Sublethal treatment with plasma-activated medium induces senescence-like growth arrest of A549 cells: involvement of intracellular mobile zinc.

Plasma-activated medium (PAM) is a solution produced by exposing a liquid medium to non-thermal atmospheric pressure plasma (NTAPP). A number of reactive molecules, such as reactive oxygen species and reactive nitrogen species, are contained in PAM. Therefore, exposure to high doses of PAM results in cell death. We previously demonstrated that intracellular zinc (Zn2+) serves as an important mediator in PAM-induced cell death; however, the effects of sublethal treatment with PAM on cell functions are not fully understood. In the present study, we found that sublethal PAM treatment suppressed cell proliferation and induced senescence-like changes in lung adenocarcinoma A549 cells. Cell cycle analysis revealed that PAM induced cell cycle arrest at the G2/M phase. PAM increased the level of intracellular free Zn2+ and the Zn2+ chelator TPEN counteracted PAM-induced growth suppression, suggesting that Zn2+ functions in PAM-induced growth suppression. In addition, sublethal treatment with PAM induced phosphorylation of ATM kinase, accumulation of p53 protein, and expression of p21 and GADD45A, which are known p53 target genes, in a Zn2+-dependent manner. These results suggest that the induction of growth arrest and cellular senescence by sublethal PAM treatment is mediated by Zn2+-dependent activation of the ATM/p53 pathway.

Pneumocystis jirovecii pneumonia developed in a patient with rheumatoid arthritis after 14 weeks of iguratimod add-on to treatment with methotrexate and etanercept.

A 66-year-old woman who had rheumatoid arthritis and underwent a long-term treatment with methotrexate and etanercept developed Pneumocystis jirovecii pneumonia (PCP) 3 months after iguratimod add-on. Although most rheumatologists might have the impression that iguratimod has less toxicity and immunosuppressive effect compared with methotrexate and biologic disease-modifying antirheumatic drugs, this case suggests that iguratimod may increase the risk of PCP, especially in combination with other drugs.

Sphingosine-1-phosphate-enhanced Wnt5a promotes osteogenic differentiation in C3H10T1/2 cells.

In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration.

Development of in vivo tissue-engineered microvascular grafts with an ultra small diameter of 0.6 mm (MicroBiotubes): acute phase evaluation by optical coherence tomography and magnetic resonance angiography.

Biotubes, i.e., in vivo tissue-engineered connective tubular tissues, are known to be effective as vascular replacement grafts with a diameter greater than several millimeters. However, the performance of biotubes with smaller diameters is less clear. In this study, MicroBiotubes with diameters <1 mm were prepared, and their patency was evaluated noninvasively by optical coherence tomography (OCT) and magnetic resonance angiography (MRA). MicroBiotube molds, containing seven stainless wires (diameter 0.5 mm) covered with silicone tubes (outer diameter 0.6 mm) per mold, were embedded into the dorsal subcutaneous pouches of rats. After 2 months, the molds were harvested with the surrounding capsular tissues to obtain seven MicroBiotubes (internal diameter 0.59 ± 0.015 mm, burst pressure 4190 ± 1117 mmHg). Ten-mm-long MicroBiotubes were allogenically implanted into the femoral arteries of rats by end-to-end anastomosis. Cross-sectional OCT imaging demonstrated the patency of the MicroBiotubes immediately after implantation. In a 1-month follow-up MRA, high patency (83.3 %, n = 6) was observed without stenosis, aneurysmal dilation, or elongation. Native-like vascular structure was reconstructed with completely endothelialized luminal surfaces, mesh-like elastin fiber networks, regular circumferential orientation of collagen fibers, and α-SMA-positive cells. Although the long-term patency of MicroBiotubes still needs to be confirmed, they may be useful as an alternative ultra-small-caliber vascular substitute.

Plasma-activated medium-induced intracellular zinc liberation causes death of SH-SY5Y cells.

Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons. Plasma-activated medium (PAM), which is prepared by the irradiation of cell-free medium with non-thermal atmospheric pressure plasma, induces cell death in various types of cancer cell. Since PAM contains reactive oxygen species (ROS), its anti-cancer effects are thought to be attributable to oxidative stress. Meanwhile, oxidative stress has been shown to induce the liberation of zinc (Zn(2+)) from intracellular Zn(2+) stores and to provoke Zn(2+)-dependent cell death. In this study, we thus examined whether Zn(2+) is involved in PAM-induced cell death using human neuroblastoma SH-SY5Y cells. Exposure to PAM triggered cell death in SH-SY5Y cells. The cell-permeable Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) protected against PAM-induced cell death. Zn(2+) imaging using the fluorescent Zn(2+) probe FluoZin-3 revealed that PAM elicited a rise of intracellular free Zn(2+). In addition, PAM stimulated PARP-1 activation, mitochondrial ROS generation, and the depletion of intracellular NAD(+) and ATP. These findings suggest that PAM-induced PARP-1 activation causes energy supply exhaustion. Moreover, TPEN suppressed all of these events elicited by PAM. Taken together, we demonstrated here that Zn(2+) released from intracellular Zn(2+) stores serves as a key mediator of PAM-induced cell death in SH-SY5Y cells.

The vomeronasal organ and incisive duct of harbor seals are modified to secrete acidic mucus into the nasal cavity.

Most terrestrial mammals have a vomeronasal system to detect specific chemicals. The peripheral organ of this system is a vomeronasal organ (VNO) opening to the incisive duct, and its primary integrative center is an accessory olfactory bulb (AOB). The VNO in seals is thought to be degenerated like whales and manatees, unlike otariids, because of the absence of the AOB. However, olfaction plays pivotal roles in seals, and thus we conducted a detailed morphological evaluation of the vomeronasal system of three harbor seals (Phoca vitulina). The VNO lumen was not found, and the incisive duct did not open into the oral cavity but was recognized as a fossa on the anteroventral side of the nasal cavity. This fossa is rich in mucous glands that secrete acidic mucopolysaccharides, which might originate from the vomeronasal glands. The olfactory bulb consisted only of a main olfactory bulb that received projections from the olfactory mucosa, but an AOB region was not evident. These findings clarified that harbor seals do not have a VNO to detect some chemicals, but the corresponding region is a specialized secretory organ.

Deterioration of Cement-Based Materials in Low-Temperature Seawater.

Cementitious materials have potential for infrastructure development in low-temperature marine environments, including in seawater at high latitudes and in deep-sea environments (water depths of >1000 m). Although the marine deterioration of cementitious materials has been widely investigated, the influence of seawater temperature has not been elucidated. In this study, to determine the effects of low-temperature seawater on the durability of cementitious materials, cement paste specimens were immersed in a seawater tank at room temperature and 2 °C for 433 days. The specimen immersed in low-temperature seawater exhibited significant deterioration with a partially collapsed surface, whereas the specimen immersed in room-temperature seawater maintained its original shape. Following low-temperature immersion, Ca dissolution was more pronounced and dissolved portlandite, decalcified calcium (alumino)silicate hydrate (C-(A-)S-H), magnesium (alumino)silicate hydrate (M-(A-)S-H), and thaumasite were observed on the collapsed surface. Such significant deterioration can be attributed to the increased solubility of portlandite under low-temperature conditions, which could promote Ca dissolution and subsequently lead to C-(A-)S-H decalcification and the formation of M-(A-)S-H and thaumasite. These insights are expected to contribute to the successful construction and maintenance of cementitious structures in low-temperature seawater.

Anti-Brucella antibodies in seals at coastal locations of Hokkaido, Japan, with focus on life stages.

The enzyme-linked immunosorbent assay (ELISA) was applied to detect antibodies against Brucella abortus in serum samples from four seal species at nine coastal locations of Hokkaido, Japan. These antibodies were detected in 27% (32/118) of Western Pacific harbor seals (Phoca vitulina stejnegeri) at Cape Erimo. The antibodies were observed in spotted seals (P. largha) in one out of six at Nemuro, in two out of three at Rebun Island, in one out of two at Bakkai, and in examined one at Soya. They were also found in respective examined one ribbon seal (Histriophoca fasciata) and one ringed seal (Pusa hispida) at Akkeshi. Harbor seals that tested positive were mostly yearlings (35%, 20/57) and juveniles (45%, 10/22), while only one pup (1/13) and one subadult (1/5) tested positive with low titers of the antibody; no antibodies were observed in adults (n=21). These results suggest that Brucella mainly infected harbor seals from the environment while weaning, and the bacteria were cleared during the early life stage of the seals. In spotted seals, however, antibodies were also detected in adults, suggesting that spotted seals could become infected with Brucella even as adults. It is also possible that a different, more persistent strain of Brucella may have infected the spotted seals.

Morphological identification in skull between spotted seal and harbor seal using geometric morphometrics.

