known as samon no kawa no shio yaki in Japan is an easy recipe that can be savoured as an appetiser or as a side dish with soup or salad This preparation is featured in Chihiro Masui’s book which contains 30 traditional recipes adapted to European ingredients by chef Masao Karasuyama In this book, journalist Chihiro Masui meets prominent names in Japanese fish cuisine, such as Hachiro Mizutani and Takeshi Ohira, a fugu specialist They detail the three fundamental Japanese techniques for fish preparation: ikejime the bleeding of the fish; and ‘fish resting.’ Set aside the carefully scaled skin of a salmon used in another preparation Cut it to the desired dimensions and clean it with a knife removing excess fat and flesh to leave only the skin Salt the skin and let it rest for a few minutes Cut the skin into narrow strips and roll them around the skewers Cook on a grill directly over a gas flame or on charcoal embers Poissons, un art du Japon (2018) (‘Fish: An Art of Japan’), a cookbook by Chihiro Masui with photographs by Richard Haugton, published by Glénat (not currently available in English) Chihiro Masui is a Japanese journalist and translator she has developed a deep knowledge of world cuisines through her travels She mainly writes about the culinary creations of renowned chefs Recipe for English Breakfast with ‘Udon’ by TheSocialFood Recipe for ‘Tai No Osumashi’ by Chihiro Masui and Masao Karasuyama Recipe for Chicken ‘Karaage’ from the ‘Midnight Diner’ Traditional tattoos were strong signifiers; murderers had head tattoos The series 'Hysteric Ten' by photographer Sawatari Hajime revisits one of the most sulphurous relationships in Japanese art Nagisa Oshima made Sada Abe the heroine of his film defying the codes of 'pinku eiga' that make men the focus of attention This rice soup seasoned with miso is served by a monk to Ashitaka one of the heroes in Hayao Miyazaki's film we interviewed its founder and chief representative Look out for your first newsletter in your inbox soon We help you navigate a myriad of possibilities Sign up for our newsletter for the best of the city By entering your email address you agree to our Terms of Use and Privacy Policy and consent to receive emails from Time Out about news, events, offers and partner promotions. Tokyo Recycle Gallery News Karasuyama is one of the larger recycle shops you’ll come across in Tokyo and offers some pretty good thrifting for those with enough patience to sift the entire store. The two-storey shop is like a giant warehouse filled with all sorts of bits and bobs ranging from used furniture to full-on working appliances like refrigerators and washing machines.  The first floor is where you'll find a large selection of antiques, ceramics, Japanese tableware and home appliances. There's also a musical instrument section and an area dedicated to secondhand clothing. Larger furniture pieces such as bed frames, tables and couches are available upstairs. If you’re someone who’s just moved to Tokyo, this would be a good spot to pick up some affordable items to fill your new home. The shop even offers a free one-hour rental of a small truck if you need some help to bring your things home with you (available only for purchases of ¥5,000 or more). The shop can be reached in under 30 minutes from Shibuya and is just a ten-minute walk from Chitose-Karasuyama Station. facebooktwitterpinterestinstagramAbout us Your browser does not support JavaScript, or it is disabled．Please check the site policy for more information Tochigi Prefecture--The mere mention of the Meguro to a diehard motorbike fan in Japan is likely to induce raptures the legendary motorcycle is making its return after 75 years to its manufacturing place here where a rare model--identical to the one featured in “Godzilla Minus One”--is being meticulously restored to like-new condition Fans across Japan are gearing up for the moment they hear its engine roar once more in its rightful setting: Karasuyama in Tochigi Prefecture named after a specific area of the capital was established in 1924 by Meguro Manufacturing Co Meguro Manufacturing relocated its factory to what is now NasuKarasuyama city to avoid air raids the Karasuyama factory took on motorcycle production fulltime Renowned for their exceptional performance and quality Meguro motorcycles were used by the Tokyo Metropolitan Police and as lead vehicles for the torch relay in the 1964 Tokyo Olympics making them dream bikes for riders at the time A project is now under way to restore a 1949 Meguro Z model which were built at the now-closed Karasuyama factory and which featured in “Godzilla Minus One,” to like-new condition within three years Fifteen companies under the umbrella of the Nasukarasuyama chamber of commerce and industry combined their expertise for the restoration the project is nearing completion with only the engine remaining to be finished The restoration has been a painstaking process due to the lack of detailed blueprints and other data are handcrafted based on surviving originals A muffler from a different model was replaced with a correct replica was scanned with a 3D scanner and recreated by machining it from a solid block of metal The paint was carefully analyzed for color and composition at the Industrial Technology Center of Tochigi Prefecture to replicate it as closely as possible to the original “I want to hear the engine roar next year,” Sawamura said Labor disputes and fierce competition with rivals such as Honda Motor Co. proved to be the death knell for Meguro Manufacturing’s operations leading to its acquisition by Kawasaki Aircraft Co. the Karasuyama factory was sold to another company Meguro regained the spotlight in February 2021 when Kawasaki Motors Corp. a member of the Kawasaki Heavy Industries Group but there’s no trace of it left here,” lamented Yoshiyuki Yamada a motorcycle enthusiast and restaurant owner He wondered if there was something he could do Yamada contacted fellow motorcycle enthusiasts and organized a fan meeting in Nasukarasuyama in November 2021 approximately 300 people from across the country gathered on their motorcycles “We started (the fan meeting) as an amateur idea but it exceeded all expectations in excitement,” Yamada said “I’m still amazed by how many fans Meguro has.” The city government and local tourism association also joined the effort to support community revitalization making the fan meeting an annual event with the goal of establishing the city as a “Meguro pilgrimage site.” it donated a massive promotional billboard for the K3 measuring 2.8 meters tall and 4.5 meters wide that was installed in front of a tourist facility in the city center “We appreciated the citizens’ efforts to preserve the Meguro’s legacy,” a Kawasaki Motors representative said The facility now features exhibits showcasing the history of Meguro Manufacturing the fourth annual fan meeting drew more than 380 people on motorcycles nationwide “It’s rare to see so many Meguro motorcycles in one place,” said a 70-year-old enthusiast who rode his 1960s Meguro Z7 from Tokyo’s Adachi Ward Participants enthusiastically shared stories about the Meguro and paraded through the city on their motorcycles they celebrated the 100th anniversary of Meguro Manufacturing’s founding with a toast of specially made cider The event also featured food trucks and play areas for children It attracted around 1,200 people and is gradually growing into a gathering that appeals to more than just Meguro fans “This is a unique attempt to combine a hobby gathering with community revitalization,” said Yamada who chairs the fan meeting organizing committee “I want locals to learn about the city’s history and the skills of those who came before us.” which used to prosper due to Meguro productions An 85-year-old resident who used to work at the Karasuyama factory said “The event brought back a wave of nostalgia Kawasaki rolls out world-first hydrogen engine motorcycle Coming to grips with limitations of electric motorbikes Enthusiasts risk life and limb to ride prewar motorcycles Giant Godzilla scarecrow roars to world record in Kyushu town Son repays dad with Honda bike he sold off to get him into baseball Smart robots do all the work at Nissan’s ‘intelligent’ plant Information on the latest cherry blossom conditions Please right click to use your browser’s translation function.) A series based on diplomatic documents declassified by Japan’s Foreign Ministry Here is a collection of first-hand accounts by “hibakusha” atomic bomb survivors chefs and others involved in the field of food introduce their special recipes intertwined with their paths in life A series about Japanese-Americans and their memories of World War II In-house News and Messages Copyright © The Asahi Shimbun Company. All rights reserved. No reproduction or republication without written permission. Volume 5 - 2024 | https://doi.org/10.3389/falgy.2024.1402841 Basophils are the least common granulocytes accounting for <1% of peripheral blood leukocytes analytical tools for mouse basophils have been developed and we now recognize that basophils play critical roles in various immune reactions including the development of allergic inflammation and protective immunity against parasites the combined use of flow cytometric analyses and knockout mice has uncovered several progenitor cells committed to basophils in mice advancements in single-cell RNA sequencing (scRNA-seq) technologies have challenged the classical view of the differentiation of various hematopoietic cell lineages This is also true for basophil differentiation and studies using scRNA-seq analysis have provided novel insights into basophil differentiation including the association of basophil differentiation with that of erythrocyte/megakaryocyte and the discovery of novel basophil progenitor cells in the mouse bone marrow we summarize the recent findings of basophil ontogeny in both mice and humans mainly focusing on studies using scRNA-seq analyses indicating the evolutional conservation of basophils and the unique role of basophils in animals we will focus on novel insights into the ontogeny of basophils mainly identified by using single-cell transcriptomic techniques Analytical tools for mouse basophil research Progenitor cells committed to basophils or mast cells Single-cell colony-forming assay revealed that ∼10% of BMCPs in the bone marrow can differentiate into both basophils and mast cells bipotential basophil and mast cell progenitor cells produce unipotent progenitor cells of each cell type which further differentiate into mature basophils and mast cells whether BMCPs and pre-BMPs are the different cell types is unclear Comprehensive single-cell transcriptomic analysis that can characterize both BMCPs and pre-BMPs is required to resolve this problem whether the differentiation of human basophils is associated with that of human mast cells is unclear in contrast to the case of mouse basophils Classical and revised model of hematopoietic cell differentiation (A) Classical model of hematopoietic cell differentiation tree Hematopoietic stem cells (HSCs) and multi-potential progenitors (MPPs) firstly differentiate into common myeloid precursors (CMPs) and common lymphoid precursors (CLPs) and CMPs further differentiate into megakaryocyte/erythrocyte precursors (MEPs) and granulocyte/macrophage precursors (GMPs) (B) The revised model of hematopoietic cell differentiation HSCs and MPPs differentiate into common myeloid precursors (CMPs) and lympho-myeloid primed progenitors (LMPPs) CMPs differentiate into MEPs and eosinophil-basophil progenitor (EoBP) while LMPPs differentiate into GMPs and lymphoid progenitor cells (C) Continuous hematopoietic differentiation landscape model emerged after the advancements in the scRNA-seq analysis Hematopoietic cell lineages continuously change their cellular states during the differentiation we summarize the recent findings in basophil differentiation mainly focusing on the following points: (1) the association of the basophil differentiation trajectory to the erythrocyte/megakaryocyte differentiation trajectory in mice and humans; (2) the association of basophil differentiation with the differentiation of mast cells or eosinophils; (3) the characterization of basophil progenitor cells located upstream of mature basophils Single-cell transcriptomic datasets for the analysis of basophil/mast cell ontogeny They conducted scRNA-seq analysis of DNA-barcoded mouse hematopoietic stem cells cultured ex vivo for 2 Lineage tracing identified the presence of progenitor cells that differentiate into basophil/mast cell/eosinophil and erythrocyte/megakaryocyte lineages Lineage hierarchy tree constructed by clonal couplings inferred that the differentiation trajectory of basophil/mast cell/eosinophil is coupled with that of erythrocyte/megakaryocyte in vitro the conclusion was different when the authors used scRNA-seq datasets obtained from in vivo differentiated DNA-barcoded hematopoietic stem cells indicating further experimental evidence to verify the coupling of differentiation pathways between basophil/mast cell and erythrocyte lineages they showed that CMP populations can be subdivided by surface expressions of two markers: CD114 (G-CSFR) and CD131 (IL-3Rβ) Single-cell culture experiments showed that CD114+ CMPs produce neutrophil and monocyte lineages whereas CD131+ CMPs produce both eosinophil/mast cell/basophil and megakaryocyte/erythrocyte lineages These results support the notion that the differentiation trajectory of basophils/eosinophils/mast cells is associated with that of erythrocytes/megakaryocytes but not that of neutrophils/monocytes the relationship among the ontogeny of eosinophil Contrary to the case of mice, the association of the basophil-differentiation trajectory with that of mast cells or eosinophils is less clear in humans. Because studies using scRNA-seq have positioned basophils close to mast cells or eosinophils (56, 57) and eosinophils may be closely coupled to each other in humans the bulk transcriptomic analysis identified that pre-basophils showed limited gene expression changes in response to antigen/IgE stimulation although FcεRIα expression on pre-basophils was higher than mature basophils Bulk transcriptomic analysis further showed that IL-3-stimulated pre-basophils and mature basophils showed distinct gene expression profiles pre-basophils and mature basophils are summarized pre-basophils reside in the bone marrow and are rarely detected outside the bone marrow 7 days after infection with intestinal helminth Nippostrongylus brasiliensis (Nb) CLEC12Ahi pre-basophils egress the bone marrow and can be detected in the spleen CLEC12Ahi pre-basophils detected in the infected lungs retained their proliferative capacity and expressed IL-4 comparable to mature basophils the scRNA-seq analysis of basophils accumulating in the skin infection site after the second Nb infection demonstrated that pre-basophils detected in the skin infection site show transcriptional profiles similar to those of pre-basophils in the bone marrow of noninfected mice IL-3 promotes blood basophilia during Nb infections (69, 70) mice treated with IL-3 complexes also showed the emergence of CLEC12Ahi pre-basophils in the peripheral blood IL-3 upregulation caused by helminth infection may promote the egress of pre-basophils from the bone marrow Further mechanistic study identified that IL-3 promotes the downregulation of chemokine receptor CXCR4 on basophils in the bone marrow which prevents the retention of pre-basophils in the bone marrow and promotes the appearance of pre-basophils in the periphery Several transcription factors (TFs) regulate the differentiation into basophils and mast cells (Figure 3 and Table 4) (16, 37–39, 42, 44, 76–83) These TFs are divided into two groups: (1) TFs important for both basophil and mast cell lineages; (2) TFs important for basophils but not for mast cells Transcription factors (TFs) regulating the differentiation of basophils and mast cells in mice For the generation of common basophil/mast cell progenitors (pre-BMPs) possibly caused by upregulation of STAT5 and/or IRF8 the STAT5–GATA2–C/EBPα signaling pathway is required the STAT5–GATA2–MITF signaling pathway is required Transcription factors (TFs) regulating the fate of basophils and mast cells in mice STAT5–GATA2 signaling is critical for both the differentiation and maintenance of basophils and mast cells in mice the factor(s) inducing STAT5 activity for basophil/mast cell differentiation under homeostatic conditions remains unclear indicating that factor(s) other than IL-3 or TSLP played critical roles for the STAT5-mediated basophil differentiation IRF8 plays critical roles in the development of basophils and mast cells. Mechanistically, IRF8 regulates GATA2 expression in granulocyte progenitors; therefore, IRF8 deficiency impairs the generation of bipotent pre-BMPs in the bone marrow, leading to impaired development of basophils and mast cells in the bone marrow (79) IRF8-deficient mice showed significantly reduced pre-BMPs IRF8-deficient mice showed a normal number of MCPs and mature mast cells in the tissues (e.g. Considering that the number of BMCPs in the spleen was unaffected by IRF8 deficiency multiple pathways might have existed to retain tissue mast cell numbers the discrepancy in the basophil numbers between ΔdblGATA mice and GATA1-low mutant mice might stem from the extent of the GATA2 expression in basophils A recent report identified that the use of the Myb-68kb enhancer region is largely restricted to basophil and mast cell lineages (16) CRISPR-mediated depletion of Myb-68 enhancer results in the reduced Myb expression and impaired generation of basophils and mast cells in vitro indicating the role of Myb-68 enhancer in basophil and mast cell differentiation When bone marrow cells from Ikaros-deficient mice were cultured with IL-3 and stem cell factor (SCF) for 6 weeks some cells showed c-Kit−FcεRIα+ basophil-like surface expression phenotype Ikaros directly binds to the promotor region of Cebpa and represses the C/EBPα expression possibly through histone modification PLZF deficiency partially reduced the number of basophil progenitors and mature basophils in the bone marrow PLZF deficiency also influenced IL-4 production of basophils in response to IgE-dependent stimulation PLZF plays key roles in the development and function of basophils Although PLZF is expressed in mature mast cells the effect of PLZF deficiency on mast cells remains unclear possibly due to the reduced IL-4 expression in skin-infiltrating basophils patients with GATA2 deficiency syndrome have decreased surface expression of FcεRIα on peripheral blood basophils patients with GATA2-deficiency syndrome had a reduced prevalence of IgE-medicated hypersensitivity syndrome possibly due to the reduced FcεRI expression on basophils and mast cells whether GATA2 deficiency in humans affects the generation of basophils and mast cells in the bone marrow remains unclear although the frequency of basophils in the peripheral blood is unaffected by GATA2 deficiency