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In euryhaline teleosts, the cystic fibrosis transmembrane conductance regulator (CFTR) in seawater (SW)-type chloride cells facilitates apical Cl - secretion for SW adaptation, while alternative Cl - excretion pathways remain understudied. This study investigates the role of the calcium-activated chloride channel, Anoctamin 1 (ANO1), in the gills of the euryhaline Japanese medaka (Oryzias latipes) under hyperosmolality and cortisol (CORT) influence. Acclimation to artificial SW, NaCl, mannitol, or glucose significantly upregulated ANO1 and CFTR mRNA expression in gills, unlike urea treatment. In situ hybridization revealed ANO1 mRNA in chloride cells co-expressing CFTR and Na + , K + -ATPase under hyperosmotic conditions. ANO1 inhibition elevated plasma Cl - concentration, indicating impaired Cl - excretion. CORT or dexamethasone administration in freshwater (FW) fish significantly increased branchial ANO1 and CFTR mRNA expression, an effect attenuated by the glucocorticoid receptor (GR) antagonist RU486. Hyperosmotic treatment of isolated gill tissues rapidly induced ANO1 mRNA expression independent of CFTR mRNA changes, and this induction was unaffected by RU486. These findings highlight the dual regulation of ANO1 expression via hyperosmolality-induced cellular response and the CORT-GR system. Thus, branchial ANO1 may likely complement CFTR in Cl⁻ excretion, playing a key role in the hyperosmotic adaptation of euryhaline teleosts., (© 2024 Wiley Periodicals LLC.)

Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possesses fluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Cigarette smoking is a preventable risk factor for various diseases such as cancer, ischemic stroke, cardiac stroke, and chronic obstructive pulmonary disease. Smoking cessation is of great importance not only for individual smokers but also for social health. Regarding current cessation therapies, the effectiveness of nicotine replacement is limited, and the cost of varenicline medication is considerable. Thus, a method for screening smokers who are responsive to cessation therapy based on the therapeutic effectiveness is required. Peripheral biomarkers reflecting smoking dependence status are necessary to establish a method for achieving effective cessation therapy., Methods: Methylation status of smokers' blood DNA was evaluated focusing on SHATI/NAT8L, an addiction-related gene. Eight CpG sites in SHATI/NAT8L were quantified by pyrosequencing., Results: There was no difference in the methylation status of this gene between smokers (n = 129) and non-smokers (n = 129) at all CpG sites. No correlations between the methylation status of SHATI/NAT8L and indicators of smoking dependence were found., Conclusions: Although the present study found no significance in the DNA methylation of SHATI/NAT8L among smokers, the exploration of predictable peripheral biomarkers for the effectiveness of smoking cessation therapy is required., (© 2023 The Authors. Neuropsychopharmacology Reports published by John Wiley & Sons Australia, Ltd on behalf of The Japanese Society of Neuropsychopharmacology.)

Depression is a frequent and serious illness, and stress is considered the main risk factor for its onset. First-line antidepressants increase serotonin (5-hydroxytryptamine; 5-HT) levels in the brain. We previously reported that an N -acetyltransferase, Shati/Nat8l, is upregulated in the dorsal striatum (dSTR) of stress-susceptible mice exposed to repeated social defeat stress (RSDS) and that dSTR Shati/Nat8l overexpression in mice (dSTR-Shati OE) induces stress vulnerability and local reduction in 5-HT content. Male mice were used in this study, and we found that dSTR 5-HT content decreased in stress-susceptible but not in resilient mice. Moreover, vulnerability to stress in dSTR-Shati OE mice was suppressed by the activation of serotonergic neurons projecting from the dorsal raphe nucleus (dRN) to the dSTR, followed by upregulation of 5-HT content in the dSTR using designer receptors exclusively activated by designer drugs (DREADD). We evaluated the role of GABA in modulating the serotonergic system in the dRN. Stress-susceptible after RSDS and dSTR-Shati OE mice exhibited an increase in dRN GABA content. Furthermore, dRN GABA content was correlated with stress sensitivity. We found that the blockade of GABA signaling in the dRN suppressed stress susceptibility in dSTR-Shati OE mice. In conclusion, we propose that dSTR 5-HT and dRN GABA, controlled by striatal Shati/Nat8l via the dSTR-dRN neuronal circuitry, critically regulate stress sensitivity. Our study provides insights into the neural processes that underlie stress and suggests that dSTR Shati/Nat8l could be a novel therapeutic target for drugs against depression, allowing direct control of the dRN serotonergic system., Competing Interests: S.-i.M. has equity with the Gene Therapy Research Institution, Co., Ltd., which commercializes AAV vectors for gene therapy applications. S.-i.M. has several conflicts of interest, to the extent that the work in this manuscript increases the value of these commercial holdings. All other authors declare no competing financial interests., (Copyright © 2023 Miyanishi et al.)

