Etablierung von Hochdurchsatz-Kultivierungs- und -Screeningmethoden für phototrophe Einzeller

Mit Hochdurchsatz-Kultivierungs- und -Screeningmethoden können viele Proben parallel, miniaturisiert und kostengünstig bearbeitet werden. Für phototrophe Organismen wie Mikroalgen und Cyanobakterien sind Hochdurchsatz-Kultivierungsverfahren jedoch bis heute kaum etabliert. Im Rahmen dieser Arbeit wurden diese Verfahren beispielhaft für das Cyanobakterium Synechocystis sp. PCC 6803 etabliert. Die benötigte technische Automatisierung wurde hierbei durch den Einsatz eines Tecan Genesis RSP 150 Pipettierroboters erreicht. Die Kultivierung erfolgte in Deepwell-Mikrotiterplatten innerhalb einer speziell angefertigten Kammer mit programmierbaren Schüttlern, einstellbarer Belichtung und CO2-Atmosphäre. Die in diesem System erreichten Wachstumsraten sind vergleichbar mit publizierten Kultivierungsmethoden. Das Hochdurchsatz-Screening wurde mit Hilfe eines in den Roboter integrierten Tecan Genios Plus Plattenreaders durchgeführt. Es wurden beispielhaft Methoden zur Bestimmung von optischer Dichte und Chlorophyllgehalt etabliert. Die hier vorgestellte Plattform kann vielseitig zur Analyse phototropher Organismen eingesetzt werden und ist durch entsprechende Assays leicht zur Messung anderer Parameter erweiterbar.High-throughput cultivation and screening methods allow a parallel, miniaturized and cost efficient processing of many samples. These methods however, have not been generally established for phototrophic organisms such as microalgae or cyanobacteria. In this work we describe and test high-throughput methods with the model organism Synechocystis sp. PC6803. The required technical automation for these processes was archived with a Tecan Genesis RSP 150 pipetting robot. The cultivation was performed in deepwell microtiter plates within a specially constructed cultivation chamber. The chamber is outfitted with programmable shaking conditions, variable illumination and an adjustable CO2 atmosphere. The growth rates archived within this system are comparable to those achieved with established methods such as bioreactors. The high-throughput screening was achieved with a Tecan Genios Plus plate reader integrated within the pipetting robot. Methods for determination of optical density and amount of chlorophyll were established within the scope of this work. The presented platform can be used for a variety of analyses of phototrophic organisms and is easily expandable with further assays to screen for additional targets

Automatisierung des Bio-Plex Pro Analyseverfahrens

Für den simultanen Nachweis mehrerer Analyten innerhalb einer Probe ist die Bead-basierte Multiplexanalytik ein häufig verwendetes Verfahren und wird beispielsweise zur Quantifizierung von Proteinen genutzt. Die aufwendige Durchführung der Assays soll durch Automatisierung einerseits dem Anwender abgenommen werden, während andererseits die Genauigkeit und Reproduzierbarkeit der Analyse gesteigert wird. Die Automatisierung des Bio-Plex Pro™ Assays ist mit dem Pipettierroboter Tecan Freedom EVO 200 umgesetzt worden. Es ist ein Skript mit der Software Freedom EVOware® entwickelt worden, welches die Probenvorbereitung des Assays vollständig übernimmt. Für einen Vergleich der manuellen und automatisierten Methode sind die humanen Zytokine IL-2, IL-4, IL-10, GM-CSF, IFN-γ und TNF-α in einer achtstufigen Standardverdünnungsreihe und in unterschiedlich konzentrierten Proben gemessen worden. Die Berechnung der Streuungen (Standardabweichung und Variationskoeffizient) der einzelnen Standardverdünnungsreihen sowie der Vergleich von gemessenen und erwarteten Konzentrationen der automatisierten und manuellen Methode zeigen, dass die Automatisierung neben der zeitlichen Optimierung auch die Genauigkeit und Reproduzierbarkeit der Analyse verbessert.Bead-based multpiplex analysis is frequently used for the simultaneous detection of multiple analytes within a sample. Such assays are commonly used to quantify proteins. The automation of the process relieves the user from the complex assay conductance and on the other hand increases the accuracy and reproducibility of the analysis. The automation of the Bio-Plex Pro™ assay has been successfully implemented with the pipetting-robot Tecan Freedom EVO 200. A script has been developed, using the Freedom EVOware® which has the ability to perform the complete assay procedure. For a comparison between manual and automated methods, the human cytokines IL-2, IL-4, IL-10, GM-CSF, IFN-γ and TNF-α were analysed by an eight-point standard dilution series and with samples of different concentrations. The calculation of the variances (standard deviation and coefficient of variation) of the single standard dilution series as well as the comparison of the observed and expected concentrations of the manual or automated method show the higher precision and reproducibility of the automated process in addition to its time-saving nature

