CELEBRATING THE BEST OF TV, MOVIES, AND COMICS The legendary Akira and Studio Ghibli animator is bringing his chaotic surreal animation style to Star Wars and we love it The Star Wars: Visions panel at Star Wars Celebration 2025 has wrapped up and it has this anime fan tingling with excitement. We got to see the first footage of several episodes in Volume 3 but none took our breath away quite like 'Black,' the upcoming episode from legendary anime director and animator Shinya Ohira which is being produced by David Production Shinya Ohira has been involved in some of the most iconic anime of all time From Akira to Studio Ghibli films to One Piece episodes so the news that he would be writing and directing an episode of Star Wars: Visions Volume 3 was exciting news to all of us we got to see the first footage of Black and it was every bit as chaotic and intense as we hoped The is creepy backward music that accompanies an intense bout dogfight between TIE Fighters and X-Wings in what looks like the Death Star trench This leads to a scene of Stormtroopers being shot and mangled to an upbeat jazz score Our eyes were firmly glued to the screen the entire time the footage was on the screen We didn't always know what was happening but it was beautiful to watch unfold but we do know that it will follow Stormtroopers closely The vibe we got was that it will frame them as somewhat tragic figures corrupted and controlled and used by the Empire as disposable foot soldiers It seems likely that the episode will try to give us a new side to Stormtroopers – before they are inevitably killed because that’s their lot in life Star Wars: Visions Volume 3 is out October 29 Fans of all ages from all over the world will come together to celebrate the cultural phenomenon, Star Wars. Star Wars Celebration revels in the excitement of new movies, television shows, books, art, and comics while also commemorating the vast history of the Star Wars universe. Star Wars Celebration has something special for every kind of Star Wars fan! Follow Popverse for upcoming event coverage and news Visit Reedpop ID if you need to resend the confirmation email Find out how we conduct our review by reading our review policy Sign in and let us help you find your new favorite thing Star Wars' Ahmed Best got to be the voice of Jar Jar Binks because his voice made Jake Lloyd laugh Just like the best parts of Star Wars, Andor season 2 episode 1 shows how easy it is to give in to fascism Star Wars' C-3PO voice came from a mixture of Doctor Who, Barbarella, and the horrible suit, says Star Wars actor Anthony Daniels Finally, fans get a chance to join the Clone Wars for themselves (with special secret missions) in upcoming Star Wars Zero Company video game Star Wars' Andor series was a joke on the set of Rogue One: A Star Wars Story according to Diego Luna, then years later it became real Return to Mos Eisley in a Star Wars meets Studio Ghibli anime coming as part of Visions Volume 3 "It really works!" Star Wars' Rosario Dawson has a new favorite cosplay mashing up her Ahsoka and DC Wonder Woman roles Popverse is owned by Reed Exhibitions, a division of RELX Inc. d/b/a ReedPop © 2025 Reed Exhibitions, a division of RELX Inc., 401 Merritt 7, 5th Floor, Norwalk CT, 06851. All rights reserved. No part of this website or its content may be reproduced without the copyright owner's permission. Your Ads Privacy ChoicesIMDb The iconic racing game Gran Turismo introduced much to the world The series’s huge popularity made heroes of many cars from the unsuspecting Suzuki Cappuccino to the mighty Nissan Skyline GT-R also exposed many to a unique version of jazz fusion was pioneered in the 1970s by groups like the Mahavishnu Orchestra and so many others helped fine-tune the sound perhaps most notably with Steely Dan and Joni Mitchell whose adventurous rhythms and harmonies far exceeded their peers musically Jazz fusion became a global phenomenon and Japan was no exception The country produced its own fusion artists most notably groups like T Square and Casiopea Ohira learned Japanese folk music on guitar I played Casiopea with my friends," he told Motor1 "Then I discovered Larry Carlton's guitar playing His searing solo on Steely Dan’s "Kid Charlemagne" epitomizes this mixing jazz sophistication with rock attitude I realized that I needed jazz knowledge and training and decided to attend the Berklee College of Music," Ohira says "That was the beginning of my musical life and career." He wrote arrangements for small jazz groups and big bands alike A perfect education for the gig he’s now known for Ohira went to high school with Yuichi Matsumo one of the original car modelers for the Gran Turismo series who tapped the composer to create menu music for this new racing game "When we discussed music for vehicle-based games we realized there wasn't a jazzy style before GT so we tried to incorporate a jazzy concept vibe into the new game," Ohira recalls His music was featured in the Japanese version of Gran Turismo alongside compositions from T-Square guitarist Masahiro Andoh and Japanese releases of the game having unique in-race music that attempted to reflect the tastes of local audiences Gran Turismo 2 through Gran Turismo 4 featured Ohira’s menu music you might’ve been rocking out to the remix of Lenny Kravitz’s “Are You Gonna Go My Way” in GT3 while running the MR Challenge in a tuned NSX Ohira’s menu music was worming its way into your brain while browsing the in-game car dealerships The technical limitations of the PlayStation 1 and 2 meant that the music for Gran Turismos 1-4 was rendered with software instruments the series did produce soundtrack albums that featured live instrumentation Ohira’s music was an integral part of the feel of these games and I think it’s worth remembering their popularity Let’s look at the games where Ohira’s music featured across the globe: GT2 sold over 9 million copies Three of the most popular jazz fusion albums of all time and Weather Report’s Heavy Weather are certified platinum but you can argue Ohira is one of the most-heard jazz fusion artists of all time his music might be the only jazz fusion they’ve ever heard A friend who I’m convinced only listens to rock music and Taylor Swift was incredibly excited when he heard I was talking with Ohira Gran Turismo 5 was the first title in the series not to feature any new or reworked music from Ohira for a Japan-only mobile title called Break My Case like the song Be-Bop Aporia sounds like an evolution of his Gran Turismo music and a guitar solo that sounds like Denny Diaz’s on Steely Dan’s “Aja.” The GT series inspires a lot of nostalgia—have you looked at R34 Skyline GT-R prices recently?—and that extends to Ohira’s music Jazz Fusion is having another moment as well We’re in the middle of a Steely Dan renaissance; Joni Mitchell is on a stunning comeback While the 1980s resurgence of acoustic jazz made fusion uncool modern artists like Thundercat are reviving the genre while old-school groups like Casiopea and T-Square are finding new Western audiences via video and streaming platforms “Light Velocity,” the car-dealership music from Gran Turismo 3 has over 1.5 million plays on YouTube "That’s just a huge surprise for me," he says "I didn’t hear so much about my music from GT initially… I was surprised to see a lot of my tunes uploaded with my name on YouTube." The Ferrari Purosangue Has Magic Suspension 2026 Jeep Compass Unveiled: Hybrid or EV With Up to 375 HP Lotus Emira With Yellow Exhaust Tips Pays Tribute to an F1 GOAT The Cadillac Celestiq Will Be as Rare as a Bugatti Annecy Festival has revealed the short films for its Official Selection 2025, and included in the Official category is “Black,” a Star Wars: Visions episode directed by veteran animator Shinya Ohira The featured still shows several stormtroopers engulfed in laser and flame while AT-STs stand silently in the background in their hangar bays Ohira whose previous director credits include Genius Party Beyond “Wanwa” and Asura’s Wrath Episode 11.5 “Forging Ahead,” is also the writer for “Black.”  The music composer is Sakura Fujiwara, whose “Scent of First Love” anime music video (directed by Maho Takagi at  CoMix Wave Films) we previously covered last year This year’s Annecy Festival will run from June 8 to 14 including the French-Japanese co-production Ordinary Life Star Wars: Visions Season 3 is slated to premiere this year it will feature contributions produced by Japanese animation studios “Black” is listed with a run time of 13 minutes Source: Annecy Festival (via Animation World Network) a cultural event held in Tokyo’s Shibuya area from February 8 (Saturday) to February 11 (Tuesday the event offers a chance to experience the latest culture it attracted around 110,000 visitors over three days The event showcased new forms of culture through an art parade on Kōen-dōri This second edition will expand opportunities for both domestic and international artists including collaborative projects with 12 selected groups from an open call The highlight of the official program is Pineapple Scramble Ohira will collaborate with students from Jinnan Elementary School in Shibuya to color the sculpture using action painting techniques The piece will be displayed as a float along Kōen-dōri from 12:00 to 15:00 on February 9 (Sunday) the piece will be showcased at Kitayama Park on February 10 (Monday) and 11 (Tuesday Ohira has expressed hopes that this project will offer a new way to rediscover Shibuya The highlight of this event is the collaboration with local elementary school students Ohira himself is unsure of the exact reason why a large pineapple needs to appear in Shibuya by adopting the perspective of children who ask honest questions about things we often take for granted walking through Shibuya may lead to new discoveries of the area Ohira hopes this will be an opportunity for everyone involved in the event to rediscover Shibuya Also included in the official program is the “Pac-Man Scavenger Hunt.” In celebration of the 45th anniversary of the iconic game Pac-Man a collaboration between the Los Angeles-based startup Flickplay and Bandai Namco Entertainment will offer participants a location-based scavenger hunt throughout Shibuya The popular “Shibuya Crossing Night Art,” which features art displays at the Shibuya Crossing late at night works from the official and open-call programs will be showcased on the large screens at the Shibuya Crossing will direct the project “BYOD² – BRING YOUR OWN DATA & DISPLAY.” This exhibition will be held at locations such as Shibuya PARCO and Miyashita Park and will focus on installing “data” in physical space a tribute festival titled “RADIO SAKAMOTO Uday -New Context Fes × DIG SHIBUYA-” will be held continuing the spirit of Ryuichi Sakamoto’s regular program “RADIO SAKAMOTO,” which aired on J-WAVE for 20 years until 2023 The event will take place on February 10 (Monday) ● Event Dates: February 8 (Saturday) to February 11 (Tuesday 2025 (tentative)● Venue: Shibuya Park Street and surrounding areas and others● Participation Fee: Free (some programs may require a fee)● Organized by: SHIBUYA CREATIVE TECH Executive Committee Independent Administrative Institution Japan Arts and Culture Foundation Agency for Cultural Affairs● Co-organized by: Shibuya Ward● Supported by: Tokyo Metropolitan Government (Public Art section) Shibuya Future Design Association● Collaboration: Civic Creative Base Tokyo [CCBT] (CCBT-hosted programs)● Commissioned by: 2024 Japan Expo 2.0 Project (Commission-based) NiEW Best Music is a playlist featuring artists leading the music scene and offering alternative styles in our rapidly evolving society the NiEW editorial team proudly curates outstanding music that transcends size Today's print edition Home Delivery Many Americans and people around the world were likely saddened to learn of the passing of former U.S. President Jimmy Carter on Sunday many associate him with the Middle East peace process which saw the signing of the Egypt-Israel Peace Treaty in 1979 Egypt’s Anwar Sadat and Israel’s Menachem Begin won the Nobel Peace Prize for negotiating and signing the treaty which has secured the peace between those two countries for more than four decades.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); Carter also developed close relations with Japan eventually visiting here twice as president once in June 1979 for the Group of Seven summit in Tokyo and a second time in July 1980 for the funeral of Prime Minister Masayoshi Ohira As American heads of state rarely traveled abroad at the time his two trips to Japan were a significant number he was only the second president to visit this allied nation (with his immediate predecessor In a time of both misinformation and too much information quality journalism is more crucial than ever.By subscribing Your subscription plan doesn't allow commenting. To learn more see our FAQ Sponsored contents planned and edited by JT Media Enterprise Division. Connecting decision makers to a dynamic network of information, people and ideas, Bloomberg quickly and accurately delivers business and financial information, news and insight around the world 2024 at 7:00 PM EDTBookmarkSaveHong Kong-based Sengu Capital Ltd is starting a new hedge fund focused on improving corporate management in Japan tapping into the country’s financial market revival The company will start trading for the fund in early October, Chief Investment Officer Yoshihiko Ohira and Chief Operating Officer Xavier Fanjaud said in an interview. The Japan fund lured multiyear backing from HS Group (Hong Kong) Ltd., a provider of strategic capital to such fledgling managers in exchange for a cut of their fee revenue. Volume 13 - 2019 | https://doi.org/10.3389/fnhum.2019.00153 Decision-making is assumed to be supported by model-free and model-based systems: the model-free system is based purely on experience while the model-based system uses a cognitive map of the environment and is more accurate The recently developed multistep decision-making task and its computational model can dissociate the contributions of the two systems and have been used widely This study used this task and model to understand our value-based learning process and tested alternative algorithms for the model-free and model-based learning systems The task used in this study had a deterministic transition structure and the degree of use of this structure in learning is estimated as the relative contribution of the model-based system to choices We obtained data from 29 participants and fitted them with various computational models that differ in the model-free and model-based assumptions The results of model comparison and parameter estimation showed that the participants update the value of action sequences and not each action the model fit was improved substantially by assuming that the learning mechanism includes a forgetting process where the values of unselected options change to a certain default value over time We also examined the relationships between the estimated parameters and psychopathology and other traits measured by self-reported questionnaires and the results suggested that the difference in model assumptions can change the conclusion inclusion of the forgetting process in the computational models had a strong impact on estimation of the weighting parameter of the model-free and model-based systems the data can be explained only by adjusting the parameters under the framework of the model improving the model data fit can diminish possible noise and biases in parameter estimation and minimize undesirable the purpose of this study is to examine alternative hypotheses regarding the model-free and model-based systems by comparing candidate computational models with different algorithms We will explain these assumptions in detail after we outline the procedure of the Kool two-step task In the Kool two-step task, participants are required to choose an action (i.e., choose a rocket) in the first stage, which is followed by a second-stage state (a screen with an alien) and a reward outcome (Figure 1) The participants' goal is to maximize the total reward amount the first stage is shown as one of the two states including two options (state A with rockets 1 and 2 and state B with rockets 3 and 4) A key feature of this task is that one of the rockets in each state is always followed by a specific second-stage state while the other rocket is always followed by the other second-stage state (rockets 1 and 3 always lead to second-stage state C and rockets 2 and 4 always leads to second-stage state D) this task encourages participants to use the task structure or “model,” to base their choices on their past outcome experiences when a participant wins a large reward in a previous trial but the current first stage is different from the previous first stage the participant must use the model of the transitions to consider which rocket leads to the previously experienced second-stage state The two-step task used in the experiment (left panel) and its outcome design (right panel) The first stage started with one of the two states: state A The participants selected one of the two rockets which deterministically led to a specific second-stage state (action 1 to State C and action 2 to State D) After the subject pressed the spacebar key in the second-stage state the outcome reward was displayed as an integer point value ranging from 1 to 9 This point value changed slowly and independently according to Gaussian random walks but the same values applied to all participants A conceptual framework for the examined assumptions (A) The standard model-free algorithm is the SARSA temporal-difference (TD) learning model where the values are computed for all state-action pairs (standard value updating) We examined another possibility in which only the action values in the choice stage are computed (parsimonious value updating) (B) The originally used model-based system assumes that the expected values for all state-action pairs are calculated anew each time using the transition-probability model of the task (the forward-looking model-based system) This system carries a high calculation cost but realizes fully model-based updating we applied model-based updating for the credit assignment problem (the backward-looking model-based system) This system updates only the state-action pairs relating to the last state that produced the outcome based on the transition-probability model of the task but it works efficiently with similar accuracy to the forward-looking model-based system when the transition probabilities are stable the values of unselected options are assumed to remain unchanged (without forgetting) We examined another possibility in which the values of unselected options change to a certain default value over time (with forgetting) The impact of the outcome reward directly updates the actions that can lead to the last state that produces this outcome The advantage of this model-based updating is that participants do not need to activate the representations of all state-action values but only the values of states and actions associated with the last state “default value,” such that we could freely set the endpoint of forgetting instead of restricting it to zero The default value represents an expected value for options in the absence of knowledge or experience related to the relationship between options and outcomes the examined computational models have some or all of these assumptions These models were compared using data from the Kool two-step task to test the effect of model construction on the parameter estimates we compared the computational models in terms of the relationship between the estimated parameter values and subjects' scores on questionnaires regarding obsessive tendencies Thirty-four undergraduate students at Nagoya University participated in the experiment The data from two participants were excluded because the participants were unable to complete the training session by themselves due to their misunderstanding of the instructions and three participants were excluded because they did not pass the exclusion criteria (see section Exclusion criteria) the data from the remaining 29 participants were analyzed (13 males All participants provided written informed consent in accordance with the Declaration of Helsinki The protocol was approved by the ethical committee of Nagoya University Participants were seated ~50 cm in front of a 21.5-inch iiyama ProLite monitor with a screen resolution of 1920 × 1080 pixels and a refresh rate of 60 Hz Instructions and stimuli were presented using the computer program Inquisit 5 Lab (2016) by Millisecond Software in