Realization of a New-to-Nature Carboxylation Pathway

Most inorganic carbon enters the biosphere via the Calvin-Benson-Bassham (CBB) cycle by its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). An unproductive side reaction of RuBisCO with oxygen leads to the formation of 2-phosphoglycolate (2-PG), which is recycled via complex pathways into 3-phosphoglycerate (3-PGA), releasing carbon dioxide in the process. The tartronyl-CoA pathway represents a synthetic pathway that was designed to recycle 2-PG more efficiently, avoiding the release of carbon dioxide, and fixing carbon dioxide instead. It consists of four main reactions steps, which are not known to take part in any natural metabolic pathway. These steps are the activation of glycolate to glycolyl-CoA, the carboxylation of glycolyl-CoA to tartronyl-CoA as its key reaction, and the subsequent two reductions giving rise to glycerate. In this work, all required enzymes were identified or established by engineering and the tartronyl-CoA pathway was realized in vitro. Promiscuous enzyme candidates performing analogous reactions with similar substrates were screened and further improved to perform their desired functions. These include engineered glycolyl-CoA synthetase and glycolyl-CoA carboxylase (GCC), as well as a tartronyl-CoA reductase. For the engineering of GCC, rational design as well as high-throughput directed evolution was applied resulting in a new-to-nature carboxylase that matches the kinetic properties of natural carboxylases. Moreover, a 1.96 Å resolution cryogenic electron microscopy (cryo-EM) structure of GCC was obtained, highlighting and corroborating the effects of the introduced mutations. The concerted function of all tartronyl-CoA pathway enzymes was confirmed in the context of photorespiration in vitro. The in vitro reconstitution also included the optimization of reaction parameters as well as efficient cofactor recycling. Besides its function as photorespiratory bypass, the tartronyl-CoA pathway was shown to be functional as an additional carbon fixing module, able to connect a synthetic carbon dioxide fixation cycle to central carbon metabolism. Furthermore, the tartronyl-CoA pathway was successfully employed for the in vitro conversion of the plastic waste component ethylene glycol into the central carbon metabolite glycerate. In an initial attempt of an in vivo implementation of the tartronyl-CoA pathway for ethylene glycol assimilation, it was shown that GCC, the key enzyme of the tartronyl-CoA pathway, can be functionally produced in Pseudomonas putida

Zirkadiane Rhythmik zirkulierender microRNA bei gesunden Probanden

Zirkadiane Rhythmik zirkulierender miRs bei gesunden Probanden: MicroRNAs (miRs) sind seit einigen Jahren Gegenstand vieler Studien, welche Hoffnung säen, in den kleinen nicht-kodierenden Nukleinsäuren neue Biomarker gefunden zu haben. Diese sollen in der Diagnostik als Verlaufs- und Prognoseparameter und darüber hinaus sogar als mögliche Targets neuer Therapieansätze genutzt werden können. Viele Studien untersuchten lediglich ausgewählte miRs in Bezug zu verschiedensten Tumorleiden, aber auch zu anderen Krankheiten - häufig mit inkonsistenten Ergebnissen. Als eines der ursächlichen Probleme gilt die bislang fehlende Kenntnis über die physiologische Funktion der miRs, wie auch deren potenziellen Einflussfaktoren beim Gesunden. Darum widmete sich unsere Untersuchung speziell dem physiologischen Verhalten von miRs im Tagesverlauf. Wir rekrutierten vier gesunde, junge, sportliche Männer zur Studie des physiologischen Verhaltens von miRs im peripheren Blut. Die miRs wurden nach mehreren präanalytischen Schritten mittels eines qPCR-basierten Arrays analysiert. Insgesamt wurden 1066 miRs untersucht. Der Fokus wurde auf miRs mit hohem Expressionsniveau gelegt (Ct<30 (Ct = cycle threshold, Maß für Menge an miRs)). Im Einzelnen waren miR-320, miR-24-3p und miR-1280 über den Tag gleichmäßig hoch exprimiert. Einzelne miRs zeigten tageszeitliche Unterschiede. So zeigte miR-365-3p eine Expressionssteigerung um das beinahe 2-Fache im Tagesverlauf, wohingegen miR-637 ein Expressionsmaximum und miR- 1207 ein Expressionsminimum am Mittag zeigten. Bei miR-365-3p ist eine circadiane Rhythmik denkbar, wohingegen bei miR-637 und miR-1207 die vorrausgegangene Nahrungsaufnahme eine entscheidende Rolle spielen könnte. Wenngleich es sich bei unserer Stichprobe um eine qualitative Untersuchung handelte, konnte gezeigt werden, dass bei gesunden Probanden nicht alle, jedoch einige miRs im peripheren Blut zuverlässig messbar waren. Zudem haben wir festgestellt, dass einzelne miRs, nicht aber das miRNom als Ganzes, einer zirkadianen Rhythmik folgen können und/oder durch Nahrungsaufnahme beeinflusst werden können. Unsere Untersuchung sollte daher durch Forschung an größeren Stichproben erhärtet werden, um einen Vergleich zu Untersuchungen bei Patienten zu ermöglichen.Circadian rhythm of circulating miRs in healthy volunteers: miRs are a major research focus for the past several years, especially in the quest to identify novel biomarkers for diagnosis and progression of various diseases and maybe as therapeutic targets. Attempts to generate miR profiles from either tissues/cells or serum/plasma that are indicative for certain diseases have yielded inconsistent results. One problem consists in the limited understanding of the presence of certain miRs for instance in blood and their functional consequences. In addition, many physiological factors such as age, gender, nutrition state and others have been shown to influence the presence of miRs in a given clinical setting that are often not taken in consideration. Therefore, the aim of the present study was to generate a physiological profile of circulating miRs in the blood of healthy subjects over the course of the day. We recruited four healthy, young, normal-weight, male adults to profile serum miRs during the day. miRs were analyzed using qPCR-based whole miRNome arrays after several pre-analytic steps A total of 1066 miRs were tested for their presence in serum. We turned attention to those miRs which were abundantly expressed in blood. Those highly expressed miRs did not reveal a consistent circadian rhythm or pattern. Several miRs (miR-320, miR-24-3p und miR-1280) had no specific fluctuation during the day but were consistently detectable. Among the miRs with variable expression levels, miR-365-3p demonstrated an expression increase during the day (average fold change 1.96) suggesting a seeming circadian rhythm. miR-637 displayed an expression maximum and miR-1207 a minimum at noon time. This may be owed to food intake. The low number of subjects in our study did only allow us to observe qualitative changes which is a limitation. In conclusion, we found miRs with and without fluctuation, but the whole miRnome has no specific pattern. Moreover fluctuation may due to foodintake/starvation. So, studies with larger cohorts on the circadian behavior of circulating miRs should be conducted to shed light on the physiological function of circulating miRs in peripheral blood and to enable comparison to our study

