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Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence which was able to fold in a typical two- or three- layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. Based on the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.

Serial repitching of brewing yeast inoculates is an important economic factor in the brewing industry, as their propagation is time and resource intensive. Here, we investigated whether replicative aging and/or the population distribution status changed during serial repitching in three different breweries with the same brewing yeast strain but different abiotic backgrounds and repitching regimes with varying numbers of reuses.Next to bud scar numbers the DNA content of the Saccharomyces pastorianus HEBRU cells was analyzed. Gene expression patterns were investigated using low-density microarrays with genes for aging, stress, storage compound metabolism and cell cycle.Two breweries showed a stable rejuvenation rate during serial repitching. In a third brewery the fraction of virgin cells varied, which could be explained with differing wort aeration rates. Furthermore, the number of bud scars per cell and cell size correlated in all 3 breweries throughout all runs. Transcriptome analyses revealed that from the 6th run on, mainly for the cells positive gene expression could be seen, for example up-regulation of trehalose and glycogen metabolism genes. Additionally, the cells’ settling in the cone was dependent on cell size, with the lowest and the uppermost cone layers showing the highest amount of dead cells.In general, cells do not progressively age during extended serial repitching.

Individuals within natural populations are highly diverse. They can vary regarding their physiological states and thus contribute differently to the performance of the whole community [1]. Hence, the characterization of microbial physiology on the single cell level is crucial for optimization and understanding of biotechnological processes. Here a protocol is presented for detecting and monitoring the functional heterogeneity in a given yeast population, on both the cellular and molecular levels, using flow cytometry and microarray analysis (first published in Nature Protocols by Achilles et al. [2]). The protocol includes staining of living S. cerevisiae cells with three different fluorescent dyes, flow cytometric analysis, sorting of live cells and stabilization of their status, the subsequent isolation of RNA from small amounts of separated cells, and finally the quantification and integrity check of this RNA. Owing to the high quality and quantity of the isolated RNA from sorted cells, gene expression analysis can be performed by using microarrays, for example.

Bottom-fermenting Saccharomyces pastorianus strains driving brewing fermentation processes are usually reused several times. It is still unclear, whether the number of successions may have an impact on cell physiology prompting consequences for brewing quality. In this study, fermentation performance of up to twenty consecutive runs in a brewery was investigated. For each run mRNA expression levels of cellular marker molecules, which are known to correlate with metabolism, hexose transport, aging processes, stress response mechanisms and flocculation capability was estimated to obtain information on changes in cell physiology over the successive runs. Low-density microarrays were used for this purpose and the resulting gene expression profiles were finally correlated with changes in the abiotic micro-environments. A surprising stability of the marker molecule expression profiles within each specific serial repitching was stated. Loss of flocculation or an advanced aging could not be detected during serial repitching in the analyzed brewery. However, certain runs of the serial repitchings showed high variation in stress response which was found to be caused by perturbations of the abiotic conditions. Regardless, the study showed that S. pastorianus can be used repeatedly in serial repitching processes without loss of prominent physiological characteristics.

Short Term Plasticity (STP) has been shown to exist extensively in synapses throughout the brain. Its function is more or less clear in the sense that it alters the probability of synaptic transmission at short time scales. However, it is still unclear what effect STP has on the dynamics of neural networks. We show, using a novel dynamic STP model, that Short Term Depression (STD) can affect the phase of frequency coded input such that small networks can perform temporal signal summation and determination with high accuracy. We show that this property of STD can readily solve the problem of the ghost frequency, the perceived pitch of a harmonic complex in absence of the base frequency. Additionally, we demonstrate that this property can explain dynamics in larger networks. By means of two models, one of chopper neurons in the Ventral Cochlear Nucleus and one of a cortical microcircuit with inhibitory Martinotti neurons, it is shown that the dynamics in these microcircuits can reliably be reproduced using STP. Our model of STP gives important insights into the potential roles of STP in self-regulation of cortical activity and long-range afferent input in neuronal microcircuits.

The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under laboratory conditions. Using microarray techniques and proteomics, the four flocculation genes Lg-FLO1, FLO1, FLO5, FLO9, and the protein Lg-Flo1p were identified as f! actors of major importance for flocculation. The expression of the genes was several times higher in flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to quantify the proportions of powdery and flocculent yeast cells in suspensions under defined cultivation conditions. However, differentiation under industrial conditions will require mRNA and protein expression profiling. (C) 2008 international Society for Advancement of Cytometry

One of the mysteries surrounding the phenomenon of chaos is that it can rarely be found in biological systems. This has led to many discussions of the possible presence and interpretation of chaos in biological signals. It has caused empirical biologists to be very sceptical of models that have chaotic properties or even employ chaos for problem solving tasks. In this paper, it is demonstrated that there exists a possible mechanism that is part of the catalytical reaction mechanisms which may be responsible for controlling enzymatic reactions such that they do not become chaotic. It is proposed that where these mechanisms are not present or not effective, chaos may still occur in biological system.

Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase over six generations of random point mutagenesis and recombination couled with screening for improved variants. The best clones identified after four generations of sequential random mutagenesis and two generations of random recombination display more than 150 times the p-nitrobenzyl esterase activity of wild type towards loracarbef-p-nitrobenzyl ester in 15% dimethylformamide. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (<2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a ‘rational’ design effort.

Das Maischen als ein Schlüsselprozeß der Bierherstellung entzieht sich bis heute weitgehend der prozeßbegleitenden Überwachung. Das im Rahmen dieser Arbeit entwickelte Meßsystem stellt einen Beitrag dar, die Informationsgewinnung aus dem Prozeß zu verbessern. Um eine kontinuierlich im Bypass arbeitende Probenahme herum ist ein Multisensorsystem aufgebaut. Die damit gewonnenen Daten und parallel dazu erfaßte Referenzwerte bilden die Grundlage für eine multivariate Prozeßanalyse. Ein Regressionsmodell auf der Basis einer nicht linearen Partial Least Squares Regression schätzt die Konzentrationen von Extrakt, FAN und β-Glucan als Maß für die enzymatischen Abbauvorgänge Amylolyse, Proteolyse und Cytolyse. Ein trilineares Tucker3-Modell klassifiziert den Maischprozeß nach der zu Grunde liegenden Malzqualität. Unfold Principal Component Anlysis erkennt Fehlchargen anhand multivariat berechneter Abweichungen von einem Referenzverlauf bereits kurz nach dem Einmaischen, so daß ein Eingreifen in den Maischprozeß noch möglich ist., Mashing represents a key process during the production of beer. Nonetheless, neither an online monitoring system nor a quality control exists. In this work a measuring system was developed that represents a contribution to improve the acquisition of information from the process. A filtering hydrocyclone was used to produce a continuous stream of cleared mash in which a multisensor system measured physical properties. Additionally, reference analyses were conducted in the laboratory. The so gathered data formed the basis for a multivariate process analysis. A non-linear Partial Least Squares regression model estimated the quantities extract, free amino nitrogen and β-Glucan as a measure for amylolysis, proteolysis and cytolysis respectively. A trilinear Tucker3-model classified batches according to the malt quality. Unfold Principal Component Analysis detected batches with poor malt quality on the basis of multivariate deviations from the data of a reference batch. As the strongest deviations occurred at the very beginning of the mashing, a correction of the process would then still be possible.