Temperature-responsive culture surfaces for insect cell sheets to fabricate a bioactuator

ABSTRACT

For the first time, we have fabricated insect-derived cell sheets by using temperature-responsive culture surfaces having a phase-transition temperature below 25°C. We prepared the temperature-responsive cell culture surfaces (tissue culture polystyrene, TCPS) by grafting a copolymeric gel consisting of hydrophobic N-tert-butylacrylamide (tBAAm) and N-isopropylacrylamide (IPAAm) units. First, to characterize the hydrophilic and hydrophobic properties of the copolymeric gel-grafted surfaces, static water contact angles of each surface were measured at various temperatures. By increasing the amount of tBAAm in the grafted copolymeric gel, the transition temperature of the gel was shifted to lower temperatures. At 25°C, the grafted copolymeric gel was dehydrated, and the insect-derived cells (AeAl2 cells) adhered on all the copolymeric gel-grafted surfaces. At 20°C, AeAl2 cells cannot adhere on the P(IPAAm-1.62tBAAm)-TCPS surface (the initial molar ratio of IPAAm and tBAAm (tBAAm?=?1.62 mol%)) better than on other surfaces (TCPS and tBAAm?=?4.88, 8.17 mol%). These two findings implied that the lower critical solution temperature of the copolymeric gel-grafted-TCPS existed from 20°C to 25°C. The laminin-coated P(IPAAm-1.62tBAAm)-TCPS surface showed temperature-dependent cell attachment and detachment properties, while AeAl2 cells were not detached from the extracellular matrix uncoated P(IPAAm-1.62tBAAm)-TCPS surface. AeAl2 cells and insect muscle cells were harvested as the respective sheets.     

Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0–7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.     

Knowledge discovery in databases for determining formulation in topology optimization

Whereas topology optimization has achieved immense success, it involves an intrinsic difficulty. That is, optimized structures obtained by topology optimization strongly depend on the settings of the objective and constraint functions, i.e., the formulation. Nevertheless, the appropriate formulation is not usually obvious when considering structural design problems. Although trial-and-error to determine appropriate formulations are implicitly performed in several studies on topology optimization, it is important to explicitly support the process of trial-and-error. Therefore, in this study, we propose a new framework for topology optimization to determine appropriate formulations. The basic idea of this framework is incorporating knowledge discovery in databases (KDD) and topology optimization. Thus, we construct a database by collecting various and numerous material distributions that are obtained by solving various structural design problems with topology optimization, and find useful knowledge with respect to appropriate formulations from the database on the basis of KDD. An issue must be resolved when realizing the above idea, namely the material distribution in the design domain of a data record must be converted to conform to the design domain of the target design problem wherein an appropriate formulation should be determined. For this purpose, we also propose a material distribution-converting method termed as design domain mapping (DDM). Several numerical examples are used to demonstrate that the proposed framework including DDM successfully and explicitly supports the process of trial-and-error to determine the appropriate formulation.

     

Exfoliation of non-swelling muscovite on dodecylammonium chloride intercalation between layers using wet-jet milling

The exfoliation of layered muscovite with non-swelling property has been performed by combining various processes, such as heating, intercalation, and wet-jet milling. The c axis of muscovite was expanded from 2004.0 to 2022.8 pm at 800 °C without the destruction of crystallinity of muscovite. The heating at 800 °C led to the weak attraction force between potassium ions and silicate layers by hydroxylation of muscovite. The muscovite heated at 800 °C progressed the intercalation of dodecylammonium chloride (DDAC) into the layers effectively. Furthermore, the DDAC molecules were inserted to the interlayer of muscovite effectively by suppressing the formation of micelle of DDAC. The sedimentation test of wet-jet milled muscovite slurry showed that the relative packing density of muscovite was decreasing with increasing the amount of the intercalated DDAC. As results, the aspect ratio of muscovite prepared with combining the heating, the intercalation and the wet-jet milling was increased by 253% as compared to the raw muscovite. The aspect ratio was calculated from laser particle size distribution and thickness size distribution which was estimated from field emission-scanning electron microscopic images. The expansion of the interlayer led to the effective exfoliation of muscovite with high aspect ratio.     

Vapor-phase carbonylation of methanol over lead on active carbon catalyst

Lead supported on active carbon showed a catalytic activity for the vapor phase carbonylation of methanol under pressurized conditions in the presence of methyl iodide promoter.     