High stocking densities have negative effects on fish. However, the mechanism mediating density perception and growth inhibition is still unknown. This study was conducted to confirm the occurrence of growth inhibition and evaluate changes in growth-related factors in fish reared under high-stocking-density conditions and to determine the role of vision in density perception of medaka. In the graduated-stocking experiment, growth inhibition was clearly observed in fish reared at higher densities, although environmental factors, such as water quality, dissolved oxygen, and feeding conditions, were the same in each experimental group. Differences in growth were observed between the 6-fish and 8-fish groups, indicating that medaka have a superior sense that allows them to accurately perceive the number of individuals in their surroundings. In the pseudo-high stocking experiment, the inner 2-L tank in both groups contained six fish; however, the outer 3-L tank in the pseudo group contained several fish, while that of the control group contained only water. Growth inhibition was observed among the fish in the inner tank of the pseudo group despite having similar spatial density with the control group. These findings suggest that vision is important for density perception. The gene expression of growth-related and metabolic-regulatory hormones decreased in the high-density group. Furthermore, neuropeptide Y expression increased, while pro-opiomelanocortin expression decreased in the high-density group. This study is the first to report that fish can visually perceive density and the resulting growth inhibition, and concluded that medaka is a suitable model for studying density effects and perception in fish.

The number of cannabis users is increasing in the world. However, the mechanisms involved in the psychiatric effects and addiction formation remain unclear. Medical treatments against cannabis addiction have not yet been established. Δ9-Tetrahydrocannabinol (THC), the main active substance in cannabis, binds and affects cannabinoid type 1 receptors (CB1R) in the brain. The mice were intraperitoneally (i.p.) administered arachidonylcyclopropylamide (ACPA), a CB1R-selective agonist, and then two behavioral experiments on anxiety and addiction were performed. Administration of ACPA caused anxiolytic-like behavior in the elevated plus maze test. In addition, ACPA increased place preference in a conditioned place preference (CPP) test. The basolateral amygdala (BLA), which is the focus of this study, is involved in anxiety-like behavior and reward and is reported to express high levels of CB1R. We aimed to reveal the role of CB1R in BLA for ACPA-induced behavior. AM251, a CB1R selective antagonist, was administered intra-BLA before i.p. administration of ACPA. Intra-BLA administration of AM251 inhibited ACPA-induced anxiolytic-like behavior and place preference. These results suggest that CB1R in the BLA contributes to behavior disorders caused by the acute or chronic use of cannabis., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As the elderly population rapidly increases worldwide, the onset of cognitive dysfunction is expected to increase. Although neuronal plasticity, neurogenesis, and mitochondrial dysfunction have been reported to be involved in cognitive function, the detailed mechanism of cognitive impairment accompanied by aging is poorly understood as there are many confounding factors associated with aging. Therefore, effective treatments for aging have not yet been developed, and the establishment of therapeutic strategies has not progressed accordingly. We have previously found a decline of cognitive function in the developmental stage in mice who lack the expression of Shati/Nat8l, an N-acetyl transferase However, the contribution of Shati/Nat8l to cognitive impairment in aged mice has not yet been investigated. In this study, we aimed to investigate the role of Shati/Nat8l in cognitive function during aging. We observed a reduction in Shati/Nat8l mRNA expression in the dorsal hippocampus of mice as a result of their aging. Moreover, the cognitive dysfunction observed in aged mice was reversed by Shati/Nat8l overexpression in the dorsal hippocampus. Shati/Nat8l overexpression in the dorsal hippocampus of mice did not alter the expression of neurotrophic factors or mitochondrial function-related genes, including Bdnf or Pgc-1α, which are suggested to be downstream genes of Shati/Nat8l. Decreased N-acetyl aspartate (NAA) in aged mice was upregulated by Shati/Nat8l overexpression, suggesting that the Shati/Nat8l-NAA pathway determines cognitive function with aging. Taken together, Shati/Nat8l and NAA in the dorsal hippocampus may be novel targets for the treatment of cognitive impairment., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In recent years, studies on circadian control in immunity have been actively conducted in mammals, but little is known about circadian rhythms in the field of fish immunology. In this study, we aimed to analyse the regulation of the diel oscillation of inflammatory cytokine interleukin-1β (il1b) gene expression by core components of the circadian clock in Japanese medaka (Oryzias latipes). The expression of il1b and clock genes (bmal1 and clock1) in medaka acclimated to a 12:12 light (L): dark (D) cycle showed diel rhythm. Additionally, higher expression of il1b was detected in medaka embryo cells (OLHdrR-e3) overexpressing bmal1 and clock1. A significant decrease in il1b expression was observed in OLHdrR-e3 cells after bmal1 knockdown using morpholino oligos. These changes may be mediated by transcriptional regulation via clock proteins, which target the E-box sequence in the cis-element of il1b as identified using luciferase reporter assays. Moreover, LPS stimulation and pathogenic bacterial infection at different zeitgeber time (ZT) under LD12:12 conditions affected the degree of il1b expression, which showed high and low responsiveness to both immuno-stimulations at ZT2 and ZT14, respectively. These results suggested that fish IL-1β exhibited diel oscillation regulated by clock proteins, and its responsiveness to immune-stimulation depends on the time of day., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century., Results: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages., Conclusions: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys., (© 2022. The Author(s).)

