Changes of Gene Expression in Euglena gracilis Obtained During the 29th DLR Parabolic Flight Campaign

Parabolic flight maneuvers of Novespace’s Airbus A310 ZERO-G produce subsequent phases of hypergravity (about 20 s), microgravity (about 22 s) and another 20 s hypergravity on experiments located in the experiment area of the aircraft. The 29th DLR parabolic flight campaign consisted of four consecutive flight days with thirty-one parabolas each day. Euglena gracilis cells were fixed with TRIzol during different acceleration conditions at the first and the last parabola of each flight. Samples were collected and analyzed with microarrays for one-color gene expression analysis. The data indicate significant changes in gene expression in E. gracilis within short time. Hierarchical clustering shows that changes induced by the different accelerations yield reproducible effects at independent flight days. Transcription differed between the first and last parabolas indicating adaptation effects in the course of the flight. Different gene groups were found to be affected in different phases of the parabolic flight, among others, genes involved in signal transduction, calcium signaling, transport mechanisms, metabolic pathways, and stress-response as well as membrane and cytoskeletal proteins. In addition, transcripts of other areas, e.g., DNA and protein modification, were altered. The study contributes to the understanding of short-term effects of microgravity and different accelerations on cells at a molecular level

Investigation of gravitaxis-dependent proteins in Euglena gracilis, particularly a certain proteinkinase A

