VFUs zur flexiblen Nutzung der Landesforschungsinfrastrukturen

Das Projekt VICE (Virtual Open Science Collaboration Environment) unterstützt Wissenschaftler unterschiedlicher Fachdisziplinen beim Aufbau und der Anpassung virtueller Forschungsumgebungen (VFU) auf moderne Basisinfrastrukturen, wie sie mit Projekten wie bwHPC, bwCloud-Scope oder bwLehrpool landesweit angeboten werden. Virtualisierung erlaubt die Ablösung von der konkreten ortsgebundenen Hardware und damit sowohl eine leichte Verschieb- und Reproduzierbarkeit. Im nächsten Schritt kann so übergreifende Kollaboration entstehen, die gleichzeitig eine langfristige Nachnutzung von Forschungsergebnissen besonders im Hinblick auf neue Fragestellungen in der Wissenschaft gewährleistet. Ziel hierbei ist, es den Wissenschaftlern zu ermöglichen, unterschiedliche Versionsstände ihrer virtuellen Forschungsumgebungen und Forschungsdaten prozessbegleitend dokumentieren und auch anderen Forschenden zur Verfügung stellen zu können. Die Plattform wird gemeinsam mit den Infrastrukturpartnern Freiburg, Tübingen und Mannheim exemplarisch für die Fach-Communities Anglistik, Wirtschaftsinformatik, Lebenswissenschaften und Teilchenphysik bereitgestellt. Erste nutzbare Prototypen von VFUs wurden für die Gebiete der Teilchenphysik (CMS und ATLAS Arbeitsgruppen) sowie der Bioinformatik (Galaxy) geschaffen. Parallel dazu finden Beratungung der Forschenden zur zukünftigen Ausgestaltung wissenschaftlicher Arbeitsabläufe statt und werden Austauschplattformen für die geschaffenen VFUs diskutiert und entwickelt. Die im Projekt beteiligten Rechenzentren bauen ihre Beratungskompetenz für VFUs aus und koordinieren ihre Serviceangebote gemeinsam mit den Wissenschafts-Communities. In diesem Zuge werden mögliche Geschäfts- und Finanzierungsmodelle erörtert, so dass die Entwicklungen zunehmend weiteren Disziplinen langfristig zur Verfügung stehen und in der Lehre und Integration des wissenschaftlichen Nachwuchses zum Einsatz kommen

Model-guided metabolic engineering of Pseudomonas taiwanensis VLB120 for the production of methyl ketones

Aliphatic methyl ketones are discussed as promising novel diesel blendstocks because of their favorable cetane numbers. To achieve sustainable production of these compounds, bio-based production in engineered microbes is followed and successful synthesis in Escherichia coli1,2,3 and Pseudomonas putida4 has recently been shown. In this presentation, we report on the metabolic engineering of Pseudomonas taiwanensis VLB1205 for the production of saturated and monounsaturated medium chain methyl ketones (C11, C13, C15, C17). Major arguments for the use of this microbe are its metabolic versatility, high tolerance of organic solvents5 and ease of cultivation. P. taiwanensis VLB120 can grow on various carbon sources besides glucose such as glycerol, an important by-product of biodiesel production, as well as on major components of biomass hydrolysate such as xylose, organic acids and aromatic compounds, e.g., 4-hydroxybenzoate4. Further, its superior redox cofactor regeneration capability6 might benefit the synthesis of the reduced, aliphatic target compounds. The transformation of P. taiwanensis VLB120 into a microbial cell factory for methyl ketone production was achieved by: (i) overproduction of the fatty acyl-CoA synthetase FadB to increase acyl-CoA availability, (ii) oxidation of acyl-CoA to a trans-2-enoyl-CoA by a heterologously expressed acyl-CoA oxidase from Micrococcus luteus, (iii) conversion of this intermediate to β-hydroxyacyl-CoA and further oxidation to a β -ketoacyl-CoA by overexpression of the native fadB gene, (iv) increased thioesterase activity by overexpression of fadM to form free β -keto acids, which spontaneously decarboxylate to methyl ketones. The 1st generation production strain yielded 550 mg L-1aq methyl ketones in a batch fermentation with in situ product extraction into a second organic layer of n-decane. Further strain optimization was guided by metabolic modeling, which suggested an additional deletion of the acyl-CoA thioesterase II (tesB). TesB hydrolyzes acyl-CoA to free fatty acids, hence, reverses the desired FadD reaction. In a simple batch fermentation, the proposed gene deletion resulted in a 2.5-fold increased product titer of 1.4 g L-1aq while 9.4 g L-1aq were reached in fed-batch cultivations. Additional, successful strategies tested in parallel were the deletion of the pha operon, responsible for polyhydroxyalkanoate synthesis and deletion of a fadA homologue in the 1st generation production strain, with the later resulting in an even 4-fold improvement of the product titer. While the production of 9.4 g L-1aq is already the highest reported titer of recombinantly produced methyl ketones so far, consolidation of all successfully tested engineering strategies holds great promise to significantly boost methyl ketone production in P. taiwanensis VLB120 to even higher titers. Overall, the results of this study underline the high potential of P. taiwanensis VLB120 for the production of methyl ketones and highlight model-guided metabolic engineering as a means to rationalize and accelerate strain optimization efforts. 1Dong et al. 2018: doi:10.1101/496497 2Goh et al. 2012: doi: 10.1128/AEM.06785-11 3Goh et al. 2014: doi: 10.1016/j.ymben.2014.09.003. 4Goh et al. 2018: doi: 10.1002/bit.26558. 5Rühl et al. 2009: doi: 10.1128/AEM.00225-09 6Blank et al. 2008: doi: 10.1111/j.1742-4658.2008.06648.x