Mucosal tissue forms the first line of defense against pathogenic microorganisms. Cellular damage in the mucosal epithelium may induce the interleukin (IL)-22-related activation of many immune cells, which are essential for maintaining the mucosal epithelial barrier. A previous study on mucosal immunity elucidated that mammalian IL-22 contributes to mucus and antimicrobial peptides (AMPs) production and anti-apoptotic function. IL-22 has been identified in several teleost species and is also induced in response to bacterial infections. However, the roles of IL-22 in teleost immunity and mucus homeostasis are poorly understood. In this study, Japanese medaka ( Oryzias latipes ) was used as a model fish. The medaka il22 , il22 receptor A1 ( il22ra1 ), and il22 binding protein ( il22bp ) were cloned and characterized. The expression of medaka il22 , il22ra1 , and il22bp in various tissues was measured using qPCR. These genes were expressed at high levels in the mucosal tissues of the intestines, gills, and skin. The localization of il22 and il22bp mRNA in the gills and intestines was confirmed by in situ hybridizations. Herein, we established IL-22-knockout (KO) medaka using the CRISPR/Cas9 system. In the IL-22-KO medaka, a 4-bp deletion caused a frameshift in il22 . To investigate the genes subject to IL-22-dependent regulation, we compared the transcripts of larval medaka between wild-type (WT) and IL-22-KO medaka using RNA-seq and qPCR analyses. The comparison was performed not only in the naïve state but also in the dextran sulfate sodium (DSS)-exposed state. At the transcriptional level, 368 genes, including immune genes, such as those encoding AMPs and cytokines, were significantly downregulated in IL-22-KO medaka compared that in WT medaka in naïve states. Gene ontology analysis revealed that upon DSS stimulation, genes associated with cell death, acute inflammatory response, cell proliferation, and others were upregulated in WT medaka. Furthermore, in DSS-stimulated IL-22-KO medaka, wound healing was delayed, the number of apoptotic cells increased, and the number of goblet cells in the intestinal epithelium decreased. These results suggested that in medaka, IL-22 is important for maintaining intestinal homeostasis, and the disruption of the IL-22 pathway is associated with the exacerbation of inflammatory pathology, as observed for mammalian IL-22., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Takahashi, Okamura, Harada, Watanabe, Miyanishi, Kono, Sakai and Hikima.)

Phospholipase A2 (PLA2), a phospholipid hydrolase, has recently attracted attention owing to its broad functionality. Immunological evidence has revealed increased susceptibility to infectious diseases and immunodeficiency in knockout (KO) mice of several pla2 genes. However, no progress has been made in terms of immunological research on any pla2 gene in fish. In this study, we focused on the intestinal immune responses of fish PLA2s. The full-length open reading frames of pla2g1b, pla2g3, pla2g10, pla2g12b1, pla2g12b2 , and pla2g15 cDNAs were cloned in Japanese medaka ( Orizias latipes ), and their gene expressions were quantified by real-time PCR (qPCR) and in situ hybridization (ISH). Characterization of pla2 genes revealed a functional domain and three-dimensional structure similar to the mammalian counterparts. In addition, expression of pla2g1b, pla2g12b1 , and pla2g12b2 was extremely high in Japanese medaka intestines. ISH detected strong expression of pla2g1b mRNAs in the basal muscle layer, and pla2g12b1 and pla2g12b2 mRNAs were detected in the epithelial cells. In the medaka exposed to Edwardsiella piscicida, pla2g12b1, pla2g12b2 and pla2g15 were significantly induced in the anterior and posterior intestines, and pla2g1b was upregulated in the anterior intestine. Furthermore, pla2g1b, pla2g3, pla2g10 , and pla2g12b2 were significantly downregulated in the IL-17A/F1 KO medaka compared to those in wild-type medaka. These results suggest that these PLA2s are involved in intestinal immunity in teleosts., Competing Interests: None., (© 2021 The Author(s).)

Depression is one of the most common mental diseases, with increasing numbers of patients globally each year. In addition, approximately 30% of patients with depression are resistant to any treatment and do not show an expected response to first-line antidepressant drugs. Therefore, novel antidepressant agents and strategies are required. Although depression is triggered by post-birth stress, while some individuals show the pathology of depression, others remain resilient. The molecular mechanisms underlying stress sensitivity remain unknown. Brain-derived neurotrophic factor (BDNF) has both pro- and anti-depressant effects, dependent on brain region. Considering the strong region-specific contribution of BDNF to depression pathogenesis, the regulation of BDNF in the whole brain is not a beneficial strategy for the treatment of depression. We reviewed a novel finding of BDNF function in the dorsal striatum, which induces vulnerability to social stress, in addition to recent research progress regarding the brain regional functions of BDNF, including the prefrontal cortex, hippocampus, and nucleus accumbens. Striatal BDNF is regulated by Shati/Nat8l, an N -acetyltransferase through epigenetic regulation. Targeting of Shati/Nat8l would allow BDNF to be striatum-specifically regulated, and the striatal Shati/Nat8l-BDNF pathway could be a promising novel therapeutic agent for the treatment of depression by modulating sensitivity to stress.