The morphology of the skull contains considerable ecological information about a species, because the skull contains sensory organs that are used to look for food, compete for mates, or to migrate. Spotted seals (Phoca largha) and harbor seals (Phoca vitulina) are similar in body size and pelage color but differ in habitat use and reproductive biology. The current study aims to clarify differences in the shapes of skulls in the spotted and harbor seals using geometric morphometrics and to discuss whether ecological differences can explain morphological differences in skulls. First, we discovered that the age at which the shape of skulls stopped changing was 7 years in both species, using the linear-threshold model. Using a total of 75 landmarks, 54 individuals (25 spotted seals, 29 harbor seals) that were older than the age at which skulls stopped changing were correctly identified at a rate of 100%. The total of 75 landmarks was narrowed down to eight key landmarks that resulted in an identification accuracy rate of 100% using random forests. Of the eight landmarks, seven were related to feeding apparatus, indicated that the harbor seal had a broader mouth and mandible than the spotted seal. Because of both species were dietary generalists and classified as pierce feeders, we suggested that the different features in the shapes of their skulls were caused not only by differences in their feeding behavior but also other differences related to reproductive behavior.

Notocotylus ikutai n. sp. (Digenea: Notocotylidae) from lymnaeid snails and anatid birds in Hokkaido, Japan.

An unknown species of the genus Notocotylus (Digenea: Notocotylidae) was found as the larval stage from the lymnaeid snail, Radix auricularia, in a static water area of the Chubetsu River, Hokkaido, the northernmost island of Japan. A DNA barcoding identification system was applied to detect the adult stage. Through the inspection of anatid game birds in Hokkaido, Anas crecca, Anas platyrhynchos, Anas zonorhyncha, and Mareca penelope were demonstrated to serve as the definitive hosts. The detailed morphological features of the species were characterized using adults raised experimentally in immunosuppressed mice and naturally developed larvae in R. auricularia. Although the species is morphologically similar to Notocotylus attenuatus and Notocotylus magniovatus in both adult and larval stages, its taxonomic independence was confirmed by a comprehensive study based on molecular phylogeny, morphology, and ecology. Here we propose Notocotylus ikutai n. sp. for this species. The migratory behavior of the anatid hosts and the North-Eurasian distribution of R. auricularia suggest that the new species is widely distributed in the northern Far East.

Foraging activity of harbour porpoises around a bottom-gillnet in a coastal fishing ground, under the risk of bycatch.

Bycatch of harbour porpoises (Phocoena phocoena) by gillnets is a recognised threat to populations. To develop effective mitigation measures, understanding the mechanics of bycatch is essential. Previous studies in experimental conditions suggested foraging activity is an important factor influencing porpoises' reaction to gillnets. We acoustically observed the behaviour of wild harbour porpoises around a bottom-gillnet set-up in a commercial fishing ground, especially foraging activity. Passive acoustic event recorders (A-tags) were fixed to the ends of the gillnet, and recorded for 1 392 hours. Although harbour porpoises frequently and repeatedly appeared around the net each day, incidental bycatch occurred only three times during the observations. The stomach contents of two individuals contained mainly Ammodytes sp., which were observable around the bottom-gillnet but not targeted by the fishery. A total of 276 foraging incidents were acoustically detected, and 78.2% of the foraging activity was in the bottom layer (deeper than 25 m). Porpoises appeared around the net with more frequency on the day of a bycatch incident than on the days without bycatch. These results suggest that the harbour porpoises appeared around the bottom-gillnet to forage on fish distributed in the fishing ground, but not captured by this bottom-gillnet. Thus, porpoises face the risk of becoming entangled when foraging near a gillnet, with the probability of bycatch simply increasing with the length of time spent near the net. Bycatch mitigation measures are discussed.

Physiological impact of intrinsic ADP inhibition of cyanobacterial FoF1 conferred by the inherent sequence inserted into the gammasubunit.

The F(o)F(1)-ATPase, which synthesizes ATP with a rotary motion, is highly regulated in vivo in order to function efficiently, although there remains a limited understanding of the physiological significance of this regulation. Compared with its bacterial and mitochondrial counterparts, the gamma subunit of cyanobacterial F(1), which makes up the central shaft of the motor enzyme, contains an additional inserted region. Although deletion of this region results in the acceleration of the rate of ATP hydrolysis, the functional significance of the region has not yet been determined. By analysis of rotation, we successfully determined that this region confers the ability to shift frequently into an ADP inhibition state; this is a highly conserved regulatory mechanism which prevents ATP synthase from carrying out the reverse reaction. We believe that the physiological significance of this increased likelihood of shifting into the ADP inhibition state allows the intracellular ATP levels to be maintained, which is especially critical for photosynthetic organisms.

