Dopaminergic (DA) neurons located in the preoptico-hypothalamic region of the brain exert a major neuroendocrine control on reproduction, growth, and homeostasis by regulating the secretion of anterior pituitary (or adenohypophysis) hormones. Here, using a retrograde tract tracing experiment, we identified the neurons playing this role in the zebrafish. The DA cells projecting directly to the anterior pituitary are localized in the most anteroventral part of the preoptic area, and we named them preoptico-hypophyseal DA (POHDA) neurons. During development, these neurons do not appear before 72 hours postfertilization (hpf) and are the last dopaminergic cell group to differentiate. We found that the number of neurons in this cell population continues to increase throughout life proportionally to the growth of the fish. 5-Bromo-2'-deoxyuridine incorporation analysis suggested that this increase is due to continuous neurogenesis and not due to a phenotypic change in already-existing neurons. Finally, expression profiles of several genes (foxg1a, dlx2a, and nr4a2a/b) were different in the POHDA compared with the adjacent suprachiasmatic DA neurons, suggesting that POHDA neurons develop as a distinct DA cell population in the preoptic area. This study offers some insights into the regional identity of the preoptic area and provides the first bases for future functional genetic studies on the development of DA neurons controlling anterior pituitary functions.

Regionalization is a critical, highly conserved step in the development of the vertebrate brain. Discrepancies exist in how regionalization of the anterior vertebrate forebrain is conceived since the "preoptic area" is proposed to be a part of the telencephalon in tetrapods but not in teleost fish. To gain insight into this complex morphogenesis, formation of the anterior forebrain was analyzed in 3D over time in zebrafish embryos, combining visualization of proliferation and differentiation markers, with that of developmental genes. We found that the region containing the preoptic area behaves as a coherent morphogenetic entity, organized around the optic recess and located between telencephalon and hypothalamus. This optic recess region (ORR) makes clear borders with its neighbor areas and expresses a specific set of genes (dlx2a, sim1a and otpb). We thus propose that the anterior forebrain (secondary prosencephalon) in teleosts contains three morphogenetic entities (telencephalon, ORR and hypothalamus), instead of two (telencephalon and hypothalamus). The ORR in teleosts could correspond to "telencephalic stalk area" and "alar hypothalamus" in tetrapods, resolving current inconsistencies in the comparison of basal forebrain among vertebrates.

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.

The lack of characteristic clinical findings and accurate diagnostic tools has made the diagnosis of enteric fever difficult. We evaluated the classic signs of relative bradycardia and eosinopenia as diagnostic predictors for enteric fever among travellers who had returned from the tropics or subtropics.

The coronavirus disease 2019 (COVID-19) continues to spread around the world. In addition to community-acquired infections, nosocomial infections are also a major social concern. The likelihood of environmental contamination and transmission of the virus based on disease severity is unknown.

A high-flow nasal cannula (HFNC) therapy plays a significant role in providing respiratory support to critically ill patients with coronavirus disease 2019 (COVID-19); however, the dispersion of the virus owing to aerosol generation is a matter of concern. This study aimed to evaluate if HFNC disperses the virus into the air. Among patients with COVID-19 admitted to private rooms with controlled negative pressure, we enrolled those admitted within 10 days of onset and requiring oxygenation through a conventional nasal cannula or HFNC therapy. Of the 17 patients enrolled, we obtained 22 samples (11 in the conventional nasal cannula group and 11 in the HFNC group). Viral RNA was detected in 20 nasopharyngeal swabs, and viable viruses were isolated from three nasopharyngeal swabs. Neither viral RNA nor viable virus was detected in the air sample at 0.5 m regardless of the oxygen-supplementation device. We detected viral RNA in two samples in the conventional nasal cannula group but not in the HFNC therapy group in gelatin filters located 3 m from the patient and the surface of the ventilation. This study directly demonstrated that despite viral RNA detection in the nasopharynx, viruses may not be dispersed by HFNC therapy. This warrants further research to determine if similar results can be obtained under different conditions.

Sphingolipidoses are inherited metabolic disorders associated with glycosphingolipids accumulation, neurodegeneration, and neuroinflammation leading to severe neurological symptoms. Lysoglycosphingolipids (lysoGSLs), also known to accumulate in the tissues of sphingolipidosis patients, exhibit cytotoxicity. LysoGSLs are the possible pathogenic cause, but the mechanisms are still unknown in detail. Here, we first show that lysoGSLs are potential inhibitors of phosphoinositide 3-kinase (PI3K) to reduce cell survival signaling. We found that phosphorylated Akt was commonly reduced in fibroblasts from patients with sphingolipidoses, including GM1/GM2 gangliosidoses and Gaucher's disease, suggesting the contribution of lysoGSLs to the pathogenesis. LysoGSLs caused cell death and decreased the level of phosphorylated Akt as in the patient fibroblasts. Extracellularly administered lysoGM1 permeated the cell membrane to diffusely distribute in the cytoplasm. LysoGM1 and lysoGM2 also inhibited the production of phosphatidylinositol-(3,4,5)-triphosphate and the translocation of Akt from the cytoplasm to the plasma membrane. We also predicted that lysoGSLs could directly bind to the catalytic domain of PI3K by in silico docking study, suggesting that lysoGSLs could inhibit PI3K by directly interacting with PI3K in the cytoplasm. Furthermore, we revealed that the increment of lysoGSLs amounts in the brain of sphingolipidosis model mice correlated with the neurodegenerative progression. Our findings suggest that the down-regulation of PI3K/Akt signaling by direct interaction of lysoGSLs with PI3K in the brains is a neurodegenerative mechanism in sphingolipidoses. Moreover, we could propose the intracellular PI3K activation or inhibition of lysoGSLs biosynthesis as novel therapeutic approaches for sphingolipidoses because lysoGSLs should be cell death mediators by directly inhibiting PI3K, especially in neurons.