in humans Recent advancements in the development of scRNA-seq techniques have brought us novel insights into the developmental pathways of hematopoietic cell lineages This is also the case in the differentiation of basophils and mast cells and recent studies using scRNA-seq analysis have elucidated the differentiation trajectory of basophils and mast cells in both mice and humans scRNA-seq analyses have confirmed the associated differentiation trajectory between basophils and mast cells in mice a series of scRNA-seq analyses have shown the potential coupling of basophil-differentiation pathway with the erythrocyte/megakaryocyte differentiation pathway in mice and humans scRNA-seq studies have identified a novel progenitor population of basophils in the mouse bone marrow which might connect to the identification of novel drug targets in basophil-related disorders including allergic diseases and parasitic infections The authors declare financial support was received for the research This work was supported by research grants from the Japanese Ministry of Education Astellas Foundation for Research on Metabolic Disorders (KM) Daiichi Sankyo Foundation of Life Science (KM) The Chemo-SeroTherapeutic Research Institute (KM) Grant-in-Aid for JSPS Fellows 23KJ0837 (JI) 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 The authors declared that they were an editorial board member of Frontiers All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher Expression profiling of constitutive mast cells reveals a unique identity within the immune system Naturally abundant basophils in the snapping turtle possess cytophilic surface antibody with reaginic function (1983) 130(1):334–40 doi: 10.4049/jimmunol.130.1.334 Teleost basophils have IgM-dependent and dual Ig-independent degranulation systems Basophils are essential initiators of a novel type of chronic allergic inflammation Basophils enhance immunological memory responses Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness Novel basophil- or eosinophil-depleted mouse models for functional analyses of allergic inflammation Basophils orchestrate chronic allergic dermatitis and protective immunity against helminths Genetic analysis of basophil function in vivo The Basoph8 mice enable an unbiased detection and a conditional depletion of basophils Basophils trigger emphysema development in a murine model of COPD through IL-4-mediated generation of MMP-12-producing macrophages in IL-4 production by IL-3-stimulated murine basophils CT-M8 mice: a new mouse model demonstrates that basophils have a nonredundant role in lupus-like disease development Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils A Myb enhancer-guided analysis of basophil and mast cell differentiation PubMed Abstract | Crossref Full Text | Google Scholar PubMed Abstract | Crossref Full Text | Google Scholar Role of basophils in a broad spectrum of disorders PubMed Abstract | Crossref Full Text | Google Scholar Basophils beyond allergic and parasitic diseases Lung single-cell signaling 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identified through single-cell RNA sequencing Single-cell molecular profiling provides a high-resolution map of basophil and mast cell development Identification of two distinct pathways of human myelopoiesis CD203c distinguishes the erythroid and mast cell-basophil differentiation trajectories among human FcεRI+ bone marrow progenitors Active eosinophils regulate host defence and immune responses in colitis TAS-seq is a robust and sensitive amplification method for bead-based scRNA-seq Functional interleukin-33 receptors are expressed in early progenitor stages of allergy-related granulocytes IL-3 is required for increases in blood basophils in nematode infection in mice and can enhance IgE-dependent IL-4 production by basophils in vitro T cell-derived IL-3 plays key role in parasite infection-induced basophil production but is dispensable for in vivo basophil survival Modulating the human basophil phenotype during its development and maturation: basophils derived from in vitro cultures of CD34(+) progenitor cells Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population Human mast cells arise from a common circulating progenitor Lin- CD34hi CD117int/hi FcepsilonRI+ cells in human blood constitute a rare population of mast cell progenitors Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors Stat5 expression is critical for mast cell development and survival IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen The STAT5-GATA2 pathway is critical in basophil and mast cell differentiation and maintenance Transcription factor IRF8 plays a critical role in the development of murine basophils and mast cells GATA-1 regulates the generation and 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mutant Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression [GATA-1(low) mice] Runx transcription factors in the development and function of the definitive hematopoietic system Regulation of hematopoietic development by ZBTB transcription factors PubMed Abstract | Crossref Full Text | Google Scholar Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms The role of GATA2 in adult hematopoiesis and cell fate determination GATA-2-deficient mast cells limit IgE-mediated immediate hypersensitivity reactions in human subjects Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq CRISPR screening in hematology research: from bulk to single-cell level Ito J and Karasuyama H (2024) Novel insights into the ontogeny of basophils Received: 18 March 2024; Accepted: 1 May 2024;Published: 13 May 2024 © 2024 Miyake, Ito and Karasuyama. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Kensuke Miyake, bWl5YWtlLm1iY2hAdG1kLmFjLmpw Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish A seven-minute walk north from Chitose-Karasuyama Station on the Keio Line brought me to a 1980s-style cafe in a residential area The sign with a cute logo reminiscent of old girls’ manga and the white lace curtains hanging over the glass door give it an air of the Showa era (1926-1989) I was drawn in by the atmosphere of Kissa Housekibako please disable the ad blocking feature and reload the page This website uses cookies to collect information about your visit for purposes such as showing you personalized ads and content By clicking “Accept all,” you will allow the use of these cookies Users accessing this site from EEA countries and UK are unable to view this site without your consent By Keito Uchiyama / Yomiuri Shimbun Staff Writer I was struck by the record covers of Showa singers Kenji Sawada and Seiko Matsuda Nostalgic items fill the shelves and walls the small cafe has only six seats and is run by Naomi Kanai alone “I’ve been collecting girly items from the 1970s and 1980s since I was a kid,” the 52-year-old owner said “That era was full of cute things that were not too sophisticated.” posed dolls in glass cases and photographic portraits of stars representing the era The tables are covered with red and white gingham tablecloths and a retro beaded curtain hangs over the aisles Napolitan (ketchup-flavored) spaghetti is the cafe’s signature dish and they also serve soda floats that come in 10 flavors named after jewels such as aquamarine or emerald A pair of women were seen looking around the interior saying “kawaii” (cute) and taking photos of the character merchandise and soda floats they ordered Kanai chooses the songs played in the cafe I ate the Napolitan spaghetti and a cream float while listening to “Kaito Ruby” sung by 1980s idol Kyoko Koizumi and felt like I was transported back to the Showa era Access: 7-minute walk from north exit of Chitose-Karasuyama Station Memo: With a maximum capacity of six people the cafe accepts groups of no more than two Our weekly ePaper presents the most noteworthy recent topics in an exciting © 2025 The Japan News - by The Yomiuri Shimbun Metrics details The light-dependent ion-transport function of microbial rhodopsin has been widely used in optogenetics for optical control of neural activity In order to increase the variety of rhodopsin proteins having a wide range of absorption wavelengths the light absorption properties of various wild-type rhodopsins and their artificially mutated variants were investigated in the literature we demonstrate that a machine-learning-based (ML-based) data-driven approach is useful for understanding and predicting the light-absorption properties of microbial rhodopsin proteins We constructed a database of 796 proteins consisting of microbial rhodopsin wildtypes and their variants We then proposed an ML method that produces a statistical model describing the relationship between amino-acid sequences and absorption wavelengths and demonstrated that the fitted statistical model is useful for understanding colour tuning rules and predicting absorption wavelengths two residues that were not considered in previous studies are newly identified to be important to colour shift we construct a machine-learning-based (ML-based) statistical model that describes the relationship between amino-acid sequences and absorption wavelengths of microbial rhodopsins based on past experimental data a tissue-directed delivery method of these analogues must be developed An overview of the machine-learning-based (ML-based) data-driven approach introduced in the present paper for functional protein studies a training protein set containing pairs of amino-acid sequence and molecular properties is first constructed an ML method is applied to the training set and an ML-based statistical model is constructed The obtained ML model can be used in understanding the relationship between amino-acid sequences and molecular properties such as the colour tuning rules in the case of microbial rhodopsins The ML model can also be used to predict the molecular properties of new uninvestigated proteins We refer to the set of new proteins as the target protein set we regard KR2 wildtype and its 118 variants as target proteins and other 677 rhodopsin proteins in the database as the training proteins We consider the following hypothetical scenario for the purpose of demonstration The database is divided into two sets: a target protein set and a training protein set The target set contains KR2 wild-type rhodopsin and its variants (which are assumed to be uninvestigated as of yet) whereas the training set contains the remaining proteins in the database We constructed an ML model using only the proteins in the training set The constructed model was then applied to the proteins in the target set for predicting the absorption wavelengths of KR2 and its variants This scenario is interpreted as a hypothetical situation where a researcher is interested in predicting the absorption wavelengths of a new group of rhodopsin proteins based on previously reported data on other groups of rhodopsin proteins The advantages of group-wise sparse learning approaches are not only predictability but also interpretability of the constructed models by using a group-wise sparse learning approach the absorption wavelengths of KR2 and its variants could be predicted from their amino-acid sequences with an average error of ±7.8 nm The residues affecting the absorption wavelength were also identified and their strength for colour shift and the effect of mutation were quantitatively investigated the effects for colour shift of which were not reported in previous studies were newly shown to significantly affect the absorption wavelengths the model constructed by a group-wise sparsity learning approach enables the identification of active residues residues for which the choice of the amino-acid species has a great influence on the absorption wavelength Although we herein focus on the prediction of absorption wavelengths of rhodopsin proteins the same ML approach can be used to predict other molecular properties in other types of functional proteins Structure of the database used in the present study The database is composed of the sequences and λmaxs of 519 previously reported proteins and 277 newly reported proteins We used 677 rhodopsin proteins other than KR2 and their variants as the training proteins (red rectangle) and 119 proteins in KR2 group as the target proteins (blue rectangle) The target set consists of 119 rhodopsin proteins in the KR2 group (KR2 wildtype and its 118 variants), whereas the training set consists of the remaining 677 rhodopsin proteins (see Figs 1 and 3) We applied an ML method to the training set and constructed a statistical model describing the relationship between the amino-acid sequences and absorption wavelengths The statistical model was then applied to the rhodopsin proteins in the target set in order to predict their absorption wavelengths This scenario assumes a hypothetical situation in which a researcher is interested in investigating a new group of rhodopsin proteins based on previously reported data on other groups of rhodopsin proteins A schematic description of the ML method introduced in the present paper for functional protein studies (a) Binary sequence representation of an amino-acid sequence Let M = 20 be the number of amino-acid species and let N be the number of residues considered in the present study the amino-acid sequence of a protein is represented by M × N binary variables each of which represents the amino-acid species at each residue (b) By writing the MN binary variables as xi,j we consider an MN-dimensional linear model The linear model has an intercept parameter β0 and MN coefficient parameters βi,j a group-wise sparsity constraint is introduced all of the corresponding M coefficients would be fitted to zero and only a small number of residues have nonzero coefficient parameters The latter residues are called active residues The choice of amino-acid species in these active residues is expected to play an important role in determining molecular properties such as absorption wavelength Coefficient parameters of the fitted statistical model Coefficients for the top 20 active residues where the activeness of each residue is defined as \({s}_{j}:=\sqrt{{\sum }_{i=1}^{M}\,{\beta }_{i,j}^{2}},j=\mathrm{1,}\ldots ,N\) red and blue indicate that the corresponding parameters are positive and negative whereas grey indicates that the amino-acid species did not exist in the training data if the value of a coefficient parameter βi,j is positive/negative (i.e then the existence of the i-th amino-acid species at the j-th residue has a red-shifting/blue-shifting effect Absorption wavelength prediction results for KR2 wildtype and its 118 variants (a–d) Absorption spectra of KR2 mutants ((a) KR2 NTQ/F72G and (d) DTD/D102N) with their absorption maxima as predicted by ML analysis (green lines) and experimentally determined (blue lines) The spectrum of KR2 wildtype is indicated by the solid grey line (e) The horizontal axis represents the experimentally observed absorption wavelengths whereas the vertical axis represents the absorption wavelengths predicted by the ML model The red points indicate the KR2 group rhodopsin proteins in the target set whereas the black points indicate other rhodopsin proteins in the training set (f) Histogram of the prediction errors for KR2 group proteins in the target set The effect of a point mutation on the absorption wavelength shift can be estimated based on the coefficient parameters βi,j 1}MN be the binary vector representation of the KR2 wild-type sequence The difference in the predicted absorption wavelengths between KR2 wildtype and a variant having amino-acid sequence x(Var) ∈ {0 The colour-shifting effect of point mutation at the j-th residue is written as Lists of sequences for the KR2 wildtype and the variants with their observed and predicted absorption wavelengths (a) KR2 and the 25 variants that have the longest observed wavelengths and (b) KR2 and the 25 variants that have the shortest observed wavelengths The residues shown here are replaced at least once among the 50 variants Boxes with thick black lines indicate positions that have different amino-acid species from the KR2 wildtype the colour indicates the wavelength change produced by the replacement of the j-th position estimated by \({\sum }_{i\mathrm{=1}}^{M}\,{\beta }_{i,j}({x}_{i,j}^{(\mathrm{Var})}-{x}_{i,j}^{(\mathrm{KR}\mathrm{2)}})\) where \({x}_{i,j}^{(\mathrm{KR}\mathrm{2)}}\) and \({x}_{i,j}^{(\mathrm{Var})}\) are the binary representation the KR2 wildtype and a variant Whereas the aspartate at the position of BR Asp85 has the second lowest coefficient value (−19.5) among all of the residues investigated herein the value of the position of BR Asp212 is moderate (−3.2) This result suggests that the former has a much stronger effect on colour tuning despite the symmetric location of these two residues relative to RSB (The distances from Asp85 and Asp212 to the N atom of RSB are 3.4 and 3.5 Å This strong interaction with retinal is consistent with the high degree of activeness of this residue and the coefficient parameter of tryptophan is a large positive value (12.0) This suggests that this tryptophan has a role in shifting the absorption wavelength to be longer in many rhodopsins GR Glu166 works as an active residue for the colour tuning in GR These results imply the usefulness of ML analysis in identifying active residues located far from retinal which are generally of less concern in experimental research on the colour tuning mechanism from a structural point of view The effects on the absorption wavelength by the mutation of these residues have not yet been reported we expect that they will be experimentally verified in the near future N can be also used as a guide for new functional protein design suppose that a researcher wants to construct a rhodopsin mutant the absorption wavelength of which is as long as possible for optogenetics application Note that positive/negative coefficient parameter values indicate that the amino-acid species at the residue have a red-shifting/blue-shifting effect on the light-absorption properties of rhodopsin proteins Consider a residue j at which there exists i1 and i2 such that \({\beta }_{{i}_{1},j} < {\beta }_{{i}_{2},j}\) If there exists a rhodopsin protein having the i1-th amino-acid species at the j-th residue by replacing this species with the i2-th amino-acid species the new protein is expected to have a longer wavelength than the original protein the basic experimental design strategy for the above-mentioned researcher would be to replace the amino-acid species having a smaller coefficient parameter with that having a larger coefficient parameter such as protein stability and functionality must be taken into account in new functional protein design the above discussion suggests that the ML-based data-driven approach enables systematic design of experiments without relying on the intuition or heuristics of researchers The errors between the predicted and the actual wavelengths are 8.04 nm in average which is close to the average error 7.8 nm for target set We believe that our result would be a significant case study for a