bc helium wasn't available, due to the 1929 US helium act. Then the major source of helium, the US forbade export. Otherwise that would have been used.

true for AI, but wasn't the case for Hindenburg I'd say.

prediction Michael Wooldridge (Oxford, AI), sees a risk at an 'Hindenburg' event. Shattering the global confidence in AI tech. I"m not sure this analogy entirely fits other than in its potential impact (AI isn't globally trusted, the Hindenburg did not fail bc of the tech itself but bc helium not being allowed to export from the US at the time. Still the Hindenburg did put an end to the entire zeppelin industry yes. No matter the causes.)

Gentoo Linux distribution is in the process of moving from GitHub to Codeberg (Germany based). Copilot the major reason. (I have codeberg account, feb 2025, not used yet)

Confusing and not easy enough to understand and read, especially if no experience with airtable. Also text is a bit to close to eachother and explanations to vague. Should explain and do everything step by step for easiest possible understanding.

In last step always add extra check step or test, even if one test is done.

Example: Make sure it now looks like this -> or make sure it says this or that...

End step missing:

Examples = -> Press confirm -> Move onto next step or move onto step 5

eLife Assessment

This useful study uses creative scalp EEG decoding methods to attempt to demonstrate that two forms of learned associations in a Stroop task are dissociable, despite sharing similar temporal dynamics. However, the evidence supporting the conclusions is incomplete due to concerns with the experimental design and methodology. This paper would be of interest to researchers studying cognitive control and adaptive behavior, if the concerns raised in the reviews can be addressed satisfactorily.

Reviewer #1 (Public review):

Summary:

This study focuses on characterizing the EEG correlates of item-specific proportion congruency effects. Two types of learned associations are characterized, one being associations between stimulus features and control states (SC), and the other being stimulus features and responses (SR). Decoding methods are used to identify time-resolved SC and SR correlates, which are used to test properties of their dynamics.

The conclusion is reached that SC and SR associations can independently and simultaneously guide behavior. This conclusion is based on results showing SC and SR correlates are: (1) not entirely overlapping in cross-decoding; (2) simultaneously observed on average over trials in overlapping time bins; (3) independently correlate with RT; and (4) have a positive within-trial correlation.

Strengths:

Fearless, creative use of EEG decoding to test tricky hypotheses regarding latent associations.

Nice idea to orthogonalize ISPC condition (MC/MI) from stimulus features.

Weaknesses:

I still have my concern from the first round that the decoders are overfit to temporally structured noise. As I wrote before, the SC and SR classes are highly confounded with phase (chunk of session). I do not see how the control analyses conducted in the revision adequately deal with this issue.

In the figures, there are several hints that these decoders are biased. Unfortunately, the figures are also constructed in such a way that hides or diminishes the salience of the clues of bias. This bias and lack of transparency discourage trust in the methods and results.

I have two main suggestions:

(1) Run a new experiment with a design that properly supports this question.

I don't make this suggestion lightly, and I understand that it may not be feasible to implement given constraints; but I feel that this suggestion is warranted. The desired inferences rely on successful identification of SC and SR representations. Solidly identifying SC and SR representations necessitates an experimental design wherein these variables are sufficiently orthogonalized, within-subject, from temporally structured noise. The experimental design reported in this paper unfortunately does not meet this bar, in my opinion (and the opinion of a colleague I solicited).

An adequate design would have enough phases to properly support "cross-phase" cross-validation. Deconfounding temporal noise is a basic requirement for decoding analyses of EEG and fMRI data (see e.g., leave-one-run-out CV that is effectively necessary in fMRI; in my experience, EEG is not much different, when the decoded classes are blocked in time, as here). In a journal with a typical acceptance-based review process, this would be grounds for rejection.

Please note that this issue of decoder bias would seem to weaken the rest of the downstream analyses that are based on the decoded values. For instance, if the decoders are biased, in the within-trial correlation analysis, how can we be sure that co-fluctuations along certain dimensions within their projected values are driven by signal or noise? A similar issue clouds the LMM decoding-RT correlations.

(2) Increase transparency in the reporting of results throughout main text.

Please do not truncate stimulus-aligned timecourses at time=0. Displaying the baseline period is very useful to identify bias, that is, to verify that stimulus-dependent conditions cannot be decoded pre-stimulus. Bias is most expected to be revealed in the baseline interval when the data are NOT baseline-corrected, which is why I previously asked to see the results omitting baseline correction. (But also note that if the decoders are biased, baseline-correcting would not remove this bias; instead, it would spread it across the rest of the epoch, while the baseline interval would, on average, be centered at zero.)

Please use a more standard p-value correction threshold, rather than Bonferroni-corrected p<0.001. This threshold is unusually conservative for this type of study. And yet, despite this conservativeness, stimulus-evoked information can be decoded from nearly every time bin, including at t=0. This does not encourage trust in the accuracy of these p-values. Instead, I suggest using permutation-based cluster correction, with corrected p<0.05. This is much more standard and would therefore allow for better comparison to many other studies.

I don't think these things should be done as control analyses, tucked away in the supplemental materials, but instead should be done as a part of the figures in the main text -- including decoding, RSA, cross-trial correlations, and RT correlations.

Other issues:

Regarding the analysis of the within-trial correlation of RSA betas, and "Cai 2019" bias:

The correction that authors perform in the revision -- estimating the correlation within the baseline time interval and subtracting this estimate from subsequent timepoints -- assumes that the "Cai 2019" bias is stationary. This is a fairly strong assumption, however, as this bias depends not only on the design matrix, but also on the structure of the noise (see the Cai paper), which can be non-stationary. No data were provided in support of stationarity. It seems safer and potentially more realistic to assume non-stationarity.

This analysis was included in the supplemental material. However, given that the correlation analysis presented in the Results is subject to the "Cai 2019" bias, it would seem to be more appropriate to replace that analysis, rather than supplement it.

Regardless, this seems to be a moot issue, given that the underlying decoders seem to be overfit to temporally structured noise (see point above regarding weakening of downstream analyses based on decoder bias).

Outliers and t-values:

More outliers with beta coefficients could be because the original SD estimates from the t-values are influenced more by extreme values. When you use a threshold on the median absolute deviation instead of mean +/-SD, do you still get more outliers with beta coefficients vs t-values?

Random slopes:

Were random slopes (by subject) for all within-subject variables included in the LMMs? If not, please include them, and report this in the Methods.

Reviewer #2 (Public review):

Summary:

In this EEG study, Huang et al. investigated the relative contribution of two accounts to the process of conflict control, namely the stimulus-control association (SC), which refers to the phenomenon that the ratio of congruent vs. incongruent trials affects the overall control demands, and the stimulus-response association (SR), stating that the frequency of stimulus-response pairings can also impact the level of control. The authors extended the Stroop task with novel manipulation of item congruencies across blocks in order to test whether both types of information are encoded and related to behaviour. Using decoding and RSA they showed that the SC and SR representations were concurrently present in voltage signals and they also positively co-varied. In addition, the variability in both of their strengths was predictive of reaction time. In general, the experiment has a sold design and the analyses are appropriate for the research questions.

Strength:

(1) The authors used an interesting task design that extended the classic Stroop paradigm and is effective in teasing apart the relative contribution of the two different accounts regarding item-specific proportion congruency effect.

(2) Linking the strength of RSA scores with behavioural measure is critical to demonstrating the functional significance of the task representations in question.

Weakness:

(1) The distinction between Phase 2 and Phase 1&3 behavioral results, specifically the opposite effect of MC/MI in congruent trials raises some concerns with regard to the effectiveness of the ISPC manipulation. Why do RTs and error rates under MC congruent condition in Phase 2 seem to be worse than MI congruent? Could there be other factors at play here, e.g. order effect? How does this potentially affect the neural analyses where trials from different phases were combined? Also, the manuscript does not mention whether there is counterbalancing for the color groups across participants, so far as I can tell.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment

This useful study uses creative scalp EEG decoding methods to attempt to demonstrate that two forms of learned associations in a Stroop task are dissociable, despite sharing similar temporal dynamics. However, the evidence supporting the conclusions is incomplete due to concerns with the experimental design and methodology. This paper would be of interest to researchers studying cognitive control and adaptive behavior, if the concerns raised in the reviews can be addressed satisfactorily.

We thank the editors and the reviewers for their positive assessment of our work and for providing us with an opportunity to strengthen this manuscript. Please see below our responses to each comment raised in the reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study focuses on characterizing the EEG correlates of item-specific proportion congruency effects. In particular, two types of learned associations are characterized. One being associations between stimulus features and control states (SC), and the other being stimulus features and responses (SR). Decoding methods are used to identify SC and SR correlates and to determine whether they have similar topographies and dynamics.

The results suggest SC and SR associations are simultaneously coactivated and have shared topographies, with the inference being that these associations may share a common generator.

Strengths:

Fearless, creative use of EEG decoding to test tricky hypotheses regarding latent associations. Nice idea to orthogonalize the ISPC condition (MC/MI) from stimulus features.

Thank you for acknowledging the strength in EEG decoding and design. We have addressed all your concerns raised below point by point.

Weaknesses:

(1a) I'm relatively concerned that these results may be spurious. I hope to be proven wrong, but I would suggest taking another look at a few things.

While a nice idea in principle, the ISPC manipulation seems to be quite confounded with the trial number. E.g., color-red is MI only during phase 2, and is MC primarily only during Phase 3 (since phase 1 is so sparsely represented). In my experience, EEG noise is highly structured across a session and easily exploited by decoders. Plus, behavior seems quite different between Phase 2 and Phase 3. So, it seems likely that the classes you are asking the decoder to separate are highly confounded with temporally structured noise.

I suggest thinking of how to handle this concern in a rigorous way. A compelling way to address this would be to perform "cross-phase" decoding, however I am not sure if that is possible given the design.

Thank you for raising this important issue. To test whether decoding might be confounded by temporally structured noise, we performed a control decoding analysis. As the reviewer correctly pointed out, cross-phase decoding is not possible due to the experimental design. Alternatively, to maximize temporal separation between the training and test data, we divided the EEG data in phase 2 and phase 1&3 into the first and second half chronologically. Phase 1 and 3 were combined because they share the same MC and MI assignments. We then trained the decoders on one half and tested them on the other half. Finally, we averaged the decoding results across all possible assignments of training and test data. The similar patterns (Supplementary Fig.1) observed confirmed that the decoding results are unlikely to be driven by temporally structured noise in the EEG data. The clarification has been added to page 13 of the revised manuscript.

(1b) The time courses also seem concerning. What are we to make of the SR and SC timecourses, which have aggregate decoding dynamics that look to be <1Hz?

As detailed in the response to your next comment, some new results using data without baseline correction show a narrower time window of above-chance decoding. We speculate that the remaining results of long-lasting above-chance decoding could be attributed to trials with slow responses (some responses were made near the response deadline of 1500 ms). Additionally, as shown in Figure 6a, the long-lasting above-chance decoding seems to be driven by color and congruency representations. Thus, it is also possible that the binding of color and congruency contributes to decoding. This interpretation has been added to page 17 of the revised manuscript.

(1c) Some sanity checks would be one place to start. Time courses were baselined, but this is often not necessary with decoding; it can cause bias (10.1016/j.jneumeth.2021.109080), and can mask deeper issues. What do things look like when not baselined? Can variables be decoded when they should not be decoded? What does cross-temporal decoding look like - everything stable across all times, etc.?

As the reviewer mentioned, baseline-corrected data may introduce bias to the decoding results. Thus, we cited the van Driel et al (2021) paper in the revised manuscript to justify the use of EEG data without baseline-correction in decoding analysis (Page 27 of the revised manuscript), and re-ran all decoding analysis accordingly. The new results revealed largely similar results (Fig. 2, 4, 6 and 8 in the revised manuscript) with the following exceptions: narrower time window for separatable SC subspace and SR subspace (Fig. 4b), narrower time window for concurrent representations of SC and SR (Fig. 6a-b), and wider time window for the correlations of SC/SR representations with RTs (Fig. 8).

(2) The nature of the shared features between SR and SC subspaces is unclear.

The simulation is framed in terms of the amount of overlap, revealing the number of shared dimensions between subspaces. In reality, it seems like it's closer to 'proportion of volume shared', i.e., a small number of dominant dimensions could drive a large degree of alignment between subspaces.

What features drive the similarity? What features drive the distinctions between SR and SC? Aside from the temporal confounds I mentioned above, is it possible that some low-dimensional feature, like EEG congruency effect (e.g., low-D ERPs associated with conflict), or RT dynamics, drives discriminability among these classes? It seems plausible to me - all one would need is non-homogeneity in the size of the congruency effect across different items (subject-level idiosyncracies could contribute: 10.1016/j.neuroimage.2013.03.039).

Thank you for this question. To test what dimensions are shared between SC and SR subspaces, we first identify which factors can be shared across SC and SR subspaces. For SC, the eight conditions are the four colors × ISPC. Thus, the possible shared dimensions are color and ISPC. Additionally, because the four colors and words are divided into two groups (e.g., red-blue and green-yellow, counterbalanced across subjects, see Methods), the group is a third potential shared dimension. Similarly, for SR decoders, potential shared dimensions are word, ISPC and group. Note that each class in SC and SR decoders has both congruent and incongruent trials. Thus, congruency is not decodable from SC/SR decoders and hence unlikely to be a shared dimension in our analysis. To test the effect of sharing for each of the potential dimensions, we performed RSA on decoding results of the SC decoder trained on SR subspace (SR | SC) (Supplementary Fig. 4a) and the SR decoder trained on SC subspace (SC | SR) (Supplementary Fig. 4b), where the decoders indicated the decoding accuracy of shared SC and SR representations. In the SC classes of SR | SC, word red and blue were mixed within the same class, same were word yellow and green. The similarity matrix for “Group” of SR | SC (Supplementary Fig. 4a) shows the comparison between two word groups (red & blue vs. yellow & green). The similarity matrix for “Group” of SC | SR (Supplementary Fig. 4b) shows the comparison between two color groups (red & blue vs. yellow & green).

The RSA results revealed that the contributions of group to the SC decoder (Supplementary Fig. 5a) and the SR decoder (Supplementary Fig. 5b) were significant. Meanwhile, a wider time window showed significant effect of color on the SC decoder (approximately 100 - 1100 ms post-stimulus onset, Supplementary Fig. 5a) and a narrower time window showed significant effect of word on SR decoder (approximately 100 - 500 ms post-stimulus onset, Supplementary Fig. 5b). However, we found no significant effect of ISPC on either SC or SR decoders. We also performed the same analyses on response-locked data from the time window -800 to 200 ms. The results showed shared representation of color in the SC decoder (Supplementary Fig. 5c) and group in both decoders (Supplementary Fig. 5c-d). Overall, the above results demonstrated that color, word and group information are shared between SC and SR subspaces.

Lastly, we would like to stress that our main hypothesis for the cross-subspace decoding analysis is that SR and SC subspaces are not identical. This hypothesis was supported by lower decoding accuracy for cross-subspace than within-subspace decoders and enables following analyses that treated SC and SR as separate representations.

We have added the interpretation to page 13-14 of the revised manuscript.

(3) The time-resolved within-trial correlation of RSA betas is a cool idea, but I am concerned it is biased. Estimating correlations among different coefficients from the same GLM design matrix is, in general, biased, i.e., when the regressors are non-orthogonal. This bias comes from the expected covariance of the betas and is discussed in detail here (10.1371/journal.pcbi.1006299). In short, correlations could be inflated due to a combination of the design matrix and the structure of the noise. The most established solution, to cross-validate across different GLM estimations, is unfortunately not available here. I would suggest that the authors think of ways to handle this issue.

Thank you for raising this important issue. Because the bias comes from the covariance between the regressors and the same GLM was applied to all time points in our analysis, we assume that the inflation would be similar at different time points. Therefore, we calculated the correlation of SC and SR betas ranging from -200 to 0 ms relative to stimulus onset as a baseline (i.e., no SC or SR representation is expected before the stimulus onset) and compared the post-stimulus onset correlation coefficients against this baseline. We hypothesized that if the positively within-trial correlation of SC and SR betas resulted from the simultaneous representation instead of inflation, we should observe significantly higher correlation when compared with the baseline. To examine this hypothesis, we first performed the linear discriminant analysis (Supplementary Fig. 7a) and RSA regression (Supplementary Fig. 7b) on the -200 - 0 ms window relative to stimulus onset. We then calculated the average r_baseline of SC and SR betas on that time window for each participant (group results at each time point are shown in Supplementary Fig. 7c) and computed the relative correlation at each post-stimulus onset time point using (fisher-z (r) - fisher-z (r_baseline)). Finally, we performed a simple t test at the group level on baseline-corrected correlation coefficients with Bonferroni correction. The results (Fig. 6c) showed significantly more positive correlation from 100 - 500 ms post-stimulus onset compared with baseline, supporting our hypothesis that the positive within-trial correlation of SC and SR betas arise from simultaneous representation rather than inflation. The related interpretation was added to page 17 of the revised manuscript.

