6 research outputs found

    Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure

    Get PDF
    The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.Das Pansenmikrobiom ist ein komplexes Ă–kosystem mit einer groĂźen Vielfalt an Organismen, die kurzkettige Fettsäuren (SCFAs) produzieren und verbrauchen. Es ist von groĂźem Interesse, diese Aktivitäten zu analysieren, da sie sowohl fĂĽr das Mikrobiom als auch fĂĽr den Wirt von Nutzen sind. Ziel dieser Arbeit ist es, das Proteom und Metabolom des vorherrschenden Pansenvertreters Prevotella bryantii B14 in Gegenwart verschiedener SCFA und unter Einwirkung des Antibiotikums Monensin in Rein- und Mischkultur (in vitro) darzustellen. Aufgrund der groĂźen Anzahl von Prevotellaceae im Pansenmikrobiom wurde der Vertreter P. bryantii B14 (DSM 11371) ausgewählt, um die biochemischen Faktoren fĂĽr die erfolgreiche Resistenz gegenĂĽber Monensin und die Auswirkungen von kurzkettigen Fettsäuren auf ihr Wachstum zu untersuchen. Die aktuelle Arbeit besteht aus zwei Publikationen [1, 2], die in Kapitel 2 und 3 eingefĂĽgt sind, sowie einem eingereichten Manuskript in Kapitel 4. Die Formatierung der Publikationen und des Manuskriptes wurde an die Dissertation angepasst. Die erste Veröffentlichung (Kapitel 2) befasste sich mit der Supplementierung verschiedener SCFAs und zeigt, dass SCFAs wachstumsfördernd, aber nicht essentiell fĂĽr P. bryantii B14 sind. Reinkulturen von P. bryantii B14 sind in Hungate-Röhrchen unter anaeroben Bedingungen durchgefĂĽhrt worden. Gaschromatographie-Flugzeit-Massen-spektrometrie (GC-ToF MS) wurde zur Quantifizierung der langkettigen Fettsäuren (LCFA) von P. bryantii B14 verwendet. Die nicht-markierte und nicht-angereicherte Proteine von P. bryantii B14 wurden mit Hilfe von Massenspektrometrie identifiziert und quantifiziert. Es wurde ein unterschiedliches Wachstumsverhalten in Abhängigkeit von der zugefĂĽhrten SCFA beobachtet. Eine Auswirkung der strukturellen Eigenschaften der SCFAs hat sich auf die LCFAs und ebenso auf die Anwesenheit verschiedener Membranproteine bemerkbar gemacht. Die Supplementierung von P. bryantii B14 mit verzweigtkettigen Fettsäuren (BCFAs), insbesondere Isovaleriansäure, hat einen Anstieg des Stoffwechsels von 3-Isopropylmalat (IPM) gezeigt, welcher Teil des Stoffwechsels von verzweigtkettigen Aminosäuren (BCAAs) ist. Die Ergebnisse deuten darauf hin, dass die Strukturähnlichkeit von Isovaleriansäure und Valin höchstwahrscheinlich die Umwandlung von BCFA in BCAA begĂĽnstigt. Der relative Anstieg der erforderlichen Enzyme BCAA-Stoffwechsel unterstĂĽtzen diese Sichtweise. Das Ionophor Monensin hat antibiotische Eigenschaften, welche in der Rindermast, aber auch zur Behandlung von Ketose und Azidose bei Wiederkäuern eingesetzt wird. Die zweiten Veröffentlichung (Kapitel 3) zeigte den Einfluss verschiedenen Konzentrationen von Monensin (0, 10, 20 und 50 μM) und verschiedenen Expositionszeiten (9, 24, 48 und 72 h) mit und ohne Monensin auf P. bryantii B14. Das Wachstumsverhalten, extrazelluläre Glukose- und intrazelluläre Natriumkonzentration wurde unter den beschriebenen Bedingungen ermittelt. Das Proteom von P. bryantii B14 wurde mittels markierungsfreier Quantifizierung nach der gleichen Methode wie in Kapitel 2 analysiert. Fluoreszenzmikroskopie wurde zur Unter-suchung der extrazellulären Polysaccharide (EPS) von P. bryantii B14 eingesetzt. Mit fortschreitender Monensin-Exposition hat sich gezeigt, dass P. bryantii B14 die Verbindung zur äuĂźeren EPS abbaute, indem es die Anzahl und Menge der kohlenhydrataktiven Enzyme (CAZyme) erhöhte. Der Anstieg an extrazellulärer Glukose bestätigte die Vermutung, dass die Opferschicht abgebaut wurde. Die beobachtete Reduktion des intrazellulären Natriums ist wahrscheinlich mit der Zunahme der Menge an Ionentransportern verbunden. Weiterhin wurde eine erhöhte Aktivität der Na+-transferrierenden NADH:Quinon-Oxidoreduktase (NQR) unter Monensin-Supplementierung dokumentiert. Die Rolle von mit Monensin supplementierten Prevotella in einer Mischkultur des Pansenmikrobioms ist im letzten Manuskript beschrieben worden (Kapitel 4). Pansensaft ist mit Hilfe der Pansensimulationstechnik (Rumen simulation technique = Rusitec) anaerob inkubiert worden. Das Proteom der festen Phase ist mit einem ähnlichen Ansatz wie in Kapitel 2 und 3 durchgefĂĽhrt worden. Das Metabolom der flĂĽssigen Phase aus dem Rusitec ist anhand der 1H-Kernresonanz (Nuclear magnetic resonance = NMR) spektroskopie ermittelt worden. Weitere Parameter wie pH-Wert, Gas- und Methan-produktion wurden im Zeitverlauf ĂĽberwacht. Das Experiment bestand aus drei Phasen, beginnend mit einer Anpassungsphase von 7 Tagen. Es folgte eine Behandlungsphase, in der Monensin ĂĽber das tägliche verabreichen der totalen Mischration (TMR) fĂĽr weitere 7 Tage durchgefĂĽhrt wurde. Die Elutionsphase war die letzte Phase, in der die Monensinzugabe eingestellt und die Ăśberwachung fĂĽr weitere 3 Tage fortgesetzt wurde. Metabolomics und Proteomics haben gezeigt, dass Mitglieder der Gattung Prevotella unter Monensinzugabe am häufigsten vorkommen. AuĂźerdem verlagerte sich der Pansenstoffwechsel auf eine erhöhte Propionatproduktion. Der Stoffwechsel von Prevotella sp. wurde auf eine erhöhte Succinat-produktion umgestellt. Die vorliegende Arbeit zeigt die Auswirkungen von SCFAs auf verschiedene Stoffwechselfunktionen von P. bryantii B14. Es wurde gezeigt, dass verschiedene Abwehrmechanismen von Prevotella sp. und insbesondere von P. bryantii B14 die anti-biotische Wirkung von Monensin verhindern

