Authorities on Tuesday said they are looking into the homicide of a man found in a car in Johnston County months earlier and are asking for help to unwind the mystery was found unconscious in February in the driver's seat of a Ford Mustang parked along Interstate 95 in Johnston County was found after a report of a car accident on I-95 near Four Oaks early in the morning of Feb the North Carolina State Highway Patrol initially charged Takashima with possession of a stolen car and other traffic-related misdemeanors troopers asked the State Bureau of Investigation for help after "being advised of inconsistencies with Mr Takashima's injuries." Neither the Highway Patrol nor SBI have said what those inconsistencies were An examination by the North Carolina Medical Examiner's Office classified his death as a homicide FOUR OAKS – A car accident in Johnston County is now being investigated as a homicide a car accident with injuries was reported at the 86 mile marker on I-95 northbound near the Keen Road exit in Four Oaks Local fire and EMS crews discovered an unconscious man in the driver’s seat of a 2017 Ford Mustang The man was transported to WakeMed Hospital for treatment The North Carolina State Highway Patrol initiated the investigation into the accident The driver was identified as 87-year-old Toshio Takashima from Virginia Injuries he suffered were reportedly inconsistent with the auto accident Delayed Investigation It took more than three weeks for the Highway Patrol to ask for assistance the State Highway Patrol in Smithfield reached out to the North Carolina State Bureau of Investigation (SBI) to request help Takashima had passed away one day before his 88th birthday His body was sent to the North Carolina Medical Examiner’s Office for a complete autopsy The initial findings of the autopsy classified Mr and Johnston County District Attorney are seeking the public’s help in gathering information related to this case Anyone with information is encouraged to contact the SBI at 919-662-4500 or the Johnston County Sheriff’s Office Criminal Investigations Division at 919-989-5010 This case is a joint investigation involving the SBI and the Johnston County Sheriff’s Office It took more than three weeks for the Highway Patrol to ask for assistance why else would they wait until he was dead The dog Joey in the photo is still missing with no credible sightings Somebody definitely dropped the ball on this case Yea caring about it and worrying about a murderer out and about isn’t that big a deal…..sarcasm intended Please log in and check your service registration status.To cancel your subscription 1) === '|') document.write("&#"+unescape(l[i].substring(1))+";");else document.write(unescape(l[i]));} 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 Takashima Ryosuke could have gone into consulting or finance Instead he embarked on an unusual career path for a Japanese youngster “Being a mayor seemed like a career that would allow me to impact society the most,” he says This article appeared in the Asia section of the print edition under the headline “Gen Z mayor” Discover stories from this section and more in the list of contents Ahead of the vote on May 3rd, politics has flipped The country is making it first big bet on semiconductors This one could be riskier than their last major crisis in 2019 After the Kashmir attack, military action is possible but comes with huge risks Yanosuke was instrumental in promoting the diversification of Mitsubishi's business thereby laying the foundations for today's Mitsubishi Group Yanosuke was 16 years younger than his brother Yataro he traveled to New York in 1872 in order to further his education the experience greatly broadened Yanosuke's knowledge of the world Yataro changed the name of the company to Mitsubishi and set out to expand its business in the area of marine transport Yanosuke subsequently received a letter from Yataro asking him to come home immediately to serve as his right-hand man Yanosuke returned to Japan in November 1873 and joined the company as vice president at the age of 22 Mitsubishi Shokai relocated its headquarters to Tokyo With a steadfast dedication to providing excellent service the company's business began to gain momentum after prevailing in a fierce struggle with a rival company Yanosuke amassed valuable experience when Mitsubishi supported marine transport operations in connection with a Japanese military expedition to Taiwan he headed up marine transport operations as the Japanese military sought to suppress an internal uprising known as the Satsuma Rebellion Yanosuke later went on to pioneer the development of various new fields including insurance warehousing and railways; these early ventures represent t he fore runners of "Mitsubishi" companies such as Tokio Marine & Nichido the Bank of Tokyo Mitsubishi UFJ and Mi tsubishi Logist ics Corporation marine transport still represented the company's core business and Yanosuke eventually joined with Yataro as Mitsubishi engaged in an intense battle for the company's survival with a rival shipping enterprise named Kyodo Unyu Kaisha (later Nippon Yusen Kaisha (NYK)) The purchase of the Takashima Coal Mine stands out as perhaps the greatest achievement of the second half of Yanosuke's tenure as vice president but it fell into dire straits in the late 1870s Yataro was approached about buying the mine Yanosuke conducted a comprehensive assessment of the mine's potential from the mine's estimated reserves and projected output to the value of equipment held by the mine and the benefits of having Mitsubishi's ships transport its cargo He developed a case in favor of purchasing the mine and presented his recommendation to Yataro The purchase of the mine remained in doubt for some time Yanosuke's assessment of the mine's potential proved to be correct it generated large profits and emerged as Mitsubishi's largest business while underpinning Mitsubishi's expansion into new industries Yanosuke studied at a small boarding school in rural Connecticut more than 200 km north of New York City where he was the only Japanese person around He spent his days studying the Bible in English when the son of a high-ranking Mitsubishi official went to study in England Yanosuke penned the following advice in a letter: "The quickest way to learn English is to avoid associating with other Japanese people; that's how I was able to master English in 16 months." Gary Takashima shakes hands with President Biden on Jan 3 after receiving the Medal of Honor awarded posthumously to his uncle With his term as the 46th president of the United States of America winding down Joe Biden as commander-in-chief of the nation’s armed forces on Jan 3 awarded seven Medals of Honor — the highest military decoration awarded only to those who “distinguish themselves conspicuously by gallantry and intrepidity at the risk of their own lives” — to U.S Five of the medals were awarded to Korean War veterans: Gen Cavazos was Mexican American; Hawaii-born Orig was Filipino American; Los Angeles-born Nakamura was Japanese American; and McGee and Johnson were African American Nakamura and Orig were made “… pursuant to laws providing for reviews to determine whether prejudice may have been involved in denying the Medal of Honor.” Two of the medals were awarded to Vietnam War veterans: Capt “I’m deeply privileged to honor seven American heroes,” Biden said to those gathered and even different generations … who all went above and beyond the call of duty.” attended the ceremony to receive the Medal of Honor — an upgrade from the Distinguished Service Cross — from Biden Prior to the Jan. 3 ceremony, the only other Japanese American to receive the Medal of Honor for service during the Korean War was Hiroshi “Hershey” Miyamura. (Pacific Citizen, Dec. 12, 2022, tinyurl.com/4rpztz69) A member of I Co., 38th Infantry Regiment, 2nd Infantry Division, Nakamura was killed in action on May 18, 1951. He was 29. Prior to America’s Dec. 8, 1941 declaration of war on Japan that marked its entry into World War II, Nakamura, the second of seven children, had lived and worked in San Francisco but would move to Arkansas to join his family, who were incarcerated at the Rohwer War Relocation Center after President Roosevelt signed Executive Order 9066 on Feb. 19, 1942. With the war still being prosecuted, Nakamura joined the Army from Rohwer in April 1944, and was assigned to K Co. of the 442nd Regimental Combat Team, the famed, segregated Army unit comprised mostly of Japanese Americans from the Hawaii territory and the U.S. mainland. In 2000, when President Clinton belatedly awarded 22 Medals of Honor to WWII veterans of Asian heritage, 20 had served with the 442nd. After the war’s end, Nakamura stayed in the Army Reserve, moved to Chicago and was called back to active duty after America entered the Korean War. June 16, 1951 Pacific Citizen article reporting that Nakamura had been killed in action while serving in the Korean War. According to the Army, Nakamura received the belated honor because he “ … singlehandedly attacked and destroyed a hostile machine-gun nest and drove the enemy from several of the bunkers they had captured. When his ammunition was depleted, he withdrew while under enemy fire. “Nakamura then met an ammunition party ascending the hill. After briefing the officer in charge, Nakamura rearmed himself and, covered by the fire of the officer and two fellow soldiers, returned to the attack. He killed three of the enemy in one bunker and killed and seriously wounded another in the last enemy-held bunker. Continuing to press the attack, he fell mortally wounded by an enemy grenade.” Volume 11 - 2021 | https://doi.org/10.3389/fcimb.2021.805482 Control measures have significantly reduced malaria morbidity and mortality in the last two decades; however the downward trends have stalled and have become complicated by the emergence of COVID-19 Significant efforts have been made to develop malaria vaccines but currently only the RTS,S/AS01 vaccine against Plasmodium falciparum has been recommended by the WHO for widespread use among children in sub-Saharan Africa and therefore the development of more efficacious vaccines is still needed the development of transmission-blocking vaccines (TBVs) to reduce the parasite transmission from humans to mosquitoes is required toward the goal of malaria elimination Few TBVs have reached clinical development and challenges include low immunogenicity or high reactogenicity in humans novel approaches to accelerate TBV research and development are urgently needed In this mini review we summarize the progress in TBV research and development and discuss how to accelerate novel TBV candidate discovery Malaria continues to be responsible for a substantial global health burden, with 409,000 malarial deaths reported in 2019 (WHO, 2020). From 2000 to 2015, malaria morbidity and mortality were significantly reduced; however, the decreasing trend stalled between 2015 and 2019 and was further complicated by the emergence of COVID-19 (Wang et al., 2020; WHO, 2020) the control and eventual eradication of this disease relies on the development of a highly effective malaria vaccine Since the RTS,S/AS01 vaccine efficacy is modest, the development of more efficacious vaccines is still needed. A number of second-generation malaria vaccines are in clinical trials, such as R21/Matrix-M (Datoo et al., 2021) the above mentioned two malaria vaccines are classified as pre-erythrocytic stage vaccines the development of erythrocytic stage vaccines to reduce morbidity and mortality and transmission-blocking vaccines (TBVs) to reduce parasite transmission from humans to mosquitoes Table 1 Discovery of malaria transmission-blocking vaccine antigens with publication yearsa The vaccine was generally well-tolerated; however the functional activity of the anti-Pfs25 antibodies induced were modest Figure 1 Expression of malaria transmission-blocking vaccine (TBV) target antigens. Sexual developmental stages of malaria parasites in humans (gametocytes) and mosquitoes (gametes, zygotes, and ookinetes) are schematically presented. The TBV candidate antigens (Table 1) are categorized as pre-fertilization antigens (mainly expressed in the sexual stages of parasites before fertilization) and post-fertilization antigens (mainly expressed in the sexual stages of parasites after fertilization) In addition to the above TBV development efforts novel TBV candidate discovery is required to accelerate the success in TBV development Most of the TBV candidates investigated to date have orthologs in rodent malaria parasites and thus the rodent malaria models are useful for the discovery and characterization of novel TBV candidates In the last decade several potential TBV candidates have been identified using rodent malaria models The general strategy of these studies is to select candidate genes from the PlasmoDB according to the following criteria: i) genes must be specifically expressed in sexual-stages; ii) they must share orthologs with human parasites vivax; and iii) the presence of a predicted signal peptide with/without transmembrane domain(s) or a GPI-anchor indicating possible protein export and exposure to inhibitory antibodies Candidate TBV genes are then expressed in one or more recombinant protein expression systems immunized mice are infected with rodent malaria parasites and then mosquitoes are fed directly on these mice; termed a direct feeding assay The transmission-blocking activity (TBA) is expressed as a percent reduction of the prevalence of infected mosquitoes; and transmission-reducing activity (TRA) is expressed as a percent reduction of oocyst density The majority of such studies were conducted with P. berghei rodent parasites because of the ease for genetic manipulation, such as the knockout of candidate genes for functional characterization of novel TBV candidates. Most such activities are listed in Table 1 and following are descriptions of examples of post-genomic studies The P. berghei ookinete-stage protein, PbPSOP12, was identified based upon annotation as a putative secreted protein and then expressed using the baculovirus dual expression system (BDES). Mouse antibodies against BDES-PbPSOP12 recognized the surface of gametes/ookinetes. Immunization of mice with BDES-PbPSOP12 conferred modest TRA (53%) (Sala et al., 2015) although the usefulness of the rodent models is clear and expansion of the repertoire of novel TBV candidates are necessary to accelerate TBV development the approach might not identify TBV candidates whose protein expression is solely in the mosquito and not in gametocytes Additional gametocyte-specific gene discovery efforts have been published (Ikadai et al., 2013; Chawla et al., 2021; Muthui et al., 2021); although antigen expression and TRA assessment of the antibodies are not completed To this end it is also essential to obtain well-characterized plasma samples from infected individuals who carry transmission-reducing antibodies All authors listed have made a substantial and intellectual contribution to the work and approved it for publication Some of the work presented here was partially supported by JSPS KAKENHI Grant (JP18H02651 Global Health Innovative Technology (GHIT) Fund (grant # G2019-111 BNK is an EDCTP Fellow under EDCTP2 program supported by the European Union (grant TMA2020CDF-3203-EndPAMAL) The funding sources had no role in study design 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 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 Templeton for critical reading of the manuscript anopheline alanyl aminopeptidase N 1; BDES ExoProtein A from Pseudomonas aeruginosa; HAP2/GCS1 RTS,S Malaria Vaccine Pilots in Three African Countries PubMed Abstract | CrossRef Full Text | Google Scholar Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium Berghei and Falciparum Recombinant Pvs48/45 Antigen Expressed in E Coli Generates Antibodies That Block Malaria Transmission in Anopheles Albimanus Mosquitoes Conserved Epitope in Anopheles APN1 Inhibit Universal Transmission of Plasmodium Falciparum and Plasmodium Vivax Malaria Evidence of Artemisinin-Resistant Malaria in Africa Immunofocusing Humoral Immunity Potentiates the Functional Efficacy of the AnAPN1 Malaria Transmission-Blocking Vaccine Antigen Current Challenges in the Identification of Pre-Erythrocytic Malaria Vaccine Candidate Antigens PubMed Abstract | CrossRef Full Text | Google Scholar Plasmodium Berghei HAP2 Induces Strong Malaria Transmission-Blocking Immunity In Vivo and In Vitro Antibody Targeting of a Specific Region of Pfs47 Blocks Plasmodium Falciparum Malaria Transmission Malaria Transmission Blocked by Immunisation With Gametes of the Malaria Parasite PubMed Abstract | CrossRef Full Text | Google Scholar Characterization of Antigens on Mosquito Midgut Stages of Plasmodium Gallinaceum Changes in Zygote Surface Proteins During Transformation to Mature Ookinete Targeting Gametocytes of the Malaria Parasite Plasmodium Falciparum in a Functional Genomics Era: Next Steps Efficacy of a Low-Dose Candidate Malaria Vaccine With Seasonal Administration to Children in Burkina Faso: A Randomised Controlled Trial Immunity Against Sexual Stage Plasmodium Falciparum and Plasmodium Vivax Parasites Artemisinin Resistance in Plasmodium Falciparum Malaria Transmission-Blocking Vaccines: Harnessing Herd Immunity for Malaria Elimination PubMed Abstract | CrossRef Full Text | Google Scholar and Pfs25 are Genetically Linked and Synergistic as Falciparum Malaria Transmission-Blocking Vaccines Google Scholar Successful Immunization Against the Sexual Stages of Plasmodium Gallinaceum PubMed Abstract | CrossRef Full Text | Google Scholar