(4) Are results robust to running response-locked analyses? Especially the EEG-behavior correlation. Could this be driven by different RTs across trials & trial-types? I.e., at 400 ms poststim onset, some trials would be near or at RT/action execution, while others may not be nearly as close, and so EEG features would differ & "predict" RT.

Thanks for this question. We now pair each of the stimulus-locked EEG analysis in the manuscript with response-locked analysis. To control for RT variations among trial types, when using the linear mixed model (LMM) to predict RTs from trial-wise RSA results, we included a separate intercept for each of the eight trial types in SC or SR. Furthermore, at each time point, we only included trials that have not generated a response (for stimulus-locked analysis) or already started (for response-locked analysis). All the results (Fig. 3, 5, 7, 9 in the revised manuscript) are in support of our hypothesis. We added these detailed to page 31 of the revised manuscript.

(5) I suggest providing more explanation about the logic of the subspace decoding method - what trialtypes exactly constitute the different classes, why we would expect this method to capture something useful regarding ISPC, & what this something might be. I felt that the first paragraph of the results breezes by a lot of important logic.

In general, this paper does not seem to be written for readers who are unfamiliar with this particular topic area. If authors think this is undesirable, I would suggest altering the text.

To improve clarity, we revised the first paragraph of the SC and SR association subspace analysis to list the conditions for each of the SC and SR decoders and explain more about how the concept of being separatable can be tested by cross-decoding between SC and SR subspaces. The revised paragraph now reads:

“Prior to testing whether controlled and non-controlled associations were represented simultaneously, we first tested whether the two representations were separable in the EEG data.

In other words, we reorganized the 16 experimental conditions into 8 conditions for SC (4 colors × MC/MI, while collapsing across SR levels) and SR (4 words × 2 possible responses per word, while collapsing across SC levels) associations separately. If SC and SR associations are not separable, it follows that they encode the same information, such that both SC and SR associations can be represented in the same subspace (i.e., by the same information encoded in both associations). For example, because (1) the word can be determined by the color and congruency and (2) the most-likely response can be determined by color and ISPC, the SR association (i.e., association between word and most-likely response) can in theory be represented using the same information as the SC association. On the other hand, if SC and SR associations are separable, they are expected to be represented in different subspaces (i.e., the information used to encode the two associations is different). Notably, if some, but not all, information is shared between SC and SR associations, they are still separable by the unique information encoded. In this case, the SC and SR subspaces will partially overlap but still differ in some dimensions. To summarize, whether SC and SR associations are separable is operationalized as whether the associations are represented in the same subspace of EEG data. To test this, we leveraged the subspace created by the LDA (see Methods). Briefly, to capture the subspace that best distinguishes our experimental conditions, we trained SC and SR decoders using their respective aforementioned 8 experimental conditions. We then projected the EEG data onto the decoding weights of the LDA for each of the SC and SR decoders to obtain its respective subspace. We hypothesized that if SC and SR subspaces are identical (i.e., not separable), SC/SR decoding accuracy should not differ by which subspace (SC or SR) the decoder is trained on. For example, SC decoders trained in SC subspace should show similar decoding performance as SC decoders trained in SR subspace. On the other hand, if SC and SR association representations are in different subspaces, the SC/SR subspace will not encode all information for SR/SC associations. As a result, decoding accuracy should be higher using its own subspace (e.g., decoding SC using the SC subspace) than using the other subspace (e.g., decoding SC using the SR subspace). We used cross-validation to avoid artificially higher decoding accuracy for decoders using their own subspace (see Methods).” (Page 11-12).

We also explicitly tested what information is shared between SC and SR representations (see response to comment #2). Lastly, to help the readers navigate the EEG results, we added a section “Overview of EEG analysis” to summarize the EEG analysis and their relations in the following manner:

“EEG analysis overview. We started by validating that the 16 experimental conditions (8 unique stimuli × MC/MI) were represented in the EEG data. Evidence of representation was provided by above-chance decoding of the experimental conditions (Fig. 2-3). We then examined whether the SC and SR associations were separable (i.e., whether SC and SR associations were different representations of equivalent information). As our results supported separable representations of SC and SR association (Fig. 4-5), we further estimated the temporal dynamics of each representation within a trial using RSA. This analysis revealed that the temporal dynamics of SC and SR association representations overlapped (Fig. 6a-b, Fig. 7a-b). To explore the potential reason behind the temporal overlap of the two representations, we investigated whether SC and SR associations were represented simultaneously as part of the task representation, independently from each other, or competitively/exclusively (e.g., on some trials only SC association was represented, while on other trials only SR association was represented). This was done by assessing the correlation between the strength of SC and SR representations across trials (Fig. 6c, Fig. 7c). Lastly, we tested how SC and SR representations facilitated performance (Fig.8-9).” (Page 8-9).

Minor suggestions:

(6) I'd suggest using single-trial RSA beta coefficients, not t-values, as they can be more stable (it's a t-value based on 16 observations against 9 or so regressors.... the SE can be tiny).

Thank you for your suggestion. To choose between using betas and t-values, we calculate the proportion of outliers (defined as values beyond mean ± 5 SD) for each predictor of the design matrix and each subject. We found that outliers were less frequent for t-values than for beta coefficients (t-values: mean = 0.07%, SD = 0.009%; beta-values: mean = 0.19%, SD = 0.033%). Thus, we decided to stay with t-values.

(7) Instead of prewhitening the RTs before the HLM with drift terms, try putting those in the HLM itself, to avoid two-stage regression bias.

Thank you for your suggestion. Because our current LMM included each of the eight trial types in SC or SR as separate predictors with their own intercepts (as mentioned above), adding regressors of trial number and mini blocks (1-100 blocks) introduced collinearity (as ISPC flipped during the experiment). We therefore excluded these regressors from the current LMM (Page 31).

(8) The text says classical MDS was performed on decoding *accuracy* - is this accurate?

We now clarify in the manuscript that it is the decoders’ probabilistic classification results (Page 28).

(9) At a few points, it was claimed that a negative correlation between SC and SR would be expected within single trials, if the two were temporally dissociable. Wouldn't it also be possible that they are not correlated/orthogonal?

We agree with the reviewer and revised the null hypothesis in the cross-trial correlation analysis to include no correlation as SC and SR association representations may be independent from each other (Page 17, 22).

Reviewer #2 (Public review):

Summary:

In this EEG study, Huang et al. investigated the relative contribution of two accounts to the process of conflict control, namely the stimulus-control association (SC), which refers to the phenomenon that the ratio of congruent vs. incongruent trials affects the overall control demands, and the stimulus-response association (SR), stating that the frequency of stimulusresponse pairings can also impact the level of control. The authors extended the Stroop task with novel manipulation of item congruencies across blocks in order to test whether both types of information are encoded and related to behaviour. Using decoding and RSA, they showed that the SC and SR representations were concurrently present in voltage signals, and they also positively co-varied. In addition, the variability in both of their strengths was predictive of reaction time. In general, the experiment has a solid design, but there are some confounding factors in the analyses that should be addressed to provide strong support for the conclusions.

Strengths:

(1) The authors used an interesting task design that extended the classic Stroop paradigm and is potentially effective in teasing apart the relative contribution of the two different accounts regarding item-specific proportion congruency effect, provided that some confounds are addressed.

(2) Linking the strength of RSA scores with behavioural measures is critical to demonstrating the functional significance of the task representations in question.

Thank you for your positive feedback. We hope our responses below address your concerns.

Weakness:

(1) While the use of RSA to model the decoding strength vector is a fitting choice, looking at the RDMs in Figure 7, it seems that SC, SR, ISPC, and Identity matrices are all somewhat correlated. I wouldn't be surprised if some correlations would be quite high if they were reported. Total orthogonality is, of course, impossible depending on the hypothesis, but from experience, having highly covaried predictors in a regression can lead to unexpected results, such as artificially boosting the significance of one predictor in one direction, and the other one to the opposite direction. Perhaps some efforts to address how stable the timed-resolved RSA correlations for SC and SR are with and without the other highly correlated predictors will be valuable to raising confidence in the findings.

Thank you for this important point. The results of proportion of variability explained shown in the Author response table 1 below, indicated relatively higher correlation of SC/SR with Color and Identity. We agree that it is impossible to fully orthogonalize them. To address the issue of collinearity, we performed a control RSA by removing predictors highly correlated with others. Specifically, we calculated the variance inflation factor (VIF) for each predictor. The Identity predictor had a high VIF of 5 and was removed from the RSA. All other predictors had VIFs < 4 and were kept in the RSA. The results (Supplementary Fig. 6) showed patterns similar to the results with the Identity predictor, suggesting that the findings are not significantly influenced by collinearity. We have added the interpretation to page 17 of the revised manuscript.

Author response table 1.

Proportion of variability explained (r²) of RSA predictors.

(2) In "task overview", SR is defined as the word-response pair; however, in the Methods, lines 495-496, the definition changed to "the pairing between word and ISPC" which is in accordance with the values in the RDMs (e.g., mccbb and mcirb have similarity of 1, but they are linked to different responses, so should they not be considered different in terms of SR?). This needs clarification as they have very different implications for the task design and interpretation of results, e.g., how correlated the SC and SR manipulations were.

Thank you for pointing out this important issue with how our operationalization captures the concept in questions. In the revised manuscript, we clarified the stimulus-response (SR) association is the link between the word and the most-likely response (i.e., not necessarily the actual response on the current trial). This association is likely to be encoded based on statistical learning over several trials. On each trial, the association is updated based on the stimulus and the actual response. Over multiple trials, the accumulated association will be driven towards the most-common (i.e., most-likely) response. In our ISPC manipulation, a color is presented in mostly congruent/incongruent (MC/MI) trials, which will also pair a word with a most-likely response. For example, if the color blue is MC, the color blue, which leads to the response blue, will co-occur with the word blue with high frequency. In other words, the SR association here is between the word blue and the response blue. As the actual response is not part of the SR association, in the RDM two trial types with different responses may share the same SR association, as long as they share the same word and the same ISPC manipulation, which, by the logic above, will produce the same most-likely response. These clarifications have been added to page 4 and 29 of the revised manuscript.

In the revised manuscript (Page 17), we addressed how much the correlated SC and SR predictors in the RDM could affect the correlation analysis between SC and SR association representation strength. Specifically, we conducted the RSA using the same GLM on EEG data prior to stimulus onset (Supplementary Fig. 7a-b). As no SC and SR associations are expected to be present before stimulus onset, the correlation between SC and SR representation would serve as a baseline of inflation due to correlated predictors in the GLM (Supplementary Fig. 7c, also see comment #3 of R1). The SC-SR correlation coefficients following stimulus onset was then compared to the baseline to control for potential inflation (Fig. 6c). Significantly above-baseline correlation was still observed between ~100-500 ms post-stimulus onset, providing support for the hypothesis that SC and SR are encoded in the same task representation.

Minor suggestions:

(3) Overall, I find that calling SC-controlled and SR-uncontrolled representations unwarranted. How is the level controlledness defined? Both are essentially types of statistical expectation that provide contextual information for the block of tasks. Is one really more automatic and requires less conscious processing than the other? More background/justification could be provided if the authors would like to use these terms.

Following your advice, we have added more discussion on how controlledness is conceptualized in this work and in the literature, which reads:

“We consider SC and SR as controlled and uncontrolled respectively based on the literature investigating the mechanism of ISPC effect. The SC account posits that the ISPC effect results from conflict and involves conflict adaptation, which requires the regulation of attention or control (Bugg & Hutchison, 2013; Bugg et al., 2011; Schmidt, 2018; Schmidt & Besner, 2008). On the other hand, the SR account argues that ISPC effect does not require conflict adaptation but instead reflects contingency leaning. That is, the response can be directly retrieved from the association between the stimulus and the most-likely response without top-down regulation of attention or control. As more empirical evidence emerged, researchers advocating control view began to acknowledge the role of associative learning in cognitive control regarding the ISPC effect (Abrahamse et al., 2016). SC association has been thought to include both automatic that is fast and resource saving and controlled processes that is flexible and generalizable (Chiu, 2019). Overall, we do not intend to claim that SC is entirely controlled or SR is completely automatic. We use SC-controlled and SR-uncontrolled representations to align with the original theoretical motivation and to highlight the conceptual difference between SC and SR associations.” (Page 24-25)

(4) Figures 3c and d: the figures could benefit from more explanation of what they try to show to the readers. Also for 3d, the dimensions were aligned with color sets and congruencies, but word identities were not linearly separable, at least for the first 3 axes. Shouldn't one expect that words can be decoded in the SR subspace if word-response pairs were decodable (e.g., Figure 3b)?

Thank you for the insightful observation. We now clarified that Fig. 3c and d in the original manuscript (Fig. 4c and d in the current manuscript) aim to show how each of the 8 trial types in the SC and SR subspaces are represented. The MDS approach we used for visualization tries to preserve dissimilarity between trial types when projecting from data from a high dimensional to a low dimensional space. However, such projection may also make patterns linearly separatable in high dimensional space not linearly separatable in low dimensional space. For example, if the word blue has two points (-1, -1) and (1, 1) and the word red has two points (-1, 1) and (1, -1), they are not linearly separatable in the 2D space. Yet, if they are projected from a 3D space with coordinates of (-1, -1, -0.1), (1, 1, -0.1), (-1, 1, 0.1) and (1, -1, 0.1), the two words can be linearly separatable using the 3^rd dimension. Thus, a better way to test whether word can be linearly separated in SR subspace is to perform RSA on the original high dimensional space. We performed the RSA with word (Supplementary Fig. 2) on the SR decoder trained on the SR subspace. Note that in Fig. 3c and d of the original script (Fig. 4c and d in the current manuscript) there are two pairs of words that are not linearly separable: red-blue and yellow-green. Thus, we specifically tested the separability within the two pairs using the one predictor for each pair, as shown in Supplementary Fig. 2. The results showed that within both word pairs individual words were presented above chance level (Supplementary Fig. 3). Considering that the decoders are linear, this finding indicates linear separability of the word pairs in the original SR subspace. The clarification has been added to page 13 (the end of the second paragraph) of the revised manuscript.

References

Abrahamse, E., Braem, S., Notebaert, W., & Verguts, T. (2016). Grounding cognitive control in associative learning. Psychological Bulletin, 142(7), 693-728.doi:10.1037/bul0000047.

Bugg, J. M., & Hutchison, K. A. (2013). Converging evidence for control of color-word Stroop interference at the item level. Journal of Experimental Psychology:Human Perception and Performance, 39(2), 433-449. doi:10.1037/a0029145.

Bugg, J. M., Jacoby, L. L., & Chanani, S. (2011). Why it is too early to lose control in accounts of item-specific proportion congruency effects. Journal of Experimental Psychology: Human Perception and Performance, 37(3), 844-859. doi:10.1037/a0019957.

Chiu, Y.-C. (2019). Automating adaptive control with item-specific learning. In Psychology of Learning and Motivation (Vol. 71, pp. 1-37).

Schmidt, J. R. (2018). Evidence against conflict monitoring and adaptation: An updated review. Psychonomic Bulletin & Review, 26(3), 753-771. doi:10.3758/s13423018-1520-z.

Schmidt, J. R., & Besner, D. (2008). The Stroop effect: Why proportion congruent has nothing to do with congruency and everything to do with contingency. Journal of Experimental Psychology: Learning, Memory, and Cognition, 34(3), 514-523. doi:10.1037/0278-7393.34.3.514.

This phrase reminded me of something. When I was in high school, I ran the Redmond [High School] Compliments Facebook page (yeah, it was 2014). People sent anonymous compliments for people at the school into a google form and I posted them anonymously onto h the page. I had the page passed down to me anonymously and I had to keep my identity a secret until the end of the year when I revealed it in the yearbook as my "senior confession". I got to watch people react to anonymous kindness all year. It's one of the reasons why I really believe in the power of social media as a possible force for good. What this phrase "beauty of online anonymity" reminded me of specifically was a quote I found at the time that said something along the lines of "we read things from others in their voice, When we read something anonymously, we read it in our own voice." It meant so much to me to be a part of having people read aloud kindness in their own voices.

I think the people have forgotten what social media was created for - a means to connect people around the world. Forgetting this core idea is probably what has made social media so harmful as now it is used for almost every aspect of our life except the simplicity of connecting with someone.

