Advancing Knowledge on Situation Comprehension in Dynamic Traffic Situations by Studying Eye Movements to Empty Spatial Locations.

OBJECTIVE: This study used the looking-at-nothing phenomenon to explore situation awareness (SA) and the effects of working memory (WM) load in driving situations. BACKGROUND: While driving, people develop a mental representation of the environment. Since errors in retrieving information from this representation can have fatal consequences, it is essential for road safety to investigate this process. During retrieval, people tend to fixate spatial positions of visually encoded information, even if it is no longer available at that location. Previous research has shown that this "looking-at-nothing" behavior can be used to trace retrieval processes. METHOD: In a video-based laboratory experiment with 2 (WM) x 3 (SA level) within-subjects design, participants (N = 33) viewed a reduced screen and evaluated auditory statements relating to different SA levels on previously seen dynamic traffic scenarios while eye movements were recorded. RESULTS: When retrieving information, subjects more frequently fixated emptied spatial locations associated with the information relevant for the probed SA level. The retrieval of anticipations (SA level 3) in contrast to the other SA level information resulted in more frequent gaze transitions that corresponded to the spatial dynamics of future driving behavior. CONCLUSION: The results support the idea that people build a visual-spatial mental image of a driving situation. Different gaze patterns when retrieving level-specific information indicate divergent retrieval processes. APPLICATION: Potential applications include developing new methodologies to assess the mental representation and SA of drivers objectively.

What's Driving Me? Exploration and Validation of a Hierarchical Personality Model for Trust in Automated Driving.

OBJECTIVE: This paper presents a comprehensive investigation of personality traits related to trust in automated vehicles. A hierarchical personality model based on Mowen's (2000) 3M model is explored in a first and replicated in a second study. BACKGROUND: Trust in automation is established in a complex psychological process involving user-, system- and situation-related variables. In this process, personality traits have been viewed as an important source of variance. METHOD: Dispositional variables on three levels were included in an exploratory, hierarchical personality model (full model) of dynamic learned trust in automation, which was refined on the basis of structural equation modeling carried out in Study 1 (final model). Study 2 replicated the final model in an independent sample. RESULTS: In both studies, the personality model showed a good fit and explained a large proportion of variance in trust in automation. The combined evidence supports the role of extraversion, neuroticism, and self-esteem at the elemental level; affinity for technology and dispositional interpersonal trust at the situational level; and propensity to trust in automation and a priori acceptability of automated driving at the surface level in the prediction of trust in automation. CONCLUSION: Findings confirm that personality plays a substantial role in trust formation and provide evidence of the involvement of user dispositions not previously investigated in relation to trust in automation: self-esteem, dispositional interpersonal trust, and affinity for technology. APPLICATION: Implications for personalization of information campaigns, driver training, and user interfaces for trust calibration in automated driving are discussed.

Get Ready for Being Chauffeured : Passenger's Preferences and Trust While Being Driven by Human and Automation.

OBJECTIVE: We investigated passenger's trust and preferences using subjective, qualitative, and psychophysiological measures while being driven either by human or automation in a field study and a driving simulator experiment. BACKGROUND: The passenger's perspective has largely been neglected in autonomous driving research, although the change of roles from an active driver to a passive passenger is incontrovertible. Investigations of passenger's appraisals on self-driving vehicles often seem convoluted with active manual driving experiences instead of comparisons with being driven by humans. METHOD: We conducted an exploratory field study using an autonomous research vehicle (N = 11) and a follow-up experimental driving simulation (N = 24). Participants were driven on the same course by a human and an autonomous agent sitting on a passenger seat. Skin conductance, trust, and qualitative characteristics of the perceived driving situation were assessed. In addition, the effect of driving style (defensive vs. sporty) was evaluated in the simulator. RESULTS: Both investigations revealed a close relation between subjective trust ratings and skin conductance, with increased trust and by trend reduced arousal for human compared with automation in control. Even though driving behavior was equivalent in the simulator when being driven by human and automation, passengers most preferred and trusted the human-defensive driver. CONCLUSION: Individual preferences for driving style and human or autonomous vehicle control influence trust and subjective driving characterizations. APPLICATION: The findings are applicable in human-automation research, reminding to not neglect subjective attributions and psychophysiological reactions as a result of ascribed control duties in relation to specific execution characteristics.

