High-resolution real-time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices

We have used improved miniaturized adenosine biosensors to measure adenosine release during hypoxia from within the CA1 region of rat hippocampal slices. These microelectrode biosensors record from the extracellular space in the vicinity of active synapses as they detect the synaptic field potentials evoked in area CA1 by stimulation of the afferent Schaffer collateral-commissural fibre pathway. Our new measurements demonstrate the rapid production of adenosine during hypoxia that precedes and accompanies depression of excitatory transmission within area CA1. Simultaneous measurement of adenosine release and synaptic transmission gives an estimated IC50 for adenosine on transmission in the low micromolar range. However, on reoxygenation, synaptic transmission recovers in the face of elevated extracellular adenosine and despite a post-hypoxic surge of adenosine release. This may indicate the occurrence of apparent adenosine A1 receptor desensitization during metabolic stress. In addition, adenosine release is unaffected by pharmacological blockade of glutamate receptors and shows depletion on repeated exposure to hypoxia. Our results thus suggest that adenosine release is not a consequence of excitotoxic glutamate release. The potential for adenosine A1 receptor desensitization during metabolic stress implies that its prevention may be beneficial in extending adenosine-mediated neuroprotection in a variety of clinically relevant conditions.     

Dynamic contact stress patterns on the tibial plateaus during simulated gait: A novel application of normalized cross correlation

The spatial distribution and pattern of local contact stresses within the knee joint during activities of daily living have not been fully investigated. The objective of this study was to determine if common contact stress patterns exist on the tibial plateaus of human knees during simulated gait. To test this hypothesis, we developed a novel normalized cross-correlation (NCC) algorithm and applied it to the contact stresses on the tibial plateaus of 12 human cadaveric knees subjected to multi-directional loads mimicking gait. The contact stress profiles at different locations on the tibial plateaus were compared, where regions with similar contact stress patterns were identified across specimens. Three consistent regional patterns were found, among them two most prominent contact stress patterns were shared by 9–12 of all the knees and the third pattern was shared by 6–8 knees. The first pattern was located at the posterior aspect of the medial tibial plateau and had a single peak stress that occurred during the early stance phase. The second pattern was located at the central-posterior aspects of the lateral plateau and consisted of two peak stresses coincident with the timing of peak axial force at early and late stance. The third pattern was found on the anterior aspect of cartilage-to-cartilage contact region on the medial plateau consisted of double peak stresses. The differences in the location and profile of the contact stress patterns suggest that the medial and lateral menisci function to carry load at different points in the gait cycle: with the posterior aspect of the medial meniscus consistently distributing load only during the early phase of stance, and the posterior aspect of the lateral meniscus consistently distributing load during both the early and late phases of stance. This novel approach can help identify abnormalities in knee contact mechanics and provide a better understanding of the mechanical pathways leading to post-traumatic osteoarthritis.     

Glucose oxidase immobilized electrode for potentiometric estimation of glucose

Glucose oxidase has been immobilized onto a thin platinum strip, by co-crosslinking with bovine serum albumin and glutaraldehyde. The retention of redox characteristics of glucose oxidase has been verified by cyclic voltammetry. The activity of the immobilized enzyme reduces to a quarter of its value when the enzyme is in solution but improves when coimmobilized with 1 urea. The potentiometric response builds up and remains stable after 100 s. It is sensitive to the thickness of the immobilizing matrix, pH and temperature. An improvement in the performance of the electrode has been achieved by coimmobilizing 2 urea and metal ions such as Mg²⁺ and Mn²⁺. The presence of Cu has been proved to be detrimental. The electrode has been calibrated in the 0.1–5.0 mM glucose concentration range. It gives a stable response for more than 50 independent assays and can be stored for 60 days without significant loss of function.     

Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane

Many plant‐ and animal‐pathogenic Gram‐negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel‐like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two‐step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two‐domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin‐rich middle lamella by the bacterial pilus. One‐domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin‐type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly.     

Techniques for Universal Evaluation of Astringency of Green Tea Infusion by the Use of a Taste Sensor System

A practical method for universal evaluation of the astringency of green tea infusion by a taste sensor system was established. The use of EGCg aqueous solution as a standard enabled analysis with high accuracy and reproducibility. The sensor output was converted into taste-intensity on the basis of Weber’s and Weber-Fechner laws, which was named the “EIT _ast ” value (“EIT” and “ast” are abbreviations for “Estimated Intensity of Taste” and “astringency” respectively). It was clarified that green tea infusion is to be classified into eight grades on the EIT _ast scale. Furthermore, the high correlation of the EIT _ast value with the human gustatory sense and the high stability of the taste sensor were proved.     

