JBB : Journal of Bioscience and Bioengineering

JBB Vol. 126表紙  



  • Tandem repeated expression of lunasin gene in Pichia pastoris and its anti-inflammatory activity in vitro
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Yingying Zhu, Everaert Nadia, Yang Yao, Zhenxing Shi, Guixing Ren

    Lunasin is a novel promising health-beneficial peptide derived from soybean. However, the application of lunasin is limited by its high cost. In this study, we developed a successful protocol for expression of a dimer formation protein containing 4 tandem repeated lunasin analogs (lunasin-4) in Pichia pastoris. The expression level at the optimal condition (initial pH 7.0, 1.0% final methanol concentration and induction for 72 h at 26 °C) was 0.24 mg/mL cell-free broth. Lunasin analog, obtained from purified lunasin-4 protein through enterokinase digestion and ultrafiltration, significantly decreased (p < 0.05) the release of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in a dose-dependent manner. In addition, intracellular signaling array analysis demonstrated down-regulated levels of phosphorylated Akt, mechanistic target of rapamycin (mTOR) and p70 s6 kinase (p70s6k) and an up-regulated level of glycogen synthase kinase-3β (GSK-3β) after lunasin analog treatment. These results suggest that lunasin analog exerted anti-inflammatory activities in LPS-stimulated RAW264.7 cells partly via inhibiting the activation of Akt/mTOR/p70s6k signaling pathway. In conclusion, this study provides a potential strategy for recombinant production of bioactive lunasin in industry.





  • N-glycan sialylation in a silkworm-baculovirus expression system
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Masatoshi Suganuma, Tsuyoshi Nomura, Yukiko Higa, Yukiko Kataoka, Shunsuke Funaguma, Hironobu Okazaki, Takeo Suzuki, Kazuhito Fujiyama, Hideki Sezutsu, Ken-ichiro Tatematsu, Toshiki Tamura

    A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm.

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  • Heterologous expression, purification and characterization of human β-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Takatsugu Miyazaki, Tatsuya Kato, Enoch Y. Park

    β-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure–function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 μg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity.





  • Evaluation of leader peptides that affect the secretory ability of a multiple bacteriocin transporter, EnkT
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Hirotoshi Sushida, Naoki Ishibashi, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto

    EnkT is a novel ATP-binding cassette (ABC) transporter responsible for secretion of four bacteriocins, enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z), produced by Enterococcus faecium NKR-5-3. It is generally recognized that the secretion of a bacteriocin requires a dedicated ABC transporter, although molecular mechanisms of this secretion are yet to be revealed. In order to characterize the unique ability of EnkT to secrete multiple bacteriocins, the role of N-terminal leader peptides of bacteriocin precursors was evaluated using Ent53C precursor as a model. The 18-amino acid leader peptide of Ent53C (Lc) was modified by site-directed mutagenesis to generate various point mutations, truncations, or extensions, and substitutions with other leader peptides. The impact of these Lc mutations on Ent53C secretion was evaluated using a quantitative antimicrobial activity assay. We observed that Ent53C production increased with Ala substitution of the highly conserved C-terminal double glycine residues that are recognized as the cleavage site. In contrast, Ent53C antimicrobial activity decreased, with decrease in the length of the putative α-helix-forming region of Lc. Furthermore, EnkT recognized and transported Ent53C of the transformants possessing heterologous leader peptides of enterocin A, pediocin PA-1, brochocins A and B, and lactococcins Qα and Qβ. These results indicated that EnkT shows significant tolerance towards the sequence and length of leader peptides, to secrete multiple bacteriocins. This further demonstrates the functional diversity of bacteriocin ABC transporters and the importance of leader peptides as their recognition motif.





  • Stochastic expression of lactate dehydrogenase A induces Escherichia coli persister formation
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Naoki Yamamoto, Rino Isshiki, Yuto Kawai, Daiki Tanaka, Tetsushi Sekiguchi, Shinya Matsumoto, Satoshi Tsuneda

    Bacterial persisters are phenotypic variants that survive the treatment of lethal doses of growth-targeting antibiotics without mutations. Although the mechanism underlying persister formation has been studied for decades, how the persister phenotype is switched on and protects itself from antibiotics has been elusive. In this study, we focused on the lactate dehydrogenase gene (ldhA) that was upregulated in an Escherichia coli persister-enriched population. A survival rate assay using an ldhA-overexpressing strain showed that ldhA expression induced persister formation. To identify ldhA-mediated persister formation at the single-cell level, time-lapse microscopy with a microfluidic device was used. Stochastic ldhA expression was found to induce dormancy and tolerance against high-dose ampicillin treatment (500 μg/ml). To better understand the underlying mechanism, we investigated the relationship between ldhA-mediated persister formation and previously reported persister formation through aerobic metabolism repression. As a result, ldhA expression enhanced the proton motive force (PMF) and ATP synthesis. These findings suggest that ldhA-mediated persister formation pathway is different from previously reported persister formation via repression of aerobic metabolism.





  • Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Kentaro Ueda, Tsubasa Nakajima, Katsunori Yoshikawa, Yoshihiro Toya, Fumio Matsuda, Hiroshi Shimizu

    The role of the oxidative pentose phosphate pathway (oxPPP) in Synechocystis sp. PCC 6803 under mixotrophic conditions was investigated by 13C metabolic flux analysis. Cells were cultured under low (10 μmol m−2 s−1) and high light intensities (100 μmol m−2 s−1) in the presence of glucose. The flux of CO2 fixation by ribulose bisphosphate carboxylase/oxygenase under the high light condition was approximately 3-fold higher than that under the low light condition. Although no flux of the oxPPP was observed under the high light condition, flux of 0.08–0.19 mmol gDCW−1 h−1 in the oxPPP was observed under the low light condition. The balance between the consumption and production of NADPH suggested that approximately 10% of the total NADPH production was generated by the oxPPP under the low light condition. The growth phenotype of a mutant with deleted zwf, which encodes glucose-6-phosphate dehydrogenase in the oxPPP, was compared to that of the parental strain under low and high light conditions. Growth of the Δzwf mutant nearly stopped during the late growth phase under the low light condition, whereas the growth rates of the two strains were identical under the high light condition. These results indicate that NADPH production in the oxPPP is essential for anabolism under low light conditions. The oxPPP appears to play an important role in producing NADPH from glucose and ATP to compensate for NADPH shortage under low light conditions.





  • Nanoparticles synthesis by Agaricus soil basidiomycetes
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Ekaterina A. Loshchinina, Elena P. Vetchinkina, Maria A. Kupryashina, Viktor F. Kursky, Valentina E. Nikitina

    We examined the effect of various concentrations of HAuCl4, AgNO3, Na2SeO3, Na2SiO3, and GeO2 on mycelial growth of the soil basidiomycetes Agaricus bisporus and A. arvensis in submerged and solid media. Fungal mycelial extracts and cell-free culture filtrates were able to reduce ions of Au, Ag, Se, Si, and Ge compounds, forming Au0, Ag0, Se0, Si0/SiO2 and Ge0/GeO2 nanoparticles. The physical characteristics of the mycogenic nanoparticles differed depending on the species of Agaricus and the type of extract. Au nanospheres obtained with cell-free culture filtrates were of 2–5 nm diameter in A. bisporus and of 2–10 nm in A. arvensis. Nanoparticles produced by extracts of mycelia were several times larger and highly heterogenous. Ag nanoparticles produced by cell-free culture filtrates were spherical or irregular-shaped and agglomerated, whereas with extracts of mycelia, small homogenous nanospheres of 1–10 nm were formed. Se nanospheres obtained with cell-free culture filtrates were of 100–250 nm diameter in A. bisporus and of 150–550 nm diameter in A. arvensis. The particles synthesized with extracts of mycelia were of 40–140 nm in A. bisporus and of 100–250 nm in A. arvensis. Incubation of Na2SiO3 with cell-free culture filtrates resulted in porous Si nanoparticles of 30–65 nm in A. bisporus and of 50–200 nm in A. arvensis. Ge nanoparticles synthesized by both Agaricus species were mostly spheres of 50–250 nm diameter.

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  • Nuclear magnetic resonance- and gas chromatography/mass spectrometry-based metabolomic characterization of water-soluble and volatile compound profiles in cabbage vinegar
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Satoru Ishihara, Takashi Inaoka, Toshihide Nakamura, Keitarou Kimura, Yasuyo Sekiyama, Satoru Tomita

