Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

Osteoclastic and Osteoblastic Responses to Hypergravity and Microgravity: Analysis Using Goldfish Scales as a Bone Model.

It is known that the bone matrix plays an important role in the response to physical stresses such as hypergravity and microgravity. In order to accurately analyze the response of bone to hypergravity and microgravity, a culture system under the conditions of coexistence of osteoclasts, osteoblasts, and bone matrix was earnestly desired. The teleost scale is a unique calcified organ in which osteoclasts, osteoblasts, and the two layers of bone matrix, i.e., a bony layer and a fibrillary layer, coexist. Therefore, we have developed in vitro organ culture systems of osteoclasts and osteoblasts with the intact bone matrix using goldfish scales. Using the scale culture system, we examined the effects of hypergravity with a centrifuge and simulated ground microgravity (g-µG) with a three-dimensional clinostat on osteoclasts and osteoblasts. Under 3-gravity (3G) loading for 1 day, osteoclastic marker mRNA expression levels decreased, while the mRNA expression of the osteoblastic marker increased. Upon 1 day of exposure, the simulated g-µG induced remarkable enhancement of osteoclastic marker mRNA expression, whereas the osteoblastic marker mRNA expression decreased. In response to these gravitational stimuli, osteoclasts underwent major morphological changes. By simulated g-µG treatments, morphological osteoclastic activation was induced, while osteoclastic deactivation was observed in the 3G-treated scales. In space experiments, the results that had been obtained with simulated g-µG were reproduced. RNA-sequencing analysis showed that osteoclastic activation was induced by the down-regulation of Wnt signaling under flight-microgravity. Thus, goldfish scales can be utilized as a bone model to analyze the responses of osteoclasts and osteoblasts to gravity.

Search for nutritional and inflammatory marker ratios to predict neutropenia in FEC therapy for breast cancer: A retrospective observational study.

OBJECTIVE: Nutritional and inflammatory marker ratios are known to predict response to chemotherapy in breast cancer, but whether they predict adverse effects caused by chemotherapy remains unclear. We investigated whether nutritional and inflammatory marker ratios before starting FEC therapy (5-fluorouracil, epirubicin, and cyclophosphamide) predict grade 4 neutropenia as a serious adverse effect. MATERIALS AND METHODS: 61 patients with breast cancer who started FEC therapy for the first time as preoperative or postoperative chemotherapy were studied. Relevant nutritional and inflammatory marker ratios were compared between patients who developed grade 4 neutropenia (n = 44) and those who did not (n = 17). RESULTS: In univariate analysis, occurrence of neutropenia was related significantly (p < 0.05) to pre-FEC-therapy white blood cell count, platelet count, neutrophil count, lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and modified Glasgow prognostic score. Analysis using cutoff values obtained from receiver operating characteristic curves showed that LMR, NLR, and PLR predicted grade 4 neutropenia. However, multivariate logistic regression analysis identified no independent factor associated with grade 4 neutropenia. A post-hoc power analysis revealed an inadequate sample size. CONCLUSION: Inflammatory marker ratios, especially PLR, may predict grade 4 neutropenia caused by FEC therapy for breast cancer. Although multivariate analysis identified no independent predictive markers in this study due to inadequate sample size, further prospective large-scale research is needed to examine the usefulness of nutritional and inflammatory marker ratios for predicting adverse effects.

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish.

To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.

Expression dynamics of the genes for the hypothalamo-pituitary-gonadal axis in tiger puffer (Takifugu rubripes) at different reproductive stages.

Tiger puffer, Takifugu rubripes, a commercially important long-distance migratory fish, return to specific spawning grounds for reproduction. To clarify reproductive neuroendocrine system of the tiger puffer, the changes in the expression levels of the genes encoding three gonadotropin-releasing hormones (GnRHs), gonadotropin-inhibitory hormone (GnIH), GnIH receptor (GnIH-R), kisspeptin and kisspeptin receptor in the brain and gonadotropin (GTH) subunits, growth hormone (GH) and prolactin (PRL) in the pituitary were examined in the tiger puffer captured in the wild at different reproductive stages, namely immature and mature fish of both sexes, and post-ovulatory females that were obtained by hormonal treatment. The amounts of three gnrh mRNAs, gnih, gnih-r, fshb and lhb were substantially increased in the mature fish compared to the immature fish, especially in the females, and these augmented expressions were drastically decreased in the post-ovulatory females. gh expression showed a slight increase in the mature males. In contrast, kiss2, kiss2r and prl did not show significant changes in the males but significantly decreased in the post-ovulatory females. The present results demonstrate the expression dynamics of the hypothalamo-pituitary-gonadal axis genes associated with the reproductive conditions and the possible involvement of the GnRH/GnIH/GTH system in the regulation of the sexual maturation and spawning in the wild tiger puffer.