data-driven analysis of functional proteins We constructed the database of absorption wavelengths of microbial rhodopsins for the first time and demonstrated that ML provides an accurate prediction model of the wavelength which can be beneficial for optogenetic applications Note that the machine-learning model employed in this paper is a simple approximation of complicated colour tuning rule of microbial rhodopsion our machine-learning model only consider additive effect of each amino-acid there is a trade-off between the database size and the model complexity If we can increase the size of our database by collecting new experimental results in the future which can incorporate combinatorial effects of multiple amino acids 1} be a binary variable that indicates the existence of the i-th amino-acid species in the j-th residue M of xi,j corresponds to one of M = 20 amino-acid species We consider predicting the absorption wavelength based on a linear model: Suppose that we have K pairs of an amino-acid sequence and its absorption wavelength \({\{({{\bf{x}}}^{(k)},{\lambda }_{{\rm{\max }}}^{(k)})\}}_{k=1}^{K}\) where x(k) ∈ \({\mathbb{R}}\) MN is the bied as a vector and \({\lambda }_{{\rm{\max }}}^{(k)}\in {\mathbb{R}}\) is the absorption wavelength of the k-th rhodopsin protein The parameters are fitted by solving the following penalized least-squares problem: where γ > 0 is a tuning parameter. This formulation is called group LASSO19 in which the first term is the sum of the squared prediction errors and the second term is the group-wise penalty for the parameters we define the M = 20 coefficient parameters (β1,j If the training set indicates that the choice of the amino-acid species at the j-th residue does not affect the colour tuning property then the group-wise sparsity penalty forces all of the M = 20 parameters (β1,j We can easily identify a set of important residues for determining the absorption wavelength by this effect because usually only a small subset of the residues have non-zero coefficient parameters the parameter γ was objectively chosen by the cross-validation procedure within the training set The difference spectra after and before the bleaching by hydroxylamine in 100 mM NaCl 2% DDM for KR2 mutants for which large red shift of the absorption was expected by ML The times after the addition of hydroxylamine are shown in each panel Our program code of the group LASSO for wavelength prediction is available at http://www-als.ics.nitech.ac.jp/~karasuyama/software/GrplassoSeq.zip The database of the amino-acid sequences and their wavelengths is provided in Supplementary Table 1 Microbial and animal rhodopsins: structures Optogenetics: 10 years of microbial opsins in neuroscience Anion-induced wavelength regulation of absorption maxima of schiff bases of retinal Independent optical excitation of distinct neural populations The photochemical reactions of bacterial sensory rhodopsin-I flash photolysis study in the one microsecond to eight second time window ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis Directed evolution of Gloeobacter violaceus rhodopsin spectral properties An external point-charge model for bacteriorhodopsin to account for its purple color A hybrid method for solutes in complex solvents: Density functional theory combined with empirical force fields Electrostatic potential at the retinal of three archaeal rhodopsins: Implications for their different absorption spectra Color tuning in rhodopsins: The mechanism for the spectral shift between bacteriorhodopsin and sensory rhodopsin II Polarization effects stabilize bacteriorhodopsin’s chromophore binding pocket: A molecular dynamics study Toward automatic rhodopsin modeling as a tool for high-throughput computational photobiology Retinal-based proton pumping in the near infrared Statistical Learning with Sparsity: The Lasso and Generalizations Regression shrinkage and selection via the lasso Model selection and estimation in regression with grouped variables Structure-function studies on bacteriorhodopsin substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin Interaction of the indole of tryptophan-182 with the 9-methyl groap of the retinal in the L intermediate of bactericrhodopsin Machine-learning-assisted materials discovery using failed experiments A blue-shifted light-driven proton pump for neural silencing Atomistic design of microbial opsin-based blue-shifted optogenetics tools Nature’s toolkit for microbial rhodopsin ion pumps A light-driven sodium ion pump in marine bacteria The role of the NDQ motif in sodium-pumping rhodopsins Effects of three characteristic amino acid residues of pharaonis phoborhodopsin on the absorption maximum Diversification and spectral tuning in marine proteorhodopsins A color-determining amino acid residue of proteorhodopsin Theoretical studies on the color-tuning mechanism in retinal proteins Role of Asn112 in a light-driven sodium ion-pumping rhodopsin Specific lipid-protein interactions in a novel honeycomb lattice structure of bacteriorhodopsin Download references We appreciate for the insightful discussion and data collection by Drs Fujiwara for ML application for the color tuning of rhodopsin The present study was financially supported by grants from the Japanese Ministry of Education from the RIKEN Center for Advanced Intelligence Project to I.T. and by the JST support program for starting up innovation-hub on materials research by information integration initiative to M.K Masayuki Karasuyama and Keiichi Inoue contributed equally Japan Science and Technological Agency (JST) Center for Materials research by Information Integration National Institute for Materials Science (NIMS) Department of Life Science and Applied Chemistry RIKEN Center for Advanced Intelligence Project constructed the database and interpreted the results conducted hydroxylamine bleaching experiments The authors declare no competing interests Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-018-33984-w Anyone you share the following link with will be able 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Volume 9 - 2018 | https://doi.org/10.3389/fphys.2018.01769 This article is part of the Research TopicTick and Tick-Borne Pathogens: Molecular and Immune Targets for Control StrategiesView all 25 articles Ticks are blood-sucking arthropods that can transmit various pathogenic organisms to host animals and humans causing serious infectious diseases including Lyme disease Tick feeding induces innate and acquired immune responses in host animals depending on the combination of different species of animals and ticks Acquired tick resistance (ATR) can diminish the chance of pathogen transmission from infected ticks to the host the elucidation of cellular and molecular mechanism underlying ATR is important for the development of efficient anti-tick vaccines we briefly overview the history of studies on ATR and summarize recent findings particularly focusing on the role for basophils in the manifestation of ATR basophil accumulation is observed at the tick re-infestation site even though the frequency of basophils among cellular infiltrates varies in different animal species ranging from approximately 3% in mice to 70% in guinea pigs memory CD4+ T cells contribute to the recruitment of basophils to the tick re-infestation site through production of IL-3 in mice Depletion of basophils before the tick re-infestation abolishes ATR in guinea pigs infested with Amblyomma americanum and mice infested with Haemaphysalis longicornis demonstrating the crucial role of basophils in the manifestation of ATR The activation of basophils via IgE and its receptor FcεRI is essential for ATR in mice Histamine released from activated basophils functions as an important effector molecule in murine ATR probably through promotion of epidermal hyperplasia which interferes with tick attachment or blood feeding in the skin Accumulating evidence suggests the following scenario The 1st tick infestation triggers the production of IgE against tick saliva antigens in the host and blood-circulating basophils bind such IgE on the cell surface via FcεRI IgE-armed basophils are recruited to tick-feeding sites and stimulated by tick saliva antigens to release histamine that promotes epidermal hyperplasia Further studies are needed to clarify whether this scenario in mice can be applied to ATR in other animal species and humans tick infestation is of medical and veterinary public health importance the elucidation of cellular and molecular mechanisms underlying ATR is important for developing efficient anti-tick vaccines that can minimize the transmission of pathogens causing serious infectious diseases Basophils contribute to protective immunity particularly to parasitic infections while they are involved in the pathogenesis of various disorders including allergic and autoimmune disorders we summarize recent advances in our understanding of the cellular and molecular mechanisms underlying ATR particularly focusing on the role of basophils identified mainly in mouse models of tick infestation even though the frequency of basophils among cellular infiltrates varied and the functional role of basophils in these animals has not yet been determined to our knowledge it remained elusive whether the important finding on basophils in guinea pig ATR can be generalized to other animal species and humans including the distinct roles played by basophils and mast cells and the influence of different genetic background of both mice and ticks remained to be clarified Basophils accumulate at the tick-feeding site during the 2nd but not 1st tick infestation Mcpt8GFP (green basophil) mice were infested with ticks one or twice and subjected to intravital fluorescence imaging analysis of green basophils at tick-feeding sites on day 2 of the 1st or 2nd infestation Basophil infiltration was detected in humans at the tick-feeding sites and in the skin lesions of scabies (Ito et al., 2011; Nakahigashi et al., 2013; Kimura et al., 2017). Of note, a patient lacking basophils and eosinophils reportedly suffered from widespread scabies (Juhlin and Michaelsson, 1977) These observations suggest the possible involvement of basophils in protective immunity to ectoparasites The 1st infestation triggers the production of IgE against tick saliva antigens and basophils and mast cells bind IgE on the cell surface via FcεRI tick saliva antigens delivered into the tick-feeding site bind to IgE on these cells leading to the cross-linking of FcεRI and hence activation of these cells that may contribute to ATR Schematic view of the proposed mechanism underlying ATR dendritic cells in the skin take up tick saliva antigens and move to the draining lymph node where they present tick antigens to naive CD4+ T cells leading to the generation of IL-4-producing T cells T cell-derived IL-4 stimulates B cells to produce tick antigen-specific IgE that in turn circulates in the peripheral blood and bind to the surface of blood-circulating basophils via FcεRI Some of tick antigen-specific CD4+ T cells generated in the lymph node migrate into the skin throughout the body and are retained as skin-resident memory CD4+ T cells are stimulated with tick antigens to produce IL-3 that in turn promotes the recruitment of IgE-armed basophils from the peripheral blood to the tick-feeding site IgE-armed basophils are activated with tick antigens to release histamine that acts on keratinocyte resulting in epidermal hyperplasia that may interfere with tick attachment or blood feeding in the skin The role of skin mast cells in ATR remains elusive These results suggested that basophils rather than mast cells play a critical role in IgE-dependent ATR through FcεRI-mediated activation even though both types of cells contribute to ATR It remains to be investigated whether anti-α-gal IgE is involved in ATR Molecular characterization of tick salivary components has demonstrated that different members among the same multi-gene family are expressed at distinct time points during tick feeding (Karim and Ribeiro, 2015). For example, two cystatin genes from Ixodes scapularis change their expression reciprocally during feeding (Kotsyfakis et al., 2007; Karim and Ribeiro, 2015) Such antigenic variation or sialome switch during tick feeding is considered as a possible mechanism by which ticks avoid host immune responses It remains to be determined whether such variation can affect the production of anti-tick IgE and hence IgE-mediated ATR and whether IgE raised against one family member is cross-reactive to other members of the same family Further studies are needed to examine whether such IgG binding proteins can interfere with IgE-mediated ATR in the host and whether the host raises antibodies against them to neutralize their activity suggesting the possible involvement of histamine to ATR the cellular source of histamine responsible for ATR and the mechanism underlying histamine-mediated ATR remained ill-defined demonstrating the crucial role of basophil-derived histamine in the manifestation of ATR Intravital imaging analysis of cells at the 2nd tick feeding site demonstrated that basophils make a cluster within the epidermis and surround a tick mouthpart. In contrast, mast cells are mostly scattered in the dermis rather than epidermis and localized more distantly from the tick mouthpart (Tabakawa et al., 2018) It is well known that histamine has a short half-life basophil-derived histamine may be much more effective than mast cell-derived in the manifestation of ATR considering the fact that higher numbers of basophils are localized closer to a tick mouthpart the concentration of histamine near tick mouthparts should be much higher during the 2nd infestation compared to the 1st infestation one may assume that HBPs might be less effective in sequestering histamine at the 2nd tick-feeding site in which ATR is executed The influence of HBPs on histamine-mediated ATR could be explored by generating HBP-deficient ticks in future studies suggesting the possibility that histamine-induced thickening of the epidermis prevents the former’s but not the latter’s mouthparts from penetrating into the dermis in order to form blood pools This may explain the differential responsiveness to histamine among tick species in terms of ATR induction it is possible that the presence or absence (or differential amounts) of HBPs in different tick species is correlated in part with differential reactivity to histamine in the induction of tick resistance leading to transendothelial migration of basophils and their accumulation in the skin one may assume that mast cells may contribute to ATR by directly or indirectly regulating basophil behavior Further studies are needed for elucidating how mast cells contribute to the manifestation of ATR Further studies on the detailed mechanism underlying ATR may help develop the strategy to prevent tick infestation and tick-borne diseases HK has substantially contributed to the literature review and drafted the manuscript SY contributed to the discussion and drafting This work was supported by research grants from Japanese Ministry of Education Science and Technology [15H05786 (HK) and 17K15719 (SY)] and TMDU President’s Young Researchers Award (SY) Expression and function of histamine and its receptors in atopic dermatitis Tick resistance: basophils in skin reactions of resistant guinea pigs CrossRef Full Text | Google Scholar Histology of bovine skin reactions to Ixodes holocyclus neumann Cutaneous basophil-associated resistance to ectoparasites (ticks) Transfer with immune serum or immune cells Google Scholar Cutaneous Immunity to the Tick Ixodes Holocyclus Google Scholar Resistance to tick-borne Francisella tularensis by tick-sensitized rabbits: allergic klendusity IL-3 augments adhesiveness for endothelium and CD11b expression in human basophils but not neutrophils basophils and eosinophils in the sequence of cellular events in the skin of infested or re-infested rabbits Google Scholar Characterization of tick antigens inducing host immune resistance Description of rabbit-acquired immunity to Amblyomma americanum ticks and identification of potential tick antigens by Western 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Evaluation of basophil infiltration into the skin lesions of tick bites Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity The role of histamine H1 and H4 receptors in atopic dermatitis: from basic research to clinical study Skin CD4+ memory T cells play an essential role in acquired anti-tick immunity through interleukin-3-mediated basophil recruitment to tick-feeding sites Tick histamine-binding proteins: isolation Ticks and tick-borne bacterial diseases in humans: an emerging infectious threat Emerging antigens involved in allergic responses the first lineage-specific differentiation marker for mouse basophils Elevated numbers of potent IL-4-producing and MMCP-8-positive cells in spleens of malaria-infected mice doi: 10.1002/1521-4141(200009)30:9<2660::AID-IMMU2660>3.0.CO;2-I A high affinity serotonin- and histamine-binding lipocalin from tick saliva Tick resistance in mast cell-deficient mice: histological studies The alpha-gal story: lessons learned 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Hajime Karasuyama, a2FyYXN1eWFtYS5tYmNoQHRtZC5hYy5qcA== Soichiro Yoshikawa, eW9zaGlzb3UubWJjaEB0bWQuYWMuanA= In a mountainous area of Tochigi Prefecture north of Tokyo tiger puffers are being farmed inland with low salinity water from local hot springs sells 25 tons a year to around 150 local stores Based in the rural town of Nasu-karasuyama Yumesozo is the first in Japan to successfully farm tiger puffers The technique of cultivating the species (Takifugu rubripes) in hot spring water was invented to help revitalize the town Now it’s spreading to other areas of Japan “People in Nasu-karasuyama began moving to the cities to work,” Yumesozo President Katsuaki Noguchi told the Advocate “This reduced the town’s population considerably Japan’s declining birthrate and ageing population are also significant issues I was born and raised in Nasu-karasuyama and wondered if there was anything I could do to revitalize it.” While running his own environmental consulting firm Noguchi began investigating Tochigi Prefecture’s chloride springs as a possible resource to develop Nasu-karasuyama He found that the Yamizo-Sanchi mountain range contained underground seawater at about 500 meters below ground level he discovered that it contained elements such as sodium and potassium “It became clear that the water could be used to cultivate fish,” said Noguchi “Its salt concentration is around 0.9 to 1.2 percent I set up Yumesozo and began a series of rearing experiments with filefish They all grew well but after comparing profitability and species value Noguchi wondered if tiger puffer were particularly suited to hot spring water a fish physiology specialist who has spent many years researching fish osmoregulation at the University of Tokyo’s Graduate School of Agricultural and Life Sciences “After receiving requests from universities