Effect of pine bark and compost on the biological denitrification process of non-hazardous landfill leachate: Focus on the microbiology

In an attempt to optimize the cost-efficiency of landfill leachate treatment by biological denitrification process, our study focused on finding low-cost alternatives to traditional expensive chemicals such as composted garden refuse and pine bark, which are both available in large amount in South African landfill sites. The overall objective was to assess the behaviour of the bacterial community in relation to each substrate while treating high strength landfill leachates. Denitrification processes in fixed bed reactors were simulated at laboratory scale using anaerobic batch tests with immature compost and pine bark. High strength leachate was simulated using a solution of water and nitrate at a concentration of 500 mg l−1. Results suggest that pine bark released large amounts of phenolic compounds and hydroxylated benzene rings, which both can delay the acclimatization time and inhibit the biological denitrification (only 30% efficiency). Furthermore, presence of potential pathogens like Enterobacter and Pantoea agglomerans prevents the applicability of the pine bark in full-scale operations. On the other hand, lightly composted garden refuse (CGR) offered an adequate substrate for the formation of a biofilm necessary to complete the denitrification process (total nitrate removal observed within 7 days). CGR further contributed to a rapid establishment of an active consortium of denitrifiers including Acinetobacter, Rhizobium, Thermomonas, Rheinheimera, Phaeospirillum and Flavobacterium. Clearly the original composition, nature, carbon to nitrogen ratio (C/N) and degree of maturity and stability of the substrates play a key role in the denitrification process, impacting directly on the development of the bacterial population and, therefore, on the long-term removal efficiency

Yeast Two-Hybrid, a Powerful Tool for Systems Biology

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided

Ubiquitinating enzymes as potential therapeutic targets to prevent muscle atrophy -The case of MuRF1/ TRIM63

International audienc

Skeletal muscle atrogenes: from rodent models to human pathologies

Epub ahead of printSkeletal muscle atrophy is a common side effect of most human diseases. Muscle loss is not only detrimental for the quality of life but it also dramatically impairs physiological processes of the organism and decreases the efficiency of medical treatments. While hypothesized for years, the existence of an atrophying programme common to all pathologies is still incompletely solved despite the discovery of several actors and key regulators of muscle atrophy. More than a decade ago, the discovery of a set of genes, whose expression at the mRNA levels were similarly altered in different catabolic situations, opened the way of a new concept: the presence of atrogenes, i.e. atrophy-related genes. Importantly, the atrogenes are referred as such on the basis of their mRNA content in atrophying muscles, the regulation at the protein level being sometimes more complicate to elucidate. It should be noticed that the atrogenes are markers of atrophy and that their implication as active inducers of atrophy is still an open question for most of them. While the atrogene family has grown over the years, it has mostly been incremented based on data coming from rodent models. Whether the rodent atrogenes are valid for humans still remain to be established. An "atrogene" was originally defined as a gene systematically up- or down-regulated in several catabolic situations. Even if recent works often restrict this notion to the up-regulation of a limited number of proteolytic enzymes, it is important to keep in mind the big picture view. In this review, we provide an update of the validated and potential rodent atrogenes and the metabolic pathways they belong, and based on recent work, their relevance in human physio-pathological situations. We also propose a more precise definition of the atrogenes that integrates rapid recovery when catabolic stimuli are stopped or replaced by anabolic ones

SNF1/AMPK/SnRK1 kinases, global regulators at the heart of energy control?

The SNF1-related kinases are considered to be crucial elements of transcriptional, metabolic and developmental regulation in response to stress. In yeast, SNF1 is one of the main regulators in the shift from fermentation to aerobic metabolism; AMPK, its mammalian counterpart, is a master metabolic regulator involved in a variety of metabolic disorders such as diabetes and obesity. The aim of this review is to examine the literature concerning SnRK1 proteins, the SNF1 homologues in plants. The remarkable structural similarities between the plant complexes and those of yeast and mammalian suggest the existence of a common ancestral function in the regulation of energy and carbon metabolism. We will also highlight some distinctive features acquired by the plant proteins during evolution