Yes, this kind of attitude drives me nuts. I hear this constantly, and as a historian it takes everything in me not to say "literally every generation fearmongered about the new technology and called the previous generation's technology wholesome!!" People were freaking out about the television's influence on children when it first got big, but it's all forgotten once something new things comes around. They did it for videogames, TV, phones, social media, even PRINTED BOOKS.

As the frequency of social media usage increases, the level of standards rises as well. People who watch social media are constantly exposed to some of the most tailored and presentable people, which raises their own perceptions of what is normal.

I think this really highlights the difficulty we're facing with social media these days as while we all know the toxic aspects of it we cannot get it our of lives as in truth it's not as toxic if used in moderation. However due to it's addictive nature most people cannot use it in moderation and therefore it only adds harm to people's life.

This quote shows why it’s so hard to address most issues around social media. It’s almost a necessary evil, as it is an essential part of all of our lives. It is difficult to keep up with real life if you’re not connected online too, so solutions to social media addiction or negative effects must be found on the other end — the developers’ end.

not much has changed since back then, there is still this decline for new immigrants now

The Harlem Renaissance was a cultural, artistic place centered in Harlem, New York, that redefined African American expression

It sucks that during these times women could not choose careers that were considered to be "masculine"

Often, this is a self reinforcing cycle. Dwelling on the fact that women aren't interested in you won't make you more appealing. Additionally with the rise in standards from social media, incels also won't go for women similar to them. Couple these factors together produces no good.

This reminds me of ED twitter a lot, where young women (usually) post their bodies and ask for honest critiques of their body to worsen their own perception of their body. I think its an incredibly harmful, and often overlooked, form of sel-fharm because people see it as just being young and on social media.

The internet certainly accelerates dangerous communities, especially when users are lonely and struggle with mental health. The incel community has only continued to expand and grow, developing into the looksmaxxing and blackpill communities today and even connecting with major right wing content creators and politicians.

Linking verbs in the thesis is the word "are'. Linking verbs don't show action, they just connect ideas. Which can make a thesis feel weak. Instead of leaving readers unsure, you should use an action verb to make the thesis stronger and show a clear opinion or stance.

The thesis could change as you write. Start with a working thesis, a rough idea to guide your writing. As you research more, your thesis gets stronger. To improve, ask yourself questions about your ideas and use the answers to make your thesis clearer and more specific.

Focuses on 1-3 main points that you'll explain in the essay. A thesis shows what your essay will argue and how it's organized.

A thesis is your main point or opinion about a topic. It's 1-2 sentences at the end of the intro and shows whatyour essay will argue.

Visual aids in the classroom are extremely helpful for students and teachers. To show and understand the point that is trying to get across. I feel that this is something that is now being enforced now more than ever with earlier grades, rather than just middle school and up.

I appreciate how this mentions accessibility. I have grown this semester to learn that accessibility is a need in the classroom. The only thing I question is, what if we have an emergent bilingual student or a student who struggles with reading and the resources of technology are limited? Or other current issue is how can we get students engaged in a reading on a technological device without having them wonder off, or retain the information that we are trying to get across.

I love this statement as a reminder to educators and as a reassurance to students. I feel as though times in high school and higher education that we are so much distress in our personal and academic lives that we feel as though we are not doing "enough" or learning "enough" because we are receiving so much content information. Now as a teaching perspective I love this reminder that brain breaks are NEEDED! There will be now growth for a student if the content is long and rigorous, rather we should engage with them, piece it up, and not be stress under a "time line" we are given.

It's also important to remember that just because a person can see an image doesn't meant that they understand what's going on in the image so keep that in mind when it some to visuals. Given students time to really understand what's in the photo.

I always want to make sure that my students can adjust the text the ways they need so they can read them more easily.

It's important to keep this in mind, because students have whole lives outside of school. Returning to school can be overwhelming for them, because they're learning what their teachers' expectations are. It might be best to give them time to adapt to the course load before we start overloading them.

Student anxiety is a big issue--and one that profs can't always solve.

This is a really important point

wonderful!

I would like to know more about what they mean by "pressure." Some stress can be useful in learning but pressure sounds negative.

I'd love to see a big ;most of renewable assignments!

I feel like social media has kind of helped me get away from whats happening in my reality by allowing me to just sit and watch a few short videos. It frees me from a sense of responsibility and gives me a break from whatever I'm thinking about in general. However, using social media in general makes me feel lazy and guilty about how I'm spending my time. There are a lot of things I could be focusing my energy on to relax, and social media just doesn't seem like the best one.

Recognizing this will help me not ramble on in my writing and get to a direct point.

Strategies to help become a strong reader will also help you become successful.

This stood out to me because we usually think writing is one skill we can use everywhere, but she’s saying writing always depends on who we’re writing to and why. Writing a text,an essay, and a job email are all different.

It's honestly quite difficult trying to find a way to automate detection. Words often overlap in contexts, and I feel like people often use these trigger words in both positive and negative ways. For example, people may make videos/posts using the word "suicide" as a way to discourage bullying, promote seeking help in forms of support lines or therapy, or keeping someone's story alive. On the other hand, people may make videos or posts also using "suicide" to encourage self-loathing or techniques to harm yourself/commit suicide. It's a double-edged sword that ends up affecting both sides no matter what the intentions are. Additionally, those who try to bypass the limitations to create positive videos are giving the other party (negative) another way to talk about it freely.

This shows how moderation and user behavior constantly adapt to each other. When platforms try to filter certain language, people often respond creatively, which makes the system harder to manage. It also raises questions about whether removing certain words actually addresses harm, or just shifts how people express it.

This is actually a really helpful thing to read right now, because for my group project I'm attempting to detect content on Bluesky that promotes eating disorders/disordered eating habits. It's a good reminder at least for my Dream version of the program I'm creating that only scanning keywords allows for loopholes.

This ending thought leaves me with questions and makes me think about how these people have been neglected.

Poughkeepsie NY

Richard khan

Richard khan

Max

Jeffery E

Name HBRK Associates Inc. 575 Lexington Avenue, 4th Floor. rk 10022

MIT University?

Nickname

Email

From jeffery

Name

Paris

Little st.

Email

Nickname?

This is actually exciting. I have been a lazy thinker prior to ENGL C1000. College is definitely expanding the mind and how we think.

This is my quealm with theory - how do we account for something and discount another aspect of a phenamenon to create a solution when we havent accounted for the entirey of it.

Relates to the epdmiological conecpt in Joralemons book

Resilience here is intellectual, not just emotional. It is rooted in reflective capacity and competence. This reframes teacher retention not as endurance alone, but as cultivated professional identity.

This is a bold claim: internal belief may override external conditions. Yet it also invites caution, how much should systems rely on individual resilience instead of structural reform?

High efficacy teachers may not quit teaching, but they will exit toxic systems. Retention, therefore, may depend less on keeping teachers in specific schools and more on ensuring ethical, supportive environments.

This complicates the dominant narrative that environment alone determines retention. It suggests an interaction between personal competencies and contextual fit, raising questions about hiring, placement, and mentorship alignment.

This critiques the “information dump” model of induction. If learning requires processing, experimentation, and dialogue, then induction programs must mirror the reflective practices we expect teachers to use with students.

This suggests that structured reflective models become internalized cognitive habits. Teacher education, then, does not simply impart knowledge, it shapes how teachers think under pressure.

Reflection is presented as empowerment rather than mere introspection. This reframes reflective practice as an act of agency, an intellectual resistance against burnout and helplessness.

Self-belief here is not naïve optimism; it is strategic endurance. The deeper question becomes: how do teacher education programs systematically cultivate belief without promoting overconfidence?

Integration between theory and practice appears central. This suggests that disconnected coursework may unintentionally undermine teacher confidence. How often do teacher candidates experience coherence rather than fragmentation?

Reflection is not accidental; it must be structured and protected. In schools dominated by urgency and pacing guides, is time for reflection treated as essential professional practice, or a luxury?

This challenges traditional metrics of teacher quality (GPA, credentials). If belief outweighs skill, then teacher education must intentionally cultivate confidence rooted in authentic mastery, not just content coverage.

This sentence reframes resilience from a personality trait to a belief system. If efficacy drives persistence, then strengthening teachers’ beliefs about their capability may be as critical as developing their technical skills.

Preparation is framed here as protective armor. If inadequate preparation predicts departure, then retention may be less about resilience alone and more about justice, ensuring teachers are not placed in environments for which they are underprepared.

A one-third attrition rate within three years suggests not merely an individual failure but a systemic one. What does it reveal about the transition from preparation to practice—and are we designing teacher education programs with survival, sustainability, and identity formation in mind?

Sally Hemings lived under slavery but also lived in a patriarchal society where women's lives were restricted compared to men's. Her experience was not only shaped by race, but also gender.

Не выводить, если нет значения

Easy way for large corporations to dodge accountability

Major child labor issue

Child labor in a company that claims zero tolerance -- What companies are being accused? Apple, Samsung, Sony

High risk industry -- Raises questions about occupational safety data and how these deaths occurred and were they preventable

DRC Cobalt mines but also what other types of mines

This sentence is important to me because it shows the fear of any parent right through is words of worry. The fear of their daughters being dishonored. In this day and age there are still cases such as where a woman is disrespected and or mistreated just because she is a woman, and this is today where society has changed its views and is less misogynistic. I can only imagine the scenarios that play in Jourdon Anderson mind back then. I believe He is a great father.

So they only care about the financial reasons for not fishing in oyster reefs

SO WHY AREN'T WE DOING ANYTHING ABOUT IT

as they are using the 2010 fmp

yet they don't know if they are overfished or not

at what stage were they sampled? right as the trawl unloads? or when the fish are actually sorted through

so given the uncertainty, why continue to trawl?

underestimate

accounts for less than have of estuarine ecosystems in nc

we know that fish outnumber shrimp in catch abundance according the the 2016 brown study.

misquoting this research, it is just polychaetes that are growing in numbers due to dead bycatch creating a trophic shift.

where is the research for this?

I believe there should be this rule for all school, Elementary, Middle School, and High school because high school students are allowed to use their phones and it makes it a distraction for them in their education

fdfd

This is not measurable.

makes the body paragraphs interesting and informative to keep the reader engaged while sharing facts or other information

keeps the essay organized and focuses on the topic of the essay to keeps the reader engaged

Answering these questions help shape the research papers that you write and it organizes all the information

Why do researcher need to revise or change their research questions instead of sticking to the original one?

yeah agree! research is better when we truly want to do it, not just we have to.

It seems like research is ongoing process instead of one time task.

This makes me realize that all the questions cannot be answer in the same way . That means research is not just finding information but finding the right information.

Auvelity

VerB　のタグはなくす

a strange conceit. original design implies that there is some authoritative consensus building strict boundaries of what writers write about and why.

"being left to itself" is too vague. Humans are a part of nature and always will be. There is no separation, therefore it cannot be left to itself. We can identify behaviors that are more or less destructive to individuals and ecosystems thriving, but we can't say nature must just be left to itself.

caused by multiple genes

I find this interesting, as it's basically Carnegie saying that the wealth and power is better served in the hands of a few select people, rather than in the hands of the masses of people. It's interesting, because it's effectively the antithesis of the United States. The U.S. was founded on the idea that common people would have a say in government and in what the government spends money on. Individuals having freedom to do as they please. Carnegie is arguing that by sacrificing the freedom to purchase things as an individual, you enable a singular person to make decisions on what's best for the community. I think it then begs to question as to why we would all want to trust one person to decide what will benefit the common people the most, rather than letting the common people decide.

The way I hear this, is it's Carnegie saying that competition leads to improvements in overall life. Analysing it from a military perspective proves that this is actually true. The Second World War had dozens of nations fighting and competing to out fight the others, with the Allied and Axis powers being full of many major nations. The technology they developed to try and defeat each other would end up leading directly to the invention of the microwave, men in space, nuclear power, jet engines, and more. These are thing that we do interact with and use frequently. You'd be hard pressed to find someone who has never had anything in their life affected by jet engines and modern aviation, be it through travel or shipping things. Same with a microwave, many people have interacted with them. A competition, not necessarily between two companies, but rather two halves of the world, did indeed produce new commodities which would help many people in the future.

The song, "Song about Life in Virgina" is about a female indentured servant who shows as a glimpse into 5 years of her life while in that time. This woman writes very minimal about what she went through while telling a lot. The woman tells us “Five Years served I, Under master guy, In the land of Virginny, O: which made me for to know, sorrow, grief, and woe”, this one part shows us without explanation that she gave 5 years of her life taking care of this master needs to make his life better to have to live a like of misery for herself. Indentured servants are those who work for a person in return for things like food and shelter and no pay, this woman in this poem shows that through out her time she had to work and live in bad conditions like little food, thin clothing, dirty areas to sleep in, etc. This shows us as readers that these people that were indentured servants gave many years of their lifes to work for these masters which make their lifes easier and better just to have to live in horrible conditions to get food, clothing, and shelter.

I love how these questions get straight to the point, but they will be beneficial for us as educators. I think when teaching in the arts, things will always have to change and be explained. These questions address them, but also are not accusatory which is also not beneficial to objectively understanding.

I appreciate having these steps to help students through the creative process. It is so important that students take ownership of their creativity, but I have often asked, " How do you do this? These steps really help lay it out and help us know how to motivate them.

Constrants represent context, and generative AI has problems observing and identifying context.

Holy real trump parallel and they dont even know it

Greivence

because the unions are gone

But labour unions are no longer mobilizing the other populations

Less democratic voting by extension

delaying the effect of deindustrialization

Which all eroded as the midwest become non-defined by industrialism

And the unification of races around labor would have eroded

Outlandish: Well yes? isn't that why there are so many genres to choose from? if a person thinks that it's not enough then pick something else. Why does there have to be hate and finding something threatening?

Outlandish: I wholeheartedly disagree with this take on games. I believe that a game doesn't have to be complex or even hard to be considered interesting. Different people have different views on what's considered interesting: there are people who'd prefer easier and light hearted games.

Outlandish: "walking simulator" being used as an insult is so stupid, like why would you be offended by that at all

In my opinion, games typically considered walking simulators are best when they do what's described in this section. I honestly think this is the only kind of story which walking simulators excel at (stories in which the horrible truth about the main character is revealed slowly).

Outlandish: This interpretation of this misleading horror element of the game 'Gone Home' is interesting for sure. But, I do wonder if the creators of the game meant for it to be intentional because I believe a majority of players felt this way.

Outlandish: It's more than easy to lose sight of what you're doing and not realize what the game is going for, I remember when I tried to play video games as a kid I wouldn't realize what I was actually supposed to do and then get mad when nothing was going my way. I felt that way when playing Gone Home at times.

Outlandish: Not sure if this qualifies as outlandish, but if this is referring to the old Atari video game, I dont think it was text.

Outlandish: If there is no hierarchy of skill, then there is no clear status structure, which might threaten players who value mastery. It makes me rethink complaints about “no gameplay” as possibly complaints about “no competition.”

OUTLANDISH .I think this is pretty stupid. I can get walking out in nature or like in real life but on a screen I'm not gonna be admiring a video game the same way I admire nature even if it's super realistic. Like it's still a game and though there could be aspects of beauty in no way do I believe I am gonna retain my humanity.

Outlandish I found this claim crazy, adding depth I had never considered to games with "no consequences". In this way, it contradicts itself. There are consequences. They' re knowing what happened is fully your fault. If anything, the consequence is more scarring than any "death" could be.

Outlandish - Personally, I think I would react the same if a game I spent hundreds of hours making got grouped into a category of games that's considered to be trash by the general public.

Outlandish: I'm surprised that someone defending walking simulators would admit that they're boring to their critics. It seems impossible to deny that they aren't as excited as action-based games, so most of the debate centers around the strength of their message instead.

OUTLANDISH: Permalife is not difficult. If the player is always alive, there are no stakes and nothing to lose. The player's instincts to run if they are seen or fight back or hide are completely neutralized. This does not benefit the player in any way. There is nothing to deal with in the face of survival because survival is guaranteed.

Outlandish: Then what's the point? Walking sims bore me without having an objective. Feels mindless.

I think it's interesting to see how participating in a walking simulator game allows you to view a story from a completely different perspective that you might be used to. In terms of Gone Home, the player is detached in terms of knowing what has been happening in their home and family across the time period they have been gone, that presents the opportunity for judgement and other emotions to arise.

This quote really makes me think about the tension between immersion and observation in games. Gone Home lets the player witness an important queer story without ever participating in it…does that create empathy, or does it keep the player at arm’s length? It also raises questions about what it means to “experience” a story through a character: can understanding and reflection replace direct involvement, or does passivity limit the emotional impact?

The way that the player become an intruder in your own home is really crazy to think about. Personally, for me it can't be a home if you don't want to be there and you don't even recognize all the character traits it makes as a home. This "long absence" that's there is something that can make it feel like are an intruder but it should be quick turnaround to feel like you're home. This makes you realize that something is really wrong in this home and that's really the horror aspect of this game.