Euglena gracilis ist ein einzelliger Süßwasserflagellat und bewegt sich vorwiegend negativ gravitaktisch (Aufwärtsbewegung der Zelle entgegen dem Schwerkraftvektor), um eine optimale Position in der Wassersäule für die Photosynthese, das Wachstum und die Reproduktion zu erreichen. Der vermutliche Signalweg für die Gravitaxis in Euglena gracilis beginnt, indem Kraft auf die untere Membran wirkt, weil die Zellen eine horizontale Schwimmbewegung durchführen. Dadurch kommt es zur Öffnung von mechano-sensitiven Kanälen und einem Ionenstrom, was wiederum eine Veränderung des Membranpotentials bewirkt. Anschließend wird Calmodulin 2 durch Calcium stimuliert, welches wiederum vermutlich eine Adenylatzyklase aktiviert, die cAMP aus ATP synthetisiert. Danach wird die Proteinkinase A durch dieses cAMP stimuliert und phosphoryliert weitere Proteine, die am Geißelschlag beteiligt sind. Aus diesem Grund wurde die Proteinkinase A im Zusammenhang mit Veränderungen der Schwerkraft untersucht. Zusätzlich erfolgten erstmalige Proteomanalysen von Euglena gracilis mittels Massenspektrometrie, um ein besseres Verständnis, was durch den Einfluss der Schwerkraft in der Zelle passiert, zu erlangen. Während des Parabelfluges kam es innerhalb der ersten Parabel ausschließlich in der zweiten Hypergravitationsphase zu Veränderungen in der Phosphorylierung der PKA, während in der letzten Parabel keine Veränderungen der Phosphorylierung mehr beobachtet werden konnten. Innerhalb des zweistündigen Parabelfluges konnte eine Abnahme der PKA und eine leichte Zunahme der Phosphorylierung der PKA nach den 31 Parabeln verzeichnet werden. Dies könnte auf mögliche Adaptationseffekte hindeuten. Bei der Proteomanalyse waren 22 Proteine signifikant verändert, wovon 8 Proteine wiederholt auftraten. Dabei zeigten sich fast identische Proteinveränderungen in den einzelnen Phasen der Parabel 1, welche primär hochreguliert waren. Diese Proteine sind an der Translation, Phosphorylierung, Citratzyklus und Zellteilung beteiligt. Ferner konnte HSP70 (herunterreguliert) identifiziert werden. Nach zwei Stunden kam es zu einer Hochregulation einer Glutamatdehydrogenase, welche als Stressindikator fungiert. Beim MAXUS-Flug waren bei 0,02 und 0,04 x g keine Veränderungen des gravitaktischen Verhaltens und der Proteinkinase A gegeben. Bei einem Beschleunigungslevel von 0,08 x g konnte eine Zunahme der Präzision der Orientierung der Zellen sowie eine Veränderung der PKA und deren Phosphorylierung beobachtet werden. Somit konnte auch der Schwellenwert von Euglena gracilis für die Wahrnehmung der Schwerkraft näher präzisiert werden. Bei 0,16 x g zeigten sich erneut keine Veränderungen im PKA-Gehalt. Bei der Proteomanalyse waren 34 Proteine signifikant verändert, davon traten 9 Proteine wiederholt auf. Unter Schwerelosigkeit Zusammenfassung 9 waren die Proteine primär hochreguliert und an der Fettsäure-Biosynthese, Intrazellulärer Proteintransport, Mannitol-Metabolismus (unterstützt bei osmotischen Stress), Translation sowie Phosphorylierung beteiligt. Ab dem niedrigsten Beschleunigungslevel über den gesamten Flug konnte eine Herunterregulation des 86 kDa Articulins in Euglena gracilis beobachtet werden. Bei 0,02 und 0,04 x g kam es zu Veränderungen von Proteinen, die an Citratzyklus, Phosphorylierung und Photosynthese beteiligt sind. Ab dem nächsten Beschleunigungslevel von 0,08 x g kam es zu einer 2-fachen Hochregulation von Proteinen für die Phosphorylierung und Photosynthese (PS II). In der letzten Beschleunigungsphase konnten am wenigsten signifikant veränderte Proteine beobachtet werden, wovon primär herunterregulierte Proteine (Translation) auftraten. Beim Hypergravitationsexperiment (1,8 x g) auf der Zentrifuge nimmt die Präzision der Orientierung der Zellen zu und es zeigte sich nach einer 10-minütigen Expositionszeit eine Veränderung im PKA-Gehalt, die sich nach 30 und 60 min nicht weiter veränderte. Bei der Proteomanalyse waren 88 Proteine verändert, wovon 36 Proteine wiederholt auftraten. Nach 5 min Hypergravitation kam es zu einem deutlichen Anstieg in der Proteinveränderung (36), wobei keine Übereinstimmungen zu den anderen untersuchten Zeitpunkten auftraten. Die herunterregulierten Proteine (11) sind an der Translation, Cytoskelett und Proteinfaltung beteiligt, aber es konnten sehr viele Proteine nicht annotiert werden. Die hochregulierten Proteine (25) spielen eine Rolle in der Translation, Zellteilung, Phosphorylierung, oxidativer Stress, mRNA-Spleißing, Citratzyklus, Gluconeogenese und Pentosephosphatweg. Das Histon H2A war 42-fach hochreguliert. Nach 10 min Hypergravitation zeigten sich 34 veränderte Proteine, wovon nur ein Protein hochreguliert war, welches eine Threonin-Ammoniak-Lyase bzw. Threonin-Desaminase ist und eine mögliche Antwort auf abiotischen Stress sein könnte. Die herunterregulierten Proteine spielen eine Rolle in der Translation, De-/Phosphorylierung, Proteinmodifikation, Protonentransport, Steuerung der Proteinlokalisierung, RNA Modifikation, mRNA-Spleißing, Fettsäure-Biosynthese und Proteinfaltung. Nach 30 und 60 min Hypergravitation gegenüber der 1 x g Kontrolle konnten zu einem hohen Prozentsatz die gleichen signifikant veränderten Proteine im Vergleich zu den 10 min Hypergravitation beobachtet werden. Somit konnte nach 5 und 10 min Hypergravitation die drastischste Veränderung in den Proteinen verzeichnet werden.Euglena gracilis is a single-cell freshwater flagellate and shows an upward movement against the gravity vector to achieve an optimal position in the water column for photosynthesis, growth and reproduction. The current model of gravitaxis in Euglena gracilis begins with a force acting on the lower membrane because the cells perform a horizontal swimming pattern. The force activates mechano-sensitive ion channels and the resulting ion influx changes the membrane potential. Subsequently, calmodulin 2 is stimulated by calcium, which in turn probably activates an adenylate cyclase, which synthesizes cAMP from ATP. Thereafter, protein kinase A is stimulated by cAMP, which phosphorylates other proteins involved in changes of the flagellar beating pattern. For this reason, protein kinase A has been studied with respect to changes in gravity. In addition, the first proteome analyzes in Euglena gracilis were performed by mass spectrometry to get a better understanding about what happens in the cell through the influence of gravity. During the parabolic flight, changes in the phosphorylation of PKA were only observed in the second hypergravity phase of the first parabola. However, no changes of the phosphorylation was detected in the last parabola. Looking at the total parabolic flight, a decrease in PKA and a slight increase in phosphorylation of PKA were observed after the 31 parabolas (2 hours). This could be due to possible adaptation effects. In proteome analysis, 22 proteins were significantly altered, of which eight proteins are occurred repeatedly. Almost identical protein changes were found in the three different phases of parabola 1, which were primarily upregulated. These proteins are involved in translation, phosphorylation, citrate cycle and cell division. HSP70 was found to be downregulated. After 2 hours, there was an upregulation of a glutamate dehydrogenase, which acts as a stress indicator. During the MAXUS flight, no changes were detected at 0.02 and 0.04 x g, neither in gravitational behavior nor in the amount of protein kinase A. At the acceleration level of 0.08 x g, an increase in the precision of orientation of the cells as well as a change in PKA and its phosphorylation could be observed. Thus, the threshold of Euglena gracilis for the perception of gravity could be specified more precisely. There was no change in the PKA content at 0.16 x g. In proteome analysis, 34 proteins were significantly altered, of which nine proteins are occurred repeatedly. Under weightlessness, the proteins were primarily upregulated and involved in fatty acid biosynthesis, intracellular protein transport, mannitol metabolism (involved in osmotic stress), translation and phosphorylation. At any acceleration level over the entire flight, a downregulation of the 86-kDa articulin in Euglena gracilis was observed. At 0.02 and 0.04 x g, protein changes are involved in Abstract 11 the citrate cycle, phosphorylation and photosynthesis. At 0.08 x g, there was a twofold upregulation of proteins for phosphorylation and photosynthesis (PS II). In the last acceleration phase, the fewest and primarily downregulated proteins (translation) were observed. In the hypergravity experiment (1.8 x g) on the centrifuge the precision of the orientation of the cells increases and after an exposure time of 10 minutes, there was a change in the PKA content, which did not change further after 30 and 60 min. In the proteome analysis, 88 proteins were altered, of which 36 proteins are occurred repeatedly. After 5 min of hypergravity, there was a strong increase in protein change (36) with no matches at the other time points. The downregulated proteins (11) are involved in translation, cytoskeleton and protein folding, but many proteins could not be annotated. The upregulated proteins (25) play a role in translation, cell division, phosphorylation, oxidative stress, mRNA splicing, citrate cycle, gluconeogenesis and pentose phosphate pathway. Histone H2A was 42-fold upregulated. After 10 minutes, 34 altered proteins were observed, of which only one protein was upregulated. This protein is a threonine ammonia lyase or threonine deaminase, respectively, and could be a response to abiotic stress. The downregulated proteins play a role in translation, dephosphorylation, protein modification, proton transport, control of protein localization, RNA modification, mRNA splicing, fatty acid biosynthesis and protein folding. After 30 and 60 min, almost the same proteins were regulated as after 10 min of hypergravity