Hyperammonemia with impaired consciousness caused by continuous 5-fluorouracil infusion for colorectal cancer: A case report.

A 66-year-old woman was diagnosed with stage IVb sigmoid colon cancer. Modified FOLFOX-6 (mFOLFOX-6; levofolinate‒fluorouracil‒oxaliplatin) plus panitumumab was selected as the chemotherapeutic regimen, but she was administered a regimen without oxaliplatin (L-OHP) or bolus 5-fluorouracil (5-FU) because of her general condition and concern about adverse effects. The patient had impaired consciousness on day 3 of chemotherapy. Computed tomography (CT) and magnetic resonance imaging (MRI) of the brain showed no findings of hemorrhage, infarction, brain metastasis, and leukoencephalopathy. Except for high blood ammonia concentration (353 µg/dL), there were no other findings that could have caused her condition. Impaired consciousness due to hyperammonemia was diagnosed. We started an intravenous drip supplemented with branched chain amino acids for liver protection. Approximately 6 hours later, blood ammonia level improved to 88 µg/dL, which approached the reference value. Consciousness level improved over time, reaching a level of alertness on day 5 after starting chemotherapy. 5-FU was suspected to be the cause of impaired consciousness due to hyperammonemia, but the exact cause could not be identified because most of the previously reported cases were given L-OHP, bolus 5-FU, and other concomitant medications. In this case, since there were no other concomitant medications, it is highly probable that continuous infusion of 5-FU alone caused impaired consciousness due to hyperammonemia. This is an important case that indicates the need to monitor carefully for the occurrence of hyperammonemia when 5-FU is administered continuously; it also proposes future issues for investigation.

Geometrical Conversion of the EGFR Extracellular Domain by Adiabatic Mapping Combining Normal Mode Analysis of the Elastic Network Model and Energy Optimization.

The activation of epidermal growth factor receptor (EGFR) involves the geometrical conversion of the extracellular domain (ECD) from the tethered to the extended forms with the dynamic rearrangement of the relative positions of four subdomains (SDs); however, this conversion process has not yet been thoroughly understood. We compare the two different forms of the X-ray crystal structures of ECD and simulate the ECD conversion process using adiabatic mapping that combines normal mode analysis of the elastic network model (ENM-NMA) and energy optimization. A comparison of the crystal structures reveals the rigidity of the intradomain geometry of the SD-I and -III backbone regardless of the form. The forward mapping from the tethered to the extended forms retains the intradomain geometry of the SD-I and -III backbone and reveals the trends to rearrange the relative positions of SD-I and -III and to dissociate the C-terminal tail of SD-IV from the hairpin loop in SD-II. The reverse mapping from the extended to the tethered forms complements the promotion of ECD conversion in the presence of epidermal growth factor (EGF).

Population pharmacokinetics and pharmacodynamics of fondaparinux in Japanese patients after artificial total knee replacement
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OBJECTIVE: This study was designed to clarify the factors affecting the efficacy, adverse events, and pharmacokinetics of fondaparinux in Japanese patients undergoing artificial knee replacement surgery. MATERIALS AND METHODS: Fondaparinux (1.5 mg/d) was administered subcutaneously to patients (n = 30) at 24 hours after surgery, and blood samples were taken at various time points thereafter. Venous thromboembolism (VTE), presence of bleeding, and pharmacokinetics were evaluated. Multivariate analysis and population pharmacokinetic analysis were performed to detect factors that necessitated withdrawal of fondaparinux and individual differences in its pharmacokinetics. RESULTS: VTE was observed in 9 patients (3 for whom administration was continued and 6 for whom withdrawal was necessary). The maximum plasma concentration of fondaparinux was found to be a significant factor determining withdrawal of the drug. Population pharmacokinetic analysis demonstrated that individual renal function and body weight were significant factors associated with apparent clearance and volume of distribution, respectively. CONCLUSIONS: A high maximum plasma concentration of fondaparinux may result in subcutaneous hemorrhage, necessitating withdrawal of fondaparinux administration. The patient's kidney function and body weight also contribute to individual differences in pharmacokinetics. We recommend considering an adjustment to the dose of fondaparinux based on body weight in patients undergoing artificial knee replacement surgery.
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A quantitative LC/MSMS method for determination of edoxaban, a Xa inhibitor and its pharmacokinetic application in patients after total knee arthroplasty.