    Non-targeted metabolomic analyses employing nuclear magnetic resonance- and gas chromatography/mass spectrometry-based techniques were applied for an in-depth characterization of cabbage vinegar, an original agricultural product made from cabbage harvested in Tsumagoi, Japan. Water-soluble and volatile metabolite profiles of cabbage vinegar were compared with those of various vinegars: rice vinegar, grain vinegar, apple vinegar, and black vinegar (Japanese kurozu made of brown rice). Principal component analysis (PCA) of the water-soluble metabolites indicated that cabbage vinegars belonged to an isolated class by the contributions of fructose, pyroglutamic acid, choline, and methiin (S-methylcysteine sulfoxide). Regarding the volatile compounds, the PCA data represented that rice, black, and apple vinegars were characterized by most of the dominant volatiles, such as acetate esters, alcohols, ketones, and acids. Cabbage and grain vinegars were included in the same class although these two vinegars have different flavors. Orthogonal partial least squares-discrimination analysis exhibited the differences in volatile compound profile between cabbage and grain vinegars, revealing that cabbage vinegars were characterized by the presence of sulfides (dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide), nitriles (allyl cyanide and 4-methylthio-butanenitrile), 3-hexene-1-ol, and crotonic acid. The time-course changes in these highlighted compounds during the acetic acid fermentation of cabbage vinegar suggested that pyroglutamic and crotonic acids were produced through fermentation, whereas choline, methiin, sulfides, nitriles, and 3-hexene-1-ol were derived from cabbage, suggesting the key role of these compounds in the unique taste and flavor of cabbage vinegar.





  • Integrated molecular analysis of the inactivation of a non-enveloped virus, feline calicivirus, by UV-C radiation
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Tsuyoshi Tanaka, Osamu Nogariya, Nozomi Shionoiri, Yoshiaki Maeda, Atsushi Arakaki

    UV-C treatment has been shown to be a powerful way to inactivate non-enveloped viruses in water samples. However, little is known about how the viruses were inactivated by UV-C radiation. In this study, we investigated the inactivation mechanism of a single-stranded RNA (ssRNA) non-enveloped virus, feline calicivirus (FCV), as a surrogate for the human norovirus, using UV-C radiation with different wavelengths. Integrated molecular analyses using RT-qPCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and mass spectrometry were employed to evaluate the extent of ssRNA genome and protein degradation. UV-C radiation of FCV efficiently impaired the infectivity of FCV in mammalian cells. We also identified degradation of the RNA genome, whose copy numbers decreased from 48% to 56% following UV255 or UV281 radiation. Significant degradation of capsid protein was not observed, whereas oxidation of amino acid residues in the major capsid protein VP-1 was determined. Our results suggest that damage to the RNA genome is primarily responsible for the observed decrease in FCV infectivity of CRFK cells. This study provides not only relevant baseline data but also an overview and possible mechanism for the disinfection of non-enveloped ssRNA viruses using UV-C radiation.





  • Synthesis of novel laccase-biotitania biocatalysts for malachite green decolorization
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Xinying Zhang, Meiyin Wang, Linlin Lin, Gao Xiao, Zhenping Tang, Xuefeng Zhu

    Biomimetic mineralization has emerged as a novel tool for generating excellent supports for enzyme stabilization. In this work, protamine was used to induce titanium (IV) bis(ammonium lactato) dihydroxide (Ti-BALDH) into titania nanoparticles. This biomimetic titanification process was adopted for laccase immobilization. Laccase-biotitania biocatalyst was prepared and the effect of different parameters (buffer solution, titania precursor concentration, protamine concentration, and enzyme loading) on the encapsulation efficiency and recovery of laccase were evaluated. Compared with free laccase, the thermal and pH stability of immobilized laccase were improved significantly. In addition, laccase loaded on titania was effective at enhancing its storage stability. After seven consecutive cycles, the immobilized laccase still retained 51% of its original activity. Finally, laccase-biotitania biocatalysts showed good performance on decolorization of malachite green (MG), which can be attributed to an adsorption and degradation effect. The intermediates of the MG degradation were identified by gas chromatography-mass spectrometry (GC–MS) analysis, and the most probable degradation pathway was proposed. This study provides deeper understanding of the laccase-biotitania particles as a fast biocatalyst for MG decolorization.