Eu:CROPIS - Euglena gracilis: Combined Regenerative Organic-food Production in Space - A Space Experiment Testing Biological Life Support Systems Under Lunar And Martian Gravity

Human space exploration needs stable life support systems for the supply of oxygen, water and food for each human explorer due to long term missions. The most promising approach for building stable life support systems is a combination of physico-chemical and biological systems. These hybrid systems combine the reliability of physico-chemical and the sustainability of biological life support systems. Also the disadvantages, which are the finite resources of physico-chemical and the imperfect reliability of biological systems, are mutually balanced. To improve the reliability of biological life support systems, a combination of different biological systems may stabilize the whole approach during long term operations. The satellite mission Eu:CROPIS (Euglena gracilis: Combined Regenerative Organic-food Production In Space) is a testbed for investigating the behavior of combined biological life support systems under the influence of altered gravity, here, Lunar and Martian gravity. The core systems are a biological trickle filter for processing urine into a fertilizer solution via nitrification and Euglena gracilis, a photosynthetic protist which is able to produce oxygen and biomass while protecting the whole system against high ammonia concentrations

The design and implementation of the immune epitope database and analysis resource

Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance is reflected in the vast amount of epitope-related information gathered, ranging from interactions between epitopes and major histocompatibility complex molecules determined by X-ray crystallography to clinical studies analyzing correlates of protection for epitope based vaccines. Our goal is to provide a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, “The Immune Epitope Database and Analysis Resource.” We describe how we plan to capture, structure, and store this information, what query interfaces we will make available to the public, and what additional predictive and analytical tools we will provide