A novel method for assessing the 3-D orientation accuracy of inertial/magnetic sensors

A novel method for assessing the accuracy of inertial/magnetic sensors is presented. The method, referred to as the “residual matrix” method, is advantageous because it decouples the sensor's error with respect to Earth's gravity vector (attitude residual error: pitch and roll) from the sensor's error with respect to magnetic north (heading residual error), while remaining insensitive to singularity problems when the second Euler rotation is close to ±90°. As a demonstration, the accuracy of an inertial/magnetic sensor mounted to a participant's forearm was evaluated during a reaching task in a laboratory. Sensor orientation was measured internally (by the inertial/magnetic sensor) and externally using an optoelectronic measurement system with a marker cluster rigidly attached to the sensor's enclosure. Roll, pitch and heading residuals were calculated using the proposed novel method, as well as using a common orientation assessment method where the residuals are defined as the difference between the Euler angles measured by the inertial sensor and those measured by the optoelectronic system. Using the proposed residual matrix method, the roll and pitch residuals remained less than 1° and, as expected, no statistically significant difference between these two measures of attitude accuracy was found; the heading residuals were significantly larger than the attitude residuals but remained below 2°. Using the direct Euler angle comparison method, the residuals were in general larger due to singularity issues, and the expected significant difference between inertial/magnetic sensor attitude and heading accuracy was not present.     

Dynamics of Nitric Oxide and Peroxynitrite During Global Brain Ischemia/Reperfusion in Rat Hippocampus: NO-sensor Measurement and Modeling Study

The dynamics of nitric oxide (NO) and peroxynitrite concentration changes during brain ischemia/reperfusion are poorly understood. In this paper, a NO-selective sensor was used to measure NO concentration changes in the rat brain hippocampus during global brain ischemia/reperfusion. Four-vessel occlusion model of transient global brain ischemia was used. Global cerebral ischemia was induced by occluding both common carotid arteries with artery nips (for 20 min) and reperfusion was induced by loosening the artery nips. Results showed that the changes of NO concentration during global brain ischemia/reperfusion could be divided into different stages. Together with the effects of O₂ tension changes and NO synthase (NOS) on nitric oxide levels, we determined five stages in the NO concentration profile: (1) acute O₂-limited decrease stage; (2) O₂-limited steady stage; (3) neuronal NOS activation stage; (4) acute O₂-recovery elevation stage; and (5) O₂-recovery steady stage. In addition, a chemical reaction network model was constructed to simulate the dynamics of peroxynitrite during the reperfusion stage, and the effects of a change in the NO formation rate on the dynamics of peroxynitrite were investigated specifically. Results show the rate of NO formation has a great influence on peroxynitrite dynamics.     

A compact multi-channel apparatus for automated real-time monitoring of bioluminescence

We have developed a multi-channel apparatus for automated monitoring of bioluminescence in real time. We designed this apparatus to be compact (230 mm wide, 600 mm deep, and 227.5 mm high) so that it can be operated in a relatively small commercially-available incubator. The apparatus can process 20 samples at maximum in a single run, providing enough processibility in small-scale experiments. We verified the reliability and sensitivity of the apparatus by observing circadian bioluminescence rhythms over one week from a bioluminescent reporter strain (E9) of the cyanobacterium Synechococcus sp. strain PCC 7942 [Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johnson, C.H., Kondo, T., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria, Science, 281 (1998) 1519-1523]. Our apparatus allows flexible experimental designs and will be effectively used for the studies of gene expression in various purposes.     

Verification of accuracy and validity of gait phase detection system using motion sensors for applying walking assistive FES

In this study, we have analysed heel strike (HS) and toe off (TO) of normal individuals and hemiplegic patients, taking advantage of output curves acquired from various sensors, and verified the validity of sensor detection methods and their effectiveness when they were used for hemiplegic gaits. Gait phase detections using three different motion sensors were valid, since they all had reliabilities more than 95%, when compared with foot velocity algorithm. Results showed that the tilt sensor and the gyrosensor could detect gait phase more accurately in normal individuals. Vertical acceleration could detect HS most accurately in hemiplegic patient group A. The gyrosensor could detect HS and TO most accurately in hemiplegic patient groups A and B. The detection of TO from all sensor signals was valid in both the patient groups A and B. However, the vertical acceleration detected HS validly in patient group A and the gyrosensor detected HS validly in patient group B.