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  • Isolation and identification of Penicillium chrysogenum strain Y5 and its copper extraction characterization from waste printed circuit boards
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Ming-Chen Xia, Peng Bao, A-Juan Liu, Shi-Shi Zhang, Tang-Jian Peng, Li Shen, Run-Lan Yu, Xue-Ling Wu, Jiao-Kun Li, Yuan-Dong Liu, Miao Chen, Guan-Zhou Qiu, Wei-Min Zeng

    Biohydrometallurgy is generally considered as a green technology for the recycling of industrial solid waste. In this study, an indigenous fungal strain named Y5 with the ability of high-yielding organic acids was isolated and applied in bioleaching of waste printed circuit boards (PCBs). The strain Y5 was identified as Penicillium chrysogenum by morphological and molecular identification. Meanwhile, we investigated that an optimal set of culturing conditions for the fungal growth and acids secretion was 15 g/L glucose with initial pH 5.0, temperature 25°C and shaking speed 120 rpm in shaken flasks culture. Moreover, three bioleaching processes such as one-step, two-step and spent medium processes were conducted to extract copper from waste PCBs. Spent medium bioleaching showed higher copper extraction percentage and it was 47% under 5%(w/v) pulp density. Transmission electron microscope (TEM) observation combining with energy dispersive analysis of X-rays (EDAX) showed that the leached metal ions did not obviously damage the hypha cells. All above results indicated that P . chrysogenum strain Y5 has the tolerance to metal ions, suggesting its potential in recycling of metals from waste PCBs in industry.





  • Nitrogen removal performance and microbial community structure in the start-up and substrate inhibition stages of an anammox reactor
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Wan Yang, Shilong He, Ming Han, Bingbing Wang, Qigui Niu, Yujia Xu, Yi Chen, Haibo Wang

    In this study, the nitrogen removal performance and microbial community structure were investigated during the start-up, instability, and recovery stages of an anaerobic ammonium oxidation (anammox) reactor loaded with compound carriers (shale ceramsite and suspended ball carrier). The results indicated that the anammox reactor successfully started up on 116th d when the nitrogen loading rate (NLR) reached 0.72 ± 0.05 kg N m−3 d−1. The anammox reactor ran well with free ammonia (FA) at 13.65 ± 2.69 mg/L and free nitrous acid (FNA) at 39.49 ± 10.95 μg/L, indicating that its tolerance for FA and FNA was higher than that of granular sludge anammox reactors. The anammox system was inhibited when FA and FNA reached 29.65 mg/L and 77.02 μg/L, respectively. The tolerance of anammox bacteria towards FA and FNA decreased after this inhibition. The nitrogen removal performance could be efficiently recovered by decreasing the influent substrate concentration and increasing the hydraulic retention time (HRT). Candidatus Brocadia and Candidatus Jettenia, two genus-level anammox bacteria, were detected in this reactor using a high-throughput sequencing technique. After high substrate shock, the abundance of Candidatus Brocadia decreased while that of Candidatus Jettenia increased, which might be due to the competition between Candidatus Jettenia and Candidatus Brocadia. The relationships between anammox communities and operational factors were investigated via redundancy analysis (RDA), which showed that FA was the principal factor affecting the microbial community structure during the operation stage.





  • Effect of intermittent opening of breathable culture plugs and aeration of headspace on the structure of microbial communities in shake-flask culture
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Masato Takahashi, Hideki Aoyagi

    In this study, we found that opening breathable culture plugs for 30 s during periodic and aseptic sampling affects the community structure of cultured soil microbes. Similar effects were observed using an automatic aeration flask system that mimics aseptic opening of the breathable culture plug during sampling, but without interruption in shaking. Thus, the observed changes in the microbial consortia appear to be due exclusively to the intermittent ventilation of the flask headspace. To elucidate the mechanism driving this phenomenon, we monitored CO2 and O2 concentrations in both headspace and culture broth using the new system termed as circulation direct monitoring and sampling system. The data show that the CO2 concentration in the culture broth temporarily decreased with the CO2 concentration in the headspace, strongly suggesting that the effect of intermittent ventilation of the headspace on the microbial consortia depends on CO2. Importantly, the data also imply that environmental variables during shake flask culture, especially CO2 concentration, is important for screening aerobic microorganisms.





  • Enhancement of glycosylation by stable co-expression of two sialylation-related enzymes on Chinese hamster ovary cells
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Nguyen Thi Sam, Ryo Misaki, Takao Ohashi, Kazuhito Fujiyama

    Sialic acid plays important roles in stabilization and modulation of the interaction of molecules and membranes in organisms. Due to its high electronegativity, sialic acid can promote binding effects of molecules and support the transportation of drugs and ions in cells. This also strengthens cells against degradation from glycosidases and proteases. Hence sialic acid helps glycoproteins extend their half-lives and bioactivity. On the other hand, Chinese hamster ovary (CHO) cells have been widely used as a workhorse in biopharmaceutical fields in part due to the similarity between their glycan properties and those in humans. Thus, a high sialylation produced by CHO host cell line is strongly desired. In this study, we simultaneously overexpressed two key sialylated-based enzymes human β-galactoside α(2,6) sialyltransferase I and UDP-GlcNAc 2-epimerase/ManNAc kinase to achieve greater sialylation pattern produced host cells. The single-cell line thus-generated produced an approximately 41.6% higher level of total free sialic acid, and the glycan profiles showed a significant increase of more than 7-fold in the relative amount of total sialylated N-glycan as compared to the wild-type. These results demonstrated that co-expression of these two sialylated-based key enzymes yielded a cell line that effectively produced glycoproteins with superior sialylation and achievable human-like glycoforms.