I do get what they’re saying, but I’m not fully convinced that walking is automatically grounds for reflection. Although slowing down can create space to think, it just ends up becoming boring for the player if there is nothing big happening. If the slowness of a game is intentional, like it is in walking simulators, then the world has to carry a lot of weight and substance.

This idea is important because it refutes the notion that games typically considered walking simulators are simply equivalent to action games without the enemies. Walking simulators place the emphasis of their interactive aspect on the features of the world around the player.

While I was playing Gone Home, I found it hard to see what the protagonist's role in the story was. Ultimately, I think their main purpose was to serve as a judge for the family's situation, since they are the most separated from Sam's issues with her parents. Similarly, our job as a player is to assess whether or not Sam made a good decision, which is why this ending is so impactful to the player.

This argument can definitely be backed by my own prior experiences. Whether it’s Gone Home or other similar games that I've played and seen–some with horror and mystery aspects–I never truly explored the environments simply for the sake of exploring my surroundings. Rather, I was always driven by a sense of curiosity to unfold the mysteries surrounding my character and the others around me.

Personally dislike this. If I want to retain humanity and walk into unseen places, why do it on a screen? Would much rather go outside and take a walk. A proper mental reset turning off screens.

When I read this portion of this passage, I was wondering what was the point of being so hateful towards alternative types of media? I feel that It is odd for gamers to be so affected by different types or games entering the gaming sphere. Shouldn't they be happy that something they love is gaining more traction and new ideas are being implemented?

I think this point of adventure games making it easy to move to the interesting parts is something I see fairly often. There's not many games I’ve played that don't have some sort of feature to skip to the interesting parts and skip over travel. I have noticed that most games do require you to usually travel to a particular location the more time-consuming way at least once before you can skip there immediately.This definitely allows the player to appreciate the action of exploration more.

The ability to interact with objects further elevates the story and environmental atmosphere of 'Gone Home', which made the walking aspect of the game much more meaningful to me.

I think this idea of Permalife is the most interesting idea in this chapter. Most games are focused on survival mechanics where death is the reset button. But permalife suggests that the challenge isn’t dying by continuing. It mirrors life, and that allows for emotional issues to really stand out through these games. But I also think that there needs to be something to keep the player moving forward. Sure some people may go on without a failure condition, but others would feel unmotivated to do so. They would need to nail down aspects like emotional weight, narrative curiosity and more for the game to really work.

I find it interesting that calmer games like walking sims originated from more violent and action-packed games like first-person shooters. Wanting to explore a world usually unavailable due to actions and conflict is common in human beings. It’s like setting a pack of cookies on a table, saying no one can eat it, and then leaving. Surely no one will miss one cookie in the pack. It’s the same concept for these shooter games. Players are busy running around, surviving, and fighting, so they don’t get to actually appreciate the world they are in (by design). It makes a part of their brain curious to experience the world they are already in without the pressures of battle, so a modder will take a cookie form the pack and eat it, opening up the rest of the pack for other people to enjoy. That’s when they realize the game they were praising for graphics and immersion is actually just a rough sketch, raisin cookies when they thought they were chocolate chips. This creates a sense of disappointment. I can see how walking sims were born from it. To cure the disappointment players felt when they realize the game they loved isn’t as polished as they thought. But without the allure of fighting, walking sims need something else, some other temptation. So, they promise ice cream with the cookies, sprinkling lore and stories into their world. Finding out pieces of the story give players their dopamine boost while satisfying their curiosity for adventure. The article then mentioned punishments within the game and how walking sims remove that and instead explore living with the consequences of your actions. This adds more depth to the ice cream and cookies players have been enjoying before, forcing them to either love or hate them more intensely.

Originally, I was not a fan of the subversion of my expectations, but in the past week since I have played it and discussed the game in class, the horror elements have grown on me. Over time, I have started to realize the purpose of subverting my expectations. The realization that Katie is too late to help her sister is cleverly implemented. Seeing satanic imagery leads the player to believe in supernatural elements, but it is just a red herring. The fear the player has allows them to relate to Sam and her experience of coming out. The lightning and creaking noises make the player anxious but not so anxious that they can’t keep playing and have to take a break. As this feeling grows, it transforms into internal fears and intensifies the feeling for the player.

People would never have thought of first-person shooters such as Doom and Unreal with walking simulators like Gone Home, but if you take out the violence in FPS games they are walking sims. Especially in open world games like Zelda you walk around the majority of the time. The only difference is that there's fights from time to time while walking sims tell stories through the environment.

Walking simulators challenge the traditional view that games were designed only for players to win. The game’s purpose shifts from “Achieve a certain number of kills” or “Capture a certain number of enemy bases” to “Explore the house” or “Check your surroundings.” Players learn how to interpret and examine their environments.

It is interesting to me that there were mods created for Quake that removed the entire main goal of the game. I have never thought about taking out a main mechanic of a videogame and then playing it. It makes me rethink all the games ive played and imagine it just like exploration. Playing games with different music can change the whole vibe of the game, I have noticed, like i remember playing horror games with my own music and it made it not as scary.

Goes against the idea that writers write well due to their spark within them. The author goes against this belief that many people have due to romanticization. Writers work hard to get their work done; it is unrealistic to believe that this is not the case.

I am someone who has been exposed to procrastination, especially when it comes to my schoolwork. I push all my work until the last minute, causing me to rush to get it done in the end.

Drinking can relieve writers of any pressure or anxiety they are feeling by doing this so that the writing can come out more natural.

It is not always about being deprived of sleep; writer's block and procrastination can come from psychological issues such as anxiety or depression. It can also come from social and physical factors.

Nathan Mortensen

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Reply to the reviewers

General Response to Review

We would like to thank all three reviewers for their encouraging comments on our manuscript. We now submit our revised study after considerable efforts to address each of the reviewer concerns. I will first provide a response related to a major change we have made in the revision that addressed a concern common to all three reviewers, followed by a point-by-point response to individual comments.

Replacing LRRK2ARM data with a LRRK2 specific type II kinase inhibitor: The most critical issue for all 3 reviewers was the use of our new CRISPR-generated truncation mutant of LRRK2 that we called LRRK2ARM. We had not provided direct evidence of the protein product of this truncation, which was a significant limitation. To address this we performed proteomics analysis of all clones, and to our surprise, we identified 7 peptides that were C-terminal to our "predicted" stop codon we had engineered into the CRISPR design. A repeat of the deep sequencing analysis in both directions then more clearly revealed site specific mutations leading to 4 amino acid changes at the junction of exon 19, without introducing a stop codon. Given that we could not detect the protein by western blot (even though proteomics now indicated the region of LRRK2 recognized by our antibodies was present) we decided to remove this clone from the manuscript. In the meantime we had compared the ineffectiveness of MLi-2 to block Rab8 phosphorylation during iron overload in the LRRK2G2019S cells with a type II kinase inhibitor called rebastinib. The data showed very clearly that treatment with rebastinib reversed the iron-induced phospho-Rab8 at the plasma membrane (and by western blot, in new Fig 3). Since this inhibitor is very broad spectrum inhibiting ~30% of the kinome we reached out to Sam Reck-Peterson and Andres Leschziner, experts in LRRK2 structure/function, who recently developed a much more selective LRRK2-specific type II kinase inhibitor they called RN341 and RN277 (developed with Stefan Knapp PMID: 40465731). These compounds effectively coupled the MLi-2 compound through an indole ring to a rebastinib type II compound to provide LRRK2 binding specificity to the efficient DYG "out" type II inhibitor. As with rebastinib, the new LRRK-specific kinase inhibitors also effectively reversed the cell surface p-Rab8 seen in LRRK2G2019S, iron loaded cells. These new data provide the first biological paradigm where the kinase activity of LRRK2 is resistant to type I MLi-2, yet remains highly sensitive to type II inhibitors. While the loss of our LRRK2ARM clone marks a significant change in the manuscript we believe the main message is stronger with the addition of the new LRRK2 specific type II kinase inhibitor. Our data show that it is indeed the active kinase function of LRRK2G2019S that is impacting the iron phenotypes we observe but highlight the conformational specificity upon iron overload such that MLi-2 is ineffective. The overall phenotypes we observe in LRRK2G2019S macrophages remain unchanged and are now expanded within the manuscript. We hope reviewers will agree that our work provides important new insights into LRRK2 function in iron homeostasis while opening new avenues of research in future studies.

Given this new information we have changed the title from "LRRK2G2019S acts as a dominant interfering mutant in the context of iron overload" to the more accurate "LRRK2G2019S interferes with NCOA4 trafficking in response to iron overload leading to oxidative stress and ferroptotic cell death."

Response to Reviewer 1

Reviewer 1 (R1): There are two major concerns with the data in their present form. In brief, first, the G2019S cells express much less LRRK2 and more Rab8 that the WT cells and this severely affects interpretability.

Heidi McBride (HM): We agree that the LRRK2G2019S lines express lower levels of LRRK2 than wild type, which is a previously documented phenomenon, presumably as the cell attempts to downregulate the increased kinase activity by reducing protein expression. However, the levels of Rab8 across 10s of experiments do not consistently show any differences between the wild type, G2019S and KO. We have provided more comprehensive quantifications of the blots in the revised version, and the Rab8 levels are consistent across all the blots presented in the manuscript (Figure 1A and 1B).

R1: Second, the investigators used CRISPR to truncate the endogenous LRRK2 locus to produce a hypothetical truncated LRRK2-ARM polypeptide. This appears to have robust effects on NCOA4, in particular, which drives the overall interpretation of the data. However, the expression of this novel LRRK2 species is not confirmed nor compared to WT or G2019S in these cells (although admittedly the investigators did seek to address this with subsequent KO in the ARM cells). It would be premature to account for the changes reported without evidence of protein expression. This latter issue may be more easily addressed and could provide very strong support for a novel function/finding, see more detailed comments below, most seeking clarifications beyond the above.

HM: As described in my common response above, we have removed the LRRK2ARM data from the manuscript.

R1: Need to make clear in the results whether the G2019S CRISPR mutant is heterozygous or homozygous (presumably homozygous, same for ARM)

HM: The RAW cell line we generated is homozygous for the G2019S and the KO alleles. We added this to the beginning of the results section and methods.

R1: The text of the results implies that MLi2 was used in both WT and G2019S Raw cells, but it's only shown for G2019S. Given the premise for the use of RAW cells, it's important to show that there is basal LRRK2 kinase activity in WT cells to go along with its high protein expression. This is particularly important as the G2019S blot suggests minor LRRK2-independent phosphorylation of Rab8a (and other detected pRabs). One would imagine that pRab8 levels in both WT and G2019S would reduce to the same base line or ratio of total Rab in the presence of MLi2, but WT untreated is similar to G2019S with MLi2. This suggests no basal LRRK2 activity in the Raw cells, but I don't think that is the case.

HM: We have included the data from MLi-2 treatment of wild type cells in Fig 3C quantified in D. Again, the baseline levels of Rab8 are unchanged across the genotypes. However, the reviewer is correct that there is some baseline LRRK2 kinase activity that is sensitive to MLi2 in wild type cells. This is seen most clearly on the autophosphorylation of LRRK2 at S1292 in Fig 3C. The pRab8 blots is not as clear in wild type cells. It is likely that LRRK2 must be actively recruited to membranes (as seen by others with LLOME, etc) to easily visualize p-Rabs in wild type cells. Nevertheless, we do clearly see the activity of autophosphorylation in wild type cells. Therefore while we understand the reviewers point that there should be some Rab8 phosphorylation in wild type cells, we don't see a significant, or very convincing, amount of it in our RAW macrophages.

R1: Also, in terms of these cells, the levels of LRRK2 are surprisingly unmatched (Fig 1A, 1D, 1H, S1D, etc.) as are total levels of Rab8 (but in opposite directions) between the WT and G2019S. This is not mentioned in the Results text and is clearly reproducible and significant. Why do the investigators think this is? If Rab8 plays a role in iron, how do these differences affect the interpretation of the G2019S cells (especially given that MLi2 does not rescue)? Are other LRRK2-related Rabs affected at the protein (not phosphorylation level)? Could reduced levels of LRRK2 or increase Rab 8 alone or together account for some of these differences? Substantial further characterization is required as this seriously affects the interpretability of the data. Since pRab8 is not normalized to total Rab8, this G2019S model may not reflect a total increase in LRRK2 kinase activity, and could in fact have both less LRRK2 protein and less cellular kinase activity than WT (in this case).

HM: In our hands, the RAW cells with homozygous LRRK2G2019S mutations show clearly that the total protein levels of LRRK2 is reduced compared to wild type, which is likely a compensatory effect to reduce cellular kinase activity overall. We understand that some of our previous blots were not so clear on the total Rab8 levels across the different experiments. We have repeated many of these experiments and hope the reviewer can see in Figs 1A, 3C, 3E, 3J, and Sup3A that the total Rab8 levels are stable across the conditions. We also present quantifications from 3 independent experiments normalizing the pRab8/Rab8 levels in all three genotypes in untreated and iron-loaded conditions (Supp Fig 3A and B), and upon MLi2 treatment (Fig 3C). In 3C and D the data show the effectiveness of MLi-2 to reduce pRab8 in control conditions, but the resistance to MLi-2 in FAS treated cells.

R1: Presumably, the blots in 1H are whole cell lysates and account for the pooled soluble and insoluble NCOA4 (increased in G2019S), as there is no difference in soluble NCOA4 (Fig 2H). I suspect the prior difference is nicely reflected in the insoluble fraction (Fig 2H). This should be better explained in the Results text. This is a very interesting finding and I wonder what the investigators believe is driving this phenotype? Is the NCOA4 partitioning into a detergent-inaccessible compartment? Does this replicate with other detergents, those perhaps better at solubilizing lipid rafts? Is this a phenotype reversible with MLi2? Very interesting data.

HM: We apologize for not being clearer in the text describing the behavior of NCOA4. The reviewer is correct that the major change in G2019S is the increased triton-X100 insoluble NCOA4. Previous work has established that NCOA4 segregates into detergent-insoluble foci upon iron overload as a way to release it from ferritin cages, and this fraction is then internalized into lysosomes through a microautophagy pathway (see Mizushima's work PMID: 36066504). In Fig 1I we show that the elevation in NCOA4 and ferritin heavy chain seen in untreated G2019S cells can be cleared upon iron chelation with DFO, indicating that the canonical NCOA4 mediated ferritinophagy (macroautophagy) pathway remains intact to recycle the iron in conditions of iron starvation. However in Figure 2 we show that conditions of iron overload, when NCOA4 segregates from ferritin (to allow cytosolic storage of iron), this form of NCOA4 cannot be degraded within the lysosome through the microautophagy pathway, and begins to accumulate. We see this with our live and fixed imaging compared to wild type cells (Fig 2A,D), and by the lack of clearance seen by western blot (Fig 2E). As for the impact of MLi-2, we observe some reversal of NCOA4 accumulation in untreated cells at 4 and 8 hrs after MLi-2 treatment (Supp Fig 2F). However, in iron loaded conditions the high NCOA4 levels in G2019S cells are MLi2 insensitive, while the elevated NCOA4 in wild type cells is reduced upon MLi2 addition (Fig. 2F, compare lates 3vs4 in wt with lanes 7vs8 in G2019S). This is consistent with a block in the microautophagy pathway of phase-separated NCOA4 degradation in G2019S cells.

R1: Figure 2 describes the increased NCOA4-positive iron structures after iron load, but does not emphasize that the G2019S cells begin preloaded with more NCOA4. How do the investigators account for differential NCOA4 in this interpretation? Is this simply a reflection of more NCOA4 available in G2019S cells? This seems reasonable.

HM: The reviewer is correct, we showed that there is some turnover of NCOA4 in untreated conditions through canonical ferritinophagy, but in iron overload this appears to be blocked, the NCOA4 segregates from ferritin and remains within insoluble, phase-separated structures that cannot be degraded through microautophagy. We have written the text to be more clear on these points.

R1: These are very long exposures to iron, some as high as 48 hr which will then take into account novel transcriptomic and protein changes. Did the investigators evaluate cell death? Iron uptake would be trackable much quicker.

