Functional analysis of small signaling molecules in jasmonate signaling.

Plants exhibit multitudes of defense mechanisms against different kinds of stress. Jasmonic acid (JA) is one of the identified signaling compounds mediating plant’s response to wounding, attack by herbivores or necrotrophic pathogens. Central parts of the JA signaling pathway have recently been unraveled by demonstrating that (+)-7-iso-JA-L-Ile is the most bioactive form of JA and that the SCF(COI1)-complex functions as its receptor. However, many other components of the JA signaling pathway remain unknown. This includes how and where protein kinases may be involved in JA signaling. Likewise, the mechanistic details of cross-talk between different hormone signaling pathways are unknown. Using a chemical biology approach, screening for selective compounds that can be used as tools in applications complementing genetic approaches, I aimed at identifying some of these unknown components. The advantage of this method is that it has the potential to circumvent redundancy of gene function, lethality of mutants and pleiotropic effects, problems generally encountered in genetic approaches. I developed a screening procedure for bioactive compounds that uses a transgenic Arabidopsis thaliana line, harboring the JA-responsive reporter gene LOX2p::LUC. This procedure allowed bidirectional screening for activators or inhibitors of reporter expression. Sifting through approx. 1,700 natural compounds, I identified one activator of reporter gene expression and 16 inhibitors of methyl jasmonate induced reporter expression. Critical validation of these primary hits revealed that the putative activator in fact interfered with the activity of the luciferase reporter. It presumably binds and stabilizes luciferase, thereby enhancing its apparent activity, whereas reporter gene expression was not affected. After validation and characterization of the inhibitors, one compound (12) was identified as selective inhibitor of JA signaling. Structure-activity relationship studies, using derivatives of the compound, defined parts of the molecule that where indispensable for its bioactivity. Based on this analysis, a derivatized probe was designed that harbors a ‘photoreactive’ benzophenone for establishing covalent binding and an alkyne residue to attach a detectable fluorophore using ‘click chemistry’. Importantly, this probe retained activity and was used in first affinity-based target identification experiments. In a second screen using a small, targeted library of 84 known protein kinase inhibitors, I identified three compounds that impaired JA signaling. This finding suggests the involvement of protein kinases in the JA signaling pathway that has been previously reported. Among the identified inhibitors was 5-iodotubercidin, a nucleoside antibiotic. A derivative of this compound, toyocamycin, was previously described to selectively impair auxin signaling, which is mechanistically related to JA signaling. Several structural analogs were investigated with respect to their effect on JA-dependent reporter expression or JA-independent readouts. Toyocamycin was considered to be the most specific derivative. To elucidate the role of toyocamycin in the Arabidopsis hormonal signaling network, I performed a microarray analysis after treatment with toyocamycin. The expression data showed that this compound modulates expression of JAZ genes, which are repressors of JA induced gene expression. Toyocamycin also modulated genes, which may be involved in hormonal crosstalk between e.g. auxin or salicylic acid signaling. The fact that toyocamycin caused a root growth phenotype, which is dependent on allene oxide synthase (AOS) and jasmonoyl-isoleucine synthetase (JAR1), indicated that multiple targets may exist, because inhibition of the LOX2 marker was independent of JAR1. Identification of the protein targets of toyocamycin and compound 12 may eventually lead to identification of yet unknown components in JA signaling or hormonal crosstalk

Considerations for designing chemical screening strategies in plant biology

Traditionally, biologists regularly used classical genetic approaches to characterize and dissect plant processes. However, this strategy is often impaired by redundancy, lethality or pleiotropy of gene functions, which prevent the isolation of viable mutants. The chemical genetic approach has been recognized as an alternative experimental strategy, which has the potential to circumvent these problems. It relies on the capacity of small molecules to modify biological processes by specific binding to protein target(s), thereby conditionally modifying protein function(s), which phenotypically resemble mutation(s) of the encoding gene(s). A successful chemical screening campaign comprises three equally important elements: (1) a reliable, robust, and quantitative bioassay, which allows to distinguish between potent and less potent compounds, (2) a rigorous validation process for candidate compounds to establish their selectivity, and (3) an experimental strategy for elucidating a compound's mode of action and molecular target. In this review we will discuss details of this general strategy and additional aspects that deserve consideration in order to take full advantage of the power provided by the chemical approach to plant biology. In addition, we will highlight some success stories of recent chemical screenings in plant systems, which may serve as teaching examples for the implementation of future chemical biology projects