The More You Know: Trust Dynamics and Calibration in Highly Automated Driving and the Effects of Take-Overs, System Malfunction, and System Transparency.

OBJECTIVE: This paper presents a theoretical model and two simulator studies on the psychological processes during early trust calibration in automated vehicles. BACKGROUND: The positive outcomes of automation can only reach their full potential if a calibrated level of trust is achieved. In this process, information on system capabilities and limitations plays a crucial role. METHOD: In two simulator experiments, trust was repeatedly measured during an automated drive. In Study 1, all participants in a two-group experiment experienced a system-initiated take-over, and the occurrence of a system malfunction was manipulated. In Study 2 in a 2 × 2 between-subject design, system transparency was manipulated as an additional factor. RESULTS: Trust was found to increase during the first interactions progressively. In Study 1, take-overs led to a temporary decrease in trust, as did malfunctions in both studies. Interestingly, trust was reestablished in the course of interaction for take-overs and malfunctions. In Study 2, the high transparency condition did not show a temporary decline in trust after a malfunction. CONCLUSION: Trust is calibrated along provided information prior to and during the initial drive with an automated vehicle. The experience of take-overs and malfunctions leads to a temporary decline in trust that was recovered in the course of error-free interaction. The temporary decrease can be prevented by providing transparent information prior to system interaction. APPLICATION: Transparency, also about potential limitations of the system, plays an important role in this process and should be considered in the design of tutorials and human-machine interaction (HMI) concepts of automated vehicles.

Strategic naps in automated driving - Sleep architecture predicts sleep inertia better than nap duration.

At higher levels of driving automation, drivers can nap during parts of the trip but must take over control in others. Awakening from a nap is marked by sleep inertia which is tackled by the NASA nap paradigm in aviation: Strategic on-flight naps are restricted to 40 min to avoid deep sleep and therefore sleep inertia. For future automated driving, there are currently no such strategies for addressing sleep inertia. Given the disparate requirements, it is uncertain whether the strategies derived from aviation can be readily applied to automated driving. Therefore, our study aimed to compare the effects of restricting the duration of nap opportunities following the NASA nap paradigm to the effects of sleep architecture on sleep inertia in takeover scenarios in automated driving. In our driving simulator study, 24 participants were invited to sleep during three automated drives. They were awakened after 20, 40, or 60 min and asked to manually complete an urban drive. We assessed how napping duration, last sleep stage before takeover, and varying proportions of light, stable, and deep sleep influenced self-reported sleepiness, takeover times, and the number of driving errors. Takeover times increased with nap duration, but sleepiness and driving errors did not. Instead, all measures were significantly influenced by sleep architecture. Sleepiness increased after awakening from light and stable sleep, and takeover times after awakening from light sleep. Takeover times also increased with higher proportions of stable sleep. The number of driving errors was significantly increased with the proportion of deep sleep and after awakenings from stable and deep sleep. These results suggest that sleep architecture, not nap duration, is crucial for predicting sleep inertia. Therefore, the NASA nap paradigm is not suitable for driving contexts. Future driver monitoring systems should assess the sleep architecture to predict and prevent sleep inertia.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose.

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

A comparative study of hydrolysis and transglycosylation activities of fungal ß-glucosidases.

ß-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10-15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.

Plant-based model for the visual evaluation of electroporated area after irreversible electroporation and its comparison to in-vivo animal data.

