Author response:

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

We thank the editor and the reviewers for the detailed and constructive comments. In revising the manuscript we have: (i) clarified what is new relative to prior stress tolerance work, (ii) made explicit that we observe phenotypic convergence without a shared genetic route, (iii) stated upfront that we evolved four independent lines plus two controls, and (iv) corrected figure legends, statistics, and the missing citations. Below we respond point-by-point.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript presents findings on the adaptation mechanisms of Saccharomyces cerevisiae under extreme stress conditions. The authors try to generalize this to adaptation to stress tolerance. A major finding is that S. cerevisiae evolves a quiescence-like state with high trehalose to adapt to freeze-thaw tolerance independent of their genetic background. The manuscript is comprehensive, and each of the conclusions is well supported by careful experiments.

Strengths:

This is excellent interdisciplinary work.

Weaknesses:

I have questions regarding the overall novelty of the proposal, which I would like the authors to explain.

(1) Earlier papers have shown that loss of ribosomal proteins, that slow growth, leads to better stress tolerance in S. cerevisiae. Given this, isn’t it expected that any adaptation that slows down growth would, overall, increase stress tolerance? Even for other systems, it has been shown that slowing down growth (by spore formation in yeast or bacteria/or dauer formation in C. elegans) is an effective strategy to combat stress and hence is a likely route to adaptation. The authors stress this as one of the primary findings. I would like the authors to explain their position, detailing how their findings are unexpected in the context of the literature.

We agree that the link between slower growth and higher stress tolerance has been well studied. What is distinctive here is that repeated, near-lethal freeze–thaw selected not only for a tolerant/quiescent-like state but also for a shorter lag on re-entry. In this regime of freeze–thaw–regrowth, cells that are tolerant but slow to restart would be outcompeted by naive fast growers. Our quiescence-based selection simulations reproduce exactly this constraint. We have added this explanation to the Results to make clear that the novelty is the co-evolution of a tolerant, trehaloserich state together with rapid regrowth under an alternating regime.

(2) Convergent evolution of traits: I find the results unsurprising. When selecting for a trait, if there is a major mode to adapt to that stress, most of the strains would adapt to that mode, independent of the route. According to me, finding out this major route was the objective of many of the previous reports on adaptive evolution. The surprising part in the previous papers (on adaptive evolution of bacteria or yeast) was the resampling of genes that acquired mutations in multiple replicates of an evolution experiments, providing a handle to understand the major genetic route or the molecular mechanism that guides the adaptation (for example in this case it would be - what guides the overaccumulation of trehalose). I fail to understand why the authors find the results surprising, and I would be happy to understand that from the authors. I may have missed something important.

Our surprise was precisely that we did not see the classical pattern of “phenotypic convergence + repeated mutations in the same locus/module.” All independently evolved lines converged on a trehalose-rich, mechanically reinforced, quiescence-like phenotype, but population sequencing across lines did not reveal a single repeatedly hit gene or small shared pathway, even when we increased selection stringency (1–3 freeze–thaw cycles per round). We have now stated in the manuscript that this decoupling (strong phenotypic convergence, non-overlapping genetic routes) is the central inference: selection is acting on a physiologically defined state that multiple genotypes can reach.

(3) Adaptive evolution would work on phenotype, as all of selective evolution is supposed to. So, given that one of the phenotypes well-known in literature to allow free-tolerance is trehalose accumulation, I think it is not surprising that this trait is selected. For me, this is not a case of ”non-genetic” adaptation as the authors point out: it is likely because perturbation of many genes can individually result in the same outcome - up-regulation of trehalose accumulation. Thereby, although the adaptation is genetic, it is not homogeneous across the evolving lines - the end result is. Do the authors check that the trait is actually a non-genetic adaptation, i.e., if they regrow the cells for a few generations without the stress, the cells fall back to being similarly only partially fit to freeze-thaw cycles? Additionally, the inability to identify a network that is conserved in the sequencing does not mean that there is no regulatory pathway. A large number of cryptic pathways may exist to alter cellular metabolic states.

This is a point in continuation of point #2, and I would like to understand what I have missed.

We agree, and we have removed the wording “non-genetic adaptation.” The evolved populations retain high survival even after regrowth for ≥25 generations without freeze–thaw, so the adaptation is clearly genetically maintained. What our data show is that there is no single genetic route to the shared phenotype; different mutations can all drive cells into the same trehalose-rich, quiescencelike, mechanochemically reinforced state. We now describe this as “genetic diversification with phenotypic convergence.”

(4) To propose the convergent nature, it would be important to check for independently evolved lines and most probably more than 2 lines. It is not clear from their results section if they have multiple lines that have evolved independently.

We indeed evolved four independent lines and maintained two independent controls. We have added this information at the start of the Results so that the level of replication is immediately clear.