Seattle The task procedure was almost the same as the two-step task originally proposed by Kool et al. (2016, 2017) although we slightly changed the cover story and settings The participants' goal was to maximize their reward by their choice of rockets which were divided into two blocks separated by 30-s breaks the participants were required to select one of two rockets (downloaded from Freepik.com) by pressing the F key for the left rocket or the J key for the right rocket within 2.5 s This stage was characterized by one of two states: state A always included rockets 1 and 2 The subsequent second-stage state was based on the first-stage choice Rockets 1 and 3 were always followed by state C in the second stage and rockets 2 and 4 were always followed by state D in the second stage each state included one unique alien (downloaded from pngtree.com) The participants were required to press the space bar within 1.5 s to obtain a reward from the alien Each alien produces a reward feedback value ranging from 1 to 9 These feedback values for each alien changed slowly over the course of the task according to a Gaussian random walk (mean = 0 σ = 0.025) with bounds of 0.25 and 0.75 and was displayed as an integer on the screen Auditory stimuli were played when participants made a choice (bell sound) and when they obtained a reward (money sound) if no response was made within the time limits “Too late!!” was presented and the participants proceeded to the next trial the participants were informed that the positions of the rockets and the response speed within time limits would have no relationship with subsequent feedback or the total experimental time and that the choice of rockets is only related to the transition to the second-stage states The participants were also repeatedly told that each rocket in each first stage was connected decisively with one of the two aliens in the second stage and that the reward from each alien would change slowly and independently over time depending on these aliens' moods within the range from 1 to 9 the participants were informed that they would obtain greater rewards by focusing on the moods of each alien The participants were also informed in advance that they could receive additional monetary rewards along with their total earned points in this task with an additional monetary reward of either ¥300 (if they earned more than 1,300 points) or ¥200 (if they earned fewer than 1,300 points) The participants also completed a training session to learn the structure of the task in advance; in this session they were required to repeatedly choose the rockets connected with one of the two aliens in the training trials without time limits or feedback and if they succeeded in more than 5 consecutive trials for each alien then they were next trained with 18 trials with time limits and feedback The stimuli used in the training session were completely different from the stimuli used in the real task The reward probabilities were the same for all participants, but the order of the first-stage state during the task was deliberately controlled in advance, and each participant was allocated to one of four sequences (see Supplementary Text 1.1, Figure S1 and Table S1) The full series of 253 trials started with one of the two first-stage states and the same first-stage state was repeated within 6 trials 180 trials started with the same first-stage state as the previous trial and the remaining 72 trials started with a different first-stage state from the previous trial participants do not need to use the transition model participants need to use the transition model if they wish to use the information from previous feedback we conveniently refer to the former trials as MF (model-free) trials and the latter trials as MB (model-based) trials The choices predicted by the model-free and model-based systems are similar in the MF trials but not in the MB trials we excluded the data from uncompleted trials (i.e. those in which the choice was not made within 2.5 s) and the data from trials in which the response time was <120 ms which were considered anticipated responses that did not reflect the stimulus types Two participants who had more than 20% of their trials omitted based on these criteria were excluded we excluded one participant who chose the same rocket in each first-stage state in more than 90% of the trials the data of 29 participants were used for the subsequent analyses (rate of excluded trials: max 8% We first describe two basic models (the parallel model and the parsimonious learning-rate adjustment model, Figure 3) as candidates to explain the data for the two-step task we introduce some variations of the forgetting process that can be combined with these models A schematic of value updating in the parallel model (left panel) and the parsimonious learning-rate adjustment model (right panel) The panels show the difference in value updating in the two models when the agent selected action 1 in state A followed by state C and an outcome The parallel model assumes that model-free values are updated for the experienced state-action pairs and that model-based values are updated for every state-action pair These values are mixed according to a model-based weighting parameter w the parsimonious learning-rate adjustment model updates only the first-stage actions relating to the state that produced the outcome based on the transition-probability model the value of action 1 in state A is updated by the outcome in state C the value of action 1 in state B is also updated but downweighted by the model-based weighting parameter w For data from the Kool two-step task, a computational model developed by Daw et al. (2011) is ordinarily used. This model uses the forward-looking model-based system and assumes that the model-free and model-based values are computed in parallel and combined as a net value for a choice weighted by the weighting parameter w (Figure 3 we call this model the parallel model (or P model) The model-free learning system uses a SARSA (λ) TD learning rule (Rummery and Niranjan, 1994) and updates state-action values two states are included in each stage (sA and sB for s1,t the selected state-action value is updated as follows: where 0 ≤ αL ≤ 1 is the learning rate parameter and 0 ≤ ri,t ≤ 1 denotes the reward in trial t which is linearly transformed from actual feedback ranging from 1 to 9 the selected first- and second-stage values The second-stage reward prediction error (RPE) which reflects the difference between the expected and actual reward also updates the first-stage value but is downweighted by the eligibility trace decay parameter λ as follows: where λ denotes the trace decay parameter that modulates the magnitude of the effect of the second-stage RPE on the first-stage value. This type of updating is called the eligibility trace rule and enables efficient value updating (Sutton and Barto, 1998) for each action are defined by the Bellman optimality equation an option value is computed anew each time as a sum of the maximum values of the possible subsequent state-action values weighted by the transition probabilities for the respective states The transition probability determines this weight a2 in the first stage and ak = a1 in the second stage ak) is a transition-probability function representing the probability of moving to state ś after choosing action ak at state sj a represents possible actions at state ś and the max operator indicates the maximum of all action values in state ś the transition probability from the first-stage action to the second-stage state is determinate and the second-stage state simply requires the subject to press the space bar the first-stage model-based values are equal to the model-based value in state ś because T(ś|sj model-based values are equivalent to model-free values because no transition to a further stage occurs Finally, QMF and QMB are integrated to generate a net value for choice with a model-based parameter 0 ≤ w ≤ 1 (Daw et al., 2011): The second-stage QNET values are equal to QMB and QMF These net values determine the first-stage choice probability of choosing action a among the candidate actions three free parameters represent particular propensities in the choice process: β adjusts how sharply the value difference between options is reflected in the choice probability; π determines the degree of perseveration in the same option; and ρ expresses the degree of key-response stickiness rep(a) is an indicator variable that equals one if a is a first-stage action and is the same as the action chosen in the previous trial and zero otherwise resp(a) is an indicator variable that equals one if a is a first-stage action using the same response key pressed in the previous trial and zero otherwise the parameters π and ρ express perseveration (when the values are positive) or switching (when the values are negative) in favor of one option or one side then calculated value differences have no influence on choice probabilities then the maximum-value option is always chosen Among these parameters, β is usually included in any RL model. In the two-step tasks, Daw et al. (2011) used π but did not use ρ, whereas Kool et al. (2016) used both π and ρ but they were not supported by model selection; therefore we will report the models using both π and ρ as free parameters the system can be implemented by adjusting the learning rate parameter we refer to this model as the parsimonious learning-rate adjustment model (the parsimonious LA model hereafter called the “LA model” for brevity) This model has another parsimonious aspect: it uses the backward-looking model-based system which assumes only one value for one action (in contrast the forward-looking model-based system calculates model-free and model-based values in parallel and combines them into a net value for each action) a deterministic action sequence followed by a choice is the unit for valuation and only the action values in the choice stage are updated these values correspond to the values of the first-stage rockets We use Q in this model because one value for one action is assumed The first actions of the deterministic state-action sequences are updated as follows: The pure model-free value calculation ends here If the backward-looking model-based system works then the other state-action pair that leads to the same second-stage state with a1,t is also updated as follows: the weight of model-based updating is adjusted by a model-based parameter 0 ≤ w ≤ 1 The pure model-based system (w = 1) equally updates all the actions eligible for the outcome if an agent receives a certain amount of reward after choosing rocket 1 then he can speculate that the same reward would have been obtained if he had chosen rocket 3 he updates the value of rocket 3 in the same manner as rocket 1 The LA model obviously has simpler calculations than the P model This process is identical to that introduced in the P model (Equation 7) The values of unselected actions (including the actions of the unvisited state) are not updated in typical RL these values can naturally be considered to decay through a forgetting process The following equation is one algorithm for this process: the values of unselected actions are updated as follows in each step when a selected action is updated by Equations 2 3 in the parallel model and by Equation 8 in the LA model: the current study examines a model without a forgetting process (where αF = 0) and three types of models with a forgetting process: the first model assumes that the values of unselected options gradually approach zero (where αF is a free parameter and μ = 0) the second model assumes that they approach 0.5 which corresponds to the least biased value (where αF is a free parameter and μ = 0.5) and the third model assumes that people have their own default value to which the values approach (where both αF and μ are free parameters) We used the R function “solnp” in the Rsolnp package (Ghalanos and Theuss, 2015) to estimate the free parameters. For a comparison of these models, we computed the Akaike information criterion [AIC; Akaike (1974)] where LL is the log likelihood and k is the number of free parameters The model with a smaller value is considered the preferred model We used this criterion to compare the predictive ability of the models which is equivalent to the parallel model in which w = 0 for comparison the best-fitting model was the LA-F05 model To examine the total improvement by the best-fitting model (the LA-F05 model) compared with the ordinarily used parallel model (the P model) we subtracted the AIC of the LA-F05 model from that of the P model for each participant This calculation showed that the data for 28 of the 29 participants supported the LA-F05 model In sections Overall model comparison and estimated parameters and Model differences and the estimated weighting parameters additional comparisons are performed to determine whether the fitting improvement was attributed to the backward-looking model-based system of the LA models Information concerning the models compared on the basis of their fit to the choices of 29 participants if αL and λ are set to one then the parallel models have a similar structure to the LA models in which αL is set to one the second-stage state values are equal to the last piece of feedback if αL = 1 and the last piece of feedback is directly reflected in the first-stage value because λ = 1 such specific parallel models behave similarly to the LA models which do not distinguish the first-stage state-action value and the following second-stage state-action value Note that these results support the parsimonious updating assumed in the LA models but provide no information on the comparison between the forward-looking and the backward-looking model-based systems the AIC values were lower in the LA models than those in the parallel models: The LA model was favored over the P model the LA-F05 model was favored over the P-F05 model and the LA-FD model was favored over the P-FD model (favored by more than 20 of 29 participants in each comparison; ps < 0.005 for the paired t-tests) no significant differences in AIC values were observed between the basic models with any forgetting process assumptions (i.e. all ps > 0.10 in the paired t-tests) the higher AIC values in the parallel models than those in the LA models among the full models are attributable to the effect of the redundant free parameters in the parallel models and the difference in the model-based system (parallel or LA) is not critical for fitting improvement Regardless of the model comparisons among the full models or those among the reduced models, the models with forgetting processes were favored. Here, we show only the results of the full models, but the similar results were obtained for the reduced models (Supplementary Text 1.2) Most participants showed reduced AIC values in the LA-FD model vs. the LA model [Figure 4A, favored by 27 of 29 participants, t(28) = −6.30, p < 0.001] and in the P-FD model vs. the P model [Figure 5A These results strongly suggest that the forgetting process cannot be neglected in constructing the framework of value-based learning Model comparison by differences in the Akaike information criterion (AIC) scores in the parsimonious learning-rate adjustment models (LA models) The AIC scores of the LA models were compared One of the models has no forgetting process (LA) and the other three have a forgetting rate parameter for the forgetting process and either a free default-value parameter (LA-FD) (A) The LA-FD model was favored over the LA model (red bars favor the LA-FD model (B) By including a default-value parameter the data fit was improved (red bars favor the LA-FD model (C) No statistically significant improvement was observed by fixing the default value at 0.5 (red bars favor the LA-F05 model Note that the scales of the vertical axis are different among the panels Model comparison by differences in the Akaike information criterion (AIC) scores in the parallel models (P models) The AIC scores of the P models were compared One of the models has no forgetting process (P) and the other three have a forgetting rate parameter for the forgetting process and either of a free default-value parameter (P-FD) (A) The P-FD model was favored over the P model (red bars favor the P-FD model the data fit was improved (red bars favor the P-FD model (C) No statistically significant improvement was observed by fixing the default value at 0.5 (red bars favor the P-F05 model although 20 participants favored the LA-F05 model over the LA-FD model and the P-F05 model over the P-FD model according to the AIC scores These results may reflect the current task setting in which the average expected outcome over the task was close to 0.5 (when the points in the task were linearly transformed to the range of 0–1); the expected outcome was 0.46 under random choice and 0.53 on average among the participants In the previous section, we reported that the model fits were improved by using the reduced models: the LA models in which αL was fixed and the P models in which αL and λ were fixed. To assess the influence of fixing these parameters on the estimation of the weighting parameter w, we conducted linear regression analyses and confirmed that the estimations of w were not different between the full models and the reduced models (Figure S2) The regression analyses revealed that the models with forgetting processes had lower estimated w values than those in the models without forgetting processes The correspondence of the estimated weighting parameter w by different models the correspondence (A) by the parallel model (P model) and the parsimonious learning-rate adjustment model (LA model) (B) by the P model with forgetting (P-FD model) and the LA model with forgetting (LA-FD model) and (D) by the LA model and the LA-FD model are shown Each panel shows the coefficient of determination (R2) Red lines indicate linear regression lines The data on the black lines indicate complete correspondence between the estimations by the two models The analyses in this section were conducted to understand the characteristics of the model parameters the P-F05 and LA-F05 models showed lower AIC values than the P-FD and LA-FD models although no significant differences were noted we mainly used the parameters estimated by the P-FD and LA-FD models to avoid possible estimation biases of w caused by fixing μ to 0.5 for some participants We also provide the results of the analyses using the P and LA models for comparison The computational models were developed supposing that the weighting parameter w reflects use of the “model,” or the transition structure; therefore we examined this assumption from the statistical characteristics of the obtained data Considering that the Kool two-step task includes trials that prompt the use of the transition structure (MB trials) and trials that do not (MF trials) if a participant uses the “model,” he or she can be predicted to behave similarly in both types of trials and this tendency is expected to be captured by the parameter w p < 0.001) as expected because only the MB trials require use of the “model” to reach a certain second-stage state based on the previous outcome (A) This boxplot shows the average probabilities of revisiting the second-stage state that was visited in the previous trial given low (1~3 points) and high (7~9 points) previous rewards This probability obviously changed depending on the previous outcome (B) This boxplot shows the average sensitivity to the previous outcomes (SPO) for choices in model-based (MB) trials and model-free (MF) trials This sensitivity is calculated as the average ratio of the probability of revisiting the same second-stage state after a high reward to the same probability after a low reward This score was significantly higher in MF trials than that in MB trials (C) This panel shows the relationship between the parameter w estimated by the LA-FD model and the difference in SPO between the MF and MB trials (ΔSPO) A negative relationship was found between these variables showing that participants with a low w were more sensitive to the previous reward in the MF trials vs indicating that participants with a high w behave the same in the MF and MB trials whereas participants with a low w base their choices on the previous outcome less often in the MB trials than in the MF trials such correlations were not observed with other parameters (all rs < 0.33) These results suggest that the model-based parameter w uniquely captures use of the “model,” or the transition structure we conducted the same analyses for the w estimated using the other models the estimated w had no significant