Genetic variation in the harbor seal (Phoca vitulina) and spotted seal (Phoca largha) around Hokkaido, Japan, based on mitochondrial cytochrome b sequences.

The harbor seal (Phoca vitulina) and spotted seal (Phoca largha) are the main seal species around Hokkaido, Japan. While some investigations have been conducted on the ecology and morphology of these two species, there is a lack of genetic information. We studied variation in mitochondrial cytochrome b sequences in the two species. Fifteen haplotypes were observed in 39 harbor seals from Erimo, Akkeshi, and Nosappu, and 23 were observed in 31 spotted seals from Erimo, Akkeshi, Nosappu, Rausu, Yagishiri Island, and Hamamasu. Phylogenetic trees showed two harbor seal lineages: Group I contained primarily haplotypes from Erimo, and Group II contained haplotypes from Akkeshi and Nosappu. Because the Erimo population had fewer haplotypes and less nucleotide diversity than the Akkeshi and Nosappu populations, we considered it to be Isolated from the others. In contrast, genetic variance within populations of spotted seals (97.3%) was far higher than that among populations (2.7%), determined by analysis of molecular variance. There were no significant difference among the spotted seal populations, indicating the absence of distinct lineages around Hokkaido. The differences in the genetic population structure between the two species could have been generated by their ecological differences. This study provides basic genetic information on these seal species and will contribute to the conservation and management of fisheries and seals throughout Hokkaido.

One-sided infections by intestinal parasites in two sympatric porpoises bycaught from the Nemuro Strait of Hokkaido, Japan.

Due to the difficulties and limitations associated with field sampling of marine mammals, quantitative surveys on their parasites are still scarce in Japan. We here report the infection statuses of intestinal parasites in harbor porpoises (n = 34) and Dall's porpoises (n = 10), bycaught between 2014 and 2018 by a bottom set-net fishery in the northern part of the Nemuro Strait, Japan. Necropsies of the harbor porpoises recovered one digenean, Synthesium nipponicum, and two acanthocephalans, Corynosoma sp. and Bolbosoma sp.; no infection was observed in the Dall's porpoises. The dominant parasites were Corynosoma sp. and S. nipponicum, with a prevalence of 73.53 and 64.71% and a mean abundance of 43.88 and 7.38 individuals, respectively. Although the two porpoise-species have a sympatric distribution in this narrow strait, the findings support the existence of trophic-link dependences, which can differentially affect vulnerability to these parasites.

Molecular markers comparing the extremely simple body plan of dicyemids to that of lophotrochozoans: insight from the expression patterns of Hox, Otx, and brachyury.

Because of their extremely simple body plan, dicyemids have long been the subject of phylogenetic controversy, regarding whether their body plan reflects their primitiveness or a degeneration from complex metazoans. Several studies have argued that the simple body plan of dicyemids are likely secondarily derived from higher lophotrochozoan animals, as a result of their endoparasitic, or endosymbiotic, lifestyle in the cephalopod kidney. To clarify the evolution of their simple body plan, we investigated the developmental expression patterns of three important regulatory genes, the central type Hox gene (DoxC), otx, and brachyury homologs in the dicyemid mesozoa, Dicyema orientale. DoxC was expressed in the trunk and tail of the asexually developing vermiform embryo, with clear anterior boundaries. Do-otx was expressed in the vegetal pole cells of the developing infusoriform embryos, suggesting that the invagination in infusoriform embryo is homologous to the gastrulation of other metazoans. Do-bra is expressed in the presumptive ventral cells, which are ventral to the opening of the urn cavity. The expression of Do-bra suggests that the urn cavity opening of the infusoriform embryo is comparable to the stomodium of trochophore larvae. These gene expression patterns provide molecular clues to trace the evolutionary history of degeneration in the dicyemid embryogenesis and life cycle from those of ancestral lophotrochozoan animals.

Growth variation in skull morphology of Kuril harbor seals (Phoca vitulina stejnegeri) and spotted seals (Phoca largha) in Hokkaido, Japan.