(5) For the genomic studies, it is not clear if the authors sequenced a pool or a single colony from the evolved strains. This is an important point, since an average sequence will miss out on many mutations and only focus on the mutations inherited from a common ancestral cell. It is also not clear from the section.

We sequenced population samples from the evolved lines. Our specific question was whether independently evolved lines would show the same high-frequency genetic solution, as is often seen in parallel evolution. Pool sequencing may under-sample rare/private variants, but it is appropriate for detecting such shared, high-frequency routes — and we do not find any. We have clarified this rationale in the Methods/Results.

Reviewer #2 (Public review):

Summary:

The authors used experimental evolution, repeatedly subjecting Saccharomyces cerevisiae populations to rapid liquid-nitrogen freeze-thaw cycles while tracking survival, cellular biophysics, metabolite levels, and whole-genome sequence changes. Within 25 cycles, viability rose from ~2 % to ~70 % in all independent lines, demonstrating rapid and highly convergent adaptation despite distinct starting genotypes. Evolved cells accumulated about threefold more intracellular trehalose, adopted a quiescence-like phenotype (smaller, denser, non-budding cells), showed cytoplasmic stiffening and reduced membrane damage, and re-entered growth with shorter lag traits that together protected them from ice-induced injury. Whole-genome sequencing indicated that multiple genetic routes can yield the same mechano-chemical survival strategy. A population model in which trehalose controls quiescence entry, growth rate, lag, and freeze-thaw survival reproduced the empirical dynamics, implicating physiological state transitions rather than specific mutations as the primary adaptive driver. The study therefore concludes that extreme-stress tolerance can evolve quickly through a convergent, trehalose-rich quiescence-like state that reinforces membrane integrity and cytoplasmic structure.

Strengths:

The strengths of the paper are the experimental design, data presentation and interpretation, and that it is well-written.

(1) While the phenotyping is thorough, a few more growth curves would be quite revealing to determine the extent of cross-stress protection. For example, comparing growth rates under YPD vs. YPEG (EtOH/glycerol), and measuring growth at 37ºC or in the presence of 0.8 M KCl.

We thank the referee for the interesting suggestions. However, growth rates alone may be difficult to interpret since WT strains also show different growth rates under these conditions. Therefore, comparing the relative fitness or survival of the evolved strains versus the WT under these stresses would be more informative. In the present study we limited growth/survival measurements to what was needed to parameterize the adaptation model in YPD under the freeze–thaw regime. We have now added a statement in the Discussion that, given the shared trehalose/mechanical mechanism, such cross-stress assays are an expected and straightforward follow-up.

(2) Is GEMS integrated prior to evolution? Are the evolved cells transformable?

Yes. GEMs were integrated prior to evolution, because the non-integrated evolved population showed low transformation efficiency, likely due to altered cell-wall properties.

(3) From the table, it looks like strains either have mutations in Ras1/2 or Vac8. Given the known requirements of Ras/PKA signaling for the G1/S checkpoint (to make sure there are enough nutrients for S phase), this seems like a pathway worth mentioning and referencing. Regarding Vac8, its emerging roles in NVJ and autophagy suggest another nutrient checkpoint, perhaps through TORC1. The common theme is rewired metabolism, which is probably influencing the carbon shuttling to trehalose synthesis.

We appreciate the reviewer’s suggestion to consider pathways like Ras/PKA (linked to Ras1/2) and autophagy/TORC1 (linked to Vac8) as potential upstream modulators. While these pathways are involved in nutrient sensing and metabolic regulation, we choose not to emphasize them specifically. This is because (i) some evolved lines lack Ras1/2 or Vac8 variants, and (ii) none of the variants lies directly in trehalose synthesis/degradation pathways. Furthermore, direct links to trehalose accumulation are not well established for these specific variants in this context, and pathways like Ras are global regulators with broad effects. Together with the strongly convergent phenotype, this supports our main inference that multiple genetic/metabolic routes can feed into the same trehalose-rich, mechanochemically reinforced, quiescence-like state. We have added a note in the discussion regarding metabolic rewiring and trehalose.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Generally, the results sections should have more details. The figures should be corrected, and the legends should be checked for correctness. The manuscript seems to have been assembled in haste?

We have expanded the relevant Results subsections with one-sentence motivations (why each measurement was performed) and we have corrected the figure legends for ordering and consistency.

Figure 3: It will be good to have the correct p-values on the figure itself. P-values are typically less than 1, unless there is some special method (here the values presented are , etc). Please explain how the P-values were obtained in the figure legend itself.

Figure 3 now shows the actual p-values. The legend specifies the details and the sample sizes used.

Figure 5: It is not clear what the error bars show in 5B, E (different evolved population/ clones/ cells?). All the figure legends are mixed up, please correct them. It is difficult to follow the paper.

Figure 5 legends now state clearly what the error bars represent (biological replicates) and which panels are from single-cell measurements. We have checked the panel lettering and legend order for consistency with the flow of the main text.