HM: We agree that many things will change after our FAS treatments and now provide a full proteomics dataset on wild type and G2019S cells with and without iron overload, which is presented in Figure 4A-B. Indeed Figure 4 is entirely new to this revised submission. The proteomics highlighted a series of cellular changes that reflect major cell stress responses including the upregulation of HMOX1 (western blots to validate in Supp Fig 4A), an NRF2 transcriptional target consistent with our observation that NRF2 is stabilized and translocated to the nucleus in G2019S iron loaded cells (Sup Fig 4B,C). There are several interesting changes, and we highlighted the three major nodes, which are changes in iron response proteins, lysosomal proteins - particularly a loss of catalytic enzymes like lysozymes and granzymes consistent with the loss of hydrolytic capacity we show in Fig. 4C,D. We also noted changes in cytoskeletal proteins we suspect is consistent with the "blebbing" of the plasma membrane we see decorated with pRab8 in Fig 3. To test the activation of lipid oxidation likely resulting from the elevation in Fe2+ and oxidation signatures we employed the C11-bodipy probe and observe strong signal specific to the G2019 iron-loaded cells, particularly labelling endocytic compartments and the cell surface (Fig. 4E-G).

Lastly, an analysis of SYTOX green uptake experiments was done to monitor the uptake of the dye into cells that have died of cell membrane rupture, commonly used to examine ferroptotic cell death. We now show the G2019S cells are very susceptible to this form of death (Fig 4H,I). These data add new functional evidence for the consequence of the G2019S mutation in an increased susceptibility to iron stress.

R1: The legend for 2F is awkward (BSADQRED)

HM: We have changed this to BSA-DQRed, which is a widely used probe to monitor the hydrolytic capacity of the lysosome.

R1: Why are WT cells not included in Fig 2G?

HM: We have now included new panels in Fig 3C,D showing wild type and G2019S +/- FAS and +/-ML-i2 with quantifications of pRab8/Rab8.

R1: The biochemical characterization of NCOA4 in the LRRK2-arm cells is a great experiment and strength of the paper. The field would benefit by a bit further interrogation, other detergents, etc.

HM: We have removed all of the LRRK2ARM data given our confusion over the impact of the 4 amino acid changes in exon 19 and our inability to monitor this protein by western blot. The concept that NCOA4 enters into TX100 insoluble, phase separated compartments has been well established, so we didn't explore other detergents at this point.

R1: Have the investigators looked for aberrant Rab trafficking to lysosomes in the LRRK2-arm cells? Is pRab8 mislocalized compared to WT? Other pRabs?

HM: We did initially show that pRab8 was also at the plasma membrane in the LRRK2ARM cells, and we still focus on this finding for the G2019S, seen in Fig 3A,B,F,H. We did try to look at other p-Rabs known to be targets of LRRK2 but none of them worked in immunofluorescence so we couldn't easily monitor specific traffic and/or localization changes for them.

R1: The expression levels and therefore stability of the ARM fragment is not shown. This is necessary for interpretation. While very intriguing, the data in Aim 3 rely on the assumption that the ARM fragment is expressed, and at comparable levels to G2019S to account for phenotypes. The generation of second clone is admirable, but the expression of the protein must be characterized. This is especially true because of the different LRRK2 levels between WT and G2019S. One could easily conceive of exogenous expression of a tagged-ARM fragment into LRRK2 KO cells, for example, as another proof-of-concept experiment. If it is truly dominant, does this effect require or benefit from some FL LRRK2? It seems easy enough to express the LRRK2-ARM in at least WT and KO RAW cells.

HM: We agree and our attempts to understand this clone resulted in its removal from the manuscript. We did also express cDNA encoding our ARM domain (up to exon 19), but it didn't phenocopy the CRISPR clone, which of course made sense once we had better proteomics and repeated our deep sequencing.

In our further efforts to understand why our phenotype was MLi-2 resistant upon iron overload we expanded to examine the impact of pan-specific TypeII kinase inhibitors, and then reached out to the Reck-Peterson and Leschziner labs to obtain a newly developed LRRK2 selective type II kinase inhibitor. These all very efficiently reversed the pRab8 signals seen at the plasma membrane of G2019S cells upon iron overload (Fig 3E-K). Therefore the G2019S is not dominant negative, as we had initially supposed, rather there is a specific conformation of LRRK2 in high iron that potentially opens the ATP binding pocket to bind the type II inhibitors, but not MLi2. We do not understand exactly what this conformation is but likely involves new protein interactions specific to high iron, or perhaps LRRK2 binds iron directly as a sensor somehow that ultimately leads to the differential sensitivity we observe between type I and type II kinase inhibitors. Our data indicate that MLi-2 treatment in clinic will not be protective against iron toxicity phenotypes that may contribute to PD, where these newer selective type II LRRK2 kinase inhibitors would be effective in this conformation-specific context of iron toxicity.

R1: Does iron overload induce Rab8a phosphorylation in a LRRK2 KO cell? This would be a solid extension on the ARM data and support the important finding that an additional kinase(s) can phosphorylate Rab8a under these conditions, and while not unexpected, this may not have been demonstrated by others as clearly. It also addresses whether the ARM domain is important to this other putative kinase(s), which may add value to the authors' model.

HM: Iron overload does not induce pRab8 in LRRK2 KO cells, as seen by immunofluorescence in Fig 3A,B, and western blot in Supp Fig 3 A,B. With our new type II kinase inhibitor data we can confirm that the plasma membrane localized Rab8 is indeed phosphorylated by LRRK2.

R1: Minor concern - the abstract but not the introduction emphasizes a hypothesis that loss of neuromelanin may promote cell loss in PD (through loss of iron chelation), while post mortem studies are by definition only correlative, early works suggested that the higher melanized DA neurons were preferentially lost when compared to poorly melanized neurons in PD. This speculation in the abstract is not necessary to the novel findings of the paper.

HM: We appreciate that the links to iron in PD are correlative, we have maintained some of our discussion on this point within the manuscript given the lack of attention the field has paid to the cell biology of iron homeostasis in PD models. If there is a cell autonomous nature to the loss of DA neurons in PD, iron is very likely to be a part of this specificity in our opinion. Most of the newer MRI studies looking at iron levels in patient brains are showing higher free iron and working on this as potential biomarkers of disease. The precise timing of this relative to the stability/loss of neuromelanin is, I agree, not really clear.

R1: (Significance (Required)): This study could shed light on a both novel and unexpected behavior of the LRRK2 protein, and open new insights into how pathogenic mutations may affect the cell. While studied in one cell line known for unusually high LRRK2 expression levels, data in this cell type have been broadly applicable elsewhere. Give the link to Parkinson's disease, Rab-dependent trafficking, and iron homeostasis, the findings could have import and relevance to a rather broad audience.

HM: We are so very appreciative that reviewer 1 feels our work will be of interest to the PD and cell biology communities.

Response to Reviewer 2

Reviewer 2 (R2): Major: Please confirm that the observed phenotype is conserved within bone marrow-derived macrophages of LRRK2 G2019S mice. These mice are widely available within the community and frozen bone marrow could be sent to the labs. The main reason for this experiment is that CRISPR macrophage cell lines do sometimes acquire weird phenotypes (at least in our lab they sometimes do!) and it would strengthen the validity of the observations.

HM: We did a series of experiments on primary BMDM derived from 3 pairs of wild type, LRRK2G2019S and LRRK2KO mice. We examined levels of ferritin heavy and light chains in steady state and withFAS treatment experiments. Unfortunately the data did not phenocopy the RAW macrophage lines we present here since FTL and FTH were mostly unchanged. We did observe an increase in NCOA4 levels, consistent with potential issues with microautophagy as observed in our RAW system.

While we understand the danger that our phenotypes are nonspecific and linked to a CRISPR-based anomaly, there are a number of arguments we would make that these data and pathways are potentially very important to our understanding of LRRK2 mutant phenotypes and pathology. The first point is that we now include a LRRK2-specific type II kinase inhibitor that reverses the iron-overload pRab8 accumulation at the plasma membrane in LRRK2G2019S cells, showing that this is at least directly linked to LRRK2 kinase activity, even though it is resistant to MLi2.

Second, Suzanne Pfeffer recently published their single cell RNAseq datasets from brains of untreated LRRK2G2019S mice (PMID: 39088390). She reported major changes in Ferritin heavy chain (it is lost) in very specific cell types of the brain, astrocytes, microglia and oligodendrocytes, with no changes in other cell types at all (her Fig 6 included left). This is consistent with a very context specific impact of LRRK2 on iron homeostasis that we don't yet understand.

Third, the labs of both Cookson, Mamais and Lavoie have been working on the impact of LRRK2 mutations on iron handling in a few different model systems, including iPSCs, and see changes in transferrin recycling and iron accumulation. Those studies did not go into much detail on ferritin, NCOA4 and other readouts of iron homeostasis but are roughly in agreement with our work here. In the last biorxiv study submitted after we sent this work for review they concluded their phenotypes were reversed by MLi2 treatment, however they required 7 days of treatment for a ~20% restoration in iron levels. Given our work it would seem the impact of LRRK2G019S in high iron conditions is also very resistant to MLi2 treatment. In all these studies we do not yet know for sure whether iron overload in the brain may be a precursor to DA neuron cell death, which could be exacerbated in G2019S carriers. But we hope the reviewer will agree that our approach and findings will be useful for the field to expand on these concepts within different models of PD.

R2: Minor comments: Supplementary Fig 1: I don't think one should normalize all controls to 1 and then do a statistical test as obviously the standard deviation of control is 0.

HM: We agree with the reviewer that statistical testing is not appropriate when the WT control is fixed to a value of 1, as this necessarily eliminates variance in that group; accordingly, we have removed both statistical comparisons and standard deviation from the WT control while retaining variability measures for all experimental conditions. Raw densitometry values could not be pooled across independent experiments due to substantial inter-blot variability, and therefore normalization to the WT control was used solely to allow relative comparison within experiments, acknowledging the inherent quantitative limitations of Western blot densitometry. Ultimately the magnitude of the changes relative to the control lanes in each biological replicate was consistent across experiments, even if the absolute density of the bands between experiments was not always the same.

R2: The raw data needs to be submitted to PRIDE or similar.

HM: All of our data is being uploaded to the GEO databases, protocols to protocols.io and raw data deposited on Zenodo site in compliance with our ASAP funding requirements and the journals.

R2: Some of the western blots could be improved. If these are the best shown, I am a little concerned about the reproducibility. How often has they been done?

HM: We now ensure there is quantification of all the blots for at least 3 independent experiments and have worked to improve the quality of them throughout the revision period.

R2: (Significance (Required)): Considering the importance of LRRK2 biology in Parkinson's and the new biology shown, this paper will be of great interest to the community and wider research fields.

HM: We are so very grateful that the reviewer appreciates that the LRRK2 and PD community will find our work of interest. We hope our revisions will prove satisfactory even in the absence of ferritin changes in primary G2019S BMDM.

Response to Reviewer 3

Reviewer 3 (R3): What is missing in the study is the physiological relevance of these findings, mainly whether this effect actually results in higher cell death during iron overload. Since iron overload is known to result in ferroptosis, it is surprising that the authors have not checked whether the LRRK2 G2019S and ARM cells undergo more ferroptosis relative to LRRK2 WT cells.

HM: We thank the reviewer for pushing us to monitor the functional implications of the iron mishandling upon iron overload in the G2019S RAW cell system. We now add a completely new Figure 4 to get to these functional points. We employed two tools to look at established aspects of ferroptosis, first the C11-bodipy probe that labels oxidized lipids and we see significant signals specific to the G2019S iron loaded cells, where it labels endocytic membranes and the cell surface (Fig 4 E-G). This is consistent with the elevation of free iron 2+. We also used the SYTOX green death assay where the dye is internalized into cells when the cell surface is ruptured and show that G2019S cells die upon iron overload, but not the LRRK2KO or wild type cells (Fig 4 H,I). Lastly, we performed full proteomics analysis of the wt and G2019S RAW cells in iron overload conditions. These data provide a better view of the full stress response initiated in the G2019S cells, including the upregulation of HMOX1 (an NRF2 target gene), changes in lysosomal hydrolytic enzymes consistent with the reduction in BSA-DQRed signals, and in cytoskeleton, which is consistent with the plasma membrane blebbing phenotypes we see in G2019S (Fig. 4A-D and Supp. Fig 4 data). We hope these new data help to position the phenotype into a more physiological output.

R3: Moreover, their conclusion of the findings as "resistant to LRRK2 kinase inhibitors" is not convincing, since in most of the studies, they have removed the kinase domain, and this description implies the use of pharmacological kinase inhibition which has not been done in this paper.

HM: We took this comment to heart and, as explained in the general response we removed the LRRK2ARM clones from the study. To understand the kinase function in the iron overload conditions we first explored the pan-specific type II kinase inhibitor rebastinib, shown to inhibit LRRK2. In contrast to MLi2, this drug effectively blocked p-Rab8 in G2019S cells exposed to high iron. However, since it is not specific and likely inhibits about 30-40% of all kinases we reached out to the Reck-Peterson and Leschziner labs who have developed a LRRK2 specific type II kinase inhibitor (published in June 2025 PMID: 40465731). They provided these to us (along with a great deal of discussion) and the two drugs both blocked the effect of LRRK2G2019 on p-Rab8 at the plasma membrane. These data show that the phenotypes we observe are indeed linked to the increased kinase activity of LRRK2, even though they are fully resistant to MLi-2. It suggests that high iron results in some alteration in LRRK2 conformation that alters the ability of MLi2 to block the kinase activity, while still allowing the type II kinase inhibitors that bind deeper in the ATP-binding pocket, to functionally block activity. We believe that these new data remove a great deal of confusion we had in the initial submission to explain the MLi-2 resistance.

R3: There is lower LRRK2 expression in LRRK2 G2019S cells, have the authors checked Rab phosphorylation to validate the mutation?

HM: We agree that the G2019S mutation leads a reduction in total LRRK2 levels in the cell, which is likely a compensatory effect to lower kinase activity in the cell. We do show that the G2019S mutation has clear activation of phosphorylation on both Rab8 and at the autophosphorylation site S1292 of LRRK2, as seen in Fig 1A, quantified in Fig 1B. In untreated conditions, these phosphorylation events are reversible upon treatment with MLi-2. We also provide the sequencing data in the supplement to confirm the presence of the G2019S mutation in this clone, shown in Supp Fig. 1A.

R3: The authors should specify if their cells are heterozygous or homozygous since they are discussing a dominant interfering mutant.

HM: The G2019S and LRRK2 KO are both homozygous. We state this early in the results section and the methods.

R3: The transferrin phenotype validated through proteomics and western blot is solid. HM: We agree, thank you very much!

R3: Quantification in figure 1F-G is problematic, not clear what they mean by "diffuse and lysosomal". Puncta is either colocalising with lysosomes or not colocalising. This needs to be clarified and re-analysed.

HM: We apologize for the confusion. In control cells the Cherry tagged FTL is efficiently cycling through the lysosomes and we don't see a strong cytosolic (diffuse) pool, which likely reflects the relatively iron-poor culture conditions. However, in G2019S cells, there is a highly elevated amount of FTL, with a strong cytosolic/diffuse stain in steady state, with some flux into lysosomes. In this experiment we chelated iron to test whether this cytosolic pool of FTL was capable of clearing through the lysosomes (ferritinophagy). While there is a cytosolic (diffuse) pool that remains, the pool that fluxes into the lysosome increases in G2019S chelated cells. This is also seen by the reduction in total FTL seen by western blot (endogenous FTL). Our conclusion here is that the general ferritinophagy machinery remains functional in G2019S cells. We have changed the term "diffuse" to "cytosolic" and improved our description of this experiment in the text.

R3: Text in the first results part called "LRRK2G2019S RAW macrophages have altered iron homeostasis" is very long. It could be divided into more sections to improve readability. HM: We have improved the text to be more descriptive of the conclusions and added new sections

R3: If the effect is armadillo-dependent, where does LRRK2 G2019S is implicated since there is no kinase domain in these cells?

HM: Our new data employing the LRRK2-specific type II kinase inhibitors now confirm that the effects of the G2019S on iron overload are indeed kinase dependent, it's just insensitive to MLi2.

R3: The authors do not show any controls (PCR, sequencing) confirming knockout or truncation. HM: We did higher resolution proteomics and deep sequencing and learned that the "Arm" mutation was not a truncation but a series of 4 point mutations around exon 19. Therefore we removed all data referring to this clone and replaced it with the use of the type II kinase inhibitor experiments. We feel this removed a lot of confusion and provides much clearer conclusions on the role of the kinase activity in iron overload. We may continue to explore what the 4 amino acid mutations created such strong phenotypes, as it could reflect a critical conformational change that impacts the kinase activity. But that is for future work. We now include the sequencing files of the G2019 and KO as Supplementary Data Files 1 and 2.

R3: The data is interesting and the image quality with the insets is very high. HM: We thank the reviewer for their positive comments!

R3: Mutant not clearly described in text, did the authors remove just the kinase and ROC-COR domains or all the domains downstream of the Armadillo domain? This is not clear. HM: We have removed the clone from the manuscript.