Etude de la structure des complexes kinases AKIN chez Arabidopsis thaliana (expression et fonctions des sous-unités de type beta)

Les complexes kinases SNF1/AMPK chez la levure et les mammifères sont considérés comme des régulateurs importants du métabolisme en réponse à des changements environnementaux et nutritionnels. SNF1 est notamment impliqué dans la réponse de S. cerevisiae à une carence en glucose et AMPK dans la réponse des cellules à un faible taux d'ATP. Les données actuelles suggèrent fortement que leur homologue chez les plantes, SnRK1, régulerait des enzymes clefs du métabolisme carboné et azoté.Chez la levure et les mammifères, le complexe fonctionne sous forme d'hétérotrimère comprenant deux sous-unités non catalytiques : une sous-unité de type b et une sous-unité de type g. Dans un premier temps, une étude fine de la structure des complexes chez A. thaliana a été réalisée par l'analyse par techniques de double-hybride et coimmunoprécipitation, des interactions entre ces différentes sous-unités ou différents domaines de ces protéines.Une étude approfondie de l'expression des gènes AKINb au cours du développement et en réponse à différents changements environnementaux a permis de montrer qu'un des niveaux de régulation du complexe pourrait se faire via une régulation transcriptionnelle différentielle des sous-unités de type b. L'utilisation de plantes transgéniques a permis de mettre en évidence des profils d'expression extrêmement variables et très spécifiques de chacune des sous-unités b, tout au long du développement de la plante. Finalement, l'utilisation de trois approches, complémentation de mutants de levure, plantes transgéniques et criblages double-hybride réalisées avec les trois sous-unités b a ouvert des pistes pour l'identification de différentes fonctions du complexe.Several evidences designate the yeast SNF1 and mammals AMPK as important regulators of the metabolism in response to environmental or nutritional changes. SNF1 is implicated in the response of S. cerevisiae to glucose starvation and AMPK in the response of cells to low ATP levels. Currently, data indicate that their plant homologues SnRK1 could regulate key enzymes of sugar synthesis and nitrate assimilation.SNF1 and AMPK have been shown to function as an heterotrimeric complex including two types of non-catalytic subunits : the b- and g-types subunits. We have first performed a detailed structural analysis of the complexes. Two-hybrid and co-immunoprecipitation experiments have been performed in order to study the interactions occurring between the different subunits and between different domains of these proteins. Interestingly, detailed expression studies of AKINb genes during development and in response to environmental changes reveal that one level of regulation of the SnRK1 kinase could be due to the a differential transcription of the ? subunits. Precisely, we have shown that the three b-type subunits present very specific and differential patterns of expression all along plant development.Finally three different approaches, yeast complementation, transgenic plants and two-hybrid screens using b1/2/3-subunits as baits, have provided different indications related to the functions of these different complexes in plants.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Role of UBE2L3 and MuRF1/TRIM63 in Skeletal Muscle Atrophy

International audienc

Mechanisms of muscle atrophy: from UPS implication in rodent models to human biomarkers

International audienceThe ubiquitin proteasome system (UPS) is a major player of skeletal muscle wasting, a common characteristic of many diseases (cancer, sepsis, heart failure, kidney diseases, etc.) that negatively impacts treatment and life prognosis. In the early 2000, the notion of "atrogenes" (atrophy-related genes) was proposed for genes that were systematically up regulated during catabolic situations in rodents, which included 2 founding members, the E3 ubiquitin ligases MAFbx/Atrogin-1 and MuRF1/TRIM63 [1]. Interestingly, MuRF1 is so far the only E3 ligase known for targeting several sarcomeric proteins (a-actin, MYHC, Troponin I, telethonin) and we recently identified the E2 ubiquitin conjugating enzymes that bring the catalytic activity for MuRF1-dependent protein degradation [2, 3]. Besides rodent models, we and others demonstrated that MAFbx and MuRF1 are also gold standard atrogenes in human pathologies and we recently found that numerous proteins may be part of a muscle atrophy program in chronic kidney disease and lung cancer patients [4]. In addition, we have identified blood markers that reflect skeletal muscle loss in patients suffering from several pathologies