Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.     

Electrostatic switching of biopolymer layers. Insights from combined electrokinetics and reflectometric interference

Structural integrity and functional characteristics of biomacromolecules are largely defined by electrostatic forces between ionized moieties, which are often altered at interfaces. Unraveling these changes requires access to charge state and structure of surface-confined biopolymers in aqueous environments. We therefore combined electrokinetic measurements of interfacial electrical potentials with the simultaneous determination of the optical layer thickness by reflectometric interference spectroscopy. Two examples are summarized to demonstrate the resulting options: The pH-switching of grafted poly(l-glutamic acid) layers caused by dissociation-dependent helix-coil transitions was studied; potential distribution and ion mobility within the grafted polyelectrolyte were unraveled using an new theoretical model for the charging of polyelectrolyte layers. The charge-driven modulation of biopolymers at interfaces was furthermore analyzed in the adsorption of fibronectin onto polymer substrates with varied charge density; the results permit us to reach a conclusion about the relevance of electrostatic matching for orientation and anchorage of the protein. Altogether, the introduced methodology was found suitable to follow the electrosurface characteristics of biomacromolecules in situ.     

Melt-mixing by novel pitched-tip kneading disks in a co-rotating twin-screw extruder

Melt-mixing in twin-screw extruders is a key process in the development of polymer composites. Quantifying the mixing performance of kneading elements based on their internal physical processes is a challenging problem. We discuss melt-mixing by novel kneading elements called “pitched-tip kneading disk (ptKD)”. The disk-stagger angle and tip angle are the main geometric parameters of the ptKDs. We investigated four typical arrangements of the ptKDs, which are forward and backward disk-staggers combined with forward and backward tips. Numerical simulations under a certain feed rate and screw revolution speed were performed, and the mixing process was investigated using Lagrangian statistics. It was found that the four types had different mixing characteristics, and their mixing processes were explained by the coupling effect of drag flow with the disk staggering and pitched-tip and pressure flows, which are controlled by operational conditions. The use of a pitched-tip effectively controls the balance of the pressurization and mixing ability.     

Flexible media — recent developments from the tribology point of view

With the increase in the recording density of hard disk systems, the demand on the increase in the data recording capacity of tape drive systems as back-up systems is increasing. One of the advantages of the tape drive systems is high volumetric recording density, which is obtained by high areal recording density and thin tapes. The areal recording density can be increased by introducing high performance tapes, such as metal evaporated tapes, with superior magnetic characteristics and smooth magnetic surface to reduce the spacing loss. However, a smoother surface often produces a higher friction coefficient, which could cause tape damage by rotary heads and unstable tape runnability in the tape drives. Adoption of MR heads to tape drive systems is also effective in increasing the areal recording density. However, the wear allowance of the MR heads (shield type) is much smaller than that of the inductive heads. Thinner tapes show lower mechanical stiffness in general, which could cause damage to tape edges easily during tape transportation. In the second or later generation of tape drive systems, a thinner tape is often introduced. These thinner tapes should also have the interchangeability of the original thickness tape. New materials for a base film, such as PEN (polyethylene naphthalate) or aramid in which the elastic moduli are larger than those of PET, are required for thinner tapes. It was found that these side effects by the increase in the volumetric recording density can be improved by tribology. The tribological improvements from the drive design side is very important, as well as from the tape design side.     

Osteocytes: Their Lacunocanalicular Structure and Mechanoresponses

Osteocytes connect with neighboring osteocytes and osteoblasts through their processes and form an osteocyte network. Shear stress on osteocytes, which is induced by fluid flow in the lacunae and canaliculi, has been proposed as an important mechanism for mechanoresponses. The lacunocanalicular structure is differentially developed in the compression and tension sides of femoral cortical bone and the compression side is more organized and has denser and thinner canaliculi. Mice with an impaired lacunocanalicular structure may be useful for evaluation of the relationship between lacunocanalicular structure and mechanoresponses, although their bone component cells are not normal. We show three examples of mice with an impaired lacunocanalicular structure. Ablation of osteocytes by diphtheria toxin caused massive osteocyte apoptosis, necrosis or secondary necrosis that occurred after apoptosis. Osteoblast-specific Bcl2 transgenic mice were found to have a reduced number of osteocyte processes and canaliculi, which caused massive osteocyte apoptosis and a completely interrupted lacunocanalicular network. Osteoblast-specific Sp7 transgenic mice were also revealed to have a reduced number of osteocyte processes and canaliculi, as well as an impaired, but functionally connected, lacunocanalicular network. Here, we show the phenotypes of these mice in physiological and unloaded conditions and deduce the relationship between lacunocanalicular structure and mechanoresponses.     