Electroporation (EP) is widely used in medicine, such as cancer treatment, in form of electrochemotherapy or irreversible electroporation (IRE). For EP device testing, living cells or tissue inside a living organism (including animals) are needed. Plant-based models seem to be a promising alternative to substitute animal models in research. The aim of this study is to find a suitable plant-based model for visual evaluation of IRE, and to compare the geometry of electroporated areas with in-vivo animal data.For this purpose, a variety of fruit and vegetables were selected and visually evaluated after 0/1/2/4/6/8/12/16/24 h post-EP. Apple and potato were found to be suitable models as they enabled a visual evaluation of the electroporated area. For these models, the size of the electroporated area was determined after 0/1/2/4/6/8/12/16/24 h. For apples, a well-defined electroporated area was visual within two hours, while in potatoes it reached a plateau after eight hours only. The electroporated area of apple, which showed the fastest visual results was then compared to a retrospectively evaluated swine liver IRE dataset which had been obtained for similar conditions. The electroporated area of the apple and swine liver both showed a spherical geometry of comparable size. For all experiments, the standard protocol for human liver IRE was followed. To conclude, potato and apple were found to be suitable plant-based models for the visual evaluation of electroporated area after irreversible EP, with apple being the best choice for fast visual results. Given the comparable range, the size of the electroporated area of the apple may be promising as a quantitative predictor in animal tissue. Even if plant-based models cannot completely replace animal experiments, they can be used in the early stages of EP device development and testing, decreasing animal experiments to the necessary minimum.

Recombinant production and characterisation of two related GH5 endo-ß-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity.

The glycoside hydrolase family 5 (GH5) endo-ß-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards ß-1,4 manno-oligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer ß-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA. Site-directed mutagenesis to ManA S289W lowered K(M) for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely, K(M) for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-ß-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity.

An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose.

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the ß-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.

In vivo MRI visualization of mesh shrinkage using surgical implants loaded with superparamagnetic iron oxides.

BACKGROUND: Prosthetic mesh implants are widely used in hernia surgery. To show long-term mesh-related complications such as shrinkage or adhesions, a precise visualization of meshes and their vicinity in vivo is important. By supplementing mesh fibers with ferro particles, magnetic resonance imaging (MRI) can help to delineate the mesh itself. This study aimed to demonstrate and quantify time-dependent mesh shrinkage in vivo by MRI. METHODS: Polyvinylidenfluoride (PVDF) meshes with incorporated superparamagnetic iron oxides (SPIOs) were implanted as an abdominal wall replacement in 30 rats. On days 1, 7, 14, or 21, MRI was performed using a gradient echo sequence with repetition time (TR)/echo time (TE) of 50/4.6 and a flip angle of 20°. The length, width, and area of the device were measured on axial, coronal, and sagittal images, and geometric deformations were assessed by surgical explantation. RESULTS: In all cases, the meshes were visualized and their area estimated by measuring the length and width of the mesh. The MRI presented a mean area shrinkage in vivo of 13% on day 7, 23% on day 14, and 23% on day 21. Postmortem measurements differed statistically from MRI, with a mean area shrinkage of 23% on day 7, 28% on day 14, and 30% on day 21. Ex vivo measurements of shrinkage showed in vivo measurements to be overestimated approximately 8%. Delineation of the mesh helped to show folding or adhesions close to the intestine. CONCLUSION: Loading of surgical meshes with SPIOs allows their precise visualization during MRI and guarantees an accurate in vivo assessment of their shrinkage. The authors' observation clearly indicates that shrinkage in vivo is remarkably less than that shown by illustrated explantation measurements. The use of MRI with such meshes could be a reliable technique for checking on proper operation of implanted meshes and showing related complications, obviating the need for exploratory open surgical revision.

Implicit intention communication as a design opportunity for automated vehicles: Understanding drivers' interpretation of vehicle trajectory at narrow passages.

