Testes-specific hemoglobins in Drosophila evolved by a combination of sub- and neofunctionalization after gene duplication

<p>Abstract</p> <p>Background</p> <p>For a long time the presence of respiratory proteins in most insects has been considered unnecessary. However, in recent years it has become evident that globins belong to the standard repertoire of the insect genome. Like most other insect globins, the <it>glob1 </it>gene of <it>Drosophila melanogaster </it>displays a conserved expression pattern in the tracheae, the fat body and the Malpighian tubules.</p> <p>Results</p> <p>Here we show that the recently discovered <it>D. melanogaster </it>globin genes <it>glob2 </it>and <it>glob3 </it>both display an unusual male-specific expression in the reproductive tract during spermatogenesis. Both paralogs are transcribed at equivalent mRNA levels and largely overlap in their cellular expression patterns during spermatogenesis. Phylogenetic analyses showed that <it>glob2 </it>and <it>glob3 </it>reflect a gene duplication event that occurred in the ancestor of the <it>Sophophora </it>subgenus at least 40 million years ago. Therefore, flies of the <it>Drosophila </it>subgenus harbor only one <it>glob2/3</it>-like gene.</p> <p>Conclusions</p> <p>Phylogenetic and sequence analyses indicate an evolution of the <it>glob2 </it>and <it>glob3 </it>duplicates by a combination of sub- and neofunctionalization. Considering their restricted, testes-specific expression, an involvement of both globins in alleviating oxidative stress during spermatogenesis is conceivable.</p

Zeit- und kosteneffiziente Prozess- und Produktentwicklung für den Hochleistungs-Faserverbundleichtbau mittels Nasspresstechnologie

Großserientaugliche Produktionsprozesse von Hochleistungs-Faserverbundkunststoffen stellen aufgrund der gewünschten Prozesseffizienz bei gleichzeitiger Realisierung herausragender gewichtsspezifischer Materialeigenschaften ein wichtiges, zukunftsträchtiges Themenfeld dar. Im Automotive-Bereich kommen diese Prozesse verstärkt zur Anwendung, insbesondere bei Premiumfahrzeugen und im Rahmen der E Mobilität. Der erheblichen Gewichtseinsparung und hohen Energieeffizienz von Leichtbaustrukturen stehen bisher jedoch noch hohe Entwicklungs- und Stückkosten (Material, Prozessaufwand) gegenüber. Neben den verschiedenen Varianten der Resin Transfer Moulding (RTM) Technologie bietet sich zur Herstellung leichter, komplex geformter Strukturbauteile das Nasspressverfahren als Großserienanwendung an. Durch Parallelisierung von Prozessschritten in Verbindung mit hochreaktiven Harzsystemen können niedrigere Zykluszeiten erreicht werden als beim RTM-Verfahren. Da für den Nasspressprozess bisher weder ein umfassendes physikalisch-basiertes Prozessverständnis, noch Methoden zur virtuellen Prozesssimulation und -optimierung existieren, besteht ein erheblicher Forschungsbedarf für eine ressourceneffiziente Prozess- und Bauteilentwicklung [1]. Die Forschungsbrücke „KIT – Uni Stuttgart“ baut auf der langjährigen wissenschaftlichen und strategischen Zusammenarbeit der Leichtbau-Institute des Karlsruher Instituts für Technologie (KIT-FAST) und der Universität Stuttgart (IFB Stuttgart) auf und widmet sich der Erforschung und Weiterentwicklung des Nasspressprozesses (vgl. Abbildung 1). Dabei werden zwei unterschiedliche Prozessrouten grundlegend untersucht, modelliert, optimiert und bewertet. Besondere Herausforderungen sind die fundierte Material- und Prozessanalyse der Nasspresstechnologie sowie die Methodenentwicklung zur effizienten, virtuellen Prozess- und Bauteilentwicklung und die ganzheitliche Optimierung

Multitrophic Interaction in the Rhizosphere of Maize: Root Feeding of Western Corn Rootworm Larvae Alters the Microbial Community Composition