I’d decided to explore a new type of aquaculture – land-based farming of marine fish in low-salinity environments – when I was contacted by Yumesozo,” he said The natural habitat of tiger puffer is seawater which has a salt concentration of around 3.5 percent Tiger puffer reduce that salt density to around 0.9 percent by absorbing water through their gills Kaneko told Noguchi that because the salt density of the hot spring water in Nasu-karasuyama is also around 0.9 percent the fish do not need to burn calories to adjust the salt content in their bodies And thanks to the amount of nutrients in the water they also grow bigger than those farmed at sea while hot spring water has very low pathogen levels making it difficult for the fish to become ill There is also no winter chill to slow down their growth as the hot spring water is used to heat the rearing tanks via a heat exchanger “The temperature of the water in our tanks is kept at around 22 degrees-C or 72 degrees-F,” said Noguchi “The hot spring water travels through the heat exchanger before returning to the hot spring facilities we exchange the water we use to farm the tiger puffer but because we have a closed circulation system Using heat from the Earth’s interior is affordable and it’s a sustainable way of reducing dependence on fossil fuels,” he said The biggest challenge in land-based farming is electricity costs and there are additional expenses for fuel and boilers – unless you are using hot spring water Tiger puffer are one of Japan’s top delicacies despite (or because of) the fact that they usually contain a deadly neurotoxin called tetrodotoxin This requires the fish to be prepared by licensed chefs But the fish farmed by Yumesozo are nontoxic “Tetrodotoxin is produced by bacteria,” said Kaneko “High concentrations accumulate mainly in organs such as the liver or ovaries but these won’t necessarily be poisonous in every fish Tetrodotoxin doesn’t accumulate in tiger puffer reared in hot spring water but their liver and ovaries cannot be supplied to shops at present.” Noguchi also carried out research to improve the taste of tiger puffer meat which can become mild and watery because of the low salt concentration of hot spring water he transferred the fish to artificial seawater the day before shipment and killed them by draining their blood from above the gills and the base of the tail which had been accustomed to lower salt concentrations of the hot spring water injected amino acids into their muscle tissue to cope with the sudden change of osmotic pressure caused by the increase in salinity This made their taste stronger and more delicious Yumesozo is now focusing on promoting tiger puffer farming firms that have franchise contracts with the company operate in 18 locations across Japan Land-based farming is also proving popular with the tiger puffer growing just as well as in ordinary seawater Although it takes a year and a half for tiger puffer raised in sea farms to be ready for market those grown in hot spring water reach market size in just one year “This is because we’re farming the fish in a low-salinity environment and increasing heat in winter,” said Noguchi “We can also guarantee a high production volume and yields of 75 to 80 percent and there are additional expenses for fuel and boilers – unless you are using hot spring water.” Farming fish on land offers protection against natural disasters such as tsunamis and has fewer environmental impacts and it allows land-locked regions to play a part in aquaculture I think that land-based marine fish farming could become more mainstream and we may see huge developments in future involving land-based farming and hot spring water,” he said Follow the Advocate on Twitter @GAA_Advocate … please consider supporting GSA’s mission to advance responsible seafood practices through education The Advocate aims to document the evolution of responsible seafood practices and share the expansive knowledge of our vast network of contributors By becoming a Global Seafood Alliance member you’re ensuring that all of the pre-competitive work we do through member benefits Individual membership costs just $50 a year Sara Rademaker launched American Unagi to shift eel farming to American soil and because she saw an opportunity to do things better.  A team of scientists recently made the case against octopus farming What lies behind this emerging aquaculture opportunity?  Bluefin tuna may be the most prized fish in the ocean closed-cycle aquaculture may help keep it on menus.  With all eyes on Japan for the 2020 Olympic Games leading advocates for responsible aquaculture there see a key opportunity to make stronger sustainability commitments.  The Responsible Seafood Advocate supports the Global Seafood Alliance’s (GSA) mission to advance responsible seafood practices through education Kodansha will publish the July issue on June 6 The manga's story centers around a Japanese high school teacher named Seiji Hasumi an accomplished Harvard College graduate who is secretly a psychopath He systematically begins murdering bullies and overbearing parents to solve these problems The manga adapts Kishi's 2010 dark crime novel of the same name. Karasuayama launched the manga adaptation in 2012 Kodansha will publish the manga's eighth volume on May 7 Japanese film director Takashi Miike also adapted the novel into a live-action movie starring Hideaki Ito AD Leave a rating/comment#Manga / Comics#StartingInApr2025RecommendedExhibits#Exhibitions to see in 2024#Exhibitions to see during Summer VacationBack to ArticlesSHARE �@�@Top > �X�^�b�t�Љ� > �G�R�@��@�v���t�B�[�� Karasuyama, H., Wada, T., Yoshikawa, S., and Obata, K.: Emerging roles of basophils in protective immunity against parasites. Trends Immunol . 32: 125-130, 2011. (PubMed) Karasuyama, H., Mukai, K., Obata, K., Tsujimura, Y., and Wada, T.: Nonredundant roles of basophils in immunity. Annu. Rev. Immunol . 29: 45-69, 2011.�@(PubMed) Saito, M., Nagasawa, M., Takada, H., Hara, T., Tsuchiya, S., Agematsu, K., Yamada, M., Kawamura, N., Ariga, T., Tsuge, I., Nonoyama, S., Karasuyama, H ., and Minegishi, Y.: Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells. J. Exp. Med . 208: 235-249, 2011. (PubMed) Wada, T., Ishiwata, K., Koseki, H., Ishikura, T., Ugajin, T., Ohnuma, N., Obata, K., Ishikawa, R., Yoshikawa, S., Mukai, K., Kawano, Y., Minegishi, Y., Yokozeki, H., Watanabe, N., and Karasuyama, H.: Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks. J. Clin. Invest . 120: 2867-2875, 2010. (PubMed) Karasuyama, H., Mukai, K., Tsujimura, Y. and Obata, K.: Newly-discovered roles for basophils: a neglected minority gains new respect. Nat. Rev. Immunol . 9: 9-13, 2009. (PubMed) Tsujimura, Y., Obata, K., Mukai, K., Shindou, H., Yoshida, M., Nishikado, H., Kawano, Y., Minegishi, Y., Shimizu, T. and Karasuyama, H .: Basophils play a pivotal role in immunoglobulin G- but not immunoglobulin E-mediated systemic anaphylaxis. Immunity 28: 581-589, 2008 (PubMed) Minegishi, Y., Saito, M., Tsuchiya, S., Tsuge, I., Takada, H., Hara T., Kawamura, N., Ariga, T., Pasic, S., Stojkovic, O., Metin, A., and Karasuyama, H .: Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature 448: 1058-1062, 2007 (PubMed) Mukai, K., Matsuoka, K., Taya, C., Suzuki, H., Yokozeki, H., Nishioka, K., Hirokawa, K., Etori, M., Yamashita, M., Kubota, T., Minegishi, Y., Yonekawa, H., and Karasuyama, H.: Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells. Immunity 23: 191-202, 2005 (PubMed) Toyama-Sorimachi, N., Tsujimura, Y., Maruya,M., Onoda, A., Kubota, T., Koyasu, S., Inaba, K. and Karasuyama, H.: Ly49Q, a novel member of Ly49 family that is selectively expressed on myeloid lineage cells and involved in regulation of cytoskeletal architecture. Proc. Natl. Acad. Sci. USA. 101: 1016-1021, 2004 (PubMed) A.: Essential role of Src-family protein tyrosine kinases in NF-kB activation during B cell development Metrics details Basophils are the rarest granulocytes and are recognized as critical cells for type 2 immune responses their differentiation pathway remains to be fully elucidated we assess the ontogenetic trajectory of basophils by single-cell RNA sequence analysis Combined with flow cytometric and functional analyses we identify c-Kit-CLEC12Ahi pre-basophils located downstream of pre-basophil and mast cell progenitors (pre-BMPs) and upstream of CLEC12Alo mature basophils The transcriptomic analysis predicts that the pre-basophil population includes previously-defined basophil progenitor (BaP)-like cells in terms of gene expression profile Pre-basophils are highly proliferative and respond better to non-IgE stimuli but less to antigen plus IgE stimulation than do mature basophils Although pre-basophils usually remain in the bone marrow probably through IL-3-mediated inhibition of their retention in the bone marrow the present study identifies pre-basophils that bridge the gap between pre-BMPs and mature basophils during basophil ontogeny and protective immunity against parasitic infections single-cell transcriptomes have shown differentiation pathways from HSCs to BaPs the continuous differentiation trajectory from progenitors to mature basophils remains to be fully defined and it is uncertain whether intermediate populations exist between progenitors and mature basophils along the basophil ontogeny we define the basophil differentiation trajectory by conducting highly sensitive scRNA-seq analysis of basophils in mice and identify pre-basophils located between pre-BMPs and mature basophils during basophil ontogeny much lower than commonly used concentration turned out to be optimal for high purity of basophils Bone marrow cells were cultured in the presence of 0.3 ng/mL of IL-3 for 7 days a CD200R3+cKit− BMBAs were gated (left panel) and the surface expression of FcεRIα and CD49b is shown (right panel) c Two subpopulations of BMBAs (FcεRIαhiCD49blo and FcεRIαloCD49bhi) were separately isolated and subjected to May-Grünwald-Giemsa staining (Scale bar 10 μm) (b) and flow cytometric analysis (c) d–f BMBAs were subjected to scRNA-seq analysis Shown are UMAP plot of basophil clusters (d) violin plots of the expression of indicated genes in each cluster (e) and volcano plot of genes differentially expressed in Baso1 versus Baso2/3 (f) Blue and pink dots indicate genes upregulated in Baso1 and Baso2/3 clusters non-parametric Wilcoxon rank sum test with Bonferonni correction was conducted g CD200R3+cKit- BMBAs were subdivided into FcεRIαhiCD49blo and FcεRIαloCD49bhi subpopulations (left panel) and the surface expression of CLEC12A and CD9 in each subpopulation is shown (right panels) h CD200R3+cKit− BMBAs were subdivided into CLEC12AloCD9hi and CLEC12AhiCD9lo subpopulations (left panel) and the surface expression of FcεRIα and CD49b in each subpopulation is shown (right panels) h) are representative of at least three independent experiments Data in (d–f) were obtained from a single experiment Data shown in (a–d) are representative of at least three independent experiments Data in (e–i) were obtained from a single experiment A small fraction (11.4%) of Clec12ahi basophils displayed low levels of Cd34 in accordance with the analysis of our scRNA-seq data suggesting this small population within Clec12ahi basophils may correspond to Cd34lo BaP-like cells a–c FcεRIαhiCD49blo and FcεRIαloCD49bhi subpopulations of basophils sort-purified from the bone marrow were separately cultured ex vivo for 1 or 2 days the time course of their surface CLEC12A and CD9 expression is shown the change in the frequency of CLEC12Ahi cells during the culture is shown (mean ± SEM the change in the number of live cells during the culture is shown The red and blue bars correspond to the CLEC12AloCD9hi and CLEC12AhiCD9lo fractions Data in (a–c) are representative of at least three independent experiments d FcεRIαhiCD49blo and FcεRIαloCD49bhi subpopulations of basophils were separately isolated from the bone marrow of CD45.2+ C57BL/6 mice (5 × 104 cells/mouse) mixed with CD45.1+ whole bone marrow cells (0.3 × 106 cells/mouse) and intravenously administered to sublethally-irradiated CD45.1+ congenic C57BL/6 mice bone marrow cells were prepared from recipient mice and subjected to flow cytometric analysis for the expression of CLEC12A and CD9 on CD45.2+ donor-derived basophils (bottom panels) the CLEC12A and CD9 expression on transferred cells (Day0) is displayed in the upper panels The frequency of CLEC12Ahi cells before and 1 day after the transfer in each experimental group is shown in the right panel where data are pooled from two experiments (mean ± SEM Two-way ANOVA with Tukey’s multiple comparisons test was used for multiple comparisons (b we concluded that CLEC12AhiCD9lo/FcεRIαhiCD49blo basophils in the bone marrow display precursor-like phenotypes in terms of surface marker expression and differentiate within the bone marrow into CLEC12AloCD9hi/FcεRIαloCD49bhi mature basophils that are also detected in the peripheral blood and spleen we designated CLEC12AhiCD9lo/FcεRIαhiCD49blo and CLEC12AloCD9hi/FcεRIαloCD49bhi basophils as pre-basophils and mature basophils CD34-CLEC12AhiCD9lo pre-basophils (CD34− pre-Baso) and CLEC12AloCD9hi mature basophils (Mature Baso) isolated from the bone marrow as well as CLEC12AloCD9hi mature basophils isolated from the spleen were subjected to bulk RNA-seq analysis (n = 3 the hierarchically clustered heatmap of DEGs is shown the top 10 enriched GO terms upregulated in pre-basophils and in mature basophils The size of the dots indicates the number of genes associated with indicated GO terms while the color of the dots indicates the adjusted P values (p adjust) calculated by one-sided Fisher’s exact test with Benjamini-Hochberg correction d CD49b+ cells isolated from the bone marrow were incubated ex vivo with EdU for 2 h EdU uptake in CLEC12Ahi and CLEC12Alo basophils is shown e and f Mice were intravenously sensitized with anti-TNP IgE 1 day prior to the experiment CLEC12Ahi and CLEC12Alo basophils isolated from the bone marrow of TNP-IgE-sensitized mice were separately cultured ex vivo in the presence of TNP-conjugated OVA (TNP-OVA) or control OVA for 1 h (e) or 6 h (f) the surface CD63 expression on CLEC12Ahi (blue shaded histogram) and CLEC12Alo (red shaded histogram) basophils is shown in the left panels (mean ± SEM namely MFI of CD63 staining divided by MFI of control staining is shown in each experimental group (middle panel The frequency of CD63hi cells in each experimental group is shown (right panel IL-4 concentration in culture supernatants in each experimental group is shown (mean ± SEM g CLEC12Ahi and CLEC12Alo basophils isolated from the bone marrow were separately cultured ex vivo in the presence of PBS The IL-4 concentration in culture supernatants is shown (mean ± SEM Data in (a–c) were obtained from a single experiment Those shown in (d–g) are representative of at least three independent experiments Unpaired Student’s t test was used for comparing two groups (d) and two-way ANOVA with Tukey’s multiple comparisons test for multiple comparisons (e–g) pre-basophils appeared to be more reactive to non-IgE stimulation whereas mature basophils are more reactive to IgE/allergen stimulation TSLP-elicited basophils do not seem to correspond to pre-basophils and can be categorized as a subpopulation of mature basophils a C57BL/6 mice were infected with Nb larvae and cells were prepared from the bone marrow The expression of CLEC12A and CD9 on cKit-CD200R3+CD49b+ basophils in each time point (left panels) and the time course of cell numbers of CLEC12Ahi pre-basophils and CLEC12Alo basophils (Mature) in each experimental group (right panels) are shown (mean ± SEM b CD49b+ cells isolated from the bone marrow and lungs on day 7 post Nb-infection were incubated ex vivo with EdU for 2 h EdU uptake in pre-basophils and mature basophils is shown in the left panels The frequency of EdU+ cells is shown in the right panel (mean ± SEM c WT mice and IL-4 reporter (G4) mice were infected with Nb larvae GFP fluorescence in pre-basophils (Pre-baso) and mature basophils on day 7 post Nb-infection is shown in the left panels MFI of GFP is shown in the right panel (mean ± SEM d–g C57BL/6 mice were infected twice with Nb larvae and on day 2 of the second infection cKit-CD200R3+CD49b+ basophils were isolated from the bone marrow and infected skin their surface expression of CLEC12A and CD9 is shown e–g They were separately subjected to scRNA-seq analysis UMAP plots show 3 basophil clusters (pre-basophil Mature 1 and 2; defined by Seurat clustering) in the left panel The frequency of each cluster in the bone marrow (BM) and skin is shown in the right panel violin plots of the expression of indicated genes in each cluster are shown the gene set enrichment analysis comparing pre-basophil and mature 1&2 was conducted GSEA enrichment plot of genes involved in the chromosome segregation is shown Normalized enrichment scores (NES) and Benjamini-Hochberg-adjusted p values (p.adjust) calculated by a permutation test are shown Data in (e–g) were obtained from a single experiment Unpaired Student’s t test was used for comparing two groups (c) and one-way ANOVA with Tukey’s multiple comparisons test (a) or two-way ANOVA with Tukey’s multiple comparisons test (b) for multiple comparisons suggesting that pre-basophils retain their high proliferative capacity in the infected skin as observed in the infected lungs a C57BL/6 mice were intraperitoneally administered with IL-3 complex or control PBS Surface expression of CLEC12A and CD9 in cKit-CD200R3+CD49b+ basophils isolated from the peripheral blood 4 days after the treatment is shown (left panels) The number of CLEC12Ahi basophils in the peripheral blood of the treated mice is shown (right panel n = 7 each) (b) BMBAs were stimulated with IL-3 or control PBS for 6 h and subjected to bulk RNA-seq analysis The MA plot of genes expressed in PBS- versus IL-3-treated BMBAs is shown Black dots indicate differentially expressed genes (DEGs) while gray dots indicate non-differentially expressed genes (non-DEGs) Bulk RNA-seq data were obtained in n = 3 cultures for each stimulation c Cell surface expression of CXCR4 in CLEC12AhiCD9lo (blue shaded histogram) and CLEC12Alo CD9hi (red shaded histogram) basophils isolated from the bone marrow of non-treated mice is shown (left panel) Open histogram indicates control staining with isotype-matched control MFI of CXCR4 is shown (right panel) (mean ± SEM d CD49b+ cells isolated from the bone marrow of non-treated mice were cultured ex vivo with IL-3 (10 ng/mL) or control PBS for 4 h CXCR4 expression on CLEC12AhiCD9lo and CLEC12Alo CD9hi basophils after the treatment with IL-3 (blue shaded) or control PBS (gray shaded) is shown (left panel) Open histograms indicate control staining with isotype-matched control e Mice were intraperitoneally treated with AMD3100 (AMD) the spleen and peripheral blood cells were separately isolated and subjected to flow cytometric analysis The surface expression of CLEC12A and CD9 in cKit-CD200R3+CD49b+ basophils and the number of CLEC12AhiCD9lo basophil