Emotional face recognition in male adolescents with autism spectrum disorder or disruptive behavior disorder: an eye-tracking study.

Autism Spectrum Disorder (ASD), Oppositional Defiant Disorder (ODD), and Conduct Disorder (CD) are often associated with emotion recognition difficulties. This is the first eye-tracking study to examine emotional face recognition (i.e., gazing behavior) in a direct comparison of male adolescents with Autism Spectrum Disorder or Oppositional Defiant Disorder/Conduct Disorder, and typically developing (TD) individuals. We also investigate the role of psychopathic traits, callous-unemotional (CU) traits, and subtypes of aggressive behavior in emotional face recognition. A total of 122 male adolescents (N = 50 ASD, N = 44 ODD/CD, and N = 28 TD) aged 12-19 years (M = 15.4 years, SD= 1.9) were included in the current study for the eye-tracking experiment. Participants were presented with neutral and emotional faces using a Tobii 1750 eye-tracking monitor to record gaze behavior. Our main dependent eye-tracking variables were: (1) fixation duration to the eyes of a face and (2) time to the first fixation to the eyes. Since distributions of eye-tracking variables were not completely Gaussian, non-parametric tests were chosen to investigate gaze behavior across the diagnostic groups with Autism Spectrum Disorder, Oppositional Defiant Disorder/Conduct Disorder, and Typically Developing individuals. Furthermore, we used Spearman correlations to investigate the links with psychopathy, callous, and unemotional traits and subtypes of aggression as assessed by questionnaires. The relative total fixation duration to the eyes was decreased in both the Autism Spectrum Disorder group and the Oppositional Defiant Disorder/Conduct Disorder group for several emotional expressions. In both the Autism Spectrum Disorder and the Oppositional Defiant Disorder/Conduct Disorder group, increased time to first fixation on the eyes of fearful faces only was nominally significant. The time to first fixation on the eyes was nominally correlated with psychopathic traits and proactive aggression. The current findings do not support strong claims for differential cross-disorder eye-gazing deficits and for a role of shared underlying psychopathic traits, callous-unemotional traits, and aggression subtypes. Our data provide valuable and novel insights into gaze timing distributions when looking at the eyes of a fearful face.

Parallel updating and weighting of multiple spatial maps for visual stability during whole body motion.

It is known that the brain uses multiple reference frames to code spatial information, including eye-centered and body-centered frames. When we move our body in space, these internal representations are no longer in register with external space, unless they are actively updated. Whether the brain updates multiple spatial representations in parallel, or whether it restricts its updating mechanisms to a single reference frame from which other representations are constructed, remains an open question. We developed an optimal integration model to simulate the updating of visual space across body motion in multiple or single reference frames. To test this model, we designed an experiment in which participants had to remember the location of a briefly presented target while being translated sideways. The behavioral responses were in agreement with a model that uses a combination of eye- and body-centered representations, weighted according to the reliability in which the target location is stored and updated in each reference frame. Our findings suggest that the brain simultaneously updates multiple spatial representations across body motion. Because both representations are kept in sync, they can be optimally combined to provide a more precise estimate of visual locations in space than based on single-frame updating mechanisms.

Gaze is driven by an internal goal trajectory in a visuomotor task.

When we make hand movements to visual targets, gaze usually leads hand position by a series of saccades to task-relevant locations. Recent research suggests that the slow smooth pursuit eye movement system may interact with the saccadic system in complex tasks, suggesting that the smooth pursuit system can receive non-retinal input. We hypothesise that a combination of saccades and smooth pursuit guides the hand movements towards a goal in a complex environment, using an internal representation of future trajectories as input to the visuomotor system. This would imply that smooth pursuit leads hand position, which is remarkable, as the general idea is that smooth pursuit is driven by retinal slip. To test this hypothesis, we designed a video-game task in which human subjects used their thumbs to move two cursors to a common goal position while avoiding stationary obstacles. We found that gaze led the cursors by a series of saccades interleaved with ocular fixation or pursuit. Smooth pursuit was correlated with neither cursor position nor cursor velocity. We conclude that a combination of fast and slow eye movements, driven by an internal goal instead of a retinal goal, led the cursor movements, and that both saccades and pursuit are driven by an internal representation of future trajectories of the hand. The lead distance of gaze relative to the hand may reflect a compromise between exploring future hand (cursor) paths and verifying that the cursors move along the desired paths.

A practical approach for exploration and modeling of the design space of a bacterial vaccine cultivation process.

A licensed pharmaceutical process is required to be executed within the validated ranges throughout the lifetime of product manufacturing. Changes to the process, especially for processes involving biological products, usually require the manufacturer to demonstrate that the safety and efficacy of the product remains unchanged by new or additional clinical testing. Recent changes in the regulations for pharmaceutical processing allow broader ranges of process settings to be submitted for regulatory approval, the so-called process design space, which means that a manufacturer can optimize his process within the submitted ranges after the product has entered the market, which allows flexible processes. In this article, the applicability of this concept of the process design space is investigated for the cultivation process step for a vaccine against whooping cough disease. An experimental design (DoE) is applied to investigate the ranges of critical process parameters that still result in a product that meets specifications. The on-line process data, including near infrared spectroscopy, are used to build a descriptive model of the processes used in the experimental design. Finally, the data of all processes are integrated in a multivariate batch monitoring model that represents the investigated process design space. This article demonstrates how the general principles of PAT and process design space can be applied for an undefined biological product such as a whole cell vaccine. The approach chosen for model development described here, allows on line monitoring and control of cultivation batches in order to assure in real time that a process is running within the process design space.

Insect cells for human food.

There is a need for novel protein sources. Insects are a possible interesting source of protein. They are nutritious in terms of protein (40-75 g/100g dry weight) and minerals. Insect protein is of high quality and has a high digestibility (77-98%) and concentration of essential amino acids (46-96% of the nutritional profile). Also insect cells may be a promising novel source of protein. Choice of cell line, growth conditions and use of the baculovirus expression system opens up possibilities to engineer the nutritional value of the biomass. The technological limits as well as consumer acceptance of insect cell based food remains to be investigated.

Biocatalysts: measurement, modelling and design of heterogeneity.

Multiple phenomena are involved in conversions by immobilized biocatalysts. A paradox is identified between analytical desires on one hand and analytical boundary conditions on the other: while the study of interdependent phenomena would call for their simultaneous analysis in an integrated context, the available experimental options may impose a series of separate and dedicated analyses. From this analysis, bottlenecks in particle performance may be identified, if possible supported by a mechanistic model and performance criteria. Subsequently, a strategy for further biocatalyst development may be chosen. Finally, possibilities for future improvement of biocatalysts are discussed for various fields of research. Some examples of recent developments in enzyme and matrix characteristics, reactor operation, and micro-technology are discussed.

Web-based education in bioprocess engineering.

The combination of web technology, knowledge of bioprocess engineering, and theories on learning and instruction might yield innovative learning material for bioprocess engineering. In this article, an overview of the characteristics of web-based learning material is given, as well as guidelines for the design of learning material from theories of learning and instruction and from the bioprocess engineering domain. A diverse body of learning material is presented, which illustrates the application of these guidelines; this material has been developed during the past six years for different courses, mostly at undergraduate level, and it illustrates how web-based learning material can enable various different approaches to learning objectives that might improve overall learning. Such learning material has been used for several years in education, it has been evaluated with positive results, and is now part of the regular learning material for bioprocess engineering at Wageningen University.

A multicomponent reaction-diffusion model of a heterogeneously distributed immobilized enzyme.

A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.

Field-emission scanning electron microscopy analysis of morphology and enzyme distribution within an industrial biocatalytic particle.

Field-emission scanning electron microscopy (FESEM) was used in a technical feasibility study to obtain insight into the internal morphology and the intraparticle enzyme distribution of Assemblase, an industrial biocatalytic particle containing immobilized penicillin-G acylase. The results were compared with previous studies based on light and transmission electron microscopic techniques. The integrated FESEM approach yielded the same quantitative results as the microscopic techniques used previously. Given this technical equivalence, the integrated approach offers several advantages. First, the single preparation method and detection system avoids interpretation discrepancies between corresponding areas that were examined for different properties with different detection techniques in different samples. Second, the specimen size suitable for whole particle study is virtually unlimited, which simplifies sectioning and puts less stringent demands on the embedding technique. Furthermore, the sensitivity toward enzyme presence and distribution increases because the epitopes inside thick sections become available for labeling. Quick and unambiguous analysis of the relation between particle morphology and enzyme distribution is important because this information may be used in the future for the design of enzyme distributions in which the particle morphology can be used as a control parameter.

Enzyme distribution and matrix characteristics in biocatalytic particles.

In a study of Assemblase, an industrial immobilized penicillin-G acylase, various electron microscopic techniques were used to relate intra-particle enzyme heterogeneity with the morphological heterogeneity of the support material at various levels of detail. Transmission electron microscopy was used for the study of intra-particle penicillin-G acylase distribution in Assemblase particles of various sizes; it revealed an abrupt increase in enzyme loading at the particle surface (1.4-fold) and in the areas (designated halo's) surrounding internal macro-voids (7.7-fold). Cryogenic field-emission scanning electron microscopy related these abrupt local enzyme heterogeneities to local heterogeneity of the support material by revealing the presence of dense top layers surrounding both the particle exterior and the internal macro-voids. Furthermore, it showed a very distinct morphological appearance of the halo. Most probably, all these regions contained relatively more chitosan than gelatin (the polymers Assemblase was constructed of), which suggested local polymer demixing during particle production. A basic thermodynamic line of reasoning suggested that a difference in hydrophilicity between the two polymers induced local demixing. In the future, thermodynamic knowledge on such polymer interactions resulting in matrix heterogeneity may be used as a tool for biocatalyst design.