The global number of patients with depression increases in correlation to exposure to social stress. Chronic stress does not trigger depression in all individuals, as some remain resilient. The underlying molecular mechanisms that contribute to stress sensitivity have been poorly understood, although revealing the regulation of stress sensitivity could help develop treatments for depression. We previously found that striatal Shati/Nat8l, an N-acetyltransferase, was increased in a depression mouse model. We investigated the roles of Shati/Nat8l in stress sensitivity in mice and found that Shati/Nat8l and brain-derived neurotrophic factor (BDNF) levels in the dorsal striatum were increased in stress-susceptible mice but not in resilient mice exposed to repeated social defeat stress (RSDS). Knockdown of Shati/Nat8l in the dorsal striatum induced resilience to RSDS. In addition, blockade of BDNF signaling in the dorsal striatum by ANA-12, a BDNF-specific receptor tropomyosin-receptor-kinase B (TrkB) inhibitor, also induced resilience to stress. Shati/Nat8l is correlated with BDNF expression after RSDS, and BDNF is downstream of Shati/Nat8l pathways in the dorsal striatum; Shati/Nat8l is epigenetically regulated by BDNF via histone acetylation. Our results demonstrate that striatal Shati/Nat8l-BDNF pathways determine stress sensitivity through epigenetic regulation. The striatal Shati/Nat8l-BDNF pathway could be a novel target for treatments of depression and could establish a novel therapeutic strategy for depression patients., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

In the intestine, the host must be able to control the gut microbiota and efficiently absorb transiently supplied metabolites, at the risk of enormous infection. In mammals, the inflammatory cytokine interleukin (IL)-17A/F is one of the key mediators in the intestinal immune system. However, many functions of IL-17 in vertebrate intestines remain unclarified. In this study, we established a gene-knockout (KO) model of IL-17 receptor A1 (IL-17RA1, an IL-17A/F receptor) in Japanese medaka (Oryzias latipes) using genome editing technique, and the phenotypes were compared to wild type (WT) based on transcriptome analyses. Upon hatching, homozygous IL-17RA1-KO medaka mutants showed no significant morphological abnormality. However, after 4 months, significant weight decreases and reduced survival rates were observed in IL-17RA1-KO medaka. Comparison of gene-expression patterns in WT and IL-17RA1-KO medaka revealed that various metabolism- and immune-related genes were significantly down-regulated in IL-17RA1-KO medaka intestine, particularly genes related to mevalonate metabolism (mvda, acat2, hmgcs1, and hmgcra) and genes related to IL-17 signaling (such as il17c, il17a/f1, and rorc) were found to be decreased. Conversely, expression of genes related to cardiovascular system development, including fli1a, sox7, and notch1b in the anterior intestine, and that of genes related to oxidation-reduction processes including ugp2a, aoc1, and nos1 in posterior intestine was up-regulated in IL-17RA1-KO medaka. These findings show that IL-17RA regulated immune- and various metabolism-related genes in the intestine for maintaining the health of Japanese medaka.

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka ( Oryzias latipes ) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1β ( IL-1 β), complement 1q subunit C ( C1qc ), transferrin a ( Tfa ), and G-type lysozyme ( LyzG ), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB ( pla2g1b ), and elastase-1-like ( CELA1 ), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome., (Copyright © 2020 Okamura, Morimoto, Ikeda, Mizusawa, Watabe, Miyanishi, Saeki, Takeyama, Aoki, Kinoshita, Kono, Sakai and Hikima.)

This study was carried out to identify and estimate physiological function of a new type of opsin subfamily present in the retina and whole brain tissues of Japanese eel using RNA-Seq transcriptome method. A total of 18 opsin subfamilies were identified through RNA-seq. The visual opsin family included Rh2, SWS2, FWO, DSO, and Exo-Rhod. The non-visual opsin family included four types of melanopsin subfamily (Opn4x1, Opn4x2, Opn4m1, and Opn4m2), peropsin, two types of neuropsin subfamily (Opn5-like, Opn5), Opn3, three types of TMT opsin subfamily (TMT1, 2, 3), VA-opsin, and parapinopsin. In terms of changes in photoreceptor gene expression in the retina of sexually mature and immature male eels, DSO mRNA increased in the maturation group. Analysis of expression of opsin family gene in male eel brain before and after maturation revealed that DSO and SWS2 expression in terms of visual opsin mRNA increased in the sexually mature group. In terms of non-visual opsin mRNA, parapinopsin mRNA increased whereas that of TMT2 decreased in the fore-brain of the sexually mature group. The mRNA for parapinopsin increased in the mid-brain of the sexually mature group, whereas those of TMT1 and TMT3 increased in the hind-brain of the sexually mature group. DSO mRNA also increased in the retina after sexual maturation, and DSO and SWS2 mRNA increased in whole brain part, suggesting that DSO and SWS2 are closely related to sexual maturation., Competing Interests: The authors declare there are no competing interests, (©2020 Byun et al.)