The HPC certification forum: toward a globally acknowledged HPC certification

The goal of the HPC Certification Forum is to categorize, define, and examine competencies expected from proficient HPC practitioners. The community-led forum is working toward establishing a globally acknowledged HPC certification process, a process that engages with HPC centres to identify gaps in users’ knowledge, and with users to identify the skills required to perform their tasks. In this article, we introduce the forum and summarize the progress made over the last two years. The release of the first officially supported certificate is planned for the second half of 2020

2017 EACTS/EACTA Guidelines on patient blood management for adult cardiac surgery

Authors/Task Force Members: Christa Boer (EACTA Chairperson)(Netherlands), Michael I. Meesters (Netherlands), Milan Milojevic (Netherlands), Umberto Benedetto (UK), Daniel Bolliger (Switzerland), Christian von Heymann (Germany), Anders Jeppsson (Sweden), Andreas Koster (Germany), Ruben L. Osnabrugge (Netherlands), Marco Ranucci (Italy), Hanne Berg Ravn (Denmark), Alexander B.A. Vonk (Netherlands), Alexander Wahba (Norway), Domenico Pagano (EACTS Chairperson)(UK),. Document Reviewers: Moritz W.V. Wyler von Ballmoos (USA), Mate Petricevic (Croatia), Arie Pieter Kappetein (Netherlands), Miguel Sousa-Uva (Portugal), Georg Trummer (Germany), Peter M. Rosseel (Netherlands), Michael Sander (Germany), Pascal Colson (France), Adrian Bauer (Germany)

Citrulline supplementation improves organ perfusion and arginine availability under conditions with enhanced arginase activity

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with l-arginine supplementation exhibited less consistent results; however, l-citrulline, the precursor of l-arginine, may be a promising alternative. In this study, we determined the effects of l-citrulline compared to l-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with l-citrulline or l-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. l-arginine and l-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that l-citrulline, and not l-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues

Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site

Genome-wide association analysis of genetic generalized epilepsies implicates susceptibility loci at 1q43, 2p16.1, 2q22.3 and 17q21.32

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% and account for 20-30% of all epilepsies. Despite their high heritability of 80%, the genetic factors predisposing to GGEs remain elusive. To identify susceptibility variants shared across common GGE syndromes, we carried out a two-stage genome-wide association study (GWAS) including 3020 patients with GGEs and 3954 controls of European ancestry. To dissect out syndrome-related variants, we also explored two distinct GGE subgroups comprising 1434 patients with genetic absence epilepsies (GAEs) and 1134 patients with juvenile myoclonic epilepsy (JME). Joint Stage-1 and 2 analyses revealed genome-wide significant associations for GGEs at 2p16.1 (rs13026414, Pmeta = 2.5 × 10−9, OR[T] = 0.81) and 17q21.32 (rs72823592, Pmeta = 9.3 × 10−9, OR[A] = 0.77). The search for syndrome-related susceptibility alleles identified significant associations for GAEs at 2q22.3 (rs10496964, Pmeta = 9.1 × 10−9, OR[T] = 0.68) and at 1q43 for JME (rs12059546, Pmeta = 4.1 × 10−8, OR[G] = 1.42). Suggestive evidence for an association with GGEs was found in the region 2q24.3 (rs11890028, Pmeta = 4.0 × 10−6) nearby the SCN1A gene, which is currently the gene with the largest number of known epilepsy-related mutations. The associated regions harbor high-ranking candidate genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. Further replication efforts are necessary to elucidate whether these positional candidate genes contribute to the heritability of the common GGE syndrome