In mammals and birds, tool-using species are characterized by their relatively large telencephalon containing a higher proportion of total brain neurons compared to other species. Some teleost species in the wrasse family have evolved tool-using abilities. In this study, we compared the brains of tool-using wrasses with various teleost species. We show that in the tool-using wrasses, the telencephalon and the ventral part of the forebrain and midbrain are significantly enlarged compared to other teleost species but do not contain a larger proportion of cells. Instead, this size difference is due to large fiber tracts connecting the dorsal part of the telencephalon (pallium) to the inferior lobe, a ventral mesencephalic structure absent in amniotes. The high degree of connectivity between these structures in tool-using wrasses suggests that the inferior lobe could contribute to higher-order cognitive functions. We conclude that the evolution of non-telencephalic structures might have been key in the emergence of these cognitive functions in teleosts.

Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.

Osteoclasts, responsible for bone resorption, are multinucleated cells formed by cell-cell fusion of mononuclear pre-osteoclasts. Although osteoclast fusion is a pivotal step for osteoclastogenesis, little is known about the mechanism involved. To clarify the underlying process, we investigated dynamics of membrane phospholipids during osteoclastogenesis in vitro. We found that the cellular content of phospholipids, phosphatidylethanolamine (PE) in particular, was increased during osteoclast differentiation. Furthermore, PE was greatly increased in the outer leaflet of the plasma membrane bilayer during osteoclastogenesis, being concentrated in filopodia involved in cell-cell fusion. Immobilisation of the cell surface PE blocked osteoclast fusion, revealing the importance of PE abundance and distribution. To identify the molecules responsible for these PE dynamics, we screened a wide array of lipid-related genes by quantitative PCR and shRNA-mediated knockdown. Among them, a PE-biosynthetic enzyme, acyl-CoA:lysophosphatidylethanolamine acyltransferase 2 (LPEAT2), and two ATP-binding cassette (ABC) transporters, ABCB4 and ABCG1, were markedly increased during osteoclastogenesis, and their knockdown in pre-osteoclasts led to reduction in PE exposure on the cell surface and subsequent osteoclast fusion. These findings demonstrate that the PE dynamics play an essential role in osteoclast fusion, in which LPEAT2, ABCB4 and ABCG1 are key players for PE biosynthesis and redistribution.

Simultaneous circulation of multiple arboviruses presents diagnostic challenges. In May 2016, chikungunya fever was diagnosed in a traveler from Angola to Japan. Travel history, incubation period, and phylogenetic analysis indicated probable infection acquisition in Angola, where a yellow fever outbreak is ongoing. Thus, local transmission of chikungunya virus probably also occurs in Angola.

Polyunsaturated fatty acids (PUFAs) confer health benefits by preventing inflammation and obesity and by increasing thermogenesis in brown and beige adipocytes. As well as being supplied exogenously as nutrients, PUFAs are largely stored in membrane glycerophospholipids and released by phospholipase A2s (PLA2s). However, the molecular identity of the PLA2 subtype(s) that supplies endogenous PUFAs for metabolic homeostasis remains unclear. Here we show that PLA2G2D, a secreted PLA2 isoform, is constitutively expressed in M2-type macrophages in white adipose tissue (WAT) and shows a reciprocal correlation with obesity. Studies using global and macrophage-specific Pla2g2d-deficient mice reveal that PLA2G2D increases energy expenditure and thermogenesis by facilitating adipocyte browning, thereby ameliorating diet-induced obesity, insulin resistance, and WAT inflammation. Mechanistically, PLA2G2D constitutively supplies a pool of PUFAs, ω3 in particular, in WAT. Thus, our present findings underscore the contribution of the macrophage-driven PLA2G2D-ω3 PUFA axis to metabolic health.

