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Purpose: Technological advancements and innovations in the use of microneedles for medicinal purposes are constantly emerging. To promote the marketing of microneedles, the safety of microneedle systems during application and storage must be highlighted. Therefore, investigating the toxicity and skin safety of these materials is important. We aimed to determine the physicochemical properties of influenza-antigen-coated microneedle (Influenza MN) and the skin irritation, acute toxicity, and specific antibody production in vivo after applying the coated microneedles to rat skin. Methods: We prepared Influenza MN patches using polylactic acid. The protein antigen coating and microneedle morphology were identified using scanning electron microscopy (SEM) and SEM–energy-dispersive X-ray spectroscopy (EDS), and the coating was assayed using a bicinchoninic acid kit. We used the Draize method to assess skin irritation after applying the patches to rat skin and serum biomarkers to assess the systemic toxicity of the patches. We used an enzyme-linked immunosorbent assay to determine the antigen-specific immune response induced by the patches in vivo. Results: The antigenic protein coated on the microneedles was visualized by SEM and confirmed by EDS. The protein content of the patches was 96.41 ± 3.18 μg. Treatment with the patches did not cause any significant skin irritation as determined by the Draize method. Applying the patch did not cause systemic toxicity in the rats according to the serum biomarkers but induced a strong antigen-specific immune response in vivo. Conclusion: Applying the Influenza MN patches did not cause significant skin irritation or dermal toxicity in rats but induced antigen-specific immune responses in vivo.

This paper proposes a dual lifting and its cooperative control system with two different kinds of floating cranes. The Mega-erection and Giga-erection in the ship building are used to handle heavier and wider blocks and modules as ships and off-shore platforms are enlarged. However, there is no equipment to handle such Tera-blocks. In order to overcome the limit on performance of existing floating cranes, the dual lifting is proposed in this research. In the dual lifting, two floating cranes are well-coordinated to add up the lift capabilities of both cranes without any loss such that virtually a single crane is lifting, maneuvering and unloading. Two main constraints for the dual lifting are as follows: First, two barges of floating cranes should be constrained as a rigid body not to cause a relative motion between two barges and main hooks of the two cranes should be controlled as main hooks of a single crane. In order words, it is necessary to develop the cooperative control of two floating cranes in order to sustain a center of gravity of the module and minimize the tilting angle during the lifting and unloading by the two floating cranes. Two floating cranes are handled as a master-slave system. The master crane is able to gather information about all working conditions and make a decision to control the individual hook speed, which communicates the slave crane by TCP/IP. The developed control system has been embedded in the real floating crane systems and the dual lifting has been demonstrated five times at SHI shipyard in 2015. The moving angles of the lifting module are analyzed and verified to be suitable for hoisting control. It is verified that the dual lifting can be applied for many heavier and wider blocks and modules to shorten the construction time of ships and off-shore platforms.

This article presents an automated thermal forming system for use in the ship fabrication process. The system consists of scanning devices for measurement of plates, heating devices for plate forming, a computational unit for heating information and a gantry robot with six axes, which moves the scanning and heating devices. Two levels of measurement, pre-scanning and post-scanning, are introduced. A pre-scanning system using a multi-LVS (laser vision sensor) is installed on the gantry for detecting the position of the plate in the working area. Once the position is determined, a post-scanning device with a Local-LVS, installed at the robot arm, is employed to measure the plate accurately. A high-frequency induction heater is installed at the robot arm as a heating device. The computational unit computes heating information including heating locations and speed. The robot controls the position and orientation of the heater and the measurement devices to heat a plate following the computed heating information and to measure the shape of the plate accurately. The system was thoroughly evaluated with diverse test plates, and has been used in the actual fabrication process at a shipyard.