relationships with the SPO in the MF trials (all rs < 0.30) but had moderate positive relationships with the SPO in the MB trials (P-F05: r = 0.42 Regarding the relationships with ΔSPO the models with forgetting processes showed relatively stronger negative correlations (P-F05: r = −0.74 p < 0.001; LA-F05: r = −0.77 p < 0.001; P-FD: r = −0.66 although the models without forgetting showed no or weak negative correlations (LA: r = −0.33 the parameter w reflects the similarity of the magnitude of the effect of the latest outcome on choices in the MB trials to that in the MF trials which is more clear when the models with forgetting processes are used many other factors may be related to total rewards other than w such as the reward schedule of the second-stage states and the contributions of other parameters to agents' choices we found a strong positive correlation between total rewards and the value-based parameter β (P: r = 0.82 We also found a moderate positive correlation between total rewards and the forgetting rate αF when using the models with forgetting processes (P-FD: r = 0.44 which may reflect the nature of this task that the immediately preceding outcome is the most informative The w estimated by the models with forgetting processes had no significant relationship with self-reported OCD tendencies as measured by the OCI contrary to our expectation (P-FD: r = −0.10 whereas the w estimated by the models without forgetting processes showed weak negative correlations (P: r = −0.31 Associations of estimated parameter values with psychopathology and other traits This change in the correlations can be explained as follows: (1) the reduction in the AIC values by using the P-FD or LA-FD model instead of the P or LA model showed a marginally significant negative correlation with OCI scores (r = −0.33 p = 0.072); (2) this negative correlation indicates that the effect of the forgetting process was greater in participants with low OCI scores than that in participants with high OCI scores; (3) the values of w estimated by the models with forgetting processes (the P-FD and LA-FD models) were lower than those estimated by the models without forgetting processes (the P or LA models); (4) therefore the w of participants with low OCI scores decreased more than that of participants with high OCI scores causing the negative correlations between w and OCI scores to nearly disappear When the models with a forgetting process were used for model fitting the weighting parameter w showed a moderate positive relationship with impulsivity as measured by the BIS 11 (P-FD: r = 0.54 which was surprising because impulsivity is generally considered negatively correlated with model-based behavior A possible explanation for this result may be related to the role of w in this task; that is a greater w can heighten the effect of the previous outcome in this study using healthy university students higher impulsivity may have heightened the effect of the last outcome on the current choice which may have been reflected by a greater w other parameters showed some correlations with OCI scores (negative correlation: αL and β; positive correlation : π) The current correlation results may not be generalizable because of the small number of participants and the restricted population the results showed how the differences between the computational models greatly affect the parameter estimates and their relationships with other indices We compared several computational models for data from a two-step task with a deterministic transition structure (Kool et al., 2016) Based on model comparisons and parameter estimations the participants appeared to use a parsimonious computational algorithm reward feedback seemed to directly update the first action of the deterministic state-action sequences model fits were improved by including forgetting processes the weighting parameter w strongly corresponded to the statistically characterized degree of “model” use we showed the possibility that the models with forgetting processes result in different conclusions compared to the original model in terms of the relationships between the model parameters and psychopathology the best method to predict the next option values is to record only the information from the immediately preceding outcome because the outcomes change randomly and slowly every trial; most of the participants may have used this type of strategy This heuristic learning process was revealed by fitting the data with the computational model Obtaining this finding only from the statistical descriptions of the data is difficult Histogram of the estimated parameter values of 29 participants The reward average in this task was 4.7 points (equal to 0.46 in the computational model) In our previous study (Toyama et al., 2017) which assumes memory decay for unselected actions improved the fit of the computational model for the data from the Daw two-step task This result was replicated in the current study using the Kool two-step task retaining all action values in a stable manner is difficult the inclusion of forgetting processes may be reasonable to express natural choice behavior by the RL model The standard RL model does not assume this process possibly because it was first developed in the field of engineering and may not need to assume that memory decay occurs This tendency can be distinguished from a mere increase in choice randomness because randomness is expressed by the inverse temperature parameter (β) μ expresses the value-based expectation assigned to the recently unselected options if we use the model without a forgetting process then choice shifts to unselected options are all erroneously expressed by decreasing β and the perseveration parameter (π) proper model construction is important to reduce the erroneous bias of the parameters Situations in which the forgetting process can affect the learning process are easy to conceptualize cognition regarding the task condition can affect the forgetting process In a situation where the reward outcomes change frequently the expectation for unselected options also becomes uncertain quickly and the agent may change options often (expressed as a high forgetting rate) in a situation where the reward outcomes are stable the expectation for unselected options is also stable (expressed as a low forgetting rate) Individual trait differences can also affect the forgetting process the difference between optimistic and pessimistic outlooks may be expressed as an individual difference in default values the computational model with a forgetting process is expected to provide new insights in research related to value-based decision-making the backward-looking model-based system may work as well as the forward-looking system or more efficiently The interpretation of the weighting parameter w is sometimes ambiguous in terms of whether it reflects the degree of learning about the transition model or the use of the transition model The Kool two-step task has the advantage of restricting the parameter's meaning Because this task has a clear deterministic transition structure w is expected to mostly reflect the difference in the degree of model use among participants we demonstrated the correspondence between the estimated w and individual differences in the model-free and model-based sensitivity to the previous outcome This correspondence was found even when the parameter was estimated by the original parallel model but the correlation was weaker than that when the parameter was estimated by the models with forgetting processes this correlation almost disappeared when the parameter w was estimated by the models with forgetting processes implying that we must be careful when interpreting this parameter considering that the model fits were improved for most of the participants by assuming that forgetting occurs their choice data included a property corresponding to the forgetting process if the data are estimated by the models without forgetting these models are forced to reflect this property only by their parameters Our result showed that the values of w were estimated in a more model-based direction compared with the values estimated by the models with forgetting processes The value changes affected the correlation between OCI scores and w which may reflect the varying effect of the forgetting process among participants the models with forgetting processes seem to lose their predictive ability for OCD tendencies or at least their interpretability for OCD became complicated OCD may require interpretation with a combination of parameters the crucial parameters should be able to be appropriately estimated even when behaviors have additional or fewer characteristics than assumed in the models task setting is also important to improve the sensitivity of the weighting parameter to individual differences more than twice as many MF trials as MB trials were included By increasing the ratio of MB trials in future studies the relationships between w and other individual traits may be clearer Although some future challenges remain, model fitting was notably improved for most of the participants by assuming a forgetting process, and as a result, the relationships between the parameters and self-reported psychopathology changed. If a data characteristic cannot be captured by a model, the model still must express the characteristic using its model parameters, which sometimes leads to misinterpretation of the parameters (Katahira, 2018) the models with forgetting processes should be examined as candidate models to understand our cognitive process in future studies The current study showed that participants favored the models with parsimonious computation which assumes that the values are updated for action sequences which assumes memory decay for unselected option values we confirmed that the estimated model-based weighting parameter could capture individual differences in “model use.” To date most learning models do not contain psychological aspects such as cognitive savings and memory decay research using the proposed model will force re-evaluation of how the features of the learning process correlate with psychopathology or abnormal decision-making and will enrich the study of the theory and neural basis of learning processes This study was carried out in accordance with the recommendations of the ethical committee of Nagoya University with written informed consent from all participants All participants gave written informed consent in accordance with the Declaration of Helsinki The participants were all Nagoya university healthy students AT collected and analyzed the data and prepared the draft KK and HO reviewed the manuscript critically and provided important intellectual input All authors contributed to the design of the work as well as the interpretation of the results This study was supported by Grant-in-Aid for Early-Career Scientists (18K13366 to AT) by Grant-in-Aid for Scientific Research (C) (18K03173 to KK) and by Grant-in-Aid for Scientific Research (B) (17H02649 to HO) They are all form Japan Society for the Promotion of Science The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnhum.2019.00153/full#supplementary-material 1. ^For example participants make two successive choices in the Daw two-step task but only one choice in the Kool two-step task There are four options with different reward outcomes in the second stage of the Daw two-step task and two options with different reward outcomes in the second stage of the Kool two-step task the current condition of each option is easy to assess in the Kool task using gradual integral point feedback whereas the Daw two-step task uses binary feedback based on hidden probability 2. ^For example considering that the final stage in the Daw two-step task has multiple options the participants may intend to visit the same final state after they are not rewarded in that state because they can try the option that they did not choose in the previous trial this strategy is not included in the existing computational models; 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Accepted: 23 April 2019; Published: 09 May 2019 Copyright © 2019 Toyama, Katahira and Ohira. 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Volume 10 - 2016 | https://doi.org/10.3389/fnhum.2016.00444 This study investigates the prolonged effects of physiological responses induced by acute stress on risk-taking in decision-making Participants were divided into a Stress group (N = 14) and a Control group (N = 12) The Trier Social Stress Test was administered as an acute stressor and reading was administered as a control task; thereafter participants performed a decision-making task in which they needed to choose a sure option or a gamble option in Gain and Loss frame trials 2 h after (non-) exposure to the stressor and subjective stress levels validated acute stress manipulation Stressed participants made fewer risky choices only in the Gain domain whereas no effect of stress was shown in the Loss domain Deceleration of HR reflecting attention was greater for Gains compared with Losses only in the Stress group Risk avoidance was determined by increased levels of cortisol caused by acute stress These results suggest that processes regarding glucocorticoid might be involved in the prolonged effects of acute stress on the evaluation of risks and the monitoring of outcomes in decision-making the investigation of physiological mechanisms underlying influences of acute stress on decision-making is only in its primary stages The present study explored this issue with an experiment measuring typical physiological indices of stress and risky choices in a lottery decision-making task performed 2 h after exposure to acute stress Acute stress can make habit action more dominant; therefore individuals might habitually prefer or avoid risk without deliberating over expected gains or losses acute stress can facilitate individuals’ strategic deliberation in preferring or avoiding risk to satisfy their inner goals we manipulated expected values (EV) of a safer and a riskier option in each decision-making trial the riskier option provided higher EV than the safer option the EV of the risker option was lower than that of the safer option If acute stress makes habit action dominant participants should show consistent within-individual tendencies of risk preference or risk avoidance regardless of differences of EVs if acute stress facilitates goal-directed action participants should become more sensitive to EVs of options and they should make risk-taking or risk-avoiding choices depending on relative comparisons of the EVs of options In the present study, 28 Japanese male undergraduates at Nagoya University participated (age range 18–22 years; mean = 19.92; SD = 1.20). They were randomly assigned to a Stress group or a Control group. This sample size was determined according to a priori analysis of statistical power using G∗power 3, version 3. 1. 9. 2 (Faul et al., 2007). A sample size adequate to detect the effect size reported by Pabst et al. (2013) indicating significant effects of acute stress on risky choice was estimated as N = 24 (alpha error = 0.05; 1-beta error = 0.95) Two participants in the Stress group were excluded from analysis because of technical problems in data collection No participants suffered from any chronic illnesses Participants were advised not to smoke or drink alcohol on days they participated in the experiments The Ethics Committee of Nagoya University approved the study (No.: 315 and its methods were conducted according to approved guidance for human subjects All participants signed an informed consent before participating in the study At the beginning of each trial, participants were shown a message indicating a starting amount of money. They had to choose a sure or a gamble option for each trial. The sure option meant keeping the amount of money given at the beginning in Gain frame trials and losing the amount given at the beginning in Loss frame trials. The gamble option was shown as a pie chart depicting the probability of Hit (red) or Miss (blue) and the amount of monetary reward or loss (see Figure 1) followed by a response cue to prompt participants to choose we manipulated EV to assess decision-makers’ sensitivity and adaptability to contingencies making a choice advantageous or disadvantageous we set conditions at three different EV levels between gamble and sure options: a large EV gamble with a larger EV of the gamble option than of the sure option; a small EV gamble with a smaller EV of the gamble option than of the sure option; and an equal EV gamble with the same EV of the gamble option as of the sure option independent variables in this task were Domain (Gain or Loss) and EV of gamble option (Large we manipulated the probability of the hit outcomes when the gamble option was chosen (see Supplement 1) Gain and Loss trials were presented as separate blocks with counterbalanced orders across participants in each group conditions of the EV gamble option were presented randomly Experimental sessions started at either 09:00 a.m To control effects of diurnal variations in cortisol secretion numbers of participants allocated into early and late experimental sessions were counterbalanced between Stress and Control groups Participants were instructed to eat a light breakfast on the morning of the experiment Participants suffering from an infectious illness within 2 weeks of the experiment were rescheduled participants were given 10 min to prepare for their speech They were then exposed to a simulated interview (5 min) in front of a video camera and conducted by two interviewers followed by a mental arithmetic task (5 min) and participants again subjectively rated their stress Participants read newspapers during the 120-min rest period participants rated their stress subjectively instructions for the decision-making task were given and participants completed the decision-making task after several practice trials Cardiodynamic activity was measured continuously throughout the experimental session and participants were fully debriefed and thanked The experimental session in the Control group was identical to that in the Stress group except that participants read newspapers for 20 min instead of focusing on the TSST Experimental protocol of the present study we determined a conditional probability of an action (a) under a state (S) the conditional probability P(a|S) is calculated as follows: where Num(a|S) is the number of choices of gamble or keep (a) under a state S and Num(k|S) is the number of total choices k under a state S The constant c was introduced to stabilize the calculated probability and was fixed to 1 here The value of entropy H was standardized from 0 to 1 by dividing by N (here Conditional entropy calculated by this formula reflects the degree of deviation from dependence of a choice on the outcome of the previous trial if a participant more often chooses gamble when hit was given in the previous trial and chooses keep when miss was given in the previous trial (Win-Stay This pattern of decision-making can be regarded as more dependent on previous outcomes and thus more experiential as a participant more often chooses gamble or keep totally independently from the previous trial’s outcome then entropy H will be larger (approaching 1) This pattern of decision-making is regarded as more independent from previous outcomes and probably stochastically determined based on an inner standard and is thus more descriptive Blood samples were collected in EDTA tubes and centrifuged at 3,000 rpm for 10 min to measure cortisol levels in plasma Plasma was then separated and stored at -80°C until analysis The plasma cortisol concentration was measured using a cortisol ELISA kit (Oxford Biochemical Research Inc. The intra-assay coefficient of variation was 3.4–3.7% and the inter-assay coefficient of variation was 3.8–6.4% The limit of detection was 0.3 μg/ml blood samples were collected in serum separator tubes and centrifuged for 15 min Serum was removed and then kept at -80°C until analysis The concentration of adrenaline in serum was measured using an HPLC-electrochemical detector (ECD) (CoulArray; ESA Biosciences The inter-assay coefficient of variation was less than 7.0% we focused on examining outcome-related HR reactivity induced by feedback signals of hit and miss When significant interactions were found in ANOVAs post hoc analyses using Bonferroni tests (p < 0.05) were conducted to examine which combinations of data points differed significantly Post hoc analyses (p < 0.05) indicated that after the TSST task adrenaline level in the Stress group was higher than that of