Working memory (WM) is a system for maintenance of and access to a limited number of goal-relevant representations in the service of higher cognition. Because of its limited capacity, WM requires interference-control processes, allowing us to avoid being distracted by irrelevant information. Recent research has proposed two interference-control processes, which are conceptually similar: (1) an active, item-wise removal process assumed to remove no-longer relevant information from WM, and (2) an inhibitory process assumed to suppress the activation of distractors against competing, goal-relevant representations. The purpose of this study was to determine the extent to which the tasks used to assess removal and inhibition measure the same interference-control construct. Results showed acceptable to good reliabilities for nearly all measures. Similar to previous studies, a structural equation modeling approach identified a reliable latent variable of removal. However, also similar to some previous studies, no latent variable of inhibition could be established. This was the case even when the correlation matrix used to compute the latent variable of inhibition was disattenuated for imperfect reliability. Critically, the individual measures of inhibition were unrelated to the latent variable of removal. These results provide tentative support for the notion that removal is not related to the interference-control processes assessed in inhibition tasks. This suggests that the removal process should be conceptualized as a process independent of the concept of inhibition, as proposed in computational WM models that implement removal as the “unbinding” of a WM item from the context in which it occurred. Working memory (WM) refers to the ability to maintain and manipulate a limited amount of information in the service of higher cognition e.g., , 2. WM rRecent WM research has proposed that active, item-wise removal is a key process underlying performance in WM tasks e.g., –9. To spThe validity of removal as a psychometric construct was further established in a recent set of individual-differences studies using structural equation modelling (SEM). Singh and colleagues  investigInhibition is often conceptualized as a general ability underlying attentional control or executive functions, viz. a set of executive abilities allowing us to supervise and control thoughts and actions in order to achieve current goals. Miyake and colleagues  reportedIt should be noted, though, that recent research has revealed some difficulties in establishing attentional control—and in particular, inhibition—as a latent variable. Some studies have pointed out low reliability estimates for inhibition measures, possibly explaining the observed low zero-order correlations between these measures e.g., –35. FurtIn summary, previous individual-differences research has put forward inhibition as a core interference-control mechanism related to WM. Nevertheless, there is some evidence that establishing inhibition as a valid and reliable construct at the latent-variable level is more difficult than previously thought. This emphasizes that inhibition might not be a general construct, thus mandating us to be very cautious when referring to the concept of “inhibition”.The purpose of the present study was to investigate the extent to which the tasks used to assess removal and inhibition measure the same construct. In addition to the conceptual similarity between removal and inhibition abilities, there is empirical support for the assumption that the tasks used to assess removal and inhibition measure similar constructs. First, the removal process has been argued to be involved in WM and updating task performance e.g., –9. This In the present study, we used a confirmatory factor analysis (CFA) approach to account for the correlational structure among the measures of removal and inhibition. Removal was measured using the updating task battery of Ecker and colleagues , 5, 9, wIt was hypothesized that if the tasks used to assess removal and inhibition measure the same cognitive process, zero-order correlations should be high between all measures. Moreover, either all measures should load on a single factor, or, if separate factors for removal and inhibition were to emerge, these two factors should be highly correlated. In contrast, if the tasks used to assess removal and inhibition measure different cognitive processes, zero-order correlations should be low between the removal measures and the inhibition measures. There should also be no significant correlation between removal and inhibition factors. Moreover, in case there is no general construct of inhibition and thus no coherent factor of inhibition were to emerge, as suggested by recent research e.g., , 14, 32,SD below the respective mean of all participants. Eight participants were removed because they had a low number of trials  in any task. In addition, the data of one participant were excluded as they were identified as a multivariate outlier  when checking for multivariate normality using Mardia’s  and run on IBM compatible computers. For each task, participants were instructed to be as fast and as accurate as possible. Additional details regarding the number of trials and block structure are provided in Removal efficiency was measured using three updating tasks based on the paradigm introduced by Ecker and colleagues , 5, 9. TAn example trial sequence for the digit updating task is illustrated in All inhibition tasks were programmed using Tscope5 . For eacAs episodic memory and associative learning impacts performance on inhibition tasks e.g., , 54, thrincongruent trials , congruent trials , and neutral trials . Inhibition is assumed to occur in incongruent trials in order to reduce the interference triggered by irrelevant features. For each task, all trial types were presented randomly and occurred with equal frequency. First-order transitions between these trials also occurred with equal frequency. The main dependent measure was the congruency effect. This was calculated by subtracting RTs on congruent trials from RTs on incongruent trials .For the congruency tasks , there were two to three trial types: For most tasks , the trial structure was similar . That is, a central fixation cross was presented for 500 ms. Then, the stimulus was presented until a response was made or 2,000 ms had elapsed. Finally, accuracy feedback was presented centrally for 500 ms, followed by a 500 ms blank screen. An example trial sequence for each inhibition task is illustrated in number-Stroop task, participants were presented with a centrally-displayed row of 1 to 4 identical digits , and were instructed to indicate the number of digits presented while ignoring their numerical value . In incongruent trials, the central and flanking arrows pointed in opposite directions ; in congruent trials, central and flanking arrows pointed in the same direction ; in neutral trials, hyphens were displayed as flankers instead of arrows . Participants pressed the A and L keys with their left and right index fingers, respectively, to provide their responses.In the global-local task, participants were presented on each trial with a large letter that was composed of smaller letters was presented centrally. Only the letters Y and V were used. Participants were instructed to perform the “local” task variant, that is, to identify the small constituent letters while ignoring the large letter . In incongruent trials, the target stimulus faced the opposite direction as the prime ; in congruent trials, the target stimulus faced the same direction as the prime ; in neutral trials, the prime was an unrelated symbol . In this task, it is assumed that the response induced by the prime is automatically suppressed and requires reactivation in order to respond to a congruent target  / RTshort CTI for the three updating tasks, [RTincongruent−RTcongruent] / RTincongruent for number-Stroop, arrow-flanker, local, and Simon tasks, [RTcongruent−RTincongruent] / RTcongruent for the negative-compatibility task, and [RTantisaccade−RTprosaccade] / RTantisaccade for the antisaccade task). (3) For each construct , we estimated bi-factor models. That is, for removal, we fit a bi-factor model, which forced performance from both CTI conditions to load on a baseline factor, and performance from the short CTI condition to load on a removal factor. For inhibition, the bi-factor model forced performance on the antisaccade and prosaccade trials of the antisaccade task, and performance on incongruent and congruent trials of the number-Stroop, arrow-flanker, Simon, local, and negative-compatibility tasks to load on a baseline factor, and performance on antisaccade trials of the antisaccade task, congruent trials of the negative-compatibility task, and incongruent trials of all other tasks to load on an inhibition factor. (4) We included participants who had missing data in less than half of the inhibition measures, and we ran the CFA with case-wise maximum-likelihood estimation. The goodness-of-fit statistics of all these model assessments and the parameter estimates can be found at https://osf.io/c8qb2. The results of these supplementary tests were either equivalent to those presented in this article or worse  had worse fit statistics or the model(s) did not converge).To test the robustness of our results, we re-ran the analyses by separately applying the following modifications. (1) For the inhibition measures, we computed two alternative dependent measures. That is, we calculated the interference effect  as well as the facilitation effect . (2) Instead of using residuals as dependent measures, we computed proportional scores for all measures  and six tasks designed to assess inhibition . The removal measures displayed good reliability estimates and high correlations. Bayesian inference provided very strong evidence in favor of the interrelations. Using CFA, a coherent latent factor of removal was established, replicating previous findings , 5, 9. IIn the present study, the internal consistency reliabilities of most tasks were similar to Rey-Mermet and colleagues , where tOverall, inhibition measures did not have any significant correlations between them. In addition, a coherent latent construct of inhibition could not be established. Even when imperfect reliability estimates were taken into account by disattenuating the correlation matrix, we were unable to establish a coherent inhibition construct. These results are in line with recent studies highlighting the difficulty of establishing inhibition as a coherent latent construct , 39, 69.As pointed out recently by Draheim and colleagues , 70, oneA further reason why the use of difference scores may impede extraction of a