BACKGROUND: Larvae of the Western Corn Rootworm (WCR) feeding on maize roots cause heavy economical losses in the US and in Europe. New or adapted pest management strategies urgently require a better understanding of the multitrophic interaction in the rhizosphere. This study aimed to investigate the effect of WCR root feeding on the microbial communities colonizing the maize rhizosphere. METHODOLOGY/PRINCIPAL FINDINGS: In a greenhouse experiment, maize lines KWS13, KWS14, KWS15 and MON88017 were grown in three different soil types in presence and in absence of WCR larvae. Bacterial and fungal community structures were analyzed by denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene and ITS fragments, PCR amplified from the total rhizosphere community DNA. DGGE bands with increased intensity were excised from the gel, cloned and sequenced in order to identify specific bacteria responding to WCR larval feeding. DGGE fingerprints showed that the soil type and the maize line influenced the fungal and bacterial communities inhabiting the maize rhizosphere. WCR larval feeding affected the rhiyosphere microbial populations in a soil type and maize line dependent manner. DGGE band sequencing revealed an increased abundance of Acinetobacter calcoaceticus in the rhizosphere of several maize lines in all soil types upon WCR larval feeding. CONCLUSION/SIGNIFICANCE: The effects of both rhizosphere and WCR larval feeding seemed to be stronger on bacterial communities than on fungi. Bacterial and fungal community shifts in response to larval feeding were most likely due to changes of root exudation patterns. The increased abundance of A. calcoaceticus suggested that phenolic compounds were released upon WCR wounding

Das Interleukin-1Beta-Rezeptor-Homolog im Modifizierten Vakziniavirus Ankara (MVA) - Charakterisierung und möglicher Einfluss auf MVA-basierte Impfvektoren