Chattonella antiquaisolated in 2010 showed extremely more potent fish-killing activities against red sea bream, Japanese horse mackerel, and blue damselfish than those of Chattonella marinaisolated in 1985. Chemiluminescence and electron spin resonance (ESR) analyses suggested greater reactive oxygen species (ROS)-producing activity of C. antiquathan that of C. marina. Sodium benzoate, a hydroxyl radical scavenger, significantly suppressed the fish-killing activity of C. antiquaon blue damselfish. The chlorophyll level in the gill tissue of blue damselfish exposed to flagellate cells increased along with the exposure time, and the cell count of gill-associated C. antiquaestimated with chlorophyll level was higher than that of C. marina. These results suggest that the ROS-producing activity and affinity of Chattonellacells to the gill surface may be important factors influencing the fish-killing activity of Chattonellaspecies.

Forming a curved plate is an essential process for building a ship. The process is currently performed manually, which is a critical bottleneck in shipbuilding. Therefore, there is a need to automate the process. In this paper, a novel process is proposed for automating the manual process. The current manual process is analysed to identify properties necessary for automation, and technical approaches for automation are presented. The proposed process consists of measurement, shape evaluation and forming, which constructs a loop that produces a curved plate iteratively. The correctness and convergence of the process are verified.

Alginate is an acidic linear polysaccharide with immune-modulating activities. In this study, we found that enzymatically digested alginate oligomer (AO) with various degrees of polymerization (DP; 2–5) induced a higher level of nitric oxide (NO) production in RAW264.7 cells than undigested alginate polymer (AP). Reverse transcription-polymerase chain reaction and western blot analyses revealed that the expression level of inducible NO synthase in AO-treated RAW264.7 cells was higher than that in AP-treated cells. AO induced nuclear translocation of nuclear factor (NF)-κB p65 subunit in RAW264.7 cells to a greater extent than AP. Although AO and AP induced similar extents of phosphorylation in three mitogen-activated protein (MAP) kinases, c-Jun N-terminal kinase inhibitor exhibited the most potent inhibitory effect on NO induction in AO- and AP-treated RAW264.7 cells, among three MAP kinase inhibitors that were tested.

Heterocapsa circularisquamashowed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquamaexhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marinawas less effective than H. circularisquama, and Heterocapsa triquetrawas almost harmless in all these criteria. These results suggest that H. circularisquamaexerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

An N-acetylgalactosamine (GalNAc)-specific Ca2+-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 μg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 μg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 μg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.

To elucidate the ichthyotoxic mechanism of the harmful dinoflagellate Cochlodinium polykrikoides, a bioassay using damselfish was conducted. After exposure to a live-cell suspension of C. polykrikoides, all the fish were died within 90 min. In the presence of catalase and superoxide dismutase (SOD), no significant reduction in the toxicity of C. polykrikoideson the fish was observed. Furthermore, no significant levels of reactive oxygen species (ROS) were detected in five strains of C. polykrikoidesisolated in Japan. Our results support the idea that certain toxic substances, rather than ROS, are mainly responsible for the fish-killing activity of C. polykrikoides.

In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric–protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)–OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric–protein complex, the effects of other ferric–protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicumand Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric–protein complex, and that an extract of C. ovatadid not. These results suggest that the presence of such a ferric–protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicumand Alexandrium affineand the raphidophycean flagellate Chattonella ovatawere examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicumagainst superoxide and hydroxyl radicals were increased by heat exposure at 100 °C and 200 °C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicumis not clear, the heat-resistance of the extract from G. impudicummight make it a desirable antioxidant.

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodiniumspp., Scrippsiella trochoidea, and Kareniasp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonellaspp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.

α-linolenic acid and linoleic acid isolated from Ulva fasciatashowed toxic effects on red tide phytoplankters in a concentration-dependent manner. Among six species tested, raphidophycean flagellate Heterosigma akashiwowas the most susceptible to these fatty acids, and 50% lethal concentrations (LC50) of α-linolenic acid and linoleic acid were estimated to be 0.58 and 1.91 μg/ml respectively, whereas dinoflagellate Gymnodinium impudicumand Heterocapsa circularisquamawere highly resistant and no significant toxic effects were observed up to 1,000 μg/ml. Both fatty acids were less toxic to fish (devil stinger), zooplankters (brine shrimp and rotifer), and mammalian cell lines (U937, HeLa, Vero, and CHO cells) than H. akashiwo.