To ensure road safety in mixed traffic, automated vehicles (AVs) must be equipped with distinct and easy-to-understand communication strategies. For this, the communication design of AVs might be oriented toward implicit communication between manually driven vehicles. This research focused on how drivers interpret observable vehicle behavior (vehicle trajectory) to predict vehicle intentions and planned maneuvers. In addition, the processing and interpretation of contradictory cues were examined as a possible cause for misunderstandings and failed coordination. Two video-based experiments investigated the presumed intention (yielding or insisting on priority), intention recognition time, distinctiveness, and cooperativeness of a vehicle's implicit communication at narrow road passages. For this, two vehicles approached a bottleneck from opposite sides, and only one vehicle could pass at a time. The driving behavior of the oncoming vehicle consisted of longitudinal (decelerating, stopping, accelerating, maintaining speed) and lateral movements (driving to the center, driving to the edge of the road, no lateral deviation) at different timings (early, late). The results indicated advantages of lateral vehicle movements for distinct intention communication. Lateral movements were interpreted the fastest, perceived more distinct, and were more decisive for the presumed intention than longitudinal movements. Early communication was preferred by drivers but increased the intention recognition time, especially for longitudinal movements. However, early lateral communication was interpreted as fast as late longitudinal communication. Furthermore, lateral and longitudinal vehicle movements within a driving pattern that contradicted each other led to ambiguous perceptions. For AVs, early distinct communication via lateral vehicle movement is recommended. Minimizing ambiguity in the interpretation of communication cues might maximize the probability of the safest response from human drivers and contribute to accident prevention in mixed traffic.

Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays.

In lignocellulosic raw materials for biomass conversion, hemicelluloses constitute a substantial fraction, with xylan being the primary part. Although many pretreatments reduce the amount or change the distribution of xylan, it is important to degrade residual xylan so as to improve the overall yield. Typically, xylanase reaction rates are measured in stopped assays by chemical quantification of the reducing ends. With isothermal titration calorimetry (ITC), the heat flow of the hydrolysis can be measured in continuous fashion, with the reaction rate being directly proportional to the heat flow. Reaction enthalpies for carbohydrate hydrolysis are typically below 5kJ/mol, which is the limiting factor for straight forward calorimetric quantification of enzymatic reaction rates using current ITC technology. To increase the apparent reaction enthalpy, we employed a subsequent oxidation of hydrolysis products by carbohydrate oxidase and catalase. Here we show that the coupled assay with carbohydrate oxidase and catalase can be used to measure enzyme kinetics of a GH10 xylanase from Aspergillus aculeatus on birch xylan and wheat arabinoxylan. Results are discussed in the light of a critical analysis of the sensitivity of four chemical-reducing-end quantification methods using well-characterized substrates.

Pedestrian assessment: Is displaying automated driving mode in self-driving vehicles as relevant as emitting an engine sound in electric vehicles?

Pedestrians rely on vehicle dynamics, engine sound, and driver cues. The lack of engine sound now constitutes an addressed pedestrian safety issue for (hybrid) electric vehicles ((H)EVs). Analogously, lacking driver cues may constitute a pedestrian safety issue for self-driving vehicles (SDVs). The purpose of this study was to systematically compare the relevance of substituting driver cues with an external human-machine interface among SDVs (no eHMI vs. eHMI) with the relevance of substituting engine sound with artificial sound among (H)EVs (no engine sound vs. engine sound). In a within-subject design, twenty-nine participants acting as pedestrians encountered a simulated SDV in a parking lot. The results revealed that both informational cues have equally large effects on subjective measures such as perceived safety. In semi-structured interviews, participants stated that it is equally crucial to equip SDVs with an eHMI as equipping (H)EVs with an artificial sound generator. We conclude that an eHMI for SDVs seems to be as relevant as an artificial sound for (H)EVs.

More Than a Feeling-Interrelation of Trust Layers in Human-Robot Interaction and the Role of User Dispositions and State Anxiety.

With service robots becoming more ubiquitous in social life, interaction design needs to adapt to novice users and the associated uncertainty in the first encounter with this technology in new emerging environments. Trust in robots is an essential psychological prerequisite to achieve safe and convenient cooperation between users and robots. This research focuses on psychological processes in which user dispositions and states affect trust in robots, which in turn is expected to impact the behavior and reactions in the interaction with robotic systems. In a laboratory experiment, the influence of propensity to trust in automation and negative attitudes toward robots on state anxiety, trust, and comfort distance toward a robot were explored. Participants were approached by a humanoid domestic robot two times and indicated their comfort distance and trust. The results favor the differentiation and interdependence of dispositional, initial, and dynamic learned trust layers. A mediation from the propensity to trust to initial learned trust by state anxiety provides an insight into the psychological processes through which personality traits might affect interindividual outcomes in human-robot interaction (HRI). The findings underline the meaningfulness of user characteristics as predictors for the initial approach to robots and the importance of considering users' individual learning history regarding technology and robots in particular.

