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Pseudomonas aeruginosa (P. aeruginosa) infection is one of the major causes of keratitis. However, effective prophylactic and therapeutic vaccines against P. aeruginosa keratitis have yet to be developed. In this study, we explored the use of P. aeruginosa membrane vesicles (MVs) as a prophylactic vaccine as well as the use of immune sera derived from P. aeruginosa MV-immunized animals as a treatment for P. aeruginosa corneal infections in C57BL/6 mice., C57BL/6 mice were intramuscularly immunized with P. aeruginosa MVs; the mouse corneas were then scarified and topically infected with several P. aeruginosa strains, followed by determination of corneal clinical score and corneal bacterial load. Next, immune sera derived from P. aeruginosa MV-immunized ICR mice were administered intraperitoneally to naïve C57BL/6 mice, followed by topical P. aeruginosa challenge. Finally, the immune sera were also used as a topical treatment in the mice with established P. aeruginosa corneal infections., P. aeruginosa-specific IgG and IgA antibodies induced by intramuscular immunization were detected not only in the sera but also in the eye-wash solution. Both active and passive immunization significantly inhibited P. aeruginosa corneal infection. Finally, topical treatment with immune sera in the mice with established P. aeruginosa corneal infections notably decreased the corneal clinical score and corneal bacterial load., P. aeruginosa keratitis can be attenuated by vaccination of P. aeruginosa MVs and topical application of P. aeruginosa MV-specific immune sera.

Diverse ascomycete fungi oxidize manganese(II) [Mn(II)] and produce Mn(III, IV) oxides in terrestrial and freshwater environments. Although multicopper oxidase (MCO) is considered to be a key catalyst in mediating Mn(II) oxidation in ascomycetes, the responsible gene and its product have not been identified. In this study, a gene, named mco1, encoding Mn(II)-oxidizing MCO from Acremonium strictum strain KR21-2 was cloned and heterologously expressed in the methylotrophic yeast Pichia pastoris. Based on the phylogenetic relationship, similarity of putative copper-binding motifs, and homology modeling, the gene product Mco1 was assigned to a bilirubin oxidase. Mature Mco1 was predicted to be composed of 565 amino acids with a molecular mass of 64.0 kDa. The recombinant enzyme oxidized Mn(II) to yield spherical Mn oxides, several micrometers in diameter. Zinc(II) ions added to the reaction mixture were incorporated by the Mn oxides at a Zn/Mn molar ratio of 0.36. The results suggested that Mco1 facilitates the growth of the micrometer-sized Mn oxides and affects metal sequestration through Mn(II) oxidation. This is the first report on heterologous expression and identification of the Mn(II) oxidase enzyme in Mn(II)-oxidizing ascomycetes. The cell-free, homogenous catalytic system with recombinant Mco1 could be useful for understanding Mn biomineralization by ascomycetes and the sequestration of metal ions in the environment

The formation of local high temperature regions, or so-called “hot spots”, in heterogeneous reaction systems has been suggested as a critical factor in the enhancement of chemical reactions using microwave heating. In this paper, we report the generation of local high temperature regions between catalyst particles under microwave heating. First, we demonstrated that reaction rate of the dehydrogenation of 2-propanol over a magnetite catalyst was enhanced 17- (250 °C) to 38- (200 °C) fold when heated with microwave irradiation rather than an electrical furnace. Subsequently, the existence of microwave-generated specific local heating was demonstrated using a coupled simulation of the electromagnetic fields and heat transfer as well as in situ emission spectroscopy. Specific high-temperature regions were generated at the vicinal contact points of the catalyst particles due to the concentrated microwave electric field. We also directly observed local high temperature regions at the contact points of the particles during microwave heating of a model silicon carbide spherical material using in situ emission spectroscopy. We conclude that the generation of local heating at the contact points between the catalyst particles is a key factor for enhancing fixed-bed flow reactions under microwave irradiation.