Low‐frequency sound absorption of organic hybrid comprised of chlorinated polyethylene and N,N′‐dicyclohexyl‐2‐benzothiazolyl sulfenamide

We investigated the sound absorption characteristics of an organic hybrid material comprised of chlorinated polyethylene (CPE) as the matrix polymer and N,N′‐dicyclohexyl‐2‐benzothiazolyl sulfenamide (DBS) as the second component of an organic low‐molecular‐weight compound. We found specific crystallites, obtained by annealing, that generated new absorption for a low‐frequency sound in a CPE/DBS blend. We observed two sound absorption peaks, around 300 and 1000 Hz, in the annealed CPE/DBS (50 : 50 w/w) blends, whereas those peaks were not observed in the untreated sample. There were two kinds of crystals with different melting points in the annealed samples. It was confirmed that the crystals with the lower melting point brought about sound absorption at a low frequency. The crystals that had the lower melting point were smaller and/or more disordered than the crystals that had the higher melting point. We calculated the fraction of these two types of crystals from differential scanning calorimetry and wide‐angle X‐ray diffraction measurements. The annealing or reannealing temperature specified the fraction of the crystal with the lower melting point, and the obtained crystal fraction characterized sound absorption frequency. Therefore, it is possible to control the sound absorption frequency of an organic hybrid by heat treatment such as annealing. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

Enrichment of arachidonic acid: Selective hydrolysis of a single-cell oil fromMortierella withCandida cylindracea lipase

Three lipases, isolated previously in our laboratory, and a known lipase fromCandida cylindracea were screened for the enrichment of arachidonic acid (AA). The enzyme fromC. cylindracea was the most effective for the production of oil with high concentration of AA. When a single-cell oil fromMortierella alpina, containing 25% AA, was hydrolyzed with this lipase for 16 h at 35°C, the resulting glycerides contained 50% AA at 52% hydrolysis. After this, no further hydrolysis occurred, even with additional lipase. However, when the glycerides were extracted from the hydrolyzate and were hydrolyzed again with new lipase, the resulting oil contained 60% AA, with a recovery of 75% of its initial AA content. Triglycerides were the main components of the resulting oil. The release of each fatty acid from the oil depended on the hydrolysis rate of its ester. The fatty acid, whose ester is the poorest substrate for the enzyme, is concentrated in the glycerides.     

Heterodimerization of Group I Ribozymes Enabling Exon Recombination through Pairs of Cooperative trans‐Splicing Reactions

Group I (GI) self‐splicing ribozymes are attractive tools for biotechnology and synthetic biology. Several trans‐splicing and related reactions based on GI ribozymes have been developed for the purpose of recombining their target mRNA sequences. By combining trans‐splicing systems with rational modular engineering of GI ribozymes it was possible to achieve more complex editing of target RNA sequences. In this study we have developed a cooperative trans‐splicing system through rational modular engineering with use of dimeric GI ribozymes derived from the Tetrahymena group I intron ribozyme. The resulting pairs of ribozymes exhibited catalytic activity depending on their selective dimerization. Rational modular redesign as performed in this study would facilitate the development of sophisticated regulation of double or multiple trans‐splicing reactions in a cooperative manner.     

Natural denitrification in the Kakamigahara groundwater basin,Gifu prefecture,central Japan

Although nitrate is recognized as the most common groundwater contaminant due to growing anthropogenic sources, such as agriculture in particular, its adverse effects on human and animal health are debatable. The current issue, however, is to control and reduce nitrate contamination with regards to the long residence time of groundwater within aquifers. Denitrification has recently been recognized for its ability to reduce high nitrate concentrations in groundwater. The Kakamigahara groundwater basin, Gifu prefecture, Japan, witnessed rising levels of nitrate (>12 mg/l NO(3)-N) originating from agricultural sources. Chemical analyses for the determination of major constituents of groundwater and delta(15)N of residual nitrate were performed on representative groundwater samples in order to fulfill two main objectives. One is to investigate the current situation of nitrate groundwater pollution. The second objective is to determine whether the denitrification is a potential natural mechanism, which eliminates nitrate pollution in the Kakamigahara aquifer. Agricultural nitrate contamination of groundwater was obvious from characteristically high concentrations of Ca(2+), Mg(2+), NO(3)(-) and SO(4)(2-). High nitrate concentrations were found on the eastern side of the basin in association with vegetable cultivation fields, and decreased gradually towards the west of the basin along the direction of groundwater flow. The decrease of nitrate concentration was conveniently coupled with increase of HCO(3)(-) (the heterotrophic denitrification product), pH and delta(15)N of residual nitrate (due to isotopic fractionation) from east to west. Therefore, denitrification in situ is continuously removing nitrate from the Kakamigahara groundwater system.