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In bioproduction processes cellular heterogeneity can cause unpredictable process outcomes or even provoke process failure. Still, cellular heterogeneity is not examined systematically in bioprocess research and development. One reason for this shortcoming are the applied average bulk analyses, which are not able to detect cell-to-cell differences. In this work we present a microfluidic tool for single-cell cultivation of mammalian suspension cells (MaSC). The design of our platform allows cultivation at highly controllable environments. As model system CHO K1 cells were cultivated over 150 h. Growth behavior was analyzed on single-cell level and resulted in growth rates between 0.85 - 1.16 d-1 , which are comparable to classical cultivation approaches such as shake flask and lab-scale bioreactor. At the same time, heterogeneous growth and division behavior, e.g., unequal division time, as well as rare cellular events like polynucleation or reversed mitosis were observed, which would have remained undetected in a standard population analysis based on average measurements. Therefore, MaSC will open the door for systematic single-cell analysis of mammalian suspension cells. Possible fields of application represent basic research topics like cell-to-cell heterogeneity studies, clonal stability, pharmaceutical drug screening and stem cell research, as well as bioprocess related topics such as media development and novel scale-down approaches. This article is protected by copyright. All rights reserved.

Motion adaptation has been attributed in flying insects a pivotal functional role in spatial vision based on optic flow. Ongoing motion enhances in the visual pathway the representation of spatial discontinuities, which manifest themselves as velocity discontinuities in the retinal optic flow pattern during translational locomotion. There is evidence for different spatial scales of motion adaptation at the different visual processing stages. Motion adaptation is supposed to take place, on the one hand, on a retinotopic basis at the level of local motion detecting neurons and, on the other hand, at the level of wide-field neurons pooling the output of many of these local motion detectors. So far, local and wide-field adaptation could not be analyzed separately, since conventional motion stimuli jointly affect both adaptive processes. Therefore, we designed a novel stimulus paradigm based on two types of motion stimuli that had the same overall strength but differed in that one led to local motion adaptation while the other did not. We recorded intracellularly the activity of a particular wide-field motion-sensitive neuron, the horizontal system equatorial cell (HSE) in blowflies. The experimental data were interpreted based on a computational model of the visual motion pathway, which included the spatially pooling HSE-cell. By comparing the difference between the recorded and modeled HSE-cell responses induced by the two types of motion adaptation, the major characteristics of local and wide-field adaptation could be pinpointed. Wide-field adaptation could be shown to strongly depend on the activation level of the cell and, thus, on the direction of motion. In contrast, the response gain is reduced by local motion adaptation to a similar extent independent of the direction of motion. This direction-independent adaptation differs fundamentally from the well-known adaptive adjustment of response gain according to the prevailing overall stimulus level that is considered essential for an efficient signal representation by neurons with a limited operating range. Direction-independent adaptation is discussed to result from the joint activity of local motion-sensitive neurons of different preferred directions and to lead to a representation of the local motion direction that is independent of the overall direction of global motion. Copyright © 2021 Li, Niemeier, Kern and Egelhaaf.

The presence of leachables in biopharmaceutical processes using single-use technologies (SUT) is well known. For the detection and quantification of the latter, extractable studies of SUT are very common nowadays. Although a mixture of compounds is regularly found in extractable studies, research has only been carried out regarding the effect of individual compounds on cell culture and the cumulative effect of a mix of leachables has not been investigated yet. In this study, a set of leachable model compounds (LMCs) was chosen and the effect of the LMCs on a Chinese hamster ovary DG44 cell line producing an IgG antibody was investigated concerning cell growth, cell cycle distribution and productivity. It was shown that even if worst-case concentrations were used, the LMCs solely impact cell growth. Additionally, interaction studies revealed that the inhibiting effect of the mix is lower than the expected cumulative effect. A strong antagonism between the antioxidant butylated hydroxytoluene and the plasticizer Tris(2-ethylhexyl)trimellitate was found using an isobologram analysis. © 2021 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6 +/- 0.2 and 2.1 +/- 0.2 A mu g/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1 +/- 4.4 to 55.0 +/- 6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5 +/- 3.3% at 5 A degrees C followed by 54.5 +/- 1.6% at 18 A degrees C and 19.4 +/- 1.3% at 25 A degrees C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3 +/- 7.6 to 98.0 +/- 1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.