  • Development of a perfusable 3D liver cell cultivation system via bundling-up assembly of cell-laden microfibers
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Yuya Yajima, Chu Ning Lee, Masumi Yamada, Rie Utoh, Minoru Seki

    Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications.

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  • In vivo tissue distribution and safety of polyacrylic acid-modified titanium peroxide nanoparticles as novel radiosensitizers
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Kenta Morita, Takahiro Suzuki, Yuya Nishimura, Kazuhisa Matsumoto, Chiya Numako, Kazuyoshi Sato, Masao Nakayama, Ryohei Sasaki, Chiaki Ogino, Akihiko Kondo

    Polyacrylic acid (PAA)-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers. PAA-TiOx NPs were synthesized from commercial TiO2 nanoparticles that were modified with PAA and functionalized by H2O2 treatment. To realize practical clinical uses for PAA-TiOx NPs, their tissue distribution and acute toxicity were evaluated using healthy mice and mice bearing tumors derived from xenografted MIAPaCa-2 human pancreatic cancer cells. Healthy mice were injected with PAA-TiOx NPs at 25 mg/kg body weight via the tail vein, and tumor-bearing mice were injected either into the tumor locally or via the tail vein. The concentration of PAA-TiOx NPs in major organs was determined over time using inductively coupled–plasma atomic emission spectrometry. After 1 h, 12% of the PAA-TiOx NP dose had accumulated in the tumor, and 2.8% of the dose remained after 1 week. Such high accumulation could be associated with enhanced permeability and retention effects of the tumor, as PAA-TiOx NPs are composed of inorganic particles and polymers, without tumor-targeting molecules. The liver accumulated the largest proportion of the injected nanoparticles, up to 42% in tumor-bearing mice. Blood biochemical parameters were also investigated after intravenous injection of PAA-TiOx NPs in healthy mice. PAA-TiOx NPs invoked a slight change in various liver-related biochemical parameters, but no liver injury was observed over the practical dose range. In the future, PAA-TiOx NPs should be modified to prevent accumulation in the liver and minimize risk to patients.

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  • Development of an analytical method for polycyclic aromatic hydrocarbons in coffee beverages and dark beer using novel high-sensitivity technique of supercritical fluid chromatography/mass spectrometry
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Toshiaki Yoshioka, Yasushi Nagatomi, Koichi Harayama, Takeshi Bamba

    Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic substances that are mainly generated during heating in food; therefore, the European Union (EU) has regulated the amount of benzo[a]pyrene and PAH4 in various types of food. In addition, the Scientific Committee on Food of the EU and the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives have recommended that 16 PAHs should be monitored. Since coffee beverages and dark beer are roasted during manufacture, monitoring these 16 PAHs is of great importance. On the other hand, supercritical fluid chromatography (SFC) is a separation method that has garnered attention in recent years as a complement for liquid and gas chromatography. Therefore, we developed a rapid high-sensitivity analytical method for the above-mentioned 16 PAHs in coffee beverages and dark beer involving supercritical fluid chromatography/atmospheric pressure chemical ionization-mass spectrometry (SFC/APCI-MS) and simple sample preparation. In this study, we developed a novel analytical technique that increased the sensitivity of MS detection by varying the back-pressure in SFC depending on the elution of PAHs. In addition, analysis of commercially available coffee and dark beer samples in Japan showed that the risk of containing the 16 PAHs may be low.