MVA ist ein attenuiertes Vakziniavirus, das durch wiederholte Passagierung von Chorioallantoisvirus Ankara auf Hühnerembryofibroblasten gewonnen wurde. Es ist bis auf wenige Ausnahmen nicht mehr in der Lage, in Säugerzellen zu replizieren, zeigt aber dennoch eine vollständige virale Proteinexpression und induziert nach Immunisierung eine zu VACV vergleichbare Immunantwort. Aus diesem Grund wurde es bereits als Impfstoff gegen die menschliche Pockenerkrankung eingesetzt, ohne hierbei die bei den klassischen Impfviren beobachtbaren Nebenwirkungen hervorzurufen. Das Genom von MVA enthält jedoch noch Immunmodulatoren, deren Deletion Ansatzpunkt für die weitere Verbesserung des Impfstoffes sein kann. Im Rahmen der vorliegenden Arbeit wurde eine Deletionsmutante untersucht, bei der das Gen für den Interleukin-1β Rezeptor (IL-1βR) deletiert ist (MVA ΔIL-1βR). Es konnte nachgewiesen werden, dass der IL-1βR in murinen als auch in humanen Zellen exprimiertes IL-1β bindet und somit inaktiviert. Des Weiteren wurde gefunden, dass MVA in der Lage ist, IL-1β in antigenpräsentierenden Zellen zu induzieren, welches aber durch die Neutralisierung aufgrund des IL-1βR nur transient nachzuweisen war. Im Gegensatz dazu induzierte MVA ΔIL-1βR eine anhaltende und um ein Vielfaches erhöhte Sekretion an IL-1β. Untersuchungen in antigenpräsentierenden Zellen aus knock-out Mäusen, die verschiedene Defizienzen in den Signalwegen zur IL-1β-Induktion trugen, zeigten, dass die Sekretion von IL-1β von Caspase-1 abhängig war, welches wahrscheinlich aus der vorgeschalteten Aktivierung der NLRP3- und/oder AIM2-Inflammasomen resultierte. Interessanterweise wurden auch Caspase-1 unabhängige Mechanismen beobachtet, die auf eine Inflammasom-unabhängige IL-1β-Induktion hinweisen könnten. In Bezug auf die Immunaktivierung führte die vermehrte Sekretion von IL-1β durch MVA ΔIL-1βR vermutlich zu einer verbesserten Antigenpräsentation, die die nachfolgende T-Zellantwort beeinflusste. In Übereinstimmung mit bereits veröffentlichten Daten wurde nach Immunisierung mit MVA ΔIL-1βR eine effektivere Gedächtnis-T-Zellantwort festgestellt, deren Charakteristika und zu Grunde liegenden Mechanismen hier untersucht wurden. Jedoch konnten weder Unterschiede in weiteren pro-inflammatorischen Zytokinmustern noch im Verlauf insbesondere der CD8+ T-Zell-Aktivierung und -erhaltung zwischen MVA und MVA ΔIL-1βR beobachtet werden. Als mögliche weitere Ursache für die veränderte Gedächtnis-T-Zellantwort könnte daher eine vermehrte Stimulation durch antigenpräsentierende Zellen und eine IL-21-vermittelte bessere Unterstützungsfunktion der CD8+ Gedächtnis-T-Zellen durch CD4+ T-Zellen in Frage kommen. Zusammenfassend konnten hier neue molekulare Mechanismen, die zur Induktion von IL-1β nach einer MVA-Infektion führen, aufgedeckt werden. Darüber hinaus existieren bereits erste Hinweise auf einen Vorteil der Deletion des IL-1βR für MVA-basierte Vektorimpfstoffe. Die vorliegende Arbeit hat weitere Daten erhoben, die das Erzielen verbesserter Immunantworten nach Immunisierung mit MVA ΔIL-1βR unterstützen, woraus sich neue Ansätze für die Entwicklung MVA-basierter Impfstoffe ergeben könnten.MVA is an attenuated Vaccinia Virus strain that was obtained by repeated passages of Chorioallantois Virus Ankara on chicken embryo fibroblasts. With a few exceptions MVA is unable to replicate in mammalian cells. Nevertheless all proteins are expressed in mammalian cells and MVA induces immune responses comparable to VACV. Therefore it has already been used as a smallpox vaccine without inducing side effects which are known from conventional smallpox vaccines. However the genome of MVA contains still some immunomodulators which are potential targets for further improvement of MVA as a vaccine. During the work presented here, a deletion mutant of the interleukin-1β receptor (IL-1βR) was analyzed. Evidence was presented, that the IL-1βR binds and inactivates IL-1β of human and murine origin. Furthermore MVA was shown to be able to transiently induce IL-1β in antigen presenting cells. In contrast, MVA ΔIL-1βR induced sustained elevated levels of IL-1β. Experiments in antigen presenting cells derived from knock out mice with deficiencies in IL-1β signalling showed, that secretion of IL-1β was dependant on caspase-1 activation, which was most probably induced by activation of the NLRP3 and AIM2 inflammasomes. Interestingly, also caspase-1 independent mechanisms for IL-1β-processing, which indicate inflammasome-independent pathways, were observed. Whith respect to immune activation, increased secretion of IL-1β induced by MVA ΔIL-1βR presumably resulted in enhanced antigen presentation, which could subsequently influence T cell responses. In line with published results, immunization with MVA ΔIL-1βR resulted in more effective memory T cell responses, whose underlying mechanisms were analyzed in this study. However, neither regarding production of proinflammatory cytokines, nor regarding CD8+ T cell activation and homeostasis significant differences between immunization with MVA or MVA ΔIL-1βR were observed. A proposed model for the observed improved memory T cell response could be an IL-21-mediated support by CD4+ T cells for the CD8+ memory T cell population. In summary, in this study new molecular mechanisms which lead to production of IL-1β after MVA infection could be uncovered. Moreover there are indications that deletion of the MVA IL-1βR could be beneficial for future vector vaccines. This study has presented further data, which support the hypothesis of an enhanced immune resonse after immunization with MVA ΔIL-1βR, thereby providing new strategies for the development of MVA-based vector vaccines

Interleukin-1 beta receptor expressed by modified vaccinia virus Ankara interferes with interleukin-1 beta activity produced in various virus-infected antigen-presenting cells