SiC fiber-MO2 (M = Ce, Zr) mixtures with various compositions were heated by applying an 80 W microwave electric field, to investigate their heating rate, maximum temperature, and dielectric constant. For the SiC fiber-CeO2 mixture, all three parameters continued to increase as the weight ratio of the SiC fiber increased, in contrast, for the SiC fiber-ZrO2 mixture, these parameters reached a maximum value at a certain composition. A thermal gradient of 500 &deg, C was observed at a microlevel in the SiC fiber-ZrO2 mixture, and hot spots were located in regions with a certain composition. This result not only contributes to designing a novel good microwave absorber but also presents new aspects with regard to high-temperature microwave processing, including the mechanism behind the high-temperature gradients on the order of micrometers as well as engineering applications that utilize these high-temperature gradients.

Chemical reactions performed under microwave irradiation often demonstrate high reaction rates, high selectivity, and low reaction temperatures, which allows for more compact reactors and more energy-efficient processes than used in conventional heating methods. In this study, we discovered a new chemical reaction and proposed a new material synthesis method, performed using a solid-state microwave source with an oscillator and a single-mode cavity. We developed a microwave heating thermogravimetry apparatus to confirm that microwave irradiation reduced the temperature of the reduction process. Next, we applied this apparatus to the reduction process of copper oxide with carbon as the reducing agent and compared microwave heating with conventional heating. We demonstrated that the reduction temperature of microwave heating was 192 °C in a magnetic (H) field and 265 °C in an electric (E) field, which were lower than those of conventional heating. For microwave heating in the E-field, plasma strongly affected the reaction process, leading to a reduction in the reaction temperature. For microwave heating in the H-field, localized heating occurred and the average reaction temperature was lower. Using the obtained results, an applicator suitable for these modes can be designed, enabling a reduction process with an additional energy-saving effect.

The polymer electrolyte fuel cell (PEFC) is an attractive power generation method from the perspective of environmental protection. Carbon is usually used as a catalyst support in PEFC, but it is oxidized under high electrical potential conditions. Ti4O7 is expected as a new catalyst support because of its high electrical conductivity and chemical resistivity. Though Ti4O7 as a catalyst support must have a high specific surface area for a high performance, it is difficult to synthesize nanostructured Ti4O7. In this research, Ti4O7 nanoparticles with a size of about 60 nm were synthesized by carbothermal reduction of TiO2 nanoparticles with polyvinylpyrrolidone (carbon source) using 2.45 GHz microwave irradiation. The experiment condition was at 950 °C for 30 min and the samples synthesized by conventional heating showed a grain growth. The findings of this study suggest that microwave processing can drastically reduce the total processing time for the synthesis of nanostructured Ti4O7.

NiMn2O4 prepared by conventional heating was irradiated with a microwave H-field using a single-mode cavity under air and magnetic properties of the microwave-irradiated material were investigated. X-ray diffraction and transmission electron microscopy demonstrated that the phase and microstructure are not affected by H-field irradiation. Measurements of the magnetization as a function of temperature revealed that the antiferromagnetic sublattice disappeared and electron spin resonance showed the existence of Mn2+, suggesting that Mn3+ is partially reduced. Moreover, the magnetization of NiMn2O4 was controlled from 35.3 to 18.2 emu/g and the coercivity from 140 to 750 Oe by changing the sample temperature during microwave irradiation. The reduction reaction of NiMn2O4 is controlled by microwave H-field irradiation, resulting in control over the magnetic properties.

Burkholderiales bacterium GJ-E10, isolated from the Tamagawa River in Akita Prefecture, Japan, is an unclassified, iron-oxidizing chemolithoautotrophic bacterium. Its single circular genome, consisting of 3,276,549 bp, was sequenced by using three types of next-generation sequencers and the sequences were then confirmed by PCR-based Sanger sequencing.