Integrated pathway modules using time-course metabolic profiles and EST data from Milnesium tardigradum

<p>Abstract</p> <p>Background</p> <p>Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade <it>Milnesium tardigradum</it> were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress.</p> <p>Results</p> <p>In this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurable metabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration.</p> <p>Conclusions</p> <p>The functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.</p

A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ

<p>Abstract</p> <p>Background</p> <p>Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating) or preparation-intensive (eg. fluorescent staining). In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation.</p> <p>Results</p> <p>The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism <it>Synechocystis </it>sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis.</p> <p>Conclusions</p> <p>The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.</p

Soluble urokinase plasminogen activator receptor levels are associated with severity of fibrosis in patients with primary sclerosing cholangitis

The soluble urokinase-type plasminogen activator receptor (suPAR) has evolved as a useful biomarker for different entities of chronic liver disease. However, its role in patients with primary sclerosing cholangitis (PSC) is obscure. We analyzed plasma levels of suPAR in 84 patients with PSC and compared them to 68 patients with inflammatory bowel disease (IBD) without PSC and to 40 healthy controls. Results are correlated with clinical records. suPAR concentrations were elevated in patients with PSC compared to patients with IBD only and to healthy controls ((p) (&)lt; 0.001). Elevated suPAR levels were associated with the presence of liver cirrhosis ((p) (&)lt; 0.001) and signs of portal hypertension ((p) (&)lt; 0.001). suPAR revealed a high accuracy for the discrimination of the presence of liver cirrhosis comparable to previously validated noninvasive fibrosis markers (area under the curve (AUC) 0.802 (95%CI: 0.702-0.902)). Further, we demonstrated that suPAR levels may indicate the presence of acute cholangitis episodes ((p) (&)lt; 0.001). Finally, despite the high proportion of PSC patients with IBD, presence of IBD and its disease activity did not influence circulating suPAR levels. suPAR represents a previously unrecognized biomarker for diagnosis and liver cirrhosis detection in patients with PSC. However, it does not appear to be confounded by intestinal inflammation in the context of IBD

Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

The association between DNA methylation status and embryogenic competency in oil palm tissue culture was examined through Representational Difference Analysis (RDA) approach, using methylation-sensitive restriction endonucleases. "Difference Products" (DPs) of RDA derived from palms of similar genetic backgrounds but exhibiting different embryogenesis rates during the regeneration process were isolated. The DPs were sequenced using a pyrosequencing platform. To our knowledge, this is the first study profiling partial HpaII methylation sites in oil palm young leaf tissues which are potentially associated with embryogenic amenability through a genomic subtractive approach. Quantitative real-time PCR analysis demonstrated that the methylation status of a novel fragment, EgNB3, was higher in highly embryogenic leaf explants compared to low embryogenesis rate materials. These differences are likely to be contributed by the 5′-mCCGG-3′ and/or 5′-mCmCGG-3′ methylation patterns. Our data suggest that the differentially methylated site in EgNB3 has potential as a molecular biomarker for the screening of oil palm leaf explants for their embryogenic potentials

Hepatobiliary long-term consequences of COVID-19: dramatically increased rate of secondary sclerosing cholangitis in critically ill COVID-19 patients

BACKGROUND: Increasing evidence suggests that secondary sclerosing cholangitis (SSC), which can lead to cirrhosis or liver failure, may be a hepatobiliary long-term complication of COVID-19. The aim of this study was to estimate the frequency and outcome of this COVID-19 sequela and to identify possible risk factors. METHODS: This observational study, conducted at University Hospital Charité Berlin and Unfallkrankenhaus Berlin, Germany, involved hospitalized patients with COVID-19 pneumonia, including 1082 ventilated COVID-19 patients. We compared COVID-19 patients who developed SSC with a COVID-19 control group by univariate and multivariate analyses. RESULTS: SSC occurrence after COVID-19 was observed exclusively in critically ill patients with invasive ventilation, albeit with extreme clustering among them. One in every 43 invasively ventilated COVID-19 patients developed this complication. Risk factors preceding the development of secondary sclerosing cholangitis in critically ill COVID-19 patients (SSC-CIP) were signs of systemic reduced blood oxygen supply (e.g., low PaO(2)/FiO(2), ischemic organ infarctions), multi-organ failure (high SOFA score) at admission, high fibrinogen levels and intravenous ketamine use. Multivariate analysis confirmed fibrinogen and increased plasma lactate dehydrogenase as independent risk factors associated with cholangiopathy onset. The 1-year transplant-free survival rate of COVID-19-associated SSC-CIP was 40%. CONCLUSIONS: COVID-19 causes SSC-CIP in a substantial proportion of critically ill patients. SSC-CIP most likely develops due to severe tissue hypoxia and fibrinogen-associated circulatory disturbances. A significant increase of patients with SSC-CIP is to be expected in the post-COVID era