHTam while no effects were observed when these chemicals were tested individually at the same concentration. Among the anticancer drugs that were tested during this thesis, imatinib was the less toxic molecule towards D. pulex. No effects on size and reproduction were observed within two generations, except for the first whose reproduction decreased at the highest test concentration, i.e., 626 µg/L. Our results also underline the need to use measured or predicted concentrations instead of the nominal during aquatic experiments, particularly when lipophilic molecules are tested. Indeed, notable differences between nominal (i.e., theoretical) and measured concentrations were found with tamoxifen, 4OHTam and endoxifen at all test concentrations. A cost and time sustainable method was proposed to predict the test exposure levels of these chemicals during long-term experiments. This predictive method was efficient particularly for low concentrations, which corresponded to the test concentrations in multigenerational tests. In the ecotoxicoproteomic experiments a total of 3940 proteins were identified and quantified in D. pulex exposed to tamoxifen. These results are currently the largest dataset from D. pulex that is published and the results of proteomic analyses are available for the scientific community. Among these 3940 proteins, 189 were significantly different from controls. After protein annotation, we assumed that treated daphnids with tamoxifen had shifted cost-energy functions, such as reproduction, to maintain their basic metabolism necessary to survive. This metabolic cost hypothesis was supported by the presence of proteins involved in oxidative stress. Biomarkers for early detection of tamoxifen harmful effects on D. pulex were not discovered but the proteins of the vitellogenin-2 family (E9H8K5) and the ryanodine receptor (E9FTU9) are promising potential biomarkers because their expression was already modified after 2 days of treatment. In this thesis, the effects of tamoxifen, 4OHTam and endoxifen on daphnids raise questions about the potential impact of tamoxifen and 4OHTam in other aquatic ecosystems, and therefore, about metabolites in ecotoxicology. Because the NOECs were environmentally relevant, these results suggest that tamoxifen and 4OHTam may be interesting pharmaceuticals to consider in risk assessment. Our findings also emphasize the importance of performing long-term experiments and of considering multi-endpoints instead of the standard reproductive endpoint. Finally, we open the discussion about the importance to measure test exposures or not, during ecotoxicological studies. -- Les milieux aquatiques sont exposés continuellement à un nombre croissant de substances chimiques, notamment les médicaments issus de la médecine vétérinaire et humaine. Chez les patients, les substances administrées sont utilisées par le corps avant d'être éliminées par l'intermédiaire des excrétas dans le système d'eaux usées de la ville. Ces eaux rejoignent ensuite une station de traitement afin d'y éliminer les déchets. Dans le cas des molécules chimiques, il arrive que les processus de traitement d'eaux usées ne soient pas suffisamment efficaces et que ces molécules ne soient pas dégradées. Elles sont alors libérées dans le milieu aquatique avec les effluents de la station d'épuration. Une fois dans l'environnement, ces résidus de médicaments sont susceptibles d'induire des effets sur la faune et la flore aquatique, dont les conséquences à long terme et à faibles concentrations sont peu connues. Les anticancéreux sont une famille de médicaments qui peuvent échapper aux traitements des stations d'épuration et qui sont retrouvées dans le milieu aquatique naturel. Parmi ces substances, le tamoxifen est une molécule utilisée dans le monde entier pour prévenir et traiter les cancers hormonaux dépendant du sein, notamment. Une fois ingéré, le tamoxifen est transformé par le foie en métabolites dont deux d'entre eux, le 4-hydroxy-tamoxifen (4OHTam) et l'endoxifen, possèdent un affinité pour les récepteurs aux estrogènes et une efficacité sur les cellules tumorales supérieure au tamoxifen lui- même. Tout comme la molécule mère, ces métabolites sont principalement éliminés par l'intermédiaire des fèces. Le tamoxifen a déjà été mesuré dans les effluents de stations d'épuration et dans les eaux naturelles, mais aucune valeur n'a été reportée pour ses métabolites jusqu'à présent. Un autre anticancéreux, également éliminé par voie biliaire et susceptible d'atteindre l'environnement, est l'imatinib. Cette récente molécule a révolutionné le traitement et la survie des patients souffrant de leucémie myéloïde chronique et de tumeur stromales gastrointestinales. Les effets du tamoxifen et de l'imatinib sur plusieurs générations d'organismes aquatiques, tels que les microcrustacés Daphnia, sont inconnus et le 4OHTam et l'endoxifen n'ont même jamais été testés en écotoxicologie. Cette thèse s'est articulée autour de trois objectifs principaux. Premièrement, la sensibilité des D. pulex exposés au tamoxifen, 4OHTam, endoxifen et imatinib a été évaluée par l'intermédiaire de tests aigus et de tests sur deux à quatre générations. La mobilité, la taille, la reproduction, la viabilité et la croissance potentielle de la population ont été relevées au cours de ces expériences. Des tests supplémentaires, à but prospectifs, ont également été réalisés afin d'évaluer 1) la capacité de récupération des daphnies, lorsque leurs descendants ont été placés dans un milieu exempté de tamoxifen ou de 4OHTam, 2) les effets chez les daphnies exposées à une solution contenant de faibles concentration de tamoxifen et de 4OHTam mélangés. Le deuxième objectif a été d'évaluer la stabilité du tamoxifen, 4OHTam et endoxifen dilué dans le milieu des daphnies. Après analyses, les concentrations mesurées ne correspondaient pas aux concentrations nominales (c.