The association between the tumor microenvironment (TME) and treatment response or survival has been a recent focus in several types of cancer. However, most study materials are resected specimens that were completely modified by prior chemotherapy; therefore, the unmodified host immune condition has not yet been clarified. The aim of the present study was to evaluate the relationship between TME assessed in pre-therapeutic biopsy samples and chemoresistance in esophageal cancer (EC). A total of 86 endoscopic biopsy samples from EC patients who received neoadjuvant chemotherapy (NAC) prior to surgery were evaluated for the number of intratumoral CD4+ lymphocytes (with/without Foxp3 expression), CD8+ lymphocytes (with/without PD-1 expression), monocytes (CD14+ ) and macrophages (CD86+ , CD163+ and CD206+ ) by multiplex immunohistochemistry (IHC). The number of tumor-infiltrating CD206+ macrophages I significantly correlated with cT, cM, cStage and neutrophil/lymphocyte ratio (NLR), whereas the number of lymphocytes (including expression of Foxp3 and PD-1) was not associated with clinico-pathological features. The high infiltration of CD163+ or CD206+ macrophages was significantly associated with poor pathological response to NAC (P = 0.0057 and 0.0196, respectively). Expression of arginase-1 in CD163+ macrophages tended to be higher in non-responders (29.4% vs 18.2%, P = 0.17). In addition, patients with high infiltration of M2 macrophages exhibited unfavorable overall survival compared to those without high infiltration of M2 macrophages (5-year overall survival 57.2% vs 71.0%, P = 0.0498). Thus, a comprehensive analysis of TME using multiplex IHC revealed that M2 macrophage infiltration would be useful in predicting the response to NAC and long-term survival in EC patients.

Cerebrospinal fluid-contacting (CSF-c) cells containing monoamines such as dopamine (DA) and serotonin (5-HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF-c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5-HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF-c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF-c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF-c cells coexpress TH2 and TPH1 and contain both DA and 5-HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF-c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF-c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF-c cells, while teleosts have increased their relative number.

Extracellular vesicles (EVs) including exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of EV lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived EVs is augmented via secreted phospholipase A2 (sPLA2)-driven lipid metabolism. Hydrolysis of EV phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids, and their metabolites. sPLA2-treated EVs were smaller and self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor-associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice, while treatment with sPLA2-modified EVs reversed this phenotype. Furthermore, sPLA2 expression in human large B cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated EV modification promotes tumor development, highlighting a non-canonical mechanistic action of EVs as an extracellular hydrolytic platform of sPLA2.

Tapeworm (cestode) infections occur worldwide even in developed countries and globalization has further complicated the epidemiology of such infections. Nonetheless, recent epidemiological data on cestode infections are limited. Our objectives were to elucidate the clinical characteristics and epidemiology of diphyllobothriosis and taeniosis in Tokyo, Japan.

Although the clinical outcomes for patients with ST-elevation myocardial infarction (STEMI) have improved significantly, some patients still experience poor clinical outcomes. The available risk classifications focus on the short-term outcomes, and it remains important to find high-risk features among patients with STEMI. In Japan, the 200 m walk electrocardiogram (ECG) test is widely performed before discharge. The purpose of this study was to investigate the association between the excessive increase in systolic blood pressure (SBP) following a 200 m walk and the long-term clinical outcomes in patients with STEMI.

Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis, catalyzing transformation of l-tyrosine to l-DOPA. Two TH genes (TH1 and TH2) have been reported to exist in the genome of some teleost fishes, TH1 being orthologous to the mammalian TH gene (Candy and Collet, 2005). Here we show that two TH genes are commonly found in genomes of jawed vertebrates. Our analyses of molecular phylogeny and gene synteny strongly suggest that the two TH genes emerged as a consequence of a whole genome duplication before the divergence of jawed vertebrates, and that TH2 was secondarily lost in eutherians (placental mammals). The distribution of TH1 and TH2 transcripts revealed that TH1 and TH2 are differentially expressed in the zebrafish adult brain, as often observed for duplicated genes. In particular we found that TH2 transcripts were much more abundant than TH1 in the hypothalamus, and that the TH2 cells along the periventricular zone are devoid of TH immunoreactivity, due to the lack of affinity of the available anti-TH antibodies. Although these neurons have been considered to be dopamine-uptaking cells in previous studies, the expression of other monoaminergic markers such as aromatic amino acid decarboxylase (AADC), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) suggests that these TH2 cells are dopamine-synthesizing neurons.

Oxidative stress and chronic low-grade inflammation in the lungs are associated with aging and may contribute to age-related immune dysfunction. To maintain lung homeostasis, chronic inflammation is countered by enhanced expression of proresolving/antiinflammatory factors. Here, we show that age-dependent increases of one such factor in the lungs, a phospholipase A2 (PLA2) group IID (PLA2G2D) with antiinflammatory properties, contributed to worse outcomes in mice infected with severe acute respiratory syndrome-coronavirus (SARS-CoV). Strikingly, infection of mice lacking PLA2G2D expression (Pla2g2d(-/-) mice) converted a uniformly lethal infection to a nonlethal one (>80% survival), subsequent to development of enhanced respiratory DC migration to the draining lymph nodes, augmented antivirus T cell responses, and diminished lung damage. We also observed similar effects in influenza A virus-infected middle-aged Pla2g2d(-/-) mice. Furthermore, oxidative stress, probably via lipid peroxidation, was found to induce PLA2G2D expression in mice and in human monocyte-derived macrophages. Thus, our results suggest that directed inhibition of a single inducible phospholipase, PLA2G2D, in the lungs of older patients with severe respiratory infections is potentially an attractive therapeutic intervention to restore immune function.