The Cytokinin Status of the Epidermis Regulates Aspects of Vegetative and Reproductive Development in Arabidopsis thaliana

The epidermal cell layer of plants has important functions in regulating plant growth and development. We have studied the impact of an altered epidermal cytokinin metabolism on Arabidopsis shoot development. Increased epidermal cytokinin synthesis or breakdown was achieved through expression of the cytokinin synthesis gene LOG4 and the cytokinin-degrading CKX1 gene, respectively, under the control of the epidermis-specific AtML1 promoter. During vegetative growth, increased epidermal cytokinin production caused an increased size of the shoot apical meristem and promoted earlier flowering. Leaves became larger and the shoots showed an earlier juvenile-to-adult transition. An increased cytokinin breakdown had the opposite effect on these phenotypic traits indicating that epidermal cytokinin metabolism can be a factor regulating these aspects of shoot development. The phenotypic consequences of abbreviated cytokinin signaling in the epidermis achieved through expression of the ARR1-SRDX repressor were generally milder or even absent indicating that the epidermal cytokinin acts, at least in part, cell non-autonomously. Enhanced epidermal cytokinin synthesis delayed cell differentiation during leaf development leading to an increased cell proliferation and leaf growth. Genetic analysis showed that this cytokinin activity was mediated mainly by the AHK3 receptor and the transcription factor ARR1. We also demonstrate that epidermal cytokinin promotes leaf growth in a largely cell-autonomous fashion. Increased cytokinin synthesis in the outer layer of reproductive tissues and in the placenta enhanced ovule formation by the placenta and caused the formation of larger siliques. This led to a higher number of seeds in larger pods resulting in an increased seed yield per plant. Collectively, the results provide evidence that the cytokinin metabolism in the epidermis is a relevant parameter determining vegetative and reproductive plant growth and development

Fluchtmigration im Kontext von 20 Jahren Zuwanderung in die Stadt Freiburg

Hinter dem Wachstum der Stadt Freiburg in den letzten 20 Jahren verbergen sich insbesondere beim Zuzug aus dem Ausland vielfältige Migrationsformen, obwohl in der medialen und öffentlichen Wahrnehmung der letzten Jahre nur die Fluchtmigration präsent war. Die Zuwanderung lässt sich dabei nach der Herkunft klassifizieren oder nach Art der Migration. Hierbei kann unterschieden werden in Arbeitsmigration (Osteuropa), Bildungsmigration, Fluchtmigration und Mischmigration (Westeuropa). Es wird deutlich gemacht, dass für Freiburg die Arbeitsmigration und Bildungsmigration relevanter sind als die Fluchtmigration. In der kleinräumigen Verteilung zeigt sich, dass die Fluchtmigration am stärksten segregiert und die Mischmigration annähernd gleichverteilt ist

Nutzung von Sensor Spots zur Bestimmung der Gelöstsauerstoffkonzentration in Single-use Systemen im Labormassstab

Einleitung: Der wirtschaftlich gewinnbringende Einsatz biotechnologischer Verfahren setzt eine explizite Kenntnis des verwendeten Systems voraus. Für aerobe Kultivierungen bedeutet dies insbesondere die Überwachung und Optimierung der Gelöstsauerstoffkonzentration im Bioreaktor. Dies gilt sowohl für die zunehmend an Bedeutung gewinnenden Single-Use Systeme als auch für klassische Bioreaktoren, wobei sich für erstere durch ihre besonderen Eigenschaften Chancen und Herausforderungen ergeben. Einsatzgebiete: Der Einsatz klassischer Messsonden in Single-Use Systemen widerspricht dem zugrundeliegenden out of the box Konzept und erhöht gleichzeitig den Aufwand in Vorbereitung und das Risiko einer Kontamination. Für Systeme wie Schüttelkolben oder Bagreaktoren ist der Einsatz dieser Messsonden aus geometrischen und strömungstechnischen Gründen zudem wenig sinnvoll. In diesem Anwendungsfeld (Laborbioreaktoren im Milliliter- und Litermaßstab) bieten Sensor Spots eine einfache und vielversprechende Alternative zur Sauerstoffüberwachung. In der vorliegenden Posterpräsentation wird der Einsatz von Sensor Spots zur Bestimmung des volumenbe-zogenen Sauerstoffübergangskoeffizienten (kLa-Wert) in verschiedenen Systemen (beispielsweise in Thomson Optimum Growth™ Schüttelkolben oder TubeSpin® Bioreaktoren) illustriert. Im Fokus steht dabei die Optimierung der Prozessparameter für pflanzliche und tierische Zellkulturen. Anwendungsbeispiele: Den Abschluss dieser Posterpräsentation bildet die Vorstellung verschiedener Auswertungsmöglichkeiten sowie die Demonstration der Einsatzmöglichkeiten unter verschiedenen Gesichtspunkten. Die Anwendung wird am Beispiel einer Kultivierungsstudie für CHO (Chinese Hamster Ovary) Zellen demonstriert