latent inhibition variable is that computing difference scores is based on the premise of additive factors , 69. HowThus, it seems that there remains only one reason to explain the failure to establish a coherent inhibition latent factor. It is possible that the selected tasks used to measure inhibition do not assess a common underlying construct, but the highly task-specific ability to reduce the interference arising in that particular task. As interference from a flanking arrow differs, for example, from interference from an irrelevant stimulus location (as in the Simon task), the abilities to minimize interference in these tasks may be task-specific. Thus, whatever is measured by the tasks that are used to assess inhibition seems highly task-specific, which thus calls into question the view of inhibition as a psychometric construct see .The present study’s results showed the difficulty of establishing a coherent factor of inhibition. It was, therefore, not possible to determine the relationship between the interference-control processes assessed in inhibition and removal tasks at the latent-variable level. As the purpose of the present study was to determine the relation between the interference-control processes measured in inhibition and removal tasks, a different strategy was then adopted by investigating the relations between the individual measures of inhibition and the latent factor of removal. None of the inhibition measures related to the removal construct. This occurred even in tasks where the chance of finding a relation was optimized due to measures being on the same scale . Thus, the results of the present study indicate that the inhibition measures assess interference-control processes that are different from removal ability. This first shows that although removal can be estimated at the latent-variable level in a valid and reliable way, removal tasks cannot be used to measure the interference-control processes assessed in inhibition tasks. Furthermore, this emphasizes the necessity of conceptualizing removal based on previous research and WM theory. One possibility is that removing an item from WM rather represents “unbinding” the item from the context in which it occurred  see , 5, 72. Together, the present results show that performance on the inhibition tasks we used relies on interference-control processes that are different from removal. On a general level, the results also provide additional evidence that we should stop using inhibition as an umbrella term for a variety of distinct processes. Thus, although the present study supports the notion that removal is not related to the interference-control processes assessed by the inhibition tasks, in order to progress, the field will need to specify how inhibition could act on irrelevant/outdated information in WM, and should attempt to build experimental paradigms that are able to disentangle inhibition from other mechanisms of conflict resolution .In the present study, we used tasks which were assumed to asses removal and inhibition, respectively. However, the reader may wonder whether our task selection can explain the present study’s results. First, one may question our decision not to use the color Stroop task, the stop-signal task, or the sustained-attention-to response task (SART) task in accordance with previous research e.g., , 37, 38.d’) do not control for variance in processing speed. If fast and slow individuals respond within about the same time, slower individuals will make more errors, thus transferring some speed variance onto accuracy and d’.We did not include the color Stroop task because our testing would have required a key-press version of the task . However, with such a task version, the set of stimulus-response mappings would have been large and each stimulus-response mapping would have been completely arbitrary. This would have increased WM requirements, thus possibly impacting the relation between task performance and WM. We also did not include the stop-signal task because participants have been found to wait for the stop signal to occur in order to stop their responses e.g., , 38, meaSecond, one may argue that we found a coherent factor for removal but not for inhibition because the tasks used to assess removal were more similar than those used to asses inhibition. In other words, a coherent inhibition factor may have emerged had we used the same inhibition task with different stimulus materials. The Stroop task may be one candidate for such a design because there exists a color version (as described earlier), a number version (such as used in the present study) and a spatial version (in which participants indicate the left-right position of the words “left” or “right” while ignoring their meaning). However, previous research has shown only small to moderate correlations between the different versions of the Stroop task  see , 55, 76.Finally, as removal is a process that reduces WM interference, one may wonder whether it would have been easier to find a relation between removal and inhibition tasks if we had used tasks assessing memory interference . However, as we aimed to determine whether the correlations between (1) WM and attentional control, and between 2) WM updating and inhibition were driven by a common removal/inhibition factor, it was imperative to use the same type of tasks as the research investigating these correlations. Moreover, it has been argued that memory-interference tasks involve delayed recall of long-term associations that are more robust than the temporary bindings created in WM for performing the inhibition and removal tasks Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Brief SummaryThe manuscript reports one study aimed to examine whether the ability to update information in working memory (as measured with three updating tasks) relates to the construct of inhibition (as measured with six very different tasks that some have claimed to tap into inhibition). While a latent variable of removal/updating was evident from the analyses, this not was the case for inhibition. Individual measures of inhibition did not even relate to removal as a construct. The authors conclude these results support the idea that removal is different from inhibition.General evaluation and commentsFirst of all, I have to say I have read the manuscript from the perspective of a researcher who is interested in inhibitory control but is also extremely critical of how some people (over)use the concept of inhibition, which has become meaningless. And I am afraid the reviewed manuscript highlights this latter issue (even when I am not putting the blame on the authors. I understand they are using tasks and measures that many others think are ‘inhibitory’ in nature).The manuscript is very well written and clear, and I do appreciate its transparency. The main goal of the study is theoretically relevant and timely and the statistical approach is appropriate and well reported.That said, I have also some  concerns with the manuscript that essentially stem from how the concept ‘inhibition’ is (mis)used (not necessarily by the authors of the manuscript but it is clear they rely on it) and mainly focus on the conclusions to be drawn from the results of the study. Actually, some of my concerns are already mentioned in the Discussion of the paper (but poorly addressed).First, the selection of the tasks noticeably did bias the latent variables that were (or not) identified. It seems obvious that the structural similarities shared by the three updating tasks maximized the high correlations between their measures. The opposite seems to be the case regarding the inhibition tasks, which only have one thing in common; the need of conflict/interference resolution in some experimental conditions. However, the conflict source, the target of control, the level of processing wherein conflict arose and the mechanisms  to be triggered to deal with the different types of conflict substantially differed across tasks. Moreover, none of tasks directly relate to managing information in working memory. Hence, even in the case inhibition was the construct underlying the six conflict resolution tasks, why should we expect performance on them correlate with performance on updating/removal tasks?I know the answer: some researchers have claimed that inhibition (as an omnipresent factor) is also involved in managing contents in working memory.My thought is that the best way to test this idea is to define/precise how inhibition could act on irrelevant/outdated information in WM and, if possible, to build up experimental procedures to disentangle inhibition from any other potential mechanism*. Otherwise, removal, updating, inhibition or any other concept will remain blurred and theoretically vague. Moreover, I do not think methodological approaches like the one used in the reviewed manuscript helps because they rely on imprecise constructs and hard to interpret findings. In fact, in my opinion the ‘main’ contribution of the reviewed manuscript is to reveal that the latent variable underlying three very similar updating tasks is not the same as the construct(s) that is(are) measured by a set of very different conflict-resolution tasks. The study says nothing about inhibition, because we do not know if different inhibitory mechanisms are involved in the used tasks . If the mechanism is not well defined in computational terms to describe what is inhibited and how, and it is not well supported by behavioral and brain-related measures to illustrate the inhibitory process and its consequences, in reality what we call inhibition could be anything.As a final point, I was shocked by the rationale for not using the Stop-Signal Task as an inhibitory task. I do not know a task that is totally free of strategical thinking (do you?). Despite this, versions of this task exist that minimizes the use of strategies. The SST is widely considered a “process pure” task to measure inhibitory control . Anyway, personally I wouldn’t expect the SSRT (the inhibitory index from the SST) to correlate with removal as a construct .*Examples of this now exist in the literature on (episodic memory) inhibition where behavioral and brain-related measures can straightforwardly be interpreted from an inhibitory view after ruling out alternative non-inhibitory mechanisms. See for example:- Weller et al. (2013). On the status of cue independence as a criterion for memory inhibition: evidence against the covert blocking hypothesis. J. Exp. Psychol. Learn. Mem. Cognit. 39 (4), 1232–1245.- Wimber et al. (2015). Retrieval induces adaptive forgetting of competing memories via cortical pattern suppression. Nat. Neurosci. 