Encapsulation of hydrolases in biopolymer-based hydrogels often suffers from low activities and encapsulation efficiencies along with high leaching and unsatisfactory recycling properties. Exemplified for the encapsulation of pig liver esterase the coating of alginate and chitosan beads have been studied by creating various biopolymer hydrogel beads. Enzyme activity and encapsulation efficiency were notably enhanced by chitosan coating of alginate beads while leaching remained nearly unchanged. This was caused by the enzymatic reaction acidifying the matrix, which increased enzyme retention through enhanced electrostatic enzyme-alginate interaction but decreased activity through enzyme deactivation. A practical and ready-to-use method for visualising pH in beads during reaction by co-encapsulation of a conventional pH indicator was also found. Our method proves that pH control inside the beads can only be realised by buffering. The resulting beads provided a specific activity of 0.267 μmol ∙ min-1 ∙ mg-1, effectiveness factor 0.88, encapsulation efficiency of 88%, 5% leaching and good recycling properties. This work will contribute towards better understanding and application of encapsulated hydrolases for enzymatic syntheses.

BACKGROUND: Wireworms (Coleoptera: Elateridae) are major insect pests of worldwide relevance. Owing to the progressive phasing-out of chemical insecticides, there is great demand for innovative control options. This study reports on the development of an attract-and-kill co-formulation based on Ca-alginate beads, which release CO2 and contain neem extract as a bioinsecticidal compound. The objectives of this study were to discover: (1) whether neem extract can be immobilized efficiently, (2)whether CO2-releasing Saccharomyces cerevisiae and neem extract are suitable for co-encapsulation, and (3) whether co-encapsulated neem extract affects the attractiveness of CO2-releasing beads towards wireworms. RESULTS: Neem extractwas co-encapsulated togetherwith S. cerevisiae, starch andamyloglucosidase with a high encapsulation efficiency of 98.6% (based onmeasurement of azadirachtin A as themain active ingredient). Even at enhanced concentrations, neemextract allowed growth of S. cerevisiae, and beads containing neem extract exhibited CO2-emission comparable with beads without neem extract. When applied to the soil, the beads established a CO2 gradient of>15 cm. The co-formulation containing neem extract showed no repellent effects and was attractive for wireworms within the first 24 h after exposure. CONCLUSION:Co-encapsulationof S. cerevisiaeandneemextract is a promising approach for the development of attract-and-kill formulations for the control of wireworms. This study offers new options for the application of neem extracts in soil.

This study reports the development of encapsulated and dried entomopathogenic fungus Metarhiuzm brunneum with reduced conidia content, increased conidiation, a high drying survival and enhanced shelf life. Dried beads prepared with the fillers corn starch, potato starch, carboxymethylcellulose or autoclaved baker’s yeast, showed enhanced survival with increasing filler content. The maximum survival of 82% was found for beads with 20% corn starch at

Adaptation in sensory and neuronal systems usually leads to reduced responses to persistent or frequently presented stimuli. In contrast to simple fatigue, adapted neurons often retain their ability to encode changes in stimulus intensity and to respond when novel stimuli appear. We investigated how the level of adaptation of a fly visual motion-sensitive neuron affects its responses to discontinuities in the stimulus, i.e. sudden brief changes in one of the stimulus parameters (velocity, contrast, grating orientation and spatial frequency). Although the neuron's overall response decreased gradually during ongoing motion stimulation, the response transients elicited by stimulus discontinuities were preserved or even enhanced with adaptation. Moreover, the enhanced sensitivity to velocity changes by adaptation was not restricted to a certain velocity range, but was present regardless of whether the neuron was adapted to a baseline velocity below or above its steady-state velocity optimum. Our results suggest that motion adaptation helps motion-sensitive neurons to preserve their sensitivity to novel stimuli even in the presence of strong tonic stimulation, for example during self-motion.