Ultrahydrophobe chitosanstabilisierte Composite-Schichten auf Aluminiumwerkstoffen

Selbstreinigende, ultrahydrophobe Oberflächen lassen sich in der Technik vielfältig einsetzen. Das ultrahydrophobe Verhalten beruht einerseits auf einer Rauigkeit im μm-Bereich und andererseits auf der chemischen Zusammensetzung der Oberfläche. Durch den gegebenen Oberflächenaufbau sind derartige Materialien jedoch empfindlich gegen Verschleiß. In diesem Beitrag wird ein Schichtverbund bestehend aus Aluminiumoxid und zwei polymeren Komponenten vorgestellt. Die Aluminiumoxidschicht wird auf dem Wege der anodischen Oxidation erzeugt. Dieses seit langem bekannte Verfahren ermöglicht nicht nur die Oxidation der Aluminiumoberfläche, sondern gestattet es, auch, definierte Oberflächenprofile einzustellen. Durch den gezielten Einbau des hochmolekularen Polymers Chitosan in die mikroprofilierte Aluminiumoxidschicht wurde eine mechanische Stabilisierung der Schicht im Sinne eines anorganisch-organischen Composites erreicht. Außerdem dienten die Amino-Seitengruppen des Chitosans als reaktives Interface für die notwendige chemische Hydrophobierung und als Reaktionszentrum für Vernetzungen, wodurch eine weitere mechanische Stabilisierung bewirkt wird. Der Schichtaufbau hat wesentliche

Immediate tumor resection in patients with locally advanced gastroesophageal adenocarcinoma with nonresponse to chemotherapy after 4 weeks of treatment versus resection after completion of chemotherapy (OPTITREAT trial, DRKS00004668): study protocol for a randomized controlled pilot trial

Background: Neoadjuvant chemotherapy is a standard of care for patients with adenocarcinoma of the esophagus and stomach in Europe, but still only 20–40 % respond to therapy and the critical issue; how to treat nonresponding patients is still unclear. So far, there is no randomized trial evaluating the impact of early termination of neoadjuvant chemotherapy and immediate tumor resection in nonresponding patients with locally advanced gastroesophageal cancer on postoperative outcome. With this exploratory pilot trial, we want to get first estimates about the effect of discontinuation of chemotherapy with the aim to plan and conduct a further definitive trial. Methods/design: OPTITREAT is designed as a single-center, randomized controlled pilot trial with two parallel study groups. Four weeks after starting neoadjuvant chemotherapy in all patients, clinical response will be assessed by endoscopy and endosonographic ultrasound. Then, nonresponding patients (n = 84) will be randomized in a 1:1 ratio to intervention group with stopping chemotherapy and immediate tumor resection or control group with completion of chemotherapy before surgery. Outcome measures are overall survival, R0 resection rate, perioperative morbidity and mortality, histopathological response, and quality of life. Statistical analysis will be based on the intention-to-treat population. Due to the study design as an explorative pilot trial, no formal sample size calculation was performed. The planned total sample size of 120 patients is considered ethical and large enough to show the feasibility and safety of the concept. First data on differences between the study groups in the defined endpoints will also be generated. Discussion: Individualized therapy is of utmost interest in the treatment of locally advanced gastroesophageal adenocarcinoma as less than half of the patients show objective response to current chemotherapy regimens. The findings of the OPTITREAT trial will help to get first data about clinical response evaluation followed by immediate tumor resection in nonresponding patients after 4 weeks of neoadjuvant chemotherapy. Based on the results of this pilot study, a future confirmatory trial will be planned to prove efficacy and evaluate significance. Trial registration: German Clinical Trial Register number: DRKS0000466