-à-d., théoriques) et il a été nécessaire de développer une méthode efficace de prédiction des niveaux d'exposition lors de tests de longue durée réalisés avec ces trois molécules. Finalement, des changements dans l'expression des protéines chez des daphnies exposées au tamoxifen ont été investigués par l'intermédiaire d'expériences écotoxicoprotéomiques avec une approche dite de shot-gun avec une étape de fractionnement des protéines. Les résultats obtenus dans cette thèse montrent que le tamoxifen, le 4OHTam et l'endoxifen induisent des effets indésirables chez les daphnies à des niveaux d'exposition proches ou identiques aux concentrations du tamoxifen mesurées dans l'environnement, c'est-à-dire 0.12 et 0.67 µg/L de tamoxifen. Ces molécules ont induit des effets inhabituels tels que la production de : nouveau-nés anormaux, avec des antennes et des queues déformées, des prématurés et des oeufs avortés. Le tamoxifen fut la molécule la plus toxique pour les D. pulex suivie du 4OHTam, de l'endoxifen et enfin de l'imatinib. Lors des expériences sur plusieurs générations, les concentrations n'ayant statistiquement pas d'effet (c.à.d. NOEC en anglais) sur la taille, la reproduction et la croissance intrinsèque de la population étaient du même ordre de grandeur que les concentrations environnementales du tamoxifen. Par exemple, les NOECs du tamoxifen calculées pour la reproduction étaient de 0.67 et 0.72 µg/L, tandis que celle calculée sur la base des anomalies chez les nouveau-nés était < 0.15 µg/L. Les NOECs du 4OHTam se situaient entre 0.16 et 1.48 µg/L et celles de l'endoxifen pour la croissance intrinsèque de la population, ainsi que pour la reproduction, étaient de 0.4 et 4.3 µg/L, respectivement. Dans l'expérience basée sur la récupération des daphnies, la taille et la reproduction ont diminué bien que la descendance fût placée dans un milieu sans substances chimiques. Les daphnies exposées au mélange de tamoxifen et de 4OHTam ont produit moins de nouveau-nés que les contrôles, alors que ces concentrations n'ont pas induit d'effets lorsque testées individuellement. Finalement, l'imatinib n'a pas montré d'effets sur les deux générations testées. Seule la première génération exposée à la plus haute concentration (626 µg/L) a montré une diminution de la reproduction. Les résultats obtenus lors de l'évaluation de la stabilité du tamoxifen, 4OHTam et endoxifen dans le milieu des daphnies ont souligné l'importance d'utiliser des concentrations mesurées ou prédites en écotoxicologie. En effet, des différences notables entre concentrations nominales et mesurées ont été observées à toutes les concentrations et l'hypothèse d'un phénomène d'adsorption sur le verre des récipients a été posée. De ce fait, il a été nécessaire d'élaborer une méthode prédictive efficace et acceptable, en terme de temps et de coûts. Une régression polynomiale basée sur des concentrations mesurées et nominales a permis de prédire avec efficacité les faibles niveaux d'exposition utilisés lors d'expériences écotoxicologiques à long terme, sur plusieurs générations. Suite aux expériences d'écotoxicoprotéomiques, un total de 3940 protéines ont été identifiées et quantifiées chez des daphnies exposées au tamoxifen. Ce nombre est actuellement la plus large série de données publiées et mises à disposition pour la communauté scientifique. Parmi ces protéines, 189 sont significatives et possiblement reliées à des processus de reproduction et de stress. Sur cette base, nous avons émis l'hypothèse que les individus subissant un stress, lié à l'exposition au tamoxifen, ont utilisé leur énergie de base pour favoriser leur survie plutôt que la reproduction. Enfin, la détermination de bio-marqueurs exprimant des dommages précoces des daphnies exposées au tamoxifen n'a pas abouti en tant que telle, mais des protéines prometteuses, telle que la famille de viellogenin-2 (E9H8K5) et le récepteur à la ryanodine (E9FTU9), ont été exprimées après deux jours d'exposition déjà. Ces protéines pourraient faire l'objet d'investigations écotoxicoprotéomiques futures. Les résultats de cette thèse posent certaines questions quant au risque du tamoxifen, du 4OHTam et de l'endoxifen sur la faune et la flore aquatique et plus particulièrement sur les anticancéreux présents dans l'environnement. Les effets toxiques de ces molécules ont été observés à des concentrations environnementales et sur plusieurs générations. La question de considérer les métabolites, et ainsi les pro-médicaments, en écotoxicologie est soulevée, notamment parce que ces molécules peuvent être plus actives et efficaces que la molécule mère. Les expériences chroniques, sur plusieurs générations sont également à favoriser car elles offrent un meilleur reflet de la réalité environnementale que des essais aigus ou d'une génération. L'utilisation de la protéomique permet d'agrandir les connaissances sur les effets des médicaments à un niveau inférieur de l'organisation biologique et ainsi, de mieux comprendre de potentiels mécanismes d'action ou de déterminer de potentiels biomarqueurs. Finalement, il semble important de discuter de l'opportunité de mesurer les concentrations qui sont testées en écotoxicologie afin de ne pas sous-estimer le risque pour la faune et la flore aquatique.