18 (4), 582–589.Reviewer #2: The authors report an experimental study aimed to test whether two distinct processes strongly related to working memory  are the same construct or not. To this purpose, the authors 1) administered various tasks assumed to tap into the two processes under investigation, and 2) adopted a confirmatory factor analysis approach to explore the possible correlational structure of the different measures. The results are interpreted as tentative evidence that removal and inhibition are independent processes.This is a study addressing a timely issue, whose results might be interesting for a wide community of cognitive scientists interested in working memory and executive functioning. The study looks well conducted overall, predictions are clear, the analyses seem elegant and informative, and writing is very clear. There are only a few points that, in my view, should be addressed in a revision.1. Methods section. More details should be provided concerning the number of trials  for each task.2. I think that the discussion section would benefit from including the analysis of a recent paper by Draheim et al. (2020) which is related to the issues addressed in the present paper. I believe the authors should acknowledge the potential problems arising from addressing individual differences investigations by means of RT tasks.https://doi.org/10.1037/xge0000783Here is the reference: Draheim, C., Tsukahara, J. S., Martin, J. D., Mashburn, C. A., & Engle, R. W. (2020). A toolbox approach to improving the measurement of attention control. Journal of Experimental Psychology: General. In press. 3. In the general discussion, I think it would be fair to cite other tasks that are often used to investigate inhibitory processing. These include tasks belonging to the go/no-go family , but also more complex paradigms addressing episodic memory such as the Retrieval Practice Paradigm.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  4 Aug 2020Reviewer AA1 “First, the selection of the tasks noticeably did bias the latent variables that were (or not) identified. It seems obvious that the structural similarities shared by the three updating tasks maximized the high correlations between their measures. The opposite seems to be the case regarding the inhibition tasks, which only have one thing in common; the need of con-flict/interference resolution in some experimental conditions. However, the conflict source, the target of control, the level of processing wherein conflict arose and the mechanisms  to be triggered to deal with the different types of conflict sub-stantially differed across tasks. Moreover, none of tasks directly relate to managing infor-mation in working memory. Hence, even in the case inhibition was the construct underlying the six conflict resolution tasks, why should we expect performance on them correlate with performance on updating/removal tasks?I know the answer: some researchers have claimed that inhibition (as an omnipresent factor) is also involved in managing contents in working memory.My thought is that the best way to test this idea is to define/precise how inhibition could act on irrelevant/outdated information in WM and, if possible, to build up experimental procedures to disentangle inhibition from any other potential mechanism*. Otherwise, removal, updating, inhibition or any other concept will remain blurred and theoretically vague. Moreover, I do not think methodological approaches like the one used in the reviewed manuscript helps because they rely on imprecise constructs and hard to interpret findings. In fact, in my opinion the ‘main’ contribution of the reviewed manuscript is to reveal that the latent variable underlying three very similar updating tasks is not the same as the construct(s) that is(are) measured by a set of very different conflict-resolution tasks. The study says nothing about inhibition, be-cause we do not know if different inhibitory mechanisms are involved in the used tasks . If the mechanism is not well defined in computational terms to describe what is inhibited and how, and it is not well supported by behavioral and brain-related measures to illustrate the inhibitory process and its consequences, in reality what we call inhibition could be anything. *Examples of this now exist in the literature on (episodic memory) inhibition where behavioral and brain-related measures can straightforwardly be interpreted from an inhibitory view after ruling out alternative non-inhibitory mechanisms. See for example:- Weller et al. (2013). On the status of cue independence as a criterion for memory inhibition: evidence against the covert blocking hypothesis. J. Exp. Psychol. Learn. Mem. Cognit. 39 (4), 1232–1245.- Wimber et al. (2015). Retrieval induces adaptive forgetting of competing memories via corti-cal pattern suppression. Nat. Neurosci. 18 (4), 582–589.”RESPONSE: In order to take Reviewer A’s point of view into account, we have clarified more explicitly in the Discussion section (see p. 34) that our main conclusion with regards to inhibi-tion is indeed that performance on the inhibition tasks we used relies on interference-resolution processes that are different from removal. We also agree with the reviewer that this provides additional evidence that we should stop calling everything inhibition, and that more work needs to be done to tease apart specific conflict-resolution processes. The new section reads: "Together, the present results show that performance on the inhibition tasks we used relies on interference-resolution processes that are different from removal. On a general level, the results also provide additional evidence that we should stop using inhibition as an umbrella term for a variety of distinct processes. Thus, although the present study sup-ports the notion that removal is not an inhibitory process, in order to progress, the field will need to specify how inhibition could act on irrelevant/outdated information in WM, and should attempt to build experimental paradigms that are able to disentangle inhibition from other mechanisms of conflict resolution .” A2 “As a final point, I was shocked by the rationale for not using the Stop-Signal Task as an inhibitory task. I do not know a task that is totally free of strategical thinking (do you?). De-spite this, versions of this task exist that minimizes the use of strategies. The SST is widely considered a “process pure” task to measure inhibitory control . Anyway, personally I wouldn’t expect the SSRT (the inhibitory index from the SST) to correlate with removal as a construct .”RESPONSE: We now address this in the Discussion, clarifying why we did not include the stop-signal task (p.35-36): “Although there exist versions of the stop-signal task that minimize the use of strategies , these versions have rarely been used in individual-differences research . As stated in the introduction, we opted for inhibition tasks that are broadly assumed to require inhibition and have been commonly used in individ-ual-differences research. Similarly, we did not include the SART because the dependent measures typically used in this task  do not control for variance in processing speed. If fast and slow individuals re-spond within about the same time, slower individuals will make more errors, thus transferring some speed variance onto accuracy and d’.”Reviewer BB1 “Methods section. More details should be provided concerning the number of trials  for each task.”RESPONSE: Details about the number of trials were initially provided in the supplementary Table S1. Nevertheless, we agree with Reviewer B that these details are sufficiently important to be provided in the main text. Therefore, we have now added this table (as the new Table 1) to the main text (p. 10).B2 “I think that the discussion section would benefit from including the analysis of a recent paper by Draheim et al. (2020) which is related to the issues addressed in the present paper. I believe the authors should acknowledge the potential problems arising from addressing indi-vidual differences investigations by means of RT tasks.”RESPONSE: We thank the reviewer for pointing this out. We now discuss Draheim et al. (2020) in the Discussion section as follows (see p. 32-33): “As pointed out recently by Draheim et al. , one possible reason for the difficulty in establishing a coherent factor of inhibition might be the use of RTs—and in particular, RT dif-ference scores—because these scores have been associated with low reliability and speed-accuracy trade-offs. Besides the substantial impact of measurement error , previous research has emphasized the low performance variability in the tasks used to assess inhibition, resulting in a low test/re-test reliability of these measures . However, both the present study and Rey-Mermet et al. (2018) showed that when computed with a sufficient number of trials, RT difference scores can be measured with reasonable internal consistency reliabilities. Perhaps more importantly, in the present study we also disattenuated the correlations for imperfect reliability in the inhibition measures. Moreover, when using a calibration procedure in order to measure inhibition through accuracy, thus pushing speed-accuracy trade-offs into accuracy scores, Rey-Mermet et al. (2019) were still unable to establish a factor of inhibition. Thus, the use of RTs and RT difference scores per se cannot be the sole reason for the difficulties to establish inhibition at the latent-variable level. A further reason why the use of difference scores may impede extraction of a latent inhibition variable is that computing difference scores is based on the premise of additive factors . However, this premise is questionable be-cause it is not known whether the durations of baseline processes and inhibitory processes combine additively to the RTs of incongruent trials. It is for this reason that both the present study and Rey-Mermet et al.  applied a bi-factor modeling approach to overcome the premise of additive factors. In this approach, all incongruent and congruent trials were forced to load on a baseline factor and the incongruent trials were forced to load on an inhibi-tion factor. However, even when using a bifactor model, no inhibition-specific variance could be established. Thus, the use of difference scores—either in RTs or accuracy rates—can also not totally explain the difficulty of establishing an inhibition factor at the latent-variable level.”B3 “In the general discussion, I think it would be fair to cite other tasks that are often used to investigate inhibitory processing. These include tasks belonging to the go/no-go family , but also more complex paradigms addressing epi-sodic memory such as the Retrieval Practice Paradigm.” RESPONSE: The Discussion now mentions the go/no-go task (or Sustained Attention to Re-sponse Task) and the reason it was not included in the present study (see p. 36): “Similarly, we did not include the SART because the dependent measures typically used in this task  do not control for variance in processing speed. If fast and slow individuals respond within about the same time, slower individuals will make more errors, thus transferring some speed variance onto accuracy and d’.”We decided not to mention the retrieval practice paradigm because this task does not belong to the tasks commonly used to assess inhibition in individual-differences research focusing on attentional control/executive functions. 