Direct electron transfer of heme- and molybdopterin cofactor-containing chicken liver sulfite oxidase on alkanethiol-modified gold electrodes

Direct heterogeneous electron transfer (ET) of sulfite oxidase (SOx), a heme- and molybdopterin cofactor-containing intermembrane enzyme, was studied on alkanethiol-modified Au electrodes both with SOx entrapped between the modified Au electrode and a permselective membrane and with SOx adsorbed at the electrode surface, in the absence of any membrane. SOx in direct electronic communication with the electrode surface gave a quasi-reversible electrochemical signal with a midpoint potential of--120 mV vs Ag/AgCl corresponding to the redox transformations of the heme domain of SOx and with a heterogeneous ET constant in the order of 15 s(-1). The efficiency of the bioelectrocatalytic 2e- oxidation of sulfite catalyzed by SOx in direct ET exchange with the electrode was shown to depend essentially on the nature of the alkanethiol layer. Adsorption and orientation of SOx on an 11-mercapto-1-undecanol (MuD-OH) self-assembled monolayer, i.e., terminally functionalized with OH groups, provided efficient catalytic oxidation of sulfite, contrary to nonfunctionalized alkanethiols, e.g., 1-decanethiol, or alkanethiol layers terminally functionalized with NH2 groups. Comparative studies with short-chain alkanethiols, e.g., cysteamine and 2-mercaptoethanol, revealed an evidently different mode of adsorption of SOx on these layers, onto which SOx was not catalytically active. Coadsorption of MuD-OH and 11-mercapto-1-undecanamine improved the surface properties of the SAM, resulting in a higher surface coverage with bioelectrocatalytically active SOx but not in an increased apparent catalytic rate constant, kcat, ranging in the order of 18-24 s(-1) at pH 7.4. The achieved efficiency of SOx bioelectrocatalysis in direct ET reaction between the modified electrode and the enzyme approached the rates characteristic for the catalysis mediated by cytochrome c, the natural redox partner of SOx, thus implying the retention of the biological function of SOx under the heterogeneous electrode reaction conditions. Results obtained enable the development of a third-generation biosensor for sulfite monitoring.     

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.     

A simple and sensitive method for lactose detection based on direct electron transfer between immobilised cellobiose dehydrogenase and screen-printed carbon electrodes

A rapid and simple approach of lactose analysis is proposed based on 3rd generation amperometric biosensors employing cellobiose dehydrogenase (CDH) from Trametes villosa or Phanerochaete sordida immobilised on screen-printed carbon electrodes (SPCEs). After optimisation of the working conditions of the biosensors - pH of the carrier buffer, flow rate and applied potential - the sensors were able to detect lactose in a concentration range between 0.5-200 μM and 0.5-100 μM employing T. villosa and P. sordida CDH, respectively. The limit of detection is 250 nM (90 μg/L) for both. Biosensors based on SPCEs modified with multiwalled carbon nanotubes showed a higher sensitivity than unmodified SPCEs. Cross-linking with glutaraldehyde or poly(ethyleneglycol)diglycidyl ether improved not only the stability but also the analytical response. The developed sensor has been successfully applied for the determination of lactose in dairy (milk with different percentages of fat, lactose-free milk and yogurt) with a good reproducibility (RSD = 1.5-2.2%). No sample preparation except a simple dilution process is needed. The biosensor is easy to make and operate, is inexpensive and reveals a high sensitivity and reliability.     

Design and implementation of a high‐performance CCA event service

Event services based on publish–subscribe architectures are well‐established components of distributed computing applications. Recently, an event service has been proposed as part of the common component architecture (CCA) for high‐performance computing (HPC) applications. In this paper we describe our implementation, experimental evaluation, and initial experience with a high‐performance CCA event service that exploits efficient communications mechanisms commonly used on HPC platforms. We describe the CCA event service model and briefly discuss the possible implementation strategies of the model. We then present the design and implementation of the event service using the aggregate remote memory copy interface as an underlying communication layer for this mechanism. Two alternative implementations are presented and evaluated on a Cray XD‐1 platform. The performance results demonstrate that event delivery latencies are low and that the event service is able to achieve high‐throughput levels. Finally, we describe the use of the event service in an application for high‐speed processing of data from a mass spectrometer and conclude by discussing some possible extensions to the event service for other HPC applications. Published in 2009 by John Wiley & Sons, Ltd.     