the Control group but no difference was observed during rest periods All these indices consistently clarified that the stress task in this study elicited typical robust psychological and physiological (SAM and HPA) acute stress responses Means (Standard Error of Mean) in the intensity of stress and results of ANCOVA Error bars indicate standard errors of means Heart rate (HR) through experimental sessions in each group Error bars indicate standard errors of mean conditional entropy showed relatively higher values (H > 0.50) in all experimental conditions Neither main effects nor interactions indicated significant levels (F < 1.60); thus acute stress showed no effect on conditional entropy ∗Significant difference between groups (p < 0.05) Means (Standard Error of Mean) in the rates of gamble choice per each previous outcome Means (Standard Error of Mean) in the conditional entropy showed relatively higher values (H > 0.50) in all experimental conditions HR time-locked to gamble outcomes in the Equal EV level showed deceleration that can be interpreted as a typical orienting response HR deceleration to outcome of a sure option was less than that to outcome of a gamble option (p < 0.01) and no difference was shown between groups (see Supplement 2) (A) Heart rate deceleration in decision-making task (B) Waveforms of time-series variations of HR deceleration There was no correlation between gamble choice rate and HR deceleration during the decision-making period in each experimental condition (r < -0.33 HR deceleration reflecting trial-by-trial attention to decision-making outcomes did not influence the rate of gamble choice Because this result indicated that gamble choice and HR deceleration are independent we conducted further analyses of these variables separately change values were calculated by subtracting values at baseline from values immediately after the TSST and those change values were used as predicted variables Structural equation modeling for rates of gamble choice and HR deceleration (A) The hypothetical model for rate of gamble choice (B) The hypothetical model for HR deceleration (C) The result of SEM for rate of gamble choice The AIC value decreased from 34.48 for Model 1 to 16.74 for Model 2 as indicated by multiple indicators of fit: ratio chi-square/df = 0.43 The AIC value comparing improvement of fit strongly decreased from 34.48 for Model 3 to 16.44 for Model 4 there might be a complicated fluctuation of preferences for risk in decision-making from risk avoidance to risk-taking and again to risk avoidance along the flow of time after exposure to acute stress This possibility should be examined more in detail by multiple manipulating temporal intervals between exposure to acute stress and the decision-making task This result means that Stress group participants did not necessarily depend more on outcomes of immediately previous trials for their decision-making results of these behavioral indices did not support the experiential account but were more compatible with the descriptive account for risk avoidance observed in the Stress group The 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Kaori Yamakawa, a2FvcmkueWFtYWthd0BnbWFpbC5jb20= Hideki Ohira, b2hpcmFAbGl0Lm5hZ295YS11LmFjLmpw Volume 8 - 2014 | https://doi.org/10.3389/fnbeh.2014.00381 We previously reported that sympathetic activity was associated with exploration in decision-making indexed by entropy which is a concept in information theory and indexes randomness of choices or the degree of deviation from sticking to recent experiences of gains and losses and that activation of the anterior insula mediated this association The current study aims to replicate and to expand these findings in a situation where contingency between options and outcomes is manipulated Sixteen participants performed a stochastic decision-making task in which we manipulated a condition with low uncertainty of gain/loss (contingent-reward condition) and a condition with high uncertainty of gain/loss (random-reward condition) Regional cerebral blood flow was measured by 15O-water positron emission tomography (PET) and cardiovascular parameters and catecholamine in the peripheral blood were measured norepinephrine as an index of sympathetic activity was positively correlated with entropy indicating exploration in decision-making Norepinephrine was negatively correlated with neural activity in the right posterior insula suggesting neural bases for detecting changes of bodily states right anterior insular activity was negatively correlated with entropy suggesting influences on exploration in decision-making entropy correlated with activity in the dorsolateral prefrontal and parietal cortices but not with sympathetic activity These findings suggest that influences of sympathetic activity on exploration in decision-making and its underlying neural mechanisms might be dependent on the degree of uncertainty of situations Exploitation is a strategy to stick to an option that has delivered reward at the highest possibility exploration is a strategy to seek for new and previously unexplored options and thus means deviations from exploitation While exploitation is more adaptive in a stable environment organisms have to take the strategy of exploration in an unstable environment the relationship between exploitation and exploration is a trade-off and the balance between these two strategies is critical for survival of animals and humans which is sometimes called “inverse temperature,” is usually sensitive and can dynamically vary in a trial-by-trial manner along the progress of learning entropy represents more tonic states of randomness of choices within relatively larger numbers of trials we adopted entropy as an index of exploration because we aimed to elucidate influences of sympathetic activity on tonic aspects of decision-making as described above Our results (Ohira et al., 2013) showed that an increase of epinephrine in the peripheral blood as an index of sympathetic activity was associated with larger values of entropy indicating greater tendency of exploration The increase of epinephrine was positively correlated with brain activity in the right anterior insula and dorsal pons [near the locus coeruleus (LC)] activity in the anterior insula mediated this correlation between epinephrine and entropy the association of sympathetic responses and exploration was found only after introduction of the reversal of the association between options and outcomes but not during the initial learning stage before the reversal This suggests that the effects of sympathetic activity were not fixed but were tuned based on evaluation of situations this was the first report of an association between peripheral sympathetic responses and exploration in decision-making further evidence is needed to support the findings Compared with the contingent-reward condition and dorsal striatum were dominantly activated in the random-reward condition where participants had to continue to seek contingency between options and outcomes we expected that the association between sympathetic activity and underlying brain activity would be observed more dominantly in the contingent-reward condition but such associations would be weakened in the random-reward condition Sixteen healthy right-handed Japanese male undergraduate and graduate students who had no past history of psychiatric and neurological illness were recruited (M ± SD; 21.69 ± 2.25 years) They gave written informed consent in accordance with the Declaration of Helsinki The present study was approved by the Ethics Committee of Kizawa Memorial Hospital The task in the control condition was identical to that in the other two conditions except that the computer made a decision on each trial and participants pressed a key that the computer indicated thus the task is object learning but not spatial learning The same pair of two stimuli was presented through blocks per each condition Time course of a trial in stochastic decision-making task Participants performed eight blocks of the decision-making task Three blocks were for the contingent-reward condition three blocks were for the random-reward condition and two blocks were for the control condition with an 11-min interval from the previous block Each condition was consisted of three continuous blocks and the order of the contingent-reward condition and the random-reward condition was counterbalanced between the participants Both in the contingent-reward condition and in the random-reward condition the advantageous and disadvantageous stimuli were counter-balanced between participants and the same stimulus was delivered as an advantageous stimulus in all blocks for a participant Blocks for the control condition were placed in the 1st and 5th block such that a control condition was followed by either blocks of the contingent-reward condition or blocks of the random-reward condition The contingency between stimuli and outcomes in each control block was matched to the in the following experimental blocks; i.e. 70:30% gain/loss mapping to stimuli in one control block and 50:50% gain/loss mapping to stimuli in the other control block Participants were told that gain and loss in the control conditions would also influence the money paid for participation PET scanning to collect rCBF data was conducted in each block and TPR) were measured for 2 min before each block as baseline and for 4 min during the task For measurement of plasma catecholamine (epinephrine and norepinephrine) blood samples were taken using a heparinized 22-gage butterfly catheter placed in the antecubital vein of the right forearm for 1 min just before the baseline period of measurement of cardiovascular parameters and for the last 1 min of each block Although participants were told that their payment would depend on their performance all participants were paid 15,000 JPY (140 USD) for participation Task performance was evaluated in two behavioral indexes: response bias and reward acquisition. Response bias means the rate of choice of the advantageous stimulus. Reward acquisition was defined as the rate of getting gain regardless of choice of advantageous or disadvantageous stimulus. Following our previous study (Ohira et al., 2013), Shannon's (1948) entropy as an index of exploration was calculated from data of participants' decisions the action is a choice of the same stimulus that was chosen in the previous trial or that of another stimulus that was not chosen in the previous trial (Stay or Shift) The state is an outcome (gain or loss) in the previous trial the conditional probability P(a|S) is calculated as follows: where Num(a|S) is the number of Stay or Shift (a) under a state S and Num(k|S) is the number of total choices k under a state S The constant c was introduced to stabilize the calculated probability four conditional probabilities were calculated: (1) Stay (choice of the same stimulus chosen in the previous trial) when gain was given in the previous trial (2) Stay when loss was given in the previous trial (3) Shift (choice of different stimulus not chosen in the previous trial) when gain was given in the previous trial and (4) Shift when loss was given in the previous trial entropy calculated by this formula reflects the degree of deviation from dependence of a choice on the outcome of the previous trial If a participant chooses the same stimulus regardless of whether it is advantageous or disadvantageous in all trials but H will not be 0 by the effect of the constant c) If a participant always chooses the same stimulus as the previous trial when gain was given in the previous trial and shifts the choice when loss was given in the previous trial (the Win-Stay These patterns of decision-making can be regarded as fixed strategies independently from task performance reflected by response bias and reward acquisition if a participant chooses a stimulus totally independently from the outcome in the previous trial in all trials (random choice) entropy H will be a maximum (approaching to 1) and entropy were determined at each block of the contingent-reward and random-reward conditions We recorded MBP and HR by using the finger cuff of a Portapres Model 2 (Finapres Medical Systems Inc. The Netherlands) which was attached to the third finger of the dominant arm of each participant HR was also measured and analyzed by using photoplethysmography using the Portapres at a sampling rate of 200 Hz and the Beatfast software using a model flow TPR was obtained by analyzing the sampled arterial pressure waveforms with the Beatfast software and TPR were calculated for 2 min just before the task as baseline and during 4 min of the task in each block We then calculated the ratio of LF to HF (LF/HF) which reflects the sympatho-vagal balance (relative increase of sympathetic activity to parasympathetic activity) (Task Force of the European Society of Cardiology The North American Society of Pacing and Electrophysiology Blood samples were anticoagulated with ethylenediamine tetra-acetate Then the plasma was removed and frozen at −80°C for storage until the analysis Epinephrine and norepinephrine in plasma were measured by using high performance liquid chromatography and the recovery rate for all amines as evaluated with a dihydroxybenzylamine standard The intra-assay coefficient of variation was less than 5% for measurement of epinephrine and the inter-assay variations were less than 6% for measurement of norepinephrine When significant interactions were found by ANOVAs post-hoc analyses using Tukey's test (p < 0.05) were performed to detect combinations of data points which showed significant differences to explore relational structures within the behavioral and autonomic indices correlations within the behavioral indices (response bias and entropy) and change scores of autonomic indices (MBP and the HF component of HRV) were examined we performed step-wise regression analyses by using change scores of autonomic indices (MBP and the HF component of HRV) as independent variables in the contingent-reward condition and random-reward condition to examine the effects of sympathetic and parasympathetic parameters on entropy To calculate the change scores of the autonomic indices subtractions of the autonomic indices at baseline from values during the task in each block were conducted first Mean scores of the subtracted values within three blocks were then calculated for each indices both in the contingent-reward condition and in the random-reward condition The distribution of rCBF was measured by using a PET scanner (General Electric Advance NXi) in a high-sensitivity three-dimensional mode at each block A venous catheter for administering the tracer was inserted in an antecubital fossa vein in the left forearm of each participant The participant's head was fixed in an inflatable plastic head-holder that prevented head movement a transmission scan using a rotating 68germanium pin source was completed for 10 min 370-MBq bolus injection was started 60 s after initiation of each block Scanning was started 30 s after initiation of bolus injection and continued for 60 s The integrated radioactivity accumulated during the scanning was measured as the index of rCBF Eight scans were performed for each participant and the 15 min interval between successive scans was placed for clearance of radioactive levels A Hanning filter was used to reconstruct images into 35 planes with 4.5 mm thickness and a resolution of 2 × 2 mm (full width half maximum) We used SPM 99 (Friston et al., 1995) implemented in Matlab (v USA) for spatial preprocessing and statistical analyses of PET images the images were realigned by using sinc interpolation to remove artifacts the images were transformed into a standard stereotactic space the images were corrected for whole brain global blood flow by proportional scaling and smoothed using a Gaussian kernel to a final in-plane resolution of 8 mm at full width at half maximum Means (Ms) and standard errors (SEs) of response bias, reward acquisition, and entropy are shown in Table 1 A main effect of Condition for response bias [F(1 but neither a main effect of Block nor an interaction of Condition and Block was significant (F < 1.78) reward acquisition showed similar results as response bias a significant main effect of Condition [F(1 No significant effect was obtained on entropy (F < 1.00 Means and standard errors of behavioral indices Entropy was not correlated with response bias and reward acquisition in either condition [see Table 3; r(14) < 0.17 indicating that entropy is independent of performance of the decision-making task Ms and SEs of autonomic indices in each condition are shown in Table 2 ANOVA showed significant main effects of Condition and Period [F(1 suggesting that MBP in the contingent-reward condition was higher than that in the random-reward condition and that MBP elevated during the task compared to the baseline a significant main effect of Condition was shown [F(1 indicating that HR was higher in the contingent-reward condition compared with that in the random-reward condition TPR showed a significant interaction of Condition and Block [F(1 indicating that TPR was markedly increased in the random-reward condition but not in the contingent-reward condition A significant main effect of Condition in the HF component of HRV [F(1 suggesting that parasympathetic activity was more enhanced in the random-reward condition compared with that in the contingent-reward condition The LF/HF ratio of HRV showed no significant effects in either condition Means and standard errors of autonomic indices epinephrine showed a significant main effect of Condition [F(1 indicating that overall concentration of epinephrine was higher in the contingent-reward condition compared with that in the random-reward condition a significant interaction of Condition and Period was observed [F(1 Further it was indicated that norepinephrine concentration did not change between baseline and task periods in the contingent-reward condition while it was reduced during the task period in the random-reward condition Table 3 shows the correlations within behavioral and autonomic indices in both conditions entropy was positively correlated with changes of norepinephrine while response bias and reward acquisition were positively correlated with the HF component of HRV The HF component of HRV and the LF/HF ratio of HRV were negatively correlated suggesting that these parasympathetic and sympathetic indices worked in opposition to each other no significant relations were found between autonomic indices and behavioral indices HR was correlated positively with norepinephrine and negatively with TPR The HF component of HRV and the LF/HF ratio of HRV were also negatively correlated in this condition Correlations among behavioral and autonomic indices In the contingent-reward condition, a hierarchical regression analysis on entropy adopted a significant model [adjusted R2 = 0.44, F(2,13) = 6.94, p < 0.01], including norepinephrine and the LF/HF ratio of HRV as independent variables. The analysis also revealed that the change of norepinephrine as an index of sympathetic activity (β = 0.65, p < 0.05), but not the LF/HF ratio of HRV, significantly and positively contributed to entropy (Figure 2) the regression model was not significant [F(7,8) = 0.53 Correlation between change of norepinephrine and entropy in decision-making in the contingent-reward condition No correlation between change of norepinephrine and entropy was observed in the random-reward condition The vertical axis of the graph represents change of norepinephrine between before and after blocks of the task (i.e. positive/negative values mean increase/decrease of norepinephrine from the baseline in each block) the HF component of HRV as an index of cardiovagal inhibitory control was positively correlated with rCBF in the rostral ACC and right DLPFC in the random-reward condition but not in the contingent-reward condition Other autonomic indices showed no significant correlations in either condition Significant negative correlations between regional cerebral blood flow and change of norepinephrine in the contingent-reward condition Significant negative correlations between rCBF and norepinephrine in contingent-reward condition (A) Significant negative correlations between regional cerebral blood flow and entropy in decision-making in the contingent-reward condition (B) Significant positive correlations between regional cerebral blood flow and entropy in decision-making in the random-reward condition Significant negative correlations between rCBF and entropy in the contingent-reward condition and positive correlations between rCBF and entropy in the