The role of fungi in the precipitation of calcite : relationships between fungal filaments, nanofibres, and needle fibre calcite

RésuméLes champignons sont impliqués dans les cycles biogéochimiques de différentes manières. En particulier, ils sont reconnus en tant qu'acteurs clés dans la dégradation de la matière organique, comme fournisseurs d'éléments nutritifs via l'altération des minéraux mais aussi comme grands producteurs d'acide oxalique et de complexes oxalo-métalliques. Toutefois, peu de choses sont connues quant à leur contribution à la genèse d'autres types de minéraux, tel que le carbonate de calcium (CaCO3). Le CaCO3 est un minéral ubiquiste dans de nombreux écosystèmes et il joue un rôle essentiel dans les cycles biogéochimiques du carbone (C) et du calcium (Ca). Le CaCO3 peut être d'origine physico-chimique ou biogénique et de nombreux organismes sont connus pour contrôler ou induire sa biominéralisation. Les champignons ont souvent été soupçonnés d'être impliqué dans ce processus, cependant il existe très peu d'informations pour étayer cette hypothèse.Cette thèse a eu pour but l'étude de cet aspect négligé de l'impact des champignons dans les cycles biogéochimiques, par l'exploration de leur implication potentielle dans la formation d'un type particulier de CaCO3 secondaires observés dans les sols et dans les grottes des environnements calcaires. Dans les grottes, ces dépôts sont appelés moonmilk, alors que dans les sols on les appelle calcite en aiguilles. Cependant ces deux descriptions correspondent en fait au même assemblage microscopique de deux habitus particulier de la calcite: la calcite en aiguilles (au sens strict du terme cette fois-ci) et les nanofibres. Ces deux éléments sont des habitus aciculaires de la calcite, mais présentent des dimensions différentes. Leur origine, physico-chimique ou biologique, est l'objet de débats intenses depuis plusieurs années déjà.L'observation d'échantillons environnementaux avec des techniques de microscopie (microscopie électronique et micromorphologie), ainsi que de la microanalyse EDX, ont démontré plusieurs relations intéressantes entre la calcite en aiguilles, les nanofibres et des éléments organiques. Premièrement, il est montré que les nanofibres peuvent être organiques ou minérales. Deuxièmement, la calcite en aiguilles et les nanofibres présentent de fortes analogies avec des structures hyphales, ce qui permet de confirmer l'hypothèse de leur origine fongique. En outre, des expériences en laboratoire ont confirmé l'origine fongique des nanofibres, par des digestions enzymatiques d'hyphes fongiques. En effet, des structures à base de nanofibres, similaires à celles observées dans des échantillons naturels, ont pu être produites par cette approche. Finalement, des enrichissements en calcium ont été mesurés dans les parois des hyphes et dans des inclusions intrahyphales provenant d'échantillons naturels de rhizomorphes. Ces résultats suggèrent une implication de la séquestration de calcium dans la formation de la calcite en aiguilles et/ou des nanofibres.Plusieurs aspects restent à élucider, en particulier la compréhension des processus physiologiques impliqués dans la nucléation de calcite dans les hyphes fongiques. Cependant, les résultats obtenus dans cette thèse ont permis de confirmer l'implication des champignons dans la formation de la calcite en aiguilles et des nanofibres. Ces découvertes sont d'une grande importance dans les cycles biogéochimiques puisqu'ils apportent de nouveaux éléments dans le cycle couplé C-Ca. Classiquement, les champignons sont considérés comme étant impliqués principalement dans la minéralisation de la matière organique et dans l'altération minérale. Cette étude démontre que les champignons doivent aussi être pris en compte en tant qu'agents majeurs de la genèse de