Heterocapsa circularisquama(Dinophyceae), a red tide dinoflagellate, is toxic to bivalve molluscs such as the pearl oyster (Pinctada fucata), but no harmful effects of this alga on fish have been observed so far. We found that 7 strains of H. circularisquamashowed hemolytic activities toward rabbit erythrocytes in a cell-density dependent manner, but to quite different extents. The strains which are known to be highly toxic to bivalves tend to show stronger hemolytic activities and vice versa, suggesting that the hemolytic activity is parallel with the shellfish toxicity of H. circularisquama.Since the hemolytic assay is simple, semiquantitative, and reproducible, this assay is useful not only to search for certain toxins responsible for the shellfish toxicity of H. circularisquamabut also to estimate the potential toxicity of a newly appeared strain of H. circularisquama.

Red tide phytoplankton, Chattonella marinaand Heterosigma akashiwo, are known to generate superoxide anion (O2-). We found that galacturonic acid (GalUA) stimulated C. marinaand H. akashiwoto generate increased amounts of O2-. Since such effect was not observed in any other monosaccharides tested, our results suggest that the binding of GalUA to specific sites on the flagellate cell surface may induce the increase of O2-production.

Chattonella marinaand Heterosigma akashiwo, known as red tide phytoplankton, are naturally wall-less and have quite fragile cell structures. In this study, we found that an equilibrium dialysis technique allowed the study of lectin binding to these flagellates. The results suggested that concanavalin A (Con A) binds to these flagellate cells through the specific carbohydrate moieties on the cell surface. Interestingly, the binding of an excess of Con A on the cell surface caused morphological changes concomitant with discharge of glycocalyx, a polysaccharide-containing common structure on the external cell surface of these flagellates. Fluorescent microscopic observation using FITC-labeled Con A (F-Con A) confirmed that F-Con A molecules are localized on the discharged glycocalyx.

Heterocapsa circularisquama(Dinophyceae), a noxious red tide dinoflagellate, is known to have a specifically lethal effect on shellfish, especially bivalves such as pearl oyster (Pinctada fucata), but no detrimental effects of this alga on fishes have not been observed so far. In this study, we found that H. circularisquamawas toxic to a microzooplankton, a rotifer (Brachionus plicatilis) in a cell concentration-dependent manner, while the cultured supernatant or ultrasonic ruptured H. circularisquamahad no significant toxic effect on the rotifer. Since no such toxic effects on the rotifer were observed in Chattonella marina, Heterosigma akashiwo, or Cochlodinium polykrikoides, other species of harmful red tide plankton, H. circularisquamamay have a strictly specific toxic mechanism against the rotifer as well as bivalves.

Chattonella marinais well known as the causative organism of red tide, and is highly toxic to fish. However, the toxic mechanism of C. marinahas not been established. Recent studies demonstrated that C. marinagenerates reactive oxygen species (ROS) such as O2-and H2O2. In this study, we attempted to establish mutant strains of C. marinausing a chemical mutagen, ethyl methane sulfonate (EMS). After 48 h of treatment with 1 mg/ml of EMS at 26°C, several viable cells were cloned, while more than 90% of C. marinacells died under these conditions. Among the strains isolated, one strain (mutant C) was significantly decreased in the production of O2-, but its growth rate was indistinguishable from the parental C. marina. Furthermore, superoxide dismutase (SOD) had almost no effect on the proliferation of mutant C, while SOD suppressed the growth of parental C. marina. In accordance with the lower level of O2-generation, mutant C was less toxic to Vibrio alginolyticusin a plankton/bacteria co-culture system as compared to parental C. marina. In contrast, no significant difference in H2O2production between mutant C and parental C. marinawas observed. These results may provide experimental evidence for a direct connection between the toxic effect of C. marinaagainst V. alginolyticusand the production of superoxide anion. Although the detailed mechanism of the production of superoxide anion by C. marinais still unclear, our mutant strain isolated in this study may be a good tool for the clarification of the mechanism of O2-generation by C. marina.