Major depressive disorder (MDD) is one of the most common psychiatric diseases. However, early detection and diagnosis of MDD is difficult, largely because there is no known biomarker or objective diagnostic examination, and its diagnosis is instead based on a clinical interview. The aim of this study was to develop a novel diagnostic tool using DNA methylation as a blood biomarker. We sought to determine whether unmedicated patients with MDD showed significant differences in DNA methylation in the promoter region of the SHATI/N-acetyltransferase 8 like (SHATI/NAT8L) gene compared to healthy controls. Sixty participants with MDD were recruited from all over Japan. They were diagnosed and assessed by at least two trained psychiatrists according to DSM-5 criteria. DNA was extracted from peripheral blood. We then assessed DNA methylation of the SHATI/NAT8L promoter regions in patients with MDD by pyrosequencing. Methylation levels of the SHATI/NAT8L promoter region at CpG sites in peripheral blood from unmedicated patients were significantly higher than in healthy controls. In contrast, medicated patients with MDD showed significantly lower methylation levels in the same region compared to healthy controls. Since previous studies of DNA methylation in MDD only assessed medicated patients, the methylation status of the SHATI/NAT8L promoter region in unmedicated patients presented herein may prove useful for the diagnosis of MDD. To our knowledge, this is the first attempt to measure methylation of the SHATI/NAT8L gene in drug-naïve patients with psychiatric diseases. Based on our findings, methylation of SHATI/NAT8L DNA might be a diagnostic biomarker of MDD.

Background: Methamphetamine (METH) is one of the most widely distributed psychostimulants worldwide. Despite active counter measures taken by different countries, neither overall usage of METH nor the frequency of repeat users has reduced over the past decade. METH induces abuse and dependence as it acts on the central nervous system and temporarily stimulates the brain. The recidivism rate for abuse of stimulants in Japan is very high and therefore prevention of repeated usage is paramount. However, we lack information about the relationship between METH users and genomic changes in humans in Japan, which would provide important information to aid such efforts., Objective: Shati/Nat8l is a METH-inducible molecule and its overexpression has protective effects on the brain upon METH usage. Here we investigated the effect of METH usage on DNA methylation rates at the promoter site of SHATI/NAT8L. We used DNA samples from human METH users, who are usually difficult to recruit in Japan., Methods: We measured DNA methylation at SHATI/NAT8L promoter sites by pyrosequencing method using 193 samples of METH users and 60 samples of healthy subjects. In this method, DNA methylation is measured by utilizing the property that only non-methylated cytosine changes to urasil after bisulfite conversion., Results: We found that the rate of DNA methylation at six CpG islands of SHATI/NAT8L promoter sites is significantly higher in METH users when compared to healthy subjects., Conclusion: These results suggest that the DNA methylation rate of SHATI/NAT8L promotor regions offers a new diagnostic method for METH usage., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The number of patients with depressive disorders is increasing. However, the mechanism of depression onsets has not been completely revealed. We previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. In this study, we revealed the involvement of Shati/Nat8l in the vulnerability to major depression. Shati/Nat8l mRNA was increased only in the striatum of mice, which were exposed to chronic social defeat stress. Shati/Nat8l-overexpressed mice showed impairment in social interaction and sucrose preference after the subthreshold social defeat (microdefeat) stress. These depression-like behaviors were restored by fluvoxamine and LY341495 injection prior to these tests. Furthermore, the intracerebral administration of only fluvoxamine, but not of LY341495, to the dorsal striatum and direct infusion of LY341495 to the dorsal raphe also rescued. Taken together, Shati/Nat8l in the striatum has an important role in the vulnerability to depression onsets by regulating the origin of serotonergic neuronal system via GABAergic projection neuron in the dorsal raphe from the dorsal striatum., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Members of the phylum Arthropoda, comprising over 80% of total animal species, have evolved regenerative abilities, but little is known about the molecular mechanisms mediating this process. Transforming growth factor β (TGF-β) signaling mediates a diverse set of essential processes in animals and is a good candidate pathway for regulation of regeneration in arthropods. In this study we investigated the role of activin signaling, a TGF-β superfamily pathway, in limb regeneration in the crayfish. We identified and cloned a downstream transcription factor in the activin pathway, Smox, and characterized its function with regard to other elements of the activin signaling pathway. Gene knockdown of Smox by RNAi induced regeneration of complete but smaller pereopods after autotomy. This indicates that activin signaling via Smox functions in regulation of pereopod growth and size. The expression levels of both Smox and the activin receptor babo were closely correlated with molting. The expression level of Smox increased when babo was knocked down by RNAi, indicating that Smox and babo transcription are linked. Our study suggests that the Babo-Smox system in activin signaling is conserved in decapods, and supports an evolutionary conservation of this aspect of molecular signaling during regeneration between protostomes and deuterostomes., (© 2018 Wiley Periodicals, Inc.)