Pfs230 Yields Higher Malaria Transmission-Blocking Vaccine Activity Than Pfs25 in Humans But Not Mice Male Fertility of Malaria Parasites Is Determined by GCS1 Antibodies to Malaria Vaccine Candidates Pvs25 and Pvs28 Completely Block the Ability of Plasmodium Vivax to Infect Mosquitoes doi: 10.1128/iai.68.12.6618-6623.2000 Changes in Infectiousness of Malarial Gametocytes Analysis of the Possible Causative Factors Transposon Mutagenesis Identifies Genes Essential for Plasmodium Falciparum Gametocytogenesis Leveraging the Wheat Germ Cell-Free Protein Synthesis System to Accelerate Malaria Vaccine Development Comprehensive Analysis of Antibody Responses to Plasmodium Falciparum Erythrocyte Membrane Protein 1 Domains Saccharomyces Cerevisiae Recombinant Pfs25 Adsorbed to Alum Elicits Antibodies That Block Transmission of Plasmodium Falciparum A Vaccine Candidate From the Sexual Stage of Human Malaria That Contains EGF-Like Domains Proteomic Analysis of the Plasmodium Berghei Gametocyte Egressome and Vesicular bioID of Osmiophilic Body Proteins Identifies Merozoite TRAP-Like Protein (MTRAP) as an Essential Factor for Parasite Transmission Cloning and Expression of the Gene Coding for the Transmission Blocking Target Antigen Pfs48/45 of Plasmodium Falciparum Characterization of a Plasmodium Berghei Sexual Stage Antigen PbPH as a New Candidate for Malaria Transmission-Blocking Vaccine Biosynthesis of Two Stage-Specific Membrane Proteins During Transformation of Plasmodium Gallinaceum Zygotes Into Ookinetes Integrated Transcriptomic and Proteomic Analyses of P Falciparum Gametocytes: Molecular Insight Into Sex-Specific Processes and Translational Repression Characterization of Plasmodium Berghei Pbg37 as Both a Pre- and Postfertilization Antigen With Transmission-Blocking Potential The Conserved Plant Sterility Gene HAP2 Functions After Attachment of Fusogenic Membranes in Chlamydomonas and Plasmodium Gametes A Conserved Malaria Parasite Antigen Pb22 Plays a Critical Role in Male Gametogenesis in Plasmodium Berghei Development and Validation of Serological Markers for Detecting Recent Plasmodium Vivax Infection Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium Falciparum Sexual Stage Protein Pfs230 Meerstein-Kessel Probabilistic Data Integration Identifies Reliable Gametocyte-Specific Proteins and Transcripts in Malaria Parasites Qualification of Standard Membrane-Feeding Assay With Plasmodium Falciparum Malaria and Potential Improvements for Future Assays Malaria Transmission-Blocking Vaccines: Wheat Germ Cell-Free Technology can Accelerate Vaccine Development Functional Comparison of Plasmodium Falciparum Transmission-Blocking Vaccine Candidates by the Standard Membrane-Feeding Assay CrossRef Full Text | Google Scholar Immunoscreening of Plasmodium Falciparum Proteins Expressed in a Wheat Germ Cell-Free System Reveals a Novel Malaria Vaccine Candidate Characterization of Naturally Acquired Immunity to a Panel of Antigens Expressed in Mature P Functional Characterization and Comparison of Plasmodium Falciparum Proteins as Targets of Transmission-Blocking Antibodies Blood-Stage Malaria Vaccines: Post-Genome Strategies for the Identification of Novel Vaccine Candidates Correctly Folded Pfs48/45 Protein of Plasmodium Falciparum Elicits Malaria Transmission-Blocking Immunity in Mice Structural Vaccinology of Malaria Transmission-Blocking Vaccines PubMed Abstract | CrossRef Full Text | Google Scholar Evaluation of Plasmodium Vivax HAP2 as a Transmission-Blocking Vaccine Candidate Efficacy and Safety of RTS,S/AS01 Malaria Vaccine With or Without a Booster Dose in Infants and Children in Africa: Final Results of a Phase 3 Safety and Immunogenicity of Pfs25H-EPA/Alhydrogel a Transmission-Blocking Vaccine Against Plasmodium Falciparum: A Randomised Dose-Escalation Study in Healthy Malian Adults The Plasmodium Berghei Sexual Stage Antigen PSOP12 Induces Anti-Malarial Transmission Blocking Immunity Both In Vivo and In Vitro Unravelling the Immune Signature of Plasmodium Falciparum Transmission-Reducing Immunity The C-Terminal Region of the Plasmodium Yoelii Microgamete Surface Antigen PyMiGS Induces Potent Anti-Malarial Transmission-Blocking Immunity in Mice A Male Gametocyte Osmiophilic Body and Microgamete Surface Protein of the Rodent Malaria Parasite Plasmodium Yoelii (PyMiGS) Plays a Critical Role in Male Osmiophilic Body Formation and Exflagellation The Plasmodium Yoelii Microgamete Surface Antigen (PyMiGS) Induces Anti-Malarial Transmission Blocking Immunity That Reduces Microgamete Motility/Release From Activated Male Gametocytes Plasmodium Vivax Gametocyte Protein Pvs230 Is a Transmission-Blocking Vaccine Candidate Induce Transmission-Reducing Antibodies by DNA Immunization Safety and Immunogenicity of Pfs25-EPA/Alhydrogel(R) a Transmission Blocking Vaccine Against Plasmodium Falciparum: An Open Label Study in Malaria Naive Adults Two Antigens on Zygotes and Ookinetes of Plasmodium Yoelii and Plasmodium Berghei That are Distinct Targets of Transmission-Blocking Immunity Primary Structure of a Novel Ookinete Surface Protein From Plasmodium Berghei Comparison of Plasmodium Yoelii Ookinete Surface Antigens With Human and Avian Malaria Parasite Homologues Reveals Two Highly Conserved Regions Sequence Polymorphism in Two Novel Plasmodium Vivax Ookinete Surface Proteins That Are Malaria Transmission-Blocking Vaccine Candidates Transmission-Blocking Vaccine of Vivax Malaria PubMed Abstract | CrossRef Full Text | Google Scholar Three Members of the 6-Cys Protein Family of Plasmodium Play a Role in Gamete Fertility Paralog of the Male Fertility Factor Pfs48/45 Is a Female Specific Surface Protein in Plasmodium Falciparum doi: 10.1016/j.molbiopara.2006.05.015 Sequential Expression of Antigens on Sexual Stages of Plasmodium Falciparum Accessible to Transmission-Blocking Antibodies in the Mosquito WHO Gives First Malaria Vaccine the Green Light PubMed Abstract | CrossRef Full Text | Google Scholar Preparedness Is Essential for Malaria-Endemic Regions During the COVID-19 Pandemic Characterization of Pb51 in Plasmodium Berghei as a Malaria Vaccine Candidate Targeting Both Asexual Erythrocytic Proliferation and Transmission Immunogenicity of the RTS,S/AS01 Malaria Vaccine and Implications for Duration of Vaccine Efficacy: Secondary Analysis of Data From a Phase 3 Randomised Controlled Trial Google Scholar Cloning and Expression of the Gene for Plasmodium Falciparum Transmission-Blocking Target Antigen a Plasmodium Falciparum Gametocyte Protein Induces Antisera That Reduce the Infectivity of Plasmodium Falciparum to Mosquitoes Identification of Three Ookinete-Specific Genes and Evaluation of Their Transmission-Blocking Potentials in Plasmodium Berghei Functional Characterization of Plasmodium Berghei PSOP25 During Ookinete Development and as a Malaria Transmission-Blocking Vaccine Candidate Kanoi BN and Tsuboi T (2021) Identification of Novel Malaria Transmission-Blocking Vaccine Candidates Received: 30 October 2021; Accepted: 16 November 2021;Published: 30 November 2021 Copyright © 2021 Takashima, Tachibana, Morita, Nagaoka, Kanoi and Tsuboi. 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: Takafumi Tsuboi, dHN1Ym9pLnRha2FmdW1pLm1iQGVoaW1lLXUuYWMuanA= Centre for Research in Infectious Diseases 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 Volume 13 - 2020 | https://doi.org/10.3389/fnmol.2020.590896 This article is part of the Research TopicTau Protein: Mechanisms from Health to DegenerationView all 22 articles Microtubule-associated protein tau is characterized by the fact that it is an intrinsically disordered protein due to its lack of a stable conformation and high flexibility Intracellular inclusions of fibrillar forms of tau with a β-sheet structure accumulate in the brain of patients with Alzheimer's disease and other tauopathies detachment of tau from microtubules and transition of tau from a disordered state to an abnormally aggregated state are essential events preceding the onset of tau-related diseases Many reports have shown that this transition is caused by post-translational modifications including hyperphosphorylation and acetylation The misfolded tau is self-assembled and forms a tau oligomer before the appearance of tau inclusions Animal and pathological studies using human samples have demonstrated that tau oligomer formation contributes to neuronal loss tau seeds are released from cells and incorporated into other cells leading to the propagation of pathological tau aggregation Accumulating evidence suggests several potential approaches for blocking tau-mediated toxicity: (1) direct inhibition of pathological tau aggregation and (2) inhibition of tau post-translational modifications that occur prior to pathological tau aggregation (3) inhibition of tau propagation and (4) stabilization of microtubules In addition to traditional low-molecular-weight compounds newer drug discovery approaches such as the development of medium-molecular-weight drugs (peptide- or oligonucleotide-based drugs) and high-molecular-weight drugs (antibody-based drugs) provide alternative pathways to preventing the formation of abnormal tau Of particular interest are recent studies suggesting that tau droplet formation by liquid-liquid phase separation may be the initial step in aberrant tau aggregation as well results that implicate roles for tau in dendritic and nuclear functions we review the mechanisms through which drugs can target tau and consider recent clinical trials for the treatment of tauopathies we discuss the utility of these newer strategies and propose future directions for research on tau-targeted therapeutics We also discuss perspectives for drug development in this area as well as oligonucleotides to reduce tau expression Tauopathies are neurological disorders (Avila et al., 2004; Götz and Götz, 2019), characterized by aberrant tau aggregates (NFT and tau inclusions) in neurons and glial cells. These aggregates represent tau gene mutations or hyperphosphorylated tau (Kovacs, 2015). The majority of tauopathic patients also show depositions of Aβ, α-synuclein, or huntingtin (Guo et al., 2017) These observations suggest that tau abnormalities have a common pathological role across neurodegenerative diseases Tau-targeted drugs may be a promising disease-modifying therapy because previous studies focusing on the correlation of AD neuropathological changes (Aβ plaques and NFT) with cognitive impairment have shown that the severity of cognitive impairment correlated best with the burden of abnormal tau (Nelson et al., 2012) many clinical trials of drugs targeting tau have been conducted Mechanism of tau post-translational modification inhibitors in clinical trials regulate the binding of tau to microtubules Microtubule instability and depolymerization are observed in tauopathies suggesting a therapeutic role for microtubule stabilizers O-GlcNAcylation at serine and threonine compete with phosphorylation of the same residues Tau degradation is inhibited by acetylation The post-translational modifications are tightly regulated by various enzymes that mediate the addition and removal of the modifying groups tau kinase inhibitors or P300 acetyltransferase inhibitors have been investigated for their ability to inhibit tau phosphorylation or tau acetylation The usefulness of O-GlcNAcase inhibitors to elevate tau O-GlcNAcylation has also been examined in clinical trials these observations suggest that tau hyperphosphorylation is involved in the development and pathogenesis of tauopathies and that its inhibition may be a therapeutic strategy suggesting that CDK5 inhibition enhances tau phosphorylation by activating GSK3β As CDK5 can phosphorylate molecules other than tau therapeutic agents targeting CDK5 should be developed with great caution These findings suggest that the development of TAOK inhibitor should proceed with caution ACI-35 has been used in a phase 1 trial (Main ID in the WHO International Clinical Trials Registry Platform: ISRCTN13033912) (see section on Tau Clearance and Immunotherapy) These facts suggest that tau acetylation may be important for tau-induced toxicity This may be explained by either the poor penetration of salsalate into the brain (<3%) or by an increase in tau aggregation following reduced tau acetylation These findings suggest that upregulation of tau O-GlcNAcylation may be a therapeutic strategy for tau-related neurodegeneration The facts suggest that compounds that directly target tau aggregation may be more effective tauopathies than tau kinase inhibitors The recombinant tau protein is polymerized in the presence of polyanion, including heparin (Goedert et al., 1996) or RNA (Kampers et al., 1996), and the aggregation level can be monitored by fluorescent dye Thioflavin-T (S). Using this experimental system, many researchers screened tau aggregation inhibitors (Taniguchi et al., 2005; Bulic et al., 2009; Crowe et al., 2009) Many of the aggregation inhibitors discovered share a common characteristic: a negative or positive charge in their structure The results of clinical trials of these drugs are expected suggesting it may be suitable for the treatment of tau-related dementia Ginseng is the root of Panax ginseng Meyer and has been used as an herbal medicine for various diseases. Red ginseng is believed to be a processed form of ginseng with enhanced pharmacological efficacy (Lee et al., 2015). Tau aggregation was inhibited by the red ginseng treatment in vitro (Shin et al., 2020b). As ginseng includes saponin and flavonoids (Choi, 2008) the inhibitory effect may be involved in the surfactant action or antioxidation by ginseng indicating that the nickel administered to humans requires careful observation Results of clinical trials on drugs against tauopathies besides AD are expected a phase-3 low-dose study of LMTM in AD (LUCIDITY; NCT03446001) was eventually launched Hyperphosphorylated tau is detached from microtubules and mislocalized in the somatodendritic compartment of neurons In vitro studies have shown that tau is self-assembled to form tau oligomers and granular tau oligomers before forming NFTs Tau aggregation inhibitors that halt these processes may be useful in the treatment of tauopathies Immunotherapy is one potential therapeutic approach for preventing tau aggregation, a line followed by a number of academic and industrial research groups. These efforts have resulted in the generation of the TOC1 (Patterson et al., 2011), TOMA (Castillo-Carranza et al., 2014), and T22 (Lasagna-Reeves et al., 2012) antibodies that recognize intermediate tau aggregates While ongoing tau immunotherapy-based clinical trials mainly targeted phosphorylated tau and aberrant conformational changes in tau (details below) it is likely that the next generation of antibodies will be directed at tau intermediate aggregates The traxane-derivative TPI287 (abeo-taxane) which has high BBB permeability proved safe and well-tolerated in a phase 1 study (NCT02133846) in patients with CBD or PSP (n = 44) and a phase 1 study (NCT01966666) in patients with mild to moderate AD (n = 33). However, the drug caused adverse effects in the AD group and worsened dementia symptoms in the CBD and PSP patients. It appears that TPI287 is no longer being developed for clinical use (VandeVrede et al., 2020a) Recent reviews propose that tau is not a stabilizer of microtubules in the axon but rather confers flexibility to the labile domain of microtubules and leads to microtubule elongation (Qiang et al., 2018; Baas and Qiang, 2019). Further, an analysis of fast single-molecule tracking showed that microtubule assembly is regulated by more rapid tau dynamics, kiss-and-hop mechanism, than previously reported (Janning et al., 2014) These observations suggest that microtubule stabilizers may not be suitable as inhibitors of tau-related dysfunction Active immunization is an attractive therapeutic approach because it can induce a sustained autoantibody response in small doses the therapeutic effects should not be limited by the production of anti-drug antibodies there are 10 tau antibodies that have entered clinical trials BIIB092 is in a phase 2 clinical trial for AD (TANGO; NCT03352557) trials with C2N-8E12 in AD patients were conducted (NCT02880956) An extension study (NCT03712787) is being conducted for patients who have successfully completed the phase 2 trial to evaluate long-term safety and tolerability The monoclonal antibody UCB0107 binds to the mid-region of tau (amino acids 235–246). Its preceding mouse version (antibody D) reportedly inhibited transneuronal propagation of pathogenic and aggregated tau in vivo (Albert et al., 2019), and seeding activity of human AD and PSP tau in vitro (Courade et al., 2018) Two phase 1 studies aimed to evaluate the safety and pharmacokinetic properties of UCB0197 in healthy adult males were conducted (NCT03464227 and NCT03605082) A phase 1 study is ongoing in patients with PSP (NCT04185415) The results of the trials are not yet available LY3303560 binds preferentially to tau aggregates rather than monomers a phase 1 trial of LY3303560 has been conducted to evaluate safety in MCI and mild to moderate AD (NCT03019536) while an ongoing phase 2 trial (NCT03518073) is being undertaken in early symptomatic AD patients The results of both studies are currently awaited BIIB076 is a human recombinant monoclonal anti-tau antibody. Although the epitope to which this antibody is directed has not been revealed, it is known that it recognizes monomeric and fibrillar tau (Alzforum.org, Therapeutics; BIIB076. https://www.alzforum.org/therapeutics/biib076) a phase 1 trial to examine the safety of BIIB076 has been conducted in healthy volunteers and AD patients (NCT03056729) JNJ-63733657 is a monoclonal antibody that recognizes the central domain of tau (Sigurdsson, 2018) but the exact epitope is unknown and results of preclinical testing are not available A phase 1 trial of JNJ-63733657 in healthy volunteers and patients with prodromal or mild AD has been conducted (NCT03375697 and NCT03689153) The monoclonal antibody Lu AF87908 binds to the phospho-S396 region of tau (Sandusky-Beltran and Sigurdsson, 2020). A preclinical study showed that the original mouse antibody inhibited tau propagation induced by exposure of tau transgenic brain lysates in vivo and in vitro (Rosenqvist et al., 2018) a phase 1 trial (NCT04149860) is assessing the tolerability of Lu AF87908 in healthy individuals and AD patients (NCT04149860) Currently a phase 1 study is being undertaken in healthy volunteers to evaluate the safety and pharmacokinetics of this antibody (NCT04096287) The trial showed that no serious adverse events occurred and RO7105705 plasma half-life was 32 days Two phase 2 trials were conducted in AD patients to evaluate efficacy and safety (NCT03289143 and NCT03828747) Genentech announced top-line results from a phase 2 trial RO7105705 did not meet the primary efficacy end-point of reducing the decline of the cognitive score (Genentech press release) such as JNJ-63733657 and UCB0107 may be more effective than N-terminal-directed antibodies at disrupting the seeding and propagation of aberrant tau Genentech showed no efficacy of the N-terminal-directed antibody close monitoring of adverse effects of oligonucleotide therapies is essential tau dissociated from microtubules migrates into the cytoplasm and forms the first aggregates the localized gathering of tau is a singularity of aggregation identification of the droplets formed specifically in tauopathies and the search for inhibitors of the droplets will be a challenge for the future Tau droplet formation by liquid-liquid phase separation (LLPS) is a key initial step in aberrant tau aggregation Tau must be abundant before it begins to aggregate LLPS-mediated formation of droplets supersaturated with tau may be a key step in the latter process A droplet is a reversible structure formed by weak interactions suggesting that tau droplets may be a better drug target than irreversible tau aggregation it appears that properly-regulated inflammation may protect against tau pathology and tau-mediated toxicity These findings indicate that tau has other physiological functions other than its better-known microtubule-stabilizing effects; therefore treatments that restore tau function may be helpful the management of tauopathies a re-focusing on the regulation of tau ubiquitination may help efforts to inhibit tau aggregation in which cDNA expressing a tau intrabody is loaded into neurons may also prove efficacious for treating tauopathies We are currently investigating the mechanism which may lead to discovering new drugs targeting tau loss-of-function Clinical trials of tau-based drugs aimed at gain-of-toxic-tau function (e.g. dysregulation of post-translational modifications and tau aggregation) or loss-of-function (microtubule instability) have been conducted in tauopathy patients Once these clinical trials have been completed the potential benefit of tau in the treatment of progressive neurodegenerative dementias may be revealed they will serve as a foundation for the next generation of tau-based drugs New tau 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Yoshiyuki Soeda, c29lZGF5NzE4OEBnbWFpbC5jb20= The Anime Japan 2025 panel for the television anime of Rocket Shokai's Sentenced to Be a Hero: The Prison Records of Penal Hero Unit 9004 (Yūsha-kei ni Shosu: Chōbatsu Yūsha 9004-tai Keimu Kiroku) light novel series unveiled a new promotional video and visual for the anime on Sunday The video reveals the anime's staff and October television and streaming premiere The previously announced cast members are Yōhei Azakami as Xylo Forbartz, Mayu Iizuka as Teoritta, Shizuka Ishigami as Patausche Kivia, Shun Horie as Dotta Luzulas, and Shunichi Toki as Venetim Leopool Shokai launched the light novel series on Kadokawa's Kakuyomu site in October 2020 and Kadokawa began publishing the series in print in September 2021 with illustrations by Mephisto Kadokawa released the seventh volume on January 17 Source: Sentenced to Be a Hero anime's AJ 2025 panel and website Misaki Emura rebounded from personal disappointment to come up big in the women's team sabre event Saturday by winning the final contest to secure bronze for Japan Emura stepped to the Grand Palais piste with Japan holding a 40-37 lead over France but with momentum against her after her teammate Seri Ozaki gave up two points to Manon Apithy-Brunet who was a shock loser in the last 16 of the individual sabre event faced off against Sara Balzer and a raucous home crowd but stood tall to win the bout 5-3 and ensure Japan would take the 45-40 win and its fourth fencing medal of the Paris Games "I haven't been able to fence the way I wanted to for a long time and I don't think my performance today was that good either," Emura said "But my teammates guided me through both mentally and fencing-wise and that enabled me to fight on till the end." "I'm really filled with joy to have been able to win the medal with these teammates," Emura one of Japan's opening ceremony flag bearers Ukraine beat South Korea 45-42 to claim gold Ozaki and their teammates Risa Takashima and Shihomi Fukushima became Japan's first sabre fencing Olympic medalists Japan has now won more at the ongoing games than it totaled in all previous Olympics with one more chance at a medal in the men's team foil event on Sunday Olympics: Japan wins 1st ever women's team foil medal in Paris Olympics: Misaki Emura a shock loser in sabre fencing last 16 To have the latest news and stories delivered to your inbox, subscribe here. Simply enter your email address below and an email will be sent through which to complete your subscription. Please check your inbox for a confirmation email. If you wish to change your message, press 'Cancel' to go back and edit. Thank you for reaching out to us.We will get back to you as soon as possible. Volume 9 - 2021 | https://doi.org/10.3389/fcell.2021.664327 Elevation of intraocular pressure is a major risk factor for glaucoma development which causes the loss of retinal ganglion cells (RGCs) Lipocalin 2 (Lcn2) is upregulated in glaucomatous retinae; however whether Lcn2 is directly involved in glaucoma is debated retinal explant cultures were subjected to increased water pressure using a two-chamber culture device and Lcn2 protein levels were examined by immunoblotting In situ TdT-mediated dUTP nick and labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemical assays were performed to assess apoptosis and gliosis The neurotoxicity of Lcn2 in the retinal explant culture was determined with exogenous administration of recombinant Lcn2 and GFAP-positive area were significantly higher in retinae cultured under 50 cm H2O pressure loads compared to those cultured under 20 cm H2O We found that Lcn2 exhibited neurotoxicity in retinae at dose of 1 μg/ml The negative effects of increased hydrostatic pressure were attenuated by the iron chelator deferoxamine This is the first report demonstrating the direct upregulation of Lcn2 by elevating hydrostatic pressure Modulating Lcn2 and iron levels may be a promising therapeutic approach for retinal degeneration Ocular hypertension is a major risk factor of glaucoma, which is characterized by the progressive loss of retinal ganglion cells (RGCs), resulting in irreversible vision loss. Although the reduction of intraocular pressure (IOP) is the only available treatment for glaucoma, the direct linkage between IOP elevation and retinal degeneration is yet to be found (Jayanetti et al., 2020; Pang, 2021) the direct involvement of Lcn2 in glaucoma caused by ocular hypertension has not been investigated we investigated the involvement of Lcn2 in retinal degeneration induced by elevated hydrostatic pressure using our two-chamber culture system C57BL/6J and DBA/2J mice were obtained from CLEA Japan Inc IOP of DBA/2J mice was measured using a rebound tonometer (TONOLAB; Icare Finland Oy Finland) according to the manufacturer’s instructions IOP measurements were performed for four times every week at 3 All animal research was conducted in accordance with the Ethical Guidelines of Japan for the use of Animals in Research and was approved by the Committee for Animal Experiments of Kindai University (protocol code KAME-29-001) The eyes of C57BL/6J mice were enucleated at 6–16 months of age and retinae were submerged in cold Dulbecco’s modified Eagle’s medium (DMEM) The retinae were placed at the bottom of a culture insert (transparent PET membrane with pore size 1.0 um; Corning United States) coated with Matrigel (Corning) Four retinae from two mice were cultured in a single culture insert with Neuronal culture medium (FUJIFILM Wako Pure Chemical Corp. Japan) containing 50 ng/ml nerve growth factor (NGF; Millipore United States) and cultured within the two-chamber culture system for pressure loading one retina was cultured per well of 12-well culture plate (Corning) in Neuronal Culture Medium containing 1% FBS and various concentrations of recombinant mouse Lcn2 (R&D systems United States) with or without 100 μM deferoxamine (Cayman Chemical the retinae were fixed with paraformaldehyde and subjected to TUNEL assessment The two-chamber culture system was set up as previously described (Yoneshige et al., 2017). Briefly, the culture insert was placed between two chambers; the upper chamber was a 50-cm-long plastic cylinder that enabled 2–50 cm H2O pressure loading, and the lower chamber consisted of a 10-cm culture dish, in which the bottom of the culture insert made contact with the medium under free gas exchange conditions (Figure 1) The upper and lower chambers were filled with DMEM containing 10% fetal bovine serum (FBS) and 1% N2 supplement (Thermo Fisher Scientific The entire two-chamber culture system was placed in a common CO2 incubator and maintained at 37°C with 5% CO2 Schematic illustration of the two-chamber culture system for pressure loading The retinae were submerged in media and placed at the bottom of a culture insert with Matrigel The culture insert was connected to the bottom of a 50-cm-long plastic cylinder that enabled 2–50 cm H2O pressure loading and was placed in a 10-cm culture dish where the culture insert made contact with the medium under free gas exchange conditions For the immunohistochemical analysis of GFAP paraformaldehyde-fixed retinae were immersed in increasing concentrations of sucrose (10–30%) and embedded in frozen medium; cryosections (5 μm thickness) were further treated with 0.1% Triton X-100 and nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories Japan) were visualized with a BZ-X710 microscope (Keyence Japan) and measured using the BZ-X analysis software (Keyence) The isolation of ganglion cell layer (GCL) from DBA/2J mouse retinae by laser-captured microdissection and the analysis of Lcn2 mRNA levels were performed as previously described (Ueno et al., 2018) the retinae of DBA/2J mice were immediately embedded in frozen embedding medium (SCEM-L1; Leica Germany) and 15-μm sections were mounted on membrane slides (2 μm-PEN-membrane; Leica) which were fixed with cold 5% acetic acid in ethanol After staining with 0.025% toluidine blue solution the GCL and inner plexiform layer (IPL) were dissected using a laser-captured microdissection system (LMD7000; Leica) and were directly captured in tissue lysis buffer Total RNA was purified using the RNeasy Plus Micro kit (Qiagen Germany) and cDNA was synthesized using Superscript IV Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturers’ instructions Expression levels of Lcn2 in the GCL were determined by quantitative PCR using the StepOnePlus real-time PCR system (StepOne software v2.3 and Power SYBR Green PCR Master Mix; Applied Biosystems Eight (20 cm and 50 cm + DFO) or ten retinae (2 and 50 cm) were used in organotypic culture for pressure loading and twelve retinae were used for each treatment in neurotoxicity assay Eight (6 and 12 months) or sixteen retinae (3 and 9 months) were used in DBA/2J mice analysis and five retinae were used in C57BL/6J mice analysis Each experiment was repeated at least 3 times and summary values were shown as mean ± SE Significant differences among three or four groups were analyzed using the Steel-Dwass test P-value ≤ 0.05 were considered to indicate statistical significance Correlations were analyzed using Spearman’s rank test and considered significant if p-value ≤ 0.05 and R2 ≥ 0.1 we collected retinae from the two-chamber culture system at 2 DIV Lipocalin 2 (Lcn2) protein levels in retinae cultured under 20 cm H2O C57BL/6J mouse retinae were cultured under medium heights of 20 cm and 50 cm retinal tissue extracts were subjected to immunoblotting for Lcn2 neuron-specific enolase (NSE) and glyceraldehyde-3-phosphate dehydrogenase (G3pdh) Graphs represent relative protein levels of Lcn2 and NSE compared to the G3pdh loading control Retinal ganglion cell (RGC) apoptosis and glial activation increased at 50 cm H2O pressure and decreased following deferoxamine (DFO) treatment green] assays were performed on frozen sections of retinae and the sections were counterstained with DAPI [(A,B) (C,D) The average percentage of TUNEL-positive nuclei in GCL (positive cell number/total cell number) and the average GFAP-positive area in retinae (positive area/horizontal length) are shown in (C,D) Correlation analyses between TUNEL-positive cells The values of TUNEL-positive cells and GFAP-positive area are represented as a scatter plot (top) The values of Lcn2 protein levels with TUNEL-positive cells (middle) or GFAP-positive area (bottom) are shown in the scatter plot the dot distribution approximates a linear function (dotted lines) Correlations and statistical significance were analyzed using Spearman’s rank test these data suggest that deferoxamine treatment attenuated pressure-induced retinal degeneration by inhibiting endogenous increase of Lcn2 but not by inhibiting retinal uptake of extracellular Lcn2 Lipocalin2 (Lcn2)-induced neurotoxicity in RGCs C57BL/6J mouse retinae were cultured in the culture medium containing 1 μg/ml of recombinant mouse Lcn2 with or without 100 μM deferoxamine (DFO) the retinae were subjected to immunoblotting analysis (A) or TUNEL assay (B) (A) Lcn2 protein levels in the retina of DBA/2J mouse at 9 months of age and the cultured retinae under atmospheric pressure with indicated treatment (0 μg/ml of recombinant Lcn2 or 1 μg/ml of recombinant Lcn2 with 100 μM deferoxamine) were estimated by immunoblotting Recombinant Lcn2 protein (rLcn2) appeared larger than native protein in immunoblotting (B) TUNEL assay (green) was performed on frozen sections of cultured retinae under atmospheric pressure with indicated treatment The sections were counterstained with DAPI (blue) Graph represents the average percentage of TUNEL-positive cells out of total cells in GCL Elevated intraocular pressure (IOP)-induced lipocalin 2 (Lcn2) upregulation in retinae of DBA/2J mice (B) mRNA levels of Lcn2 in dissected ganglion cell layer (GCL) (upper) and residual retinal layers (lower) Expression levels are represented as the mean ± standard error of fold changes compared to the level of the GCL at 3 months (C) Correlation analyses between IOP and Lcn2 mRNA levels Correlations and statistical significance were performed using Spearman’s rank test A straight line of best fit (dotted lines) (D) Lcn2 protein levels in the retinae of DBA/2J mice Retinal tissue extracts of DBA/2J mice at each age were subjected to immunoblotting for Lcn2 Retinae of C57BL/6J (B6) mice were used as an aging control Glial activation was observed in the retinae of DBA/2J mice after 9 months of age GFAP immunohistochemistry (green) was performed on frozen sections of DBA/2J mice retinae at 3 The average GFAP-positive area in retinae (positive area/horizontal length) is shown in a graph suggesting that the neurotoxicity of iron overload was not specific to RGC Whether iron dysregulation occurred within the pressure-treated retinae in our system and how this relates to Lcn2 upregulation should be determined in future studies alongside investigations into the potential therapeutic application of iron chelators in the treatment of glaucoma The raw data supporting the conclusions of this article will be made available by the authors The animal study was reviewed and approved by the Committee for Animal Experiments of Kindai University (protocol code KAME-29-001) and writing—original draft preparation AI: supervision and project administration This study was supported by the Japan Society for the Promotion of Science Kakenhi (19K07857 to AY We thank Yoshihiro Mine and Yasumitsu Akahoshi (Kindai University Life Science Research Institute The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.664327/full#supplementary-material Lipocalin-2 is increased in progressive multiple sclerosis and inhibits remyelination Viability of mouse retinal explant cultures assessed by preservation of functionality and morphology Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice Mechanosensitive release of adenosine 5’-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain Role of Hippocampal Lipocalin-2 in Experimental Diabetic Encephalopathy Satellite glia as a critical component of diabetic neuropathy: Role of lipocalin-2 and pyruvate dehydrogenase kinase-2 axis in the dorsal root ganglion Deferiprone rescues behavioral deficits induced by mild