23 Sep 2020PONE-D-20-13452R1Removal of information from working memory is not related to inhibitionPLOS ONEDear Dr. Alodie Rey-MermetThank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not yet meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================ACADEMIC EDITOR: Dear Dr. Alodie Rey-Mermet,as you will see, the reviews are mixed. In particular, one of the Reviewer is satisfied with the changes you have made. The other one still see some substantial -theoretical-  weakness in your study. I have  also looked at the revised version of your interesting paper. I have to say that I agree with the comments done by Reviewer 1 . I am thus asking you to revise the paper carefully taking into consideration the points made by Reviewer 1 that mainly regards the introduction and the discussion section. The points the Reviewer raised are important from a theoretical point of view,  and by considering  and "including" them in your revision , your paper will have a strong impact in the literature on inhibition. ==============================plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by 15th of November . 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: These are my thoughts after reading the revised manuscript carefully.To be honest, I thought my comments to the previous version of the manuscript would prompt the authors to somehow refocus the introduction/discussion . This has not been the case, however, with the revised manuscript only including a new few lines in the discussion. Well, this is their manuscript, not mine. However, I do not think the manuscript has been significantly improved.If the authors (like me) believe that their ‘inhibition’ tasks mostly tap into (mechanism-free) interference control (and the discussion now seems to endorse this idea), the introduction should also bring this into focus to increase coherence and consistency throughout the text. Potential readers would benefit from it.By the way, the new paragraph in the discussion includes the following statement: ‘Thus, although the present study supports the notion that removal is not an inhibitory process…’. Again, I DO NOT think this is true. In fact, it seems quite contradictory to the idea that maybe the used ‘inhibition’ tasks do not measure inhibition.Related to this, it is now clear to me that the title of the manuscript should be changed. I cannot see the point of having “inhibition” in it.To conclude, a couple of notes aimed to encourage the authors to rethink some of the arguments they deploy on the manuscript/response letter:https://www.jneurosci.org/content/38/36/7887). Thus, to justify that you did not pick this task for your study, you better use a different reason.1. The Stop-Signal Task has been widely used to investigate individual differences (a recent example here 2. In response to a (very smart) suggestion by the other reviewer, the authors write: ‘We decided not to mention the retrieval practice paradigm because this task does nothttps://pubmed.ncbi.nlm.nih.gov/25180807/].belong to the tasks commonly used to assess inhibition in individual-differences’. Well, the authors could want to know that the manuscript already includes mentions to the retrieval practice paradigm . 73 and 74 refer to retrieval practice studies. Also, and despite the authors think, the RP  paradigm has been extensively used to explore individual differences in inhibitory control . Obviously, I totally endorse the original suggestion made by the other reviewer.”A5 “In response to a (very smart) suggestion by the other reviewer, the authors write: ‘We decided not to mention the retrieval practice paradigm because this task does not belong to the tasks commonly used to assess inhibition in individual-differences’. Well, the authors could want to know that the manuscript already includes mentions to the retrieval practice paradigm . 73 and 74 refer to retrieval practice studies. Also, and despite the authors think, the RP  paradigm has been extensively used to explore individual differences in inhibitory control [over episodic memory, of course; have a look at the meta-analytic review by Murayama et al. (2014) here RESPONSE: We again thank Reviewer A for pointing out another imprecision. Previous research has clearly shown that inhibition in episodic memory – as assessed with the retrieval practice paradigm – differs from the interference-control processes measured in tasks such as the Stroop and flanker tasks . Based on this research, we made a distinction between the interference-control processes measured with paradigms such as the retrieval practice paradigm (which we refer to as “inhibition in episodic memory”) and the interference-control processes measured with the inhibition tasks, such as those used in the present study. Therefore, when we wrote in our previous response that the retrieval practice paradigm “does not belong to the tasks commonly used to assess inhibition in individual-differences research”, we meant that this paradigm does not belong to the tasks typically called “inhibition tasks” .AttachmentResponse To Reviewers.docxSubmitted filename: Click here for additional data file. 16 Nov 2020Interference control in working memory: Evidence for discriminant validity between removal and inhibition tasksPONE-D-20-13452R2Dear Dr. Alodie Rey-Mermet,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Erika Borella, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :I am pleased to accept this interesting paper.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: No 20 Nov 2020PONE-D-20-13452R2 Interference control in working memory:Evidence for discriminant validity between removal and inhibition tasks Dear Dr. Rey-Mermet:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Tau protein abnormally aggregates in tauopathies, a diverse group of neurologic diseases that includes Alzheimer’s disease (AD). In early stages of disease, tau becomes hyperphosphorylated and mislocalized, which can contribute to its aggregation and toxicity. We demonstrate that tau phosphorylation at Ser208 (pSer208) promotes microtubule dysfunction and tau aggregation in cultured cells. Comparative assessment of the epitopes recognized by antibodies AT8, CP13, and 7F2 demonstrates that CP13 and 7F2 are specific for tau phosphorylation at Ser202 and Thr205, respectively, independently of the phosphorylation state of adjacent phosphorylation sites. Supporting the involvement of pSer208 in tau pathology, a novel monoclonal antibody 3G12 specific for tau phosphorylation at Ser208 revealed strong reactivity of tau inclusions in the brains of PS19 and rTg4510 transgenic mouse models of tauopathy. 3G12 also labelled neurofibrillary tangles in brains of patients with AD but revealed differential staining compared to CP13 and 7F2 for other types of tau pathologies such as in neuropil threads and neuritic plaques in AD, tufted astrocytes in progressive supranuclear palsy and astrocytic plaques in corticobasal degeneration. These results support the hypothesis that tau phosphorylation at Ser208 strongly contributes to unique types of tau aggregation and may be a reliable marker for the presence of mature neurofibrillary tangles. Alzheimer’s disease (AD) is the most common form of age-related dementia and affects over 25 million people worldwide . AD is pTau is a microtubule-associated protein that is highly expressed in the distal axons of neurons in the central nervous system , 80. PhyIn AD, all six major tau isoforms aggregate to form pathological inclusions in a hyperphosphorylated state , 80. OveAlthough the AT8 antibody is very useful diagnostic tool, the exact epitope recognized by this antibody has not been completely resolved, since AT8 was originally created by immunizing mice with paired helical filaments of tau (PHF-tau) purified from AD brains . Early cIn this study, we modeled tau phosphorylation at Ser208 and nearby phosphorylation sites with phosphomimetics to determine its role in tau aggregation and MT binding in cultured cells. To confirm the in vivo relevance of pSer208, we also created a novel monoclonal antibody 3G12 specific for pSer208 and demonstrated its strong reactivity for tau inclusions in transgenic mouse models of tauopathies and in postmortem brain samples of patients with AD and other tauopathies.Escherichia coli . Recombinant K18 tau protein was purified as previous described [The K18 tau fragment consists of the MT binding repeats of the 2N4R human tau protein isoform, which includes amino acid residues Q244 to E372. An additional N-terminal methionine was added to K18 protein and expressed under the bacterial plasmid pRK172 in BL21 (DE3)/RIL escribed . ProteinPurified K18 tau protein was dissolved in PBS at a concentration of 1 mg/mL and 50 μM of heparin and was placed in a shaking incubator at 1050 RPM and 37 °C for at least 2 days. As previously described, the presence of polymerized amyloidogenic K18 fibrils structure was confirmed by K114 or thioflavin T assays . To remoThe 2N4R human tau isoform cDNA was cloned into the pcDNA3.1 mammalian expression vector. Phosphomimetic mutations were introduced by QuikChange site-directed mutagenesis  with customized oligonucleotides. The sequence of all constructs with the entire tau sequence was verified by Sanger sequencing performed by Genewiz .2 in Dulbecco’s modified Eagle’s media and 10% fetal bovine serum (FBS) supplemented with antibiotics . Calcium phosphate precipitation was used to transfect HEK293T cells with various plasmid constructs. Cells were split into 12-well plates at 20–40% confluency. For each well, 1.5 μg of DNA was mixed with 18.75 μL of 0.25 M CaCl2. This mixture was added to an equivalent of 2X BES buffer  and incubated at room temperature for 15–20 min. The final solution was placed dropwise to each well. For tau seeding experiments, 1 μM of purified K18 tau fibrils was added an hour post-transfection [HEK293T cells were maintained at 37 °C and 5% COsfection , 84. 