A Suppression Strategy for Antibiotic Discovery

AbstractHigh-throughput phenotype screening and target identification have been combined in an effort to isolate antimicrobial, small-molecule therapeutics [1]. This approach, developed by Brown and colleagues and reported in this issue, is a major technological advance for antimicrobial drug discovery

Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting

Directed evolution enables the improvement and optimisation of enzymes for particular applications and is a valuable tool for biotechnology and synthetic biology. However, studies are often limited in their scope by the inability to screen very large numbers of variants to identify improved enzymes. One class of enzyme for which a universal, operationally simple ultra-high throughput (>106 variants per day) assay is not available is flavin adenine dinucleotide (FAD) dependent oxidases. The current high throughput assay involves a visual, colourimetric, colony-based screen, however this is not suitable for very large libraries and does not enable quantification of the relative fitness of variants. To address this, we describe an optimised method for the sensitive detection of oxidase activity within single Escherichia coli (E. coli) cells, using the monoamine oxidase from Aspergillus niger, MAO-N, as a model system. In contrast to other methods for the screening of oxidase activity in vivo, this method does not require cell surface expression, emulsion formation or the addition of an extracellular peroxidase. Furthermore, we show that fluorescence activated cell sorting (FACS) of large libraries derived from MAO-N under the assay conditions can enrich the library in functional variants at much higher rates than via the colony-based method. We demonstrate its use for directed evolution by identifying a new mutant of MAO-N with improved activity towards a novel secondary amine substrate. This work demonstrates, for the first time, an ultra-high throughput screening methodology widely applicable for the directed evolution of FAD dependent oxidases in E. coli

Comportement à haute température des bétons à haute performance évolution des principales propriétés mécaniques

The presented work aims at a better understanding of the behaviour of high performance concretes (HPC) subjected to high temperatures. We were more particularly interested in the mechanical behaviour of concretes during the heating. To realize this study a new experimental device was developed, allowing testing the compressive strength and the direct tensile strength at "hot" stage. The major part of this study was to establish the relations between the mechanical properties and the temperature of different concrete types. The compression strength and the modulus of elasticity change not only with the exposure temperature of the material, but also with the heating scenario (heating ratio, hot/residual tests, etc.) In this study the influence of the water / cement ratio was studied as well as the influence of the addition of the polypropylene fibres on the hot tested mechanical properties of the HPC. It can be deduced from our studies that the evolution of mechanical properties is related to the presence of free water in the material, especially in the range of temperatures until 300°C. An important part of this study was dedicated to the study of transient thermal strains. This behaviour, called "transient thermal creep" was examined on three high performance concretes (HPC) and one ordinary concrete (OC). Among the parameters influencing the transient thermal strain we can mention: the loading rate, the history of the thermo-mechanical loading, the heating rate or the moisture content of the concrete. Furthermore, we were study an existence of the phenomena under tension load. Those results were completed by SEM observations of the microstructure, porosity measurements and weight variations with temperature changes.Le travail présenté vise une meilleure compréhension des phénomènes mis en jeu lors de l'exposition à haute température des bétons à haute performance. Nous nous sommes plus particulièrement intéressés au comportement mécanique. La première partie de ce document présente une synthèse des travaux réalisés sur l'évolution des propriétés physiques et mécaniques des bétons soumis à haute température. Les résultats présents dans la littérature sont variés, parfois contradictoires et confus notamment ceux concernant l'évolution des propriétés mécaniques au cours de l'échauffement. Les évolutions de la résistance en compression, résistance en traction et module d'élasticité sont présentés en fonction des différents paramètres influençant ces comportements. L'influence des conditions de réalisation des essais est notamment discutée. En outre, le comportement au feu est abordé sous l'angle du phénomène de l'écaillage. Nous présentons ainsi le rôle des fibres polypropylène, un moyen efficace de réduire les risques d'apparition du comportement explosif. La deuxième partie, consacrée à l'étude expérimentale. Dans cette partie nous exposons les matériaux testés, leurs compositions et les constituants utilisés, ainsi que la fabrication de ces matériaux et leur conservation. Ensuite nous présentons le banc d'essais développé, permettant de tester le comportement mécanique à chaud des bétons. Ce nouveau dispositif expérimental spécifique et relativement complexe, a été spécialement mis au point afin de réaliser l'étude de la déformation thermique libre, de la déformation thermique sous charge mécanique, et du comportement en compression et en traction à chaud. Les résultats expérimentaux obtenus, sont ensuite présentés et discutés. La majeure partie de cette étude a été consacrée à l'établissement des relations entre le comportement mécanique et la température des différents types de béton. La résistance en compression et le module d'élasticité varient non seulement avec la température d'exposition du matériau, mais aussi avec le scénario d'échauffement (vitesse de montée en température, essais à chaud/après refroidissement, etc.). Dans les observations que nous avons réalisé, l'influence du rapport E/C des bétons testés a été étudiée ainsi que l'influence de l'ajout des fibres polypropylène sur les propriétés mécanique des BHP testés à chaud. Les observations de l'évolution des propriétés mécaniques réalisées nous ont permis de constater que les résultats du comportement à haute température sont fortement influencés par la présence de l'eau dans le matériau, surtout dans la gamme de températures jusqu'à 300°C. En complément de l'étude du comportement en compression, nous avons étudié la possibilité de réalisation des essais de traction directe sur le matériau béton "à chaud". Diverses solutions ont été envisagées et testées, afin de retenir la technique plus adaptée à nos besoins. Les premiers résultats sont très prometteurs. Il faut souligner que le nombre d'études expérimentales du comportement en traction directe "à chaud" est extrêmement faible. Une grande partie de la recherche a été consacrée à l'étude de la déformation thermique transitoire (DTT). Ce comportement, encore mal connu et souvent controversé, appelé "fluage thermique transitoire" a été étudié sur trois bétons à haute performance (BHP) et un béton ordinaire (BO). Parmi les paramètres influençant la DTT nous pouvons citer: le taux de chargement, l'histoire du chargement thermo mécanique, la vitesse de montée an température ou la teneur en eau du béton. De plus, nous avons étudié de l'existence du phénomène de la DTT sous charge mécanique en traction