minéraux, en particulier de CaCO3. Ceci représente une toute nouvelle perspective en géomycologie quant à la participation des champignons au cycle biologique du C. En effet, la présence de ces précipitations de CaCO3 secondaires représente un court-circuit dans le cycle biologique du C puisque du C inorganique du sol se retrouve piégé dans de la calcite plutôt que d'être retourné dans l'atmosphère.AbstractFungi are known to be involved in biogeochemical cycles in numerous ways. In particular, they are recognized as key players in organic matter recycling, as nutrient suppliers via mineral weathering, as well as large producers of oxalic acid and metal-oxalate. However, little is known about their contribution to the genesis of other types of minerals such as calcium carbonate (CaCO3). Yet, CaC03 are ubiquitous minerals in many ecosystems and play an essential role in the biogeochemical cycles of both carbon (C) and calcium (Ca). CaC03 may be physicochemical or biogenic in origin and numerous organisms have been recognized to control or induce calcite biomineralization. While fungi have often been suspected to be involved in this process, only scarce information support this hypothesis.This Ph.D. thesis aims at investigating this disregarded aspect of fungal impact on biogeochemical cycles by exploring their possible implication in the formation of a particular type of secondary CaC03 deposit ubiquitously observed in soils and caves from calcareous environments. In caves, these deposits are known as moonmilk, whereas in soils, they are known as Needle Fibre Calcite (NFC - sensu lato). However, they both correspond to the same microscopic assemblage of two distinct and unusual habits of calcite: NFC {sensu stricto) and nanofibres. Both features are acicular habits of calcite displaying different dimensions. Whether these habits are physicochemical or biogenic in origin has been under discussion for a long time.Observations of natural samples using microscopic techniques (electron microscopy and micromorphology) and EDX microanalyses have demonstrated several interesting relationships between NFC, nanofibres, and organic features. First, it has shown that nanofibres can be either organic or minera! in nature. Second, both nanofibres and NFC display strong structural analogies with fungal hyphal features, supporting their fungal origin. Furthermore, laboratory experiments have confirmed the fungal origin of nanofibres through an enzymatic digestion of fungal hyphae. Indeed, structures made of nanofibres with similar features as those observed in natural samples have been produced. Finally, calcium enrichments have been measured in both cell walls and intrahyphal inclusions of hyphae from rhizomorphs sampled in the natural environment. These results point out an involvement of calcium sequestration in nanofibres and/or NFC genesis.Several aspects need further investigation, in particular the understanding of the physiological processes involved in hyphal calcite nucleation. However, the results obtained during this study have allowed the confirmation of the implication of fungi in the formation of both NFC and nanofibres. These findings are of great importance regarding global biogeochemical cycles as they bring new insights into the coupled C and Ca cycles. Conventionally, fungi are considered to be involved in organic matter mineralization and mineral weathering. In this study, we demonstrate that they must also be considered as major agents in mineral genesis, in particular CaC03. This is a completely new perspective in geomycology regarding the role of fungi in the short-term (or biological) C cycle. Indeed, the presence of these secondary CaC03 precipitations represents a bypass in the short- term carbon cycle, as soil inorganic C is not readily returned to the atmosphere.

Arbuscular mycorrhizal fungal communities and soil aggregation as affected by cultivation of various crops during the sugarcane fallow period

Management systems involving crop rotation, ground cover species and reduced soil tillage can improve the soil physical and biological properties and reduce degradation. The primary purpose of this study was to assess the effect of various crops grown during the sugarcane fallow period on the production of glomalin and arbuscular mycorrhizal fungi in two Latosols, as well as their influence on soil aggregation. The experiment was conducted on an eutroferric Red Latosol with high-clay texture (680 g clay kg-1) and an acric Red Latosol with clayey texture (440 g kg-1 clay) in Jaboticabal (São Paulo State, Brazil). A randomized block design involving five blocks and four crops [soybean (S), soybean/fallow/soybean (SFS), soybean/millet/soybean (SMS) and soybean/sunn hemp/soybean (SHS)] was used to this end. Soil samples for analysis were collected in June 2011. No significant differences in total glomalin production were detected between the soils after the different crops. However, total external mycelium length was greater in the soils under SMS and SHS. Also, there were differences in easily extractable glomalin, total glomalin and aggregate stability, which were all greater in the eutroferric Red Latosol than in the acric Red Latosol. None of the cover crops planted in the fallow period of sugarcane improved aggregate stability in either Latosol.

Nursery Practices and Management of Fungal Diseases in Forest Nurseries in Finland : a review

Summary

Using flow cytometry for in situ monitoring of antimicrobial compound production in the biocontrol bacterium Pseudomonas fluorescens CHA0

Pseudomonas fluorescens strain CHA0 is able to protect plants against a variety of pathogens, notably by producing the two antimicrobial compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the expression of these compounds is affected by many biotic factors, such as fungal pathogens, rhizosphere bacteria as well as plant species. Therefore, the influence of some plant phenolic compounds on the expression of DAPG and PLT biosynthetic genes has been tested using GFP-based reporter, monitored by standard fluometry and flow cytometry. In situ experiments were also performed with cucumber plants. We found that several plant metabolites such as IAA and umbelliferone are able to modify significantly the expression of DAPG and PLT. The use of flow cytometry with autofluorescents proteins seems to be a promising method to study rhizobacteria-plant interactions.

Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC-MS.

A simple and sensitive LC-MS method was developed and validated for the simultaneous quantification of aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL), clozapine (CLO), olanzapine (OLA), sertindole (STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) in human plasma. The above mentioned compounds and the internal standard (remoxipride) were extracted from 0.5 mL plasma by solid-phase extraction (mix mode support). The analytical separation was carried out on a reverse phase liquid chromatography at basic pH (pH 8.1) in gradient mode. All analytes were monitored by MS detection in the single ion monitoring mode and the method was validated covering the corresponding therapeutic range: 2-200 ng/mL for DUL, OLA, and STN, 4-200 ng/mL for DSTN, 5-1000 ng/mL for ARI, DARI and finally 2-1000 ng/mL for ATO, CLO, NCLO, VEN, OVEN. For all investigated compounds, good performance in terms of recoveries, selectivity, stability, repeatability, intermediate precision, trueness and accuracy, was obtained. Real patient plasma samples were then successfully analysed.

Occurrence and Structure of Arbuscular Mycorrhizal Fungal Communities in Cassava after Cultivation of Cover Crops as Observed by the “PCR-DGGE” Technique

ABSTRACT Cassava (Manihot esculenta Crantz) is a highly mycotrophic crop, and prior soil cover may affect the density of arbuscular mycorrhizal fungi (AMFs), as well as the composition of the AMFs community in the soil. The aim of this study was to evaluate the occurrence and the structure of AMFs communities in cassava grown after different cover crops, and the effect of the cover crop on mineral nutrition and cassava yield under an organic farming system. The occurrence and structure of the AMFs community was evaluated through polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A randomized block experimental design was used with four replications. Six different cover crop management systems before cassava were evaluated: black oats, vetch, oilseed radish, intercropped oats + vetch, intercropped oats + vetch + oilseed radish, plus a control (fallow) treatment mowed every 15 days. Oats as a single crop or oats intercropped with vetch or with oilseed radish increased AMFs inoculum potential in soil with a low number of propagules, thus benefiting mycorrhizal colonization of cassava root. The treatments did not affect the structure of AMFs communities in the soil since the AMFs communities were similar in cassava roots in succession to different cover crops. AMFs colonization was high despite high P availability in the soil. The cassava crop yield was above the regional average, and P levels in the leaves were adequate, regardless of which cover crop treatments were used. One cover crop cycle prior to the cassava crop was not enough to observe a significant response in variables, P in plant tissue, crop yield, and occurrence and structure of AMFs communities in the soil. In the cassava roots in succession, the plant developmental stage affected the groupings of the structure of the AMF community.

Density-based hierarchical clustering of pyro-sequences on a large scale--the case of fungal ITS1.

MOTIVATION: Analysis of millions of pyro-sequences is currently playing a crucial role in the advance of environmental microbiology. Taxonomy-independent, i.e. unsupervised, clustering of these sequences is essential for the definition of Operational Taxonomic Units. For this application, reproducibility and robustness should be the most sought after qualities, but have thus far largely been overlooked. RESULTS: More than 1 million hyper-variable internal transcribed spacer 1 (ITS1) sequences of fungal origin have been analyzed. The ITS1 sequences were first properly extracted from 454 reads using generalized profiles. Then, otupipe, cd-hit-454, ESPRIT-Tree and DBC454, a new algorithm presented here, were used to analyze the sequences. A numerical assay was developed to measure the reproducibility and robustness of these algorithms. DBC454 was the most robust, closely followed by ESPRIT-Tree. DBC454 features density-based hierarchical clustering, which complements the other methods by providing insights into the structure of the data. AVAILABILITY: An executable is freely available for non-commercial users at ftp://ftp.vital-it.ch/tools/dbc454. It is designed to run under MPI on a cluster of 64-bit Linux machines running Red Hat 4.x, or on a multi-core OSX system. CONTACT: dbc454@vital-it.ch or nicolas.guex@isb-sib.ch.