Background: Modified vaccinia virus Ankara (MVA) is a highly attenuated virus and a promising vaccine vector with potent immune stimulating properties. Deletion of the gene encoding the viral interleukin-1beta receptor (vIL-1 beta R) in MVA (MVA Delta IL-1 beta R) was previously shown to enhance memory T cell function. Here, we investigated the influence of vIL-1 beta R on blocking interleukin-1beta (IL-1 beta) upon MVA infection in various antigen presenting cells of murine and human origin, and analyzed whether inflammasome function contributes to IL-1 beta production in different cell types. Findings: Extending previous studies, immunizing mice with low doses of MVA Delta IL-1 beta R still showed enhanced memory CD8(+) T cell activation compared to MVA wild-type (MVAwt) immunization. In vitro, murine myeloid dendritic cells, and activated, but not naive primary macrophages were identified as potent producers of IL-1 beta upon infection with MVA. Importantly, free IL-1 beta was only detected in the absence of vIL-1 beta R. Moreover, MVA Delta IL-1 beta R increased amounts of bioactive IL-1 beta compared to MVAwt after infection of human THP-1 cells, as detected using a reporter system that only responds to active and free IL-1 beta. The MVA-mediated induction of IL-1 beta was confirmed to depend on inflammasome function in human and murine cells, however in murine cells this apparently involves caspase-1-independent pathways. Conclusions: MVA lacking IL-1 beta blocking activity leads to increased concentrations of free IL-1 beta upon infection of murine and human antigen presenting cells; this is likely responsible for enhanced memory T cell activation upon MVA Delta IL-1 beta R immunization of mice. Moreover, our results suggest that MVA-mediated IL-1 beta induction is a multifactorial process

Co-metabolic formation of substituted phenylacetic acids by styrene-degrading bacteria

Some soil bacteria are able to metabolize styrene via initial side-chain oxygenation. This catabolic route is of potential biotechnological relevance due to the occurrence of phenylacetic acid as a central metabolite. The styrene-degrading strains Rhodococcus opacus 1CP, Pseudomonas fluorescens ST, and the novel isolates Sphingopyxis sp. Kp5.2 and Gordonia sp. CWB2 were investigated with respect to their applicability to co-metabolically produce substituted phenylacetic acids. Isolates were found to differ significantly in substrate tolerance and biotransformation yields. Especially, P. fluorescens ST was identified as a promising candidate for the production of several phenylacetic acids. The biotransformation of 4-chlorostyrene with cells of strain ST was shown to be stable over a period of more than 200 days and yielded about 38 mmolproduct gcelldryweight−1 after nearly 350 days. Moreover, 4-chloro-α-methylstyrene was predominantly converted to the (S)-enantiomer of the acid with 40% enantiomeric excess

DGGE fingerprints of ITS fragments PCR-amplified from TC DNA extracted from soil and rhizosphere samples and corresponding UPGMA dendrogram.

<p>(A) DGGE fingerprints of dominant fungal populations in Haplic Chernozem (HC) soil and in the maize rhizosphere of KWS13, KWS14, KWS15 and MON88017 grown in the same soil type. The independent replicates are labeled 1 to 4. M: fungal marker prepared with the ITS fragments amplified from <i>Verticillium nigrescens</i>, <i>Basidiomycete</i> sp., <i>Trichoderma</i> sp., <i>Doratomyces</i> sp., <i>Verticillium dahliae</i>, <i>Penicillium canescens</i>, <i>Fusarium graminearum</i>, <i>Nectria haematococca</i>, <i>Fusarium solani</i>, <i>Fusarium redolens</i>, and <i>Sclerotinia sclerotiorum</i>. Arrows indicate maize genotype effects. (B) UPGMA dendrogram constructed using the Pearson correlation coefficient. The scale shows similarity values. Rh: rhizosphere samples.</p

Percentage dissimilarity (<i>D</i>) and significant values (<i>P</i>) of rhizosphere fungal or bacterial fingerprints between different maize lines (KWS13, KWS14, KWS15 and MON88017) grown in the soil types Haplic Chernozem, Haplic Luvisol, and Eutric Vertisol.

<p>Values of <i>P</i><0.05 indicate significant differences between rhizosphere samples of different maize lines grown in the same soil type. Permutation testing was done with 10.000 simulations. Bold values indicate significant differences.</p

Percentage dissimilarity (<i>D</i>) and significance values (<i>P</i>) of rhizosphere fungal or bacterial fingerprints between maize lines in presence and in absence of WCR larval feeding (Larvae+/−), in the soil types Haplic Chernozem, Haplic Luvisol, and Eutric Vertisol.

<p><i>P</i> values<0.05 indicate significant differences between rhizosphere samples of the same maize line grown with and without larval feeding in the same soil type. Values obtained by Permutation testing using 10.000 simulations. Values in bold show significant values.</p