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.

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.

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.

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.

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.

Mitochondrial DNA reveals secondary contact in Japanese harbour seals, the southernmost population in the western Pacific.

In this study, we used relatively large number of samples (n = 178) and control region of mtDNA (454bp) to clearify the divergence history of Japanese harbour seals (Phoca vitulina stejnegeri) and phylogenetic relationship between the seals in Japan and other countries. Our results suggested that Japanese harbour seals possibly consisted of more than two lineages and secondary contact of populations after a long isolation. Furthermore, one of the lineage was made only by Japanese harbour seals (Group P1). The proportion of Group P1 was the highest at the South West and gradually decreased towards the North East of Hokkaido, Japan. On the other hand, the haplotypes do not belonged to Group P1 showed close relationship to the seals in the North Pacific. Based on the fossil record of harbour seal in Japan and the range of sea ice during the Last Glacial Maximum (LGM), Group P1 might have entered Japan before the LGM and became isolated due to the geographical boundary, and gradually extended its range from the South West towards the North East of Hokkaido after the disappearance of the sea ice, while the seals which are not in Group P1 immigrated into Japan from the North Pacific.

Host characteristics and infection level of an intestinal parasite Corynosoma strumosum (Acanthocephala) in the Kuril harbor seal of Erimo Cape, Hokkaido, Japan.

The Kuril harbor seal around Hokkaido is presently recovering from a resource crisis while conflicts with local fisheries have become a concern. However, its feeding habits, which are fundamental information for taking proper preventive measures, are still poorly understood. We thus examined the infection status of a trophically-transmitted parasite, Corynosoma strumosum in the seals of Erimo Cape, to assess the host's feeding habits with a practical view of the parasite as a biological indicator. A total of 2802 worms were found from 20 male and 20 female by-caught animals in salmon set nets within local fisheries during August to November 2014. The parasite abundance was explained mainly by the host's developmental stage and intestinal length while weakly affected by gender and body size, through an estimation of generalized linear models combined with hierarchical partitioning. Considering the past records that demersal fishes are the probable main sources of infection, the infection level may owe to individual host differences regarding these sources and/or feeding grounds with relating the host characteristics. This supports that the resource management of Kuril harbor seals requires careful consideration of the individual differences in feeding behavior.

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.

Three-Dimensional Human Cardiac Tissue Engineered by Centrifugation of Stacked Cell Sheets and Cross-Sectional Observation of Its Synchronous Beatings by Optical Coherence Tomography.

Three-dimensional (3D) tissues are engineered by stacking cell sheets, and these tissues have been applied in clinical regenerative therapies. The optimal fabrication technique of 3D human tissues and the real-time observation system for these tissues are important in tissue engineering, regenerative medicine, cardiac physiology, and the safety testing of candidate chemicals. In this study, for aiming the clinical application, 3D human cardiac tissues were rapidly fabricated by human induced pluripotent stem (iPS) cell-derived cardiac cell sheets with centrifugation, and the structures and beatings in the cardiac tissues were observed cross-sectionally and noninvasively by two optical coherence tomography (OCT) systems. The fabrication time was reduced to approximately one-quarter by centrifugation. The cross-sectional observation showed that multilayered cardiac cell sheets adhered tightly just after centrifugation. Additionally, the cross-sectional transmissions of beatings within multilayered human cardiac tissues were clearly detected by OCT. The observation showed the synchronous beatings of the thicker 3D human cardiac tissues, which were fabricated rapidly by cell sheet technology and centrifugation. The rapid tissue-fabrication technique and OCT technology will show a powerful potential in cardiac tissue engineering, regenerative medicine, and drug discovery research.

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.

Real-time, noninvasive optical coherence tomography of cross-sectional living cell-sheets in vitro and in vivo.

Cell sheet technology has a history of application in regenerating various tissues, having successfully completed several clinical trials using autologous cell sheets. Tomographic analysis of living cell sheets is an important tool in the field of cell sheet-based regenerative medicine and tissue engineering to analyze the inner structure of layered living cells. Optical coherence tomography (OCT) is commonly used in ophthalmology to noninvasively analyze cross-sections of target tissues at high resolution. This study used OCT to conduct real-time, noninvasive analysis of living cell sheet cross sections. OCT showed the internal structure of cell sheets in tomographic images synthesized with backscatter signals from inside the living cell sheet without invasion or damage. OCT observations were used to analyze the static and dynamic behaviors of living cell sheets in vitro and in vivo including (1) the harvesting process of a C2C12 mouse skeletal myoblast sheet from a temperature-responsive culture surface; (2) cell-sheet adhesion onto various surfaces including a culture surface, a synthetic rubber glove, and the dorsal subcutaneous tissue of rats; and (3) the real-time propagation of beating rat cardiac cells within cardiac cell sheets. This study showed that OCT technology is a powerful tool in the field of cell sheet-based regenerative medicine and tissue engineering.

Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography.

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.

The long-term effects of pitavastatin on blood lipids and platelet activation markers in stroke patients: impact of the homocysteine level.

To examine the impact of the plasma homocysteine level on the anti-atherosclerotic effects of pitavastatin treatment, we retrospectively examined 59 patients who had a history of stroke and had been prescribed pitavastatin for the treatment of dyslipidemia at the Neurology department of Toho University Ohashi Medical Center Hospital. The patients were classified into two groups according to their homocysteine levels. Carotid artery plaque progression was determined before and after pitavastatin treatment. Plasma levels of high-sensitivity C-reactive protein, platelet molecular markers, and von Willebrand factor were measured. Pitavastatin treatment had beneficial effects on the lipid profiles of these patients and slowed atherosclerosis progression. These effects were observed in both the high and low homocysteine groups. Proactive lipid intervention using pitavastatin may inhibit the progression of atherosclerosis and contribute to secondary prevention of stroke in high-risk patients. We conclude that this statin could inhibit progression at any stage of disease and should therefore be proactively administered to these patient groups, regardless of disease severity.