iron exposure in a mouse model of Alpha-Synuclein aggregation Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice Astrocytes in the optic nerve head express putative mechanosensitive channels Google Scholar Pathological involvement of astrocyte-derived lipocalin-2 in the demyelinating optic neuritis Crapper McLachlan Intramuscular desferrioxamine in patients with Alzheimer’s disease Oral administration of the iron chelator deferiprone protects against loss of retinal ganglion cells in a mouse model of glaucoma Iron chelators inhibit amyloid-beta-induced production of lipocalin 2 in cultured astrocytes Neutrophil gelatinase-associated lipocalin and its receptors in Alzheimer’s disease (AD) brain regions: differential findings in ad with and without depression A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake Targeting chelatable iron as a therapeutic modality in Parkinson’s disease Google Scholar Role of iron in brain lipocalin 2 upregulation after intracerebral hemorrhage in rats Plasma lipocalin-2 levels in the preclinical stage of Alzheimer’s disease Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors From the periphery to the brain: Lipocalin-2 Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation Impairment of membrane repolarization accompanies axon transport deficits in glaucoma Inflammatory responses of astrocytes are independent from lipocalin 2 Early gene expression changes in the retinal ganglion cell layer of a rat glaucoma model Modest static pressure suppresses columnar epithelial cell growth in association with cell shape and cytoskeletal modifications Effects of acutely elevated hydrostatic pressure in a rat ex vivo retinal preparation Experimentally induced mammalian models of glaucoma Google Scholar 24(S)-Hydroxycholesterol protects the ex vivo rat retina from injury by elevated hydrostatic pressure Google Scholar Phenotypic polarization of activated astrocytes: the critical role of lipocalin-2 in the classical inflammatory activation of astrocytes The latest drugs in development that reduce intraocular pressure in ocular hypertension and glaucoma Lipocalin-2 deficiency attenuates neuroinflammation and brain injury after transient middle cerebral artery occlusion in mice PubMed Abstract | CrossRef Full Text | Google Scholar The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma Pathogenic upregulation of glial lipocalin-2 in the Parkinsonian dopaminergic system Astrocyte-derived lipocalin-2 mediates hippocampal damage and cognitive deficits in experimental models of vascular dementia Lipocalin-2 is an autocrine mediator of reactive astrocytosis Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration Hydrogen sulfide protects retinal ganglion cells against glaucomatous injury In Vitro and I High hydrostatic pressure induces apoptosis of retinal ganglion cells via regulation of the NGF signalling pathway Google Scholar Cerebrospinal fluid lipocalin 2 as a novel biomarker for the differential diagnosis of vascular dementia Google Scholar Role of lipocalin-2 in thrombin-induced brain injury Lipocalin 2 modulates the cellular response to amyloid beta Piezo channel plays a part in retinal ganglion cell damage Lipocalin 2: novel component of proinflammatory signaling in Alzheimer’s disease Role of lipocalin-2 in brain injury after intracerebral hemorrhage Hydrostatic pressure does not cause detectable changes in survival of human retinal ganglion cells Gene expression changes in areas of focal loss of retinal ganglion cells in the retina of DBA/2J mice and elasticity in pressure-induced retinal diseases Deferiprone reduces amyloid-beta and tau phosphorylation levels but not reactive oxygen species generation in hippocampus of rabbits fed a cholesterol-enriched diet Lipocalin 2 plays an immunomodulatory role and has detrimental effects after spinal cord injury Glaucoma: validated and facile In Vivo experimental models of a chronic neurodegenerative disease for drug development The polymodal ion channel transient receptor potential vanilloid 4 modulates calcium flux and apoptosis of mouse retinal ganglion cells TRPV1: contribution to retinal ganglion cell apoptosis and increased intracellular Ca2+ with exposure to hydrostatic pressure Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric retinal explants and animal models for common diseases of the retina Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice Shiratori-Hayashi STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch Shiratori-Hayashi Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca(2+) signals in mice Microarray analysis of retinal gene expression in the DBA/2J model of glaucoma What animal models can tell us about glaucoma Increased production of tumor necrosis factor-alpha by glial cells exposed to simulated ischemia or elevated hydrostatic pressure induces apoptosis in cocultured retinal ganglion cells Early gene expression profile in retinal ganglion cell layer after optic nerve crush in mice Changes in gene expression in experimental glaucoma and optic nerve transection: the equilibrium between protective and detrimental mechanisms Transcriptome profiling of the rat retina after optic nerve transection Google Scholar Modest static pressure can cause enteric nerve degeneration through ectodomain shedding of cell adhesion molecule 1 Role of lipocalin-2 in extracellular peroxiredoxin 2-induced brain swelling Deferoxamine attenuated the upregulation of lipocalin-2 induced by traumatic brain injury in rats Lipocalin-2 may produce damaging effect after cerebral ischemia by inducing astrocytes classical activation Google Scholar Transgenic mice expressing the Tyr437His mutant of human myocilin protein develop glaucoma Koriyama Y and Ito A (2021) Elevated Hydrostatic Pressure Causes Retinal Degeneration Through Upregulating Lipocalin-2 Copyright © 2021 Yoneshige, Hagiyama, Takashima, Ueno, Inoue, Kimura, Koriyama and Ito. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Azusa Yoneshige, YXp1c2E2MThAbWVkLmtpbmRhaS5hYy5qcA== Community Safety Department Director To Resign Amid Tension With Cambridge Police Department From Lab to Startup: Harvard’s Office of Technology Development Paves the Way for Research Commercialization People’s Forum on Graduation Readiness Held After Vote to Eliminate MCAS 8 Takeaways From Harvard’s Task Force Reports An interview with Ryosuke Takashima ’19-’22 in this article was translated from Japanese by Crimson News editor Ayumi Nagatomi ’26 Following a decisive win in a unified local election on April 23 Harvard College alum Ryosuke Takashima ’19-’22 became the youngest mayor in Japanese history assumed the mayoral seat for the city of Ashiya in Hyogo Prefecture one incumbent and two with prior experience in city government Takashima lived in Eliot House and concentrated in Environmental Engineering with a secondary in Environmental Science and Public Policy while exploring various ways to engage in politics He also worked as a Multimedia editor for The Crimson and as director of multimedia for the Harvard Project for Asian and International Relations “Harvard is a university that nurtures leaders of society,” Takashima said in an interview with The Crimson last week “I was always thinking about how I could give back to society what I had learned.” Takashima said he did not plan to run for office right after graduating from the College It is uncommon for young politicians to win elected office in Japan with the average age of mayors elected this cycle at 59 years old The minimum age to hold office in Japan is 25 “It just so happened that the timing was right after graduation,” Takashima said “When I saw that the current situation in Ashiya was very bad Takashima described Ashiya — home to 93,000 citizens — as a residential city just a little smaller than Cambridge While Ashiya is known to be a wealthy city in Japan it faces issues of an aging population and low birth rate which are more pronounced than in nearby municipalities Though more than 80 percent of couples in Ashiya report they want to have two or more children the average number of children per household in the city remains at 1.3 Along with setting up an improved system for extended daycare Takashima said he wants to implement free medical care for children an initiative being pursued by an increasing number of municipalities Harvard Undergraduate Association Co-President Shikoh Misu Hirabayashi ’24 who is from Japan and received mentorship from Takashima during the college application process said he was pleasantly surprised by the election win Hirabayashi said did not initially expect Takashima to win because older candidates are often perceived in Japan as more competent with “much more work experience and connections.” “He was also fighting against an incumbent but still had some sort of election voting base,” Hirabayashi said Takashima said holding consistent dialogue with citizens was key to his win he organized public forums across the city and distributed a booklet containing his policy proposals to households in Ashiya “Many people understood that although I am young I have a good understanding of the current situation and am trying to make effective policies,” Takashima said who attended the same middle school and high school as Takashima called the newly elected mayor a “local leader.” Yanaizu said he remembered how Takashima organized a volunteer group during high school for a summer festival held in Ashiya “He cares a lot about the community he is in,” Yanaizu said “I think he genuinely likes Ashiya as a city and is committed to making Ashiya a better place Takashima said he has always been interested in city-level politics his hometown drastically changed after the election of a new and younger mayor “I thought that the city government has a great deal of influence and is very closely connected to the lives of the citizens,” Takashima said Takashima also took multiple gap years during his time at the College giving him the opportunity to travel the world and speak with local officials and citizens about urban planning Takashima enrolled in a Harvard Kennedy School workshop called I’m Running for Office,” which helped him learn more about participating in politics Each session focused on different aspects of running for office and governing I learned how to raise money and how I should work on my policies after taking office,” Takashima said I was glad to meet many people who were thinking of running for office in the future.” remembered Takashima’s participation in events related to politics like a Public Narrative event hosted at the house and led by organizer and HKS lecturer Marshall L I can see he was looking for opportunities to develop these sorts of political skills,” Paulsell said Hirabayashi said he hopes Takashima’s victory can inspire the younger generation even as Japan has struggled with civic participation in politics in recent years “I really wanted him to set a precedent for other young people to also run for politics but also to become interested in politics,” he said A previous version of this article incorrectly stated that more than 80 percent of couples in Japan report they want to have two or more children more than 80 percent of couples in the city of Ashiya report wanting two or more children —Staff writer Ayumi Nagatomi can be reached at ayumi.nagatomi@thecrimson.com. Follow her on Twitter @ayumi_nagatomi Want to keep up with breaking news? Subscribe to our email newsletter. Your browser does not support JavaScript, or it is disabled．Please check the site policy for more information Parents in Japan often try to give their offspring a leg up by ensuring they learn early on how to use the abacus While the sound of sliding beads making clacking sounds on a small wooden frame may seem incongruous with strides in technology surveys have repeatedly shown that children proficient in old-school “soroban” abacus techniques can quickly solve complicated mathematical problems with the growth of generative artificial intelligence (AI) and computer programming educational institutions are keen to adopt the latest technologies to teach skills the abacus is enjoying a resurgence with parents in the Reiwa Era that started in 2019 At the Takashima Soroban School in the Akabane district of Tokyo’s Kita Ward students diligently get to work solving multiplication and division problems as soon as a staff member signals the start of calculations The children also perform calculations without soroban and practice flash mental arithmetic to swiftly process numbers displayed on PC screens At a 20-student class held from around 4 p.m most of the kids were from lower grades of elementary school: six “This course is challenging but fun,” said Rento Sasamoto expects her son to “improve calculation skills and other basic abilities” in preparation for entrance exams to private junior highs later on Of the 220 children enrolled in the Takashima Soroban School at present “Many parents want their offspring to obtain soroban skills during the lower grades at elementary school before they fully gear up for junior high entrance tests at dedicated cram schools,” said the principal “There is strong demand for soroban education among far younger children Takashima added that Saturday classes and weekday programs held from around 6 p.m since the hours make it easier for working fathers and mothers to pick up their children Established by Takashima’s grandfather in 1939 Takashima Soroban School “used to have hundreds of students.” By 2010 Takashima said the school started becoming packed with children again in recent years due to an influx of students who “hope to enter private junior highs.” mental arithmetic and concentration can be nurtured through soroban classes with a focus on junior high entrance exams,” she stressed noting that the enthusiasm for soroban learning is growing every year “Our hope is to produce students good at mathematics.” was part of the curriculum at “terakoya” private educational institutes during the Edo Period (1603-1867) How to use the abacus became a compulsory subject at elementary schools in Japan in 1935 soroban lessons are included in math classes for third- and fourth-graders there were 13,010 specialized soroban schools across Japan according to the Establishment and Enterprise Census But their number dropped to 5,227 in the 2021 Economic Census for Business Activity due not only to the spread of pocket calculators and personal computers but also a lack of successors The educational benefits of soroban are gaining renewed attention as a “tool for enhancing concentration and stimulating the brain.” A survey conducted between 2015 and 2023 by the Benesse Educational Research and Development Institute and the University of Tokyo found that the soroban placed fourth on the ranking of non-science after-school activities for elementary school children just behind music and instruments A high-profile school combines soroban with information technology so students can learn mental arithmetic on tablet devices An educational app called Soro Touch shows abacus beads on the liquid display panel allowing users to perform calculations in the same way as the soroban The mental arithmetic mode of Soro Touch inactivates the coloring feature to make students imagine beads instead even small children can master soroban techniques while enjoying videos “Children can continue learning spontaneously in a playful manner though our tool does not provide users with the tactile experience of wood like soroban,” said Chika Yamauchi Another advantage of Soro Touch is that it allows students to check their learning status Classes are now offered at 300 schools in 19 countries including those receiving lessons via the internet at home The school operator recommends that children begin Soro Touch at ages 5 through 8 as this age group can easily acquire the skills for mental arithmetic by imagining beads “Learning on our tool for 30 minutes every day will rapidly improve mental calculation skills,” Yamauchi said It fosters skills that are not only valuable for junior high entrance exams but throughout life.” the Kyoto-based League for Soroban Education of Japan publicized the results of its study on 348 children of elementary school age or younger who attended soroban classes in Tokyo’s 23 wards It found that three in four children were initiated by their parents or guardians in soroban lessons during their first year at elementary school or earlier The league shone the spotlight on the soroban for its “benefits in preparing for junior high entrance exams” and the fact that increasingly younger learners are turning to the traditional calculation method About 90 percent of the 77 children planning to take junior high entrance tests began studying the soroban in their first grade at elementary school or earlier the rate of students who advance to private junior high schools is rising in recent years Data compiled by the capital’s board of education reveal that a record 20.1 percent of students at public elementary schools in Tokyo entered private junior highs within the capital last year head of the soroban league’s committee on training and school education suggested that one reason lay in the priority put on arithmetic these days “It is said that winners in arithmetic will be winners in junior high entrance examinations,” he added “As admission tests become increasingly harder the soroban is being re-evaluated for its role in polishing fundamental calculation skills.” Educational facilities look to younger children for new students Schools test shift from whole-class lessons to self-paced learning New curriculum for English learning called a ‘big failure’ Night junior highs offer rare opportunity to foreign nationals 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 No reproduction or republication without written permission Shiga Prefecture--Giants sheets of colorful fabric billowing in the cool breeze at the Biwako Hakodateyama resort here are wowing visitors eager to escape the intense summer heat About 200 “Rainbow Curtains" are set up at an elevation of more than 600 meters above sea level People have been visiting the recreational spot in droves to seek respite from the heat Takashima is known as “the city of rainbows” because the natural phenomenon appears so often The “Rainbow Curtain” event was created from Takashima chijimi Visitors often snap commemorative photos as they wait for the right moment to capture the curtains unfurling in the breeze a giant trampoline and swings facing Lake Biwako The admission fee is 2,500 yen for adults and 1,200 yen for children of elementary school age and younger There is a flat fee of 1,000 yen for parking Details are on the website at https://www.hakodateyama.com/green/ Nuisances in Lake Biwako turned into leather goods Oxygen levels near zero in bottom areas of Lake Biwako Drought changes Lake Biwako island into ‘Mont Saint-Michel’ 10th Biwako Biennale art festival now running in Shiga Preservation of underwater sites widens for pirate castle Alarm bells ring in Kinki region as Lake Biwako keeps shrinking associate MD of content business at Nippon TV reveals the Japanese broadcaster’s plans for the London TV Screenings and the new properties it will be showcasing and also discusses how global demand for J-content is changing What is Nippon TV’s mission for the London TV Screenings week is a sci-fi dramedy series from the creators of the award-winning hit series Rebooting with award-winning director Itaru Mizuno attached to direct The Hot Spot began airing on Nippon TV in January with the first episode topping the ratings among teenagers and female viewers (35-49) It recorded 2.68 million views on the national VoD platform TVer in its first week The original version of The Hot Spot has been acquired by Netflix and is currently streaming having ranked number one in the list of top 10 TV shows in Japan The second scripted format series is romance drama Ensemble which is a legal love story that follows the journey of a down-to-earth female lawyer and an idealistic male rookie lawyer Ensemble premiered on Nippon TV in January with the first episode topping its time slot for female viewers (35-49) and recording about two million views on TVer in its first week We will also be introducing one new unscripted gameshow format in which six ‘spies’ must outsmart high-tech facial recognition cameras by hilariously contorting their faces and tackling thrilling challenges to escape elimination with viewers commenting in real time on social media about its unique concept and how hilarious it was How is global demand for Japanese content changing and what genres are travelling the most Global clients are increasingly seeking successful shows with proven track records Scripted formats with innovative storytelling and unique unscripted gameshow concepts from Nippon TV continue to be in high demand we have adjusted the launch timing of our new formats from April to February and these three new formats were meticulously crafted based on valuable feedback received from our clients Regarding the international formats market it continues to be sustainable and profitable How important is Europe to your business and what recent success have you had there The compatibility between European audiences and Japanese formats has been reaffirmed recently our format Red Carpet Survival was produced in Italy by Blu Yazmin and is now streaming on Prime Video Italy European audiences have consistently embraced hilarious celebrity-driven gameshows and Funny Face Spy has the potential to redefine the genre It offers a fresh take on gameshows while maintaining a strong focus on comedy and entertainment How is demand for J-content changing in the UK in particular The demand is always very high because of the stiff competition within the UK industry The UK audience was one of the first to appreciate our Old Enough format when it was produced locally in 2009 long before it became a global phenomenon when the readymade episodes streamed on Netflix in 2022 but the UK is always one of the first to reach out to us when we announce our innovative formats and we look forward to discussing them in person in London How did the huge global success of Mother change your international business We learned that the more local the story is the more it relates to global audiences facing similar situations but creators start out by telling a story about one person in hopes to share the message behind it and viewers are awakened by how relevant it actually is to them Nippon TV has been creating series for decades and our original scripted formats all have the opportunity to be adapted globally That is what we learned from the success of Mother and we owe that to each one of the 11 adaptations How is your coproduction strategy changing in 2025 We have several on-going scripted copros: Connected with ZDF Studios and Studio Zentral; How to be a Sensei with Anyway Content UK; and gameshow Koso Koso Development of each of them is progressing and additional partners are being sought All have unique styles with different target audiences and our partners value the original ideas that Nippon TV can provide How is the growth of YouTube changing things for Nippon TV’s distribution business Social media and the rise of shortform video have certainly transformed the way people consume content and have the potential to positively impact our industry One of the greatest successes in our format business has been Dragons’ Den/Shark Tank which has over 50 local versions worldwide some of which also have dedicated YouTube channels The viral nature of videos on YouTube has significantly increased the show’s fanbase CLICK HERE FOR MORE INFORMATION AND TO SUBSCRIBE This C21Screenings show is password protected.If you have a show password enter it here and click 'Go': Angelica Christine Acaylar-Takashima joined the US-ASEAN Business Council in July 2022 and is currently the Senior Manager for the Philippines With a background as an Environmental and Social Considerations Specialist she brings extensive experience in infrastructure development and policy engagement Angelica played a pivotal role in social infrastructure projects under the Official Development Assistance (ODA) program including the Metro Manila Subway Project in the Philippines funded by the Japan International Cooperation Agency (JICA) She holds a bachelor’s degree in Environmental Science and Management from Thai Nguyen University of Agriculture and Forestry and UC Davis made possible through the Vietnam Ministry of Education and Training's scholarship program Her undergraduate research focused on climate change and indigenous communities in Northern Vietnam Angelica is currently pursuing a master’s degree in ASEAN Studies at the University of the Philippines (Open University) and serves as a Board Trustee at the Lighthouse Club International – Manila Branch a non-profit supporting construction industry members through financial Angelica is fluent in Filipino and English and speaks conversational Vietnamese and Japanese There are no statistics available for this player Thanks for visiting The use of software that blocks ads hinders our ability to serve you the content you came here to enjoy We ask that you consider turning off your ad blocker so we can deliver you the best experience possible while you are here Akita Prefecture—A rookie candidate who turned to insect breeding after struggling to find work in his hometown is now the youngest mayor in Japan He vowed to address the urgent challenge of halting the rapid decline in population in this northern Japan city Ryosuke Takashima remains Japan’s youngest-ever mayor according to the Japan Association of City Mayors Ishida was asked what it means to become the youngest mayor in the nation so I will take advantage of that to promote Odate’s attraction across the country.” surrounded by cheering supporters: “Winning is not the goal I will work together with residents to gradually improve the city of Odate.” the city’s population stood at around 66,000 down by more than 10,000 from 10 years ago More than 40 percent of Odate’s residents are 65 or older the population is expected to drop below 40,000 He moved to Aomori at the age of 6 because of his father’s job and lived there until high school He returned to Odate seven years ago after working in Tokyo Ishida spent six months looking for work in the city through an employment service center He and his twin brother later founded a company that raises beetles by feeding them organic waste Ishida ran in the city assembly election in spring last year and secured the most votes he felt limited in what he could achieve as an assembly member “I want to become mayor so I can influence the budget and make personnel decisions for real change in Odate,” he said Ishida announced his candidacy for mayor in December last year and ran as an independent he focused on tackling population decline and the challenges facing an aging society with a low birthrate He also proposed policies to create jobs that would appeal to young people and bring in outside talent to alleviate the city’s labor shortage Ishida’s youth was a key part of his appeal “Being young means having the drive to act,” he told voters on the streets (This article was written by Takashi Takizawa and Hiroaki Abe.) ‘Green frog’ train exhibited in Akita before 3rd life as tourist attraction Tsukuba mayor to let residents vote on his retirement bonus online Chichibu mayor’s race comes down to pledge to slash salary the most South Korea’s opposition party wins 2 key local elections Ryosuke Takashima was elected mayor of Ashiya, Hyogo Prefecture in the second half of the local elections on April 23 he became the youngest mayor ever in Japan.  A graduate of the elite preparatory school Nada Junior and Senior High School in Higashinada-ku, Kobe, he went on to study at Harvard University his decision to run against the political big guns was deemed reckless The three other candidates were incumbent Mai Ito a former member of the prefectural assembly and a former member of the municipal assembly.  his vote count was nearly double of Ito's.  and what do we know about the youngest mayor in Japanese history "Many people told me that I was too young and being reckless," Takashima explained in an interview before the election He added that he was fully aware that his youth could be a disadvantage in the political arena.  Takashima brought his youthfulness to the forefront of his campaign he always disclosed his age when speaking on the streets 26 years old." He was following the wise counsel of his campaign supporters One of them was Kunio Yamada, a fellow Nada High School alumnus and chairman of Rohto Pharmaceutical. Takashima recalls, "Chairman Yamada encouraged me by saying, 'During the Meiji Restoration young people played an active role in ushering in the new era I hope you will also do big things to bring change to Japan.'"  Ashiya has one of Japan's most exclusive residential areas and is home to many wealthy people. However, the city's population continues to decline which has become a significant issue for the municipal government is that "many Ashiya citizens chose to entrust their future to this outstanding young man." When Takashima heard the news of his victory I will do my best to live up to your expectations." What kind of person is Takashima? Kenji Yamada, who gave pointers for his campaign, is a local Liberal Democratic Party (LDP) member of the House of Representatives and State Minister for Foreign Affairs "[Takashima] is not just a member of the elite," Yamada explains "He is a young man who shows his human side and charisma that will make anyone who meets him immediately like him and become a fan."  Takashima was born in February 1997 in Minoh City, Osaka he was a shy kindergartener who struggled to make friends "I tried to praise him to build his confidence," she said.  "I praised him for even the smallest things like greeting a friend's mother or properly lining up his shoes," she added Takashima grew into an optimistic child who openly shared his ideas as he achieved little victories the Takashimas regularly held family meetings The most memorable family meeting was when they were brainstorming names for Takashima's second brother.  "My eldest two were named 'Ryosuke' and 'Shusuke,' so I was thinking 'Shosuke' But 8-year-old Ryosuke pointed out that the names combined would make the word 'ryoshusho' [receipt] and suggested 'Jyosuke' instead."  they chose this name for his newborn brother Another interesting chapter from Takashima's life is from his time in Nada Junior and Senior High School His classmates used to fight over Takashima's notes which were well-organized and neatly written The notes even documented the teachers' small talk This earned him the nickname "the god of notes." Takashima also played scrum-half for the rugby club and was the president of the student council He recalls how organizing a choral competition as a member of the student council taught him valuable leadership skills "We had a disagreement with the teacher over how the singers should enter and exit the stage the teacher said that the class that finished singing should leave as the next class entered so we insisted on keeping the timing of entry and exit separate During the rehearsal on the day before the event the student council agreed to go through with what they thought was right The competition finish on time and it was also a huge success "The best way for everyone to enjoy an event is to do it the way they want to," Takashima explains people won't be convinced." It was during this time that he started thinking about the qualities that make a good leader.  During his time as the school council president Takashima organized events and participated in festivals in Ashiya City This connection was what would eventually lead him to run for mayor of Ashiya.  he was accepted to the University of Tokyo and Harvard University He first enrolled in the University of Tokyo but left in September for Harvard.  "Our professors often gave us reading assignments of 100 pages and my English at the time wasn't good enough thanks to my classmates who offered to help." Takashima learned the importance of asking for help rather than trying to do everything himself.  He majored in environmental engineering at Harvard and conducted research at renewable energy installations across the world He hopes to introduce these cutting-edge technologies to Ashiya paved the way for Takashima to become the youngest mayor in Japanese history (Read the article in Japanese.) You must be logged in to post a comment ' + scriptOptions._localizedStrings.webview_notification_text + ' " + scriptOptions._localizedStrings.redirect_overlay_title + " " + scriptOptions._localizedStrings.redirect_overlay_text + " « Back AnimeJapan 2025 took place at Tokyo Big Sight on March 22-23 Erina Takashima attended dressed as Kirari Momobami from "Kakegurui." Along with sharing her photos we interviewed her about key points she focused on in her cosplay and her future plans Enjoy a Bath Together in “Newlywed Roleplay”! Takamine-san” Episode 6 Synopsis & Preview Images Released Anime "From Old Country Bumpkin to Master Swordsman" Episode 5 Synopsis & Advance Cut Released "Machi★Asobi" Talk Event and Retrospective Screening Details Announced Anime 'From Old Country Bumpkin to Master Swordsman' Episode 4 Synopsis and Preview Still Released Episode 1 Free Limited-Time Release on YouTube 【Setlist Included】 MyGO!!!!!