random-reward condition Correlational activity between the right insula (red circle) and other brain regions in the contingent-reward condition and entropy described above were examined in each condition formal statistical tests did not show any significant differences of the correlations between the contingent-reward condition and the random-reward condition results of the present study must be interpreted with a caution suggesting reliability of this index of exploration while the somatosensory signals driven by cardiovascular responses might play relatively minor roles in modulation of exploration catecholamine and other sympathetic indices did not affect response bias or reward acquisition suggesting that sympathetic activity is associated with exploration in decision-making but not with currently appropriate strategies (exploitation) suggesting that participants did not abandon efforts for the task and did not just adopt simple strategies of decision-making (e.g. Such neural processes likely canceled the effects of sympathetic activity on exploration in the random-reward condition It has been well known that activity of dopamine neurons in the midbrain-striatum neural circuit is the largest when uncertainty of delivery of reward is the highest (Fiorillo et al., 2003). This classical finding is consistent with the result of our previous study (Ohira et al., 2010) showing that activation of the dorsal striatum which is a main target area of projection of midbrain dopamine neurons was higher in the random-reward condition (higher uncertainty) than in the contingent-reward condition (lower uncertainty) entropy showed no correlation with activation of the midbrain-striatum dopamine circuit in both conditions in this study while activity in dopamine neurons might involve coding and evaluation of uncertainty in decision-making the neural networks including the insula and DLPFC might involve modulation of exploration in decision-making on the basis of such coding and evaluation of uncertainty The present study suggested that uncertainty of the situation of decision-making might also be an additional moderator of the relationship and Kenta Kimura contributed to study design Naho Ichikawa and Kenta Kimura contributed to measurements and analyses of behavioral and autonomic data Seisuke Fukuyama was responsible for data-acquisition in neuroimaging using PET with the supervision of Jun Shinoda and Jitsuhiro Yamada Hideki Ohira interpreted the data with helps of Naho Ichikawa and Kenta Kimura All authors approved the final version of the paper This study was supported by a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (No 16330136) and a Grant-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Research Area) 2010 (No 4102-21119003) from the Ministry of Education Circuitry and functional aspects of the insular lobe in primates including humans Rostrolateral prefrontal cortex and individual differences in uncertainty-driven exploration Response randomization of one- and two-person rock-paper-scissors games in individuals with schizophrenia The somatic marker hypothesis: a neural theory of economic decision CrossRef Full Text | Google Scholar Different contributions of the human 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exploration in decision-making: further evidence for involvement of insula Received: 10 July 2014; Accepted: 16 October 2014; Published online: 10 November 2014 Copyright © 2014 Ohira, Ichikawa, Kimura, Fukuyama, Shinoda and Yamada. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Hideki Ohira, Department of Psychology, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan e-mail:b2hpcmFAbGl0Lm5hZ295YS11LmFjLmpw 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 SBI Holdings announced Friday the dissolution of its partnership with Taiwan's Powerchip Semiconductor Manufacturing Corp for the construction of a chip factory in Miyagi Prefecture The ailing major Taiwanese foundry has told that maintaining the tie-up became too difficult SBI said.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); But the Japanese online financial group vowed to carry out the semiconductor plant project by finding new partners PSMC and the Miyagi Prefectural Government came up with a basic agreement to construct a chipmaking plant in the village of Ohira using central government subsidies and aiming to launch production of semiconductors chiefly for automobiles in 2027 but we want to launch a semiconductor business in Miyagi that is bigger than initially planned," SBI said "I could not believe for a while the dissolution of the tie-up as I had watched the relationship of trust between the two companies closely since the prefecture started promoting the plant construction." Sponsored contents planned and edited by JT Media Enterprise Division 2024年6月12日（水）、東京都内にて第40回大平正芳記念賞贈呈式が行われ、帝京大学法学部政治学科講師 山口航の著書「冷戦終焉期の日米関係－分化する総合安全保障－」が大平正芳記念賞を受賞しました。 本賞は、公益財団法人大平正芳記念財団によって1985年に創設され、「環太平洋連帯構想」の発展に貢献する政治･経済･文化･科学技術に関する優れた著作に授与されるものです。山口講師の今後のますますの活躍が期待されます。 大平正芳記念賞の詳細はこちら 法学部についてはこちら 山口講師についてはこちら トピックス一覧へ While Masaryk might have never set foot in Mexico Ohira did become the first Japanese Prime Minister to do so in 1980 the park already existed and was known as Parque de la Pagoda (Park of the Pagoda) the park featured a "pagoda"-like structure as well as an East-Asian-inspired bridge after the park had been subject to general abandonment the park was renamed and remodeled in his honor that same year The project created more buildings that contained more specific Japanese features such as torii gates until the park's most recent reconstruction in 2015 Tomonori Ohira attended the rededication of the park Along with other members of the Japanese-Mexican community they donated one of the park's newest features: several cherry trees Given its location near Mexico City's major filmmaking center known as Churubusco Studios the park was popular with film stars and even appeared in some movies in the 1940s and 1950s the park will be able to have a second life after many-storied decades an engineering marvel from the early 20th-century still stands Constructed from the ruins of a former glass factory this park incorporates the old with the new The tabby ruin is Savannah’s oldest surviving structure fish-shaped water spouts guard Savannah’s historic facades A historic Puebla estate featuring a striking castle A century-old ancestral home was home to some of the key figures of the Philippine Revolution The World Heritage-designated site of Japan’s industrial revolution Malta’s largest cemetery is a Victorian-era architectural masterpiece We are excited to announce that the 2018 Masayoshi Ohira Memorial Prize has been awarded to “Dilemmas of a Trading Nation” by Foreign Policy Senior Fellow Mireya Solís published by the Brookings Institution Press in August 2017 Debate over the future direction of trade has catapulted to the top of national discourse in both Japan and the United States and each country will face difficult choices in charting their paths ahead as trading partners In “Dilemmas of a Trading Nation,” Solís chronicles those challenges and describes how Japan and the U.S has long aimed for greater decisiveness that would allow it to move away from a fragmented policymaking system favoring the status quo and acquire a larger voice in trade negotiations must confront a fraying domestic consensus in favor of internationalism that is essential to sustain its leadership role as a champion of free trade At stake could be a bilateral alliance central to America’s diplomacy in Asia and the reaffirmation of a rules-based international order that has been a source of postwar stability you can watch Mireya Solís discuss her book at a Brookings event with Foreign Policy Senior Fellow Richard C vice president and managing director of the Asia Society Policy Institute For more information on “Dilemmas of a Trading Nation” and to purchase your own copy, click here Leaf KYOTO Store Locations The owner of that famous restaurant has opened a counter barbecue restaurant in Gojo that attracts the enthusiastic attention of meat lovers [NIKUNINCHU] a meat professional who runs a butcher store and a popular yakiniku restaurant [Taishoen] in Kyoto opened this restaurant as his "ultimate goal We have created the ideal of what we would like to have in a restaurant like this Ohira selects a wide variety of delicious meats without sticking to any particular brand of beef Some parts of the meat are almost always served allowing diners to experience the full appeal of beef Since the restaurant is only open to customers seated at the counter you can enjoy the meat while listening to Mr you can add hormone (hormone) to your meal but the full course with its excellent cost performance will satisfy your appetite All photos are from the omakase course at market prices (approximate prices range from 8,000 yen to 10,000 yen) a part of thigh meat cooked slowly at low temperature to prevent the flavor from escaping It is sliced thin and has a moderate chewy texture and rich flavor making it a perfect accompaniment to alcoholic beverages while the namul is a perfect chopstick rest The melt-in-your-mouth harami yukaye matches the thick sauce Meat lovers love the fact that they can enjoy many rare meats as appetizers 8 to 10 different parts of the restaurant appear a lineup recommended by the owner who purchased the items that day This is in part because Ohira wasn't as much around for the Super Street Fighter 2 Turbo days (despite being exceptional and winning a major ST tournament before his sudden retirement) but instead remembers glory days from Hyper Fighting and Champion Edition Ohira was the absolute best in the United States, untouchable even by contenders like Watson and Jeff Schaefer, both of whom help to tell the tale of their unbeatable rival in Great Big Story's "The King of Street Fighter II Who Disappeared" mini documentary We've seen dominant players over the years TSM|ZeRo and PG|Punk may not have quite the same unquestionable aura that surrounded Ohira's Imagine how the fighting game community might we heard that either of the two players mentioned above suddenly stopped going to events altogether That was the reality for the community back in the 90's when the man to beat suddenly disappeared from all competition legacy and why he decided to leave it all on a dime in Great Big Story's video below Easy Reader News & Peninsula Magazine is your trusted source for hyperlocal news and stories from the South Bay and Palos Verdes Peninsula Stay informed—get the top local stories delivered straight to your inbox Terms of Service | Privacy Policy | Legal Notices Metrics details Here we identified 2′-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea Up47 confers thermal stability and nuclease resistance to tRNAs Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47 The 2′-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation which encodes an archaeal RNA kinase responsible for Up47 formation Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions Source data Here we report the identification of 2′-phosphouridine (Up) in tRNAs is the first known instance of internal RNA phosphorylation structural and genetic studies showed that Up47 is a reversible RNA modification and confers thermal stability to tRNA thereby contributing to cellular thermotolerance the tRNA without Up47 gradually melted at around 65 °C while its hyperchromicity increased with temperature whereas the tRNA with Up47 remained stable even at 70 °C The Tm values of the tRNA with and without Up47 were 85.8 ± 0.5 °C and 79.2 ± 0.5 °C These observations clearly demonstrate that a single Up47 modification increases the thermal stability of tRNAVal3 by 6.6 °C We next performed an RNase probing experiment to examine the nuclease resistance of tRNA with and without Up47. S. tokodaii tRNAVal3 and its Tpt1p-treated form were labelled with 32P at their 3′ termini and were probed with RNase I at 65 °C (Fig. 1g) the intact tRNAs were gradually degraded into RNA fragments the Tpt1p-treated tRNA was degraded more rapidly indicating that the tRNA without Up47 was highly sensitive to RNase I This observation demonstrates that Up47 stabilizes and protects tRNAs from nucleolytic degradation The residues at position 47 are shown in stick representation The V-loop structures of intact tRNA molecule A (blue) Tpt1p-treated tRNA molecule A (magenta) and Tpt1p-treated tRNA molecule B (orange) are overlaid These findings imply that Up47 stabilizes the metastable tRNA core structure with a non-canonical base triple during thermal denaturation Venn diagram showing unique and shared genes among the Bacteria (E cerevisiae and Arabidopsis thaliana) and Archaea (Methanosarcina acetivorans kodakarensis and Nitrososphaera viennensis) domains possessing (+) or lacking (–) Up47 The pale red area includes genes unique to archaea having Up47 Comparative genomic analysis performed to narrow down the candidate genes responsible for Up47 modification LC–MS nucleotide analysis of tRNA fractions from wild-type (WT KU216) (left) and Δtk2051 (right) strains of T The upper panel shows the UV trace at 254 nm The lower panel shows the XIC for the proton adduct of the dimer pUpm5C (m/z 724.1 Growth measurement (OD600) of wild-type (KU216) (open circles) ΔarkI (closed circles) and ΔarkIΔqueE (triangles) strains of T Data represent the average values of technical triplicates ± s.d In vitro reconstitution of Up47 with recombinant TkArkI in the presence (right panels) or absence (left panels) of ATP XICs show the sum of monovalent and divalent negative ions from RNase T1-digested fragments containing Up47 (upper panels) or U47 (lower panels) Kinetic measurements of in vitro Up47 formation by TkArkI The initial velocity (Vi) of the phosphorylation reaction was measured at the indicated concentrations of tRNA (left) and ATP (right) The Km and Vmax values are shown below each graph Source data This finding indicates that Up47 and G+15 cooperatively stabilize the tRNA core structure at high temperatures Overall structure of TkArkI with five features highlighted: the N-terminal lobe (residues 1–30 and 39–109; blue) residues 128–140; pink) and subdomain VII (metal-binding loop Guanosine observed in a putative ATP-binding pocket is shown in ball-and-stick representation Subdomains of TkArkI showing the locations of mutations examined in this study Colour codes for each feature are the same as in a Close-up view of the putative ATP-binding pocket in TkArkI Residues for which mutations were examined in this study are indicated Guanosine is shown in ball-and-stick representation Schematic diagram of guanosine binding in the putative ATP-binding pocket Predicted interactions are indicated with dashed lines catalytic loop and metal-binding loop are shown with bold lines Positively and negatively charged areas are coloured in blue and red The surface potential is described as dimensionless numbers Positions of mutation sites indicated in the crystal structure Relative activities of a series of TkArkI mutants normalized against the activity of wild-type TkArkI Source data TkArkI might sense the cellular energy status and guanosine binding to TkArkI might have a regulatory role in Up47 formation Given that TkArkI was a recombinant protein expressed in Escherichia coli we cannot rule out the possibility that guanosine was an artificial ligand bound to the inactive form of TkArkI It is unclear whether guanosine actually binds to TkArkI within archaeal cells at high growth temperatures Instead of the missing subdomain VIII involved in recognition of substrate peptide in ePKs (mouse PRKACA) the basic surface in the C-terminal lobe might bind substrate tRNA through electrostatic interaction K51A and E65A substitutions markedly reduced activity whereas the R95A substitution caused a mild reduction in activity a severe reduction in activity was observed in the H130A Q132A and K137A mutants with substitutions in the catalytic loop No activity was detected for the D149A mutant in which the mutated residue is in subdomain VII involved in metal binding These results clearly confirm the critical role of catalytic residues in kinase activity The N160A and T162A substitutions in subdomain IX led to decreased activity finding a severe reduction in activity with the K201A substitution and a mild reduction with the Y200A and R202A substitutions These observations indicate the importance of the conserved residues and positively charged surface in the C-terminal lobe natural RNA substrates with 2′-phosphate have not been identified UpCACAGp (fourth panels) and m5UΨCGp as a control fragment (bottom panels) Relative abundance of the Up47-containing fragments was measured in E coli strains in which TkKptA was not expressed (left panels) or where TkKptA expression was induced with 10 μM (middle panels) or 100 μM (right panels) IPTG Relative peak intensity of each Up47-containing fragment detected in the tRNA fraction from E Source data these data demonstrate that Tpt1/KptA homologues dephosphorylate Up47 of tRNAs in vivo It is interesting that similar functions are evolutionarily conserved in different V-loop modifications across the domains of life Up47 does not fix the tRNA rigidly but rather maintains a metastable structure when the tRNA core thermally fluctuates thereby preventing further collapse of the core structure as well as increasing the chance of return to the canonical structure The bacterial AQ578 family might have been acquired by horizontal gene transfer of the archaeal homologue suggesting that the strategy of stabilizing tRNA by internal phosphorylation might have spread across the domains of life we propose a new mechanism of tRNA stabilization mediated by two distinct but concerted actions of tRNA modification Reversible Up47 modification would be beneficial for hyperthermophilic organisms in extremely harsh environments Future studies will be necessary to investigate Up47 frequency and the expression levels of ArkI and KptA under various growth conditions including during rapid changes in growth temperature and introduction of environmental stresses Methanosarcina acetivorans C2A and Thermoplasma acidophilum were kindly provided by T 19564) were obtained from Japan Collection of Microorganisms RIKEN BRC which is participating in the National BioResource Project of the MEXT acidocaldarius were cultured at 80 °C in JCM medium no 0.03 mg l–1 VOSO4·H2O and 0.01 mg l–1 CoSO4·7H2O (adjusted to pH 2.5 with H2SO4) solfataricus was cultured at 80 °C in JCM medium no 0.03 mg l–1 VOSO4·H2O and 0.01 mg l–1 CoSO4·7H2O (adjusted to pH 4.0 with H2SO4) pernix was cultured at 90 °C in JCM medium no 0.7 g l–1 KCl and 0.1 g l–1 CaCl2·2H2O (adjusted to pH 7.0 with NaOH) oguniense was cultured at 90 °C in JCM medium no 165 with addition of 1.0 g l–1 Na2S2O3·5H2O (adjusted to pH 7.25 with NaOH) viennensis was cultured at 42 °C in JCM medium no 1.0 ml l–1 modified trace element mixture (30 mg l–1 H3BO3 1.0 ml l–1 vitamin solution (20 mg l–1 biotin 50 mg l–1 p-aminobenzoic acid and 2 g l–1 choline chloride (adjusted to pH 7.0 with KOH)) 1.0 ml l–1 7.5 mM EDTA·Na·Fe(III) solution (pH 7.0) 10 ml l–1 HEPES solution (238.4 g l–1 HEPES (free acid) and 24 g l–1 NaOH) 1.0 ml l–1 1 M NH4Cl solution and 1.0 ml l–1 1 M sodium pyruvate solution (adjusted to pH 7.6 with NaOH) 2.0 g l–1 elemental sulfur and 0.1% (wt/vol) resazurin 2.0 ml l–1 polysulfide solution (20% elemental sulfur in 67% Na2S·9H2O solution) was added instead of elemental sulfur and the media were solidified with 1.0% Gelrite (Fujifilm Wako Pure Chemical Corporation) When pyrF-negative transformants were selected0 We used ASW-YT-S0 medium for standard cultivation MA-YT-Pyr medium for growth comparisons and ASW-AA-S0 medium for construction of the gene knockout strain For small-scale preparation (~100-ml culture) archaeal cells were resuspended in 3 ml solution D (4 M guanidine thiocyanate 0.5% (wt/vol) N-lauroylsarcosine sodium salt and 1 mM 2-mercaptoethanol) and mixed with an equal volume of water-saturated phenol and 1/10 volume of 3 M sodium acetate (pH 5.3) The mixture was shaken for 1 h on ice and mixed with 1/5 volume of chloroform followed by centrifugation at 8,000g for 10 min at 4 °C The supernatant was collected and mixed with an equal volume of chloroform Total RNA was obtained from the resultant supernatant by isopropanol precipitation The total RNA prepared in this manner was separated by 10% denaturing PAGE followed by staining with SYBR Gold or toluidine blue The visualized tRNA fraction including class I and class II tRNAs was cut out and eluted from the gel slice with elution buffer (0.3 M sodium acetate (pH 5.3) and 