Changes in mastitis-causing pathogens, pH and water content in composted manure solids (CMS) prepared from digested slurry were evaluated during turning at 2-day intervals for 8 days (C1-C4). The numbers of streptococci, coagulase-negative staphylococci and coliforms were 2.6 × 10 1 , 1.7 × 10 2 and 1.0 × 10 1  colony-forming units (cfu)/g in CMS (C4) (summer), and these counts were markedly lower (P < 0.05) than those in CMS (C0 and C1). The bacterial counts ranged from 10 1 to 1.7 × 10 2  cfu/g in CMS (C4) (summer) and were within approved levels, <1 × 10 6  cfu/g, indicating a minimal mastitis risk. The temperatures in CMS (C1-C4) increased to 63°C-74°C in summer and 67°C-70°C in winter. The mean pH values in CMS (C0-C4) were 9.2 in summer and 8.7 in winter, and water contents ranged from 61.7% to 69.6% in summer and 73.2% to 66.2% in winter. The significant decrease of pathogenic bacteria in CMS appears to be closely related to temperature >63°C for 8 days, pH 8.7-9.2, and water content 62% to 73%. This study demonstrates that prepared CMS has value as a recycled material with the potential to alleviate udder health issues in dairy cows., (© 2017 Japanese Society of Animal Science.)

Spatiotemporal changes in branchial ionocyte distribution were investigated following transfer from seawater (SW) to freshwater (FW) in Japanese seabass. The mRNA expression levels of cystic fibrosis transmembrane conductance regulator (CFTR) and Na + /K + /2Cl - cotransporter 1a (NKCC1a) in the gills rapidly decreased after transfer to FW, whereas Na + /H + exchanger 3 (NHE3) and Na + /Cl - cotransporter 2 (NCC2) expression were upregulated following the transfer. Using quadruple-color whole-mount immunofluorescence staining with anti-Na + /K + -ATPase, anti-NHE3, anti-CFTR and T4 (anti-NKCC1a/NCC2) antibodies, we classified ionocytes into one SW type and two FW types: NHE3 cell and NCC2 cell. Time course observation after transfer revealed an intermediate type between SW-type and FW-type NHE3 ionocytes, suggesting functional plasticity of ionocytes. Finally, on the basis of the ionocyte classification of Japanese seabass, we observed the location of ionocyte subtypes on frozen sections of the gill filaments stained by triple-color immunofluorescence staining. Our observation indicated that SW-type ionocytes transformed into FW-type NHE3 ionocytes and at the same time shifted their distribution from filaments to lamellae. However, FW-specific NCC2 ionocytes appeared mainly in the filaments. Taken together, these findings indicate that ionocytes originated from undifferentiated cells in the filaments and expanded their distribution to the lamellae during FW acclimation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Background: During the course of evolution, fishes have acquired adaptability to various salinity environments, and acquirement of seawater (SW) adaptability has played important roles in fish evolution and diversity. However, little is known about how saline environments influence the acquirement of SW adaptability. The Japanese medaka Oryzias latipes is a euryhaline species that usually inhabits freshwater (FW), but is also adaptable to full-strength SW when transferred through diluted SW. In the present study, we examined how past SW experience affects hyposmoregulatory ability in Japanese medaka., Results: For the preparation of SW-experienced fish, FW medaka were acclimated to SW after pre-acclimation to 1/2 SW, and the SW-acclimated fish were transferred back to FW. The SW-experienced fish and control FW fish (SW-inexperienced fish) were transferred directly to SW. Whereas control FW fish did not survive direct transfer to SW, 1/4 of SW-experienced fish adapted successfully to SW. Although there were no significant differences in blood osmolality and plasma Na(+) and Cl(-) concentrations between SW-experienced and control FW medaka in FW, increments in these parameters following SW transfer were lower in SW-experienced fish than in control FW fish. The gene expression of SW-type Na(+), K(+)-ATPase (NKA) in the gills of SW-experienced medaka increased more quickly after direct SW transfer compared with the expression in control FW fish. Prior to SW transfer, the density of NKA-immunoreactive ionocytes in the gills was higher in SW-experienced fish than in control FW fish. Ionocytes expressing CFTR Cl(-) channel at the apical membrane and those forming multicellular complexes, both of which were characteristic of SW-type ionocytes, were also increased in SW-experienced fish., Conclusion: These results indicate that past SW experience enhances the capacity of Na(+) and Cl(-) secretion in ionocytes and thus hypoosmoregulatory ability of Japanese medaka, suggesting the presence of epigenetic mechanisms involved in seawater adaptation.