×Ave Mujica Joint Live "Wakaremichi no Sonosakie" Performance Report 'The Apothecary Diaries' and 'Re:ZERO' Lead in Views and Comments Final Winter 2025 Anime Rankings Announced What Appeared in the Trap Set for Skinner.. "LAZARUS" Episode 5 Preview and Synopsis Released Latest Work from "Cowboy Bebop" Director Shinichiro Watanabe Voice Actor and Artist Active in 'Love Live!' The Ultimate Shooting Display by Amuro in "Mobile Suit Gundam: Char's Counterattack" That Defies Belief Original Menu Items Reflecting the World of Gege Akutami's 'Jujutsu Kaisen' Exhibition Collaboration Cafe Including Satoru Gojo's Slightly Wild Pancakes Metrics details A Publisher Correction to this article was published on 12 February 2024 This article has been updated we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))—known to give rise to one of the two extraembryonic tissues essential for embryonic development—from naive human pluripotent stem cells (hPSCs) Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue the efficiency of bilaminoid formation increases from 20% to 40% and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6 we show that bilaminoids robustly recapitulate the patterning of the anterior–posterior axis and the formation of cells reflecting the pregastrula stage the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis Early blastocysts of the pre-implantation human embryos are composed of trophectoderm and inner cell mass (ICM) a process completed in the late blastocyst stage these two tissues form a bilaminar disc that functions as a developmental template for the embryo Despite the importance of early human development our knowledge of human peri-implantation development is limited owing to ethical and legal restrictions alternative approaches for analysing this developmentally critical period are necessary and the capture of in vitro pre-implantation hypoblast has not been achieved it remains unclear whether extraembryonic tissues support the development of pre-implantation epiblast Here we induced human pre-implantation hypoblast from naive hPSCs by either transgene overexpression or chemical induction which guides the epiblast to form the first embryonic cavity establishes the anterior–posterior axis and together with the second extraembryonic tissue supports the establishment of the post-implantation embryonic state we concluded that GATA6 promotes naive hPSC differentiation into the hypoblast lineage while primed hPSCs adopt a post-implantation embryonic fate Signalling pathways to specify the three cell types of blastocyst n values show biologically independent experiments Source Data We concluded that naive hPSC-derived PDGFRA+ cells overexpressing GATA6 or manipulated chemically to activate relevant signalling pathways progress into a hypoblast-like state in contrast to transcription factors of which the hierarchy and functions appear to be conserved between humans and mice signalling may be common between humans and marmosets but differs with mice suggesting that nHyCs closely resemble the pre-implantation a tissue that supports the epiblast development Bilaminar embryo-like aggregates (bilaminoids) generated by the mixture of naive hPSCs and nHyCs IL-6 was added from day 0 to 4 where indicated GFP-expressing naive hPSCs expressing GATA6 under DOX treatment Immunofluorescence images of cell aggregates qPCR analysis of aggregates on days 2 and 4 Cell aggregates were sorted by GFP on days 2 and 4 mixed aggregates of Naive(WT) and Naive-GFP(G6-OE) cells; WT Immunofluorescence images of polarization markers in aggregates on day 4 Source Data We therefore concluded that nHyCs and nEpiCs self-organize and express markers like the late human blastocysts n = 4 biologically independent experiments The efficiency of cavity formation on day 6 as in f Statistical analysis was performed using two-tailed Fisher’s exact tests The volume of the amniotic cavity of each aggregate on day 6 as in f Statistical analysis was performed using Kruskal–Wallis and Dunn’s multiple-comparisons test The efficiency of cavity formation in bilaminoids after STAT3 activation on day 4 GP130/GCSFR chimeric gene (Y118F) activates STAT3 signalling by adding G-CSF n = 3 biologically independent experiments Immunofluorescence images of aggregates with nTB on day 6 n = 5 biologically independent experiments the number of aggregates analysed for each group is shown the centre line shows the median; the box limits show the 25th and 75th percentile range and the whiskers show 1.5 × interquartile range (IQR) Source Data We concluded that nHyCs have a crucial role in regulating the expression of gastrulation-related genes in nEpiCs the number of aggregates analysed for each group is shown at the bottom Source Data indicating that nHyCs control anterior–posterior axis formation and patterns epiblast differentiation We further concluded that a subpopulation of nHyCs inhibits and thereby patterns the expression of gastrulation-related genes in nEpiCs Relative expression values of each tissue-specific marker gene in each cell type Here we highlight the crucial mechanistic roles of the two extraembryonic tissues—hypoblast and TB—to guide the progression and patterning of naive hPSCs into the post-implantation epiblast stage thereby enabling them to generate subsequent lineages (for example PGC-like and HEP cells) in a manner mimicking human embryogenesis akin to the differences in trophectoderm induction However, hypoblast induction with the transcription factors GATA6 and GATA4 induces naive hPSCs to hypoblast, similar to in mice. Although transgene copy numbers and insertion sites may be variable because we used the PB system, we reproducibly obtained more than 80% PDGFRA+ cells from five independently established DOX-inducible GATA6 H9 hPSCs (Extended Data Fig. 2a) our data show that the levels and duration of GATA6 overexpression are critical Although we titrated the DOX concentration high levels of GATA6 mRNA may have resulted in off-target effects and caused some functional disadvantages As 7F induction is a non-genetic chemical induction method 7F may enable naive hPSCs to differentiate into hypoblast under more physiologically relevant conditions compared with GATA6 overexpression further studies may confirm an IL-6 dependency functional assays with genetic modifications are almost impossible in human embryos but we performed several lineage-specific gene modifications and identified interactions between these lineages a limitation of our bilaminoids is that the amnion is covered by hypoblast when it should be in direct contact with the TB together with the other human stem cell-based embryo models will drive scientific discoveries in biomedical science The investigators were not blinded to the group allocation of experimental samples or the outcome assessment No statistical methods were used to predetermine sample sizes Our embryo model lacks TBs and does not intend to recapitulate the full conceptus our models are considered to be non-integrated embryo models and are not considered to be human embryos according to the ISSCR Our work fully complies with current ISSCR 2016 and 2021 guidelines and follows the Guidelines on the Utilization of Human Embryonic Stem Cells in Japan approved our research plan for human ES cell research (CiRA08-08) human iPSC research (CiRA18-21) and recombinant DNA experiments (190438) The WiCell lines H1 and H9 were used under agreements 10-WO-0098 and 10-WO-0099 for a research program entitled “Understanding mechanisms of pluripotency” Bilaminoid models were generated using H9 ES cells These cell lines were consented for use in this study Human-to-mouse interspecies chimera research was approved by the Research Ethics Committee of the University of Tokyo and was conducted after receiving approval from the Ministry of Education Science and Technology (MEXT) Japan after confirmation of compliance by the Specified Embryo Expert Committee This approval includes the establishment of human iPSCs from peripheral blood samples Signed informed consent was obtained from the volunteers before human peripheral blood samples were collected to establish iPSCs Primed hPSCs were maintained in DMEM/F12 (08460-95 Nacalai Tesque) containing 20% Knockout Serum Replacement (10828028 4 ng ml−1 recombinant human basic fibroblast growth factor 2 (bFGF; NIB 47079000 Oriental Yeast) and 0.1 mM 2-mercaptoethanol (M3148 Cultures were passaged every 5–7 days as small clumps using dissociation buffer containing 0.025% trypsin (15090-046 20% Knockout Serum Replacement and 1 µM CaCl2 Naive hPSCs were passaged every 3–5 days using Accutase (A6964 Resetting primed hPSCs to naive hPSCs by NANOG and KLF2 overexpression was performed as previously described18 PB vectors (2 µg) carrying DOX-inducible KLF2 or NANOG and a PB-M2rtTA expression vector (2 µg) were co-transfected with pBase helper plasmid (4 µg) using the Neon Transfection System (Program 14 The medium was switched to t2iL plus DOX (1 µM) for resetting Cells were split every 5–7 days after dissociation with Accutase and the PKC inhibitor Go6983 (3 µM) was added (t2iLGo) Cells were maintained on MEF feeders throughout 2 × 105 cells per cm2 were seeded on MEF feeder cells under primed hPSC medium with 10 µM Y-27632 The medium was switched the next day to 5iLA medium (N2B27 plus 1 µM PD03 10 ng ml−1 hLIF and 20 ng ml−1 activin A (338-AC-010 the cells were maintained under t2iLGo on MEF feeder cells Mouse ES cells were cultured on a gelatine-coated dish in 2iL (N2B27 Cells were passaged every 2–3 days using Accutase All cell lines were routinely checked for mycoplasma contamination (Lonza–MycoAlert) and all samples analysed in this study were not contaminated 25 ng ml−1 recombinant human FGF4 (FGF4; 100-31) and 1 µg ml−1 heparin sodium (081-00131 5 × 104 per cm2 naive hPSCs were seeded onto laminin511-E8 in the N2B27 medium 25 ng ml−1 FGF4 (+1 µg ml−1 heparin sodium) 10 ng ml−1 recombinant human BMP4 (BMP4; 314-BP 10 ng ml−1 recombinant human PDGF-AA (Peprotech the medium was switched to seven factors (six factors and 10 ng ml−1 recombinant human IL-6) (IL-6; 47066000 500 ng ml−1 recombinant human BMP2 (BMP2; 47304000 Oriental Yeast) or 50 ng ml−1 recombinant human BMP6 (BMP6; 120-06 N2B27 medium without vitamin A was made in house The medium was changed every day in both conditions Embryos (morulae or blastocyst) were cultured under Sequential Blast (Origio the zona pellucidae were removed using acidic Tyrode’s solution (Sigma-Aldrich) and the embryos were processed for immunosurgery using a custom rabbit polyclonal anti-marmoset antibody ICM were seeded on laminin511-E8 under N2B27 plus 7F fixed and analysed using anti-SOX17 antibodies Corning) under t2iLGo plus 10 μM Y27632 without Matrigel or Geltrex the medium was switched to N2B27 with 0.1 μM DOX Bilaminoids were cultured under N2B27 until day 10 To identify the signalling pathways involved 1 µM XAV and 1 µM CHIR were added from day 4 to day 6 Sigma-Aldrich) or 10 ng ml−1 PDGF-AA was added from day 0 to day 4 bilaminoids were cultured under N2B27 + 200 ng ml−1 BMP4 from day 5 to day 9 Bilaminoid and nTB were co-cultured using a cell culture insert (Transwell) nTB was induced from naive hPSCs on the Transwell Bilaminoids were generated by culturing a mixture of 10 naive hPSCs (Naive(WT)) and 40 naive hPSCs or GFP-expressing naive hPSCs expressing GATA6 under DOX treatment (Naive(G6-OE) or Naive-GFP(G6-OE)) in each well of an Elplasia plate under t2iLGo plus 10 µM Y27632 nTB on the Transwell was placed on the Elplasia plate under N2B27 with 0.1 µM DOX A mixture of 100 naive or primed hPSCs and 100 sorted cells expressing GFP (naive 7F- and CXCR4+CDH1+ definitive endoderm cells) were seeded in each well of an Elplasia plate under N2B27 plus 10 μM Y27632 pSpCas9/gRNA plasmid (5 μg) was electroporated into primed H9 human ES cells (Neon Program 14) mCherry+ cells were sorted by flow cytometry and seeded at a low density About 10 colonies were picked 7–8 days after seeding DNA was amplified and sequenced using the following primers: gRNA 1 Rv 5′-TCACCTGCAAGTGGCTGACGATACAG-3′; and gRNA 2 The generated LAMB1-KO primed hPSCs were reset to naive hPSCs CER1-H2B-GFP reporter cells were generated from primed H9 human ES cells by replacing the endogenous stop codon of the CER1 gene with a T2A-H2B-GFP-LoxP-SV40-NeoR-LoxP cassette using CRISPR–Cas9 homology-directed repair (Extended Data Fig. 8i) gRNA targeting the stop codon of human CER1 was designed and inserted into pX330-U6-Chimaeric_BB-CBh-hSpCas9: gRNA approximately 1,000 bp upstream and downstream of the CRISP–Cas9 cleavage site was prepared by long PCR fused with a T2A-H2B-GFP-LoxP-SV40-NeoR-LoxP cassette and cloned into a TOPO vector pSpCas9/gRNA and the donor vector (1 μg each) were electroporated into primed H9 human ES cells (Neon Program 14) G418 was added (200 µg ml−1) for about 2 weeks The cells were collected and seeded on MEFs at a low density Colonies were picked 7–8 days after seeding The SV40-NeoR gene was deleted from the CER1-H2B-GFP line by the transient introduction of a cre-expressing vector The generated CER1-H2B-GFP-primed hPSCs were reset to naive hPSCs LEFTY or DKK1 nuclei on sections of bilaminoids were analysed The centre of the T+ nuclei was defined as 0° To KO the IL6 gene, two sgRNAs that targeting exon 2 (sgRNA 1) and exon 3 (sgRNA 2) of human IL6 were designed and inserted into pSpCas9(BB)-2A-mCherry (Extended Data Fig. 7j): sgRNA 1 pSpCas9/sgRNA plasmid (5 μg) was electroporated into primed H9 human ES cells (Neon Program 14) Colonies were picked 7–8 days after seeding DNA was amplified and sequenced using the following primers: sgRNA 1 Rv 5′-GGCAGAACCAGAATTCGAGTGTGGGCTC-3′; and sgRNA 2 The generated IL6-KO primed hPSCs were reset to naive hPSCs Naive hPSCs were plated (1.5 × 105 cells per cm2) on iMatrix-coated Transwell plates and differentiated into nTB as described above (day 0) the nTB induction medium was replaced with NDiff 227 hPSCs were plated (1.5 × 105 cells per cm2) on iMatrix-coated Transwell plates under each medium (naive hPSCs the hPSC medium was replaced with NDiff 227 The cell culture supernatants were collected on day 5 and centrifuged to remove debris The levels of IL-6 were quantified using an IL-6 ELISA kit (Abcam ab178013) according to the manufacturer’s protocol The absorbance at 450 nm was measured using a plate reader (TECAN OTX2 fused to ERT2 was inserted into the PB vector (PB-OTX2-ERT2) The PB-OTX2-ERT2 vector and pBase helper plasmid were transfected into naive hPSCs expressing GATA6 under DOX treatment (Naive(G6-OE)) OTX2-ERT2 was activated by treatment with 100 nM 4-hydroxytamoxifen (tamoxifen) from day 4 to day 6 DKK1 fused to destabilizing domain (DD) was cloned into the PB vector (PB-DD-DKK1) The PB-DD-DKK1 vector and pBase helper plasmid were transfected into naive hPSCs expressing GATA6 under DOX treatment (Naive(G6-OE)) DD-DKK1 was activated by treatment with 500 nM Shield1 (Takara GP130/GCSFR chimeric receptor (Y118F) cDNA was inserted into the PB vector (PB-Y118F) The PB-Y118F vector and pBase helper plasmid were transfected into naive hPSCs (Naive(WT) or Naive(G6-OE)) STAT3 signalling was activated the treatment with G-CSF from day 0 to day 4 The cells were plated (5 × 104 per cm2) onto MEF feeder cells and cultured under RACL medium composed of RPMI 1640 medium with GlutaMAX (61870036 Anti-feeder antibody was used to remove the MEF feeder cells Primed hPSCs were differentiated into definitive endoderm as described previously63 Primed hPSCs were seeded on an uncoated bacterial dish to form EBs under StemFit AK02N (AK02N the EBs were washed and cultured under N2B27 with 200 ng ml−1 activin A and 3 µM CHIR the medium was replaced with N2B27 and 200 ng ml−1 activin A and cultured for 2 more days and CXCR4+CDH1+ definitive endoderm cells were sorted and used for the experiments The membrane inserts were coated with 1% Geltrex diluted in DMEM/F12 for 1 h primed hPSCs were seeded on membrane inserts at a density of 3 × 104 cells per cm2 under mTeSR plus 10 μM Y27632 the medium was switched to E6 supplemented with bFGF (20 ng ml−1) and BMP4 (50 ng ml−1) and cultured for 48 h day 3 PDGFRA+ cells were sorted and recultured on membrane inserts at a density of 9 × 104 cells per cm2 overnight Primed hPSCs were collected as small clumps and seeded onto the membrane inserts under E6 medium supplemented with bFGF (20 ng ml−1) The cells were cultured for another 48 or 96 h before analysis Total RNA was extracted using the RNeasy Kit (74106 Total RNA (0.5 µg) was reverse-transcribed into cDNA with an oligo-dT primer using SuperScriptIV (18090050 qPCR was performed using QuantStudio3 (Thermo Fisher Scientific) and QuantStudio12K (Thermo Fisher Scientific) with TaqMan Fast Universal Master Mix (4364103 Thermo Fisher Scientific) and TaqMan probe or PowerUP SYBR Green Master Mix (A25743 Thermo Fisher Scientific) according to the manufacturer’s instructions The results were analysed using QuantStudio Design & Analysis v.1.4.1 (Thermo Fisher Scientific) PAR6 and F-actin images were used to quantify cavity volume with the Surfaces program Bilaminoids (D6) were manually picked. Before sampling bilaminoids, aggregates surrounded by nHyCs were identified by stereomicroscope and amniotic cavity formation was additionally confirmed by microscopy using the Celldicoverer 7 (Zeiss) system (Extended Data Fig. 8a) The choice of the right bilaminoids that contain an amniotic cavity is critical Each bilaminoid was transferred in a drop of Accutase and incubated at 37 °C for 15–20 min then dissociated into single cells by repeated pipetting using glass capillaries Each single cell was transferred into individual PCR tubes and immediately frozen in Smart-seq HT lysis buffer bilaminoids on day 9 were dissociated by Accutase TFAP2C-GFP+BLIMP1-tdTomato+ cells (PGCLCs) or CD34+ (HEPLCs) were sorted as single cells and immediately frozen in Smart-seq HT lysis buffer The libraries for scRNA-seq were prepared using the SMART-seq HT kit (Z4436N Takara) according to the manufacturer’s instructions The libraries were then sequenced on the NovaSeq 6000 or NextSeq 500 (Illumina) system with paired-end sequencing The downloaded datasets and our Smart-seq HT data were preprocessed and quantified for gene expression as described in the above scripts based on each single-cell method 10x Genomics) for all human 10x Genomics single-cell datasets and STAR aligner (v.2.5.1b) and RSEM (v.1.3.1) tool for Smart-Seq datasets downloaded raw sequencing data were mapped to the same human reference genome (refdata-cellranger-GRCh38-3.0.0 downloaded from the 10x Genomics website) and quantified for gene expression in the same computational environment According to the above CheckBlastoids scripts with the gene expression matrices clustering and visualization using the R Seurat package (v.4.0.4) The count matrix of SMARTer v.2 scRNA-seq data and our bulk RNA-seq were imported into R (v.3.5.1) using DeSeq2 v.1.22.2 and the expression levels were calculated as transcripts per million (TPM) Low-expression genes (TPM < 5 in all samples) were excluded and log-scaled TPM values were used to perform the PCA analysis using R (v.3.5.1) following approval by the ethics committee at the University of Tokyo and by MEXT (Ministry of Education was used for interspecies chimera experiments Ten cells of naive hPSCs or nHyC induced by 7F or GATA6 and sorted by PDGFRA on day 3 were microinjected into mouse morula embryos 7F-nHyCs and naive hPSCs were confirmed to have contributed to late morulae-early blastocysts of mouse embryos BDF1xB6 mouse embryos were collected in M2 medium (M7167 Sigma-Aldrich) at the eight-cell or morula stage A piezo-driven micro-manipulator (Primetech) was used to drill into the zona pellucida under a microscope and 10 naive hPSCs or nHyCs were introduced into the subzonal space of each embryo the embryos underwent follow-up culture in N2B27 medium until the blastomere stage They were then transferred into the uteri of pseudopregnant recipient ICR mice for in vivo chimera assays chimeric embryos were cultured under N2B27 medium for 2 days Statistical tests were performed using GraphPad Prism v.9.4.1 and v.10.0.3 from a minimum of three independent experiments the number of biologically independent experiments is indicated in the caption n values in the figure panels represent the number of aggregates analysed All of the experiments were performed independently at least twice Data were tested for normality using the Shapiro–Wilk test Normally distributed data were analysed using parametric tests (unpaired t-test or analysis of variance) and non-normally distributed data were analysed using nonparametric tests (Mann–Whitney U-tests or Kruskal–Wallis test) as indicated in figure legends Regarding the efficiency of generating aggregates significant differences among conditions were evaluated using Fisher’s exact test based on the total number of aggregates analysed representative images of three biologically independent experiments are shown (n = 3) Approximately 42.8% of D9 bilaminoids had CD34 and ERG double-positive cells representative images of n = 4 (7F) and n = 2 (G6) independent experiments are shown Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article No custom code or algorithms were developed for the data analysis in this study A Correction to this paper has been published: https://doi.org/10.1038/s41586-024-07166-w Single-cell RNA-seq profiling of human preimplantation embryos and embryonic stem cells Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos Defining the three cell lineages of the human blastocyst by single-cell RNA-seq Reconstituting the transcriptome and DNA methylome landscapes of human implantation A developmental landscape of 3D-cultured human pre-gastrulation embryos Self-organization of the in vitro attached human embryo Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast Tyser, R. C. V. et al. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature https://doi.org/10.1038/s41586-021-04158-y (2021) A method to recapitulate early embryonic spatial patterning in human embryonic stem cells Pluripotent state transitions coordinate morphogenesis in mouse and human embryos Self-organization of a human organizer by combined Wnt and Nodal signalling Controlled modelling of human epiblast and amnion development using stem cells An in vitro model of early anteroposterior organization during human development Liu, X. et al. Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature https://doi.org/10.1038/s41586-021-03372-y (2021) Blastocyst-like structures generated from human pluripotent stem cells Yanagida, A. et al. Naive stem cell blastocyst model captures human embryo lineage segregation. Cell Stem Cell https://doi.org/10.1016/j.stem.2021.04.031 (2021) Kagawa, H. et al. Human blastoids model blastocyst development and implantation. Nature https://doi.org/10.1038/s41586-021-04267-8 (2021) Resetting transcription factor control circuitry toward ground-state pluripotency in human Systematic identification of culture conditions for induction and maintenance of naive human pluripotency Epigenetic resetting of human pluripotency Capturing human trophoblast development with naive pluripotent stem cells in vitro Human naive epiblast cells possess unrestricted lineage potential Linneberg-Agerholm, M. et al. Naive human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable naive extra-embryonic endoderm. Development https://doi.org/10.1242/dev.180620 (2019) GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst Differentiation of embryonic stem cells is induced by GATA factors Sox17-mediated XEN cell conversion identifies dynamic networks controlling cell-fate decisions in embryo-derived stem cells c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development A developmental coordinate of pluripotency among mice Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states A role for PDGF signaling in expansion of the extra-embryonic endoderm lineage of the mouse blastocyst Vrij, E. J. et al. A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression. Development https://doi.org/10.1242/dev.192310 (2022) The primitive endoderm lineage of the mouse blastocyst: sequential transcription factor activation and regulation of differentiation by Sox17 Anderson, K. G. V. et al. Insulin fine-tunes self-renewal pathways governing naive pluripotency and extra-embryonic endoderm. Nat. Cell Biol. https://doi.org/10.1038/ncb3617 (2017) Single-cell RNA-seq reveals novel regulators of human embryonic stem cell differentiation to definitive endoderm Blastocyst-like structures generated solely from stem cells Self-organization of the human embryo in the absence of maternal tissues Human hypoblast formation is not dependent on FGF signalling Lifeact: a versatile marker to visualize F-actin Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells Meistermann, D. et al. Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification. Cell Stem Cell https://doi.org/10.1016/j.stem.2021.04.027 (2021) Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3 Zhao, C. et al. Reprogrammed iBlastoids contain amnion-like cells but not trophectoderm. Preprint at bioRxiv https://doi.org/10.1101/2021.05.07.442980 (2021) Nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway Form and function: the laminin family of heterotrimers Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation Amniogenesis occurs in two independent waves in primates Rouillard, A. D. et al. The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database https://doi.org/10.1093/database/baw100 (2016) Weatherbee, B. A. T. et al. Pluripotent stem cell-derived model of the post-implantation human embryo. Nature https://doi.org/10.1038/s41586-023-06368-y (2023) Pedroza, M. et al. Self-patterning of human stem cells into post-implantation lineages. Nature https://doi.org/10.1038/s41586-023-06354-4 (2023) Liu, L. et al. Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids. 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Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. https://doi.org/10.14806/ej.17.1.200 (2011) TopHat2: accurate alignment of transcriptomes in the presence of insertions GENCODE reference annotation for the human and mouse genomes Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations TFAP2C regulates transcription in human naive pluripotency by opening enhancers MotifMap: integrative genome-wide maps of regulatory motif sites for model species Download references Kalkan for providing the Rex1-GFP(D2) mouse ES cells; A Kono for their technical support regarding the marmoset embryo work and the cell culture experiments; S Sakurai and the members of SignAC (single-cell genome information analysis core) at WPI-ASHBi Mizuno for the Smart-seq library construction; S Goulas for his advice and suggestions on our manuscript; all of the members of the Takashima laboratory for their comments and support; P Hui for reading and English editing of the manuscript; K Mitsunaga for the flow cytometry analysis; and S This work was supported by MEXT KAKENHI (grant numbers: JP16H02465 JP23K18340) and AMED (grant numbers: JP21bm0704035 JP23bm1323001) to Y.T.; MEXT KAKENHI (grant number: JP19H03418) and the JST FOREST Program (grant number: JPMJFR206C) JST CREST (grant number: JPMJCR2023) and AMED (grant numbers: JP17gm1110004 JP21gm1310011) to T.Y.; MEXT KAKENHI (grant numbers: JP15H02360 JP19H05759) to E.S.; AMED (grant number: JP22bm1004002) to H.N.; the Takeda Science Foundation to Y.T.; the Naito Foundation to Y.T.; the Mochida Memorial Foundation for Medical and Pharmaceutical Research to Y.T.; and the Astellas Foundation for Research on Metabolic Disorders to Y.T Center for iPS Cell Research and Application Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) Central Institute for Experimental Animals Institute for Stem Cell Biology and Regenerative Medicine Institute for the Advanced Study of Human Biology (WPI-ASHBi) Medical-risk Avoidance Based on iPS Cells Team RIKEN Center for Advanced Intelligence Project (AIP) performed and interpreted the cell culture experiments with contributions from N.R. performed the interspecies chimera experiments The following authors are listed as co-inventors on a patent for the generation of hypoblast-like cells from naive hPSCs (JP7176764B2 and Y.T.) and the generation of bilaminoids (WO2022/114188A1; T.O The other authors declare no competing interests 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 Primed hPSCs were seeded on Laminin511-E8 under N2B27 or serum-containing medium (N=) shows biologically independent experiments Source Data Expression patterns are shown as box-and-whisker plots the 25th and 75th percentiles range; whiskers Source Data (Steps 2 and 3) Twenty-five transcriptional factors were identified as upregulated genes in nHyC (H9 AdiPS) compared with naïve hPSCs and primed hPSCs (Log2Fold change > 2) (Step 4) To identify stably expressed transcriptional factors and 16 genes were extracted (-2<Log2Fold change < 2) (Step 5) nHyC-marker transcription factors were identified by comparing nHyC and primed PDGFRA+ cells were expressed more than 4-fold higher in nHyC; HNF1B Source Data Naïve hPSCs were cultured in 7F or 7F minus one factor PDGFRA expression was measured on D3 by flow cytometry Almost no PDGFRA expression was detected in cells cultured in 7F minus FGF4 or BMP4 PDGFRA expression in 7F minus one factor plus one opposing factor 7F minus A83 or XAV but with Activin A or CHIR (CH) reduced the PDGFRA expression to around 0% while 7F minus IL6 plus JAKi killed half of the cells during differentiation Flow cytometry of PDGFRA and CEACAM1 or ANPEP in cells cultured in 6F (-RA) medium N2B27 medium without vitamin A (retinyl acetate) was used as the basal medium (N2B27*) Minimum essential growth factors with A83 and XAV for hypoblast induction Naïve hPSCs were cultured in growth factor(s) with A83 and XAV and PDGFRA when grown in FGF4 and BMP4 (4F) Colour blocks indicate the percentage of the protein expression qPCR for hypoblast markers in PDGFRA+ cells on D3 after 7F Flow cytometry for PDGFRA and CEACAM1 or ANPEP in cells treated with A83 and XAV N2B27*: N2B27 medium without vitamin A (retinyl acetate) (a) 1.5 × IQR; (f) the minimum and maximum values Source Data Source Data Aggregates of Naïve-Lifeact (nEpiC) and Naïve-DsRed (G6-OE) (nHyC(G6-OE)) were cultured for 4 days The time-lapse images show an accumulation of Lifeact in the centre starting at around 64 h Aggregates were composed of naïve or primed hPSCs and naïve PDGFRA+ cells induced by GATA6 Aggregates were fixed and stained with F-actin (Purple) on D4 Although all sorted cell types surrounded Epi cells aggregates with a pro-amniotic cavity were formed only by 7/4F- and G6-nHyC Aggregates made by the mixture of Naïve (WT) and Naïve-GFP (G6-OE) formed pro-amniotic cavities most efficiently Naïve and primed PDGFRA+ cells were sorted by PDGFRA on D3 Representative images of aggregates are shown on the right definitive endoderm cells sorted by ECAD+CXCR4+ on D3 Efficiency of bilaminoid formation on D4 extracted from (n) The efficiency of aggregates containing more than 10 nEpiC cells and 10 nHyC with spatial separation (inner-outer) is shown The efficiency of bilaminoids where nEpiC were surrounded by nHyC(G6-OE) and had an accumulation of F-actin on D4 is shown Immunofluorescent images of aggregates on D6 Epiblasts in incomplete aggregates without an amniotic cavity partially expressed GATA2 while there were no GATA2+ cells in bilaminoids Induction efficiency to HAVCR1+ENPEP+ trophoblast Cell aggregates of H9 Naïve (WT) or Naïve (WT) + Naïve (G6-OE) were cultured under trophoblast induction medium for 3 days Immunofluorescent images of aggregates on D4 Bilaminoids were induced from 558B1 or 1390G3 naïve hPSCs Cell aggregates of 585B1 or 1390G3 Naïve (WT) + Naïve (G6-OE) were cultured under trophoblast induction medium for 3 days (n=) at the top shows the number of aggregates analysed for each group Source Data and primed hPSCs + nHyC (G6 or 7F) were cultured on Transwell plates for 4 days Immunofluorescent images for amnion markers of bilaminoids on D6 Aggregates were generated by Naïve (WT) + Naïve (G6-OE) together with nTB Source Data Percentage of each cell type in bilaminoids on D6 and nine bilaminoids were independently collected and analysed by Smart-seq Unsupervised hierarchical clustering (UHC) of nHyCs of D6 bilaminoids and relative gene expression of anterior visceral endoderm (AVE) marker genes One cluster of nHyC strongly expresses AVE marker genes PCA analysis of nHyCs of D6 bilaminoids (Left) and loadings (right) Design of the CRISPR targets for CER1-H2B-GFP knock-in (KI) The neo cassette was removed by Cre expression Representative Z-series images of an aggregate on D6 A series of confocal Z-sections of the aggregates of Naïve (WT) + Naïve (G6-OE) on D6 stained for OTX2 (Purple) Bilaminoids were generated by Naïve (WT) + Naïve (G6-OE) Yellow arrowheads indicate LEFTY+ or DKK1+ cells Angles between T+ nuclei and LEFTY+ or DKK1+ cells on sections of bilaminoids were measured The centre of T+ nuclei was defined as 0 degrees LEFTY: 21 aggregates surrounded by nHyC (G6-OE) and expressing LEFTY and T were counted DKK1: 14 aggregates surrounded by nHyC (G6-OE) and expressing DKK1 and T were counted Overexpression of DKK1 in Naïve (G6-OE) suppressed T expression in the nEpiC of D6 bilaminoids DKK1 with Destabilized domain (DD-DKK1) was introduced into Naïve (G6-OE) and was stabilized by Shield1 Bilaminoids were generated by Naïve (WT) + Naïve-GFP (G6-OE) that contain DD-DKK1 The proportion of T-expressing bilaminoids was counted under Shield1 (−) or (+) conditions Bilaminoids were generated by Naïve (WT) + Naïve-GFP (G6-OE) that contain tamoxifen-inducible OTX2 (OTX2-ERT2) RNA was collected under tamoxifen (−) or (+) conditions RNA was collected under Shield1 (−) or (+) Source Data PDGFRA expression was measured by flow cytometry on D3 Hypoblast marker genes in PDGFRA+ cells on D3 were measured by qPCR Only cells from bilaminoids were extracted and analysed Expression levels are shown as violin plots Gene expression patterns in nEpiC of bilaminoids cultured under N2B27 medium with BMP or WNT signalling ligands or inhibitors on D6 Bilaminoids were generated by the mixture of Naïve (WT) and Naïve-GFP (G6-OE) Source Data A series of confocal Z-sections of a bilaminoid of Naïve (WT) and Naïve (G6-OE) on D9 stained for ISL1 (Blue) Anterior-posterior axis of bilaminoids on D9 PGC marker gene expression in bilaminoids on D9 Bilaminoids were generated by BLIMP1-tdTomato and TFAP2C-GFP double knock-in Naïve (BTAG) and Naïve (G6-OE) BTAG- and SOX17-triple-positive cells are indicated by yellow arrowheads or CD34+ cells in D9 bilaminoids were sorted as single cells by flow cytometry and used for Smart-seq libraries The efficiency and workflow to obtain bilaminoids IL6 improved cavity formation similar to the co-cultures of nTB Embryonic day (E) 4.5 hatched mouse-human chimaera blastocysts Naïve hPSCs or 7F- or G6-nHyC were injected into mouse morula embryos the embryos were cultured in vitro for two more days Human cells are marked by tdTomato (Purple) The efficiency of mouse-human chimaera blastocysts at E4.5 Interspecies chimaera embryos developed in utero at E6.5 7F-nHyC (tdTomato)-injected embryos were transplanted into pseudopregnant mice on E3.5 and collected at E6.5 Control indicates non-chimaeric littermate Confocal images of mouse-human chimaera embryo developed in utero at E6.5 7F-nHyC-injected embryos were collected at E6.5 and stained for SOX17 (Green) and DAPI (White) nHyC-derived cells marked by tdTomato were detected in the visceral endoderm lesion (yellow arrowhead) and extraembryonic lesion (red arrows) Confocal images of mouse-human chimaera embryo developed in utero (E6.5) G6-nHyC-injected embryos were collected at E6.5 and stained by SOX17 (Green) and DAPI (White) nHyC-derived cells marked by tdTomato were detected in the visceral endoderm lesion (yellow arrowhead) A summary of mouse-human chimaera embryos at E6.5 in utero 7F- and G6-nHyC-derived cells contributed to visceral endoderm in post-implantation embryos 1–3 and legends for Supplementary Tables 1–8 Download citation DOI: https://doi.org/10.1038/s41586-023-06871-2 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science A Discussion with Ellie Omiya: What Lessons Can We Learn from the Storybook “Swimmy” How Tuna Farming is Changing the Future Landscape of Marine Resources 10 Questions Answered about DX in the Medical and Healthcare Fields In Search of Shape and Color: A New Kind of Journey through the Sky A Discussion with Eisuke Tachikawa of Nosigner: How Eco-Friendly Materials Can Expand Design Camper Vans: The Road to Creative Work and Recreation HomeSpecialRunning Out of Tuna Delicious Tuna: A Positive Food Cycle through Sustainable Farming Sojitz Tuna Farm Takashima is located a 1.5-hour drive from Fukuoka Airport on Takashima Island in Matsuura Takashima Island has a population of approximately 2,000 people and the island is well known to historians as the site marking the end of the Mongol invasion Matsuura is one of