R3: The authors cannot conclude that their phenotype is due to the independence of the kinase domain specifically as they are also interfering with the GTPase activity by removing the ROC-COR domains. HM: We agree and our new drugs allow us to confirm that the phenotypes are due to kinase activity, but there is a new conformation of LRRK2 induced in high iron that renders the kinase domain resistant to MLi-2 inhibition. We discuss this in the manuscript now.

R3: In Figure 3E, is the difference between the "ARM CTRL" and the "ARM FAS" conditions significant? A trend appears to be there, but the p-value is not shown. HM: these data are now removed.

R3: In figure 4A, it would have been important to check if Rab8 phosphorylation is also observed in LRRK2 KO cells after administration of FAS to further evaluate the mechanism through which this Rab8 phosphorylation is occurring.

HM: We show that the pRab8 is specific to the G2019S lines and not seen in LRRK2 KO (Fig 3A,B, Supp. Fig. 3A,B).

R3: The vinculin bands in figure 4A are misaligned with the rest of the bands.

HM: We now provide new blots for all of these experiments (in Fig 3) as we removed the LRRK2ARM data from the manuscript and the appropriate loading controls are all included.

R3: The authors do not have any controls to validate the pRab8 staining in IF. This is an important caveat and needs to be addressed. HM: We now include siRNA validation of Rab8 (vs Rab10) to confirm the specificity of the antibody to pRab8 in IF where it labels the plasma membrane in G2019S iron loaded cells.

R3: The authors should have checked if FAS administration in the LRRK2 G2019S and the ARM cells is leading to ferroptotic cell death (or cell death in general). This is key to validate the link between the altered iron homeostasis in LRRK2 G2019S cells and increased cytotoxicity observed during neurodegeneration.

HM: As mentioned above, we have added extensively to our new Fig 4 to include full proteomics analysis of the changes in iron loaded G2019S cells, we use C11-Bodipy probes to monitor lipid oxidation, and SYTOX green assays to monitor cell death through cell surface rupture (consistent with ferroptosis). We thank the reviewer for pushing us to do these experiments and provide further relevance to the potential for LRRK2 mutations to promote cell toxicity during neurodegeneration.

R3: Regarding the literature, the authors are missing some important papers that are preprinted and these studies need to be discussed. This includes a report with opposite findingshttps://www.biorxiv.org/content/10.1101/2025.09.26.678370v1.full and a report showing kinase independent cell death in macrophages https://www.biorxiv.org/content/10.1101/2023.09.27.559807v1.abstract

HM: We thank the reviewers for alerting us to the biorxiv papers, one of which was submitted after we sent our manuscript to review. We are excited to see the growing interest in the impact of LRRK2 function in iron homeostasis and hope our work will contribute to this. Upon reading the study from the LaVoie lab they do show some sensitivity of the iron loaded phenotype in G2019S cells, however they see a ~20% reduction in lysosomal iron after 7 days of MLi treatment in Astrocytes (their Fig 2L). To us, this is very likely an indication of a relatively high resistance to the drug. I'm sure if they tried these new Type II inhibitors the iron load would be much more rapidly reversed. The specificity of their phenotype to Rab8 is also very interesting considering the cell surface localization we see for pRab8 in our iron loaded system. Similar comments for the Guttierez study in macrophages. We have included the findings of these papers within the manuscript and thank the reviewer for pointing them out.

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Referee #3

Evidence, reproducibility and clarity

In this paper, the authors report an interesting phenotype of the LRRK2 G2019S mutation on iron homeostasis in RAW264.7 macrophages. The phenotype is well characterised through proteomic and western blot approaches investigating transferrin and ferritin trafficking. The study is well conducted and data of high quality. The authors also appear to have discovered a cellular context where Rab8 is phosphorylated independently of LRRK2. This is a major finding which can potentially have an important impact in the LRRK2 field. What is missing in the study is the physiological relevance of these findings, mainly whether this effect actually results in higher cell death during iron overload. Since iron overload is known to result in ferroptosis, it is surprising that the authors have not checked whether the LRRK2 G2019S and ARM cells undergo more ferroptosis relative to LRRK2 WT cells. Moreover, their conclusion of the findings as "resistant to LRRK2 kinase inhibitors" is not convincing, since in most of the studies, they have removed the kinase domain, and this description implies the use of pharmacological kinase inhibition which has not been done in this paper.

Significance

Major comments

In Figure 1:

• There is lower LRRK2 expression in LRRK2 G2019S cells, have the authors checked Rab phosphorylation to validate the mutation?
• The authors should specify if their cells are heterozygous or homozygous since they are discussing a dominant interfering mutant.
• The transferrin phenotype validated through proteomics and western blot is solid.
• Quantification in figure 1F-G is problematic, not clear what they mean by "diffuse and lysosomal". Puncta is either colocalising with lysosomes or not colocalising. This needs to be clarified and re-analysed.
• Text in the first results part called "LRRK2G2019S RAW macrophages have altered iron homeostasis" is very long. It could be divided into more sections to improve readability.

In Figure 2:

• If the effect is armadillo-dependent, where does LRRK2 G2019S is implicated since there is no kinase domain in these cells?
• The authors do not show any controls (PCR, sequencing) confirming knockout or truncation.
• The data is interesting and the image quality with the insets is very high.

In Figure 3:

• Mutant not clearly described in text, did the authors remove just the kinase and ROC-COR domains or all the domains downstream of the Armadillo domain? This is not clear.
• The authors cannot conclude that their phenotype is due to the independence of the kinase domain specifically as they are also interfering with the GTPase activity by removing the ROC-COR domains.
• In Figure 3E, is the difference between the "ARM CTRL" and the "ARM FAS" conditions significant? A trend appears to be there, but the p-value is not shown.

In Figure 4:

• In figure 4A, it would have been important to check if Rab8 phosphorylation is also observed in LRRK2 KO cells after administration of FAS to further evaluate the mechanism through which this Rab8 phosphorylation is occurring.
• The vinculin bands in figure 4A are misaligned with the rest of the bands.
• The authors do not have any controls to validate the pRab8 staining in IF. This is an important caveat and needs to be addressed.
• The authors should have checked if FAS administration in the LRRK2 G2019S and the ARM cells is leading to ferroptotic cell death (or cell death in general). This is key to validate the link between the altered iron homeostasis in LRRK2 G2019S cells and increased cytotoxicity observed during neurodegeneration. Regarding the literature, the authors are missing some important papers that are preprinted and these studies need to be discussed. This includes a report with opposite findings https://www.biorxiv.org/content/10.1101/2025.09.26.678370v1.full and a report showing kinase independent cell death in macrophages https://www.biorxiv.org/content/10.1101/2023.09.27.559807v1.abstract

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Referee #2

Evidence, reproducibility and clarity

In this manuscript the authors describe an interesting connection between the Parkinson's kinase LRRK2 and iron trafficking in RAW macrophages. Expression of the LRRK2 G2029S mutation affects the abundance of ferritin heavy and light chains and therefore the uptake and storage of iron. Interestingly, the loss of the kinase domain still had a strong phenotype, suggesting that this is independent of the kinase function.

The paper is well written and excellently cited. The data is convincing and of good quality.

I have only one request and else very minor comments:

Major: Please confirm that the observed phenotype is conserved within bone marrow-derived macrophages of LRRK2 G2019S mice. These mice are widely available within the community and frozen bone marrow could be sent to the labs.

The main reason for this experiment is that CRISPR macrophage cell lines do sometimes acquire weird phenotypes (at least in our lab they sometimes do!) and it would strengthen the validity of the observations.

Minor comments:

Supplementary Fig 1: I don't think one should normalize all controls to 1 and then do a statistical test as obviously the standard deviation of control is 0. I would normalize to the average of the control, which will provide an error for the control.

The raw data needs to be submitted to PRIDE or similar. This has not happened yet.

Some of the western blots could be improved. If these are the best shown, I am a little concerned about the reproducibility. How often has they been done?

Significance

Considering the importance of LRRK2 biology in Parkinson's and the new biology shown, this paper will be of great interest to the community and wider research fields.

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Referee #1

Evidence, reproducibility and clarity

Goldman et al describe some novel findings with respect to LRRK and iron handling in a series of RAW macrophage cell lines. This cell background is chosen for its recognized high levels of endogenous LRRK2 protein expression, its somewhat broad use in the field, and the investigators add its relevance due to phagocytosis of red blood cells, thus requiring iron robust metabolic processes. Proteomic analyses of WT and G2019S RAW cells revealed multiple iron-related proteins affected by LRRK2 mutation. A deeper candidate-based analysis revealed complex changes in ferritin heavy and light chain and changes in ferric and ferrous iron. Notably, reliable changes in the levels and/or solubility of NCOA4 result from this pathogenic LRRK2 mutation. Unexpectedly, however, these changes were not sensitive to LRRK2 kinase inhibitor treatment. The investigators suggest a dominant effect rather than loss-of-function as subsequent experiments revealed that these effects could be replicated with a LRRK2 variant lacking the kinase domain (LRRK2-ARM) and were not replicated by LRRK2 KO. The data are internally consistent throughout and could certainly shed new important light onto unique and unexpected effects of this LRRK2 mutation.

There are two major concerns with the data in their present form. In brief, first, the G2019S cells express much less LRRK2 and more Rab8 that the WT cells and this severely affects interpretability. Second, the investigators used CRISPR to truncate the endogenous LRRK2 locus to produce a hypothetical truncated LRRK2-ARM polypeptide. This appears to have robust effects on NCOA4, in particular, which drives the overall interpretation of the data. However, the expression of this novel LRRK2 specie is not confirmed nor compared to WT or G2019S in these cells (although admittedly the investigators did seek to address this with subsequent KO in the ARM cells). It would be premature to account for the changes reported without evidence of protein expression. This latter issue may be more easily addressed and could provide very strong support for a novel function/finding, see more detailed comments below, most seeking clarifications beyond the above.

• Need to make clear in the results whether the G2019S CRISPR mutant is heterozygous or homozygous (presumably homozygous, same for ARM)
• The text of the results implies that MLi2 was used in both WT and G2019S Raw cells, but it's only shown for G2019S. Given the premise for the use of RAW cells, it's important to show that there is basal LRRK2 kinase activity in WT cells to go along with its high protein expression. This is particularly important as the G2019S blot suggests minor LRRK2-independent phosphorylation of Rab8a (and other detected pRabs). One would imagine that pRab8 levels in both WT and G2019S would reduce to the same base line or ratio of total Rab in the presence of MLi2, but WT untreated is similar to G2019S with MLi2. This suggests no basal LRRK2 activity in the Raw cells, but I don't think that is the case.
• Also, in terms of these cells, the levels of LRRK2 are surprisingly unmatched (Fig 1A, 1D, 1H, S1D, etc.) as are total levels of Rab8 (but in opposite directions) between the WT and G2019S. This is not mentioned in the Results text and is clearly reproducible and significant. Why do the investigators think this is? If Rab8 plays a role in iron, how do these differences affect the interpretation of the G2019S cells (especially given that MLi2 does not rescue)? Are other LRRK2-related Rabs affected at the protein (not phosphorylation level)? Could reduced levels of LRRK2 or increase Rab 8 alone or together account for some of these differences? Substantial further characterization is required as this seriously affects the interpretability of the data. Since pRab8 is not normalized to total Rab8, this G2019S model may not reflect a total increase in LRRK2 kinase activity, and could in fact have both less LRRK2 protein and less cellular kinase activity than WT (in this case).
• Presumably, the blots in 1H are whole cell lysates and account for the pooled soluble and insoluble NCOA4 (increased in G2019S), as there is no difference in soluble NCOA4 (Fig 2H). I suspect the prior difference is nicely reflected in the insoluble fraction (Fig 2H). This should be better explained in the Results text. This is a very interesting finding and I wonder what the investigators believe is driving this phenotype? Is the NCOA4 partitioning into a detergent-inaccessible compartment? Does this replicate with other detergents, those perhaps better at solubilizing lipid rafts? Is this a phenotype reversible with MLi2? Very interesting data.
• Figure 2 describes the increased NCOA4-positive iron structures after iron load, but does not emphasize that the G2019S cells begin preloaded with more NCOA4. How do the investigators account for differential NCOA4 in this interpretation? Is this simply a reflection of more NCOA4 available in G2019S cells? This seems reasonable.
• These are very long exposures to iron, some as high as 48 hr which will then take into account novel transcriptomic and protein changes. Did the investigators evaluate cell death? Iron uptake would be trackable much quicker.
• The legend for 2F is awkward (BSADQRED)
• Why are WT cells not included in Fig 2G?
• The biochemical characterization of NCOA4 in the LRRK2-arm cells is a great experiment and strength of the paper. The field would benefit by a bit further interrogation, other detergents, etc.
• Have the investigators looked for aberrant Rab trafficking to lysosomes in the LRRK2-arm cells? Is pRab8 mislocalized compared to WT? Other pRabs?
• The expression levels and therefore stability of the ARM fragment is not shown. This is necessary for interpretation. While very intriguing, the data in Aim 3 rely on the assumption that the ARM fragment is expressed, and at comparable levels to G2019S to account for phenotypes. The generation of second clone is admirable, but the expression of the protein must be characterized. This is especially true because of the different LRRK2 levels between WT and G2019S. One could easily conceive of exogenous expression of a tagged-ARM fragment into LRRK2 KO cells, for example, as another proof-of-concept experiment. If it is truly dominant, does this effect require or benefit from some FL LRRK2? It seems easy enough to express the LRRK2-ARM in at least WT and KO RAW cells.
• Does iron overload induce Rab8a phosphorylation in a LRRK2 KO cell? This would be a solid extension on the ARM data and support the important finding that an additional kinase(s) can phosphorylate Rab8a under these conditions, and while not unexpected, this may not have been demonstrated by others as clearly. It also addresses whether the ARM domain is important to this other putative kinase(s), which may add value to the authors' model.

Minor concern - the abstract but not the introduction emphasizes a hypothesis that loss of neuromelanin may promote cell loss in PD (through loss of iron chelation), while post mortem studies are by definition only correlative, early works suggested that the higher melanized DA neurons were preferentially lost when compared to poorly melanized neurons in PD. This speculation in the abstract is not necessary to the novel findings of the paper.

Significance

This study could shed light on a both novel and unexpected behavior of the LRRK2 protein, and open new insights into how pathogenic mutations may affect the cell. While studied in one cell line known for unusually high LRRK2 expression levels, data in this cell type have been broadly applicable elsewhere. Give the link to Parkinson's disease, Rab-dependent trafficking, and iron homeostasis, the findings could have import and relevance to a rather broad audience.

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Reply to the reviewers

We would like to thank all the reviewers for their comments and suggestions.

Please find below our point-by-point response to the Reviewers' comments, which details the corrections already made and outlines the planned revisions, experiments, and analyses.

Reviewer 1

Major comments:

• Reviewer 1 commented that the 'manuscript would greatly benefit from having someone spend time on the figures, and associated text, to ensure they are fully comprehensible'. We agree wholeheartedly with the reviewer and apologise. We have now revisited the text, figures, and associated figure legends to ensure that they are more easily accessible and fully comprehensible to readers from across disciplines. This includes adding labels to point out specific anatomical features on images, and ensuring figures and text align. Further specific examples are included in the points below.
• In response to concerns raised by Reviewer 1 relating to: Figure 1 and the lack of figure citations; 'the persistence of mCherry in the H2B Fucci'; how mCherry seems to persist longer in H1 (compare Figs 1D and 1G)':
• We apologise for the lack of figure citations in the text. We have now reworked the figures relating to the constructs (original Figures 1 and S1) and have made these Figures 1, 2 and S1 in our updated version.
• Figure 1 is now an introductory background figure which illustrates the differences between Fucci(SA) and Fucci(CA) reporters, with additional details provided in the associated legend, and call outs to the figure starting in the introduction.
• Regarding 'the persistence of mCherry in the H2B Fucci', what we are trying to articulate is that the mCherry degradation that we observed in the Fucci(2A) expressing DF1 cells extended beyond the end of S phase and into G2/M, compared with what would be expected (Revised Figure 2H, arrows).
• We have now replaced these montages with a more representative example. Additionally, the new images (Figures 2C and 2G) are synchronised (both starting at G2/M), restricted to a single cell cycle, are larger in size, and have the cell cycle stage labelled. We believe these changes will aid interpretation.
• Specifically relating to the lack of labelling in Figure 3A, we agree that this figure was not labelled sufficiently, and neither was there enough detail included in the text or figure legend for readers to follow easily and make their own conclusions. We have now added additional labels to this figure, broken the figure down into more panels (Figures 4A-4D in revised manuscript), and included more detailed descriptions in the associated figure legend and text.
• We thank the reviewer for making the important point that it is 'hard to know where the biosensor is reporting patterns that are already well established (eg neural tube), and where the biosensor is reporting patterns that are novel - and if so, what these patterns are' which was made more challenging by insufficient references to previous studies.
• Firstly, as for the point above, we have now added labels to many of the panels (Figure 4 in revision), including highlighting features such as the non-proliferative dermal condensates and demarcating the proliferative retinal pigmented epithelium (Figures 4F and 4G in revision). Secondly, we have also now included additional references in the text, specifically relating to the neural tube, digits, and forming feathers, where our proliferation profiles are consistent with previous literature.
• With regards to the Reviewer's comment regarding the difficulty in drawing conclusions 'about cell cycle in different tissue layers without sectioning' in original Figure 3B we will include more sections of FuChi embryos which include structures such as mesenchymal condensates.
• To make our data on cell cycle stages as 'cells egress from the primitive streak, to form prechordal plate' clearer we have added additional labels to the figures (Figures 4B and 6E in revised manuscript). We will complement this adding sections of gastrulating FuChi embryos to further demonstrate the cell cycle status of cells that form the pre-chordal plates.