Parallel program design using high-level Petri nets

Petri nets are proposed as a general-purpose design and modelling tool for parallel programs. The advantages of Petri nets for this purpose are discussed, and a solution to the Dining Philosophers problem is developed using simple Place-Transition nets. The limitations of Place-Transition nets are described, and the Dining Philosophers problem is used to illustrate how Coloured Petri nets can overcome these limitations. A more complex example of a Coloured Petri net is then given, and it is shown how a collection of processes in the Occam programming language can be developed directly from the properties of the net. Another Petri net model of a simple process farm is given, and a solution is developed in Parallel C: this further highlights the suitability of Petri nets as a design tool for parallel programs.     

A Flow Injection System for the Determination of Starch in Starch from Different Origins with Immobilized α-Amylase and Amyloglucosidase Reactors

A heat stable α-amylase (Termamyl) was immobilized on controlled pore glass (CPG) with a pore size of 1 489Å and amyloglucosidase (AMG) was immobilized on a ceramic silica support (Micropil A) with a pore size of 300Å. These enzyme supports were packed into two separate immobilized enzyme reactors (IMERs) which together with a third reactor containing co-immobilized glucose dehydrogenase/mutarotase were incorporated into a flow injection (FI) system for the determination of the total glucose content of starch related poly- and oligosaccharides. Samples of maltose, a maltooligosaccharide mixture, three soluble starches, three amylopectins, two amyloses, glycogen, and native starches from different origins were injected. The degree of hydrolysis was determined by comparing the produced amount of glucose in the FI system with the calculated amount of glucose in the samples and with samples to which had been added and let to react for six hours soluble α-amylase and AMG before injected into the FI system also containing the IMERs. Virtually complete conversion to glucose was obtained for maltose, maltooligosaccharides, two soluble starches and native potato starch. Maximum enzymatic degradation by the starch hydrolyzing enzyme reactors was obtained in most instances except for glycogen (96%), native wheat (88%), rice (93%), and corn starch (83%).     

Determination of the Degree of Branching in Normal and Amylopectin Type Potato Starch with 1H-NMR Spectroscopy Improved resolution and two-dimensional spectroscopy

Starch from genetically modified potatoes was found to be highly branched compared with normal potato varieties through the use of ¹H-NRM spectroscopy. The average chain length, blue-value, and the wavelength at maximum absorptivity clearly show that the new potato varieties produce amylopectin starch. Correlation between the degree of branching as determined by ¹H-NMR and starch-iodine complexation, expressed as blue-value, was good and the NMR-method gives low standard deviation. For the first time, the anomeric proton, H-1, of a (1→4)-α-linked D-glucose residue and the H-1 of the glucose residue of a non-reducing end have been assigned separate chemical shifts in starch. Assignments were made as determined from two-dimensional homonuclear and ¹H-¹³C heteronuclear spectroscopy (COSY, HMQC, and HMBC). The molecular weight in degraded starch and pullulan were determined by means of NMR-spectroscopy. These results were in accordance with determinations by size exclusion chromatography and with the known molecular weights of pullulan standards.     

Evaluating the performance of EJB components

As part of the Middleware Technology Evaluation (MTE) project, we conducted several experiments to explore the performance implications of two common application architectures supported by J2EE's enterprise JavaBean (EJB) component technology. One architecture promises simpler engineering and maintenance of the resulting component collection. For applications that require high performance and scalability, however, the alternative architecture might offer a better solution. Such knowledge is crucial to software architects, who must make initial design decisions early in a project, before extensive engineering has begun. An examination of two EJB-based architectures reveals differences that can significantly affect the performance and scalability of applications built on them.     

Software Architecture Review: The State of Practice

Architecture reviews are an effective way of ensuring design quality and addressing architectural concerns. However, the software engineering community rarely adopts the methods and techniques available to support disciplined architecture review processes.     

Hydrolysis of Maltoheptaose in Flow through Silicon Wafer Microreactors Containing Immobilised α‐Amylase and Glycoamylase

In this study a silicon micro immobilised enzyme reactor (μIMER) has been applied for hydrolysis of maltoheptaose as a model maltodextrin and starch using immobilised μ‐amylase (from Aspergillus oryzae) and glycoamylase (from Aspergillus niger). The influence of several parameters was investigated such as immobilisation chemistry, buffer constituents, pH, temperature, flow rate and substrate concentration. The conversion efficiency profile of the substrate was measured and the long‐term stability of the reactor was tested. For separation and detection of the formed hydrolysis products, high‐performance anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) was used. The results show that the μIMERs can also be used for hydrolysis of starch and also additionally be connected directly on‐line with, e.g., liquid chromatography, making it possible to perform on‐line characterisation and analysis of starch hydrolysis products.