Firing of an individual neuron is determined by the activity of its presynaptic input ensemble. In this study we analyzed how presynaptic signals with different dynamics interact to control postsynaptic activity. In the blowfly's visual system we simultaneously recorded in vivo from an identified motion-sensitive neuron and from elements of the presynaptic ensemble. The presynaptic cells themselves are mutually electrically coupled and convey both graded and spike signals to their common postsynaptic target. We elicited spikes in the postsynaptic neuron by voltage-clamping one of the presynaptic neurons to various holding potentials and then analyzed the time course of the holding current. Current transients in the clamped presynaptic cell were found to coincide with postsynaptic spikes. The current transients were highly variable in amplitude and occasionally absent during postsynaptic spiking. These characteristics indicate that the current transients in the voltage-clamped neuron result from spikes in electrically coupled co-members of the presynaptic ensemble. Our results suggest that electrical coupling among presynaptic neurons mediates synchronization of spikes within the cell ensemble. Moreover, our findings demonstrate that the graded response component of the presynaptic cells effectively controls the postsynaptic firing rate on a coarse scale while the precise timing of the postsynaptic spikes is a consequence of spikes superimposed on the graded signals of the presynaptic neurons.

BACKGROUND: Western corn rootworm larvae use CO2 to locate maize roots. However, the importance of CO2 as a specific orientation cue close to maize roots has not been investigated unequivocally. This study aimed at elucidating the effect of CO2-emitting capsules in combination with a soil insecticide (Tefluthrin=attract and kill) within the root system.We hypothesized that the capsules would result in aggregation of the larvae at the soil insecticide, thus increasing its efficacy. A nondestructive observation device was used to study larval distribution and behaviour. RESULTS: Spatial analysis of distance indices (SADIE) revealed aggregation of the larvae around the capsules in an attractand-kill treatment after 4 h, which was not found with the conventional treatment without the capsules. However, larval mortality did not differ between treatments. CONCLUSION: CO2 is aweak attractant for western corn rootworm larvae within the root system. Consequently, an attract-and-kill strategy based on a CO2 product will not contribute to better control compared with conventional Tefluthrin applications. Host-specific compounds, combined with a CO2 source, should be used to target more larvae, making attract and kill a feasible management option against this pest.

For monitoring of recombinant human antithrombin III during cell culture processes and subsequent purification steps a rapid method for quantitative determination was developed. The need for the introduction of this rapid method came from the limited availability of a quantitative enzyme-linked immunosorbent assay (ELISA) and the very time-consuming ELISA procedure. The developed method is based on reversed-phase high-performance liquid chromatography using a C 4 column. The separation by gradient elution using water and acetonitrile takes less than 20 min even when complex samples, such as serum containing cell culture samples, have to be analyzed. Automation and a high sample throughput are possible with this reliable method. If necessary, insulin, transferrin and albumin can also be quantified with minor changes of the elution profile.

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristic under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristic of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran(R) flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO2 conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTR(max)) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR(max) was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.

In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the development of complex small scale fermentation setups. The use of continuous fermentation and cell retention devices requires appropriate process control systems. An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented. The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation. The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture. Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of kLa- or OTR-values. Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation. Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions.