Edoxaban was extracted from human plasma by simple protein precipitation with acetonitrile, followed by quantitative determination using a liquid chromatography-mass spectrometry method. The recoveries of edoxaban and the internal standard (ticlopidine) from human plasma were >85%, and the within- and between-day coefficients of variation were within 15%. The limit of quantification in human plasma was 1 ng/mL. The concentration of edoxaban in blood decreased at room temperature, but remained unchanged for 1 week at 4°C. On the other hand, the concentration in plasma at both -20 and -80°C remained unchanged for 5 months. These results indicated that blood samples should be centrifuged immediately or stored at 4°C, and that plasma samples should be stored below -20°C until analysis. This method was applied to human plasma obtained from four patients after total knee arthroplasty. Analysis of edoxaban pharmacokinetics demonstrated an absorption time lag of 4h, a maximum concentration of 110 ± 26 ng/mL and an oral clearance of 37 ± 16 L/h. The analytical methods established in this study will be suitable for determining the concentrations of edoxaban in human plasma.

Neofunctionalization of Androgen Receptor by Gain-of-Function Mutations in Teleost Fish Lineage.

Steroid hormone receptor family provides an example of evolution of diverse transcription factors through whole-genome duplication (WGD). However, little is known about how their functions have been evolved after the duplication. Teleosts present a good model to investigate an accurate evolutionary history of protein function after WGD, because a teleost-specific WGD (TSGD) resulted in a variety of duplicated genes in modern fishes. This study focused on the evolution of androgen receptor (AR) gene, as two distinct paralogs, ARα and ARß, have evolved in teleost lineage after TSGD. ARα showed a unique intracellular localization with a higher transactivation response than that of ARß. Using site-directed mutagenesis and computational prediction of protein-ligand interactions, we identified two key substitutions generating a new functionality of euteleost ARα. The substitution in the hinge region contributes to the unique intracellular localization of ARα. The substitution on helices 10/11 in the ligand-binding domain possibly modulates hydrogen bonds that stabilize the receptor-ligand complex leading to the higher transactivation response of ARα. These substitutions were conserved in Acanthomorpha (spiny-rayed fish) ARαs, but not in an earlier branching lineage among teleosts, Japanese eel. Insertion of these substitutions into ARs from Japanese eel recapitulates the evolutionary novelty of euteleost ARα. These findings together indicate that the substitutions generating a new functionality of teleost ARα were fixed in teleost genome after the divergence of the Elopomorpha lineage. Our findings provide a molecular explanation for an adaptation process leading to generation of the hyperactive AR subtype after TSGD.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

Delayed de-induction of CYP2C9 compared to CYP3A after discontinuation of rifampicin: Report of two cases
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OBJECTIVE: Timely dose reduction of concomitant medications is important after withdrawal of rifampicin, a CYP inducer. However, little is known about the differences in the time course of deinduction for various CYP isoforms. To clarify the time courses of deinduction of CYP2C9 and -CYP3A activities after rifampicin withdrawal, we monitored these enzyme activities in 2 patients over time after discontinuing rifampicin. MATERIALS AND METHODS: Two patients (aged 70 and 80 years) received warfarin and rifampicin for anticoagulation and antituberculosis therapy, respectively. Warfarin doses were increased due to rifampicin-induced CYP activity. Upon completion of antituberculosis therapy, rifampicin was discontinued and warfarin doses were titrated downward according to prothrombin time. We monitored CYP2C9 and CYP3A activities over their clinical courses by measuring the metabolic clearance of S-warfarin to S-7-hydroxywarfarin and that of cortisol to 6ß-hydroxycortisol, respectively. RESULTS: In both patients, the time courses of CYP2C9 deinduction appeared to be delayed compared to CYP3A. CONCLUSION: Our findings suggest that a uniform dose reduction protocol for drugs metabolized by different CYP isoforms may be unsafe after rifampicin withdrawal.
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Comprehensive analysis of the Co-structures of dipeptidyl peptidase IV and its inhibitor.