The CRH family of neuropeptides, including CRH and urocortins, plays pivotal roles in the regulation of physiological and behavioral stress responses in vertebrates. In this study, we identified a previously undescribed member of the CRH family of peptides in a teleost fish species (medaka; Oryzias latipes) and named this peptide teleocortin (Tcn). Medaka Tcn is a 41-amino acid polypeptide derived from the C terminus of a larger precursor protein that is encoded by a 2-exon gene, thus sharing common structural features with known CRH family peptides. tcn was found exclusively in teleost fish. Phylogenetic analysis suggested that tcn probably has an ancient origin but was lost from the tetrapod lineage shortly after the divergence of the teleost and tetrapod lineages. In the medaka brain, tcn was expressed in nuclei of the telencephalon, preoptic area, hypothalamus, tegmentum, and isthmic region. Because none of these nuclei have been implicated in the control of ACTH secretion from the pituitary, Tcn may exert its effects centrally in the brain rather than via stimulation of the pituitary-adrenal/interrenal axis. Most, if not all, tcn-expressing neurons also expressed crh, suggesting that Tcn and Crh share common physiological functions. Moreover, Tcn activated Crh receptors 1 and 2 with equivalent or slightly higher potency than Crh, further suggesting that these peptides share common functions. Taken together, these data identified Tcn as a novel, teleost-specific member of the CRH family of peptides that may act centrally with Crh to regulate physiological and behavioral stress responses.

Cardiac natriuretic peptides (atrial natriuretic peptide, ANP; b-type natriuretic peptide, BNP; ventricular natriuretic peptide, VNP) and their direct ancestor C-type natriuretic peptide 3 (CNP3) exert potent osmoregulatory actions in fish. However, very little is known about their roles in embryonic osmoregulation. In this study, we performed loss-of-function analysis using euryhaline medaka (Oryzias latipes), which has lost ANP and VNP during evolution and thus possesses only BNP and CNP3. We found that the maintenance of whole-body osmolality in seawater embryos was impaired by the knockdown of BNP+OLGC7 (BNP receptor) or CNP3 alone from 1 day postfertilization, and the CNP3 knockdown was accompanied by greater water loss. The impaired osmoregulation in the knockdown embryos was not due to the suppressed expression of major transporters for NaCl excretion via ionocytes or of key enzyme genes for metabolic water production, but to the impaired blood circulation to the yolk-sac membrane caused by abnormal heart development. We detected a strong positive correlation between impaired blood circulation and increased body fluid osmolality and pharmacological blockade of blood flow increased body fluid osmolality in seawater embryos. We also found that the exaggerated water loss in CNP3 knockdown embryos is related to the failure to suppress aquaporin (AQP3, AQP4, and AQP9) gene expression. These results show that CNP3 decrease water permeability of body surfaces and that both BNP and CNP3 ensure sufficient blood flow to the yolk-sac membrane for efficient salt excretion by ionocytes and sufficient water production by yolk metabolism to promote seawater adaptation during early development in medaka.

Purpose: To investigate crystallization behavior on the surface of amorphous solid dispersion powder using inverse gas chromatography (IGC) and to predict the physical stability at temperatures below the glass transition temperature (T (g))., Methods: Amorphous solid dispersion powder was prepared by melt-quenching of a mixture of crystalline nifedipine and polyvinylpyrrolidon (PVP) K-30. IGC was conducted by injecting undecane (probe gas) and methane (reference gas) repeatedly to the solid dispersion at temperatures below T (g). Surface crystallization was evaluated by the retention volume change of undecane based on the observation that the surface of the solid dispersion with crystallized nifedipine gives an increased retention volume., Results: On applying the retention volume change to the Hancock-Sharp equation, surface crystallization was found to follow a two-dimensional growth of nuclei mechanism. Estimation of the crystallization rates at temperatures far below T (g) using the Avrami-Erofeev equation and Arrhenius equation showed that, to maintain its quality for at least three years, the solid dispersion should be stored at -20°C (T (g) - 65°C)., Conclusions: IGC can be used to evaluate crystallization behavior on the surface of a solid dispersion powder, and, unlike traditional techniques, can also predict the stability of the solid dispersion based on the surface crystallization behavior.