Association of angiogenic factors with prognosis in esophageal cancer

Background: Despite multimodal therapy esophageal cancer often presents with poor prognosis. To improve outcome, tumor angiogenesis and anti-angiogenic therapeutic agents have recently gained importance. However, patient subgroups who benefit from anti-angiogenic therapy are not yet defined. In this retrospective exploratory study we investigated 9 angiogenic factors in patients’ serum and tissue samples with regard to their association with clinicopathological parameters, prognosis and response in patients with locally advanced preoperatively treated esophageal cancer. Methods: From 2007 to 2012 preoperative serum and corresponding tumor tissue (n = 54), only serum (n = 20) or only tumor tissue (n = 4) were collected from esophageal squamous cell carcinoma (SCC) (n = 34) and adenocarcinoma of the esophagogastric junction (AEG) (n = 44) staged cT3/4NanyM0/x after preoperative chemo(radio)therapy. Angiogenic cytokine levels in both tissue and serum were measured by multiplex immunoassay. Results: Median survival in all patients was 28.49 months. No significant difference was found in survival between SCC and AEG (p = 0.90). 26 patients were histopathological responders. Histopathological response was associated with prognosis (p = 0.05). Angiogenic factors were associated with the following clinicopathological factors: tumor tissue expression of Angiopoietin-2 and Follistatin was higher in SCC compared to AEG (p = 0.022 and p = 0.001). High HGF and Follistatin expression in the tumor tissue was associated with poor prognosis in all patients (p = 0.037 and p = 0.036). No association with prognosis was found in the patients’ serum. Neither patients’ serum nor tumor tissue showed an association between angiogenic factors and response to neoadjuvant therapy. Conclusion: Two angiogenic factors (HGF and Follistatin) in posttherapeutic tumor tissue are associated with prognosis in esophageal cancer patients. Biological differences of AEG and SCC with respect to angiogenesis were evident by the different expression of 2 angiogenic factors. Results are promising and should be pursued prospectively, optimally sequentially pre- and posttherapeutically

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

The human pathogenic bacterium Pseudomonasaeruginosa produces rhamnolipids, glycolipids with functionsfor bacterial motility, biofilm formation, and uptake of hydrophobicsubstrates. Rhamnolipids represent a chemically heterogeneousgroup of secondary metabolites composed of one ortwo rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosyntheticpathway involves rhamnosyltransferase I encoded by the rhlABoperon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoicacids (HAAs) followed by their coupling to one rhamnose moiety.The resulting mono-rhamnolipids are converted to dirhamnolipidsin a third reaction catalyzed by therhamnosyltransferase II RhlC. However, the mechanism behindthe biosynthesis of rhamnolipids containing only a singlefatty acid is still unknown. To understand the role of proteinsinvolved in rhamnolipid biosynthesis the heterologous expressionof rhl-genes in non-pathogenic Pseudomonas putidaKT2440 strains was used in this study to circumvent the complexquorum sensing regulation in P. aeruginosa. Our resultsreveal that RhlA and RhlB are independently involved inrhamnolipid biosynthesis and not in the form of a RhlAB heterodimercomplex as it has been previously postulated.Furthermore, we demonstrate that mono-rhamnolipids providedextracellularly as well as HAAs as their precursors are generallytaken up into the cell and are subsequently converted todi-rhamnolipids by P. putida and the native host P. aeruginosa.Finally, our results throw light on the biosynthesis ofrhamnolipids containing one fatty acid,which occurs by hydrolyzationof typical rhamnolipids containing two fatty acids,valuable for the production of designer rhamnolipids with desiredphysicochemical properties

Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

<p>Abstract</p> <p>Background</p> <p>Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen <it>Pseudomonas aeruginosa </it>during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular <it>P. aeruginosa</it>. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.</p> <p>Results</p> <p>Here, the metabolic engineering of a rhamnolipid producing <it>Pseudomonas putida </it>KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, <it>P. putida </it>KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria <it>Bacillus subtilis, Corynebacterium glutamicum</it>, and <it>Escherichia coli. P. putida </it>KT2440 expressing the <it>rhlAB</it>-genes from <it>P. aeruginosa </it>PAO1 produces mono-rhamnolipids of <it>P. aeruginosa </it>PAO1 type (mainly C₁₀:C₁₀). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of <it>P. aeruginosa </it>PAO1 at yields of about 0.15 g/g_glucosecorresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.</p> <p>Conclusions</p> <p>A functional alternative to the pathogenic rhamnolipid producer <it>P. aeruginosa </it>was constructed and characterized. <it>P. putida </it>KT24C1 pVLT31_<it>rhlAB </it>featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer <it>P. aeruginosa</it>.</p

Superhydrophobic Aluminum Surfaces: Preparation Routes, Properties and Artificial Weathering Impact