BACKGROUND: We comprehensively analyzed X-ray cocrystal structures of dipeptidyl peptidase IV (DPP-4) and its inhibitor to clarify whether DPP-4 alters its general or partial structure according to the inhibitor used and whether DPP-4 has a common rule for inhibitor binding. RESULTS: All the main and side chains in the inhibitor binding area were minimally altered, except for a few side chains, despite binding to inhibitors of various shapes. Some residues (Arg125, Glu205, Glu206, Tyr662 and Asn710) in the area had binding modes to fix a specific atom of inhibitor to a particular spatial position in DPP-4. We found two specific water molecules that were common to 92 DPP-4 structures. The two water molecules were close to many inhibitors, and seemed to play two roles: maintaining the orientation of the Glu205 and Glu206 side chains through a network via the water molecules, and arranging the inhibitor appropriately at the S2 subsite. CONCLUSIONS: Our study based on high-quality resources may provide a necessary minimum consensus to help in the discovery of a novel DPP-4 inhibitor that is commercially useful.

Intraperitoneal administration of arginine vasotocin (AVT) induces anorexigenic and anxiogenic actions via the brain V1a receptor-signaling pathway in the tiger puffer, Takifugu rubripes.

Arginine vasotocin (AVT) is produced mainly in the hypothalamus and as a neurohypophyseal hormone peripherally regulates water-mineral balance in sub-mammals. In addition, AVT-containing neurons innervate several areas of the brain, and AVT also acts centrally as both an anorexigenic and anxiogenic factor in goldfish. However, it is unclear whether these central effects operate in fish in general. In the present study, therefore, we investigated AVT-like immunoreactivity in the brain of the tiger puffer, a cultured fish with a high market value in Japan and also a representative marine teleost species, focusing particularly on whether AVT affects food intake and psychomotor activity. AVT-like immunoreactivity was distributed higher in the ventral region of the telencephalon, the hypothalamus and midbrain. Intraperitoneal (IP) administration of AVT at 100 pmol g-1 body weight (BW) increased the immunoreactivity of phosphorylated ribosomal proteinS6 (RPS6), a neuronal activation marker, in the telencephalon and diencephalon, decreased food consumption and enhanced thigmotaxis. AVT-induced anorexigenic and anxiogenic actions were blocked by IP co-injection of a V1a receptor (V1aR) antagonist, Manning compound (MC) at 300 pmol g-1 BW. These results suggest that AVT acts as an anorexigenic and anxiogenic factor via the V1aR-signaling pathway in the tiger puffer brain.

Sperm attractant in the micropyle region of fish and insect eggs.

In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal-a micropyle-through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance-a glycoprotein based on lectin staining-is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm "attraction" to the micropyle opening is species specific and is dependent on extracellular Ca(2+). Eggs of some insects, including Drosophila, have distinct micropyle caps with CB affinity, which also may prove to assist sperm entry. Our attempts to fertilize fly eggs in vitro were not successful.

Data on plasma cortisol levels in nibbler fish Girella punctata reared under high-density conditions in either surface seawater or deep ocean water.