0.1% (wt/vol) SDS) followed by filtration to remove the gel pieces and ethanol precipitation for RNA-MS analysis of the tRNA fraction For large-scale preparation of tRNA fractions from S cell pellets (53 g) were resuspended in 530 ml solution D and then mixed with 53 ml of 3 M sodium acetate (pH 5.3) and 425 ml neutralized phenol The mixture was shaken for 1 h on ice to which 106 ml chloroform/isoamyl alcohol (49:1) was added followed by centrifugation at 4,500g for 20 min at 4 °C The supernatant was collected and mixed with 106 ml chloroform/isoamyl alcohol (49:1) followed by centrifugation at 4,500g for 15 min at 4 °C The aqueous phase was collected and then subjected to isopropanol precipitation The collected RNA was resuspended in 53 ml water and mixed with 80 ml TriPure Isolation Reagent (Roche) followed by centrifugation at 10,000g for 20 min at 4 °C The supernatant was collected and mixed with 36 ml chloroform/isoamyl alcohol (49:1) followed by centrifugation at 10,000g for 10 min at 4 °C The aqueous phase was collected and precipitated with isopropanol The prepared total RNA (608 mg) was dissolved in 250 ml of buffer consisting of 20 mM HEPES-KOH (pH 7.6) 200 mM NaCl and 1 mM DTT and then loaded on a DEAE Sepharose Fast Flow column (320-ml beads) and fractionated with a gradient of NaCl from 200 to 500 mM Fractions containing tRNA were collected by isopropanol precipitation Approximately 200 absorbance at 260 nm (A260) units of the S tokodaii tRNA fraction was subjected to RCC The isolation procedure was carried out as follows: hybridization at 66 °C in 6× NHE buffer (30 mM HEPES-KOH (pH 7.5) washing at 50 °C with 0.1× NHE buffer (0.5 mM HEPES-KOH (pH 7.5) 0.5 mM DTT) and elution at 72 °C with 0.1× NHE buffer Eluted tRNAs were recovered by ethanol precipitation Mature and precursor tRNAs were separated by 10% denaturing PAGE and stained with SYBR Gold Visualized bands of mature and precursor tRNAs were cut out and eluted from the gel slices with elution buffer followed by filtration to remove the gel pieces and precipitation with ethanol The isolated tRNAVal3 was further purified by anion exchange chromatography to completely remove tRNAVal2 The samples were chromatographed with a ZIC-cHILIC column (3-μm particle size 2.1 × 150 mm; Merck) and eluted with 5 mM ammonium acetate (pH 5.3) (solvent A) and acetonitrile (solvent B) at a flow rate of 100 μl min–1 with a multistep linear gradient: 90–50% solvent B for 30 min 50–90% solvent B for 5 min and then initialization with 90% solvent B The chromatographed eluent was directly introduced into the electrospray ionization source of the Q Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Fisher Scientific) 800 ng (24 pmol) of the tRNA fraction or individual tRNA was digested with 0.09 U nuclease P1 in 20 mM ammonium acetate (pH 5.2) at 50 °C for 1 h and then mixed with 9 volumes of acetonitrile for LC–MS The digests were chromatographed with a ZIC-cHILIC column and analysed by Q Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Fisher Scientific) or LTQ Orbitrap XL (Thermo Fisher Scientific) with a multistep linear gradient: 90–50% solvent B for 30 min The acquired LC–MS data were analysed using Xcalibur 4.1 (Thermo Fisher Scientific) and were visualized with Canvas X (Nihon poladigital k.k) tokodaii tRNA fraction was completely digested with nuclease P1 Digests containing pN324m5C dinucleotide were subjected to periodate oxidation with 10 mM NaIO4 for 1 h on ice in the dark The reaction was stopped by addition of 1 M l-rhamnose and incubation for 30 min an equal volume of 2 M lysine-HCl (pH 8.5) was added and the sample was incubated at 45 °C for 90 min The product containing pN324p was then subjected to anion exchange chromatography with a Q Sepharose Fast Flow column (GE Healthcare) equilibrated with 20 mM triethylammonium bicarbonate (TEAB) (pH 8.2) The eluate with 2 M TEAB was collected and dried by evaporation in vacuo The pellet was dissolved with water and mixed with an equal volume of chloroform followed by centrifugation at 20,000g for 5 min at 4 °C The supernatant was recovered and dried again The resultant digest was mixed with 9 volumes of acetonitrile and subjected to LC–MS/MS using an LCQ-Advantage ion trap mass spectrometer (Thermo Scientific) equipped with an electrospray ionization source and an HP1100 LC system (Agilent Technologies) the digest was chromatographed with a ZIC-HILIC column (3.5 μm; pore size 2.1 × 150 mm; Merck) and eluted with 5 mM formic acid (pH 3.4) (solvent A) and acetonitrile (solvent B) at a flow rate of 100 μl min–1 with a multistep gradient: 90–70% solvent B for 25 min 10% solvent B for 5 min and then initialized with 90% solvent B coli BL21(DE3) or Rosetta2(DE3) cells transformed with the pE-SUMO-TEV vector carrying each arkI gene were cultured in 250 ml or 1 l of LB containing 50 μg ml–1 kanamycin and 20 μg ml–1 chloramphenicol when necessary His6–SUMO-tagged recombinant protein was expressed at 37 °C for 3–4 h by induction with 0.1 or 1 mM IPTG or 2% (wt/vol) lactose when the cells reached OD610 = 0.4–0.6 oguniense ArkI was expressed in cells cultured overnight at 18 °C The collected cells were resuspended in lysis buffer (50 mM HEPES-KOH (pH 8.0) 1 mM 2-mercaptoethanol and 1 mM PMSF) and disrupted by sonication followed by centrifugation at 15,000g for 15 min at 4 °C The supernatant was boiled at 60 °C for 20 min (for ArkI homologues from T aeolicus) and centrifuged at 15,000g for 15 min at 4 °C The recombinant protein was affinity captured on an Ni-Sepharose 6 Fast Flow column (GE Healthcare) and then eluted with lysis buffer containing 300 mM imidazole followed by gel filtration with a PD-10 column (GE Healthcare) to remove the imidazole viennensis ArkI was purified using a HisTrap column (GE Healthcare) with a linear gradient of 0–500 mM imidazole followed by dialysis using a Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific) to remove imidazole The purified protein was subjected to Ulp1 digestion at 4 °C overnight to cleave the His6–SUMO tag and then passed through a Ni-Sepharose 6 Fast Flow column to remove the tag PCC7376 (LeArkI) aggregated following tag removal His6–SUMO tag-fused proteins of these homologues were used for the phosphorylation assay Purified protein was quantified by the Bradford method using BSA as a standard coli BL21(DE3) strain carrying pE-SUMO-TkArkI was cultured in 2 l of LB containing 50 μg ml–1 kanamycin and TkArkI was expressed at 25 °C overnight by induction with 0.1 mM IPTG when the cells reached OD610 = 0.4 The cells were collected and disrupted by sonication in lysis buffer (50 mM HEPES-KOH (pH 8.0) The protein was purified using a HisTrap column with a linear gradient of 20–520 mM imidazole Fractions containing TkArkI were pooled and subjected to Ulp1 digestion at 4 °C overnight to cleave the tag followed by passage through a Ni-Sepharose 6 Fast Flow column to remove the tag fragment The flow-through fraction was filtered through a 0.45-μm PVDF membrane to remove the resin The protein was further purified by affinity chromatography with a HiTrap Heparin HP column (GE Healthcare) using a linear gradient of 150–1,150 mM KCl TkArkI was further purified by size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) with buffer containing 20 mM Tris-HCl (pH 8.0) 150 mM NaCl and 10 mM 2-mercaptoethanol and then concentrated to 5.74 mg ml–1 and stored at –80 °C Recombinant Tpt1p was purified as described above Individual tRNAs or the tRNA fraction was incubated for 3 h at 30 °C in a reaction mixture (25 μl) consisting of 20 mM Tris-HCl (pH 7.4) 0.9 μM tRNA and 0.1 μg μl–1 recombinant Tpt1p The tRNA was extracted by phenol/chloroform treatment and recovered by ethanol precipitation followed by desalting with Centri-Sep spin columns (Princeton Separations) tokodaii tRNAVal3 (202.5 μg) was dephosphorylated by yeast Tpt1p in a 200-μl reaction mixture tokodaii tRNAVal3 (25 pmol) with or without Up47 was dissolved in degassed buffer consisting of 50 mM Tris-HCl (pH 7.4) 100 mM NaCl and 1 mM MgCl2 and incubated at 80 °C for 5 min followed by cooling to 25 °C at a rate of 0.1 °C s–1 The samples were placed onto a Type 8 multi-micro UV quartz cell (path length The hyperchromicity of tRNA was monitored on a UV–visible light spectrophotometer (V-630 The gradients were as follows: 25 °C for 30 s 40 °C for 5 min and 40–105 °C at 0.5 °C min–1 The Tm was calculated using Spectra Manager v2 (JASCO) Melting curves were generated using Microsoft Excel tokodaii tRNAVal3 (25 pmol) with or without Up47 was labelled with 32P at the 3′ terminus by ligation with [5′-32P]cytidine 3′,5′-bisphosphate (PerkinElmer) The labelled tRNA was separated on a 7.5% (wt/vol) polyacrylamide gel containing 7 M urea 1× TBE and 10% (vol/vol) glycerol and was purified by gel extraction tokodaii tRNA fraction as a carrier to a concentration of 100,000 counts per minute (c.p.m.) per A260 unit and was precipitated with ethanol The pellet was dissolved in water to a concentration of 0.1 A260 units per μl 10,000 c.p.m.) was incubated at 65 °C in a reaction mixture consisting of 10 mM HEPES-KOH (pH 7.6) 100 mM NaCl and 0.1 U μl–1 RNase I (Promega) aliquots were taken from the mixture and mixed well with chilled phenol/chloroform/isoamyl alcohol (25:24:1 followed by centrifugation at 15,000g for 15 min at 4 °C The supernatant was collected and treated with an equal volume of chloroform followed by centrifugation at 15,000g for 5 min at 4 °C The supernatant was mixed with 2× loading solution (2× TBE 0.05% (wt/vol) xylene cyanol and 0.05% (wt/vol) bromophenol blue) and subjected to 10% denaturing PAGE and radioactivity was visualized by using an FLA-7000 imaging analyser (Fujifilm) Graphs were generated using Microsoft Excel was refolded in annealing buffer (50 mM HEPES-KOH (pH 7.6) 5 mM MgCl2 and 1 mM DTT) by incubation for 5 min at 80 °C and cooling to 25 °C with a rate of 0.1 °C s–1 tRNAVal3 was further purified by anion exchange chromatography using a Mono Q 5/50 GL column (GE Healthcare) with a linear gradient of 200–1,000 mM NaCl dissolved in water and precipitated with ethanol Tpt1p-treated tRNAVal3 was prepared with the same procedure as described above The purified tRNA was dissolved in buffer consisting of 10 mM Tris-HCl (pH 7.1) and 5 mM MgCl2 to a concentration of 50 μM One microlitre of tRNA solution was mixed with 1 μl Natrix 2 no 40 mM sodium cacodylate·3H2O (pH 7.0) and 30% (vol/vol) MPD) (Hampton Research) on silicon-coated glass and crystalized by the hanging drop vapor diffusion method at 20 °C The concentration of TkArkI was adjusted to 5 mg ml–1 before crystallization One microlitre of the protein solution was mixed with 0.5 μl reservoir solution TkArkI was crystallized by the hanging drop vapor diffusion method at 20 °C TkArkI purified by affinity chromatography with a HiTrap Heparin HP column (GE Healthcare) (100 pmol) was mixed with [15N]adenosine (10 pmol) and [15N]guanosine (10 pmol) as tracer molecules followed by addition of 4 volumes of methanol an equal volume of chloroform and 3 volumes of water and vigorous mixing The denatured protein was removed by centrifugation at 15,000g for 1 min at 4 °C The supernatant was dried in vacuo and dissolved in 20 μl water The tracer molecules were prepared by dephosphorylation of [15N]ATP and [15N]GTP as follows: 1,000 pmol each of [15N]ATP (Silantes) and [15N]GTP (Silantes) was treated with 0.04 U alkaline phosphatase (PAP BioDynamics Laboratory) in 20 mM ammonium acetate (pH 8.0) at 60 °C for 30 min ΔarkI and ΔarkI/queE::Tn strains were precultured in MA-YT-Pyr medium at 83 °C overnight and inoculated into 8 ml fresh MA-YT-Pyr medium with an initial OD600 of 0.01 and cell growth was monitored every 2 h by measuring OD600 with an S1200 diode array spectrophotometer The tRNAs were transcribed at 37 °C overnight in a reaction mixture consisting of 40 mM Tris-HCl (pH 7.5) followed by extraction with phenol/chloroform treatment and desalting with PD-10 columns (GE Healthcare) In vitro transcripts prepared in this way were separated by 10% denaturing PAGE The stained bands were cut out and eluted from the gel slice with elution buffer followed by filtration to remove the gel pieces and ethanol precipitation Up47 formation by TkArkI was carried out at 70 °C for 20 min in a reaction mixture (30 μl) containing 50 mM HEPES-KOH (pH 7.5) kodakarensis ΔarkI strain) and 1 μM TkArkI the tRNA was extracted by acidic phenol/chloroform desalted on a NAP-5 column (GE Healthcare) and precipitated with isopropanol the prepared tRNA was dialysed against water on a nitrocellulose membrane (0.025-μm VSWP 0.5 mM ATP or GTP was added to the reaction mixture and Up47 formation was performed with 0.5 μM TkArkI for 5 min The activities of TkArkI variants were measured by γ-phosphate transfer from [γ-32P]ATP to tRNA similarly to the kinetic studies of TkArkI (see below) tRNA phosphorylation was performed at 70 °C for 15 min in an 8-μl reaction mixture 4 μl of the reaction mixture was mixed with 4 μl of 2× loading solution resolved by 10% denaturing PAGE and exposed to an imaging plate to visualize radiolabelled RNA with an FLA-9000 imaging analyser (Fujifilm) The gel image was analysed using Multi Gauge (Fujifilm) Bar graphs with independent plots were prepared with R (R Foundation) the reaction was performed at 70 °C for 30 min in an 8-μl reaction mixture consisting of 50 mM HEPES-NaOH (pH 7.5) 100 μM [γ-32P]ATP (3,000 mCi mmol–1; PerkinElmer) 1.8 μM TkArkI and 50 ng μl–1 total RNA fraction (from the T and 4 μl of reaction mixture was mixed with 2× loading solution resolved by 10% denaturing PAGE and visualized as described above Formation of Up47 by other ArkI homologues was carried out at 70 °C for 30 min in a reaction mixture (30 μl) containing 50 mM PIPES-NaOH (pH 6.9) 1 μM tRNA transcript and 0.5 μM ArkI protein the reaction was carried out at 50 °C for 60 min Up47 formation was carried out in a reaction mixture (8 μl) containing 50 mM PIPES-NaOH (pH 6.9) 0.75 μM recombinant ArkI homologue (NpArkI the reaction mixture was mixed with 2× loading solution Dephosphorylation of Up47 by TkKptA was carried out at 60 °C for 1 h in a reaction mixture (30 μl) containing 20 mM Tris-HCl (pH 7.4) kodakarensis tRNA fraction and 0.1 μg μl–1 recombinant TkKptA the prepared tRNA was desalted by drop dialysis as described above TkArkI-mediated Up47 formation was quantified by γ-phosphate transfer from [γ-32P]ATP to tRNA For kinetic measurement of the tRNA substrate tRNA phosphorylation was performed at 70 °C in a reaction mixture (25 μl) consisting of 50 mM PIPES-NaOH (pH 6.9) 100 μM [γ-32P]ATP (1,500 mCi mmol–1; PerkinElmer) 0.05 μM TkArkI and 0.1–5.0 μM of in vitro-transcribed T For kinetic measurement of the ATP substrate the ATP concentration was altered from 15.6 to 1,000 μM [γ-32P]ATP (750 mCi mmol–1; PerkinElmer) and the tRNA concentration was increased to 1.0 μM 8-μl aliquots were taken and mixed with an equal volume of 2× loading solution (7 M urea 0.2% (wt/vol) xylene cyanol and 50 mM EDTA (pH 8.0)) to quench the reaction Each sample was subjected to 10% denaturing PAGE The gel was exposed on an imaging plate to measure radiolabelled tRNAs using an FLA-9000 imaging analyser Kinetic parameters were calculated using Prism 7 (GraphPad) TkKptA-mediated dephosphorylation of Up47 was quantified by measuring the reduction in radioactivity for tRNA kodakarensis tRNAVal3 was phosphorylated by TkArkI with [γ-32P]ATP as described above and then purified by gel extraction and isopropanol precipitation the same tRNA was phosphorylated by TkArkI with unlabelled ATP the specific activity of the labelled tRNA was adjusted to 6,250 c.p.m per pmol in buffer consisting of 50 mM HEPES-KOH (pH 7.6) The labelled tRNA was incubated at 80 °C for 5 min and then cooled at room temperature The labelled tRNA was dissolved in water to a concentration of 8 μM (50,000 c.p.m Dephosphorylation of the labelled tRNA by TkKptA was performed at 70 °C in a reaction mixture (30 μl) consisting of 50 mM PIPES-NaOH (pH 6.9) 1 nM TkKptA and 12.5–800 nM 32P-labelled tRNA 8-μl aliquots were spotted on Whatman 3MM filter paper which was immediately soaked in 5% (wt/vol) trichloroacetic acid The filter paper was washed three times for 15 min with ice-cold 5% (wt/vol) trichloroacetic acid rinsed for 5 min with ice-cold ethanol and dried in air Radioactivity on the filter paper was measured by liquid scintillation counting (Tri-Carb 2910TR Kinetic parameters were calculated using Prism 7 Chemical structures were drawn with chemical structure drawing tools including ACD/ChemSketch (ACD/Labs) or ChemDraw (PerkinElmer) Further information on research design is available in the Nature Research Reporting Summary linked to this paper Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 7VNV, 7VNW and 7VNX. Source data are provided with this paper The 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Thermus thermophilus HB 8 depending on the environmental temperature Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 Media for Thermophiles (Cold Spring Harbor Laboratory Press Chaplet column chromatography: isolation of a large set of individual RNAs in a single step Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry A cyclic form of N6-threonylcarbamoyladenosine as a widely distributed tRNA hypermodification SLiCE: a novel bacterial cell extract-based DNA cloning method Mechanism of molecular interactions for tRNAVal recognition by valyl-tRNA synthetase PHENIX: a comprehensive Python-based system for macromolecular structure solution DSSR: an integrated software tool for dissecting the spatial structure of RNA Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis Characterization of NADH oxidase/NADPH polysulfide oxidoreductase and its unexpected participation in oxygen sensitivity in an anaerobic hyperthermophilic archaeon Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro Measuring the activity of BioBrick promoters using an in vivo reference standard Download references We thank the members of the Suzuki laboratory for their continuous technical assistance and fruitful discussion We also thank the beamline staff at BL-17A of the Photon Factory for technical assistance during data collection coli ΔtrmBΔtapT strain were kindly provided by T This work was carried out with the support of the Isotope Science Center This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education Sports and Culture of Japan; Research Fellowships for Young Scientists from the Japan Society for the Promotion of Science (26113003 26220205 and 18H05272 to T.S.; 26113002 and 18H03980 to K.T.; 26840005 and 17H04997 to T.O.; and 19J20723 to K Minowa); and Exploratory Research for Advanced Technology (ERATO JPMJER2002 to T.S.) from the Japan Science and Technology Agency (JST) These authors contributed equally: Takayuki Ohira Department of Computational Biology and Medical Sciences mainly performed the series of experiments conducted LC–MS analyses and biochemical and thermodynamic analyses of S Crystal structure analysis of tRNA was performed by K.S. Gene identification by comparative genomics was conducted by K Biochemical characterization of ArkI proteins was performed by K Minowa performed genetic work assisted by A.K. Minowa performed structural studies of ArkI assisted by S.Y All authors discussed the results and revised the manuscript designed the studies and wrote the manuscript The authors declare no competing interests Nature thanks Mark Helm, Kathy Fange Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations The lower panel shows the XIC of the divalent negative ion of the fragment containing N324 XICs show negative ions of the RNA fragments derived from V-loop containing N324 (red line) or U (black line) at position 47 Modification frequency of N324 indicated in each tRNA was calculated from relative peak intensities of the modified and unmodified fragments (a) RNA-MS of the N324-containing fragment of tRNAVal3 digested with RNase T1 XICs show the divalent negative ions of N324m5Cm5CUGp (m/z 845.59 Two phosphates were removed by this treatment (b) CID spectrum of the N324-containing fragment treated with BAP The product ions are assigned on Um5Cm5CUG-OH tokodaii tRNA fraction is digested with nuclease P1 The digests were subjected to periodate oxidation and β-elimination to remove the 3′ terminal residue The resultant pN324p was purified by anion exchange chromatography and subjected to LC/MS/MS analysis (d) LC/MS nucleotide analysis of the nuclease P1 digest of S UV trace at 254 nm (upper panel) and XIC of the negatively charged ion of pN324m5C (m/z 722 The product ions were assigned on the predicted chemical structure of pN324m5C The phosphate group of N324 is shown in