We tested the hypothesis that cationic peptides (polyarginine; poly-argn) and eosinophil-derived major basic protein (MaBP) increase permeability by stimulating the release of histamine and/or nitric oxide. We used intravital microscopy, clearance, and integrated optical intensity (IOI), using FITC-dextran 150 (FITC-dx 150) as a tracer, to evaluate changes in microvascular permeability in the hamster cheek pouch. Poly-argnat 1 μM(topical) increased the clearance of FITC-dx 150 from 610 to 3240 nl/60 min/g. In contrast, 1 μMpolyglutamic acid (poly-glun; and anionic peptide) did not affect the clearance of FITC-dx 150 (605 nl/60 min/g). At 0.5 and 1.0 nM, poly-argnincreased the clearance of FITC-dx 150 from 610 to 1722 and 2396 n1/60 min/g (P< 0.05). Similarly, 0.5 nMMaBP increased clearance from 591 ± 38 to 1967 ± 168 n1/60 min/g. L-NAME at 10-4Mdid not prevent the 0.5 nMMaBP-induced elevation in clearance (1784 ± 350). Poly-argnat 1 nMincreased net mean IOI by 21.5 ± 7.2 units. This elevation was not inhibited by topical 10-4ML-NMMA (27.5 ± 8.4). Using 0.5 nMpoly-argnas agonist, we assayed suffusate samples for their histamine concentration using a competitive enzyme immunoassay and found no detectable histamine. Pyrilamine, an H1antagonist, did not inhibit the 0.5 nMpoly-argn-induced elevation in clearance of FITC-dx 150. We conclude that (1) cationic peptides and MaBP increase microvascular permeability and (2) the increase in microvascular permeability produced by low concentrations of poly-argnand by MaBP is independent of the release of histamine and does not require nitric oxide.

We used the hamster cheek pouch microcirculation to investigate by intravital microscopy the effects of neuropeptide Y (NPY) on arteriolar diameter, leukocyte adhesion to microvascular endothelium, and postcapillary venular permeability. We applied NPY topically for 3 min at concentration of 10-7, 10-9, and 10-11M. We quantified arteriolar diameter and permeability changes by digital image analysis. We used the mass of fluorescein isothiocyanate-Dextran 150 accumulated around postcapillary venules (10-30 μm) to calculate extravasation rates of macromolecules. We also measured the number of adhering white cells per 100-μm length of postcapillary venules using acridine orange to label white blood cells. At the applied doses, NPY did not alter either microvascular permeability to macromolecules or leukocyte adhesion to microvascular walls. NPY, in a dose-dependent manner, constricted arterioles ranging in control diameter from 10 to 60 μm. Vasoconstriction was strongest in arterioles ranging in diameter from 30 to 39 μm at a concentration of NPY of 10-7M. The Y1-type NPY receptor agonist, Leu31, Pro34-NPY, was as potent as NPY, whereas the carboxy-terminal fragment NPY 13-36 had no activity, indicating that the hamster cheek pouch microvasculature expresses the Y1 type of NPY receptor. We also blocked α-adrenergic receptors to test whether norepinephrine is required for NPY-induced vasoconstriction. This blockade did not inhibit the vasoconstriction caused by exogenous NPY. Our results demonstrate that (1) NPY modulates microvascular hemodynamics by changes in arteriolar diameter, (2) the NPY receptor on the hamster cheek pouch microvasculature is of the Y1 type, and (3) exogenous NPY-induced vasoconstriction is independent of the activity of endogenous norepinephrine.