subpopulation are shown (mean ± SEM Data shown in (a) and (c–e) are representative of at least three independent experiments Data in (b) were obtained from a single experiment Unpaired Student’s t test was used for comparing two groups (a e) and two-way ANOVA with Tukey’s multiple comparisons test for multiple comparisons (d) Advancements of scRNA-seq technology have enabled us to understand the differentiation trajectory of various hematopoietic cell lineages and revisited the classical view of hematopoietic cell differentiation we took advantage of this technology combined with flow cytometric and functional analyses and succeeded in the identification of CLEC12AhiCD9lo pre-basophils that are developmentally located downstream of pre-basophil and mast cell progenitors (pre-BMPs) and upstream of CLEC12AloCD9hi mature basophils in mice The transcriptomic analysis predicted that the pre-basophil population include previously-defined basophil-progenitor (BaP)-like cells in terms of the gene expression profile suggesting the possible contribution of FcεRI++ pre-basophils to the regulation of colitis its function in the basophil egress from the bone marrow remained unexplored we demonstrated that the inhibition of CXCR4 by AMD3100 promotes the egress of pre-basophils from the bone marrow the ex vivo stimulation with IL-3 reduced the expression of CXCR4 on pre-basophils suggesting the possibility that the systemic upregulation of IL-3 leads to the egress of pre-basophils from the bone marrow A similar mechanism may operate during Nb infection to promote the emergence of pre-basophils in peripheral tissues Mice were maintained under specific pathogen-free conditions in our animal facilities and humidity is maintained between 30% and 70% Animals are housed in individually ventilated cages (Innovive Caging System) and are fed with natural ingredient chow diet ad libitum (Japan CLEA: Rodent Diet CE-2) Cages are bedded with cloth material (Japan SLC: Q-pura chip) Animals are group housed whenever possible All animal studies were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (No The following antibodies were purchased from BioLegend: biotinylated anti-B220 (clone: RA3-6B2 dilution 1:400); FITC-conjugated anti-CD49b (clone: HMα2 dilution 1:400); Alexa Fluor 488-conjugated anti-CD45.2 (clone: 104 dilution 1:400); PE-conjugated anti-CD34 (clone: SA376A4 dilution 1:400) and rat IgG2b,κ Isotype Ctrl (clone: RTK4530 dilution 1:400); PE-Cy7-conjugated anti-CD49b (clone: HMα2 dilution 1:400) and anti-CD200R3 (clone: Ba13 dilution 1:400); APC-conjugated anti-CD200R3 (clone: Ba13 dilution 1:400) and anti-CLEC12A (clone: 5D3/CLEC12A dilution 1:400); APC/Cy7-conjugated anti-CD16/32 (clone: S17011E dilution 1:400); APC/Fire750-conjugated anti-CD9 (clone: MZ3 dilution 1:400); Pacific Blue-conjugated anti-c-Kit (clone: 2B8 dilution 1:400) and anti-CD45.1 (clone: A20 dilution 1:400); BV421-conjugated anti-CD34 (clone: SA376A4 dilution 1:400) and anti-CD45 (clone: 30-F11 dilution 1:400); BV510-conjugated anti-FcεRIα (clone: MAR-1 dilution 1:400); BV605-conjugated anti-c-Kit (clone: 2B8 dilution 1:400); BV711-conjugated anti-CXCR4 (clone: L276F12 dilution 1:400); BV786-conjugated anti-IL-7Rα (clone: A7R34 dilution 1:400); PerCP/Cy5.5-conjugated anti-TER119 (clone: TER-119 BD OptiBuild BV711-conjugated anti-CD49b (clone: HMα2 dilution 1:400) was purchased from BD Biosciences FITC-conjugated Streptavidin (catalog #: 11-4317-87 dilution 1:400) was purchased from Thermo Fisher Scientific purchased from ADCC) was prepared in our laboratory 0.3 ng/mL of IL-3 were used for generating BMBAs Single cell suspensions were prepared from the bone marrow and spleen followed by red blood cell (RBC) lysis by using RBC Lysis Buffer (BioLegend) for 5 min For the flow cytometric analysis of peripheral blood leukocytes from the heparinized blood sample were isolated from the interphase of 25% and 65% Percoll PLUS (cytiva) after gradient centrifugation For the flow cytometric analysis of lung and skin samples tissues were minced by razors and dissociated by using gentleMACS dissociator (Miltenyi Biotec) and Lung Dissociation Kit (Miltenyi Biotec catalog #: 130-095-927) or Multi Tissue Dissociation Kit 1 (Miltenyi Biotec Cells were stained with the indicated antibodies after treatment with normal rat serum (Merck Millipore) and TrueStain FcX PLUS (2.5 μg/mL; BioLegend; clone: S17011E dilution 1:200) to prevent non-specific binding Stained cells were analyzed with FACSLyric (BD Biosciences) and FlowJo software ver 10.8.1 (BD Biosciences) or sorted with FACS AriaIII (BD Biosciences) cells collected from the bone marrow or lung were cultured for 2 h with 10 μM EdU (5-ethynyl-2′-deoxyuridine) EdU staining was performed by using Click-iT Plus EdU flow cytometry assay kits (ThermoFisher Scientific catalog #: C10633) according to the manufacturer’s protocol Cytospin samples of sort-purified basophils were stained with May-Grünwald and Giemsa stain (FUJIFILM Wako Pure Chemical) single-cell suspensions were prepared from the bone marrow and infected skin and subjected to positive selection of CD49b-positive cells by using biotinylated anti-CD49b antibody (BioLegend; clone: DX5 dilution 1:400) and EasySep mouse Biotin Positive Selection Kit II (Stemcell Technologies catalog #: ST-17665) followed by the isolation of CD200R3+cKit- cells by cell sorting Basophils isolated from 5 mice were pooled to generate scRNA-seq data BD Rhapsody beads were subjected to TdT (Roche catalog #: 3333574001) and RNase H (Enzymatics followed by second-strand synthesis and 1st round of whole-transcriptome amplification (WTA) cDNA product was purified by 0.65× AmPure XP beads (Beckman Coulter and Hashtag product was isolated from unbounded fraction by adding additional 0.7× AmPure XP beads (final 1.35×) cDNA and hashtag libraries were then subjected to 2nd round of WTA and 2nd round of hashtag-amplification followed by purification by AmPure XP beads Illumina cDNA libraries were constructed from amplified cDNA libraries by using NEBNext Ultra II FS library prep kit for Illumina (New England Biolabs Sequencing was performed by using an Ion Hi-Q Chef kit (Thermo Fisher Scientific an Ion PI v3 Chip kit (Thermo Fisher Scientific and an Ion Proton Sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions Pre-basophils and mature basophils (5 × 104 cells) sort-purified from the bone marrow of CD45.2+ C57BL/6 J wild-type mice together with unsorted CD45.1+ BM cells (0.3 × 106 cells) were intravenously injected into 9-Gy X-ray irradiated CD45.1 congenic mice the bone marrow cells were isolated and subjected to flow cytometric analysis CD34+ cells were not excluded from CLEC12Ahi basophils The IL-3 complex or control PBS was then intraperitoneally administered to mice Single cell suspensions were prepared from the peripheral blood of mice 4 days after administration and subjected to flow cytometric analysis mice were intraperitoneally treated with a single administration of AMD3100 (200 μg per mouse; abcam) or control PBS Single cell suspensions were prepared from the spleen and peripheral blood 1 h after treatment and subjected to flow-cytometric analysis Mice were subcutaneously infected once or twice with third-stage larvae (L3) of Nippostrongylus brasiliensis (Nb) mice were injected with 500 L3 in the tail base infected mice were injected with 500 L3 in the flank 18 days after the first infection the bone marrow and skin at the inoculation site were harvested pre-basophils and mature basophils (2 × 104 cells) were isolated from the bone marrow of non-sensitized mice and stimulated with the following reagents at 37 °C for 6 h: IL-3 (BioLegend: 10 ng/mL) The concentration of IL-4 in culture supernatants was measured by using ELISA MAX Standard Set Mouse IL-4 (BioLegend Statistical analyses were conducted by using GraphPad Prism (ver 7.0.3) or R software (ver 4.1.0) Comparisons between two groups were performed using unpaired Student’s t test Comparisons among multiple groups were performed using one-way or two-way analysis of variance (ANOVA) with post hoc Tukey’s HSD test A P value < 0.05 was considered statistically significant Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The code is available at GitHub (https://github.com/KensukeMiyake/Pre-basophil-paper, https://doi.org/10.5281/zenodo.7769735) Mast cells and basophils in allergic inflammation Recent advancement in the mechanism of basophil activation Basophils and their effector molecules in allergic disorders Antagonistic regulation by the transcription factors C/EBPα and MITF specifies basophil and mast cell fates Epigenetic Regulators as the Gatekeepers of Hematopoiesis A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice Generation and characterization of mouse basophils from bone marrow and purification of basophils from spleen Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells Basophils prime group 2 innate lymphoid cells for neuropeptide-mediated inhibition The skin is an important bulwark of acquired immunity against intestinal helminths Basophil modulation by cytokine instruction Neutrophil chemoattractant receptors in health and disease: double-edged swords CXCL12/CXCR4 signal transduction in diseases and its molecular approaches in targeted-therapy Splenic Ly6G(high) mature and Ly6G(int) immature neutrophils contribute to eradication of S An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection Basophils control T-cell responses and limit disease activity in experimental murine colitis Polyfunctionality of the CXCR4/CXCL12 axis in health and disease: Implications for therapeutic interventions in cancer and immune-mediated diseases Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs Inflammation-induced IgE promotes epithelial hyperplasia and tumour growth The CLEC12A receptor marks human basophils: Potential implications for minimal residual disease detection in acute myeloid leukemia Regulation of expression of IL-4 alleles: Analysis using a chimeric GFP/IL-4 gene TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq Cutadapt removes adapter sequences from high-throughput sequencing reads EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data Integrated analysis of multimodal single-cell data clusterProfiler 4.0: A universal enrichment tool for interpreting omics data The single-cell transcriptional landscape of mammalian organogenesis Generalizing RNA velocity to transient cell states through dynamical modeling Combining an Alarmin HMGN1 Peptide with PD-L1 Blockade Results in Robust Antitumor Effects with a Concomitant Increase of Stem-Like/Progenitor Exhausted CD8(+) T Cells Complement protein C1q activates lung fibroblasts and exacerbates silica-induced pulmonary fibrosis in mice TCC: an R package for comparing tag count data with robust normalization strategies Download references Paul for providing IL-4 reporter G4 (Il4GFP/+) mice; ImmunoGeneTeqs Yoshikawa and all the members of the Karasuyama laboratory for helpful discussions Cell sorting by using FACS AriaIII and scRNA-seq analysis by using BD Rhapsody system were performed in part at Research Core of Tokyo Medical and Dental University (TMDU) KANAE Foundation for the Promotion of Medical Science (K.M.) and Ohyama Health Foundation (K.M.) JST SPRING Grant Number JPMJSP2120 (J.I.) the Japan Agency for Medical Research and Development PRIME program (Grant Number JP21gm6210025) (S.S.) Young Innovative Medical Scientist Unit by Tokyo Medical and Dental University (K.M.) and TMDU priority research areas grant (K.M.) These authors contributed equally: Kensuke Miyake Tokyo Medical and Dental University (TMDU) Division of Molecular Regulation of Inflammatory and Immune Diseases performed most of the experiments and analyzed data All authors provided critical review of the paper The remaining authors declare no competing interests Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-023-38356-1 Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1292279 This article is part of the Research TopicThe Fundamental Biology of Basophils in Health and DiseaseView all 14 articles Editorial on the Research TopicThe fundamental biology of basophils in health and disease Figure 1 Evolution of publications from 1950 to 2019 for each main immune cell type (A) The number of publications per year retrieved from the Pubmed database from 1950 to 2019 is depicted for each query (B) Percentage of publications retrieved from each query from the sum of all the queries represented in A for the year 2019 The year 2019 was chosen as an endpoint as the volume and dynamics of publications changed drastically from 2020 due to the COVID-19 pandemic Despite these exciting discoveries, basophils remain the least studied of the main immune cells, representing less than 1% of these publications in 2019 (Figure 1B) the aim of this Research Topic was to aggregate original manuscripts exploring emerging hot topics in basophils research IgE crosslinking induces several signaling events controlling intracellular calcium mobilization and degranulation. Hansen Selnø et al. explored the expression of the sarcoplasmic reticulum Ca2+ ATPase (SERCA2) in human basophils and its function SERCA2 expression is strongly inversely correlated with anti-IgE-induced histamine release and pharmaceutical inhibition or activation of SERCA proteins controls the amplitude of basophil histamine release SERCA2 appears as a new negative regulator of basophil degranulation Basophil responsiveness to IgE decreases among patients suffering from chronic spontaneous urticaria (CSU), supposedly due to the presence of autoreactive antibodies against IgE or its receptor. However, Matsubara et al. showed that the response of CSU patients’ basophils to the anaphylatoxin C5a is unaltered This suggests targeting the C5a/C5aR axis may be of critical value in patients refractory to anti-FcϵRIα treatments In chronic inducible urticaria, urticaria is induced by specific stimuli, such as ultraviolet light exposure. Mizuno et al. showed that the circulating basophils of these patients are more activated than those of healthy controls (as CSU patients) but without any IgE hyporesponsiveness This highlights differences in the pathophysiology of these distinct conditions Peripheral basopenia in CSU patients is associated with disease activity, and basophils are found in patients’ skin lesions. Kishimoto et al. confirmed previous reports underlining a reversal of basopenia upon treatment of CSU patients with Omalizumab or anti-histamine using an oxazolone-induced contact dermatitis model they demonstrated that the migration of circulating basophils to skin lesions provoked a transient basopenia This supports the concept that clinical observations of basopenia reflect an active basophils extravasation El Hachem et al. explored the mechanisms governing basophils extravasation in the skin in FITC-induced dermatitis They revealed that basophil migration was critically dependent on the secretion of IL-3 by T cells They also demonstrated that IL-3-stimulated human and mice basophils relied on an autocrine retinoid acid production to drive their expression of specific integrins and mice basophil extravasation these results strongly suggest that T cell IL-3 drives basophils autocrine secretion of retinoic acid to enable their extravasation in the inflamed skin The unique properties of skin-homing basophils have been described in a hypothesis and theory article by Shibuya and Kim, which suggests these basophils may have a unique identity, acquired during hematopoiesis and/or through late imprinting by the action of TSLP and epithelial-derived alarmins as mice lung basophils do under the control of IL-33 and GM-CSF (38) Skin-infiltrating basophils may externalize MRGPRX2 a receptor involved in pseudoallergic reactions and neuroimmune interactions MRGPRX2 expression by basophils has been the subject of some controversy. In this Research Topic, Toscano et al. explored the expression and function of this receptor on basophils from patients allergic to birch pollen or hypersensitive to moxifloxacin Circulating basophils express only very low levels of functional surface MRGPRX2 but this is very quickly externalized by specific activation (anti-IgE the reactivity of patients’ basophils to MRGPRX2 ligands can be studied but only when using carefully controlled conditions Basophils are known to participate in allergic airway inflammation and allergic asthma but have mainly been studied following allergen challenge or asthma exacerbation. Here, Iype et al. analyzed the expression of activation markers on stable asthmatics basophils and reported that they express more surface CD25 but no other activation markers As human basophils activated by IL-2 secrete type 2 cytokines this pathway seems important in asthma chronicity and pathophysiology The development of efficient helminth vaccines is an ongoing challenge in immunology. Thuma et al. cloned a new immunogenic protein secreted by the model helminth Nippostrongylus brasiliensis (Nb) during infection Immunization with Nb-LSA1a induces specific IgG1 and protective immunity against Nb infection in wild-type but not basophil-deficient mice This strongly suggests helminth vaccination strategies should benefit from inducing basophil-dependent immunity the manuscripts submitted to this Research Topic underline current and emerging trends in basophils immunology: the regulation of their degranulation via FcϵRIα or MRGPRX2; their roles in chronic urticaria or cancer; the controversies surrounding their regulation of T cell polarization and their potency in promoting anti-helminth protective immunity; the mechanisms controlling their extravasation and peripheral basopenia; and the concept of mature basophils harboring distinct specific identities The author(s) declare that no financial support was received for the research The author(s) declared that they were an editorial board member of Frontiers This had no impact on the peer review process and the final decision Ultrastructural localization of histamine in human basophils and mast cells; changes associated with anaphylactic degranulation and recovery demonstrated with a diamine oxidase-gold probe A role for vesicles in human basophil secretion PubMed Abstract | CrossRef Full Text | Google Scholar Metabolic comparison between basophils and other leukocytes from human blood PubMed Abstract | CrossRef Full Text | Google Scholar Releasability of human basophils: Cellular sensitivity and maximal histamine release are independent variables J Allergy Clin Immunol (1993) 91:605–15 A comparative study of releasing and nonreleasing human basophils: Nonreleasing basophils lack an early component of the signal transduction pathway that follows IgE cross-linking J Allergy Clin Immunol (1990) 85:1020–9 Monitoring human basophil activation via CD63 monoclonal antibody 435 J Allergy Clin Immunol (1991) 88:328–38 Human peripheral blood basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils Induction of human IgE synthesis in B cells by mast cells and basophils The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3 