Insights into the mechanism and substrate specificity of human lysine -specific demethylase-1

Histones are small basic proteins that function to organize DNA in cells. The nucleosomal core particle, the fundamental unit of chromatin, consists of 146-147 base pairs of DNA wound around a complex of two H2A-H2B histone dimers and an H3-H4 tetramer. Histones are subject to a myriad of post-translational modifications, including methylation, phosphorylation, acetylation, and ubiquitination, which function in the regulation of various cell processes, including gene transcription. Lysine-Specific Demethylase-1 (LSD1), a member of the monoamine oxidase (MAO) family of flavoprotein amine oxidases, has been shown to remove methyl groups from lysine residues four and nine of histone H3 (H3K4 and H3K9), as well as lysine 370 of the tumor suppressor protein p53. The enzyme has been shown to play a role in controlling cell differentiation, and its expression correlates with neuroblastoma and prostate cancer progression; thus, selective inhibitors of LSD1 may be beneficial for treatment of various diseases. Thorough characterization of the mechanism and substrate specificity of LSD1 are essential for the development of such inhibitors, as well as for increasing the understanding of transcriptional regulation and cancer proliferation. The substrate specificity of LSD1 has been studied in vitro by utilizing a series of peptide substrates corresponding to the amino acid sequence of the N-terminal tail of histone H3. Recombinant human LSD1 was shown to act specifically on the dimethylated H3K4 residue in vitro, with arginine residues in the peptide substrate being essential for recognition of the substrate by the enzyme. Steady-state and transient kinetic studies have shown that C-H bond cleavage is rate-limiting in oxidation of a peptide substrate by LSD1. Furthermore, the redox potential of LSD1, significantly higher than that of free flavin, demonstrates that the enzyme provides a more favorable environment for flavin-catalyzed oxidation. Finally, evidence suggests that a lysine residue conserved in the MAO family, Lys661 in LSD1, may play a role in enzyme stability. These studies contribute to the overall understanding of the mechanism and substrate-specificity of LSD1

Insights into the mechanism and substrate specificity of human lysine -specific demethylase-1

Histones are small basic proteins that function to organize DNA in cells. The nucleosomal core particle, the fundamental unit of chromatin, consists of 146-147 base pairs of DNA wound around a complex of two H2A-H2B histone dimers and an H3-H4 tetramer. Histones are subject to a myriad of post-translational modifications, including methylation, phosphorylation, acetylation, and ubiquitination, which function in the regulation of various cell processes, including gene transcription. Lysine-Specific Demethylase-1 (LSD1), a member of the monoamine oxidase (MAO) family of flavoprotein amine oxidases, has been shown to remove methyl groups from lysine residues four and nine of histone H3 (H3K4 and H3K9), as well as lysine 370 of the tumor suppressor protein p53. The enzyme has been shown to play a role in controlling cell differentiation, and its expression correlates with neuroblastoma and prostate cancer progression; thus, selective inhibitors of LSD1 may be beneficial for treatment of various diseases. Thorough characterization of the mechanism and substrate specificity of LSD1 are essential for the development of such inhibitors, as well as for increasing the understanding of transcriptional regulation and cancer proliferation. The substrate specificity of LSD1 has been studied in vitro by utilizing a series of peptide substrates corresponding to the amino acid sequence of the N-terminal tail of histone H3. Recombinant human LSD1 was shown to act specifically on the dimethylated H3K4 residue in vitro, with arginine residues in the peptide substrate being essential for recognition of the substrate by the enzyme. Steady-state and transient kinetic studies have shown that C-H bond cleavage is rate-limiting in oxidation of a peptide substrate by LSD1. Furthermore, the redox potential of LSD1, significantly higher than that of free flavin, demonstrates that the enzyme provides a more favorable environment for flavin-catalyzed oxidation. Finally, evidence suggests that a lysine residue conserved in the MAO family, Lys661 in LSD1, may play a role in enzyme stability. These studies contribute to the overall understanding of the mechanism and substrate-specificity of LSD1

Isotope Effects Suggest a Stepwise Mechanism for Berberine Bridge Enzyme

The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (<i>S</i>)-reticuline to (<i>S</i>)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on <i>k</i>_red, the rate constant for flavin reduction, is pH-independent, indicating that C–H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on <i>k</i>_red are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived