Visualisierung chemischer Prozesse mittels Einzelmolekül-Fluoreszenz-Mikroskopie

Chemische Systeme werden aus Milliarden einzelner Reaktionsereignisse mit oft komplexen molekularen Zusammenhängen gebildet. Mit einem über das Gesamtsystem gemittelten Signal können überlagerte, heterogene Prozesse übersehen werden. In der Einzelmolekülchemie (single-molecule chemistry – SMC) werden Reaktionsprozesse mikroskopisch betrachtet und einzelne Ereignisse miteinander verglichen. So werden zusätzlich zu gemittelten kinetischen Parametern, mechanistische Subspezies, z.B. seltene Intermediate, und kinetische Verteilungen aufgedeckt. Die vorliegende Arbeit schlägt die Brücke von der experimentellen Planung, der Probenvorbereitung bis hin zur Ausführung und Datenauswertung eines Einzelmolekülversuchs anhand eines metallorganischen Reaktionssystems. Zunächst wurde ein Prismen-basiertes internes Totalreflexionsfluoreszenzmikroskop (TIRFM) für Einzelmolekül-(SM-)Experimente konzipiert und validiert. Anschließend wurde eine Probenvorbereitungsroutine etabliert, mit welcher immobile Sensoren homogener Reaktivität, vergleichbar zu Sensoren in Lösung, generiert werden konnten. Die Pd-katalysierte Tsuji-Trost Deallylierung wurde an den eingeführten dual-emissiven Sensoren im Ensemble und im SM-Experiment studiert. Aus den erhaltenen Daten wurden anhand automatischer Auswertungsroutinen einzelne Reaktionsschritte und deren Kinetiken extrahiert. Dabei konnte die Bildung eines kurzlebigen Intermediats der Katalyse aufgedeckt werden, welches unter Ensemblebedingungen verborgen blieb.Chemical systems consist of billions of individual reaction events with often complex molecular relationships. A signal, averaged over the entire system can also originate from superimposed, heterogeneous processes. Single-molecule chemistry (SMC) is designated to inspect reaction processes microscopically and compare the extracted reaction events. This way, mechanistic subspecies, e.g. rare intermediates, and distributions of kinetic behavior in addition to the mean kinetic parameters are obtained. The presented work enfolds experimental design, substrate preparation as well as execution and data analysis of a single-molecule experiment on a metal-organic showcase reaction. At first, a prism-based total internal reflection fluorescence microscope (TIRFM) was implemented and validated for recording single dye-molecules. Second, a surface preparation routine was established, which allowed for generation of immobile, homogeneously reactive single-molecule species, comparable to their counterparts in solution. Third, the Pd-catalyzed Tsuji-Trost deallylation of the introduced dual-emissive sensor was studied and compared in ensemble and single-molecule measurements. The individual reaction steps and their kinetics were derived by means of automated data analyzation routines. As a result, the formation of a short-lived intermediate, hidden in ensemble measurements, was deciphered.Deutsche Forschungsgesellschaf

mPies: a novel metaproteomics tool for the creation of relevant protein databases and automatized protein annotation

Metaproteomics allows to decipher the structure and functionality of microbial communities. Despite its rapid development, crucial steps such as the creation of standardized protein search databases and reliable protein annotation remain challenging. To overcome those critical steps, we developed a new program named mPies (metaProteomics in environmental sciences). mPies allows the creation of protein databases derived from assembled or unassembled metagenomes, and/or public repositories based on taxon IDs, gene or protein names. For the first time, mPies facilitates the automatization of reliable taxonomic and functional consensus annotations at the protein group level, minimizing the well-known protein inference issue, which is commonly encountered in metaproteomics. mPies’ workflow is highly customizable with regards to input data, workflow steps, and parameter adjustment. mPies is implemented in Python 3/Snakemake and freely available on GitHub: https://github.com/johanneswerner/mPies/

Scientific cruise report Elisabeth Mann-Borgese SUMMIX-MESO

Objectives: It was intended to investigate the meso-scale and sub-meso-scale dynamics of the upper layers (upper 80 m) in the central Baltic Sea, using towed instruments and acoustic profilers, to better understand the physical conditions for cyanobacteria blooms. Under optimal weather conditions, we intended to carry out 10 one-day quasi-synoptic surveys by cruising in large meandering patterns (see fig. 1) covering areas of 15 X 15 nautical miles or 8 X 8 nautical miles, depending on the survey mode, see below. This cruise was the meso-scale component of the two-ship SUMMIX experiment together with RV Meteor (Physical and biochemical exchange-, mixing- and transformation processes in the central Baltic Sea during summer stratification and their controls on the cyanobacterial summer bloom) which was intended to be located at a fixed position nearby RV Elisabeth Mann Borgese in order to survey the water column in high vertical, spatial and parameter resolution, including biogeochemical experiments on board. In addition to the physical parameters, also vertical and horizontal zooplankton net tows as well as water samples taken by CTD bottles were planned

Deciphering the Functioning of Microbial Communities: Shedding Light on the Critical Steps in Metaproteomics

Unraveling the complex structure and functioning of microbial communities is essential to accurately predict the impact of perturbations and/or environmental changes. From all molecular tools available today to resolve the dynamics of microbial communities, metaproteomics stands out, allowing the establishment of phenotype–genotype linkages. Despite its rapid development, this technology has faced many technical challenges that still hamper its potential power. How to maximize the number of protein identification, improve quality of protein annotation, and provide reliable ecological interpretation are questions of immediate urgency. In our study, we used a robust metaproteomic workflow combining two protein fractionation approaches (gel-based versus gel-free) and four protein search databases derived from the same metagenome to analyze the same seawater sample. The resulting eight metaproteomes provided different outcomes in terms of (i) total protein numbers, (ii) taxonomic structures, and (iii) protein functions. The characterization and/or representativeness of numerous proteins from ecologically relevant taxa such as Pelagibacterales, Rhodobacterales, and Synechococcales, as well as crucial environmental processes, such as nutrient uptake, nitrogen assimilation, light harvesting, and oxidative stress response, were found to be particularly affected by the methodology. Our results provide clear evidences that the use of different protein search databases significantly alters the biological conclusions in both gel-free and gel-based approaches. Our findings emphasize the importance of diversifying the experimental workflow for a comprehensive metaproteomic study

Towards the discovery of novel molecular clocks in Prokaryotes

Diel cycle is of enormous biological importance as it imposes daily oscillation in environmental conditions, which temporally structures most ecosystems. Organisms developed biological time-keeping mechanisms – circadian clocks – that provide a significant fitness advantage over competitors by optimising the synchronisation of their biological activities. while circadian clocks are ubiquitous in eukaryotes, they are so far only characterised in Cyanobacteria within Prokaryotes. However, growing evidence suggests that circadian clocks are widespread in the bacterial and archaeal domains. As Prokaryotes are at the heart of crucial environmental processes and are essential to human health, unravelling their time-keeping systems provides numerous applications in medical research, environmental sciences, and biotechnology. in this review, we elaborate on how novel circadian clocks in Prokaryotes offer research and development perspectives. we compare and contrast the different circadian systems in Cyanobacteria and discuss about their evolution and taxonomic distribution. we necessarily provide an updated phylogenetic analysis of bacterial and archaeal species that harbour homologs of the main cyanobacterial clock components. Finally, we elaborate on new potential clock-controlled microorganisms that represent opportunities of ecological and industrial relevance in prokaryotic groups such as anoxygenic photosynthetic bacteria, methanogenic archaea, methanotrophs or sulphate-reducing bacteria

Diel Protein Regulation of Marine Picoplanktonic Communities Assessed by Metaproteomics

The diel cycle is of enormous biological importance in that it imposes temporal structure on ecosystem productivity. In the world’s oceans, microorganisms form complex communities that carry out about half of photosynthesis and the bulk of life-sustaining nutrient cycling. How the functioning of microbial communities is impacted by day and night periods in surface seawater remains to be elucidated. In this study, we compared the day and night metaproteomes of the free-living and the particle-attached bacterial fractions from picoplanktonic communities sampled from the northwest Mediterranean Sea surface. Our results showed similar taxonomic distribution of free-living and particle-attached bacterial populations, with Alphaproteobacteria, Gammaproteobacteria and Cyanobacteria being the most active members. Comparison of the day and night metaproteomes revealed that free-living and particle-attached bacteria were more active during the day and the night, respectively. Interestingly, protein diel variations were observed in the photoautotroph Synechococcales and in (photo)-heterotrophic bacteria such as Flavobacteriales, Pelagibacterales and Rhodobacterales. Moreover, our data demonstrated that diel cycle impacts light-dependent processes such as photosynthesis and UV-stress response in Synechococcales and Rhodobacterales, respectively, while the protein regulation from the ubiquitous Pelagibacterales remained stable over time. This study unravels, for the first time, the diel variation in the protein expression of major free-living and particle-attached microbial players at the sea surface, totaling an analysis of eight metaproteomes