16 HEK293T cells were harvested in 200 μL of Triton Lysis Buffer  with a mix of different protease inhibitors as previously described , 84. Cel2) supplemented with 0.1% Triton X-100, 2 mM GTP, 20 μM paclitaxel, and protease inhibitors as previously described [HEK293T cells were lysed in 200 μL of PEM buffer  were coated with 100 ng in 100 μL PBS per well of each peptide , which corresponds to tau phosphorylated at Ser208, conjugated to KLH as previously described [BALC/c mice  were immunized with a synthetic peptide Cys-escribed , 73, 74.escribed , 73. MonProtein samples were loaded on 10% polyacrylamide gels for SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked in 5% milk in TBS for 1 h at room temperature and incubated in primary antibodies overnight at 4 °C at 1:1000 dilutions for 3026 total tau antibody , β-tubulBrain samples from non-transgenic (nTG), tau knockout (KO)  and rTg4Formalin-fixed brain samples of patients with AD, PSP, and CBD were provided by the University of Florida Neuromedicine Human Brain and Tissue Bank (UF HBTB) following institutional regulations. See Table For AD and dementia with Lewy body (DLB) cases , interrater reliability was assessed by calculating the intraclass correlation coefficient (ICC) with a two-way mixed model for consistency between three different raters . For couA previous in vitro study indicated that the combined phosphorylation of Ser202, Thr205, and Ser208 residues promotes the polymerization of tau into filaments . To asseAs previously shown , 84, WT Prior in vitro studies have suggested that phosphorylation of Ser202 and Thr205 can decrease tau’s ability to promote MT polymerization , but theTo study tau phosphorylation of Ser208 (pSer208) in human tauopathies, a new monoclonal antibody specific for pSer208 was generated by immunizing mice as described in Material and Methods using the synthetic pSer208 peptide Table a. Since To test the specificity of antibody 3G12 and the presence of tau Ser208 phosphorylation within aggregated tau in disease models, immunoblot analysis was performed using the Triton-insoluble fraction of brain lysates of tau KO, nTG mice, and rTg4510 transgenic mice that overexpress 0N4R isoform of P301L tau mutation , 69. 3G1Using ELISA and different synthetic phosphorylated peptides Table , the epiWe previously made antibody 2D1 against the pSer199/pSer202/pThr205 peptide, but it was not phosphorylation specific . We confIn the brains of two transgenic tau mouse models, 3G12 immunoreactivity was compared to other antibodies  against adjacent or overlapping phospho-epitopes. Adjacent brain sections from two mouse models were stained: rTg4510 transgenic mice that overexpress 0N4R human tau isoform with the P301L mutation specifically in the forebrain , 69 and Next, we assessed if pSer208 is found in tau inclusions of post-mortem human brains from patients with different tauopathies. Brain tissue sections containing pathological tau inclusions from a series of patients with either AD or dementia with Lewy Bodies (DLB) with AD-tau pathology  specific to tau phosphorylated at Ser208 was generated to allow to monitor this modification in animal models and human brain tissue. The specificity of the 3G12 antibody was compared to the AT8 antibody and other related antibodies against nearby sites. Although AT8 antibody binds around the center of pSer202/pThr205 , 28, pasIn two different mouse models of tauopathies, our 3G12 antibody robustly stained tau inclusions in cortex and hippocampus of rTg4510 mice, and in thalamus and brainstem of PS19 mice. These two mouse models expressed different tau mutations and variants: PS19 mice overexpress the 1N4R human isoform with the P301S mutation  and rTg4In AD and DLB patients with AD-tau pathology, it is shown that 3G12 antibody reveals strong preference for mature tangles relative to pre-tangles and other unstructured aggregates of tau compared to AT8. This observation was consistent across multiple regions of the medial temporal lobe. pSer208 is unique as a marker of aggregation and mature NFT compared to the other phosphorylation sites that stain all forms of tau. Phosphorylation at Ser208 might occur sequentially after Ser202 and Thr205 phosphorylation as the S208E phosphomimetic significantly reduces phosphorylation of these sites as determined by phosphorylation-specific antibodies AT8, CP13, and 7F2, at least in HEK293T cells  , cyclin-Besides histopathological diagnosis, an antibody specific for mature tangles such as 3G12 might provide high potential diagnostic value as a blood and cerebrospinal fluid (CSF) biomarker for NFT progression in AD patients, as pSer208 was reported to be distinctively elevated in the CSF of AD patients compared to healthy controls . AntibodAdditional file 1: Figure S1. Soluble fractions of WT tau, tau mutant P301L, and tau phosphomimetics have similar expression levels. Soluble fractions of WT tau, P301L, and phosphomimetics S208E, S202E/T205E, S202E/T205E/S208E, S202E/T205E/S208E/ P301L were immunoblotted with a total tau antibody 3026. The relative molecular masses of protein markers are indicated on the left.Additional file 2: Figure S2. Tau S208E phosphomimetic presents significantly reduced phosphorylation of nearby sites Ser202 and Thr205. HEK293T cells were transfected to express WT tau or S208E tau phosphomimetic 2N4R isoform. Whole cell lysates were immunoblotted with a total tau antibody 3026 and phosphorylation specific antibodies CP13, 7F2, and AT8. The relative molecular masses of protein markers are indicated on the left.


Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness.2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05.In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells.Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked CaTaken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity. It is equally unclear the structure–function relationship between an altered E–C coupling and obesity-associated AHR.Obesity contributes to asthma diathesis by enhancing airway hyperresponsiveness (AHR) and attenuating the response to standard asthma therapy –3. Humanortening . In a paortening , 9. Addiortening . How obeq i in HASM cells was determined as previously described with some modifications i in HASM cells. HASM cells were pre-treated with vehicle (DMSO), GW9508 (10 μM), or TAK875 (10 μM) for 30 min, then stimulated with carbachol 25 μM or histamine 2.5 μM. Agonists-induced [Ca2+]i were determined for 100 s. (A-C) GW9508 or TAK875 pre-treatment has little effect on CCh-induced calcium mobilization. .


Curling is a sport in which players deliver a cylindrical granite stone on an ice sheet in a curling hall toward a circular target located 28.35 m away. The stone gradually moves laterally, or curls, as it slides on ice. Although several papers have been published to propose a mechanism of the curling phenomenon for the last 100 years, no established theory exists on the subject, because detailed measurements on a pebbled ice surface and a curling stone sliding on ice and detailed theoretical model calculations have yet to be available. Here we show using our precise experimental data that the curl distance is primarily determined by the surface roughness and the surface area of the running band on the bottom of a stone and that the ice surface condition has smaller effects on the curl distance. We also propose a possible mechanism affecting the curling phenomena of a curing stone based on our results. We expect that our findings will form the basis of future curling theories and model calculations regarding the curling phenomenon of curling stones. Using the relation between the curl distance and the surface roughness of the running band in this study, the curl distance of a stone sliding on ice in every curling hall can be adjusted to an appropriate value by changing the surface roughness of the running band on the bottom of a stone. The first written evidence of curling appeared in 1540 when a notary in Scotland recorded in his protocol book a challenge between two persons who threw a stone on ice3. Curling in its early days was played on frozen lochs and ponds during a harsh European winter. Curling has now evolved into a popular modern sport that takes place on indoor ice rinks with the condition and temperature of the ice carefully controlled.Curling is a sport in which players deliver a cylindrical granite stone (about 28 cm in diameter and 18 to 19 kg in weight) on an ice sheet in a curling hall toward a circular target located 28.35 m away. The stone gradually moves laterally, or curls, as it slides on ice. The exact origin of curling is unknown, but an old curling stone engraved with the date 1511 was discovered in Scotland4, several papers27 have been published to propose a mechanism of the curling phenomenon. However, no established theory exists on the subject, because detailed measurements on a pebbled ice surface and a curling stone sliding on ice and detailed theoretical model calculations have yet to be available.A curling player releases a stone with a small degree of clockwise rotation on ice, and the stone gradually curls toward the right, while a small anticlockwise rotation allows it to curl toward the left. When a player releases a stone without any rotation, the stone usually starts to rotate by itself and the trajectories are different every time. The curling phenomenon is primarily important in curling games because the amount of lateral displacement, called the curl distance, contributes to the strategy of games. Since the first scientific paper by HarringtonThe surface conditions of ice and a stone in curling games have distinctive features. The ice surface is not flat but has small ice pebbles attached to a flat ice surface by spraying water droplets onto the ice. The tops of the pebbles are cut off with a blade called a nipper. The bottom of a curling stone is concave at its centre, with only a running band, an annulus with an inner diameter of about 125 mm and a width of about 3−7 mm, touching the ice. These features reduce the contact area between the ice surface and the stone.7, a front-back asymmetry model 24 and a pivot-slide model 27.The curling behaviour of a stone sliding on ice has been explained by three models: a left–right asymmetry model 8, the FB and the PS models have been developed. The FB models are divided into six models, in which different mechanisms have been proposed for different front and back frictional forces: a pressure difference model (a larger pressure works on the front running band than on the back running band)11, a water layer model 14, a snowplow model 15, an evaporation-abrasion model 18, a scratch-guiding model 22 and an edge model (with different rake angles of a stone at the front and back of the stone)24. Recent papers discuss a possibility of the PS model27, which was originally suggested by Penner8. Scientific discussions