Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.     

Detection of Leuconostoc strains at a meat processing plant using polymerase chain reaction

To simplify the labor-intensive conventional routine testing of samples to detect Leuconostoc at a meat processing plant, we developed polymerase chain reaction (PCR) primers specific for Leuconostoc from 16S rRNA gene sequences. These primers did not detect other common lactic acid bacteria such as Lactobacillus plantarum, Lact. sake, Lact. fermentum, Lact. acidophilus and Weissella viridescens. PCR with this primer detected all Leuconostoc species tested (Leu. mesenteroides subsp. mesenteroides, Leu. pseudomesenteroides, Leu. carnosum, Leu. lactic, Leu. citreum, Leu. amelibiosum, Leu. gelidum), except for Leu. fallax, and no other lactic acid bacteria on agarose gel electrophoresis. The method could identify areas contaminated with Leuconostoc in a large-scale industrial meat processing plant. Of 69 samples analyzed, 34 were positive for Leuconostoc according to the conventional culture method (isolation of LAB producing dextran) and PCR, whereas 29 were negative according to both. Six samples were culture-negative but positive by PCR. No false negative results were generated by PCR. The method is rapid and simple, is useful for routinely monitoring areas contaminated with Leuconostoc in meat processing plants, and could help to prevent the spoilage of meat products.     

Tracking topic evolution via salient keyword matching with consideration of semantic broadness for Web video discovery

A method to track topic evolution via salient keyword matching with consideration of semantic broadness for Web video discovery is presented in this paper. The proposed method enables users to understand the evolution of topics over time for discovering Web videos in which they are interested. A framework that enables extraction and tracking of the hierarchical structure, which contains Web video groups with various degrees of semantic broadness, is newly derived as follows: Based on network analysis using multimodal features, i.e., features of video contents and metadata, our method extracts the hierarchical structure and salient keywords that represent contents of each Web video group. Moreover, salient keyword matching, which is newly developed by considering salient keyword distribution, semantic broadness of each Web video group and initial topic relevance, is applied to each hierarchical structure obtained in different time stamps. Unlike methods in previous works, by considering the semantic broadness as well as the salient keyword distribution, our method can overcome the problem of the desired semantic broadness of topics being different depending on each user. Also, the initial topic relevance enables correction of the gap from an initial topic at the start of tracking. Consequently, it becomes feasible to track the evolution of topics over time for finding Web videos in which the users are interested. Experimental results for real-world datasets containing YouTube videos verify the effectiveness of the proposed method.     

Ultrastructure of Paneth cells in the intestine of various mammals

Paneth cells in the following species were observed under an electron microscope: human, rhesus monkey, hare, guinea pig, rat, nude rat, mouse, golden hamster, and insect feeder bat. Secretory granules containing homogeneous electron-dense materials were observed in the Paneth cells of humans, monkeys, hares, guinea pigs, and bats; mouse Paneth-cell granules were bipartite (central core and peripheral halo), and the Paneth cells in rats and golden hamsters had secretory granules showing various electron densities. In humans, monkeys, and bats, immature granules near the Golgi apparatus sometimes showed bipartite substructure. The number and size of secretory granules were also diverse among various animal species. Some lysosome-like bodies were commonly observed in peri- or supranuclear regions, though the size and shape of the bodies differed from cell to cell. In apical cytoplasm, small clear vesicles (100–200 nm diameter) were more-or-less observed in all species examined, and it was especially note that rat Paneth cells contained many clear vesicles. Small dense-cored vesicles (150–200 nm diameter) were rare. It is unlikely that the various ultrastructural features of Paneth cells correlate with the phylogenetical classification.     

Nanostructure of pure iron anodically oxidized in borate buffer solution and annealed by infrared radiation

The behavior of oxide film on pure iron passivated in a borate buffer solution and subsequently radiated by infrared light (IR) was investigated in comparing to that by just IR annealing without passivation, and was evaluated by film structure, etc. The effect of thermal annealing over 250 degrees C was observed with gamma-Fe2O3 grain growth and sharp increase in surface roughness, film thickness and oxygen content. An ellipsometric parameter of tan psi was sensitively reflected by annealing effect, and tan psi curve had a shoulder at 150 degrees C for 5 min and a peak of tan psi was shifted from 350 nm to 450 nm in wavelength. This shift was also caused by the formation of gamma-Fe2O3, because the peak was also observed in tan psi of the bulk Fe2O3 family. Passivation effects at 800 mV prior to IR annealing on thickness and oxygen content changed at 150 degrees C, and decreased tan psi at 350 nm and excessive film growth over 250 degrees C, and increased oxygen content under 100 degrees C and surface roughness at 50-250 degrees C. The terrace width with atomic scale flatness was slightly increase by passivation prior to IR annealing at 50-250 degrees C, and the maximum terrace width reached larger than 10 nm by passivation and IR annealing at 100 degrees C for 30 min.     