Biochemical signaling determines the specific action of vasomediators in the control of microvascular permeability and tone. We tested the hypothesis that nitric oxide (NO) synthesis is involved in the biochemical signaling pathway of platelet activating factor (PAF). The cheek pouch of anesthetized male Syrian hamsters was used as a microvascular model. Vessel diameter [expressed as the ratio of the experimental to the control (e/c) diameter, with control diameter normalized to I] and extravasation of FITC-dextran 150 by integrated optical intensity (IOI) were determined using intravital fluorescent microscopy and computer-assisted digital image analysis. N-Nitro-L-arginine methyl ester (L-NAME) at 10^-5 and 10^-6 M and N-nitro-L-monomethyl arginine (L-NMMA) at 10^-4 and 10^-5 M were used as inhibitors of NO synthase (NOS). Acetylcholine (ACh) and bradykinin were used as indirect indices of NOS activation. L NAME and L-NMMA attenuated both ACh and bradykinin vasodilatory effects as well as the bradykinin-induced increase in vascular permeability. Topical PAF (10^-7 M) caused vasoconstriction (mean ± SEM e/c ratio = 0.3 ± 0.1) and increased IOI from a normalized baseline of 0 to 67.4 ± 12.8. Topical administration of L-NAME produced differential effects on the series-arranged arterioles but had no effect on postcapillary venular permeability. L-NMMA did not influence the basal arteriolar diameter, but at 10^-5 M it caused a small increase in permeability (IOI = 14.3 ± 4.2). In the presence of NOS inhibitors, PAF caused a reduced arteriolar constriction (e/c ratio = 0.6 ± 0.1) relative to PAF alone. Both NOS inhibitors reduced the PAF-stimulated increase in vasopermeability. At 10^-5 M L-NMMA, the PAF stimulated IOI mean value was 26.1 ± 5.2, while at 10^-4 M L-NMMA the PAF-stimulated IOI was 15.2 ± 2.6 compared to 10^-7 M PAF (67.4 ± 12.8). These results support our hypothesis that NO synthesis is a step in the biochemical signaling pathway of the postcapillary cellular responses to PAF. Copyright 1995, 1999 Academic Press

We investigated the efficacy of novel neuropeptide Y (NPY) antagonists to inhibit the microcirculatory dynamics of NPY in the hamster cheek pouch microcirculation using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter served as an index of vasomotor alterations. Fluorescein isothiocyanate-labeled Dextran 150 served as a tracer for measurements of macromolecular transport. GW 383 and GW 1229, two novel NPY receptor antagonists, were applied topically in separate experiments. Pretreatment with 10−5, 10−6and 10−7MGW 383 and with 10−6and 10−8MGW 1229 attenuated the vasoconstriction induced by 10−7MNPY in a dose-dependent manner. Furthermore, pretreatment with 10−7and 10−8MGW 1229 significantly inhibited the 10−9MNPY-induced vasoconstriction. At these doses, the NPY antagonists did not alter microvascular permeability. Our results demonstrate that the novel NPY antagonists inhibit the vasoconstriction induced by NPY in the hamster cheek pouch microcirculation. We suggest that the inhibition is due to binding of antagonists to Y1-type NPY receptors.

Chick embryos were incubated using standard shell-less techniques for microscopic observations at Days 4.5, 5.0, and 5.5 of the normal 21-day gestation. The chorioallantoic membrane (CAM) was prepared for intravital fluorescence confocal microscopy. A graded series of FITC-dextrans (20, 40, 70, and 150 kDa) was injected via vitelline vein. The changes in interstitial optical intensity due to FITC-dextran extravasation were evaluated by computer-assisted image analysis. Apparent permeability coefficients (P_s) were calculated for first order postcapillary vessels from the changes in intensity as a function of time. On Day 4.5, P_s values (means ± SE × 10^-7 cm/sec) in the CAM microvessels for FITC-Dextran 20, 40, 70, and 150 were 11.8 ± 1.0, 6.4 ± 0.4, 3.1 ± 0.5, and 1.5 ± 0.5, respectively. The respective P_s values fell dramatically on Day 5.0 to 2.2 ± 0.5, 0.7 ± 0.2, 0.6 ± 0.2, and 0.6 ± 0.2. On Day 5.5, P_s values for all these FITC-dextrans were equal to 0.7 ± 0.3. The evaluation of FITC-Dextran 10 on Day 5.5 yielded a P_s value of 1.9 ± 0.3. Our data demonstrate a rapid reduction in microvascular permeability to macromolecules during normal angiogenesis in the early stages of CAM development. Our data also suggest that these changes in permeability may reflect functional adaptations of the CAM. A comparison of our data to those available in the literature for adult and tumoral tissues demonstrates that microvascular permeability properties in these tissues are different from those of the CAM. Copyright 1995, 1999 Academic Press