Reliability of ultrasonographic measurements of bovine sole structures in relation to sole horn thickness,measured by computed tomography,and sole horn hardness

The goal of the present study was to determine the effect of sole horn thickness (SHT) and sole horn hardness (SHD) on ultrasonographic visualization of sole structures in the inner and outer claws of 150 Holstein-Friesian cows, and to evaluate different ultrasound frequencies for this purpose. Ultrasonographic views of the sole structure were considered complete when 3 echogenic lines, representing the ventral surface of the sole horn, the borders of the sole horn and soft-tissue layer, and the ventral surface of the distal phalanx, were seen. The proportion of complete ultrasonographic views of the sole structures, designated as the ultrasonographic visualization proportion (UVP), and the measurement errors of SHT were evaluated by comparing images from computed tomography (CT) and ultrasonography. The latter images were generated using 3 different probes, frequencies of 6.5 and 5.0 MHz, and 2 different ultrasound machines (#1 and #2) to assess the apex, middle, and heel regions of the claws. The UVP were 60.8 to 77.9% for the 6.5-MHz probe in ultrasound machine #1 (probe A), which were lower than those (>90%) for both the 5.0-MHz probe in ultrasound machine #1 (probe B) and the 5.0-MHz probe in ultrasound machine #2 (probe C). The UVP was significantly lower in claws with an SHD ≥50 units than in claws with an SHD <40 or 40 to <50 units (UVP: 77.1% compared with 93.7 and 91.4%, respectively) when measured with probe B. In claws with an SHT <10 mm, the UVP was significantly lower when SHD was ≥50 units compared with <40 or 40 to >50 units; the values were 69.0% versus 91.3 and 85.9%, respectively, for probe A, and 89.7% versus 100 and 100%, respectively, for probe B. When SHT were measured by either probes A or B in ultrasound machine #1, the proportions of claws in which ultrasonographic values were within a ±1 mm range compared with the values obtained by CT were 84.9 to 91.3% for CT-determined SHT <5 mm, 66.7 to 71.9% for CT-determined SHT 5 to <7 mm, 28.9 to 51.2% for CT-determined SHT 7 to <10 mm, and 6.2 to 19.7% for CT-determined SHT ≥10 mm. The data indicated that increased SHT was associated with a decrease in ultrasonographic measurement accuracy. In claws with an SHT <5 mm, the high proportion of ultrasonographic values that were accurate within a ±1 mm range suggests that this imaging modality would be useful in cows with thin soles.     

Cost-Reliability Optimal Release Policies for Software Systems

This paper extends an optimal software release problem to both cost and reliability requirements. The optimum software release time is determined both by minimizing a total average software cost and satisfying a software reliability requirement. The underlying model is software reliability growth described by a nonhomogeneous Poisson process.     

A note on optimum checkpointing policies

In this paper, we treat checkpointing policies. We derive the total expected loss time and obtain the optimum checkpointing policy which minimizes that expected loss time. We further present the numerical examples using the exponential and the Weibull distributions. First, we discuss the model in which the intervals between checkpointing completion times may vary, and secondly the model with constant intervals.     

Quality assessment models for initial production process control based on stochastic differential equations

It is of great importance to assess an initial production process prior to the regular mass-production. For this purpose, many statistical methods have been proposed for practical use. In this paper, we propose two stochastic models for an assessment method of the initial production process control: a Markov process model and a Markov approximation model. These models are continuous state space models and formulated by applying mathematical techniques of stochastic differential equations. Based on each model, we derive several quantitative assessment measures for initial production process control.