  • Sensitive typing of reverse ABO blood groups with a waveguide-mode sensor
    Publication date: July 2018
    Source:Journal of Bioscience and Bioengineering, Volume 126, Issue 1

    Author(s): Shigeyuki Uno, Torahiko Tanaka, Hiroki Ashiba, Makoto Fujimaki, Mutsuo Tanaka, Yoshihiro Hatta, Masami Takei, Koichi Awazu, Makoto Makishima

    Portable, on-site blood typing methods will help provide life-saving blood transfusions to patients during an emergency or natural calamity, such as significant earthquakes. We have previously developed waveguide-mode (WM) sensors for forward ABO and Rh(D) blood typing and detection of antibodies against hepatitis B virus and hepatitis C virus. In this study, we evaluated a WM-sensor for reverse ABO blood typing. Since reverse ABO blood typing is a method for detection of antibodies against type A and type B oligosaccharide antigens on the surface of red blood cells (RBCs), we fixed a synthetic type A or type B trisaccharide antigen on the sensor chip of the WM sensor. We obtained significant changes in the reflectance spectra from a WM sensor on type A antigen with type B plasma and type O plasma and on type B antigen with type A plasma and type O plasma, and no spectrum changes on type A antigen or type B antigen with type AB plasma. Signal enhancement with the addition of a peroxidase reaction failed to increase the sensitivity for detection on oligosaccharide chips. By utilizing hemagglutination detection using regent type A and type B RBCs, we successfully determined reverse ABO blood groups with higher sensitivity compared to a method using oligosaccharide antigens. Thus, functionality of a portable device utilizing a WM sensor can be expanded to include reverse ABO blood typing and, in combination with forward ABO typing and antivirus antibody detection, may be useful for on-site blood testing in emergency settings.





  • Kinetic analysis on precursors for iturin A production from Bacillus amyloliquefaciens BPD1
    Publication date: Available online 19 June 2018
    Source:Journal of Bioscience and Bioengineering

    Author(s): Jiun-Yan Wu, Jen-Hung Liao, Chwen-Jen Shieh, Feng-Chia Hsieh, Yung-Chuan Liu

    In this study, the precursor effect for iturin A production was quantitatively analyzed. A strain identified as Bacillus amyloliquefaciens BPD1 (Ba-BPD1) was selected due to its ability to produce iturin A. The enhancement of iturin A production in a submerged culture was tested using various additives, including palmitic acid, oils, and complex amino acids. Among these, complex amino acids triggered the highest yield at 559 mg/L. The respective amino acids that contribute to the structure of iturin A were used as precursors. In fact, it was found that the addition of l-proline, l-glutamine, l-asparagine and l-serine could improve iturin A yield in the defined medium. However, during the kinetic analysis, all the amino acids exhibited a lower saturation level than l-serine, which exhibited a high saturation level at 1.2% resulting in an iturin A yield of 914 mg/L. In contrast, a negative effect was observed following the addition of l-tyrosine. To analyze the kinetic behavior of l-serine, three kinetic models were adopted: the kinetic order equation, the Langmuir kinetic equation, and a modified logistic equation. The regression results showed that the modified logistic model was the best fit for the kinetic behavior of l-serine as the major precursor, which could be further referred to the biosynthesis pathway of iturin A. Among the proposed processes for iturin A production, this study achieved the highest iturin A levels as a result of the addition of precursors.

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  • Transcriptome analysis of the cyanobacterium Synechocystis sp. PCC 6803 and mechanisms of photoinhibition tolerance under extreme high light conditions
    Publication date: Available online 19 June 2018
    Source:Journal of Bioscience and Bioengineering

    Author(s): Kenichi Ogawa, Katsunori Yoshikawa, Fumio Matsuda, Yoshihiro Toya, Hiroshi Shimizu

    Photoinhibition, or cell damage caused by excessively intense light is a major issue for the industrial use of cyanobacteria. To investigate the mechanism of responses to extreme high light intensity, gene expression analysis was performed using the model cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803) cultured under various light intensities. The culture profile data demonstrated that, in contrast to the slow cell growth observed under low light intensities (30 and 50 μmol m−2 s−1), maximal cell growth was observed under mid light conditions (300 and 1000 μmol m−2 s−1). PCC 6803 cells exhibited photoinhibition when cultured under excessive high light intensities of 1100 and 1300 μmol m−2 s−1. From the low to the mid light conditions, the expression of genes related to light harvesting systems was repressed, whereas that of CO2 fixation and of D1 protein turnover-related genes was induced. Gene expression data also revealed that the down-regulation of genes related to flagellum synthesis (pilA2), pyridine nucleotide transhydrogenase (pntA and pntB), and sigma factor (sigA and sigF) represents the key responses of PCC 6803 under excessive high light conditions. The results obtained in this study provide further understanding of high light tolerance mechanisms and should help to improve the productivity of bioprocess using cyanobacteria.