Novel approach to quantify immobilized-enzyme distributions.

The quantitative intraparticle enzyme distribution of Assemblase, an industrially employed polydisperse immobilized penicillin-G acylase, was measured. Because of strong autofluorescence of the carrier, the generally applied technique of confocal scanning microscopy could not be used; light microscopy was our method of choice. To do so, Assemblase particles of various sizes were sectioned, labeled with antibodies specifically against the enzyme, and analyzed light microscopically. Image analysis software was developed and used to determine the intraparticle enzyme distribution, which was found to be heterogeneous, with most enzyme located in the outer regions of the particles. Larger particles showed steeper gradients than smaller ones. A mathematical representation of the intraparticle profiles, based on in-stationary enzyme diffusion into the particles, was validated successfully for a broad range of particle sizes using data for volume-averaged particle size and enzyme loading. The enzyme gradients determined in this work will be used as input for a physical model that quantitatively describes the complex behavior of Assemblase. Such a physical model will lead to identification of the current bottlenecks in Assemblase and can serve as a starting point for the design of improved biocatalysts that also may be based on intelligent use of enzyme gradients.

The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage.

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.

Limitations of membrane cultures as a model solid-state fermentation system.

AIMS: To examine the reliability of membrane cultures as a model solid-state fermentation (SSF) system. METHODS AND RESULTS: In overcultures of Aspergillus oryzae on sterilized wheat flour discs overlaid with a polycarbonate membrane, we demonstrated that the presence of membrane filters reduced the maximum respiration rate (up to 50%), and biomass and alpha-amylase production. We also show that the advantage of membrane cultures, i.e. total recovery of biomass, is not very evident for the system used, while the changes in metabolism and kinetics are serious drawbacks. CONCLUSIONS: The use of membrane cultures is artificial and without substantial benefits and therefore has to be carefully considered. SIGNIFICANCE AND IMPACT OF THE STUDY: In future studies on kinetics and stoichiometry of SSF, one should not completely rely on experiments using membrane cultures as a model SSF system.

Effect of oxygen tension on adult articular chondrocytes in microcarrier bioreactor culture.

Tissue-engineering approaches for cartilage repair hold promise for the treatment of cartilage defects. Various methods to prevent or reduce dedifferentiation during chondrocyte expansion are currently under investigation. In the present study we evaluated the effect of oxygen on chondrocyte proliferation, as oxygen has received increased attention as a possible regulator of chondrocyte differentiation and its effect during expansion is uncertain. Therefore, the effect of three oxygen tensions (4, 10.5, and 21%) was investigated in a bioreactor microcarrier culture, which allows precise control of the oxygen tension in the liquid phase. During culture cells acquired a round shape on microcarriers. No differences in proliferation rate of chondrocytes were observed within the range of oxygen tensions evaluated. Cells exhibited predominantly anaerobic metabolism and, per mole of glucose, approximately 2 mol of lactate was produced independent of oxygen tension. Cellular oxygen consumption was comparable for all bioreactor cultures. Nevertheless, specific consumption rates were relatively high (2-4 x 10(-17) mol. cell(-1). s(-1)), in comparison with chondrocytes in cartilage (0.8-2.2 x 10(-18) mol. cell(-1)). Subsequent cartilaginous tissue formation in pellets was not affected as qualitatively assessed by safranin-O staining. At the oxygen concentrations evaluated, no effect of oxygen tension was observed on proliferation, oxygen consumption, and yield of lactate on glucose administration. For future investigations of chondrocytes and oxygen, the bioreactor system, which allows precise control and monitoring of oxygen tension, holds promise.

The effect of PEGT/PBT scaffold architecture on oxygen gradients in tissue engineered cartilaginous constructs.