Variations in seasonal solar insolation are associated with a history of suicide attempts in bipolar I disorder

Background: Bipolar disorder is associated with circadian disruption and a high risk of suicidal behavior. In a previous exploratory study of patients with bipolar I disorder, we found that a history of suicide attempts was associated with differences between winter and summer levels of solar insolation. The purpose of this study was to confirm this finding using international data from 42% more collection sites and 25% more countries. Methods: Data analyzed were from 71 prior and new collection sites in 40 countries at a wide range of latitudes. The analysis included 4876 patients with bipolar I disorder, 45% more data than previously analyzed. Of the patients, 1496 (30.7%) had a history of suicide attempt. Solar insolation data, the amount of the sun’s electromagnetic energy striking the surface of the earth, was obtained for each onset location (479 locations in 64 countries). Results: This analysis confirmed the results of the exploratory study with the same best model and slightly better statistical significance. There was a significant inverse association between a history of suicide attempts and the ratio of mean winter insolation to mean summer insolation (mean winter insolation/mean summer insolation). This ratio is largest near the equator which has little change in solar insolation over the year, and smallest near the poles where the winter insolation is very small compared to the summer insolation. Other variables in the model associated with an increased risk of suicide attempts were a history of alcohol or substance abuse, female gender, and younger birth cohort. The winter/summer insolation ratio was also replaced with the ratio of minimum mean monthly insolation to the maximum mean monthly insolation to accommodate insolation patterns in the tropics, and nearly identical results were found. All estimated coefficients were significant at p < 0.01. Conclusion: A large change in solar insolation, both between winter and summer and between the minimum and maximum monthly values, may increase the risk of suicide attempts in bipolar I disorder. With frequent circadian rhythm dysfunction and suicidal behavior in bipolar disorder, greater understanding of the optimal roles of daylight and electric lighting in circadian entrainment is needed

Die Verbindung von Kleinwinkelstreuung und in silico-Methodik zur Aufklärung von Konformationswechseln großer Proteinkomplexe