We examined morphological growth variations in skull features between the Kuril harbor seal and the spotted seal in Hokkaido, Japan. Skulls from 80 Kuril harbor seals and 41 spotted seals were collected, and we measured 29 metric and 6 non-metric cranial characteristics. Three growth classes were defined according to the postnatal developmental stage: pups (0 year), subadults (1-4 years old) and adults (more than 5 years old). We detected sexual dimorphism in Kuril harbor seal pups, subadults, and adults. Although interspecies differences were detected in each growth class, Kuril harbor seals were larger and more massive than spotted seals; this feature was already detectable in pups. We did not detect certain cranial characteristics with which to identify the two species, but it was possible to identify any unknown specimens to their species, sex, and growth class using the cranial data generated in this study. Using 6 non-metric cranial characteristics, we identified significant interspecies differences with regard to the shape of the temporozygomatic suture and the extent of the nasal-incisive suture; the shape of the temporozygomatic suture and the shape of the nares were indicators of growth class in Kuril harbor seals. Although non-metric cranial characteristics have a lower discriminating power than metric characteristics, they are easy to use in the field even by inexperienced researchers.

Surveillance of amyloidosis in stranded and bycaught cetaceans off Hokkaido, Japan.

Systemic amyloidosis is rarely reported among cetaceans, and a surveillance dedicated for its occurrence across a certain geographic location has not been reported. Between 2013 and 2018, comprehensive gross and histopathologic examinations were conducted on 54 animals comprising 11 species of stranded and bycaught cetaceans in Hokkaido, Japan. Systemic amyloidosis was diagnosed in 2 out of 3 Stejneger's beaked whales (Mesoplodon stejnegeri), through Congo red staining and immunohistochemistry for amyloid A. The kidney and gastrointestinal tract had the largest amounts of amyloid deposits, representing a previously undescribed organ distribution in the species. The current study demonstrates the possibility of Stejneger's beaked whales being prone to the development of systemic amyloidosis, and highlights the need for further investigations.

Infection status of commercial fish with cystacanth larvae of the genus Corynosoma (Acanthocephala: Polymorphidae) in Hokkaido, Japan.

Acanthocephalans of the genus Corynosoma are known as intestinal parasites, mainly of pinnipeds. Human corynosomiasis has been reported as an infrequent foodborne disease in Hokkaido, the northernmost island of Japan. Potential sources of the human infection are marine fish, because they are paratenic hosts of these parasites. In this study, the prevalence and intensity of larval Corynosoma in commercial fish from 17 fishing ports of Hokkaido were examined from April 2016 to January 2019. Out of a total of 1217 fish examined, 122 (10.0%) were infected with cystacanth larvae. The infected fish assemblage was composed of 7 families and 13 species from all the coastal seas of Hokkaido (the Pacific Ocean, Okhotsk Sea, and Japan Sea), showing that commercial fish can be source of human infection when eaten raw. Flatfish of the family Pleuronectidae showed the highest intensity of cystacanths, ranging from 1 to 56. A DNA barcoding system was developed in this study, based on the standard mitochondrial cox1 sequences of morphologically identified adults of Corynosoma spp. from pinnipeds in Hokkaido. By using the DNA barcoding, most of the fish-derived cystacanths were identified as either C. strumosum or C. villosum, and furthermore, a clinical isolate from human as C. villosum. Both of the species were commonly detected from various fish of Hokkaido, irrespective of the coastal seas. Flatfish frequently harbored C. villosum. Considering the wide range of commercial fish in Hokkaido and the advanced transportation system of fresh fish, there is a possibility that human corynosomiasis will occur everywhere in Japan.

Expression of Sox and fibrillar collagen genes in lamprey larval chondrogenesis with implications for the evolution of vertebrate cartilage.

Lampreys possess unique types of cartilage in which elastin-like proteins are the dominant matrix component, whereas gnathostome cartilage is mainly composed of fibrillar collagen. Despite the differences in protein composition, the Sox-col2a1 genetic cascade was suggested to be conserved between lamprey pharyngeal cartilage and gnathostome cartilage. We examined whether the cascade is conserved in another type of lamprey cartilage, the trabecular cartilage. We found that SoxD and SoxE are expressed in both trabecular and pharyngeal cartilages. However, trabecular cartilage shows no clade A fibrillar collagen gene expression, including genes expressed in pharyngeal cartilage of this animal. On the basis of these observations, we propose that lampreys possess an ancestral type of cartilage that is similar to amphioxus gill cartilage, and in this respect, gnathostome cartilage can be regarded as derived for the loss of elastin-like protein as a cartilage component and recruitment of fibrillar collagen, which is included as a minor component in the ancestral cartilage, as the main component.