red (f) CID spectrum of the N324 nucleotide (pN324p; m/z 483 The product ions are assigned in the predicted chemical structure of pN324p (g) RNA-MS of the V-loop-containing RNA fragment with (+) or without (-) Tpt1p treatment before RNase T1 digestion XICs show the divalent negative ions of Upm5Cm5CUGp (m/z 845.59 (h) CID spectrum of the dephosphorylated fragment by Tpt1p The Tpt1p-treated tRNAVal3 was digested with RNase T1 and analyzed by RNA-MS The V-loop containing fragment was selected as a precursor for CID The product ions are assigned on the sequence as indicated (a,b) Schematic views of Mol. A (a) and B (b) of S. tokodaii tRNAVal3. Each residue is shown as a box. Color codes for the base pairs and base triples are the same as those in Fig. 2b,c Tertiary interactions are shown as blue dashed lines (c,d) Simulated annealing-omit Fo-Fc map contoured at 3.0 sigma around 2′-phosphate of Mol (e) Levitt base pair of G+15 and m5C48 with neighboring residues shown in stick representation with electron density map 2Fo-Fc electron density map contoured at 0.76 sigma around G+15–m5C48 is shown in the lower panel (f) Close-up view of the tRNA core around the G+15–m5C48 base pair and neighboring residues are shown in stick representation Hydrogen bonds are indicated by yellow dash lines (g) Base pair of ac4C6 with G67 in stick representation with electron density map 2Fo-Fc electron density map contoured at 0.76 sigma around ac4C6–G67 is shown in the lower panel (h) Close-up view of the acceptor stem including ac4C6 and neighboring base-pairs and nucleotides (i) Electron density map of m1I57 and m1A58 2Fo-Fc electron density maps contoured at 0.76 sigma for m1I57 and 1.02 sigma for m1A58 are shown (j) Close-up view of T-loop including m1I57 (k) Base pair of m2,2G26 with A44 in stick representation with electron density map 2Fo-Fc electron density map contoured at 0.76 sigma around m2,2G26–A44 is shown in the lower panel (l) Close-up view of D- and anticodon-stems including m2G10 Torsion angle changes from yeast tRNAPhe to Mol A) are shown on Tpt1p-treated tRNA as curved arrows The lower panels show the 90 degree-rotated models (a) Overviews of crystal structure of Tpt1p-treated S. tokodaii tRNAVal3 with stick representation. Molecules A (left) and B (right) are shown in stick representation in pink and orange, respectively. U47 is colored in red. (b) Close-up views of the core structure of Mol. A (left) and B (right). Color code is the same as in Fig. 2b (c) Schematic views of the core structure of Mol f) Atomic structures of the base triples s4U8–A14–A21 (top) Ψ13–G22–G46 (middle) and C12–G23–C9 (bottom) Dashed lines indicate predicted interactions (a) LC/MS nucleotide analyses of tRNA fractions from S UV trace at 254 nm (upper panels) and XICs of the proton adducts of pN324m5C (m/z 724 viennensis tRNA fraction treated with (right panel) or without (left panel) Tpt1p before RNase T1 digestion XICs show the RNA fragments containing Up47 (UpCGp; m/z 1,053.08 The product ions in the CID spectrum are assigned on UpCGp oguniense tRNA fraction treated with (right panel) or without (left panel) Tpt1p before RNase T1 digestion XICs show the RNA fragments containing Up47 (Upm5Cm5Cac4CGp; m/z 866.10 The product ions in the CID spectrum are assigned on Upm5Cm5Cac4CGp acidophilum tRNA fraction digested by RNase T1 XICs show the expected RNA fragments containing Up47 (left panels) or U47 (right panels) as indicated ArkI homologs and PRKACA (PRKACA_MOUSE, PDB: 1ATP) as a canonical ePK are aligned based on structure comparison using DALI (http://ekhidna2.biocenter.helsinki.fi/dali/). Bacterial and archaeal homologs of ArkI are added using MAFFT (https://mafft.cbrc.jp/alignment/server/) Black and gray boxes indicate the degree of sequence similarity Residues mutated in TkArkI are indicated as red letters The alpha helices and beta strands observed in the TkArkI structure are depicted on top of alignments as helices and arrows Those observed in PRKACA are depicted under the alignments Subdomains (I to XI) and representative motifs (P-loop and APE) in ePK are underlined and featured (a, c) In vivo dephosphorylation of Up47 by EcKptA (a) or ScTpt1p (c). XICs show Up47-containing fragments from various E. coli tRNA species (Supplementary Table 5) isolated from E and m5UΨCGp as a control fragment (bottom panels) coli strain in which EcKptA (a) or ScTpt1p (c) is not expressed (left panels) or induced by 10 μM (middle panels) or 100 μM IPTG (right panels) d) Quantification of Up47 dephosphorylation in E Peak intensity is shown for each Up47-containing fragment detected in tRNA fractions from E Data represent average values of technical triplicates ± s.d Source data This file contains Supplementary Notes 1–7 Supplementary Tables 1–7 and the legend for Supplementary Video 1 Download citation DOI: https://doi.org/10.1038/s41586-022-04677-2 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Nature Structural & Molecular Biology (2024) Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. by Owen S. Good LinkOwen S. Good is a longtime veteran of video games writing, well known for his coverage of sports and racing games.Tomo Ohira was 13 years old, probably 90 pounds, and fought utterly mistake-proof in Street Fighter 2 He is widely regarded as the natural of his sport “Hands down the number one player at the time,” says Jeff Schaefer “His name rung out though the arcades in Southern California,” said Mike Watson one of the greatest Street Fighter 2 champions of all time Great Big Story of YouTube found him and caught up with what has happened in the 25 years since “The feeling was just incredible” Ohira says in an era when it was just local multiplayer boy and a girl) and when they are old enough he will give them a game pad and introduce them to Street Fighter Tomo Ohira really isn’t Salinger-esque recluse He’s been found and interviewed repeatedly over the years He also gave expert instruction in Street Fighter 2, in a VHS video revealing the secrets and strengths of ever character in the roster. All of that can be viewed here. But it’s still fascinating to see the guy in present day, decades after he went out on top. The best of Polygon in your inbox, every Friday. By subscribing, you agree to our Terms of Use and Privacy Policy There is no rhyme or reason to what Ryuichi Ohira creates. Pineapples, muscle cars, reclining nudes, wooden bitcoin reliefs — if art is supposed to have a prescribed meaning or set of values — than the Japanese sculptor takes an antithetical approach. For his latest solo exhibition, Ohira unveiled SYNDROME at NANZUKA UNDERGROUND in Tokyo The show presents a series of totemic sculptures all made in his choice material of wood This article is written by well-known “Corolla geek” Atsushi Kobayashi who had met and chatted with the Corolla master So do both the user and Toyota’s representative “Corolla master” share similar ideas about the car Dating back to when my family first owned a Corolla I’ve been riding in and driving Corolla cars for roughly 30 years during which I’ve owned 10 Corolla sedans (including one Corolla Ceres) So others see me not just as a Toyota or Corolla fan but a real “Corolla obsessive.” Now I am fortunate enough to have the opportunity to talk to Toyota’s representative who has been dedicated to developing successive generations of Corolla models for the past 38 years This is definitely the highlight of my career as a freelance writer Kobayashi (on the right) feels joyful in meeting with the “Corolla master” (on the left) for the first time Since transferring from the Prototype Division to the Product Planning Division in 1977 Ohira worked solely on developing the Corolla until 2015 He joined the development team from around the launch of the fourth generation Corolla (sedan) was my first Corolla - the first car I drove after getting my driver’s license So I’ll now share some of the highlights of my chat with Mr mainly about the fourth generation Corolla While I was delighted to have the chance to meet Mr I was worried that he would listen to my views of successive Corollas which are nothing more than arbitrary assumptions I realized that there weren’t any vast differences between Mr Ohira’s views on the Corolla as one of its engineers we were mostly in agreement on what the standout Corolla models are within its successive generations Although I may be somewhat of an eccentric when it comes to cars the reason I’ve continued to drive a Corolla for the past 30 years is most likely because what the engineers are aiming for and my ideas of what I want in a car are largely in sync The engineers have been able to clearly convey their intentions to the users in each model probably because they really understand the needs of Corolla drivers and are working day and night to develop new models The fourth-generation Corolla equipped with four round lamps The first aspect of the fourth generation Corolla which I had asked Mr was whether or not the headlights featuring four round lamps were the only kind in the series I was in elementary school when this model debuted I still remember the powerful impact the fourth generation Corolla had on me when I first laid eyes on it; no other rival cars at the time had this style of headlights Just prior to some minor changes being made to the fourth generation Corolla (before the model had adopted the front engine/front drive (FF) style which many rival cars were already using then) and when my father was hurriedly trying to decide whether to buy the Sprinter or Corolla Sedan - although he was leaning towards the angular Sprinter - he was swayed by my suggestion that “you should give the fourth generation Corolla a try.” That was the start of my and my family’s long relationship with the Corolla After I started working in the automobile-related media and learned that until the fourth generation Corolla had debuted four round lamp-style headlights were a special fitting that could even be said to denote a “luxury vehicle,” while two round lamp-style headlights were the norm for an affordable car right from the start the developers decided to use four round lamp-style headlights in the fourth generation Corolla to create an impact Even though two round lamp-style headlights were the standard grade for cars in the 60s the fact that there was also a model featuring four round lamp-style headlights as a luxury fitting showed just how special a feature it was Toyota narrowed down the target model for a “fixed-type” glove box (leftside in the picture) to low-priced cars The next aspect I was curious about was the glove box some low-priced cars with a 1300cc engine had a “fixed-type” glove box with no door that left the inside exposed For a long time I thought this was a cost-cutting measure for such cars; but Mr These types of low-priced cars with a “fixed-type” glove box were also used by salespeople to do their rounds of customer visits and so forth In addition to vehicle inspection certificates those users also have to fit various work-related documents and forms inside the glove box; so when considering the ease of use for customers Toyota narrowed down the target model for a “fixed-type” glove box to low-priced cars I recalled my father saying that he couldn’t fit the vehicle inspection certificate folder in the glove box so he stuffed it into the car’s trunk instead it seems that the developers collected these folders from Corolla dealerships nationwide and developed the car while checking if the folder could actually fit in the glove box the vehicle inspection certificate folder we received from our Corolla dealership was by chance quite large and not the same size as the folder used during the test drive which is why it wouldn’t fit in the glove box Ohira then told me that Toyota set a standard size for the vehicle inspection certificate folder used in the glove box capacity test during the Corolla’s development so that all Toyota dealerships across Japan would use a uniform-sized folder I was convinced that the reason why the vehicle inspection certificate folder didn’t fit in the glove box is because it was designed overseas So I was really glad to have that misconception corrected and realized once again about the detailed work that went into developing the new Corolla to the point that Toyota thoroughly checked if the vehicle inspection certificate folder could fit in the glove box The fourth-generation Corolla sedan 1500GL had a full-trim door with no exposed steel Another aspect of the Corolla that impressed me was the sound of the door opening and closing our family drove a 1976 Publica Starlet Sedan Deluxe and its doors featured a semi-trim lining with part of the steel exposed the fourth-generation Corolla sedan 1500GL that we purchased next had a full-trim door with no exposed steel which definitely reduced the loud sound of metal against steel and created a similar rich and deep sound of the door closing like it does in luxury vehicles An air exhaust in the C pillar made the door easier to close and created a pleasant sound when the door opens and closes The C pillar in the fourth-generation Corolla has an air exhaust for ventilating the air within the car “Naturally the air inside the car becomes compressed once the door closes so we made the door easier to close in order to quickly release the air outside and worked on creating a pleasant sound when the door opens and closes.” Although this time I’ve only written about aspects of the fourth-generation Corolla that I asked about Ohira also shared with me various detailed features and fittings of other generations of Corollas He was able to immediately answer any question on the finest details of all Corolla models in any generation as he truly fits his title as the “Corolla master” Each generation of the Corolla has been developed by also reflecting the views of its users who has worked on the development of successive models provides advice that is sprinkled like a spice on the views of users which is then blended together to bring out the “flavor” of the car and to improve the performance of each new model What has given me joy as a long-time Corolla driver is not so much the major technical advancements in each new model but rather the moment I realize that the minor complaints I had with older models have been resolved by minor adjustments or full model changes I was curious about what kind of person a Corolla master is after actually meeting with him and talking to him briefly I understood that it’s because people like Mr Ohira have been a part of the development team that the Corolla has remained at the core of Toyota’s lineup for 50 years Developing new cars from the user’s perspective is definitely not as easy as it sounds who really understands what drivers want in a car Kobayashi grew up in a household with a fourth generation Corolla and the vehicle turned both him and his parents into Corolla fans his infatuation with Corollas led him to find employment at a Corolla dealership While delving more and more deeply into his research into Corollas he suddenly found himself switching to a job at an automobile magazine publisher If one includes the Corollas his parents owned he has been the owner of ten consecutive Corollas His wide-ranging knowledge of Corollas covers not only domestic Japanese models but also Southeast Asian and North American models After his assignment at the Prototype department he was transferred to Product Planning in 1977 and started working on product development of the Corolla Ohira had dedicated himself to the product planning of the Corolla for approximately 38 years from the fourth-generation Corolla through to the eleventh-generation Corolla He retired at the legal retirement age on August 2015 Metrics details Short-chain fatty acids produced by the gut bacterial fermentation of non-digestible carbohydrates contribute to the maintenance of skeletal muscle mass and oxidative metabolic capacity We evaluated the effect of FOS ingestion on protein expression of soleus (Sol) and extensor digitorum longus muscles in mice exposed to microgravity (μ-g) Twelve 9-week-old male C57BL/6J mice were raised individually on the International Space Station under μ-g or artificial 1-g and fed a diet with or without FOS (n = 3/group) the absolute wet weights of both muscles tended to decrease and the fiber phenotype in Sol muscles shifted toward fast-twitch type following μ-g exposure FOS ingestion tended to mitigate the μ-g-exposure-related decrease in oxidative metabolism and enhance glutathione redox detoxification in Sol muscles These results indicate that FOS ingestion mildly suppresses metabolic changes and oxidative stress in antigravity Sol muscles during spaceflight astronauts newly landed on Moon and Mars following their stay in a microgravity (μ-g) environment may have trouble carrying out daily activities since they cannot be directly supported by the local ground staff This is a serious issue in terms of mission success and crew safety crucial to understand the mechanism of skeletal muscle adaptation to gravitational unloading and establish an efficient countermeasure for its prevention during μ-g exposure Adopting mice raised under A1-g on the ISS as a control group may provide another option to determine specific responses of skeletal muscles to μ-g exposure during spaceflight FOS ingestion could be a countermeasure to prevent the deterioration of antigravity muscle properties in a μ-g environment the efficacy of FOS ingestion during spaceflight has not been verified and proteome alterations of the muscles in response to μ-g exposure were investigated by comparing data obtained from the mice raised under μ-g and A1-g without FOS ingestion the efficacy of prebiotic FOS ingestion during spaceflight against the antigravity muscle property deterioration due to μ-g exposure was evaluated by comparing the μ-g-exposure-related proteome alterations in the muscles of mice fed a diet with and without FOS The data are presented as the mean ± standard deviation (n = 3) absolute wet weight of soleus (Sol) and extensor digitorum longus (EDL) muscles and the wet weight of Sol and EDL muscles relative to body weight are depicted in panels (a−c) Ground control (GC): group of mice raised in the ground model of the Multiple Artificial-gravity Research System habitat and transportation cage units; artificial 1-g (A1-g): group of mice raised under artificial 1-g on the International Space Station (ISS); microgravity (μ-g): group of mice raised under μ-g on the ISS; and FOS (+) or (−): groups of mice fed a diet with or without FOS These results indicated that comparisons between the GC and μ-g groups would highlight not only the effect of μ-g exposure but also the effect of decreased food intake on skeletal muscles mice raised under A1-g on the ISS would serve as a more suitable control group for determining the specific responses of the skeletal muscle proteome to μ-g exposure during spaceflight Heatmaps and clustering of analyzed samples according to the abundance of 1,253 proteins demonstrated that both Sol and EDL muscles responded to μ-g exposure (Fig. 2). Additionally, Sol muscles from mice exposed to μ-g and A1-g were affected by FOS ingestion. The abundance of each protein is depicted in a gradient from blue (low) to orange (high) the artificial 1-g (A1-g) and fructo-oligosaccharide (FOS) (+) None of the proteins in the group exposed to A1-g with FOS ingestion meet these criteria to μ-g exposure was mitigated by FOS ingestion the response of other proteins was enhanced by the combined effect of μ-g exposure and FOS ingestion These results indicated that the effect of FOS ingestion was not sufficient to suppress the phenotype shift toward fast-twitch type in Sol muscles of mice exposed to μ-g but the effect may be different in mice exposed to μ-g or A1-g we could not determine whether the effect of FOS ingestion is beneficial for skeletal muscles in this study These results suggested that Sol muscles of mice exposed to μ-g were injured by gravitational reloading This study was performed under certain limitations A maximum of only 12 mice can be raised on the ISS using the MARS 12 mice were randomly divided into two groups and individually raised under µ-g or A1-g and 3 mice in each group were fed a diet containing 5% FOS The skeletal muscles sampled from this mission were analyzed in this study It is not feasible to collect additional Sol and EDL muscle samples from space-flown mice the effect of type II error could not be eliminated owing to the small sample size (n = 3/group) the mice in the µ-g group floated in each MARS habitat cage but the mice in the A1-g group used only the floor in addition to gravitational