Spawning networks

The deployment of new network architectures, services, and protocols is often manual, ad hoc, and time-consuming. We introduce “spawning networks,” a new class of programmable networks that automate the life cycle process for the creation, deployment, and management of network architectures. These networks are capable of spawning distinct “child” virtual networks with their own transport, “parent's” network resources and in isolation from other spawned networks. Spawned child networks represent programmable virtual networks and support the controlled access to communities at users with specific connectivity, security, and quality of service requirements. In this article we present a framework for the realization of spawning networks based on the notion of the Genesis Kernel, a virtual network operating system capable of creating distinct virtual network architectures on the fly. We discuss the motivation and principles that underpin spawning networks and focus on the design of the transport, programming and life cycle environments, which comprise the main architectural components of the Genesis Kernel     

Enzymatic and genetic bases on assimilation, depolymerization, and transport of heteropolysaccharides in bacteria

When microorganisms utilize macromolecules for their growth, they commonly produce extracellular depolymerization enzymes and then incorporate the depolymerized low-molecular-weight products. Assimilation of heteropolysaccharides (gellan and xanthan) by Bacillus sp. GL1 depends on this generally accepted mechanism. On the other hand, Sphingomonas sp. A1 represents an unexplored specific and interesting system for macromolecule assimilation. In the presence of heteropolysaccharide (alginate), the bacterium forms a mouthlike pit on its cell surface and directly incorporates the macromolecule using a novel ATP-binding cassette transporter (ABC transporter). In this review, we discuss enzymatic and genetic bases on the depolymerization and assimilation routes of heteropolysaccharides in bacteria, with particular emphasis on the novel incorporation system for macromolecules, characteristic post-translational modification processes of polysaccharide lyases and on the mouthlike pit structure on the bacterial cell surface.     

Activity measurement of the constituents in molten Fe–B and Fe–B–C alloys

The distribution ratios of Fe and B between molten Fe–B alloy and molten Ag were measured at temperatures between 1573 and 1923 K. Also, distribution ratios of Fe and B between molten Fe–B–C_satd. alloys and molten Ag were measured at 1873 K. It was found that the excess Gibbs free energy of mixing in molten Fe–B and Fe–B–C alloys can be expressed by utilizing the Redlich–Kister polynomial. The activity curves of the elements in molten Fe–B alloy and Fe–B–C alloy were estimated.     

Deposition and structural analyses of molybdenum-disulfide (MoS2)–amorphous hydrogenated carbon (a-C:H) composite coatings

In this study we developed composite coatings consisting of amorphous hydrogenated carbon (a-C:H) and molybdenum-disulfide (MoS₂), and clarified their microstructure. In addition, we interpreted the tribological properties of the composite coatings in the viewpoint of a deposition-induced microstructural modification. The coatings were produced by the hybrid deposition technique of RF-generated methane and argon plasma and DC magnetron co-sputtering of MoS₂ target. The deposition parameter investigated in this study was methane flow rate. Structural analyses were performed using a transmission electron microscope (TEM) and an atomic force microscope (AFM). Friction tests were conducted using a ball-on-disk type tribometer. From an electron micrograph, it was confirmed that nano-clusters were embedded into an amorphous carbon host matrix. Surface roughness of the composite coating was ～ 0.25 nm in R_a compared to 5.0 nm in R_a of sputtered MoS₂. The concentration measurements were performed, and the results show that the sulfur and molybdenum concentration ratio, [S]/[Mo], is ～ 0.9, which indicates that the amount of sulfur was reduced due to the discharged plasma. In friction tests, composite coatings showed high friction in a vacuum condition. It was considered that lubricant MoS₂ lamellar structures showing super-low friction in a vacuum condition during friction could not be formed between ball and coating during friction because of the lack of sulfur in embedded clusters.