Among the materials that can be treated in order to impart superhydrophobic properties are many originally hydrophilic metals. For this, they must undergo a sequential treatment, including roughening and hydrophobic coating. This contribution presents various preparation routes along with various characterization methods, such as dynamic contact angle (DCA) measurements, scanning electron microscopy (SEM) and spectroscopic techniques (FT–IRRAS, XPS, EIS). Micro-rough surfaces of pure and alloyed aluminum were generated most easily by using a modifie Sulfuric Acid Anodization under Intensifie conditions (SAAi). This produces a micro-mountain-like oxide morphology with peak-to-valley heights of 2 μm and sub-μm roughness components. Additionally, micro-embossed and micro-blasted surfaces were investigated. These micro-roughened initial states were chemically modifie with a solution of a hydrophobic compound, such as the reactive f uoroalkylsilane PFATES, the reactive alkyl group containing polymer POMA, or the polymer Teflo ® AF. Alternatively, the chemical modificatio was made by a Hot Filament Chemical Vapor Deposition (HFCVD) of a PTFE layer. The latter can form a considerably higher thickness than the wet-deposited coatings, without detrimental leveling effects being observed in comparison with the original micro-rough surface. The inherent and controllable morphology of the PTFE layers represents an important feature. The impacts of a standardized artificia weathering (WTH) on the wetting behavior and the surface-chemical properties were studied and discussed in terms of possible damage mechanisms. A very high stability of the superhydrophobicity was observed for the f uorinated wet-deposited PFATES and Teflo ® AF coatings as well as for some of the PTFE layer variants, all on SAAi-pretreated substrates. Very good results were also obtained for specimens produced by appropriate mechanical roughening and PTFE coating

Analysis of a wild mouse promoter variant reveals a novel role for FcγRIIb in the control of the germinal center and autoimmunity.

Genetic variants of the inhibitory Fc receptor FcγRIIb have been associated with systemic lupus erythematosus in humans and mice. The mechanism by which Fcgr2b variants contribute to the development of autoimmunity is unknown and was investigated by knocking in the most commonly conserved wild mouse Fcgr2b promoter haplotype, also associated with autoimmune-prone mouse strains, into the C57BL/6 background. We found that in the absence of an AP-1-binding site in its promoter, FcγRIIb failed to be up-regulated on activated and germinal center (GC) B cells. This resulted in enhanced GC responses, increased affinity maturation, and autoantibody production. Accordingly, in the absence of FcγRIIb activation-induced up-regulation, mice developed more severe collagen-induced arthritis and spontaneous glomerular immune complex deposition. Our data highlight how natural variation in Fcgr2b drives the development of autoimmune disease. They also show how the study of such variants using a knockin approach can provide insight into immune mechanisms not possible using conventional genetic manipulation, in this case demonstrating an unexpected critical role for the activation-induced up-regulation of FcγRIIb in controlling affinity maturation, autoantibody production, and autoimmunity

The influence of age, gender and socio-economic status on multimorbidity patterns in primary care. first results from the multicare cohort study

Background: Multimorbidity is a phenomenon with high burden and high prevalence in the elderly. Our previous research has shown that multimorbidity can be divided into the multimorbidity patterns of 1) anxiety, depression, somatoform disorders (ADS) and pain, and 2) cardiovascular and metabolic disorders. However, it is not yet known, how these patterns are influenced by patient characteristics. The objective of this paper is to analyze the association of socio-demographic variables, and especially socio-economic status with multimorbidity in general and with each multimorbidity pattern. Methods: The MultiCare Cohort Study is a multicentre, prospective, observational cohort study of 3.189 multimorbid patients aged 65+ randomly selected from 158 GP practices. Data were collected in GP interviews and comprehensive patient interviews. Missing values have been imputed by hot deck imputation based on Gower distance in morbidity and other variables. The association of patient characteristics with the number of chronic conditions is analysed by multilevel mixed-effects linear regression analyses. Results: Multimorbidity in general is associated with age (+0.07 chronic conditions per year), gender (-0.27 conditions for female), education (-0.26 conditions for medium and -0.29 conditions for high level vs. low level) and income (-0.27 conditions per logarithmic unit). The pattern of cardiovascular and metabolic disorders shows comparable associations with a higher coefficient for gender (-1.29 conditions for female), while multimorbidity within the pattern of ADS and pain correlates with gender (+0.79 conditions for female), but not with age or socioeconomic status. Conclusions: Our study confirms that the morbidity load of multimorbid patients is associated with age, gender and the socioeconomic status of the patients, but there were no effects of living arrangements and marital status. We could also show that the influence of patient characteristics is dependent on the multimorbidity pattern concerned, i.e. there seem to be at least two types of elderly multimorbid patients. First, there are patients with mainly cardiovascular and metabolic disorders, who are more often male, have an older age and a lower socio-economic status. Second, there are patients mainly with ADS and pain-related morbidity, who are more often female and equally distributed across age and socio-economic groups