Effects of lactation upon circulating plasma metabolites in cafeteria-fed rats

1. The effects of "cafeteria feeding" on primiparous Wistar rats during lactation have been studied by measuring circulating levels of glucose, amino acids, lactate, urea and ammonia as well as glycogen levels in liver and muscle. 2. No significant changes in glucose levels were observed despite alterations in blood glucose compartmentation. 3. Compared with controls, the dams given the cafeteria diet had higher liver glycogen stores which were more easily mobilized at the peak of lactation. 4. Rats given the cafeteria diet showed a lower amino acid utilization than controls and adequately maintained circulating levels, as determined by the lower circulating levels of ammonia and urea. 5. No significant differences in body-weight were observed in the period studied despite increasing dam weight after weaning in the cafeteria-fed group. 6. The size of pups of cafeteria-fed dams was greater than that of controls, and the differences were marked after weaning, when the metabolic machinery of the cafeteria pup maintained high protein accretion and body build-up using fat as the main energy substrate characteristic of the preweaning stage. The controls, however, changed to greater utilization of amino acids as an energy substrate and adapted to high-protein (lowbiological-quality) diets with a significantly different pattern of circulating nitrogen distribution.

An ultrastructural approach to analogies between fungal structures and needle fibre calcite

Needle fiber calcite (NFC) is an ubiquitous terrestrial secondary calcium carbonate mineral often associated with calcitic nanofibers. NFC's origin has been debated for a long time and a fungal origin is often proposed. Fungi are known to be involved in mineral weathering and production of metal oxalate, but little information exists regarding the genesis of other minerals, such as calcite. In this study, a comparison of similar ultrastructural characteristics of fungal hyphae and NFC has been performed to highlight analogies between both features. These analogies clearly demonstrate the probable close relationship between fungal filaments (hyphae and rhizomorphs) and NFC and its associated nanofibers.

The ecological significance of arbuscular mycorrhizal fungal effects on clonal reproduction in plants

The population ecology of clonal plants depends on the number and distribution of ramets formed during growth. Variation in clonal reproduction has previously been explained by variation in effects of abiotic resource heterogeneity and by plant genotypic variation. Different co-occurring species of the mutualistic arbuscular mycorrhizal fungi (AMF) have been shown to differentially alter growth traits of Prunella vulgaris which we hypothesize would lead to changes in clonal reproduction. Two experiments were carried out to test whether different co-occurring mycorrhizal fungi significantly influence clonal reproduction of P. vulgaris whether this effect also occurs when P. vulgaris is growing in an artificial plant community and how the effects compare with plant genotype effects on clonal growth of P. vulgaris. In the first experiment the number of ramets of P. vulgaris growing in a plant community of simulated calcareous grassland was significantly affected by inoculation with different mycorrhizal fungi. The number of ramets produced by P. vulgaris differed by a factor of up to 1.8 with different mycorrhizal fungi. The fungal effects on the number of new ramets were independent of their effects on the biomass of P. vulgaris. In a second experiment 17 different genotypes of P. vulgaris were inoculated with different mycorrhizal fungi. There were significant main effects of genotypes and mycorrhizal fungi on clonal reproduction of P. vulgaris. The effect of different mycorrhizal fungi contributed more than the effect of plant genotype to variation in size and ramet production. However mean stolon length and spacer length which determine the spatial arrangement of ramets were only significantly affected by plant genotype. There were no mycorrhizal fungal X plant genotype interactions on clonal growth of P. vulgaris indicating that there is no obvious evidence that selection pressures would favor further coevolution between P. vulgaris and mycorrhizal fungal species. In natural communities plants can be colonized by several different AMF at the same time. The effect of the mixed AMF treatment on the growth and clonal reproduction of P. vulgaris could not be predicted from the responses of the plants to the single AMF To what extent however the patterns of colonization by different AMF differ among plants in a natural community is unknown. Since the effects of AMF on growth and clonal reproduction occur on a population of P. vulgaris in a microcosm plant community and because the effects are also as great as those caused by plant genotypic variation we conclude that the effects are strong enough to potentially affect population size and variation of clonal plants in communities.