Hymenoptera-venom-induced upregulation of the basophil activation marker ecto-nucleotide pyrophosphatase/phosphodiesterase 3 in sensitized individuals Int Arch Allergy Immunol (2001) 126:335–42 Basophil activation test: Mechanisms and considerations for use in clinical trials and clinical practice A mechanism for the initiation of allergen-induced T helper type 2 responses Basophils play a pivotal role in immunoglobulin-G-mediated but not immunoglobulin-E-mediated systemic anaphylaxis Basophils can directly present or cross-present antigen to CD8 lymphocytes and alter CD8 T cell differentiation into IL-10-producing phenotypes J Immunol Baltim Md (2009) 183:3033–9 CrossRef Full Text | Google Scholar a constitutive component of the IL-3 receptor is required for IL-3-induced IL-4 production in basophils Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation Immunoglobulin D enhances immune surveillance by activating antimicrobial proinflammatory and B cell-stimulating programs in basophils Basophils contribute to TH2-IgE responses in vivo via IL-4 production and presentation of peptide–MHC class II complexes to CD4+ T cells CD200R surface expression as a marker of murine basophil activation Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33 Basophil effector function and homeostasis during helminth infection PubMed Abstract | CrossRef Full Text | Google Scholar Human basophils secrete IL-3: evidence of autocrine priming for phenotypic and functional responses in allergic disease J Immunol Baltim Md (2009) 182:2432–8 CrossRef Full Text | Google Scholar IL-18 and IL-33 elicit Th2 cytokines from basophils via a MyD88- and p38alpha-dependent pathway Role of mast cells and basophils in igE responses and in allergic airway hyperresponsiveness PubMed Abstract | CrossRef Full Text | Google Scholar Mass cytometry phenotyping of human granulocytes reveals novel basophil functional heterogeneity Diverse innate stimuli activate basophils through pathways involving Syk and IκB kinases Activation of human basophils by combined toll-like receptor- and FcϵRI-triggering can promote Th2 skewing of naive T helper cells FcϵRI-activated basophils express CCR4 Chemokine receptors in human basophils: inducible expression of functional CXCR4 Basophils are required for the induction of Th2 immunity to haptens and peptide antigens CrossRef Full Text | Google Scholar Trogocytosis of peptide–MHC class II complexes from dendritic cells confers antigen-presentinzg ability on basophils Proc Natl Acad Sci (2017) 114(5):1111–6 Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid Human basophils release the anti-inflammatory cytokine IL-10 following stimulation with α-melanocyte–stimulating hormone J Allergy Clin Immunol (2021) 147(4):1521–3.E3 The ectoenzyme E-NPP3 negatively regulates ATP-dependent chronic allergic responses by basophils and mast cells Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice The basophil IL-18 receptor precisely regulates the host immune response and malaria-induced intestinal permeability and alters parasite transmission to mosquitoes without effect on gametocytemia and mammalian host-to-mosquito transmission in malaria Citation: Pellefigues C and Karasuyama H (2023) Editorial: The fundamental biology of basophils in health and disease Received: 11 September 2023; Accepted: 06 October 2023;Published: 18 October 2023 Copyright © 2023 Pellefigues and Karasuyama. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Christophe Pellefigues, Q2hyaXN0b3BoZS5wZWxsZWZpZ3Vlc0BpbnNlcm0uZnI= By entering your email address you agree to our Terms of Use and Privacy Policy and consent to receive emails from Time Out about news, events, offers and partner promotions. But while it’s nice to browse the many outlets in the city specialising in pre-loved items sometimes the selections can feel a bit picked over many stores tend to be on the smaller side due to space constraints That’s where mega thrift stores come in: if you have time to venture a little further out into Tokyo’s suburbs or neighbouring prefectures you’ll come across these giants of secondhand retail that make you feel as if you’re shopping at Costco rather than rummaging around some tiny boutique some of these massive thrift shops have even developed cult followings with visitors making purposeful trips out of the city centre to scour stores that compete with sheer selection and amazing prices Plan a trip to one of these massive secondhand stores offering everything from designer fashion and books to homewares and electronics RECOMMENDED: one-of-a-kind concept stores you'll only find in Tokyo Visitors: Be sure to bring your passport with you as Bookoff offers tax free services for tourists. Photo: 2nd StreetYou’ll want to give yourself ample time to browse this Super 2nd Street in Saitama, a colossal version of 2nd Street, which is another popular consignment and used clothing store found across Japan. While 2nd Streets in the city often have a narrow focus on clothing, shoes and accessories, Super 2nd Street offers a much more varied selection.  Omiya Nisshin branch of Super 2nd Street still stocks a plentiful selection of fashion, shoes and accessories, but there’s also a wide range of tableware, used furniture, musical instruments, collectables and children’s toys. They’re good with designer goods, too: you can browse a great assortment of handbags and jewellery as well as many luxury watches.  Super 2nd Street Omiya Nisshin Store is about an 18-minute walk from Miyahara Station and can be reached in about an hour from central Tokyo.  Photo: Recycle Gallery News KarasuyamaRecycle Gallery News Karasuyama is one of the larger recycle shops you’ll come across in Tokyo and offers some pretty good thrifting for those with enough patience to sift the entire store. The two-storey shop is like a giant warehouse filled with all sorts of bits and bobs ranging from used furniture to full-on working appliances like refrigerators and washing machines.  Photo: Treasure Style FactoryTreasure Factory is another recognisable name in the second-hand retail business, and the company has multiple versions of its store around Japan. These include Trefac Style specialising in fashion, and Trefac Sports Outdoor selling sporting goods. The massive Treasure Factory in Nerima is like the mothership of all the stores combined, offering a mix of clothing, designer goods, home appliances, instruments, electronics and more. Treasure Factory Nerima is about a 20-minute walk from Shakujii-koen Station and can be reached in just over 40 minutes from Shinjuku. Photo: Gotemba Premium OutletsLove a good shopping spree? Chiba, Kanagawa and Saitama have discount stores offering big brands at low prices Photo: Nihombashi Mitsukoshi Main StoreFrom food to fashion – everything you need can be found at Tokyo’s vast variety of department stores These flagship stores are home to top Japanese and international brands from Muji to Gucci Thanks for subscribing! Look out for your first newsletter in your inbox soon! Metrics details An Author Correction to this article was published on 30 April 2021 This article has been updated Microbial rhodopsins are photoreceptive membrane proteins which are used as molecular tools in optogenetics a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases the ML-based method selected 65 candidate rhodopsins The wavelengths of 39 of them were able to be experimentally determined by expressing proteins with the Escherichia coli system p = 7.025 × 10−5) actually showed red-shift gains and two were found to have desirable ion-transporting properties indicating that they would be potentially useful in optogenetics These findings suggest that data-driven ML-based approaches play effective roles in the experimental design of rhodopsin and other photobiological studies a Schematic structure of microbial rhodopsins The subfamilies of light-driven ion-pump rhodopsins targeted in this study are differently colored; non-ion-pump microbial rhodopsins and ion-pumping microbial rhodopsins from eukaryotic and giant viral origins are shown in gray The development of next-generation sequencing technology is expected to continue to more rapidly identify a large number of new rhodopsin genes including proteins with even longer wavelength-shifted absorption screening of all of them either by experimental or theoretical methods would be very costly a less expensive and more efficient approach to screen red-shifted rhodopsins is needed and data-driven study is expected as the third class of approach to investigate the color-tuning rule of rhodopsins at low cost Considering the computational cost of both approaches the data-driven approach was found to be much more efficient than the QM/MM approach while the latter provides insights on the physical origin controlling λmax We conducted experiments by introducing the synthesized rhodopsin genes into E coli to measure the absorption wavelengths of 65 candidates for which the ML-based experimental design method predicted that the expected gains were >10 nm 7.025 × 10−5) of the selected candidates showed actual red-shift gains We then investigated the ion-transportation properties of the rhodopsins for which the red-shift gains were >20 nm and found that some actually had desirable ion-transporting properties suggesting that they (and their variants) could potentially be used as new optogenetics tools the differences in the amino acid sequences of the newly examined rhodopsins and the representative ones in the same subfamily could be used for further investigation of the red-shifting mechanisms This result suggests that it should be possible to find rhodopsins that have desired properties without conducting exhaustive biological experiments and suggests that data-driven ML-based approaches should play effective roles in the experimental design of rhodopsin and other photobiological studies a Bayesian prediction model constructed using the current training data (black crosses) The prediction model is represented by the predictive mean and predictive standard deviation (SD) The horizontal axis schematically illustrates the space of proteins defined through physicochemical features The four vertical dotted lines indicate target proteins (candidates to synthesize) This function is defined as the expected value of the probabilistic prediction by the Bayesian model Since the predictive SD represents the uncertainty of the prediction it has a larger value when the training data points do not exist nearby d The distributions on the vertical dotted lines represent the predictive distributions and the horizontal dashed lines are the base wavelengths of the target points The base wavelength is different for each target point because it depends on the subfamily of the protein e The density of the predictive distribution of each target protein on its red-shift gain value The gain is defined as the predicted wavelength subtracted by the base wavelength This can be seen as a “benefit” that can be obtained by observing the target protein This provides a ranking list from which the next candidates to be experimentally investigated can be determined although target #1 has the largest increase in the predictive mean compared with base wavelength in e target #4 is probabilistically expected to have a larger gain than the other targets a training data set consisting of pairs of a wavelength λmax and an amino acid sequence was constructed A particular focus was placed on the 24 amino acid residues around the retinal chromophore to build an ML-based prediction model A set of protein sequences with no known wavelength was also collected as target proteins b All amino acid sequences were transformed into physicochemical features leading to 24 ×18 = 432 dimensional numerical representations of each protein c A linear regression model was constructed using the Bayesian approach Each regression coefficient βi,j was estimated as a distribution (shown as a gray region) The broadness of these distributions represent the uncertainty of the current estimation d The expected red-shift gain values were evaluated for the target proteins The green region is the standard deviation of the prediction The red shaded region in the vertical distribution corresponds to the probability that the wavelength is larger than the base wavelength (dashed line) which is determined by the subfamily of the microbial rhodopsin The bar represents the expected red-shift gain defined by the expected value of the increase from the base wavelength This encourages the exploration of rhodopsin candidates having large uncertainty (for exploration) as opposed to only those having longer wavelengths with high confidence (for exploitation) and then selected 65 genes of putative light-driven ion-pump rhodopsins showing an \({\Bbb E}[{\mathrm{gain}}]\) >10 nm for further experimental evaluation as ion-pump rhodopsins can be used as new optogenetics tools The difference absorption spectra between before and after hydroxylamine bleaching reaction of microbial rhodopsins in solubilized E. coli membrane. The λmax of each rhodopsin was determined by the peak positions of the absorption spectra of the original proteins, and the absorption of retinal oxime produced by the reaction of retinal Schiff base and hydroxylamine was observed as a negative peak at ~360–370 nm. The predicted and observed red-shift (and blue-shift) gains for the 39 candidate rhodopsins that showed substantial coloring in E Differences between observed and base wavelengths are shown by the bars The red bars indicate red-shift from the base wavelength while the blue bars indicate observed wavelengths that were shorter than the base wavelengths Proteins are sorted in the descending order by \({\Bbb E}[{\mathrm{gain}}]\) suggesting that the proposed ML-based model can screen red-shifted rhodopsins more efficiently than random choice a The light-induced pH change in the external solvent of E coli cells expressing four microbial rhodopsins that showed a λmax  ≥ 20 nm longer than the base wavelength of the subfamily The data obtained without and with 10 μM CCCP are indicated by the blue and green lines Light was illuminated for 150 s (yellow solid lines) b Rubrivirga marina BacHR or PR (ECV93033.1 metagenome) were expressed in the membrane of ND7/23 cells (top image) and generated positive photocurrent in response to a green light pulse (200 ms The traces in the bottom are typical records at a holding potential of 0  mV Microbial rhodopsins show a wide variety of λmax by changing steric and electrostatic interactions between all-trans retinal chromophores and surrounding amino acid residues An understanding of the color-tuning rule enables more efficient screening and the design of new red-shifted rhodopsins that have value as optogenetics tools and our ML-based data-driven approach therefore provides a new basis to identify color-regulating factors without assumptions suggesting that our data-driven ML approach can screen red-shifted microbial rhodopsin genes more efficiently than random choice (p = 7.025 × 10−5) the red points have longer observed wavelengths than the base wavelength λbase while the blue points have shorter observed wavelengths than λbase a ML-based prediction of λmax (horizontal axis) vs experimentally observed λmax (vertical axis) b Expected red-shift gain (horizontal axis) vs Since we selected rhodopsins having expected red-shift gains of ≥10 nm all the points on the horizontal axis are ≥10 nm For blue points whose observed gain is equal to 0 the value of λmax−λbase is also shown as blue outlined circles The green and orange dashed lines are the averages of the horizontal and vertical axes (19.2 nm and 9.5 nm) The results indicate that the observed wavelengths and red-shift gains tended to be smaller than the predicted ones We conjecture that these differences between the observed and predicted wavelengths are due to modeling errors (see the Discussion for details) These BacHRs are highly similar to each other (55.2% identity and 70.6% similarity) and only four of 24 amino acid residues around the retinal chromophore differ marina evolved two BacHRs with 29-nm different λmax by a small number of amino acid replacements; the amino acid residue(s) responsible for this color-tuning should be investigated in the future We identified sodium pump rhodopsin (NaR) from Parvularcula oceani which also has a threonine at this position and showed 10-nm longer absorption than the base wavelength Although genes having non-proline amino acids are rare in nature it would be beneficial to identify new red-shifted rhodopsins These results indicate that ML-based modeling can provide insights for identifying new functional tuning rules for proteins based on specific amino acid residues our ML-based model could be expected to reduce the costs associated with identifying red-shifted rhodopsins from upcoming genomic data we expect that our ML-based model could be applied to ion channel and enzymatic rhodopsins which were not a focus of this study because of their eukaryotic origins; however their use in optogenetics research could help identify more useful optogenetics tools with red-shifted absorption in the future The objective of this study was to introduce and demonstrate the effectiveness of a data-driven experimental design method to screen candidates for rhodopsin proteins with desired properties from more than several thousand candidates identified in various microbial species we constructed a training dataset for developing a ML model and a target dataset for screening targets (Construction of training and target data sets) A machine learning model was constructed using the training dataset (ML modeling) which was used to select the 65 candidates from 3022 in the target dataset The protein expressions of selected candidates were induced (Protein expression) and the absorption spectra and λmax of the selected rhodopsins were measured (Measurement of the absorption spectra and λmax of rhodopsins by bleaching with hydroxylamine) we investigated the ion-transportation properties of the rhodopsins that showed large red-shift gains (Ion-transport assay of rhodopsins in E Statistical significance of the effectiveness of the data-driven experimental design method was assessed by a binomial test The aligned sequences were then used for ML-based modeling Suppose that we have K pairs of an amino acid sequence and an absorption wavelength \(\left\{ {\left( {{\boldsymbol{x}}^{\left( k \right)},\lambda _{{\mathrm{max}}}^{(k)}} \right)} \right\}_{k = 1}^K\) where x(k) ∈\({\Bbb R}\) MN is the feature vector of the k-th amino acid sequence and \(\lambda _{{\mathrm{max}}}^{(k)} \in {\Bbb R}\) is the absorption wavelength of the k-th rhodopsin protein The least-absolute shrinkage selection operator (LASSO) is a standard regression model in which important regression coefficients can be automatically selected by the penalty on the absolute value of the coefficient where \({\boldsymbol{\beta}} \in {\Bbb R}^{MN}\) is a vector of βi,j and γ > 0 is the regularization parameter