Folk perceptions and patterns of use of orchid species in Benin, West Africa

In Benin haben die Menschen ein reiches ethnobotanisches Wissen über, das die kulturelle und ökologische Vielfalt ihrer Umwelt widerspiegelt. Mehrere Studien befassten sich mit der Frage, wie wertvoll Pflanzenarten für lo abunkale Gemeinschaften sind. Trotz der Bedeutung der Orchideen für den Lebensunterhalt der lokalen Bevölkerung gab es aber nur wenig Forschungsinteresse an den Orchideenarten. Die vorliegende Studie untersuchte die Unterschiede im Gebrauch und im Wissen der lokalen Leute von Orchideen in der sudanischen Zone von Benin. Die Studie wurde unter den vier wichtigsten sozioökonomischen und ethnischen Gruppen aus sechs Dörfern rund um das Biosphärenreservat Pendjari in Benin durchgeführt. Einhundertsechzig Personen nahmen an dieser Studie teil. Die Daten wurden mit Hilfe von semi-strukturierten Einzelinterviews erhoben und mittels quantitativer ethnobotanischer Methoden analysiert. 29 verschiedene Anwendungsarten wurden erfasst und können in vier Hauptnutzungskategorien unterteilt werden: Arzneimittel, Veterinärmedizin, Spirituelles und Lebensmittel. Es gab Unterschiede in der Verwendung von Orchideen unter den ethnischen Gruppen, Geschlecht und Alter. Das Wissen über die Verwendung von Orchideen wurde maßgeblich von der ethnischen Gruppe und dem Alter der Befragten beeinflusst. Anders als junge gebildete Generationen hatten die meisten Erwachsenen und Ältesten, insbesondere Frauen, ein umfassenderes Wissen über die Verwendung von Orchideen. Calyptrochilum christianum, die am häufigsten verwendete Orchidee, wurde in mehr als 50% der Orchideenarten erwähnt. Der Stamm der Gourmantché und Waama hatte mehr Wissen über die Verwendung von Orchideen, während der Berba Stamm weniger Wissen besaß. Drei Orchideenarten (Habenaria cirrhata, Eulophia horsfallii und Nervilia bicarinata) wurden als Nahrung gemeldet. Orchideen hatten einen niedrigen Nutzwert im Bereich von 0,01 (Eulophia spp) bis 0,2 (C. christianum). Der kontrollierte Zugang zum Biosphärenreservat und die Landflucht können den Mangel an Weitergabe des Wissens der Älteren über die Orchideennutzung und deren Wert des Wissens an die jüngere Generation erklären.In Benin, people have a rich ethnobotanical knowledge of plant species, reflecting the cultural and ecological diversity of their environment. Several studies were focused on the question of how valuable are plant species for local communities. However, there has been very little research interest in the orchid species in spite of the importance of orchids in the livelihood of the local people. This study examined the use and differences in knowledge of local people of orchids in the Sudanian zone of Benin. An ethnobotanical study was conducted amongst the four main socio-economic and ethnic groups from six villages around the Pendjari Biosphere Reserve in Benin. One hundred and sixty people participated in this study. Data were gathered using semi-structured individual interviews and analysed using quantitative ethnobotanical methods. 29 different types of use were recorded and can be grouped into four main use categories: medicinal, veterinary, spiritual and food. There were differences in orchid utilization among the ethnic groups, gender and age. The knowledge of orchid uses was significantly affected by the ethnic group and the age of the respondent. Unlike young educated generations, most adults and elders, especially women, had a more comprehensive knowledge of orchid uses. Calyptrochilum christianum, the most used orchid, was mentioned in more than 50% of the types of orchid use. The Gourmantché and Waama tribe had more knowledge on orchid use whereas the Berba tribe had less knowledge. Three orchid species (Habenaria cirrhata, Eulophia horsfallii and Nervilia bicarinata) were reported as food. Orchids had low use value ranging from 0.01 (Eulophia spp) to 0.2 (C. christianum). The controlled access to the biosphere reserve and rural exodus can explain the lack of indigenous knowledge transfer of orchid use and value from elders to the young generation.Au Bénin, les connaissances ethnobotaniques sont riches et variées, reflétant la diversité culturelle et écologique en place. De nombreuses études ont porté sur la question de l’utilité des plantes pour les communautés locales. Cependant, peu de travaux ce sont intéressés aux orchidées, malgré leur importance pour le bien-être des populations locales. Cette étude a examiné l'importance et la variation des connaissances locales sur l’usage des orchidées dans la zone soudanienne au Bénin. Une étude ethnobotanique a été conduite dans les quatre principaux groupes socioéconomiques et ethniques représentés par six villages autour de la Réserve de Biosphère de la Pendjari. Cent soixante personnes ont été enquêtées. Les données ont été recueillies à l'aide d'entrevues individuelles semi-structurées et analysées suivant les méthodes ethnobotaniques quantitatives. Au total, 29 différents types d'usages ont été enregistrées et peuvent être regroupées en quatre catégories: médecine, vétérinaire, spirituel et alimentaire. Il y a une variation des usages faits des orchidées aussi bien suivant les groupes ethniques, le genre que de l'âge. Le niveau de connaissance des usages faits des orchidées est significativement influencé par le groupe ethnique et l'âge du répondant. Contrairement aux jeunes générations scolarisées, la plupart des adultes et personnes âgées, en particulier les femmes, ont une plus grande connaissance des usages possible des orchidées. Calyptrochilum christianum, l'orchidée la plus utilisée, a été mentionnée dans plus de 50% des types d’usages. Les Gourmantché et les Waama ont plus de connaissances sur l'utilisation des orchidées tandis que les Berba en ont moins. Trois espèces d’orchidées (Habenaria cirrhata, Eulophia horsfallii et Nervilia bicarinata) ont été rapportées comme utilisées dans l’alimentation. Les orchidées en générale ont une faible valeur d'usage, valeurs allant de 0,01 (Eulophia spp) à 0,2 (C. christianum). L'accès contrôlé à la réserve de biosphère et l’exode rural pourraient expliquer la rupture apparente du transfert des savoirs sur les usages et valeur des orchidées des anciens aux jeunes générations