Repair of articular cartilage defects using tissue engineered constructs composed of a scaffold and cultured autologous cells holds promise for future treatments. However, nutrient limitation (e.g. oxygen) has been suggested as a cause of the onset of chondrogenesis solely within the peripheral boundaries of larger constructs. In the present study, oxygen gradients were evaluated by microelectrode measurements in two porous polyethylene glycol terephthalate/polybutylene terephthalate (PEGT/PBT) scaffold architectures, a compression-molded and particle-leached sponge (CM) and a 3D-deposited fiber (3DF) scaffold. During the first 14 days in vitro, gradients intensified, after which a gradual decrease of the gradients was observed in vitro. In vivo, however, gradients changed instantly and became less pronounced. Although similar gradients were observed regardless of scaffold type, significantly more cells were present in the center of 3DF constructs after 2 weeks of in vivo culture. Our results stress the importance of a rationally designed scaffold for tissue-engineering applications. Organized structures, such as the 3DF PEGT/PBT polymer scaffolds, offer possibilities for regulation of nutrient supply and, therefore, hold promise for clinical approaches for cartilage repair.

Membrane-aerated biofilm reactor for the removal of 1,2-dichloroethane by Pseudomonas sp. strain DCA1.

A membrane-aerated biofilm reactor (MBR) with a biofilm of Pseudomonas sp. strain DCA1 was studied for the removal of 1,2-dichloroethane (DCA) from water. A hydrophobic membrane was used to create a barrier between the liquid and the gas phase. Inoculation of the MBR with cells of strain DCA1 grown in a continuous culture resulted in the formation of a stable and active DCA-degrading biofilm on the membrane. The maximum removal rate of the MBR was reached at a DCA concentration of approximately 80 micro M. Simulation of the DCA fluxes into the biofilm showed that the MBR performance at lower concentrations was limited by the DCA diffusion rate rather than by kinetic constraints of strain DCA1. Aerobic biodegradation of DCA present in anoxic water could be achieved by supplying oxygen solely from the gas phase to the biofilm grown on the liquid side of the membrane. As a result, direct aeration of the water, which leads to undesired coagulation of iron oxides, could be avoided.

Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes.

OBJECTIVE: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. DESIGN: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation. Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase. Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau). In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically. RESULTS: In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold. Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I. After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO. After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO. Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture. After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions. The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions. CONCLUSION: These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures.

Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling.

The supply of oxygen within three-dimensional tissue-engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2-5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 x 10(-9)) + (0.4 x 10(-9) m(2) s(-1)), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 x 10(-10) m(2) s(-1)). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 x 10(-19) mol m(-3) s(-1)), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis.

Monte Carlo simulation of the alpha-amylolysis of amylopectin potato starch. 2. alpha-amylolysis of amylopectin.

A model is presented that describes all the saccharides that are produced during the hydrolysis of starch by an alpha-amylase. Potato amylopectin, the substrate of the hydrolysis reaction, was modeled in a computer matrix. The four different subsite maps presented in literature for alpha-amylase originating from Bacillus amyloliquefaciens were used to describe the hydrolysis reaction in a Monte Carlo simulation. The saccharide composition predicted by the model was evaluated with experimental values. Overall, the model predictions were acceptable, but no single subsite map gave the best predictions for all saccharides produced. The influence of an alpha(1-->6) linkage on the rate of hydrolysis of nearby alpha(1-->4) linkages by the alpha-amylase was evaluated using various inhibition constants. For all the subsites considered the use of inhibition constants led to an improvement in the predictions (a decrease of residual sum of squares), indicating the validity of inhibition constants as such. As without inhibition constants, no single subsite map gave the best fit for all saccharides. The possibility of generating a hypothetical subsite map by fitting was therefore investigated. With a genetic algorithm it was possible to construct hypothetical subsite maps (with inhibition constants) that gave further improvements in the average prediction for all saccharides. The advantage of this type of modeling over a regular fit is the additional information about all the saccharides produced during hydrolysis, including the ones that are difficult to measure experimentally.

Expansion of bovine chondrocytes on microcarriers enhances redifferentiation.

Functional cartilage implants for orthopedic surgery or in vitro tissue evaluation can be created from expanded chondrocytes and biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems results in their dedifferentiation. The hallmark of this process is the switch of collagen synthesis from type II to type I. The aim of this study was to evaluate the postexpansion chondrogenic potential of microcarrier-expanded bovine articular chondrocytes in pellet cultures. A selection of microcarriers was screened for initial attachment of chondrocytes. On the basis of those results and additional selection criteria related to clinical application, Cytodex-1 microcarriers were selected for further investigation. Comparable doubling times were obtained in T-flask and microcarrier cultures. During propagation on Cytodex-1 microcarriers, cells acquired a spherical-like morphology and the presence of collagen type II was detected. Both observations are indicative of a differentiated chondrocyte. Pellet cultures of microcarrier-expanded cells showed cartilage-like morphology and staining for proteoglycans and collagen type II after 14 days. In contrast, pellets of T-flask-expanded cells had a fibrous appearance and showed abundant staining only for collagen type I. Therefore, culture of chondrocytes on microcarriers may offer useful and cost-effective cell expansion opportunities in the field of cartilage tissue engineering.