the largest fishing sites for mackerel and horse mackerel in Japan as well as for Japanese pufferfish farming Sojitz Tuna Farm Takashima is located in a fishing ground with abundant fishery resources A worldwide sushi craze began in the early 2000s The popularity of sushi shows the appeal of Japanese food but widespread overfishing has led to bluefin tuna becoming an endangered species Sojitz wondered if it could "create” marine resources by farming tuna in Japan Sojitz Tuna Farm Takashima was established in 2008 with the aim of delivering a stable supply of quality tuna to tables around the world Sojitz Tuna Farm Takashima started with a limited number of aquaculture cages and tuna cultivation was a challenge requiring much trial and error Sojitz Tuna Farm Takashima has 33 fish cages to cultivate 40,000 bluefin tuna and the farm has grown to such a large scale that it ships 10,000 fish a year Takashima Bluefin Tuna are farmed from juveniles for three and a half years until the fish weighs an average of 60kg Fully mature tuna are then sold to fresh fish specialty stores and restaurants as well as revolving sushi chains and supermarkets It’s possible that you have already eaten tuna farmed by Sojitz Tuna Farm Takashima without even knowing it Sojitz Tuna Farm Takashima carefully monitors the environment of the tuna so that the fish has a deep umami flavor with the optimal balance of red meat and fat Takashima Bluefin Tuna is in high demand in Japan as well as widely exported overseas significant differences in quality emerge based on freshness management processes from the time a fish is caught until it arrives on your plate Sojitz Tuna Farm Takashima’s current freshness management methods are a product of its continued search for excellence developed overs years of experimentation and refinement The answer to freshness management lies on deck Board the boat from Takashima’s fishing port and in 15 minutes you arrive at Sojitz Tuna Farm Takashima’s aquaculture cages and each fish cage is 40m in diameter and 20m deep Fishing for tuna is often associated with the thrill of pole fishing or fishing with a net such as purse seine fishing nationally certified divers go underwater to catch tuna that meet the desired size specifications from customers These licensed divers use an electric harpoon to quickly capture and bring the fish on board the crew waits on standby to bleed and gut the tuna before placing the fish in an ice bath The freshness and quality of the fish deteriorates if the blood and intestines are not removed right away There are close to 90 seconds from the moment the tuna is lifted on deck to the time it is placed in ice water The secret to maintaining freshness lies in the speed with which the crew works The single most important factor for preserving the quality of tuna is to prevent lactic acid buildup in the fish’s muscles Pictured: A new female diver who joined Sojitz Tuna Farm Takashima in December 2021 Sojitz Tuna Farm Takashima is putting effort into training young new divers tuna can have lactic acid buildup in their muscles from physical activity Floundering should be prevented during the capture process to avoid lactic acid buildup and the crew bleeds and guts the fish swiftly before the fish has time to flounder Tuna also have body temperatures higher than that of ambient water the fish reportedly feels warm during the gutting process and removal of gills the tuna’s body temperature rises and this increase in lactic acid causes the fish flesh to deteriorate—a form of deterioration known as “burnt tuna”—which can cause the fish to lose commercial value The electric harpoon is a fishing method that does not take the same time and effort as pole fishing where the crew must wait until the fish takes the bait Fishing with electric harpoons calls for a team of skilled and practiced divers who can quickly and carefully catch the tuna with an electric harpoon Since it is difficult to secure experienced divers many companies cannot employ this fishing method despite understanding its benefits Electric harpoon fishing allows the tuna to be preserved in optimal condition Sojitz Tuna Farm Takashima’s aquaculture techniques incorporate a good balance of technology staff track water temperatures by integrating smartphone technology to continually monitor fish cage conditions Sojitz Tuna Farm Takashima is also working on constructing a tech system to assess the condition of the tuna swimming around in the cages the next stop is the tuna processing plant where tuna is processed with meticulous management the tuna catch is brought to the processing plant The tuna then goes through a series of processing steps prior to being shipped to customers The small bits of intestine that were not removed on the ship are carefully scraped clean by professional processing plant workers the fish is cleaned and trimmed so only the necessary parts remain Few tuna farms go to the same lengths to trim the fish so thoroughly as trimming the tuna reduces the overall weight of the fish profit therefore depends on maintaining the weight of the fish All tuna parts that appear likely to lose freshness are removed and this process helps to maintain the freshness of the fish for a longer period Sojitz Tuna Farm Takashima is dedicated to continually maintaining and improving quality Did you know that only 50% of a tuna’s full body weight is edible as raw fish then a whole tuna will yield 1250 cuts of sashimi the tuna is placed in ice water once again The next steps resemble a tuna cutting demonstration Experts use a large knife to slice the tuna into three sections While some customers prefer a half cut of tuna The tuna is cut into different sizes based on the requested specifications from customers Roughly 90% of customers ask for the full fish which can be used in tuna cutting demonstrations the whole fish are packaged in Styrofoam boxes or put in tanks with ice water for shipping there is no fishy smell that you might expect at a plant handling seafood Fishy odors are caused by bacteria that builds up on fish flesh or fish scraps during processing bacteria can begin to grow on the surface of the fish’s skin At Sojitz Tuna Farm Takashima’s processing plant and floors are sanitized using slightly acidic electrolyzed water that is manufactured within the processing plant This special water is the secret behind preventing bacterial growth from starting or spreading this sanitizing water also prevents odors and is safe for humans even if consumed Sojitz Tuna Farm Takashima often ships fish overseas While you might assume the fish needs to be frozen in these cases Thanks to careful processing and treatment with slightly acidic electrolyzed water the fresh tuna can be shipped and there is no need for freezing as the fish can be preserved in this state with a two-week expiration date While the processing plant uses cranes and other equipment the trimming and carving process cannot be automated due to individual differences in tuna size Sojitz Tuna Farm Takashima is working to maximize efficiency while realizing the highest quality through skilled manual processing In order to meet rising tuna demand and maintain and improve quality Sojitz Tuna Farm Takashima processing plant employs local staff and creates new employment opportunities in the region The world currently faces a myriad of issues including ocean pollution conditions surrounding our food supply will likely change In the event that marine resources run low nobody 20 years ago could have imagined the high price of Pacific saury today Depending on the power of nature alone will not ensure a stable supply of food or recovery of marine resources By leveraging the best technologies for farming a stable supply of food can be provided without creating a burden on the environment while also reducing overfishing to bring tuna back to the oceans Sojitz Tuna Farm Takashima believes that tuna farming is one way to steadily contribute towards a sustainable society Japan previously sourced tuna from other countries around the world Now delicious farmed tuna will become more readily available from Japan to the rest of the world After working through an initial period of trial and error Sojitz Tuna Farm Takashima realized its idea to cultivate tuna independently in Japan Sustainably farmed tuna from Nagasaki will offer the taste of delicious tuna to the next generation Established in 2008 as a specialized producer of bluefin tuna Sojitz Tuna Farm Takashima is located off on the coast of Nagasaki on Matsuura’s Takashima Island Sojitz Tuna Farm Takashima currently cultivates close to 40,000 bluefin tuna on its fishing grounds The bluefin tuna are raised for three and a half years until the fish reach full maturity with an average weight of 60kg Sojitz Tuna Farm Takashima’s top-quality tuna is then sold to fresh fish specialty stores and restaurants ${val.cf_art_title.replaceAll(/(\r\n|\r|\n)/g,"")} ${val.cf_art_title.replaceAll(/(\r\n|\r|\n)/g,"")} KCAW’s Katherine Rose continues her assignment in Japan this week where she’s looking into the industry and culture of herring Rose visits Takashima Fishing Village in the city of Otaru and learns about one family’s gillnetting operation Rose will be sharing more stories from her travels over the next week Her trip is a project of KCAW and the Alaska Center for Excellence in Journalism KCAW Prize Drawings: click on the links for rules and winner info Yesterday, Hawaii’s most beloved surf artist, Clark Takashima, passed away due to a heart attack. I found out late this morning after a morning scope of my Instagram feed led me to Sunny Garcia’s page who dedicated a post to Clark and he will be missed by all who knew him and/or his artwork Clark was known for both his impressive artwork based mainly around waves and his surroundings on Hawaii he couldn’t have been a more enthusiastic He radiated positive vibes and seemed to embody the aloha spirit originally from Pearl City but lived on the North Shore he occasionally gave me a little update on how the waves were by him and always asked This generosity was also exemplified in his project called “The Plant to Pupukea,” which was established with the support of the 9th Wave Gallery Clark decided to dedicate himself to help other artists to aspire to their potential Clark and fellow artist Patrick Parker joined forces to one day maintain a large working studio to be a creative hotspot for artists “We open our doors to visiting and local professional artists to paint discuss the importance of sharing information on marketing painting techniques and creating the best works possible We encourage our peers to look deeper into how the Old Masters set up their compositions using information incorporating numbers those visiting will do the same in their communities welcomes amateur artists to paint and enjoy the creative process I think it’s essential for artists (or any creative person) to surround themselves in a creative environment The last two years have been amazing and incredibly inspiring with many notable artists clients and kids experiencing this energetically charged space.” Clark is now reunited with his wife and friends Andy Irons He was open about the heartache it left him with and he put that energy into his work Instead of mourning the passing of a friend let’s celebrate the life he lived while he was here his generosity and know he’s in a better place even if we may not be Clark was also a gracious contributor to The Inertia 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 Japanese version Copyright THE MAINICHI NEWSPAPERS. All rights reserved. Copyright © Japan International Cooperation Agency Volume 3 - 2022 | https://doi.org/10.3389/fitd.2022.988284 This article is part of the Research TopicRising Stars in Vaccines for Tropical Diseases ResearchView all 4 articles Pregnant women are particularly susceptible to Plasmodium falciparum malaria leading to substantial maternal and infant morbidity and mortality While highly effective malaria vaccines are considered an essential component towards malaria elimination strides towards development of vaccines for pregnant women have been minimal has modest efficacy in children suggesting that it needs to be strengthened and optimized if it is to be beneficial for pregnant women Clinical trials against pregnancy-associated malaria (PAM) focused on the classical VAR2CSA antigen are ongoing additional antigens have not been identified to supplement these initiatives despite the new evidence that VAR2CSA is not the only molecule involved in pregnancy-associated naturally acquired immunity This is mainly due to a lack of understanding of the immune complexities in pregnancy coupled with difficulties associated with expression of malaria recombinant proteins and the anticipated complications in conducting and implementing a vaccine to protect pregnant women With the accelerated evolution of molecular technologies catapulted by the global pandemic identification of novel alternative vaccine antigens is timely and feasible we discuss approaches towards novel antigen discovery to support PAM vaccine studies falciparum causes the majority of malaria infections and fatalities with most occurring in sub-Saharan Africa The major disease burden is in children less than 5 years of age Taken together, VAR2CSA appears to be an important target of protective immunity to PAM (25) and protective mechanisms could also exist have focused on understanding and/or evaluating the role played by VAR2CSA; this is the only antigen under consideration as a PAM vaccine currently Surface expression of VAR2CSA is restricted to CSA-adhering iRBCs, prompting development of vaccines targeting this protein (26). The leading VAR2CSA-based vaccines, PAMVAC and PRIMVAC (27) are designed to generate functional antibodies capable of blocking the binding of infected cells to CSA on placental syncytiotrophoblast as well as opsonizing iRBCs expressing VAR2CSA Such a vaccine would protect pregnant women These studies have clearly demonstrated genetic variation in var2csa cementing the need to develop broad multivalent vaccine candidates that could be efficacious across different variants While most vaccines against PAM focus on VAR2CSA, vaccines that prevent liver stage infection would also have a profound impact on pregnant women. One vaccine, the radiation attenuated P. falciparum NF54 sporozoites (PfSPZ Vaccine), which demonstrated significant protection in African adults (35) and potential protective efficacy in women of childbearing potential (clinicaltrials.gov; NCT03989102) Although these proteins are functionally unrelated they have a common homologous ancestry and share conserved epitopes that can be exploited in vaccine design Antibodies can activate the complement system to mediate effector functions against myriad developmental stages of the parasite life cycle (47) that are linked to protection against clinical malaria (48). New evidence suggests that these mechanisms may also contribute to vaccine-induced immunity. For example, the RTS,S vaccine can induce complement-fixing antibodies against the central repeat and C-terminal regions of CSP (49) the proportion of VAR2CSA-specific IgG in plasma that is involved in complement fixation relative to other effector functions remains to be determined these studies have demonstrated that elevated complement-fixing antibodies are associated with a reduced risk of placental parasitemia probably by enhanced inhibition of iRBC adhesion to CSA thus the role of complement in protection against PAM deserve further investigations Discovery and characterization of additional antigens that interact with VAR2CSA is an area that deserves prioritization for vaccine development Table 1 List of proteins over-expressed in placental malaria associated parasites Figure 1 An illustration of an immunoscreening pipeline to identify targets of potentially protective antibodies from pregnant women with and without PAM Unique proteins are represented by colored dots The antigens may also be identified using complex genomic assays statistical approaches such as artificial intelligence (AI) machine learning and expressed using robust platforms such as the wheat germ cell free system for functional studies Such statistical approaches can be exploited in PAM to enhance the search for novel antigens Concurrently, numerous mRNA vaccine platforms have been developed and evaluated for immunogenicity (84–86). The ability to genetically modify the RNA sequences has hastened the production of translatable non-toxic synthetic mRNA and delivery systems (87). This approach was applied recently to the production of coronavirus vaccines by Moderna and Pfizer-BioNTech (88) These examples raise optimism for adoption of this technology in the production of vaccines for PAM Understanding the complexity of naturally acquired immunity against PAM will inform antigen discovery and vaccine development It is now clear that VAR2CSA is not the only antigen that is involved in PAM but there are other antigens that may be targets of protective immunity it is plausible to further query the plasmodium genome using emerging bioinformatics tools such as machine learning and artificial intelligence to identify possible conserved antigens that can be interrogated for development of broadly neutralizing vaccines Application of eukaryotic WGCFS for generation of the recombinant proteins that induce immunogenic and protective antibodies need to be granted critical consideration the advent of mRNA-based vaccine development platforms comes with high flexibility and 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Bernard N. Kanoi, Ymthbm9pQG1rdS5hYy5rZQ==