Trace gas/aerosol boundary concentrations and their impacts on continental-scale AQMEII modeling domains

Copyright 2011 Elsevier B.V., All rights reserved.Over twenty modeling groups are participating in the Air Quality Model Evaluation International Initiative (AQMEII) in which a variety of mesoscale photochemical and aerosol air quality modeling systems are being applied to continental-scale domains in North America and Europe for 2006 full-year simulations for model inter-comparisons and evaluations. To better understand the reasons for differences in model results among these participating groups, each group was asked to use the same source of emissions and boundary concentration data for their simulations. This paper describes the development and application of the boundary concentration data for this AQMEII modeling exercise. The European project known as GEMS (Global and regional Earth-system Monitoring using Satellite and in-situ data) has produced global-scale re-analyses of air quality for several years, including 2006 (http://gems.ecmwf.int). The GEMS trace gas and aerosol data were made available at 3-hourly intervals on a regular latitude/longitude grid of approximately 1.9° resolution within 2 "cut-outs" from the global model domain. One cut-out was centered over North America and the other over Europe, covering sufficient spatial domain for each modeling group to extract the necessary time- and space-varying (horizontal and vertical) concentrations for their mesoscale model boundaries. Examples of the impact of these boundary concentrations on the AQMEII continental simulations are presented to quantify the sensitivity of the simulations to boundary concentrations. In addition, some participating groups were not able to use the GEMS data and instead relied upon other sources for their boundary concentration specifications. These are noted, and the contrasting impacts of other data sources for boundary data are presented. How one specifies four-dimensional boundary concentrations for mesoscale air quality simulations can have a profound impact on the model results, and hence, this aspect of data preparation must be performed with considerable care.Peer reviewedFinal Accepted Versio

ABC3 Consensus: Assessment by a German Group of Experts

The Advanced Breast Cancer Third International Consensus Conference on the diagnosis and treatment of advanced breast cancer took place in Lisbon, Portugal, on November 5-7, 2015. This year's conference (ABC3) was focused on the treatment of metastatic breast cancer (stage IV), as it was 4 years ago at the first consensus meeting (ABC1). A matter of particular interest was the patients' perspective. Thus, patient-relevant issues were addressed by the consensus discussions, such as those on treatment goals, quality of life, care of long-term survivors ('survivorship issues'), and coping with disease-related symptoms and the side effects of treatment. Further important issues on the agenda were the use of standardized instruments for the assessment of individual treatment success ('patient-reported outcome measures') and the evaluation of the benefit of novel drugs (e.g. the European Society for Medical Oncology (ESMO) Magnitude of Clinical Benefit Scale). Diagnosis and treatment of inoperable locally advanced breast cancer had already been discussed 2 years earlier at the ABC2 Consensus and were not dealt with in the framework of this year's ABC3 Consensus. With regard to country-specific peculiarities, which unavoidably found their way into the ABC Consensus, a working group of German breast cancer experts commented on the voting results of the ABC panelists. As for the past consensus, the group specially considered the German guidelines for the diagnosis and treatment of breast cancer (AGO (Gyneco-Oncology Working Group), S3, DGHO (German Society of Hematology and Medical Oncology)) in order to adapt the ABC3 consensus for everyday therapy in Germany. (C) 2016 S. Karger GmbH, Freibur