Even if a stimulus pattern moves at a constant velocity across the receptive field of motion-sensitive neurons, such as lobula plate tangential cells (LPTCs) of flies, the response amplitude modulates over time. The amplitude of these response modulations is related to local pattern properties of the moving retinal image. On the one hand, pattern-dependent response modulations have previously been interpreted as 'pattern-noise', because they deteriorate the neuron's ability to provide unambiguous velocity information. On the other hand, these modulations might also provide the system with valuable information about the textural properties of the environment. We analyzed the influence of the size and shape of receptive fields by simulations of four versions of LPTC models consisting of arrays of elementary motion detectors of the correlation type (EMDs). These models have previously been suggested to account for many aspects of LPTC response properties. Pattern-dependent response modulations decrease with an increasing number of EMDs included in the receptive field of the LPTC models, since spatial changes within the visual field are smoothed out by the summation of spatially displaced EMD responses. This effect depends on the shape of the receptive field, being the more pronounced - for a given total size - the more elongated the receptive field is along the direction of motion. Large elongated receptive fields improve the quality of velocity signals. However, if motion signals need to be localized the velocity coding is only poor but the signal provides – potentially useful – local pattern information. These modelling results suggest that motion vision by correlation type movement detectors is subject to uncertainty: you cannot obtain both an unambiguous and a localized velocity signal from the output of a single cell. Hence, the size and shape of receptive fields of motion sensitive neurons should be matched to their potential computational task.

Optic flow, i.e., retinal image movement resulting from ego-motion, is a crucial source of information used for obstacle avoidance and course control in flying insects. Optic flow analysis may prove promising for mobile robotics although it is currently not among the standard techniques. Insects have developed a computationally cheap analysis mechanism for image motion. Detailed computational models, the so-called elementary motion detectors (EMDs), describe motion detection in insects. However, the technical application of EMDs is complicated by the strong effect of local pattern contrast on their motion response. Here we present augmented versions of an EMD, the (s)cc-EMDs, which normalise their responses for contrast and there by reduce the sensitivity to contrast changes. Thus, velocity changes of moving natural images are reflected more reliably in the detect or response. The (s)cc-EMDs can easily be implemented in hardware and software and can be a valuable novel visual motion sensor for mobile robots.

It is still unclear how sensory systems efficiently encode signals with statistics as experienced by animals in the real world and what role adaptation plays during normal behavior. Therefore, we studied the performance of visual motion-sensitive neurons of blowflies, the horizontal system neurons, with optic flow that was reconstructed from the head trajectories of semi-free-flying flies. To test how motion adaptation is affected by optic flow dynamics, we manipulated the seminatural optic flow by targeted modifications of the flight trajectories and assessed to what extent neuronal responses to an object located close to the flight trajectory depend on adaptation dynamics. For all types of adapting optic flow object-induced response increments were stronger in the adapted compared with the nonadapted state. Adaptation with optic flow characterized by the typical alternation between translational and rotational segments produced this effect but also adaptation with optic flow that lacked these distinguishing features and even pure rotation at a constant angular velocity. The enhancement of object-induced response increments had a direction-selective component because preferred-direction rotation and natural optic flow were more efficient adaptors than null-direction rotation. These results indicate that natural dynamics of optic flow is not a basic requirement to adapt neurons in a specific, presumably functionally beneficial way. Our findings are discussed in the light of adaptation mechanisms proposed on the basis of experiments previously done with conventional experimenter-defined stimuli.

We present a small single camera imaging system that provides a continuous 280 degrees field of view (FOV) inspired by the large FOV of insect eyes. This is achieved by combining a curved reflective surface that is machined into acrylic glass with lenses covering the frontal field that otherwise would have been obstructed by the mirror. Based on the work of Seidl (1982 PhD Thesis Technische Hochschule Darmstadt), we describe an extension of the 'bee eye optics simulation' (BEOS) model by Giger (1996 PhD Thesis Australian National University) to the full FOV which enables us to remap camera images according to the spatial resolution of honeybee eyes. This model is also useful for simulating the visual input of a bee-like agent in a virtual environment. The imaging system in combination with our bee eye model can serve as a tool for assessing the visual world from a bee's perspective which is particularly helpful for experimental setups. It is also well suited for mobile robots, in particular on flying vehicles that need light-weight sensors.