    Sarcoma treatment in the era of molecular medicine

    Get PDF
    Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.Peer reviewe

    Short-Chain Fatty Acids Modulate Metabolic Pathways and Membrane Lipids in Prevotella bryantii B14

    No full text
    Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B14 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid

    A Sodium-Translocating Module Linking Succinate Production to Formation of Membrane Potential in Prevotella bryantii

    No full text
    Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff which is accomplished by the microbial community in the rumen. Roughly 40% of the rumen microbiota belong to the family of which ferment sugars to organic acids such as acetate, propionate as well as succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na -translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different species. Here we show that fumarate reduction to succinate in anaerobically growing is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This odium-translocating ADH:umarate oxidoeductase (SNFR) supercomplex was enriched by BN-PAGE and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min mg), quinone reduction (490 nmol min mg) and fumarate reduction (1200 nmol min mg) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in . Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the FAD cofactor in NQR to heme cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD and succinate. We propose that the regeneration of NAD in is intimately linked to the build-up of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by sp. We show that succinate formation by is coupled to NADH oxidation and sodium-gradient formation by a newly described supercomplex consisting of Na-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the odium-translocating ADH:umarate oxidoeductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in . Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen

    Dense Nuclear Matter Equation of State from Heavy-Ion Collisions

    No full text
    The nuclear equation of state (EOS) is at the center of numerous theoretical and experimental efforts in nuclear physics. With advances in microscopic theories for nuclear interactions, the availability of experiments probing nuclear matter under conditions not reached before, endeavors to develop sophisticated and reliable transport simulations to interpret these experiments, and the advent of multi-messenger astronomy, the next decade will bring new opportunities for determining the nuclear matter EOS, elucidating its dependence on density, temperature, and isospin asymmetry. Among controlled terrestrial experiments, collisions of heavy nuclei at intermediate beam energies (from a few tens of MeV/nucleon to about 25 GeV/nucleon in the fixed-target frame) probe the widest ranges of baryon density and temperature, enabling studies of nuclear matter from a few tenths to about 5 times the nuclear saturation density and for temperatures from a few to well above a hundred MeV, respectively. Collisions of neutron-rich isotopes further bring the opportunity to probe effects due to the isospin asymmetry. However, capitalizing on the enormous scientific effort aimed at uncovering the dense nuclear matter EOS, both at RHIC and at FRIB as well as at other international facilities, depends on the continued development of state-of-the-art hadronic transport simulations. This white paper highlights the role that heavy-ion collision experiments and hadronic transport simulations play in understanding strong interactions in dense nuclear matter, with an emphasis on how these efforts can be used together with microscopic approaches and neutron star studies to uncover the nuclear EOS

    Dense nuclear matter equation of state from heavy-ion collisions

    No full text
    International audienceThe nuclear equation of state (EOS) is at the center of numerous theoretical and experimental efforts in nuclear physics. With advances in microscopic theories for nuclear interactions, the availability of experiments probing nuclear matter under conditions not reached before, endeavors to develop sophisticated and reliable transport simulations to interpret these experiments, and the advent of multi-messenger astronomy, the next decade will bring new opportunities for determining the nuclear matter EOS, elucidating its dependence on density, temperature, and isospin asymmetry. Among controlled terrestrial experiments, collisions of heavy nuclei at intermediate beam energies (from a few tens of MeV/nucleon to about 25 GeV/nucleon in the fixed-target frame) probe the widest ranges of baryon density and temperature, enabling studies of nuclear matter from a few tenths to about 5 times the nuclear saturation density and for temperatures from a few to well above a hundred MeV, respectively. Collisions of neutron-rich isotopes further bring the opportunity to probe effects due to the isospin asymmetry. However, capitalizing on the enormous scientific effort aimed at uncovering the dense nuclear matter EOS, both at RHIC and at FRIB as well as at other international facilities, depends on the continued development of state-of-the-art hadronic transport simulations. This white paper highlights the essential role that heavy-ion collision experiments and hadronic transport simulations play in understanding strong interactions in dense nuclear matter, with an emphasis on how these efforts can be used together with microscopic approaches and neutron star studies to uncover the nuclear EOS
    corecore