N-acylhomoserine lactone (AHSL, autoinducer) is capable of regulating a set of genes by sensing cell density and developing an intercellular communication in Pseudomonas aeruginosa. Addition of AHSL in the exponential growth phase, regardless of cell density, induces a repression of cell growth of P. aeruginosa, an expression of stationary phase specific factor sigma 8 in vivo and a morphological change into smaller spherical shape indistinguishable from that in the stationary phase. It is demonstrated that AHSL can trigger an entry of bacteria into stationary phase as a growth controlling signal.

We have developed a simple process for sintering of soft magnetization materials using microwave sintering. The saturated magnetization (Ms) of sintered magnetite was 85.6 emu/g, which was as high as 95% of magnetite before heating (90.4 emu/g). On the other hand, the averaged remanence (Mr) and coercivity (Hc) of the magnetite after heating were 0.17 emu/g and 1.12 Oe under measuring limit of SQUID, respectively. For the sintering process of soft magnetic materials, magnetic fields of microwave have been performed in nitrogen atmosphere. Therefore, a microwave single-mode system operating at a frequency of 2.45 GHz and with a maximum power level of 1.5 kW was used. We can sinter the good soft magnetic material in microwave magnetic field. The sample shrank to 82% theoretical density (TD) from 45%TD of green body. The sintered sample was observed the microstructure by TEM and the crystal size was estimated the approximate average size is 10 nm.

SUMMARYThe efficacy of DNA-based immunization in conferring protective immunity against certain microbial pathogens including human immunodeficiency virus type 1 (HIV-1) has been described. The potential advantage of DNA-based immunization over the traditional vaccines largely results from its capacity to efficiently induce Th1-biased immune responses against an encoded antigen. We describe how Th1-biased immune responses are induced by DNA-based immunization, using a DNA vaccine construct encoding HIV-1 gp160 cDNA and an eukaryotic expression plasmid carrying murine IFN-γ cDNA. Transfection of an eukaryotic expression plasmid carrying immunostimulatory sequences (ISS) as well as a gene of interest (DNA vaccine) into professional antigen presenting cells (APC) induced transactivation of IL-12 mRNA, which resulted in antigen-specific Th1-biased immune responses against the encoded antigen. Th1-biased immune responses induced by DNA-based immunization were substantially upregulated by a codelivery of an ectopic IFN-γ expression system, and this augmentation was mediated via action on professional antigen presenting cells to upregulate IL-12 production. Taken together, it appears likely that Th1-biased immune responses induced by DNA-based immunization are mediated via action on professional antigen-presenting cells to produce IL-12. Interestingly, the model provided strikingly resembles that previously described in infection with Listeria monocytogenes, an intracellular Gram-positive bacterium that induces strong Th1-biased immune responses. The result suggests that DNA-based immunization mimics certain aspects of natural infection with microbial organisms like attenuated vaccines, which in turn provides a rationale to the question of why DNA-based immunization so efficiently induces protective immunity against these microbial pathogens.

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-γ) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-γ monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.

We have cloned and sequenced the lasR gene, which is involved in the transcriptional activation of several pathogenic factors, from Pseudomonas aeruginosa IFO3455 and PA103. These clones were predicted to be an open reading frame of 239 amino acids as reported for the PAO1 strain. There is only a single base change resulting in an amino acid exchange from M145 (PAO1) to I (IFO3455). PA103 DNA differs with PAO1 DNA in two bases resulting in only a single amino acid substitution from R179 to W. When the IFO3455 LasR was expressed in a PA103 strain which is known to be LasR defective, proteinase gene activation was detected, however, when PA103 LasR was expressed, no enhancement was measurable. From these results, it appears that the amino acid substitution of R179 to W inactivated LasR activity. This substitution is located in the highly conserved sequence found in many transcription factors, including sigma factors, and may disrupt amphipathic alpha-helix, predicted for the 176 to 189 region, which precedes the putative helix-turn-helix DNA binding motif. We presumed that these three helices may contribute to specific DNA binding.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.