Honeybees turn their thorax and thus their flight motor to change direction or to fly sideways. If the bee's head were fixed to its thorax, such movements would have great impact on vision. Head movements independent of thorax orientation can stabilize gaze and thus play an important and active role in shaping the structure of the visual input the animal receives. Here, we investigate how gaze and flight control interact in a homing task. We use high-speed video equipment to record the head and body movements of honeybees approaching and departing from a food source that was located between three landmarks in an indoor flight arena. During these flights, the bees' trajectories consist of straight flight segments combined with rapid turns. These short and fast yaw turns (‘saccades’) are in most cases accompanied by even faster head yaw turns that start about 8 ms earlier than the body saccades. Between saccades, gaze stabilization leads to a behavioural elimination of rotational components from the optical flow pattern, which facilitates depth perception from motion parallax.

SUMMARYHoverflies such as Eristalis tenax Linnaeus are known for their distinctive flight style. They can hover on the same spot for several seconds and then burst into movement in apparently any possible direction. In order to determine a quantitative and structured description of complex flight manoeuvres, we searched for a set of repeatedly occurring prototypical movements (PMs) and a set of rules for their ordering. PMs were identified by applying clustering algorithms to the translational and rotational velocities of the body of Eristalis during free-flight sequences. This approach led to nine stable and reliable PMs, and thus provided a tremendous reduction in the complexity of behavioural description. This set of PMs together with the probabilities of transition between them constitute a syntactical description of flight behaviour. The PMs themselves can be roughly segregated into fast rotational turns (saccades) and a variety of distinct translational movements (intersaccadic intervals). We interpret this segregation as reflecting an active sensing strategy which facilitates the extraction of spatial information from retinal image displacements. Detailed analysis of saccades shows that they are performed around all rotational axes individually and in all possible combinations. We found the probability of occurrence of a given saccade type to depend on parameters such as the angle between the long body axis and the direction of flight.

SUMMARYVisual landmarks guide humans and animals including insects to a goal location. Insects, with their miniature brains, have evolved a simple strategy to find their nests or profitable food sources; they approach a goal by finding a close match between the current view and a memorised retinotopic representation of the landmark constellation around the goal. Recent implementations of such a matching scheme use raw panoramic images (‘image matching’) and show that it is well suited to work on robots and even in natural environments. However, this matching scheme works only if relevant landmarks can be detected by their contrast and texture. Therefore, we tested how honeybees perform in localising a goal if the landmarks can hardly be distinguished from the background by such cues. We recorded the honeybees' flight behaviour with high-speed cameras and compared the search behaviour with computer simulations. We show that honeybees are able to use landmarks that have the same contrast and texture as the background and suggest that the bees use relative motion cues between the landmark and the background. These cues are generated on the eyes when the bee moves in a characteristic way in the vicinity of the landmarks. This extraordinary navigation performance can be explained by a matching scheme that includes snapshots based on optic flow amplitudes (‘optic flow matching’). This new matching scheme provides a robust strategy for navigation, as it depends primarily on the depth structure of the environment.

SUMMARYThe strength of stimulus-induced responses at the neuronal and the behavioural level often depends on the internal state of an animal. Within pathways processing sensory information and eventually controlling behavioural responses, such gain changes can originate at several sites. Using motion-sensitive lobula plate tangential cells (LPTCs) of blowflies, we address whether and in which way information processing changes for two different states of motor activity. We distinguish between the two states on the basis of haltere movements. Halteres are the evolutionarily transformed hindwings of flies. They oscillate when the animals walk or fly. LPTCs mediate, amongst other behaviours, head optomotor responses. These are either of large or small amplitude depending on the state of motor activity. Here we find that LPTC responses also depend on the motor activity of flies. In particular, LPTC responses are enhanced when halteres oscillate. Nevertheless, the response changes of LPTCs do not account for the corresponding large gain changes of head movements. Moreover, haltere activity itself does not change the activity of LPTCs. Instead, we propose that a central signal associated with motor activity changes the gain of head optomotor responses and the response properties of LPTCs.