Minor comments

• We have added additional references relating to the data in original Figure 3 (now Figure 4 see above), and any new descriptions of known proliferation profiles that we include will have appropriate citations.
• In this current revision we have addressed figure call out issues, and added labels to enhance readability, clarity and data interpretation. Reviewer 2

Major comments

• Reviewer 2 rightly pointed out that the 'description of the bicistronic tandem-Fucci(CA) system in paragraph 6 is not consistent with what is described in the original bibliographic reference indicated by the authors'. We have now added additional text to properly explain the CDT1 probe dynamics, as per the cited manuscript, and also referenced the schematics to help readers.
• To address whether the FuChi model can be accurately 'used to study embryogenesis' and following up on the suggestion to 'indicate if the size of the embryos is comparable to the wildtype' we have now included size comparisons of FuChi and wild-type/non-transgenic embryos at mid (E9) and late (E18) gestational stages demonstrating that there is no significant difference between genotypes during embryogenesis (Figure 3D in revised manuscript). For all earlier stages, we did not see any developmental or size differences. We believe if there were any differences, these would be reflected in size at the mid and late gestational stages we analysed.
• Reviewer 2 made very valuable observations and suggestions regarding our data and interpretation of somitogenesis, specifically in response to our sentence saying that "the mesenchyme, which is predominantly in G1 as they undergo condensation". Furthermore, they noted that Supplementary Video 4 "shows distinct green fluorescence (S) in the presomitic mesoderm for the first hour or so, only then turning to magenta (G1)". We were asked to review the sentence/video to clarify if this is a significant finding or if this is not representative of their observations.
• We thank the reviewer for this suggestion. From looking again at our timelapse movies, and also analysing additional static images, we agree that presomitic mesoderm (PSM) does appear to be green (S phase), which then may transition to G1 as the somites form. To address this, we plan to quantify cell cycle status in the PSM on embryos to see if this is a significant finding.
• We hope this quantification of the PSM may also enable us to include discussion on how our findings relate to the Cell Cycle model for somitogenesis proposed in the Collier et al, 2000 paper suggested by the Reviewer.
• We agree with the Reviewer that "the fluorescence profiles in original Figure 4C do not seem similar regarding the Myc-tag epitope" and believe this difference is likely just a reflection of the part of the image we used. We will include a more representative image once we have repeated the staining.
• Reviewer 2 has asked for quantitative support for our fluorescence-based interpretations. We thank the reviewer for this suggestion and are now planning to perform quantitative analyses of different tissues (similar to our quantification in germ cells) and in embryos to support our observations. These will include the PSM (see above), neural tube, intestine, and early embryos (also see Reviewer 3 response for blastoderm quantification).
• Since our original submission, we have further refined our in situ hybridisation protocol on FuChi embryos (Figures 5A & B in revision), finding that strong reporter expression is maintained for all the fluorescent proteins of the H1-Fucci(CA)2 reporter. Therefore, the "notably fainter" appearance of the hGMNN-mVenus in Figure 4A from the first version of the paper was likely a result of the experimental protocol not being 100% optimal.
• *

Minor comments

• We have reordered the paragraphs relating to the different Fucci versions in the introduction as per the suggestions by the reviewer for better clarity.
• To address the issues with Fucci system nomenclatures which made reading difficult, we have now added a background figure (new Figure 1 in revised draft) which is cited in the introduction, made sure constructs are introduced appropriately, and ensured we are consistent with our nomenclature.
• Supplementary Figure lettering corrected.
• All figure panels are now mentioned in the main text, and the incorrect call outs noted by the Reviewer have been corrected
• Removed period and included clarifying statement in the figure legend relating to the comment regarding the extraembryonic region in Figure 5 (original) / Figure 6 (revised).
• Other issues raised relating to reference duplication and missing words have been resolved.
• We have corrected the legend of Figure 1 of the original paper, see related Reviewer 1 response provided above.

Reviewer #3

Minor comments

• We have corrected all the figure call outs (see responses to similar comments by Reviewers 1 and 2) to ensure that all data presented is accurately reported.
• We would like to thank the reviewer for suggesting modifications to the cell cycle montages (original figures 1D, 1G and 2F). We agree it would help the reader to enlarge the image, and therefore reduced the montage to include just one cell cycle, and have also included annotations of cell cycle stages in Figures 2C and 2G of the revised manuscript. We have also added some labels to Figure 3E (original figure 2F) and enlarged this.
• In response to Reviewer 3's comment regarding fluorescent intensity. We quantified fluorescence levels in multiple individual DF1 cells expressing either the H1.0-Fucci(CA)2 or H2B-Fucci(SA)2 reporters, and this is shown as the fluorescent index in Figures 2D, 2E, 2H and 2I of the revised manuscript, where reporter levels were measured across time. In terms of overall mean intensity levels of the reporters, we found the reporters to be comparable in brightness and have similar mean intensity levels across the cell populations in the flow cytometry data (Figures 2F and 2J).
• To enhance speedy interpretation, we will also process our supplementary videos to include annotations and arrows to highlight key cells and events (e.g. a cell undergoing mitosis).
• As recommended by Reviewer 3, we have now quantified cell cycle status in blastoderm cells, confirming that a high proportion are in the G2/M phase. We will include these data in the final revision, which will complement our planned quantification of cell cycle status in other tissues (see response to Reviewer 2).
• For our final revision, we will include higher magnification/zoomed in images of selected regions of the somites, neural tube (lumen) and retina (epithelium). Revisiting our images of the neural tube showed that dividing cells lumen did so in the perpendicular plane and we will include these images in our revision to provide further evidence of the fidelity of the FuChi reporter. We thank the reviewer for this excellent idea to show the efficacy of our system.
• To address the levels of proliferation in somites, we plan to generate a cropped video with a fixed ROI to enable proliferation in individual cells of the forming somites to be more readily visualised. This will be further complemented by the quantification of cell cycle status in forming somites (see responses to other reviewers).
• We have added lines to the discussion regarding the use of our reporter in other conventional model systems.

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Referee #3

Evidence, reproducibility and clarity

The manuscript by Sudderick and colleagues describes the development and characterisation of a new generation of cell cycle reporter that can distinguish between cells in G1, S, G2 and M phases. Furthermore, the authors have developed a transgenic chicken line incorporating this reporter and demonstrated faithful discrimination of cell cycle stages in the in vivo context of developing transgenic embryos. Of note is the addition of epitope tags, which facilitate discrimination of cell cycle stages in tissue fixed using various techniques. This is a very important paper for the following reasons:

• The authors have achieved faithful discrimination of all four cell cycle stages, which is a major advance in itself.
• This generation of the FuChi transgenic chick is of enormous importance. This will facilitate accurate in vivo studies in a broad range of fixed and living tissue types and is a major milestone in the further establishment of the chick as a transgenic model system.

Th characterisation of the cell cycle reporter as presented is robust and convincing. The authors further demonstrate the potential utility of the FuChi chickens through their observation of partial cell cycle synchrony during onset of development. I therefore only have minor suggestions that may facilitate easier interpretation of their data.

Results 2

• I can't see any mention of Figures 1C and D. Presumably the authors have carried out fluorescence intensity measurements using the two cell cycle reporters here, but this is not mentioned in the main text.
• Figure 1D&G: I find these difficult to follow given the small size of the cells as presented. The authors may consider enlarging these and clearly annotating for cell cycle stage. They may find it helpful to focus on a single cell cycle, although I appreciate that displaying two cell cycles strengthens the claim of efficacy of the newly developed sensor. The supplementary videos associated with these figure panels are excellent as they display several cells with faithful reporter activity, but again, the authors may wish to annotate a few of these cells to enhance speedy interpretation. I have similar comments for Figure 2F and the associated movie.

Results 4

• The authors state that a large proportion of blastoderm cells were in G2/M. They may wish to formally quantify this, perhaps by performing simple cell counts in designated regions of interest. A similar quantification for gastrulating embryos would also be helpful.
• It would be helpful to see zoomed in images of selected regions of the somites, neural tube and retina displayed in Figure 3B. This would be particularly appropriate in the context of the neural tube and retina (which are not discussed in the main text) as the positioning of the nucleus is defined by the stage of the cell cycle and should therefore serve to highlight the efficacy of the reporter.
• Video 4 beautifully demonstrates the high levels of proliferation in somites, but again, it would be useful to have a zoomed in view. I appreciate the difficulty involved in doing this, given the movement of the embryo, but perhaps the authors could focus on a fixed ROI or present a separate movie of a few cells undergoing a full cell cycle.

Discussion

• The authors could perhaps expand on their discussion about potential utility in other conventional model systems (e.g. mouse, fish, etc).

Significance

General assessment: A timely piece of work that introduces a faithful cell cycle reporter that will be of broad interest.

Advance: The ability to discriminate between all four stages of the cell cycle is a clear advance here.

Audience: Broad interest, including those studying cell cycle and embryonic development in several tissue contexts.

Expertise: Chick embryology, in vivo live imaging, neurogenesis, cellular developmental biology

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Referee #2

Evidence, reproducibility and clarity

Summary:

This work presents a novel transgenic chicken model with fluorescent reporters that allow in vivo monitoring of the four phases of the cell cycle. To achieve this, the authors clearly identify the limitations of previous Fucci systems and developed an optimised reporter construct that overcomes the major technical challenges identified. Addition of epitope tags to cell cycle stage-specific markers further enables antibody detection in fixed tissues. Proof of concept is provided by live imaging of chick embryos in early developmental stages, evidencing dynamic cell cycle states in tissues and migrating cells.

Major comments:

1. Introduction: Description of the bicistronic tandem-Fucci(CA) system in paragraph 6 is not consistent with what is described in the original bibliographic reference indicated by the authors. Namely: "...accumulation of the CTD1 probe..." should be expected in the G1-S transition (not S-G2) and the yellow reporter should be expected in G2 and M phases (not S and G2, as described). Please review this portion of the text.
2. The authors state that "Of note, hatched FuChi chicks are initially smaller than wild type counterparts but grow at comparative rates and are fertile". If the model is to be used to study embryogenesis, it would be useful to indicate if the size of the embryos is comparable to the wildtype, at least for the major developmental stages mentioned in the manuscript.
3. When referring to somitogenesis, the authors state "...the mesenchyme, which is predominantly in G1 as they undergo condensation". Suppl Video 4, however, shows distinct green fluorescence (S) in the presomitic mesoderm for the first hour or so, only then turning to magenta (G1). The authors should review the sentence/video to clarify if this is a significant finding or if this is not representative of their observations.
4. (Optional) It would be interesting to describe if the authors' observations of cell cycle dynamics in the presomitic mesoderm support the proposed Cell Cycle model for somitogenesis (Collier et al., J.Theor.Biol.2000).
5. The fluorescence profiles in Figure 4C do not seem similar regarding the Myc-tag epitope (contrarily to what is stated). The authors should rephrase or revisit this image to clarify their findings.
6. Quantitative support for several fluorescence-based interpretations made throughout the manuscript. In some instances, conclusions are drawn from qualitative differences in signal intensity. For example, the statement in Fig. 4A that hGMNN-mVenus appears "notably fainter" than the other reporters. Incorporating simple quantitative analyses would strengthen these claims and ensure that observed differences reflect biological behaviour rather than technical or optical factors.

Minor comments:

1. Organization of the information in the Introduction: Paragraphs 3-5 introduce sequentially improved versions of the Fucci system. Then, paragraph 6 returns to the system described in the 4th paragraph. Authors should consider including paragraph 5 (description of Fucci4 and its limitations) just prior to the description of chickens as valuable developmental models (current paragraph 8) for clarity of the text.
2. Fucci system nomenclature. Many different Fucci systems are mentioned, but nomenclature consistency throughout the manuscript is lacking, which makes reading difficult. For example, the terms "Fucci(SA)2" and "Fucci(CA)2" should be defined in the introduction, as they are employed to describe the construction of the new biosensor in the following sections.
3. Some figure panels are not mentioned in the main text (for ex. Figures 1B and C, Figure 2C)
4. The legend of Figure 1 (D & G) mentions "denoted by *", but the * seems to be missing in the figure.
5. Supplementary Figure 1 has two D panels (and is missing the E).
6. In the main text, where it reads "...Flow cytometry analysis of three independent PGC lines... (Figures 2G & S2E)", S2E should be replaced by S1E.
7. In the Figure 4A legend, hCDT1-mVenus should be corrected to hCDT1-mcherry. Also, it is not clear why the authors state that "hGMNN-mVenus expression is notably fainter compared with hCDT1-mVenus and H1.0-mCerulean expression".
8. In Figure 5E, the optical sections "i" seem to pertain to the extraembryonic tissue/area opaca and not to anterior mesoderm, as stated in the figure legend. Also, there is a period between "prechordal plate" and "and" in the legend's last sentence.
9. Discussion: The last sentence of the third paragraph lacks "to" between "used" and "interrogate".
10. References 10 and 23 are identical.

Referee cross-commenting

I agree with all comments from reviewers 1 and 3

Significance

This is a beautiful paper, describing a long sought-after model system to study cell cycle dynamics in vivo. The methodological details are thorough, and the results obtained are clearly presented, highlighting the utility of the new model in various embryonic stages and tissues/organs.

This work is of pivotal importance to the developmental/stem cell biology community, as well as to the wider community that employs the chicken embryo as a preclinical model to assess therapeutic or teratogenic potential of biologically- or chemically-derived products.

My expertise is in chicken embryo development, namely gastrulation, somitogenesis and limb bud outgrowth.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The manuscript reports the development of a novel Fucci (Fluorescent Ubiquitination-based cell cycle indicator) system for analysing cell cycle analysis, including live imaging of cell cycle. The novel biosensor (H1.0-Fucci(CA)2) has been developed for analyses of chick cells and tissues: chick embryos are a valuable developmental model that have (and in the future, will) particularly informed our understanding of early stages of embryogenesis, and of development of numerous tissues, including the neural tube, somites, limb bud. The authors conclude that the novel system has advantages over previous Fucci systems, including faithful labelling of all four cell cycle phases. Importantly, the authors have generated a stable germline of H1.0-Fucci(CA)2 transgenic chicks, enabling, for the first time, the discrimination and tracking of cells in all 4 phases of the cell cycle - i.e. in vivo studies of cell cycle progression in vivo, in intact tissues and organs. Additional epitope tags mean that the biosensor can be detected in fixed tissues, enabling comparison of cell cycle with expression of mRNA and proteins that mediate other aspects of development/label particular cells and tissues. The authors map proliferation dynamics across numerous tissues in the developing chick, at numerous stages of development, and conclude in particular that transition from S phase may be a key morphogenetic event in gastrulation, as mesendoderm cells leave the primitive streak to form embryonic stuctures such as prechordal plate

Major comments:

The novel biosensor looks to be an incredibly useful tool, and the manuscript suggests patterns of cell cycle progression in different tissues, and at different points in time, that look intriguing. But it is sometimes difficult to draw the strong conclusions suggested by the authors because the text and figures are sometimes difficult to follow. The manuscript would greatly benefit from having someone spend time on the figures, and associated text, to ensure they are fully comprehensible.

Specifically:

Conclusion1: That the new FUCCI biosensor is a superior cell cycle probe, better at discriminating all cell cycle phases than previous versions. I was very convinced by the vidoes (video 1 and 2) but had problems with Figure 1. Potentially, this is because I am not an expert in these types of analyses - but it was not helped by the fact that components of the figure were not cited in the text. I was particularly confused by the statement remarking on 'the persistence of mCherry in the H2B Fucci' as mCherry seems to persist longer in H1 (compare Figs 1D and 1G). Please explain, in the Figure legend, why this appears to be the case.

Conclusion 2: that the FuChi chicks are the first viable stably expressing avian cell cycle biosensor model. I agree, and the authors should be congratulated on the development of this important tool.