Deep ocean water (DOW) is the water obtained from depth of >200 m below the surface of Earth's oceans and is characterized by rich nutrients and cleanliness [1,2]. We have recently reported that DOW suppresses the high-density-induced increase of plasma cortisol levels (i.e., a stress marker) in Japanese flounder (Paralichthys olivaceus) [1]. The current study aimed to examine whether the cortisol-reducing effect of DOW was observed in other marine organisms as well by comparing the plasma cortisol levels of nibbler fish Girella punctata reared under high-density conditions between surface seawater (SSW) and DOW. The nibbler fish were caught from Tsukumo Bay of Noto Peninsula (Ishikawa Prefecture, Japan). The DOW was obtained from seawater 320 m below the Noto Bay surface at a facility (Aquas Noto, Ishikawa Prefecture, Japan), whereas SSW was obtained from Tsukumo Bay (Noto Peninsula, Ishikawa Prefecture). The dissolved oxygen was maintained at approximately 7 mg/L in DOW as well as in SSW. Before they were transferred to the high-density condition, nibbler fish were acclimated in SSW at 20°C for 1 week at a mean density of 100 g/62.5 L. To expose them to the high-density stress, each of fish was kept at a density of 10 kg/m3 in a single aquarium (60 × 25 × 30 cm) containing either SSW or DOW (n = 8). Subsequently, the fish were reared with SSW or DOW for 10 days at 20°C ± 1°C under a 12:12-h light-dark cycle. A heparin containing syringe was used to obtain the blood samples from the caudal vessels of the fish anesthetized with a 0.04% 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation). The blood sampling was performed on days 0, 5, and 10 after rearing in the small aquaria. The plasma samples were prepared from the collected blood by centrifuging it at 5200 × g for 5 min and the cortisol concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cosmo Bio Co. Ltd., Tokyo, Japan) from those samples. The plasma cortisol concentration of nibbler fish reared in SSW on day 10 was significantly higher than that on day 0, whereas those reared in DOW did not show significant difference on the respective days. The current data contributes to the generalization of the cortisol-reducing effect of DOW on fish, which has been proposed in Japanese flounder [1]. These data could be used for developing and designing experiments to analyze the mechanisms underlying the cortisol-reducing effects by using small fish such as zebrafish, a well-established animal model.

Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss.

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

Tetrodotoxin Detection in Japanese Bivalves: Toxification Status of Scallops.

Tetrodotoxin (TTX), or pufferfish toxin, has been frequently detected in edible bivalves around the world during the last decade and is problematic in food hygiene and safety. It was reported recently that highly concentrated TTX was detected in the midgut gland of the akazara scallop Chlamys (Azumapecten) farreri subsp. akazara collected in coastal areas of the northern Japanese archipelago. The toxification of the bivalve was likely to involve the larvae of the flatworm, Planocera multitentaculata. However, the overall status of bivalve TTX toxification has not been elucidated. In this study, 14 species/subspecies of bivalves from various Japanese waters were subjected to LC-MS/MS analysis to reveal TTX toxification state, demonstrating that the Pectinidae, including C. farreri akazara, Chlamys farreri nipponensis, Chlamys (Mimachlamys) nobilis, and Mizuhopecten yessoensis, accumulated TTX in their midgut gland. Many individuals of C. farreri akazara and C. farreri nipponensis were found with high concentrations of TTX, while C. nobilis and M. yessoensis exhibited low concentrations. The extent of TTX accumulation in C. farreri akazara and C. farreri nipponensis varied widely by region and season. Curiously, no other bivalve species investigated in this study showed evidence of TTX. These results suggest that monitoring for TTX, like other shellfish toxins, is necessary to ensure that pectinid bivalves are a safe food resource.

Adaptation to the shallow sea floor environment of a species of marine worms, Oligobrachia mashikoi, generally inhabiting deep-sea water.

Beard worms from the family Siboglinidae, are peculiar animals and are known for their symbiotic relationships with sulfur bacteria. Most Siboglinids inhabit the deep-sea floor, thus making difficult to make any observations in situ. One species, Oligobrachia mashikoi, occurs in the shallow depths (24.5 m) of the Sea of Japan. Taking advantage of its shallow-water habitat, the first ecological survey of O. mashikoi was performed over a course of 7 years, which revealed that its tentacle-expanding behavior was dependent on the temperature and illuminance of the sea water. Furthermore, there were significantly more O. mashikoi with expanding tentacles during the nighttime than during the daytime, and the prevention of light eliminated these differences in the number of expending tentacles. These results confirmed that the tentacle-expanding behavior is controlled by environmental light signals. Consistent with this, we identified a gene encoding a photoreceptor molecule, neuropsin, in O. mashikoi, and the expression thereof is dependent on the time of day. We assume that the described behavioral response of O. mashikoi to light signals represent an adaptation to a shallow-water environment within the predominantly deep-sea taxon.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.