We present a novel approach toward evolving artificial embryogenies, which omits the graph representation of gene regulatory networks and directly shapes the dynamics of a system, i.e., its phase space. We show the feasibility of the approach by evolving cellular differentiation, a basic feature of both biological and artificial development. We demonstrate how a spatial hierarchy formulation can be integrated into the framework and investigate the evolution of a hierarchical system. Finally, we show how the framework allows the investigation of allometry, a biological phenomenon, and its role for evolution. We find that direct evolution of allometric change, i.e., the evolutionary adaptation of the speed of system states on transient trajectories in phase space, is advantageous for a cellular differentiation task.

Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR) data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs) from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets) were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

SUMMARYBehavioural responses of an animal are variable even when the animal experiences the same sensory input several times. This variability can arise from stochastic processes inherent to the nervous system. Also, the internal state of an animal may influence a particular behavioural response. In the present study, we analyse the variability of visually induced head pitch responses of tethered blowflies by high-speed cinematography. We found these optomotor responses to be highly variable in amplitude. Most of the variability can be attributed to two different internal states of the flies with high and low optomotor gain, respectively. Even within a given activity state, there is some variability of head optomotor responses. The amount of this variability differs for the two optomotor gain states. Moreover, these two activity states can be distinguished on a fine timescale and without visual stimulation, on the basis of the occurrence of peculiar head jitter movements. Head jitter goes along with high gain optomotor responses and haltere oscillations. Halteres are evolutionary transformed hindwings that oscillate when blowflies walk or fly. Their main function is to serve as equilibrium organs by detecting Coriolis forces and to mediate gaze stabilisation. However, their basic oscillating activity was also suggested to provide a gain-modulating signal. Our experiments demonstrate that halteres are not necessary for high gain head pitch to occur. Nevertheless, we find the halteres to be responsible for one component of head jitter movements. This component may be the inevitable consequence of their function as equilibrium and gaze-stabilising organs.

Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.

Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-l pilot scale bioreactor. The cell cultivation has been done as a repeated batch procedure after the initial batch in the following four repeated batches spent medium from the previous batch was partially re-used. After microfiltration and ultrafiltration a part of the filtrate was mixed with a concentrate of amino acids and glucose, sterile filtered and subsequently filled back into the bioreactor. Up to 65% of the harvested cell- and product-free spent medium was re-used in each repeated batch. This procedure results in a saving of pure and waste water volume and saving of supplemented proteins as transferrin, insulin and lipoproteins and, therefore, also in a reduction of the production costs. A strongly acidic membrane ion exchanger was evaluated for the ability to purify the monoclonal antibodies from the pilot scale cultivation. Within minutes, gram quantities of product could be purified in a high flux system, especially developed for this purpose, achieving purities of 80%. The capacity of the acidic membrane ion exchanger was found in former investigations to be 1 mg cm -2 with recoveries up to 96%. Final purification was carried out by gel column filtration.

B lymphocytes from CBA/J mice were stimulated in splenocyte cultures for 72 h with various endotoxins. Bisphosphoryl lipid A from Escherichia coli had the highest stimulatory effect followed by LPS of Citrobacter freundii and Salmonella minnesota as measured by [H-3]thymidine uptake. Gangliosides of stimulated B cells (metabolically labeled with D-[1-C-14]galactose and D-[1-C-14]glucosamine) and unlabeled gangliosides from resting B cells (prepared from spleens without stimulus) were analyzed by high-performance TLC, DEAE anion-exchange HPLC, and immunostaining procedures. Contents of ganglioside-derived sialic acids, quantified by HPLC as their fluorescent derivatives, decreased from stimulated to resident B lymphocytes in the following order: LPS S. minnesota > LPS C.freundii > bisphosphoryl lipid A E. coli > resting B cells. Gangliosides of resting B cells contained more N-glycolyl- than N-acetylneuraminic acid, whereas inverse ratios were found in activated cells, indicating a shift from N-glycolyl- to N-acetylneuraminic acid due to stimulation. Furthermore, a higher disialoganglioside content was characteristic for activated B cells. Fast atom bombardment mass spectrometry was performed with permethylated mono- and disialoganglioside fractions of LPS S. minnesota and LPS C.freundii stimulated B cells. Major gangliosides were G(M1a) and G(D1a) beside minute amounts of G(D1b). The structural heterogeneity in the gangliosides was caused by (a) N-substitution of the sialic acids with either acetyl or glycolyl groups, (b) variation in the long-chain base (sphingosine, sphinganine), and (c) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C-16:0, C24:0,24:1). In summary, these findings indicate the predominance of the G(M1a) pathway in murine B lymphocytes whereas G(M1b)-type gangliosides are preferentially expressed in T lymphocytes as well as macrophages.