International Consensus Conference for Advanced Breast Cancer, Lisbon 2019: ABC5 Consensus – Assessment by a German Group of Experts

The 5th International Consensus Conference for Advanced Breast Cancer (ABC5) took place on November 14–16, 2019, in Lisbon, Portugal. Its aim is to standardize the treatment of advanced breast cancer based on the available evidence and to ensure that all breast cancer patients worldwide receive adequate treatment and access to new therapies. This year, the conference focused on developments and study results in the treatment of patients with hormone receptor-positive/HER2-negative breast cancer as well as precision medicine. As in previous years, patient advocates from around the world were integrated into the ABC conference and had seats on the ABC consensus panel. In the present paper, a working group of German breast cancer experts comments on the results of the on-site ABC5 consensus votes by ABC panelists regarding their applicability for routine treatment in Germany. These comments take the recommendations of the Breast Committee of the Gynecological Oncology Working Group (Arbeitsgemeinschaft Gynäkologische Onkologie; AGO) into account. The report and assessment presented here pertain to the preliminary results of the ABC5 consensus. The final version of the statements will be published in Annals of Oncology and The Breast

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax)

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29 Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29 Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates. (C) 2017 Elsevier Inc. All rights reserved.European Union Seventh Framework Programme [262336]Spanish Ministry of Science and Innovation (MICINN)Spanish Ministry of the Economy and Competitiveness (MINECO) [AGL2009-11086]Spanish Ministry of the Economy and CompetitivenessRegional Government of Valencia [PROME-TEOH/2014/051]info:eu-repo/semantics/acceptedVersio

Guinea pig models for translation of the developmental origins of health and disease hypothesis into the clinic

Over 30 years ago Professor David Barker first proposed the theory that events in early life could explain an individual\u27s risk of non-communicable disease in later life: the developmental origins of health and disease (DOHaD) hypothesis. During the 1990s the validity of the DOHaD hypothesis was extensively tested in a number of human populations and the mechanisms underpinning it characterised in a range of experimental animal models. Over the past decade, researchers have sought to use this mechanistic understanding of DOHaD to develop therapeutic interventions during pregnancy and early life to improve adult health. A variety of animal models have been used to develop and evaluate interventions, each with strengths and limitations. It is becoming apparent that effective translational research requires that the animal paradigm selected mirrors the tempo of human fetal growth and development as closely as possible so that the effect of a perinatal insult and/or therapeutic intervention can be fully assessed. The guinea pig is one such animal model that over the past two decades has demonstrated itself to be a very useful platform for these important reproductive studies. This review highlights similarities in the in utero development between humans and guinea pigs, the strengths and limitations of the guinea pig as an experimental model of DOHaD and the guinea pig\u27s potential to enhance clinical therapeutic innovation to improve human health. (Figure presented.)