between different modellers have been raised and published35. However, no established theory exists, because detailed measurements on a pebbled ice surface and a curling stone sliding on ice and detailed theoretical model calculations have yet to be available, as partially explained in review papers37.Because the LR models failed to explain why a curling stone curlsThe curl distance for a stone with a typical angular velocity  ranges from about 0.5 to 1.5 m at a circular target located 28.35 m away on ice. The differences in ice surface conditions  and curling stones are the factors for controlling the curl distances. Thus, to determine the factors affecting the curling distances by precisely measuring these conditions, we carried out our measurements at ADVICS Tokoro curling hall in Kitami, Japan.The room temperature at a height of 1.5 m in the ADVICS Tokoro curling hall was 5 to 7 °C, and the ice surface temperature was − 3.5 to − 2.5 °C during our experiments.We used four instruments in this study: an automatic tracking total station , a probe-type surface roughness meter , a small digital camera , and a confocal laser scanning microscope .The ATTS was used for measuring the trajectory of the centre of a curling stone sliding on ice as shown in Fig. z-direction) is 0.02 μm. The arithmetical mean of the surface roughness (Ra), defined by Eq.  with ultraviolet light on the pebbled ice surface in the rink and used for the microscope. The accuracy of the height measurements was  ±0.8 μm. Because the replica samples shrink at 1.1% in the parallel direction of the specimens and 2.7% in the vertical direction of the specimens during solidificationWe used four stones (A to D) with different surface roughness conditions. The surface roughness of the running band of a stone was adjusted using a sheet of sandpaper as follows. A sheet of sandpaper (23 by 28 cm) was secured on a flat desk with a double sided tape, and each stone was moved on the paper forth and back in a straight line by a distance of about 10 cm. This procedure was carried out four times each with a stone rotation angle of 45°. When the stone was rotated by 45°, the stone was lifted. A total of four forth-and-back movements were carried out with the same rotation angle on the same sandpaper.We use a sheet of sandpaper of P80 grit number as a reference. We selected rougher sandpaper (P40) for A2 to increase the surface roughness of A2. We selected smoother sandpaper (P120) for C1 to slightly adjust the surface roughness of C1. For B2 and D1, we use the original surface roughness of the running band, and did not use the above sandpaper operations. Thus, sandpapers with the following grit numbers were used for the respective running bands: P80 for A1, B1, C2, and D2; P40 for A2; and P120 for C1.https://iceking.ca/web/en/). In this paper, we used stone trajectory data measured on August 17 and 18 in 2019, and a total of 201 trajectory data sets were recorded on the days. All stones were released by skilled members of the curling club at Kitami Institute of Technology.Because each stone has two running bands on its top and bottom, we measured the eight running bands of the four curling stones. We used two types of ice surface in our experiments in ADVICS Tokoro curling hall: a typical ice surface with pebbles Fig.  and a fly-axis is parallel to the longitudinal direction of the ice rink, and x-axis is vertical to the y-axis. A curling stone was normally delivered along the y-axis in our experiments; however, it was difficult to perfectly deliver the stone along the y-axis. Thus, we corrected the stone’s trajectory data as follows, outlined in Fig. A least square method by a linear approximation was applied to the first 3 to 8 positional data sets of the stone after its delivery and the linear lines were obtained, because a stone moves almost lineally for a few seconds after its delivery.The maximum distances for each data set were calculated from the lines.The most appropriate linear line was selected with the following two criteria: the maximum distance between the lines and the positional data is less than 2 mm (measurement accuracy of the ATTS), and the linear line uses the maximum number of positional data points.xi′, yi′) of a stone, and y′ is the original direction of a stone. A tilt angle (θ) between y′ and y axes expressed in Eq.  shown by solid circles in Fig. x and y are the lateral and longitudinal displacements, respectively.The ing Eqs.  and 5),,x and y For the positional data of a curling stone measured by the ATTS, we defined that y-direction, the trajectories of the stone were pulled back by the distances along the y-direction, and the curl distances at 28.35 m were corrected by the x position at y = 0.We defined two types of curl distance for our experimental data. If a stone stopped within 1 m from the centre of the house, 28.35 m away, the curl distance calculated in Eq.  was usedAll experiments were carried out in accordance with relevant guidelines, regulations and ethics approval by ADVICS Tokoro curing hall in Kitami, Japan. Permission was individually obtained from two persons who appear in Fig. Figure −2. Figure The diameter of the upper surface ranged from 0.36 to 2.18 mm, and the average diameter was 0.97 ± 0.63 mm for seven samples . The diameter of the lower base of pebbles ranged from 1.57 to 6.84 mm, and the average diameter was 3.58 ± 1.62 mm. The height of pebbles ranged from 0.11 to 0.16 mm, and the average height was 0.13 ± 0.02 mm. We found that the upper surface was slightly enlarged and the height was slightly shortened after stones passed on the pebbles. The average number density of pebbles ranged from 2 to 5 cmRa ranges from 0.389 ± 0.099 μm to 3.106 ± 0.258 μm as shown in Fig. We measured the surface roughness profiles of each running band at four places at 90° intervals of the four stones using the probe type surface roughness meter. Thus, the total of 32 surface roughness profiles of the running bands (4 stones × 2 surfaces × 4 places) were measured. Figure Ra: 2.772 ± 0.195 μm) and stone B with the running band B2 (Ra: 0.526 ± 0.070 μm), which were obtained after the coordinate rotations described in Eqs. ..Ra: 2.77We found that stone A1 moves along the almost straight trajectory within 2 m after delivery and gradually curls to the right, with the curl distance of 1.1 m at 28.35 m away. On the other hand, the curl distance of stone B2 is 0.2 m, which is 1/5 of that of stone A1, and stone B2 moves with a complex trajectory as shown in Fig. Ra for different curling stones and the curl distance for an ordinary pebbled ice surface shown by solid circles and a solid line, and for a flat ice surface shown by open circles and a broken line at a typical angular velocity . A total of 43 trajectory data sets were used in Fig. xp and xf denote the curl distances for the normal pebbled ice surface and for the flat ice surface, respectively. The correlation coefficient (r) and the level of significance (p) are 0.88 (p < 0.01) for the normal pebbled ice surface, and 0.97 (p < 0.01) for the flat ice surface.Figure xp and xf was 0.273 m for Ra = 3 μm using Eqs. (The maximum difference between the curl distances ing Eqs.  and 7)..xp and xd) is slightly different for each stone, we define the surface roughness area (SRA) for each running band using Eq.  to 126 mm , and the value d ranged from 3.05 ± 0.02 (D1) to 6.20 ± 0.00 mm (A2).Because the width of the eight running bands  and the level of significance (p) are 0.97 (p < 0.01) and 0.98 (p < 0.01). Because the SRA is corrected for the different width of the running band for each stone, the relationship between the curl distance and the surface roughness condition of running bands is better as shown in Fig. We find that the relationship is better than the relationship in Fig. SRA from 10 to 150 μm cm2. Thus, the ice surface condition has much smaller effects on the curl distance than the surface roughness condition of curling stones, because the flat ice surface condition in Fig. We also find that the different ice surface condition contributes to the curl distances of 0.13 to 0.15 m for different values of As already mentioned, the curling distances of curling stones are different on every curling rink. The reason has been unknown to curling players and to scientists studying the curling phenomena of a stone sliding on ice. Most curling players may consider that the difference is primarily caused by ice surface conditions, including ice surface temperature, the number density of pebbles, and the diameter and height conditions of pebbles. However, our study clearly demonstrates that the curl distance is primarily determined by the surface roughness and the surface area of the running band on the bottom of a stone, and the ice surface condition has smaller effects on the curl distance. We expect that our findings will form the basis of future curling theories and model calculations regarding the curling phenomenon of curling stones and will clarify requirements of the running band on the bottom of a stone to achieve an appropriate curl distance in curling games.Ra) of a running band to 2.32 μm for a curling distance of 1.0 m and to 3.54 µm for a curling distance of 1.5 m, according to Eq.  by setting the average surface roughness , the relationship was improved. This shows the importance of the surface roughness and the area of the running band for the curling phenomenon of a stone moving on ice.The curl distance of a stone is primarily determined by the surface roughness of a running band of a stone. Using the products of the surface roughness and the area of the running band (With the increase of the surface roughness of the running band, erosion depths at the surface of pebbles will be deeper, and larger ploughing forces are generated at the contact points between the running band and the surface of pebbles. Inhomogeneous distribution of the ploughing forces around the running band or continuous small pivoting around the contact points will possibly cause the curling phenomena of a curling stone moving on ice. The former is the reason for the FB model, and the latter is the reason for the PS model.The curl distance of a stone sliding on ice in every curling hall can be adjusted to an appropriate value by changing the surface roughness of the running band on the bottom of a stone, under the same conditions as described in this paper for the surface ice temperature and the pebbled ice surface conditions in curling rinks.SRA of a stone.The ice surface conditions contribute to the curling distance of a stone. However, the effect was smaller than that of the surface roughness and the In this paper, we clarified the following relating to the curl distance of a stone moving on ice.