Sleep inertia in automated driving: Post-sleep take-over and driving performance.

Sleep is emerging as a new driver state in automated driving. Post-sleep performance impairments due to sleep inertia, the transitional phase from sleep to wakefulness that can take up to 30 min, are a potential safety issue. Take-over performance immediately after sleep is impaired and drivers perceive the take-over as critical. The aim of the presented study was to assess take-over behavior immediately after sleep and driving behavior during the 10 min after sleep. A study with N = 31 drivers was conducted in a high-fidelity driving simulator. Take-over performance and driving performance were assessed a) under alert baseline conditions and b) after awakening from electroencephalography-confirmed stable sleep. Take-over performance 15 s after awakening was impaired resulting in more driving errors compared to the alert baseline. Lane keeping was dramatically impaired in the first 3 min after sleep and recovered rapidly. Drivers drove slower after sleep and speed keeping was less stable for at least 10 min. The results suggest that human-machine interaction design should account for the drivers' impaired post-sleep driving performance.

Navigating with Augmented Reality - How does it affect drivers' mental load?

Drivers have been proven to easily understand Augmented Reality (AR) information. Especially in an ambiguous navigation task, drivers are expected to benefit from AR information. The driving simulator study was aimed at examining differences in mental load while navigating in an urban area with ambiguous intersection situations (N = 59). The navigation information was presented to the driver through a head-up display (HUD): a conventional HUD or an AR display, which relates information to the surroundings. Additionally, the driver had to solve a non-driving-related task (NDRT) which was an auditory cognitive, spatial task. Results showed that while driving with the AR display, participants performed better in the NDRT, which indicates a reduced mental load compared with the HUD. Participants drove on average 3 km/h slower with the HUD, showing compensation behaviour.

Cardiac overexpression of the human 5-HT4 receptor in mice.

Serotonin (5-HT) exerts pleiotropic effects in the human cardiovascular system. Some of the effects are thought to be mediated via 5-HT(4) receptors, which are expressed in the human atrium and in ventricular tissue. However, a true animal model to study these receptors in more detail has been hitherto lacking. Therefore, we generated, for the first time, a transgenic (TG) mouse with cardiac myocyte-specific expression of the human 5-HT(4) receptor. RT-PCR and immunohistochemistry revealed expression of the receptor at the mRNA and protein levels. Stimulation of isolated cardiac preparations by isoproterenol increased phospholamban phosphorylation at Ser(16) and Thr(17) sites. 5-HT increased phosphorylation only in TG mice but not in wild-type (WT) mice. Furthermore, 5-HT increased contractility in isolated perfused hearts from TG mice but not WT mice. These effects of 5-HT could be blocked by the 5-HT(4) receptor-selective antagonist GR-125487. An intravenous infusion of 5-HT increased left ventricular contractility in TG mice but not in WT mice. Similarly, the increase in contractility by 5-HT in isolated cardiomyocytes from TG mice was accompanied by and probably mediated through an increase in L-type Ca(2+) channel current and in Ca(2+) transients. In intact animals, echocardiography revealed an inotropic and chronotropic effect of subcutaneously injected 5-HT in TG mice but not in WT mice. In isolated hearts from TG mice, spontaneous polymorphic atrial arrhythmias were noted. These findings demonstrate the functional expression of 5-HT(4) receptors in the heart of TG mice, and a potential proarrhythmic effect in the atrium. Therefore, 5-HT(4) receptor-expressing mice might be a useful model to mimic the human heart, where 5-HT(4) receptors are present and functional in the atrium and ventricle of the healthy and failing heart, and to investigate the influence of 5-HT in the development of cardiac arrhythmias and heart failure.

A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases.

The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and fluorogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of -2.5 kJ mol(-1), an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.