Recommendations for the engineering characterization of single-use bioreactors

Single-use bioreactors have been available for more than 15 years and are nowadays widely accepted for a broad range of applications. However, process engineering data for these bioreactors, including volumetric mass transfer coefficients, mixing times and power inputs, are still limited. Furthermore, these data are often generated using a number of different methods, making comparisons difficult. In addition, the large variety of bioreactor types and their mixing principles (stirred, wave-mixed, orbitally-shaken, etc.) increases the difficulty in comparing the engineering data. In order to facilitate the usage of single-use bioreactors, the DECHEMA Upstream Processing (USP) expert group on ‘Single-use technology in biopharmaceutical manufacturing’ has developed recommendations for the process engineering characterization of single-use bioreactors. The methods are based on procedures that were previously developed for multi-use bioreactors and were tested in universities as well as companies (both from the supplier and user sides) for their robustness. The validated recommendations now include measurements of volumetric mass transfer coefficient, mixing time and power input. These parameters are experimentally determined using the dynamic gassing-out method, the decolorization method or sensor method, and the torque method respectively. The poster gives an overview of the fundamentals and procedures of the methods applied, and current results from the interlaboratory tests. Recent foci include the measurement of carbon dioxide gas-liquid mass transfer and the determination of mechanical stress due to hydrodynamics

The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory. RESULTS: We demonstrate that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described here. We also provide evidence for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood. CONCLUSION: Similar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory.Published versio

CFD for stirred bioreactors : analysis of different multiphase models to determine oxygen mass transfer

Oxygen supply of cells has a significant influence on cell growth and product yield. For this reason, biopharmaceutical manufacturers and their bioreactor suppliers are interested in optimizing the oxygen supply. In this context, computational fluid dynamics (CFD) can be used to calculate the kLa value in stirred bioreactors. First investigations have shown that both the volume-of-fluid (VOF) model and the compressible VOF model are unsuitable for the calculation of kLa values due to their lack of accuracy. Simulations with the Euler-Euler model showed significantly better agreement with experimental data, but the calculated kLa values were still about 50% higher than in the experiments. One reason could be the missing consideration of gas bubble breakup and coalescence. Therefore, various investigations were carried out with a population balance model (PBM)-coupled Euler-Euler model, which all were in better agreement with the experimental data, compared to the Euler-Euler model. With the help of the successfully established PBM-coupled CFD model it is now possible to calculate the oxygen mass transfer for stirred bioreactors. Thus, bioreactor and process design can be accomplished before prototyping, and time-consuming as well as cost-intensive laboratory experiments can be reduced. In order to verify the generality for all sizes of bioreactors, further validation work will be carried out

Cultivation of CHO Cells in Thomson Optimum Growth™ Shake Flasks and Scale-up

In biotechnology, the usage of shaking flasks in upstream processing is widely common due to the easy handling. Frequent applications are process screening and optimization. Thereby, the focus lies mostly on a homogeneous and fast distribution of substrates and gases whilst power consumption and shearing force are meant to be kept low, which ideally results in high biomass concentrations and product titers. For characterization purposes, the mixing time and the oxygen mass transfer coefficient (kLa) were measured in 5 L and 500 mL Thomson Optimal Growth™ shaking flasks, using the de-colorization or the dynamic gassing-out method, respectively. Those geometrical optimized bioreactors are promising higher space-time yields compared to the predominant Erlenmeyer-shake flask design. According to the results of the procedural experiments, CHO (Chinese hamster ovary) cells were cultivated at selected, auspicious parameter combinations. The effectiveness of the predetermined parameters was evaluated and a scale-up method elaborated. The results can be summarized as follows: The key parameter for all experimental setups is the shaking rate. In contrast, the filling volume showed to have a more ambiguous role. Modeling results in the 500 mL flasks showed no significant influence of the filling volume to the maximal cell density, in contrast to the 5L flask (both shaken at 50 mm throw). The highest viable cell density (up to 4.8∙10⁶ cells mL⁻¹) was reached using the 500 mL flask with high shaking rates at 50 mm shaking diameter. Thereby, a µmax of 0.038 h-1 was achieved that correlates with a td of less than 19 h. All in all, the highest µmax of 0.055 h⁻¹ was reached during the scale-up process, whereby higher viable cell densities were reached compared to the batch cultivations using the same parameter settings. In addition to the experiments performed to date, simulations with computational fluid dynamics and experimental determination of specific power consumption rates are already in progress, increasing the range of applicability and the validity of the proposed model correlations