Y376GRAF3 reverses an auto-inhibitory interaction between the GAP and BAR-PH domains. Y376 is located in a linker between the PH and GAP domains and is invariant in GRAF3 homologues and a phosphomimetic E376GRAF3 variant exhibited elevated GAP activity. Collectively, these data provide strong support for the future identification of allosteric activators of GRAF3 for targeted anti-hypertensive therapies.Anti-hypertensive therapies are usually prescribed empirically and are often ineffective. Given the prevalence and deleterious outcomes of hypertension (HTN), improved strategies are needed. We reported that the Rho-GAP GRAF3 is selectively expressed in smooth muscle cells (SMC) and controls blood pressure (BP) by limiting the RhoA-dependent contractility of resistance arterioles. Importantly, genetic variants at the GRAF3 locus controls BP in patients. The goal of this study was to validate GRAF3 as a druggable candidate for future anti-HTN therapies. Importantly, using a novel mouse model, we found that modest induction of GRAF3 in SMC significantly decreased basal and vasoconstrictor-induced BP. Moreover, we found that GRAF3 protein toggles between inactive and active states by processes controlled by the mechano-sensing kinase, focal adhesion kinase (FAK). Using resonance energy transfer methods, we showed that agonist-induced FAK-dependent phosphorylation at  High blood pressure, known clinically as hypertension (HTN), is a highly prevalent and relevant disease in the Western world and is a major risk factor for myocardial infarction, stroke, and kidney failure. It is estimated that 1 in every 3 adults in the US has HTN and another 1 in 3 has prehypertension ,3. In fa2+-calmodulin dependent activation of myosin light chain (MLC) kinase and SMC tension is directly proportional to MLC phosphorylation [While BP regulation involves the integrated control of many different organ systems and the interplay of many genes ,6, one orylation ,10. SMC rylation ,12,13,14rylation . Thus, arylation ,11,13 anrylation , and ROCrylation .RhoA is activated by guanine nucleotide exchange factors (GEFs) that facilitate GDP-GTP exchange and is inhibited by GTPase Activating Proteins (GAPs) that facilitate RhoA’s intrinsic GTPase activity. Our recent collaborative efforts have demonstrated that the Rho-specific GTPase activating protein GRAF3 (GTPase Regulator Associated with FAK) is highly and selectively expressed in SMC in mice and humans and controls blood pressure by inhibiting RhoA-mediated SMC contractility. Specifically, we showed that GRAF3-deficient mice exhibit significant hypertension and increased pressor responses to Angiotensin II and endothelin 1; an effect that can be prevented by treatment with the Rho kinase inhibitor Y-27632 . Both laIn spite of the strong link between the RhoA signaling axis and the development of HTN, surprisingly few treatments are available that directly target this pathway. Herein, we developed and utilized a novel transgenic mouse line to show that modest ectopic expression of GRAF3 in SMC has the capacity to stably reduce BP. Importantly, we provide supportive evidence that endogenous GRAF3 protein toggles between inactive and active states that are controlled by post-translational modifications. Collectively these findings support the possibility that pharmacological targeting of GRAF3 could lead to a new class of therapeutics to treat hypertension and to reduce the morbidity and mortality associated with this disease.RQGRAF3 ES cells into the blastocoel cavity of mouse blastocysts by standard procedures. The RQGRAF3 strain was established using two independent chimeras that demonstrated germline transmission when bred to wild-type C57bl6 mice. RQ SMMHC-CreERT2GRAF3 mice were generated by crossing RQGRAF3 female mice with T2SMMHC-CreER male mice. All experiments were performed using age and sex-matched genetic controls. The T2SMMHC-CreER line is currently the most specific and robust SMC-specific Cre line available. However, because this BAC transgene was randomly incorporated into to the Y chromosome, we were limited to using male mice for our studies. Genotyping was performed using DNA isolated from tail biopsies using locus-specific primers . Expression of the transgene was assessed by Western analysis and by quantifying levels of GRAF3, GAPDH (reference gene), ACTB (reference gene) and SM22 (GRAF3 target gene) in bladders and aortas isolated from 8-month-old RQ SMMHC-CreERT2GRAF3 and genetic control mice. Forty-eight hours later after the last dose of tamoxifen, bladders were isolated and flash frozen while thoracic aorta segments were isolated and RNA was extracted using Qiagen RNeasy fibrous tissue kit . Semi quantitative RT–PCR or quantitative RT-PCR as indicated was performed with the following primers: GRAF3 exons 1–4, 5′-CTGCCCACTCTGGAGTTCAGCG, 3′-GCTGCACCGATCTGTTCTTTTCG; GAPDH, 5′-ATGGGTGTGAACCACGAGAA, 3′-GGCATGGACTGTGGTCATGA; SM22, 5’-TGGGCGGCCTACATCAGGGC, 3’-CGGGGTGGTGAGCCAAGCAGA; ACTB, 5’-AGAGCTATGAGCTGCCTGACGGC, GGATGCCACAGGATTCCATACCC. Animal husbandry was provided by staff within the University of North Carolina Division of Comparative Medicine and all animal procedures were approved by our accredited American Association for Accreditation of Laboratory Animal Care committee and the Institutional Animal Care and Use Committee #329.Chimeric mice were produced in-house by injection of l-arginine methyl ester hydrochloride (L-NAME) salt   were added to drinking water for 7 days (per dose). Mice were maintained in a 12:12 h light/dark cycle. All blood pressure parameters were telemetrically recorded and stored with the Ponemah data acquisition system . Recordings were collected for 5 min every 30 minutes throughout the study and averaged over a 24-h period for each day.Conscious blood pressure was measured in male mice aged 12–16 weeks using radiotelemetry . Implantable mouse BP transmitters (PA-C10) were used to record arterial pressure in conscious and freely moving mice. In brief, the mice were anaesthetized with 2% isoflurane, the telemetry catheter was inserted into the left carotid artery of the mouse and the catheter tip was advanced into the thoracic aorta. The catheter was fixed in the left carotid artery and the transmitter was inserted subcutaneously along the right flank. Mice were allowed 7 days of recovery following transmitter implantation and were housed individually in a standard polypropylene cage placed on a radio receiver. Following baseline readings, mice were treated with tamoxifen (100 mg/kg) for 3 consecutive days via oral gavage. Increasing doses of Nω-Nitro-Cos cells and rat aortic SMCs (RaAoSMCs) were maintained in DMEM  or DMEM-F12 media , respectively, supplemented with 10% fetal bovine serum and 0.5% penicillin/streptomycin. Cells were transfected with plasmids using Trans-IT  transfection reagents according to the manufacturer’s protocol. Myc-GRAF3 was made by cloning GRAF3 into a pCMV-Myc vector . Flag-GRAF3 Bar-PH was made by cloning into a pcDNA3 vector. GST-GRAF3-BAR-PH-GAP was made by in-fusion cloning  into a pGEX6.1 vector . All phosphomimetic and phosphodeficient mutations were made by site-directed mutagenesis . Where indicated, cos cells were infected with LacZ/Cre adenovirus  for 24 h.www.rcsb.org). The GAP domain was then docked onto the BAR-PH domain using the Clus Pro protein-protein docking server [Molecular models of GRAF3 were built using PyMol to combine the BAR-PH domains of Appl1  and the GAP domain of GRAF1 . The domains from these proteins were chosen because they were the most similar and highly conserved proteins/domains (compared to GRAF3) that had solved experimental structures available on the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB)  and starved for 4 h prior to treatment with 10 μM Sphingosine 1 phosphate (S1P). CFP and FRET signals were excited at 440 nm and emission signals were collected at 470 nm and 535 nm, respectively, using an Olympus IX-81 inverted microscope. Image J software was used to calculate FRET signals. The FRET/CFP ratio of each cell was normalized to the FRET/CFP value before S1P stimulation.Cells or tissues were lysed in RIPA buffer + 0.5% Triton with protease and phosphatase inhibitors. Protein concentration was determined by using a colorimetric BCA assay . Lysates were electrophoresed on 10–15% SDS-polyacrylamide gels, transferred to PVDF membrane, and immunoblotted with specific antibodies as indicated using a 1:1000 dilution. Myc, Src, pMLC, MLC and GRAF3 pY376 antibodies were from Cell Signaling ; Flag antibody from Sigma ; pTyr antibody from EMD Milipore ; GAPDH from Novus Biologicals ; GFP antibody from Meridian Life Sciences . Blots were washed in TBST (TBS plus 0.05% Triton X-100), followed by incubation with horseradish peroxidase conjugated secondary antibodies  at a 1:2000 dilution. Blots were visualized after incubation with chemiluminescence reagents .Cos cells were transfected with myc-GRAF3 variants and either Src or Flag-superFAK plasmid constructs. After 24 h, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer + 0.5% Triton X-100 and myc-tagged GRAF3 was immunoprecipitated overnight from cell lysate using a myc antibody  conjugated to Dynabeads Protein G . Immunoprecipitates and lysates were electrophoresed on a 10% SDS-PAGE gel and immunoblotted with indicated antibodies.E. coli and expressed by IPTG induction at 16 °C overnight. E.coli were lysed with a lysozyme-buffer , sonicated and then protein was purified using glutathione-sepharose beads . For GAP assay, constructs were dissociated from beads via reduced glutathione and GST was removed via PreScission Protease  according to the manufacturer’s protocol.GST-GRAF3 constructs were transformed into BL-21 competent 2, 5 mM EGTA, 2 mM EDTA, 12.5 mM β-glycero-phosphate, 2.5 mM DTT) with either active FAK  or Src  kinase and radioactive γ-32P  at 30°C for 15 min. Reaction mixture was run on a 10% SDS-PAGE gel, gel was dried and phosphorylation was assessed by radiograph. Gel was rehydrated and stained with Coomassie Blue to assess total protein concentration.Purified GST-GRAF3-BAR-PH-GAP or GST-GRAF3 BAR-PH-GAP-Y376F were incubated in kinase buffer . After 30 min incubation on ice, the TR-FRET signals were measured at 