Comparative Genomics Suggests that the Fungal Pathogen Pneumocystis Is an Obligate Parasite Scavenging Amino Acids from Its Host's Lungs.

Pneumocystis jirovecii is a fungus causing severe pneumonia in immuno-compromised patients. Progress in understanding its pathogenicity and epidemiology has been hampered by the lack of a long-term in vitro culture method. Obligate parasitism of this pathogen has been suggested on the basis of various features but remains controversial. We analysed the 7.0 Mb draft genome sequence of the closely related species Pneumocystis carinii infecting rats, which is a well established experimental model of the disease. We predicted 8'085 (redundant) peptides and 14.9% of them were mapped onto the KEGG biochemical pathways. The proteome of the closely related yeast Schizosaccharomyces pombe was used as a control for the annotation procedure (4'974 genes, 14.1% mapped). About two thirds of the mapped peptides of each organism (65.7% and 73.2%, respectively) corresponded to crucial enzymes for the basal metabolism and standard cellular processes. However, the proportion of P. carinii genes relative to those of S. pombe was significantly smaller for the "amino acid metabolism" category of pathways than for all other categories taken together (40 versus 114 against 278 versus 427, P<0.002). Importantly, we identified in P. carinii only 2 enzymes specifically dedicated to the synthesis of the 20 standard amino acids. By contrast all the 54 enzymes dedicated to this synthesis reported in the KEGG atlas for S. pombe were detected upon reannotation of S. pombe proteome (2 versus 54 against 278 versus 427, P<0.0001). This finding strongly suggests that species of the genus Pneumocystis are scavenging amino acids from their host's lung environment. Consequently, they would have no form able to live independently from another organism, and these parasites would be obligate in addition to being opportunistic. These findings have implications for the management of patients susceptible to P. jirovecii infection given that the only source of infection would be other humans.

SARM-S4 and metabolites detection in sports drug testing: a case report.

Recently, pharmaceutical industry developed a new class of therapeutics called Selective Androgen Receptor Modulator (SARM) to substitute the synthetic anabolic drugs used in medical treatments. Since the beginning of the anti-doping testing in sports in the 1970s, steroids have been the most frequently detected drugs mainly used for their anabolic properties. The major advantage of SARMs is the reduced androgenic activities which are the main source of side effects following anabolic agents' administration. In 2010, the Swiss laboratory for doping analyses reported the first case of SARMs abuse during in-competition testing. The analytical steps leading to this finding are described in this paper. Screening and confirmation results were obtained based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Additional information regarding the SARM S-4 metabolism was investigated by ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS).

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

Sensing of mammalian IL-17A regulates fungal adaptation and virulence.

Infections by opportunistic fungi have traditionally been viewed as the gross result of a pathogenic automatism, which makes a weakened host more vulnerable to microbial insults. However, fungal sensing of a host's immune environment might render this process more elaborate than previously appreciated. Here we show that interleukin (IL)-17A binds fungal cells, thus tackling both sides of the host-pathogen interaction in experimental settings of host colonization and/or chronic infection. Global transcriptional profiling reveals that IL-17A induces artificial nutrient starvation conditions in Candida albicans, resulting in a downregulation of the target of rapamycin signalling pathway and in an increase in autophagic responses and intracellular cAMP. The augmented adhesion and filamentous growth, also observed with Aspergillus fumigatus, eventually translates into enhanced biofilm formation and resistance to local antifungal defenses. This might exemplify a mechanism whereby fungi have evolved a means of sensing host immunity to ensure their own persistence in an immunologically dynamic environment.