BLASSO is a Bayesian extension of LASSO for which the model is defined through the following random variables: We used the “monomvn” package of R in our implementation The prediction f (x) was sampled through the Gibbs sampler of β and μ The number of samplings was set as T = 10,000 times we approximately obtain \({\Bbb E}[{\mathrm{gain}}]\) by where μ(t) and β(t) are the t-th sampled parameters. The parameters of the trained model is provided in Supplementary Data 8 The synthesized genes of microbial rhodopsins codon-optimized for E NJ) were incorporated into the multi-cloning site in the pET21a(+) vector (Novagen The plasmids carrying the microbial rhodopsin genes were transformed into the E Protein expression was induced by 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) in the presence of 10 μM all-trans retinal for 4 h coli cells expressing rhodopsins were washed three times with a solution containing 100 mM NaCl and 50 mM Na2HPO4 (pH 7) The washed cells were treated with 1 mM lysozyme for 1 h and then disrupted by sonication for 5 min (VP-300N; TAITEC and the samples were stirred for overnight at 4 °C The rhodopsins were bleached with 500 mM hydroxylamine and subjected to yellow light illumination (λ > 500 nm) from the output of a 1-kW tungsten−halogen projector lamp (Master HILUX-HR; Rikagaku) through colored glass (Y-52; AGC Techno Glass Japan) and heat-absorbing filters (HAF-50S-15H; SIGMA KOKI The absorption change upon bleaching was measured by a UV-visible spectrometer (V-730; JASCO the cells carrying expressed rhodopsin were washed three times and resuspended in unbuffered 100 mM NaCl A cell suspension of 7.5 mL at OD660 = 2 was placed in the dark in a glass cell at 20 °C and illuminated at λ > 500 nm from the output of a 1-kW tungsten–halogen projector lamp (Rikagaku Japan) through a long-pass filter (Y-52; AGC Techno Glass Japan) and a heat-absorbing filter (HAF-50S-50H; SIGMA KOKI The light-induced pH changes were measured using a pH electrode (9618S-10D; HORIBA All measurements were repeated under the same conditions after the addition of 10 μM CCCP EYFP fluorescence (543 nm) in the ND7/23 cells expressing the rhodopsins were imaged under a confocal laser scanning microscopy (LSM510 Germany) at 512 × 512 pixels using a water-immersion objective (×63/0.95 Currents were recorded using an EPC-8 amplifier (HEKA Electronic Germany) under a whole-cell patch clamp configuration while a 200 ms pulse illuminations at 549 ± 15 (nm >90% of the maximum) and 28 mW‧mm−2 was given at 0.1 Hz using a SpectraX light engine (Lumencor Inc. The internal pipette solution contained (in mM) 121.2 KOH The extracellular Tyrode’s solution contained (in mM): 138 NaCl it is reasonable to assume that the probability of observing red-shift gains from randomly selected rhodopsins must be smaller than or equal to 0.50 For statistical analysis of the ML model building and the evaluation of its performance Further information on research design is available in the Nature Research Reporting Summary linked to this article All data shown in main figures were deposited in Supplementary Data 9 Data supporting the findings are available from the corresponding authors upon reasonable request The computational code of this manuscript is available at http://www-als.ics.nitech.ac.jp/~karasuyama/BLASSO-for-Rhodopsins/ A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-02090-5 Microbial and animal rhodopsins: Structures Rhodopsin-like protein from the purple membrane of Halobacterium halobium Environmental genome shotgun sequencing of the Sargasso Sea Light-driven ion-translocating rhodopsins in marine bacteria Channelrhodopsin-1: a light-gated proton channel in green algae Demonstration of a light-driven SO42- transporter and its spectroscopic characteristics Optogenetics 10 years of microbial opsins in neuroscience Optogenetic stimulation of a hippocampal engram activates fear memory recall Creating a false memory in the hippocampus Neocortical excitation/inhibition balance in information processing and social dysfunction Cortical layer-specific critical dynamics triggering perception Red-shifting mutation of light-driven sodium-pump rhodopsin Structural determinants of spectral tuning in retinal proteins-bacteriorhodopsin vs sensory rhodopsin II a-ARM: automatic rhodopsin modeling with chromophore cavity generation Mechanism of absorption wavelength shifts in anion channelrhodopsin-1 mutants step-functional anion channelrhodopsin-2 variant as a high-potential neural silencing tool The use of a chimeric rhodopsin vector for the detection of new proteorhodopsins based on color Crystal structure of the red light-activated channelrhodopsin Chrimson nonalgal anion channelrhodopsins with highly red-shifted absorption Understanding colour tuning rules and predicting absorption wavelengths of microbial rhodopsins by data-driven machine-learning approach Gene: a gene-centered information resource at NCBI Structure and function of the global ocean microbiome Pattern Recognition And Machine Learning (Springer Advances in Neural Information Processing Systems 25 (NIPS 2012) Engineered functional recovery of microbial rhodopsin without retinal-binding lysine Characterization of a cyanobacterial chloride-pumping rhodopsin and its conversion into a proton pump Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea Environment around the chromophore in pharaonis phoborhodopsin: Mutation analysis of the retinal binding site Converting a light-driven proton pump into a light-gated proton channel The development and application of optogenetics Clustal-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice A distinct abundant group of microbial rhodopsins discovered using functional metagenomics The neighbor-joining method: A new method for reconstructing phylogenetic trees Structural basis for Na+ transport mechanism by a light-driven Na+ pump Gate-keeper of ion transport-a highly conserved helix-3 tryptophan in a channelrhodopsin chimera Download references This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS) for Scientific Research (KAKENHI grant Nos the Japan Science and Technology Agency (JST) received support from the Louis and Lyra Richmond Memorial Chair in Life Sciences These authors contributed equally: Keiichi Inoue Graduate School of Humanities and Sciences Center for Interdisciplinary AI and Data Science School of Advanced Science and Engineering conducted the phylogenetic analysis of rhodopsins and the construction of training data constructed the ML model and calculated \({\Bbb E}[{\mathrm{gain}}]\) constructed the DNA plasmids of rhodopsin genes and introduced them into E measured λmax of rhodopsins by bleaching proteins with hydroxylamine M.Konno conducted the pump activity assay of rhodopsins in E conducted the electrophysiological measurement of rhodopsins in mammalian cells All authors discussed and commented on the manuscript Download citation DOI: https://doi.org/10.1038/s42003-021-01878-9 a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science ‘I have a fairly problematic noodle addiction – in all honesty, it’s hard for me to go more than a day or two without consuming some variety of noodle’, confesses Tim Anderson, author of JapanEasy a recipe book in which he seeks to debunk the idea that Japanese cuisine is particularly difficult to reproduce at home The chef is careful to create recipes with ingredients that are easy to find in a supermarket with a common base of seven ingredients: soy sauce he proposes a recipe for a salad made with ramen (wheat noodles) Tim Anderson is an American chef who won the 2011 series of MasterChef UK. Having graduated in 2006 from Occidental College in Los Angeles, where he studied the history of Japanese cuisine, he then lived in Japan for two years, in Fukuoka. He now lives in London, where he runs two Japanese restaurants: Nanban Brixton and Nanban Central 100 g mixed salad leaves (try to get a mix of peppery/mild and crunchy/tender – I like pea shoots and rocket) thinned with a little water and salt or soy sauce  cooked al dente and rinsed under cold water Mix the sesame sauce into the ramen and portion into wide bowls Add protein sources if desired to serve as a meal for dinner rather than lunch JapanEasy (2017), by Tim Anderson is published by Hardie Grant Recipe for Grilled Salmon Skin by Masao Karasuyama This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Please upgrade to a more modern version to fully experience JapanToday site and for security reasons the most populous of Tokyo's 23 central wards Some of the city's wealthiest individuals -- including many businessmen professional athletes and show business figures -- built spacious and comfortable homes there Less affluent suburbanites can enjoy shopping and gourmet dining in trendy locales like Seijo Gakuen But in an article subtitled "Urban Collapse," Spa (July 30) looked at what is claims to be Setagaya's deteriorating image a large percentage of provisionally designated urban farms which for the last 30 years were subject to special tax breaks which means owners of many lots will put them on the market simultaneously (Setagaya has 910,000 square meters of such land making it second in the city after Nerima Ward with 1.85 million square meters.) In addition tells the magazine that with the squeeze from rising property taxes and inheritance taxes more residents are putting their holdings on the market "Property values in areas outside Ring Road Number 8 like Okamoto and Kitami which have poor access to public transport are likely to plummet in value," he predicts Another factor influencing home purchases has been a change in lifestyle perceptions "There are quite a few areas in the ward's western parts that are more than 20 minutes' walk from a rail station," Sakaki points out these homes were promoted as being in 'quiet residential areas.' But these days people in their 30s and 40s find it more appealing to be able to live closer to their place of employment."  sex-related businesses and crime groups have also been making inroads into Setagaya there's been a crackdown on illegal sex businesses in the city's central areas," the editor of a magazine that covers the flesh trade tells Spa some places offering sex around Chitose Karasuyama (an express stop on the Keio commuter line) have sprung up along with outcall centers where 'delivery health' masseuses wait to be summoned."  The area around Futako Tamagawa station on the Denentoshi Line has said to have attracted quasi-gangsters who have been active in so-called "it's me send money" scams that defraud mostly elderly victims According to Tokyo Metropolitan Police statistics last year some 6,035 crimes were reported in the ward making it the capital's second highest after Shinjuku A sidebar identified Setagaya's top five "dangerous" spots as Seijo Gakuen district including nine cases of rape; Futako Tamagawa which is said to be on the receiving end of criminal elements from across the Tama River in mostly blue-collar Kawasaki City; Chitose Karasuyama where cabaret clubs and underground massage parlors have sprung up; Yoga where fights between rival biker gang members have been known to break out at night spots; and Shimokitazawa said to be the best place in the city to score some marijuana "Shimokita" also has a reputation as being a place where "an unlocked bicycle parked on the street will disappear within 30 minutes." Part of Setagaya Ward's woes are due to an anticipated tax shortfall of 5.3 billion yen this fiscal year -- up sharply from 3.1 billion yen in 2017 It's been blamed at least in part by Setagaya's failure to harness the Furusato Nozei Program (hometown tax donation program) a tax incentive scheme aimed at supporting development of smaller New arrivals to the ward have been disappointed "We moved from Minato Ward because discipline at my son's primary school was collapsing," relates a housewife in her 30s "We enrolled him in a school here with a good reputation I checked out three other schools in other districts and found that the environment everywhere else was just as bad with bullies and 'monster parents' proliferating "People told me that the school quality started deteriorating five or six years ago when the number of 'primary school immigrants' from outside the district began to increase."  Setagaya's medical services also rate poorly in terms of the per capita availability of hospital beds and hospital floor space; the ward is also said to have dragged its heels on welfare facilities for seniors Join the leading Asian tourist attraction to assist our diverse range of customers Go over to Meguro Ward and you'll see graffiti all over the place Akabane in Kita Ward is turning into a slum things like law enforcement and rubbish collection are still better in Tokyo than in Chiba or Saitama These are some of the safest areas in the entire developed world concocted around a couple of dubious statistics and some hearsay whining I never understood why Setagaya was such a desirable place to live it's a huge ward with large amounts of lacking rail networks and since there are only a couple of train lines that also cross into Kanagawa or far west Tokyo you're guaranteed to enter a packed train on your way to Tokyo and if you work in the Marunouchi business district then you've still got a long ways to go I suppose that what's the article is getting at perhaps back in the day this kind of suburban sprawl was desirable I won't be crying for all of those stuck up "old money" complaining about the younger generation looking for better things Especially that woman cited in the article just casually moving from Minato to Setagaya like nothing and then complaining about "those people" being in the classroom Guess the only remaining option is to spend a ton of money on elite private schools assuming her kid really belongs in that "society" Like the murder in Setagaya of a couple and their two small children in Setagaya on Dec Attacking the messenger isn't necessarily a smart approach Spa has a pretty good focus on changing social trends and occasionally they scoop the competition Property values in areas outside Ring Road Number 8 like Okamoto and Kitami Those are the areas the Japanese government need to keep a close eye on or they will develop in to ghetto's very soon especially with the new policies starting to take effect People told me that the school quality started deteriorating five or six years ago Is it because the capacity of the schools in the area is not sufficient Some more clarification is needed why exactly school quality is starting to detoriate only idiots would leave their bicycles unlocked those bicycles could have just been taken away by Setagaya ward bicycle collectors - if anything that reputation may speak to the efficiency of the Setagaya ward bicycle collectors :) The real question is whether you can leave your umbrella outside your door to dry off but we have had umbrellas taken on at least two occasions.. Part of Setagaya Ward's woes are due to an anticipated tax shortfall of 5.3 billion yen this fiscal year  just means they need to stop some wasteful spending My ward complains about furusato nozei too but still has money to waste as I have seen with my own two eyes Like the murder in Setagaya of a couple and their two small children in Setagaya on Dec I remember it well - we got a visit from the cops as our car had been on one of the CCTV cameras in the area I think we'd been visiting friends after Christmas Setagaya has over 900,000 residents now - it would be interesting to see how it compares to other places with a similar population for crime rate but the Kuchikomi articles featured here often seem to be constructed from nothing more than rumour The LDP has a policy where they are welcoming hundreds of thousands of low paid immigrants in the next few years and they are known to settle in areas of any big city where real estate becomes significant cheaper because they simply can't afford better housing The Japanese government has to do something about that and give them the proper tools to integrate and mix in to Japanese society if not these immigrants will form concentrated communities which keep largely to themselves and this will create all kind of problems The article speaks about school quality started to deteriorate and I don't know if this is the case in Setagaya but in my country the school quality is dropping in some areas of big cities because the children of immigrants who live in concentrated communties don't speak the host country's langauge at home so they have a serious handicap Furthermore areas of any big city with cheap real estate are know to attract criminals and they are known to prey on said immigrants if no proper plan from the government is in place You have just described Japan's weekly tabloids on which Kuchikomi articles have been based for the past -- what is it now which even Japanese agree tend to be dreadfully boring you can see a lot of that in the up and coming neighborhood close to the NHK Studio Park in Shibuya I do too and am almost tempted to check out Chitose Karasuma but it's bound to fall short of expectations Having farms inside such a crowded metropolis that's surrounded by lots of farmland on the outskirts is a really dumb idea And how does the ending of an extensive tax break for farmers raise taxes for everyone else And what's the deal with inheritance taxes About 10 years ago there were a couple of kyabakura-style places in Chitose Karasuyama I knew a few people who went there occasionally after work but I've no idea if they're still there now there were a couple of places opened in Shimokita a few years back There are a real lot of international school students running around as well Probably the thickest enclave of foreign men too For the same reason people with new money will pay $180 for a $4 t-shirt that says 'Emporio Armani' on it Because all their friends are so they feel like they have to not so far from Shibuya but mostly low rise I heard a massive block is going up in Sangenjaya I believe Setagaya is right up there with the most people on waiting lists for nursery places It is also one of the country's largest losers from Furusato Nouzei Furusato Nouzei is a braindead scheme for diverting your local taxes to another town whose services you don't use in return for gifts Lots of places in inaka compete to attract money by giving the biggest gifts sometimes even things which have nothing to do with that town At one stage it was Ipads and game consoles the net outflow of tax from Setagaya is something like 15 million USD a year You can fund a lot of nursery places with that I’ve always enjoyed the presence of these market gardens in Nerima-ku Better than jamming more people into the area Kyodo Station is just down the street from me My husband and I are residence of Fukuoka City and live in Kyodo It’s sort of the situation many Japan residences experience about work availability for those 60 and over Companies typically reduce pay and medical benefits when you are older which means working professionally