Conclusion 3: the authors monitor cell cycle progression in chicks, in vivo, looking at stages from blastoderm, through gastrulation, and into organogenesis, and draw various conclusions

For example: Fig 3A and text: 'as gastrulation progresses, the primitive streak an presomitic mesoderm display...., whereas the .... And neural plate contains...'

Figure 3A covers an enormous range of stages and tissues. The figure is barely labelled. The text and figure need to better align, and key features in each figure panel need to be labelled so that the reader can better follow, and draw conclusions.

Fig 3B: Reports expression in numerous tissues. There are some beautiful examples of cells segregating relative to cell cycle - for instance, in the neural tube. But I found it hard to know where the biosensor is reporting patterns that are already well established (eg neural tube), and where the biosensor is reporting patterns that are novel - and if so, what these patterns are. Again, this is not described adequately in the text (for instance, there is no mention of the neural tube). And in some cases, references are provided (allowing comparison with previous studies) - but in other cases, there are no references to previous studies. The reader must be given the opportunity to compare this study with previous studies.

Overall - I can appreciate that there are some fascinating patterns, but it is very difficult to draw the conclusions suggested by the authors. Primarily this is due to poor labelling of figures, and lack of clarity between figures and text, and poor referencing. Additionally, it is not clear that strong conclusions can be drawn about cell cycle in different tissue layers without sectioning some embryos.

Fig 3C: The authors remark 'The results confirm that the ... FuChi embryos recapitulate known cell cycle profiles of those tissues'. See my comments in 3B.

Conclusion 4: Robust stability of biosensor in fixed tissues. I agree, and the authors should be congratulated for having made a construct that can be paired with in situ hybridisation and immunohistochemistry - this is invaluable.

Conclusion 5: The authors investigate the potential of the new system for live imaging, and focus on a couple of novel dynamic examples.

The data indicating that PGCs at initial migratory stages are not undergoing frequent cell division is clear.

However, the data indicating that cell cycle status changes as cells egress form the primitive streak, to form prechordal plate, is not clear. The figures need to be better labelled, and the text needs to be more clear (eg ' and prechordal plate. and anterior mesoderm'..

Minor comments:

• Specific experimental issues that are easily addressable.

I would recommend that the authors section some embryos, to better support key conclusions (eg in figure 3 and 5) - Are prior studies referenced appropriately?

Not always - see comment above (Fig 3) - Are the text and figures clear and accurate?

No - this needs work. Not all figures cited in text, or cited in wrong order; Figures are poorly labelled - making it hard to follow - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Label figures more carefully and ensure figures and text align

Referee cross-commenting

I agree with all comments from reviewers 2 and 3

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Technically this is a fantastic resource. As detailed above, the novel biosensor (H1.0-Fucci(CA)2) has been developed for analyses of chick cells and tissues: chick embryos are a valuable developmental model that have (and in the future, will) particularly informed our understanding of early stages of embryogenesis, and of development of numerous tissues, including the neural tube, somites, limb bud. Increasingly, studies show the importance of cell cycle for development, differentiation and morphogenesis - it is a huge breakthrough to be able to perform in vivo studies of cell cycle progression in intact tissues and organs.<br /> - State what audience might be interested in and influenced by the reported findings.

Broad basic research, including developmental biologists, stem cell biologists, modellers. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Developmental biologist, with expertise in chick

There should be an option for this!

In the meantime, a workaround: How to get a serial comma: Use en or en-US instead of en-GB.

new Intl.ListFormat("en-US", { style: "long", type: "conjunction" })

It is always valuable to give the option to developers to use it how they want. The developer may want to use it without necessarily making it a user-controllable UI preference!!

Me gusta en un 50/50 esta forma de pensar, si bien nada es perfecto y todo puede mejorar y evolucionar con el tiempo, no me cerraría a pensar de que estoy lejos o que no podría llegar a la meta que me propuse en cierto momento. Todo en la vida es resiliencia y mejora continua de procesos de manera progresiva y alcanzable.

Me parece algo totalmente genial (dejando de lado de que no todo el mundo tiene un récord guinness y todos los requisitos que solicitan para tenerlo) sino por el hecho de que no es algo aleatorio.

Recuerdo haber leído sobre ese récord hace un tiempo y si no me equivoco era sobre recorrer cierta cantidad de estaciones de metro (unas 400 y algo) en un tiempo de 24 horas ("pasaditas") y que estuvo vigente hasta cierto tiempo, lo que me parece fascinante es que cómo dije anteriormente, no fue algo esporádico, fue algo planeado, algo planificado en equipo

Cómo toda gran idea novedosa o innovadora que nace de la incomodidad ...

Ser disruptivo y crear algo que cambie y mejore las reglas convencionales es algo que siempre he de admirar. Tener la convicción de diseñar algo que se sabe que reúne lo mejor de varios sistemas es algo que no todo el mundo hace, si bien quisieron hacer algo más "pequeño, propio y privado" (que se entiende muy bien, no por el tema de envidia o privatización sino porque quizá uno cómo persona no dimensiona el impacto de sus creaciones), algo que me llamó la atención es que fueron de lleno a crear algo a la altura de los lenguajes de alto nivel (básicamente que se pueden hacer más y mejores cosas sin tantas líneas de código), ósea que simplemente no fue un típico proyecto que ya existía, sino que intentaron ir más allá de una vez, simplemente adelantados a su tiempo, es increíble

GENDER, SEXUALITY, AND SHIFTING STYLES: KNOWLEDGE PRODUCTION AND CODIFYING LANGUAGE USE IN STYLE GUIDES

CLEAVAGE THEORY ORIGINALLY says that national politics is representation not of party organisation, but of "UNDERLYING SOCIAL CONFLICTS" and therefore input from electorate.

Seems remarkably democratic in that electorate are ultimately the ones who are steering the electoral ship / [arty platform directions.

PAST examples of cleavages (which, again, in this context simply means disruption or shock) include industrialisatioon and the urban/rural divide -> nationalism vs obligations to the supranational Church, etc. -> In other words, the cleavage NOW is between supranational supporters (EU) and NATIONAL supporters reacting to perceived encroachments on EU identity.

TO CONCLUDE -> authors aregue that transnational cleavage is EXPLOITED BY FAR RIGHT PARTIES BECAUSE THEY BECOME SINGLE ISSUE AND FOCUS ON THESE ISSUES VERY KEY TO VOTERS, while mainstream parties are not primarily devoted to immigration, etc -> are at a disadvantage -> like that thesis that says that actors in referendums who CONTROL THE NARRATIVE of referendums have the advantage -> can make it about a single issue, etc.

"Radical right parties take more extreme positions on these issues, place more salience on them, and exhibit greater internal unity than mainstream parties. By virtue of their commitment to GAL values, green parties are located at the alter-pole. Just as the religious cleavage and the class cleavage were raised by Catholic and socialist parties on one side of the divide, so the transnational cleavage is mobilized by radical right parties at one extreme. As the transnational divide has become salient, mainstream parties have been compelled to compete on issues that lie far from their programmatic core."

Whole idea of cleavage theory is that parties and their platforms respond to CRISES and a shift in voter attitude as opposed to any internal decision making process. THEREFORE -> policy shifts by parties are SUDDEN as opposed to INCREMENTAL

"They suggest that party systems are subject to discontinuities rather than to incremental change, and that the response of a party system to exogenous change comes from voters rather than parties."

Again, all of this IS REACTION TO immigrqtion, increased supranational govt / control by "foreigners," Eurozone, etc. All about loss of sovereignty basically.

"as a reaction to reforms that have weakened national sovereignty, promoted international economic exchange, increased immigration and exacerbated cultural and economic insecurity."

TRANSNATIONAL CLEAVAGE: - i.e., the cleavage is the term for the DISRUPTOR / SHOCK - THIS shock is transnational -> i.e., EROSION OF NATION STATE SOVEREIGNTY (This is the shock) - Penetration of nation state politics by "external actors" (immigratants, "foreign" governments, etc.)

Es bastante bueno, diferencial e innovador que se permita crear y dirigir la creación de páginas web de acuerdo a lo que desean y quieren las personas. Mientras que otros servicios se encargan de ser generadores netamente de sitios web empresariales o de market, esta permite dirigir su servicio a otro del mercado, cómo el de los videojuegos

Si bien esto puede ser un ganchazo comercial muy bueno, es una razón valida para optar por herramientas gratuitas y propias que de verdad se preocupen y sean garantes de la protección de los datos, datos personales y contenidos de las páginas web que creen las personas

Si bien comparten ciertas características cómo algunos referentes que son lideres en la generación rápida de sitios web cómo GoDaddy o Wix, me llamó la atención que sean laxos en cuanto a funciones, contenidos e infraestructura, mientras que otros sitios que de cara ya son de pago o restringen el uso de cierto tipo de contenido, esta promesa que compite contra estos lideres potencia y libera la capacidad creativa de los usuarios de forma gratuita, propia e intuitiva

NOTE DE SYNTHÈSE : Dans la tête d'un colérique

Introduction

Cette note de synthèse explore les facettes de la colère présentées dans les extraits de l'émission "Dans la tête d'un colérique".

Le documentaire examine la nature de cette émotion souvent perçue négativement, sa gestion individuelle et collective, ses manifestations, ses fonctions insoupçonnées, et les conséquences de son refoulement ou de son expression violente.

À travers des témoignages, des analyses de spécialistes et des expériences, l'émission offre un éclairage nuancé sur une émotion complexe et puissante.

Thèmes Principaux

La perception sociale de la colère et son contrôle: La colère est largement considérée comme une émotion négative et "mal vue".

L'éducation et les règles de bienséance nous incitent à la contrôler.

La fonction intrinsèque et l'utilité de la colère: Malgré sa mauvaise réputation, les spécialistes affirment que la colère est nécessaire.

Elle peut nous protéger et, de manière surprenante, augmenter considérablement nos performances physiques et cognitives.

Les manifestations et mécanismes de la colère individuelle:

Le témoignage d'Eduardo illustre comment la colère peut être déclenchée par la peur (face à une agression) ou la frustration (avec ses enfants), entraînant des réactions physiques intenses ("tout ton corps qui change si ce mr bouillir... les yeux qui devient pour les rouges tout ça").

Chez un colérique, la tension monte vite et très haut, rendant difficile la prise en compte d'autres perspectives ("il est plus capable de penser... il n'ya plus que son point de vue qui compte").

La colère dans les relations proches:

Les émotions, y compris la colère, débordent davantage avec ceux qu'on aime.

L'anticipation des réactions de l'autre ("je savais que tu allais faire ça") et le système d'attachement (sécurisé ou insécure) influencent la gestion de la colère dans les relations intimes.

Un système d'attachement insécure peut amplifier les émotions négatives ("ça va alimenter active est encore plus mes émotions de tristesse de colère").

La réaction face à la colère d'autrui:

Être confronté à la colère d'un inconnu provoque souvent la "pétrification" ou la "sidération".

La colère fait peur car elle "menace potentiellement l'intégrité d'autrui", mais aussi "l'intégrité des règles de civilité".

On ne sait jamais quelles sont les "limites" de la personne en colère, d'où la peur et le retrait.

Les conséquences de la colère non maîtrisée et violente:

La colère, surtout lorsqu'elle devient violente, fait souffrir non seulement les proches mais aussi les personnes colériques elles-mêmes.

Dans les cas extrêmes, elle peut empêcher de vivre et mener à des comportements autodestructeurs ou hétéro-agressifs.

Le témoignage de Mischa, une femme violente, met en lumière la honte et le caractère dévastateur de sa colère ("c'est très violent c'est c'est comme un tremblement de terre").

La gestion et la canalisation de la colère:

Refouler la colère est comparé à un "cancer" qui peut créer des "pathologies physiques".

Apprendre à réguler sa colère ne signifie pas l'éradiquer, mais trouver d'autres stratégies pour soulager la tension interne.

L'utilité fonctionnelle de la colère: Communication et positionnement:

La colère est un "outil de communication" dès l'enfance et permet, à l'âge adulte, de "mettre des limites" et de ne pas se faire "bouffer par les autres".

Performance physique: Une expérience a démontré que la colère (induite par un sentiment d'injustice) peut augmenter la force physique ("augmentent leur force physique").

La colère agit comme un "moteur", un "booster" qui "permet d'optimiser les performances" physiques car elle est associée à un niveau d'"activation" et d'"éveil" élevé.

Performance cognitive (inconsciente):

Une autre expérience suggère que l'activation inconsciente du concept de colère (par des images subliminales) peut faciliter les performances cognitives et rendre le cœur plus efficace ("économise son énergie et devient plus efficace").

Ce processus doit être inconscient pour fonctionner.

Performance sociale et politique: La colère peut être utilisée consciemment pour "faire passer un message", comme le fait l'entraîneur Bernard Challandes.

La "colère sociale" est perçue comme un "moteur" essentiel pour "changer les choses" et maintenir l'engagement dans les mouvements collectifs (comme la grève du climat).

Les colères collectives ont historiquement fait peur car elles sont "synonyme d'émeutes" et de "révolution", mobilisant les individus en une force collective qui peut ébranler l'ordre établi.

Le traitement de la colère:

L'approche pour gérer la colère dépend de sa nature.

La psychiatrie peut être pertinente si la difficulté à contrôler la colère est constante, survient dans différents contextes et est associée à des comportements délétères, suggérant un trouble psychiatrique sous-jacent (comme une dépression).

Cependant, pour des difficultés comportementales, des approches se concentrant sur l'apprentissage de la gestion des émotions et le renforcement de l'estime de soi sont également efficaces, notamment pour les femmes violentes qui recherchent souvent de l'aide comportementale plutôt que psychiatrique.

Idées ou Faits Importants et Citations Clés

La colère, une émotion nécessaire:

"pourtant les spécialistes l'affirment nous ne pourrions pas vivre sans colère elle nous protège et vous le verrez dans cette émission elle augmente considérablement nos performances physiques et cognitives".

La montée rapide de la colère chez certains:

"[chez Eduardo] la tension monte très vite un très rapidement mais aussi très haut et qu'à ce moment là sur une émotion qui semble être de la colère il est plus capable de penser".

La colère dans les relations proches:

"les psychologues le disent les émotions déborde davantage avec ceux qu'on aime".

L'influence du système d'attachement:

Un système d'attachement insécure "va très vite interpréter les signaux que me donne l'autre comme il est en train de me laisser tomber... ça va alimenter active est encore plus mes émotions de tristesse de colère ou tout autre émotion négative".

La peur face à la colère d'autrui:

"les émotions de colère elles elles font peur parce que menace potentiellement l'intégrité d'autrui... face à la colère d'autrui surtout des personnes inconnues on est plutôt dans un état de pétrification de sidération".

Les conséquences du refoulement:

"je pense que la colère c'est comme un cancer c'est que si on la garde à l'intérieur qu'on laval caen laval caen laval... réellement je pense que oui ça rend malade sa c'est sûr ça j'en suis sûre et certaine".

La fonction de communication de la colère:

"c'est un outil de communication déjà depuis la petite la prime enfance c'est un outil qui me sera utile aussi plus tard à l'âge adulte pour me positionner dans la vie mettre des limites... Si je me mets jamais en colère je me fais bouffer par les autres".

La colère comme moteur de performance physique:

"la colère c'est une sorte de moteur un booster qui va permettre d'optimiser les performances mais certaines performances performance physique".

La colère et la performance cognitive inconsciente:

"cette activation d'une idée de la colère de penser à la colère peut influencer comment je m'applique pendant une tâche... la colère peut faciliter nos actions". "tout ça doit être inconscient".

La colère collective comme moteur du changement social: "il faut une colère sociale pour changer les choses Sinon c'est quoi le moteur". "les colères collective elles ont toujours fait extrêmement peur parce que ça a été synonyme d' émeutes synonyme de l'annoncé de révolution".

Conclusion

L'émission "Dans la tête d'un colérique" démystifie la colère en la présentant non pas uniquement comme une émotion destructive, mais aussi comme une force intrinsèque et potentiellement utile.

Si sa manifestation violente ou constante peut avoir des conséquences dévastatrices pour l'individu et ses proches, la colère, lorsqu'elle est comprise et gérée de manière appropriée, peut servir d'outil de communication, de motivation et même de catalyseur pour le changement social.

Il est crucial d'apprendre à canaliser cette énergie plutôt que de la refouler ou de la laisser déborder de manière incontrôlée.

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Microbiology.<br /> See the peer reviews and the recommendation.

term

a bar!

thats crazy

interesting

!!! important in a way that I cannot describe at the moment

heuristics for personal responsibiity

that's why I resonate with Hermetic principles

hermeticism is cosmic heuirtics

myth metaphor and mysticism

heuristic

dweb.archive

ipfs.indy0.net