The task of purifying monoclonal antibodies (MAbs) and human recombinant antithrombin III (rATIII) from cell culture supernatant was carried out using two different approaches, both based on the use of membraneous matrices. The first approach employed a strongly acidic and a strongly basic membrane ion exchanger, which were evaluated for their ability to purify monoclonal antibodies and the human active recombinant antithrombin III from cell culture supernatant. Within minutes gram amounts of product could be purified in a high-flux system, specially developed for this purpose, achieving purities of 80% for MAbs and 75% for rATIII, respectively. The capacity of the acidic membrane ion exchanger for MAbs was found to be 1 mg mg/cm 2 with recoveries up to 96% and that of the basic membrane ion exchanger for rATIII was 0.15 mg/cm 2 with recoveries up to 91%. The second approach consisted of using heparin, a mucopolysaccharide with a strong affinity towards ATIII, coupled to amine-modified or epoxy-activated membranes by reductive amination, for the purification of rATIII. The ATIII binding capacities of the membranes were found to be 91 μg/cm 2 for the amine-modified and 39 μg/cm 2 for the epoxy-activated membrane, achieving purities of 75%. The coupling proved to be fairly stable over a period of 5 months and the membranes remained operable even after steam sterilization and treatment with sodium dodecyl sulphate. Final purification in both instances was carried out by gel filtration.

Quantitative analysis of animal behaviour is a requirement to understand the task solving strategies of animals and the underlying control mechanisms. The identification of repeatedly occurring behavioural components is thereby a key element of a structured quantitative description. However, the complexity of most behaviours makes the identification of such behavioural components a challenging problem. We propose an automatic and objective approach for determining and evaluating prototypical behavioural components. Behavioural prototypes are identified using clustering algorithms and finally evaluated with respect to their ability to represent the whole behavioural data set. The prototypes allow for a meaningful segmentation of behavioural sequences. We applied our clustering approach to identify prototypical movements of the head of blowflies during cruising flight. The results confirm the previously established saccadic gaze strategy by the set of prototypes being divided into either predominantly translational or rotational movements, respectively. The prototypes reveal additional details about the saccadic and intersaccadic flight sections that could not be unravelled so far. Successful application of the proposed approach to behavioural data shows its ability to automatically identify prototypical behavioural components within a large and noisy database and to evaluate these with respect to their quality and stability. Hence, this approach might be applied to a broad range of behavioural and neural data obtained from different animals and in different contexts.

For maintenance of high cell density in continuous perfusion processes not only feeding with substrates but also removal of inhibitors and toxic waste products are of special interest. High perfusion rates cause large volumes of product containing medium which have to be processed in product isolation. In order to minimize these volumes concentrated feed solutions of optimized medium are used. On the other hand, such media may cause high concentrations of toxic or inhibitory metabolites which can negatively influence cell growth and product formation. Especially, if the spent medium (or special parts of it) is used again after product isolation, the removal or even better the control of inhibitor production is of highest importance. We have developed a continuous fermentation concept and system (continuous medium cycle bioreactor, cMCB) in which both limitation and inhibition effects can be generated to identify special substances as limiting or inhibitory components. With the results from those experiments it was possible to lower the total perfusion rate during serum-free perfusion cultures of hybridoma cells and to obtain an optimal substrate utilization. The advantages for decreasing the production costs (for media, special supplements and product isolation) are obvious. The other aim of this study was to identify secreted metabolic waste products as inhibitor or toxic metabolite.