Cardiac natriuretic peptides (NPs), atrial NP (ANP) and B-type NP (BNP), and their receptor, guanylyl cyclase (GC)-A have attracted attention of many basic and clinical researchers because of their potent renal and cardiovascular actions. In this study, we used medaka, Oryzias latipes, as a model species to pursue the physiological functions of NPs because it is a suitable model for developmental analyses. Medaka has two ligands, BNP and C-type NP3 (CNP3) (but not ANP), that have greater affinity for the two O. latipes GC-A receptors (OLGC), OLGC7 and OLGC2, respectively. CNP3 is the ancestral molecule of cardiac NPs. Initially, we examined developmental expression of cardiac NP/receptor combinations, BNP/OLGC7 and CNP3/OLGC2, using quantitative real-time PCR and in situ hybridization. BNP and CNP3 mRNA increased at stages 25 (onset of ventricular formation) and 22 (appearance of heart anlage), respectively, whereas both receptor mRNAs increased at as early as stage 12. BNP/OLGC7 transcripts were found in arterial/ventricular tissues and CNP3/OLGC2 transcripts in venous/atrial tissues by in situ hybridization. Thus, BNP and CNP3 can act locally on cardiac myocytes in a paracrine/autocrine fashion. Double knockdown of BNP/OLGC7 genes impaired ventricular development by causing hypoplasia of ventricular myocytes as evidenced by reduced bromodeoxyuridine incorporation. CNP3 knockdown induced hypertrophy of atria and activated the renin-angiotensin system. Collectively, it appears that BNP is important for normal ventricular, whereas CNP3 is important for normal atrial development and performance, a role usually taken by ANP in other vertebrates. The current study provides new insights into the role of cardiac NPs in cardiac development in vertebrates.

Atrial natriuretic peptide (ANP) exhibits a potent antidipsogenic effect in seawater (SW) eels to limit excess Na(+) uptake, thereby effectively promoting SW adaptation. Recently, cardiac ANP, BNP and VNP and brain CNP1, 3 and 4, have been identified in eels. We examined the antidipsogenic effect of all homologous NPs using conscious, cannulated eels in both FW and SW together with parameters that affect drinking. A dose-response study (0.01-1 nmol/kg) in SW eels showed the relative potency of the antidipsogenic effect was in the order ANP ≥ VNP > BNP = CNP3 > CNP1 ≥ CNP4, while the order was ANP = VNP = BNP > CNP3 = CNP1 = CNP4 for the vasodepressor effect. The minimum effective dose of ANP for the antidipsogenic effect is much lower than that in mammals. ANP, BNP and VNP at 0.3 nmol/kg decreased drinking, plasma Na(+) concentration and aortic pressure and increased hematocrit in SW eels. The cardiac NPs induced similar changes in drinking, aortic pressure and hematocrit in FW eels, but aside from BNP no change in plasma Na(+) concentration. CNPs had no effect on drinking, plasma Na(+) concentration and hematocrit but induced mild hypotension in both FW and SW eels, except for CNP3 that inhibited drinking in SW eels. These results show that ANP, BNP and VNP are potent antidipsogenic hormones in eels in spite of other regulatory factors working to induce drinking, and that CNPs are without effects on drinking except for the ancestor of the cardiac NPs, CNP3.

Hydrophobic drug substances can be formulated as a solid dispersion or solution using macromolecular matrices with high glass transition temperatures to attain satisfactory dissolution. However, very few marketed products have previously relied on solid dispersion technology due to physical and chemical instability problems, and processing difficulties. In the present study, a modified release product of a therapeutic drug for hypertension, Barnidipine hydrochloride, was developed. The drug product consisted of solid dispersion based on a matrix of carboxymethylethylcellulose (CMEC), which was produced using the spray-coating method. An enteric coat layer was sprayed on the surface of the solid dispersion to control drug release. Interestingly, the release rate accelerated as the drug product aged, while there were no indications of deceleration of the release rate which was due to crystallization of the drug substance. To prevent changes in the dissolution kinetics during storage periods, a variety of processing conditions were tried. It was found that not only use of non-aqueous solvents but also a reduction in coating temperatures consistently resulted in stable solid dispersions. The molecular bases of dissolution of the drug substance from those matrices were investigated. The molecular weight of CMEC was found to be a dominant factor that determined dissolution kinetics, which followed zero-order release, suggesting an involvement of an osmotic pumping mechanism. While dissolution was faster using a higher molecular weight CMEC, the molecular weight of CMEC in the drug product slowly increased with aging (solid phase reaction) depending on the processing conditions, causing the time-induced elevation of dissolution. While no crystalline components were found in the solid dispersion, the amorphous structure maintained a degree of non-equilibrium by nature. Plasticization by water in the coating solution relaxed the amorphous system and facilitated phase separation of the drug substance and CMEC upon production. The solid phase reaction advanced differentially in the solid dispersion depending on the degree of phase separation set initially. The use of non-aqueous solvents and/or a decrease in the coating temperatures inhibited the occurrence of phase separation upon production, thereby preventing the formation of CMEC-rich phases where the solid phase reaction occurred during storage.

A method to design novel molecules that specifically recognize a structured RNA would be a promising tool for the development of drugs or probes targeting RNA. In this study, the de novo design of the alpha-helical peptides having L-alpha-amino acids with nucleobases (nucleobase amino acids, NBAs) was carried out. Binding affinities of the peptides for a hairpin RNA derived from P22 phage were dependent on the types and positions of the NBA units they have. Some NBA peptides bound to the wild-type RNA or its mutant with high affinity and high specificity compared with the native P22 N peptide. These results indicate that the NBA units on the peptides interact with the RNA bases in a specific manner. It is demonstrated that the de novo design of peptides with the NBA units is an effective way to construct novel RNA-binding molecules.