under a contracted company provides better pay and benefits Setagaya in a loft apartment that looks pretty good and the neighborhood and area very Suburbia Our street and neighborhood in Kyodo is very family oriented families going along their everyday business of life of raising a family and the elderly proudly taking care of their beloved potted plants Our loft dwelling apartment is an addition to the area and such loft apartments are growing bringing outside people like ourselves to the area I feel like a participant observer and honored to be part of their neighborhood Our train ride are the Odakyu Line and the Chiyoda Line Odakyu Line for work and the Chiyoda Line to visit family just take the time to enjoy the familial all season atmosphere during Spring and there would be less "jamming" of residents Use your Facebook account to login or register with JapanToday you will also receive an email inviting you to receive our news alerts A mix of what's trending on our other sites It's a neighbourhood watch with a difference sniffing out trouble and keeping the streets safe for school kids: meet the pups of Tokyo’s real-life Paw Patrol They might not have the gadgets and gear of their beloved cartoon counterparts but the dogs of the Wan-Wan (bow-wow) Patrol are firm favourites in Tokyo’s Karasuyama neighbourhood Twelve-year-old Yurika Igarashi counts on seeing Sakura “Sometimes I’m scared when I’m coming home alone but I feel okay when I’m walking with Wan-Wan Patrol,” she said Sakura is one of 150 pups who form the Wan-Wan Patrol a programme that enlists dogs and their owners to turn their daily walks into a neighbourhood watch The Karasuyama programme is one of the oldest such community initiatives in Japan and was founded by a local police officer “Individual owners walk whenever they can and have their dog wear the same scarf,” said Keiko Shimizu Clearly marked canines patrolling the streets means “we can help make the neighbourhood less prone to crime,” she added Many of the dog owners are parents of current and former students of the local school but others just participate in the community watch as residents five pooches in Wan-Wan Patrol’s lime-green neckerchiefs wagged their tails as they accompanied children going to elementary school is one of the oldest members of the patrol and can no longer walk by herself But she proudly participates from the comfort of a doggy stroller pushed by her owner “The programme helps us get to know each other and become good neighbours and I feel this place stays safe that way,” said Michiko Takeuchi “We had a member who found an elderly person who had passed away alone,” patrol leader Shimizu said “They realised by noticing a room light still on in the morning We walk at around the same hours on the same route everyday so it’s easier for us to notice if there is something unusual.” Japan has a reputation as one of the world’s safest countries including a devastating accident last month east of Tokyo in which a drunk driver crashed his truck into a group of schoolchildren principal at the local Kyuden Elementary School said the neighbourhood watch helps reassure parents and children alike “Parents appreciate that many people in the neighbourhood care and make sure the environment is safe especially these days when we hear a lot of reports about crimes involving children,” he said “I’m sure that children can play carefree outside of school and anywhere in the neighbourhood because they know many adults keep their eyes on their safety,” Ameie added the cuddly canines are a reliable source of comfort Fill your home — or empty it — at Tokyo’s network of secondhand shops By the rising influx of foreign residents in Tokyo has made it somewhat easier to find long-term accommodation Some real estate companies are slowly adapting their services to be more foreigner-friendly by offering fully furnished accommodations And if you don’t mind living in a share house those who prefer to live on their own might find it challenging to furnish their new home in Japan If you’re asking yourself the question “Where on Earth am I supposed to find a desk?” then here’s a place to get you started: Recycle Gallery NEWS is a collaboration of several refurbishing and recycle shops spread throughout Tokyo They specialize in finding furniture that incorporates practicality Products are carefully examined by the staff before being put for sale and all appliances come with a 30-day warranty period.  Of course, IKEA and OFF HOUSE have become well-known solutions but furniture is one of those pesky things that you usually invest in once or twice at most If you are looking for something with a bit more character you will find it in Gallery NEWS in abundance The Karasuyama shop is by far the largest of the Gallery NEWS stores and entering the shop is an experience on its own At first glance you might describe this store as organized chaos.  Each item is carefully placed to the one adjacent creating beautiful displays filled with the most unexpected items Karasuyama Gallery NEWS is two whole floors of thrifting paradise The first floor displays a mix and match of everything from clothes and knick-knacks to musical instruments You can also find any type of appliance that you may need for your life in Japan — no more coin laundry machines.  For those living on the east side of Tokyo the Edogawa Gallery NEWS is your store of choice it’s about as big as the Karasuyama store and offers nearly twice as many electrical appliances Why settle for a regular refrigerator when you can find a one-of-a-kind design Or a cute vintage baby blue fridge with high resale value so make sure to check the selection every once in a while.  the Nishi-ogikubo branch is an excellent middle ground While it’s nowhere near the size of its larger sisters Karasuyama and Edogawa Nishiogi still manages to shine through with its small selection of carefully selected The store has a quiet and low lit atmosphere so you can relax while searching for that one item to make your 1K studio apartment feel like home Nishiogi still manages to tuck in a few affordable fridges and washing machines but if you live near the Kichijoji or Kokubunji area Nishiogi Gallery NEWS will offer you the best delivery prices.  Are you living closer to the Shinjuku and Shibuya area affiliated shop that still offers a good selection of furniture (tables as well as a few sofas and a variety of electrical appliances) lighting it up with more than 20 hanging lights and chandeliers.  Here you will find a good mix of modern Scandinavian designs and heavy antique woodwork as well as an abundance of Asian and American retro items such as a fantastic wood-carved chess board made in Bali some might have a minimum purchase requirement of ¥3,000 or so while others will happily deliver anything Delivery will cost you about ¥2,000 up to two kilometers So make sure to find the shop that is closest to your area.  A post shared by News Edogawa (@news_edogawa) on Nov 13 The Karasuyama and Edogawa shops both have a small delivery truck which can be rented free of charge for about an hour Usage after the first hour will be charged at ¥500 per 30 minutes or know someone who is familiar with the streets of Tokyo save yourself the delivery costs and drive your new couch home For those who wish to get rid of their unwanted furniture and other items the Recycle Gallery NEWS shops would gladly consider purchasing them from you as long as they’re in good condition and spark the owner’s interest When it comes to larger furniture or appliances it’s recommended that you first bring a photo of the items and provide a detailed description of their condition There’s nothing more stressful than trying to empty out your apartment a month prior to your departure.   From purple sweet potatoes to wild horse sightings here’s how to make the most of Okinawa’s tropical escape Explore the best local brands shaping Tokyo’s fashion scene From vintage clothing to Hawaiian-themed goods here’s what’s worth checking out before it’s gone From buying and registering a bike to key rules of the road Collagen staples for beauty and health in Japan Our handpicked list of the best events going on this month and the untold tales of Japan’s queer community Everything you need to know before and during a natural disaster Stay up to date with Tokyo news and events Leave a rating/comment#Manga / ComicsBack to ArticlesSHARE The Setagaya Ward in western Tokyo is full of little-known places to visit – 100 to be exact visiting each of The 100 Views of Setagaya can be achieved relatively easily It is a rather eclectic list that was curated by residents who submitted their selections of Setagaya’s “desirable landscapes” to the city office From an initial pool of around 200 suggestions There are also seasonal events like markets I have chosen 10 lesser known places that serve as oases from the noise and crowds found in more central areas of Tokyo Todoroki Valley is the most well-known place on this list Nestled at the edge of Setagaya near the Tama River this green oasis is the only gorge in Tokyo’s 23 wards The gorge is famous for its dense foliage and its cool temperatures; however this beautiful and immediately impressive sight is also one of the most popular on this list so be sure to go early in the day to beat the crowds Where to find it: ///behind.ambition.left Nomigawa Waterpath and Green Road is a path along a brook that covers a lot of ground from near Sakura-Shinmachi Station all the way into Meguro ward The little waterway is shallow and slow moving so children often play under the sakura trees in the brook catching insects and fish The whole area is peaceful and reminiscent of a small town It is the perfect place for a leisurely walk in any season Where to find it: ///adjusted.miss.breathed Where to find it: ///inherit.spirit.surviving These cliffs cover a wide area that actually extends beyond Setagaya but the section near Seijo is particularly rich with places to explore The extravagant dwellings at the top of the cliff make good fodder for daydreaming but it is the natural areas of the cliffside that are most impressive the sense of exploration you get while navigating the small paths and parks that weave their way up and down the cliffside will definitely make you feel like you’ve been transported out of Tokyo Where to find it: ///evening.sharper.brave While you are in Seijo visiting the cliffline but it is one place within these hedges that makes this sight standout from the other 99 on the list the Inomata House and Garden is a must-see The traditional design exudes a refined extravagance and sitting next to the home’s wide open windows while gazing out into the garden can only be described as tranquil Where to find it: ///taxi.match.floating Upon visiting Karasuyama Duck Pond (Karasuyama Kamoike) I was surprised that I hadn’t seen it featured in any English tourism material for Tokyo This unknown gem sits just at the edge of Setagaya’s north side near Suginami and Mitaka The lush pond features a small red shrine in its center that contrasts beautifully against the deep green summer foliage Take your time here and enjoy the nature; each season offers its own surprises Where to find it: ///lavender.moment.resting Karasuyama Temple Town (Karasuyama Teramachi) is one of the most expansive sights on this list so make sure to give yourself enough time to explore it all The 26 temples are located in close proximity to each other making the area another good pick for exploring and the area has very few pedestrians or traffic so don’t worry about crowds Visiting each of the 26 temples isn’t essential but don’t miss Myouji Temple as it has some of the largest temple buildings and residences Where to find it: ///bottled.fetch.token This difficult to find place truly feels like a secret A narrow path stuck between a rather gaudy apartment building and a forgettable residential home leads to this hidden grove At the entrance to the path you will see a small plaque labeling the thicket as one of the 100 sights of Setagaya; follow the path and you will be led around a small square grove of bamboo On a breezy day it is easy to listen to the sounds of the bamboo and imagine you are somewhere much more remote Where to find it: ///crowbar.pancakes.acting This walled temple ground is located near Kamimachi Station on the Tokyu Setagaya Line but the detail put into every aspect makes this place worth visiting fountains and rock formations are hidden by bamboo groves A two-tiered pagoda stands in the middle of the grounds; its fresh wood gives a modern look to the traditional architecture Take your time here and look around every corner Where to find it: ///encoder.gravy.presses the cute two-car Setagaya Line trams feel quaint and relaxed The tram frequently passes charmingly small and picturesque crossings In June the tracks are lined with hydrangeas making the Setagaya Line even more photogenic In Sangenjaya you can head to the free observatory in the Carrot Tower and get a birds-eye view of this unique line curving through the city Where to find it: ///then.flanks.meanings A complete list of the 100 views of Setagaya is available here (Japanese only) D-Frag! centers around a notorious delinquent named Kenji Kazama. One day, he unexpectedly stumbles across a "game-making club" (working title) with club president Roka Shibasaki (game attribute: flame/darkness) club member and student council president Chitose Karasuyama (attribute: ground) club member and first-year student Sakura Mizukami (attribute: water) and club adviser/second-year teacher Minami Ohsawa (attribute: lightning) and the onetime gang member finds himself in an even stranger gathering of girls The series premiered in January 2014, and Funimation streamed the series as it aired in Japan Funimation will release the complete series in a limited edition Blu-ray and DVD combo pack on April 28 for US$69.98 D-Frag! centers around a notorious delinquent named Kenji Kazama he unexpectedly stumbles across a "game-making club" (working title) with club president Roka Shibasaki (game attribute: flame/darkness) Today's print edition Home Delivery Working parents in Japan not only face long waiting lists when they want to enroll their children in day care centers they also find themselves looking at equally long lists for "gakudo," or after-school clubs when their children take the next step and enter elementary school as of last April there were 8,689 children nationwide on waiting lists for such clubs where mostly first- through third-grade elementary school students spend their time after school and during school breaks — up from 7,521 around the same time a year earlier.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); According to a survey in December by the International Affairs and Communications Ministry more than 70 percent of women between the ages of 35 and 44 are either working or seeking employment In a time of both misinformation and too much information quality journalism is more crucial than ever.By subscribing Your subscription plan doesn't allow commenting. To learn more see our FAQ Sponsored contents planned and edited by JT Media Enterprise Division Fixtures & Results, Trainings Players & Staff Stadium Hometown Activities Frequently Asked Questions To commemorate the display of the FC Tokyo collaboration flag on the streetlights of Chitosekarasuyama TOKYO DOROMPA will participate in a ceremony on November 23 (Saturday) □Event NameFC Tokyo × Chitose Karasuyama Collaboration Flag Completion Ceremony□Time & Location15:00～15:20Chitose Karasuyama Collaboration Flag Completion Ceremony (Karasuyama Community Center Front Square)16:00 to 16:20Participate in the Chitose Karasuyama Illumination Lighting Parade*Please note that the schedule may change unexpectedly □DetailsA ceremony will be held to commemorate the display of the collaboration flag completed with the cooperation of the Chitosekarasuyama Shopping Street community  □NotesRegarding interactions with TOKYO DOROMPA autographs and individual photo sessions will only be accommodated if circumstances allow it will generally be limited to one time per person.We would like to accommodate as many people as possible there may be cases where we cannot meet your request and we may have to decline 6/14 (Sat) C Osaka Match "Brazil Festa" Held 5/10 (Sat) Kobe Match "Komae City PR Booth" Exhibition Announcement 5/10 (Sat) Kobe Match Announcement of "Soccer Experience & Stadium Exploration Tour" for Truant Children [Update 4/26] Announcement of "Amino Vital® presents FC Tokyo Relay Marathon at Ajinomoto Stadium" [4/25 Update] Announcement of 'AO-AKA LIGHT THE NIGHT' to enhance the night games of the 2025 season Holiday) Shimizu Match 'TOKYO DOROMPA' Appearance Information Holiday) Shimizu Match Attraction Information Announcement of the "Blue-Red App Lottery" for visitors where you can win "OHYA Original Package OHYA President Curry (retort curry)" North American anime distributor Funimation is announcing at its Otakon Vegas panel on Friday that it will stream the D-Frag! television anime series as it airs in Japan The story centers around a notorious delinquent named Kenji Kazama. One day, he unexpectedly stumbles across a "game-making club" (working title) with club president Roka Shibasaki (game attribute: flame/darkness) Kana Hanazawa (Coppelion, Guilty Crown) plays Roka Shibasaki the president of the "game-making club" (working title) that Kazama stumbles across Shizuka Itou plays Takao-buchō Chiwa Saitō plays Chitose Karasuyama a member of the "game-making club" and the current student council president Mikako Takahashi plays Sakura Mizukami a member of the "game-making club" and first-year student Ami Koshimizu plays Minami Ohsawa the "game-making club's" advisor who is often in jerseys Jun Fukuyama (Code Geass Kazama's childhood friend and bad influence Hiroki Gotō plays Yokoshima Kenichirō Matsuda plays Hiroshi Nagayama Funimation will announce another anime that it will stream as it airs in Japan this winter Images © Tomoya Haruno/Kadokawa's Media Factory/D-Frag! Production Committee Please view the main text area of the page by skipping the main menu. The page may not be displayed properly if the JavaScript is deactivated on your browser The May issue of Kadokawa's Monthly Comic Alive magazine is announcing on Wednesday that the ninth special-edition volume of Tomoya Haruno's D-Frag! manga will bundle a original anime DVD (OAD) The DVD will adapt the popular Shrine Water arc and ship on September 22 アライブに闇属性のストラップは付かないけど「ディーふらぐ!」コミックス9巻はOADが付くらしいぞ http://t.co/fVQ1ZX9kEC The manga and subsequent television anime center around a notorious delinquent named Kenji Kazama. One day, he unexpectedly stumbles across a "game-making club" (working title) with club president Roka Shibasaki (game attribute: flame/darkness) The OAD will share the same cast as the television anime: Emiri Katō as Noe Kazama Ryōka Yuzuki as Takao's Mother Jouji Nakata as Sean Konekone   [Via earl.box] Image © Tomoya Haruno/Kadokawa Media Factory/D-Frag! Production Committee