In dieser Dissertation wurden die Methoden Homologiemodellierung und Molekulardynamik genutzt, um die Struktur und das Verhalten von Proteinen in Lösung zu beschreiben. Mit Hilfe der Röntgenkleinwinkelstreuung wurden die mit den Computermethoden erzeugten Vorhersagen verifiziert. Für das alpha-Hämolysin, ein Toxin von Staphylococcus aureus, das eine heptamere Pore formen kann, wurde erstmalig die monomere Struktur des Protein in Lösung beschrieben. Homologiemodellierung auf Basis verwandter Proteine, deren monomere Struktur bekannt war, wurde verwendet, um die monomere Struktur des Toxins vorherzusagen. Flexibilität von Strukturelementen in einer Molekulardynamiksimulation konnte mit der Funktionalität des Proteines korreliert werden: Intrinsische Flexibilität versetzt das Protein in die Lage den Konformationswechsel zur Pore nach Assemblierung zu vollziehen. Röntgenkleinwinkelstreuung bewies die Unterschiede der monomeren Struktur zu den Strukturen der verwandten Proteine und belegt den eigenen Vorschlag zur Struktur. Überdies konnten Arbeiten an einer Mutante, die in einer sogenannten Präporenkonformation arretiert und nicht in der Lage ist eine Pore zu formen, zeigen, dass dieser Übergangszustand mit der Rotationsachse senkrecht zur Membran gelagert ist. Eine geometrische Analyse beweist, dass es sterisch möglich ist ausgehend von dieser Konformation die Konformation der Pore zu erreichen. Eine energetische und kinetische Analyse dieses Konformationswechsels steht noch aus. Ein weiterer Teil der Arbeit befasst sich mit den Konformationswechseln von Hämocyaninen. Diese wurden experimentell mittels Röntgenkleinwinkelstreuung verfolgt. Konformationswechsel im Zusammenhang mit der Oxygenierung konnten für die 24meren Hämocyanine von Eurypelma californicum und Pandinus imperator beschrieben werden. Für eine Reihe von Hämocyaninen ist nachgewiesen, dass sie unter Einfluss des Agenz SDS Tyrosinaseaktivität entfalten können. Der Konformationswechsel der Hämocyanine von E. californicum und P. imperator bei der Aktivierung zur Tyrosinase mittels SDS wurde experimentell bestätigt und die Stellung der Dodekamere der Hämocyanine als wesentlich bei der Aktivierung festgestellt. Im Zusammenhang mit anderen Arbeiten gilt damit die Relaxierung der Struktur unter SDS-Einfluss und der sterische Einfluss auf die verbindenden Untereinheiten b & c als wahrscheinliche Ursache für die Aktivierung zur Tyrosinase. Eigene Software zum sogenannten rigid body-Modellierung auf der Basis von Röntgenkleinwinkelstreudaten wurde erstellt, um die Streudaten des hexameren Hämocyanins von Palinurus elephas und Palinurus argus unter Einfluss der Effektoren Urat und Koffein strukturell zu interpretieren. Die Software ist die erste Implementierung eines Monte Carlo-Algorithmus zum rigid body-Modelling. Sie beherrscht zwei Varianten des Algorithmus: In Verbindung mit simulated annealing können wahrscheinliche Konformationen ausgefiltert werden und in einer anschließenden systematischen Analyse kann eine Konformation geometrisch beschrieben werden. Andererseits ist ein weiterer, reiner Monte Carlo-Algorithmus in der Lage die Konformation als Dichteverteilung zu beschreiben.In this work homology modeling and molecular dynamics simulations were used to describe the structure and behavior of protein complexes in solution. With the aid of small angle scattering such computer generated models were verified. The monomeric alpha-hemolysin, a toxin from Staphylococcus aureus, which forms heptameric pores, was described for the first time. Homologymodeling based on structural homologues, for which the monomeric structures were known, was used to predict the monomeric structure of the alpha-hemolysin. Flexibility of structural elements could be shown by means of molecular dynamics simulations and correlated to functionality: Intrinsic flexibility enables the protein to undergo the conformational change towards the pore state after assembly onto a membrane. Small Angle X-ray Scattering (SAXS) proved differences to the structure of the homologues proteins and verified the own model. In addition work on a mutant, which is arrested in the so-called pre-pore state and cannot form the pore structure, could provide evidence that the axis of symmetry of the pre-pore complex is perpendicular to the membrane plane. Geometric analysis proves the steric possibility to reach the pore conformation from this pre-pore complex without loosing integrity of the complex. Further analysis with regard to energy and kinetics of this conformational change is pending. A second part of this work considers the conformational changes of hemocyanins. These changes were followed using SAXS as well. Conformational changes as a consequence of oxygenation could be described for the 24mers of Eurypelma californicum and Pandinus imperator. For some hemocyanins it has been shown that the addition of SDS can trigger tyrosinase activity. The conformational change for the hemocyanins E. californicum and P. imperator upon the addition of SDS has been shown experimentally and could be connected to the relative position of the 12meric half-molecules. In connection with other work the steric influence on the subunits b & c is to be considered the most likely cause for the enzymatic activity. An own software for rigid body-modeling on the basis of small angle scattering data was developed to analyze the SAXS data of the hemocyanins from Palinurus elephas and Palinurus argus in presence of ligands such as urate and coffein. This software is the first implementation of a Monte Carlo algorithm for rigid body-modeling. It provides two different approaches: In connection with simulated annealing a likely conformation can be selected and a subsequent systematic screen is able to describe a particular conformation. In addition a different Monte Carlo algorithm is able to describe a conformation as a distribution of sub-conformation in a likelyhood space