unloading and reloading the different behavior and physical activity of mice in µ-g and A1-g groups due to the different living space within the cage may have affected the proteome of Sol and EDL muscles we revealed the effects of μ-g exposure and/or FOS ingestion on the proteome of Sol and EDL muscles in developing mice from 9 to 12 weeks old The alteration of the homeostatic proteome of these muscles in adult mice needs to be investigated in the future we could not perform transcardial perfusion before isolating the skeletal muscles as the dissection protocol was strictly controlled due to space experiment restrictions the abundance profiles of the skeletal muscle proteins were also affected by proteins in the blood The abundance profiles of proteins in skeletal muscles and blood need to be individually determined in the future The protein abundance profiles in Sol and EDL muscles were altered in response to both µ-g exposure and FOS ingestion our results indicate that ingesting a diet containing 5% FOS is insufficient to suppress the atrophy and fiber-phenotype-shift in antigravity Sol muscles during spaceflight The shift in fiber phenotype from slow- to fast-twitch type was prominent in Sol muscles in response to µ-g exposure Ingestion of FOS tended to mitigate the decrease in proteins involved in oxidative metabolism in association with the shift of fiber phenotype the changes in abundance of proteins composing sarcomere and affecting muscle contractile properties were significant even in mice fed a diet containing FOS the abundance of proteins involved in the mitochondrial tricarboxylic acid cycle increased in EDL muscles following μ-g exposure and this response tended to be enhanced by FOS ingestion the abundance of blood microparticle and extracellular exosome proteins in Sol and EDL muscles was significantly altered by μ-g exposure and the responses of several proteins were also affected by FOS ingestion FOS ingestion may have affected extracellular vesicle components produced by skeletal muscles blood coagulation and innate immune response were activated in Sol muscles following μ-g exposure which suggested injury by acute gravitational reloading The study findings indicated that the effect of gravitational reloading on Sol muscles in mice exposed to μ-g cannot be eliminated even comparing with the data obtained from mice raised under μ-g and A1-g conditions on the ISS All of the experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Physiological Society and the National Institutes of Health This study was approved by the Committees on Animal Care and Use of JAXA (accreditation no.: 016-018) Yokohama City University (accreditation no.: T-A-15-005) National Aeronautics and Space Administration (accreditation no.: FLT-17-106) and Explora Biolabs (accreditation no.: EB15-010) which were individually raised in the ground model of the MARS habitat and transportation cage units 3 mice in the GC group were fed a diet containing 5% FOS while the remaining 3 mice were fed an energy-equivalent diet containing 5% cellulose Venous blood was collected from the mice under isoflurane anesthesia The mice were subsequently euthanized by exsanguination under isoflurane anesthesia The wet weight of the Sol and EDL muscles was measured and the samples were snap-frozen with liquid nitrogen and stored at –80 °C for subsequent analyses The mice in the GC group (n = 6) were dissected in the aforementioned manner All data in Figs. 1 and 2 are presented as the mean ± SD Owing to the small sample size in each group (n = 3) the effects of spaceflight and FOS ingestion on the body weight and wet weight of the muscles were evaluated by Kruskal−Wallis and Dunn’s multiple comparison tests using GraphPad Prism version 7.02 software (GraphPad Software Differences with p values < 0.05 were considered statistically significant The effects of μ-g exposure and FOS ingestion on the abundance of proteins in Sol and EDL muscles (n = 3/group) were individually evaluated by ANOVA performed using the Progenesis QI for proteomics software differences with p values < 0.01 were considered statistically significant Further information on research design is available in the Nature Research Reporting Summary linked to this article The data that support the findings of this study are available from the corresponding authors upon reasonable request. The raw MS data and files obtained after analyses were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the jPOST partner repository (https://jpostdb.org) with the data set identifier PXD021550 Skeletal muscle unweighting: spaceflight and ground-based models Prolonged space flight-induced alterations in the structure and function of human skeletal muscle fibres Disuse of the musculo-skeletal system in space and on earth Responses of skeletal muscles to gravitational unloading and/or reloading Region-specific responses of adductor longus muscle to gravitational load-dependent activity in Wistar Hannover rats Muscle unloading: a comparison between spaceflight and ground-based models Exercise in space: human skeletal muscle after 6 months aboard the International Space Station Effects of prolonged space flight on human skeletal muscle enzyme and substrate profiles Metabolic consequences of muscle disuse atrophy Recovery of the soleus muscle 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Download references Shinsuke Kataoka (Yokohama City University) for their technical assistance This study was supported by funding provided by the Japan Aerospace Exploration Agency (JAXA) for research selected for life science to H.H Japan Society for the Promotion of Science KAKENHI to T.O the Special Coordination Fund for Promoting Science and Technology “Creation and Innovation Centers for Advanced Interdisciplinary Research Areas” to H.H Science and Technology/Japan Science and Technology Agency The MS analyses were performed at Clinical Mass Spectrometer Platform (Yokohama City University) supported by the Ministry of Education These authors contributed equally: Takashi Ohira Research Center for Space and Medical Sciences and Organization for Research Initiatives and Development Department of Physiology and Regenerative Medicine contributed to the conception and design of the current study; C.M managed the second mission using the MARS; T.O. interpreted results; all authors revised and approved the final version of the paper Download citation DOI: https://doi.org/10.1038/s41526-021-00164-6 a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science What was supposed to be a tidy daily driver and parts hauler becomes a fully built SEMA sensation RodrezWriterMarvin RecinosPhotographerMar 31 Rather than being packed with boxes and leftover drive-thru containers it stood proudly in the Cusco booth of SEMA 2021 and helped generate parts for the U.S a brand that imports high quality automotive goods from Japan the exclusive distributor of Spirit Rei and Exarts exhaust components and exporting of U.S.-spec vehicles to Japan learned quickly that current pricing doesn't favor the buyer After losing his GS-R Integra to an accident he began searching for a replacement and wanted something with 3-doors again like a Nissan Silvia S-chassis or fifth-generation Honda Civic to stuff it full of parts boxes coming from and going to Japan Takashi wanted a manual transmission and around 200 horsepower on tap "We get a lot of requests and questions from left hand drive car owners on the U.S side that want to know if certain JDM parts will work on their car," he says and Nissan 180SX already have vast compatibility and data on what will fit." One request that popped up during his vehicle search involved whether or not a TEC-ART'S exhaust manifold would fit a left-hand drive layout and if an A/C kit was available and compatible Kamata who assured him that the parts could be developed for the U.S but he'd need an actual car in their garage to make it happen I began looking for an AE86 hatchback and it was very difficult to find a decent Corolla at a reasonable price Since it was going to be used for research and development damage and rust would have to be at a minimum." Takashi finally got a solid lead from a neighbor that sent him to Lance Freeman Takashi went through the car and found it had solid bones and a relatively straight chassis After a few months of waiting for the title to be issued the 86 left Long Beach and headed for TEC-ART'S Japan parts began pouring in to support the project and any thoughts of a daily driver and parts hauler quickly dissolved new front and rear fenders from Restoreparts.com were installed along with the brand's back panel The classic two-tone exterior treatment is a bit deceiving in this instance in that the Impulse dry carbon fiber hood and headlight covers flow with the car's lower half but there's quite a bit more carbon involved and the entire roof are also produced in the lightweight composite material Watanabe are the wheel of choice but this more modern version of the original 86 uses Work Meister CR01 in 15x8 +3 with 195/50 Toyo R1Rs KW Suspension coilovers replaced the archaic stock set up you'll find Cusco pillow tensions rods and a series of adjustable arms and links Under that lightweight hood now rests a 20-valve 7AG sporting 12.8:1 compression and TEC-ART'S rods A WeldSpeed billet aluminum ITB plenum is fed by a short-ram HKS filter a combination that takes up most of the space on the passenger side of the bay while the driver's side is home to a Fujitsubo header that leads to a Sard high-flow cat The ignition has been upgraded to a coil-on-plug set up for modernization along with MoTeC's M130 management wired in a drive-by-wire system is used to actuate the throttles with touches of gold anodizing set against a black valve cover and TEC-ART'S radiator as well as purple engine mounts to bring together a very cohesive and purposeful look the thorough massaging of the power plant applied by TEC-ART'S is top notch and power isn't lost in translation as it's sent through a Toda clutch and TRD 2-way limited slip That attention to detail is also exploited with the Corolla's reworked interior Cusco issue Bride Stradia III seats are the focal point and supported with matching Bride upholstery used for the door cards with that pattern spilling into the cargo area as well a TEC-ART'S leather version that uses red baseball stitching to match the seats was based on a factory dash donated to the project by friend Jacob Gioffrre Sport pedals and a TRD shifter and horn button-equipped Momo steering wheel finish off a pristine cabin Beyond working on projects at TEC-ART'S Kamata also serves as an event organizer and judge for the AE86 Drift Championship of Japan but somehow found enough time to complete this massive makeover in just six months soil before heading out to Las Vegas for SEMA and is brought to various events to highlight the left-hand-drive compatible parts that were developed during its build up Takashi tells us that with the drive-by-wire and MoTeC tuning "Usually people think that a car built by a tuning shop will be hard to drive and difficult to operate Even a beginning driver can feel how easy this is to drive." Of course it's fully capable of being put to task by a skilled wheelman and just before the car left Japan for its return to the U.S. Tsuchiya-san jumped behind the wheel for some "spirited testing." An incredible series of events that transpired after a customer's inquiry "I don't daily this car…therefore I still do not have a daily." Instagram @mastermind_north_america Engine 7AG 20 valve head; forged pistons 12.8:1; TEC-ART'S 7AG-specific rods, crankshaft, cams, copper 3-row radiator, Type NA fuel pump, WeldSpeed intake manifold; coil-on-plug ignition, Type L Fujitsubo exhaust manifold, Sard sports catalytic converter; Toda valve springs; 1.2mm metal head gasket; HKS 15 row oil cooler, air filter; electric water pump; 400cc injectors; Bosch electronic drive; MoTeC M130 management; Motul 300V oil; Cusco oil catch can Suspension KW Suspensions coil overs; Cusco pillow tension rod, lower arm, control arm N1 link, lateral rod Braking Endless MX72 brake pads; TEC-ART'S super slit rotor, mesh hose; Motul brake fluid Wheels & Tires Work Meister CR01 15x8 +3; Toyo Proxes R1R 195/50 Exterior Impulse dry carbon hood, doors, roof, hatch, headlight covers; Restoreparts.com front bumper, front/rear fenders, back panel, headlight bracket; OEM front lip; Cruise LED head lights; Coolverre infrared/UV cut glass Interior Cusco x Bride Stradia III front seats; Bride seat material floor mats, door cards, rear seats, rear cargo mat; TEC-ART'S leather dash panel Subscribe to our newsletters to get the latest in car news and have editor curated stories sent directly to your inbox Courtesy of the artist and Kosaku Kanechika; Charlotte Keates Courtesy of the artist and Asia Art Center; Anselm Reyle Tokyo Gendai is scheduled to be held from July 7 to July 9 at the Pacific Convention Plaza Yokohama (Pacifico Yokohama) Here is what to expect from this new world-class international art fair AD Tokyo Gendai is Japan’s first international art fair to obtain permission to use the entire fairgrounds as a bonded area Pacifico Yokohama exhibition space will allow overseas exhibitors to import and display works of art free of customs and other restrictions which significantly improves the accessibility and efficiency of the Japanese art market is one of the reasons why the fair is described as “world-class.”Participating galleries include 313 Art Project (Seoul) Fox Jensen & Fox Jensen McCrory (Sydney GALLERY SIDE 2 (Tokyo) and 74 other galleries in Japan and abroad The fair program also includes exciting performances Artist Ryuichi Ohira will present a new large-scale installation The installation plays on the many definitions of the term ‘circuit’: race tracks; electrical circuits; the philosophical concept of a non-hierarchical and interconnected system; and a synapse in neurology The work comprises a large slot car racetrack and features a series of objects and sculptures some of which refer to a traditional Japanese and Chinese festival Tanabata (Star Festival) Curated by Michiko Kasahara (Deputy Director a new program strand entitled TSUBOMI (‘flower bud’) Life Actually: The Work of Contemporary Japanese Women Artists The featured artists are Leiko Ikemura (ShugoArts) The additional NE (‘root’) section will feature the collection of several leading local foundations The fair also offers special talk programs designed to foster conversations on contemporary culture in Japan and beyond and to discuss key trends Among the speakers are leading figures in Japanese art Hirosaki Museum of Contemporary Art; Executive Director View upcoming auction estimates and receive personalized email alerts for the artists you follow Spring 2024 Men's Fashion Show Celebrities All of the influencers and celebrities at Silvia Venturini Fendi’s Fendi Spring 2024 Men’s Fashion Show from the houses factory near Florence ahead of Milan Men’s Fashion Week (June 2024).Photo Courtesy Fendi – by Pietro S This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Please enable JS and disable any ad blocker 本サービスをお楽しみいただくには、JavaScript を有効にする必要があります。 アニメーション映画『映画 えんとつ町のプペル』 大平祥生が声優として出演決定！※12/23 舞台挨拶生中継 概要追加 お笑いコンビ・キングコングの西野亮廣が監督を務め、2016年の発売以降、ロングラン大ヒットを記録し、累計発行部数50万部という驚異的な数字を叩き出した話題の絵本「えんとつ町のプペル」。その絵本を原作とした、アニメーション映画『映画 えんとつ町のプペル』が、12月25日（金）に公開が決定！ 窪田正孝さん、芦田愛菜さんを始めとした豪華声優陣に加わり、大平祥生が初の声の演技に挑戦させていただくことになりました。 全国同時生中継 劇場一覧 厚い煙に覆われた“えんとつ町”。煙の向こうに“星”があるなんて誰も想像すらしなかった。この町でただ一人、紙芝居に託して“星”を語っていたブルーノの息子・ルビッチは、父の教えを守り“星”を信じ続けていた。しかし、ルビッチは町のみんなに嘘つきと後ろ指をさされ、ひとりぼっちになってしまう。そしてハロウィンの夜、ゴミから生まれたゴミ人間・プペルが現れ、のけもの同士、二人は友達となり、ルビッチとプペルは「星を見つける」旅に出ると決意する。父を信じて、互いを信じあって飛び出した二人が、大冒険の先に見た、えんとつ町に隠された驚きの秘密とは？ ■声のキャスト：窪田正孝、芦田愛菜、立川志の輔、小池栄子、藤森慎吾、野間口徹、伊藤沙莉、宮根誠司、大平祥生（JO1）、飯尾和樹（ずん）、山内圭哉／ 國村隼 ■OP 主題歌：「HALLOWEEN PARTY -プペル Ver.-」 HYDE (Virgin Music) ■ED 主題歌：「えんとつ町のプペル」ロザリーナ（ソニー・ミュージックレーベルズ） ■アニメーション制作：STUDIO4℃ ■製作：吉本興業株式会社 ■配給：東宝=吉本興業 JO1として初の文化観光大使として京都府の魅力をＰＲする「京都府文化観光大使」に大平祥生が就任致しました。 大平は、「京都府文化観光大使にはずっとなりたいと思っていたので、信じられない気持ちです。頑張ります」 https://youtu.be/GhH0uHLFYMg The second volume features Licaxxx (Licaxxx)RIMOWA's new project "THE NEW NORMAL," was launched at the RIMOWA Japan office in Tokyo in May 2011 this is a Japan-specific project to cover artists and athletes who are pursuing their goals in the difficult situation of the Corona Disaster Ohira has more than 4 million followers on TikTok The first video he uploaded by chance suddenly became a buzzword and his name became well known This led to his appearance in TV dramas and runway shows for famous brands He talks about how his situation has changed dramatically since he started "TikTok," his unchanging thoughts since then he packs his luggage in the new color of the Rimowa Essential Cabin The beautiful pale-toned suitcase looks great in the city of Tokyo From the "TikTok" stage, Ohira has begun to spread her wings in the real world.Interview posted on the official siteHe says Is he still 20 years old or is he already 20 The young talent is moving toward the future Your browser does not support JavaScript, or it is disabled．Please check the site policy for more information Vox Populi Vox Dei is a daily column that runs on Page 1 of The Asahi Shimbun With the novel coronavirus pandemic still raging the autumn term has already started in late August at schools in some parts of Japan although the summer holiday is being extended in other areas This is perhaps a cause for concern among many parents who don't want their children's daily routine being disrupted more than in any normal year here are some research findings they may find relevant led a study to find out what tone of voice will wake sleeping children the fastest Ohira and her team invited 13 elementary school students to their lab After checking the child's brainwave to confirm they were in a deep sleep They then played three sounds for the same duration to compare the time each took to wake the subject An electronic beep took an average of 310 seconds to wake the youngsters But the average shrank to 31 seconds when each child heard their own mother's pre-recorded voice and to 26 seconds if the voice was that of a woman they didn't know The difference between these last two numbers fell within the statistical margin of error Who would have thought how ineffective the "trusty" alarm clock could be "We learned that the human voice packs a greater punch than a non-human sound and that calling the person's name worked better than just the human voice," Ohira noted Whether the voice is that of the child's father or of a voice actor Japanese children have long been considered "night owls" compared to their peers overseas Kids who struggle with drowsiness in the classroom are not rare One survey even found that one in five suffer from sleep disorders Ohira let me listen to the voices of mothers used in the experiment But every mother was asked to refrain from letting loose her usual outburst of exasperation "Are you ever going to wake up?" or "Enough here's my two cents to all parents out there: There's no need for nagging in the morning Just calling out your kid's name over and over should be sufficient It works 10 times better than the electronic beep of the alarm clock Vox Dei is a popular daily column that takes up a wide range of topics the column provides useful perspectives on and insights into contemporary Japan and its culture education officials oppose inviting children to Paralympics POINT OF VIEW/ Klaus Schwab: Young people hold the key to creating a better future Ship remembered for saving 800 Russian children a century ago Temperature in school bus where boy died tested at over 50 degrees 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 Planetarium Creator, Takayuki Ohira will have a live interactive planetarium show using “MEGASTAR” in the Sakado Children’s Center in Saitama without audiences Takayuki Ohira’s live presentation is one of the most popular event in the Children’s Festival of Sakado Children’s Center held on 5th May every year But unfortunately Sakado Children’s Center is temporarily closed to the public due to ongoing concerns related to COVID-19 this year So we have decided to have his live interactive show without audience and stream it we will stream the normal images inside the dome we will stream VR 360°images from MEGASTAR-FUSION with Takayuki’s voices Viewers can enjoy planetarium shows without coming to the site We will deliver the images that make you feel you are really in the planetarium dome *We will do this event with minimum staff and follow the infection control guidance postponed or changed depending on the situations PDF download of flyer