670 ± 10 nm and 620 ± 10 nm on a FRET setting on a CLARIOstar  plate reader.The Transcreener GDP TR-FRET Red enzyme assay was optimized according to the manufacturer’s instructions . Ten μL of GRAF3 (0.2 nM) in a reaction buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM MgCl® (Promega) was added to the C-terminal of a pNLF1-N (CMV/Hygro) vector. Then WT or Y376E mutated GRAF3 BAR-PH-GAP was inserted between a NanoLuc®  tag and a HaloTag®. RaAoSMCs were transfected with HaloTag® control and either GRAF3 WT or GRAF3 Y376E tagged plasmids. BRET signal was detected 48 h later by the NanoBRET™ Nano-Glo® Detection System . Dual-filtered luminescence signals were measured on a CLARIOstar  plate reader using a 450 ± 40 nm donor band pass filer and a 600 ± 10 nm long pass filter.A HaloTagp-values. A p-value < 0.05 was considered significant. Sample sizes were chosen based on an extensive literature search and standard exclusion criterion of two standard deviations from the mean were applied. All statistics were calculated in Excel or GraphPad Prism8.Unless stated otherwise, all data represent at least three individual experiments and are presented as means ± standard error of the mean (SEM) or ± standard deviation (SD). Means of normally distributed data were compared by two-tailed Student’s t-test, one-way ANOVA (followed by Tukey’s post-hoc correction) or linear regression where indicated and statistical significance is reported as Our previous data using GRAF3 hypomorphic mice clearly demonstrated that SMC GRAF3 is required for normal BP homeostasis . Importap < 0.05). As shown in RQ protein was induced 48 h following tamoxifen treatment of RQ SM MHC-CreERT2GRAF3 mice relative to similarly treated genetic controls. This resulted in an approximate 3-fold increase in RQGRAF3 over endogenous GRAF3 levels, as assessed by semi-quantitative RT-PCR using primers that recognize endogenous mouse and transgenic human GRAF3  relative to similarly treated RQGRAF3 mice  are not ideal for drug discovery. However, it is feasible to identify highly potent and selective compounds that mediate allosteric regulation of enzymes . Intereswww.rcsb.org) [Since structural information for GRAF3 (or any other BAR-PH-GAP containing protein) is not yet available, we next employed molecular modeling strategies to gain insight into putative mechanisms that might control such auto-regulation. To this end, we first used PyMol to build a predictive molecular model of GRAF3 based on the solved crystal structures of the Appl1 BAR-PH domain  and the csb.org) . These dcsb.org) .Using this structural information, we developed a novel GRAF3 biosensor to determine if GRAF3 undergoes dynamic allosteric regulation in response to physiologically relevant cellular stimuli. In brief, we engineered a truncated version of GRAF3 with a cyan fluorescent protein moiety (CFP) fused N-terminal to the BAR domain and a yellow fluorescent protein moiety (YFP) fused C-terminal to the GAP domain. Based on our model, the CFP and YFP moieties would be in close proximity (less than 20Å) when the protein is in a closed (auto-inhibited) state, thus permitting FRET between the two fluorophores; while relief of auto-inhibition would increase the distance between the fluorophores and prohibit FRET A. In strUndergoes dynamic allosteric regulation in cells, we found that GRAF3 FRET/CFP ratios were spatially and temporally regulated in SMC following treatment with the contractile agonist, Sphingosine 1 phosphate (S1P) B,C. SpatSince our FRET studies revealed that the temporal dynamics of GRAF3 allosteric modulation occurred in a time-frame consistent with post-translational modification, we next performed an in silico screen to identify putative phosphorylation sites in exposed regions that would be predicted to alter steric inhibition by the BAR-PH domain. We identified 15 putative phosphorylation sites that had a NetPhos 2.0 prediction scores of 0.9 or greater and that were positioned near the interface between the BAR-PH and GAP domains. One such site, Y376, is located in the 10 AA linker between the PH and GAP domain, is invariant in GRAF3 homologues and was previously identified as a phosphorylation site for pp60Src , which w529FSrc or superFAK  physically and functionally interact and because GRAF family members associate with FAK, we tested the possibility that GRAF3 was also a substrate for FAK. Indeed, we found that co-expression of GRAF3 with either constitutively active activity ) led to activity ; howeverWt GRAF3 . The capproteins F,G.Y376EGRAF3 variant. As shown in Because the above findings indicate that GRAF3 is a FAK substrate and since once activated, FAK induces RhoA inhibition ,43, we pY376EGRAF3 expression was significantly lower. These findings are consistent with the idea that phosphorylation of Y376 induces activation of GRAF3 through an allosteric mechanism and highlight the exciting possibility that small molecules that stabilize the active conformation could prove useful as anti-hypertensive therapies.We next sought to test our postulate that Y376 phosphorylation activates GRAF3 by altering BAR-PH/GAP interactions. To this end, we modified our GRAF3 biosensor to include a bioluminescent donor that enables precise quantification of conformational rearrangements of proteins in cells as the donor, acceptor and bioluminescent resonance energy transfer (BRET) signals provide internal controls for protein expression  Figure D. As shoGRAF3 gene are associated with human hypertension [RQ in SMC significantly decreased basal and vasoconstrictor-induced BP and that endogenous GRAF3 can be activated by phosphorylation-induced conformational changes provide strong support for the feasibility of GRAF3 as a druggable target.Vascular resistance is a major determinant of BP and is controlled, in large part, by RhoA-dependent smooth muscle cell (SMC) contraction within small peripheral arterioles ,6,7,8,45rtension ,47,48,49Our new findings support the postulate that GRAF3 serves as a mechanical strain-sensitive rheostat to prevent excessive feed-forward activation of the RhoA signaling axis and to limit SMC contractility and BP. RhoA activity is elevated by graded increases in intraluminal perfusion pressure and RhoA-dependent SMC contraction is important to shield downstream capillaries from the damaging effects of high flow . HoweverThe ability of induced SMC-specific expression of GRAF3 to lower BP in mice, adds to a growing body of literature supporting the promising clinical utility of inhibiting the Rho pathway for BP control ,52,53,54Targeting GAPs for therapeutic advances for cancer treatment has been largely overlooked because oncogenic mutations in Ras and Rho generally render GAPs ineffective in promoting GTP hydrolysis . While tInterestingly, in our molecular models, the nucleophilic water coordinating residue, R417 lies on the RhoA interacting face of the GAP domain and is surface exposed in the open “active” model of GRAF3 and while it has reduced activity, is not expected to impact the active conformation. However, in our model of the inactive conformation, R417 is predicted to form an electrostatic pair with residue E245 on the BAR-PH domain. Interestingly, like R417, E245 is evolutionally invariant and is conserved in other GRAF family members, indicating the likely importance of this site. Thus, it is formally possible that the Q417 variant might disrupt the inactive conformation and that the ability to do so contributes in part to its capacity to promote RhoA GTPase activity in spite of its reduced capacity to coordinate the nucleophilic water molecule for GTP hydrolysis. It will be of future interest to determine the importance of such electrostatic pairs in the modulation of GRAF family member activities.Y376GRAF3 reverses an auto-inhibitory interaction between the GAP and BAR-PH domains. Y376 is located in a linker between the PH and GAP domain, is invariant in GRAF3 homologues and a phospho-mimetic E376GRAF3 variant exhibited elevated GAP activity in vitro and in SMC. Collectively, these findings support the feasibility of GRAF3 as a druggable target.Moreover, we found that GRAF3 protein toggles between inactive and active states by processes controlled by the mechano-sensing kinase, focal adhesion kinase (FAK). Using resonance energy transfer methods, we showed that agonist-induced FAK-dependent phosphorylation at While our data reveal that phosphorylation of Y376 promotes allosteric activation of GRAF3, there are likely additional post-translational modifications that contribute to its auto-regulation. Indeed, while our BRET assays indicate that the Y376 phosphomimetic variant significantly alters BAR-PH-GAP conformations, the BAR-PH deficient variant exhibits higher activity than the Y376E variant in our cellular assays (data not shown), indicating that phosphorylation of this site does not fully relieve steric inhibition. Moreover, these data do not rule out the possibility that phosphorylation of this site alters the affinity of the RhoA/GRAF3 interaction. Also, while we show that Y376 is the major site of FAK and Src-dependent GRAF3 phosphorylation, both our phospho-tyrosine immunoprecipitations and radioactive kinase assay data indicate that Src phosphorylates GRAF3 at additional sites. However, it is certainly feasible that additional cryptic sites were exposed in the purified truncated version of GRAF3 utilized for the in vitro kinase assays. Nonetheless, discovery-based mass-spectrometry data provided on PhoshoSitePlus database (phosphosite.org) predicts 4 potential tyrosine phosphorylation sites on GRAF3 including . To date, tyrosines 376 and 792 have been identified as sites of post-translational modification in 244 and 642 independent mass spec studies, respectively, while Y142 and Y870 have each been reported once . With reIn summary, our findings presented herein and elsewhere provide strong support for a model in which GRAF3 serves as a mechanical strain-sensitive rheostat to tightly couple SMC tone to intraluminal pressure and that induced expression of GRAF3 has the capacity to have a long-lasting impact on BP control. As myogenic dysfunction has been linked to not only hypertension but also stroke, diabetes, and Alzheimer’s disease , it woul