Développement d'un test standardisé de quantification de l'expression de la myéloperoxydase des polynucléaires neutrophiles du sang périphérique par cytométrie en flux pour le diagnostic d'exclusion des syndromes myélodysplasiques : évaluation de différentes méthodes de perméabilisation

Mémoire de Diplôme d'Etudes Spécialisées (DES) tenant lieu de thèse d'exercice.Myelodysplastic syndromes (MDS) are clonal bone marrow neoplasms that predominate in the elderly whose diagnostic work-up relies on bone marrow aspiration. To prevent the risks and discomfort associated with bone marrow aspiration, a peripheral blood assay based on the semi-quantification of myeloperoxidase (MPO) in neutrophils by flow cytometry was jointly developed by three university-affiliated hospitals (Grenoble, Clermont-Ferrand and Saint-Etienne). An intra-individual robust coefficient of variation (RCV) value for peripheral blood neutrophil MPO expression lower than 30% accurately rules out MDS with a sensitivity estimate of 100%. The concept has been validated at Grenoble University Hospital and an inter-regional project has been accepted for multicenter validation. To facilitate the use of the test in other laboratories, it is important to ensure the possibility of using reagents other than those we have used, especially for membrane permeabilization. Indeed, many commercial kits are available in the commercial market.The aim of this study was to examine performances of two permeabilization kits (Dako Intrastain™ and BD FACS™ Permeabilizing Solution 2) compared to the method reference (BD Intrasure™ kit). We compared the RCV values for peripheral blood neutrophil MPO expression obtained with the 3 permeabilization methods for 10 controls and 10 cases. Controls were adults with unconfirmed suspicions of MDS. Cases were adults with established diagnosis of MDS or CMML, as defined by current guidelines. The RCV values with BD FACS™Permeabilizing Solution 2 were quantitatively comparable to the reference protocol (mean relative change, -1%). All cases with established MDS diagnosis had RCV values higher than 30%. However, RCV of neutrophil MPO expression using Dako InstraStain Kit appears to be different (mean relative change, +64%) and without discrimination between cases and controls. In conclusion, our findings suggest that BD FACS™ Permeabilizing Solution 2 represents a good technique for RCV of neutrophil MPO expression in peripheral blood compare with reference protocol. A standardization of different fixation/permeabilization methods is needed in order to use the published cut off 30%.Les syndromes myélodysplasiques (SMD) sont des hémopathies clonales qui surviennent chez des sujets âgés dont le diagnostic repose sur le myélogramme. Pour limiter les risques et l’inconfort liés aux prélèvements de moelle, un test à partir de sang périphérique basé sur la semi-quantification de la myéloperoxydase (MPO) dans les polynucléaires neutrophiles (PN) par cytométrie en flux (CMF) a été développé conjointement par les CHU de Grenoble, Clermont-Ferrand et Saint-Etienne. Un coefficient de variation robuste (CVR) intra-individuel de la distribution de la MPO inférieur à 30% exclut le diagnostic avec une sensibilité proche de 100%. Le concept a été validé au CHU de Grenoble et un PHRC inter-régional a été accepté pour une validation multicentrique. Pour faciliter l’usage du test en routine dans d’autres laboratoires, il est important de s’assurer de la possibilité d’utiliser d’autres réactifs que ceux que nous avons employés, notamment pour la perméabilisation membranaire. De nombreux kits commerciaux sont en effet disponibles sur le marché. L’objectif de ce travail a été d’évaluer les performances de deux kits de perméabilisation, à savoir Intrastain™ (Dako, Denmark) et BD FACS™ Permeabilizing Solution 2 (BD Bioscience, San Jose, CA, USA), par rapport à la méthode de référence utilisant le kit Intrasure™ (BD Biosciences, San Jose, CA, USA). Nous avons comparé les CVR de l’expression de la MPO des PN sanguins obtenus avec les 3 méthodes de perméabilisation pour 10 patients suspects non SMD et 10 patients SMD. Les CVR obtenus avec la Permeabilizing Solution 2 étaient quantitativement comparables à la méthode de référence (changement relatif de -1% en moyenne). Vis-à-vis du seuil prédéfini à 30%, les interprétations étaient toutes concordantes entre les deux protocoles. Le protocole Intrastain générait des CVR très hétérogènes et plus élevés que la méthode de référence (changement relatif de +64% en moyenne) et sans discrimination entre SMD et non SMD. En conclusion, notre étude indique que le kit Permeabilizing Solution 2 pourrait être employé en tant que réactif de perméabilisation dans notre protocole. En revanche, l’hétérogénéité des CVR obtenus avec le kit Intrastain suggère qu’il ne convient pas dans le cadre de notre test. Cette étude est un travail préliminaire qui sera complétée par une étude de plus grande envergure avec d’autres kits commerciaux. A terme, elle permettrait de valider l’emploi de plusieurs kits de perméabilisation compatibles avec le test

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