Reviewer #3 (Recommendations for the authors):

Overall, the paper is outstanding, well-written, and insightful.

A point to address is that there are missing citations on lines 60, 91.

We have added the missing citations at both locations. We apologize for the omission, which was due to a compilation error. This error has been fixed, and the bibliography has been corrected (now containing 74 references).

Reviewer #2 (Public review):

The substantially revised paper has increased in clarity and is much more accessibe and straightforward than the first version. The analyses are now clearer and support the conclusions better. There are however some remaining methodological weakness, which in my mind still renders the evidence to not be entirely convincing.

(1) The temporal autocorrelation concern is not fully convincingly addressed. The temporal autocorrelation curves supplied in the supplements are really helpful, but linearly regressing out the temporal distance from the neural distance clearly does not work, as one can see from the right panel of supplementary Figure 1. If the method had worked correctly the line should have been flat. The analysis however shows that decision trials with a lag > 2 are basically independent - so a simple way to address this is to restrict the RSA analysis to trials with a decision lag of > 2. This analysis would strengthen the paper a lot.

(2) In the final analysis, the authors use all the trials to make the claim that the hippocampus represents the characters in a shared social space. However, as within-character distances are still included in the analysis, this result could still be driven by the effects of within-character representations that are not shared across characters. A simple way of addressing this concern would be to only include between-character distances in this analysis, making it truly complementary to the previous within-character analysis. It would also be very interesting to compare the the within- and between-character analyses in the hippocampus directly.

(3) Overall, the correction for multiple comparisons in the fMRI and the resulting corrected p-values are not sufficiently explained and documented in the paper. What was exactly permuted in the tests? Was correction applied in a voxel-wise or cluster-wise fashion? If cluster-wise, the cluster-wise p-values need to be reported.

Author response:

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

Public reviews:

Reviewer #1 (Public review):

Summary:

Schafer et al. tested whether the hippocampus tracks social interactions as sequences of neural states within an abstract social space defined by dimensions of affiliation and power, using a task in which participants engaged in narrative-based social interactions. The findings of this study revealed that individual social relationships are represented by unique sequences of hippocampal activity patterns. These neural trajectories corresponded to the history of trial-to-trial affiliation and power dynamics between participants and each character, suggesting an extended role of the hippocampus in encoding sequences of events beyond spatial relationships.

The current version has limited information on details in decoding and clustering analyses which can be improved in the future revision.

Strengths:

(1) Robust Analysis: The research combined representational similarity analysis with manifold analyses, enhancing the robustness of the findings and the interpretation of the hippocampus's role in social cognition.

(2) Replicability: The study included two independent samples, which strengthens the generalizability and reliability of the results.

Weaknesses:

I appreciate the authors for utilizing contemporary machine-learning techniques to analyze neuroimaging data and examine the intricacies of human cognition. However, the manuscript would benefit from a more detailed explanation of the rationale behind the selection of each method and a thorough description of the validation procedures. Such clarifications are essential to understand the true impact of the research. Moreover, refining these areas will broaden the manuscript's accessibility to a diverse audience.

We thank the reviewer for these comments and have addressed them in various ways.

First, we removed the spline-based decoding and spectral clustering analyses. As we detail in our response to the recommendations, these approaches were complex and raised legitimate interpretational concerns, making it unclear how they supported our core claims. The revised manuscript now focuses on a set of representational similarity analyses to show representations consistent with social dimension similarity (affiliation vs. power decision trials) and social location similarity (trajectory/map-like coding based on participant choices).

Second, we expanded the Methods and Results to more clearly explain the analyses, the questions they address, and associated controls and robustness tests. The dimension similarity analysis tests whether hippocampal patterns differentiate affiliation and power decisions in a way consistent with an abstract dimension representation. The location similarity RSAs test whether within-character neural pattern distances scale with Euclidean distance in social space (relationship-specific trajectories), and whether pattern distances across all characters scale with location distances when distances are globally standardized, consistent with a shared map-like coordinate system.

Third, we emphasize new controls. For the dimension similarity RSA, we test for potential confounds such as word count, text sentiment, and reaction time differences between affiliation and power trials. For the location similarity RSA, we control for temporal distance between trials and show (in the Supplement) that the reported effects cannot be explained by temporal autocorrelation in the fMRI data or by the relationship between temporal distance and behavioral location distance.

We believe that these changes address the reviewer’s request for clearer rationale and validation.

Reviewer #2 (Public review):

Summary:

Using an innovative task design and analysis approach, the authors set out to show that the activity patterns in the hippocampus related to the development of social relationships with multiple partners in a virtual game. While I found the paper highly interesting (and would be thrilled if the claims made in the paper turned out to be true), I found many of the analyses presented either unconvincing or slightly unconnected to the claims that they were supposed to support. I very much hope the authors can alleviate these concerns in a revision of the paper.

Strengths & Weaknesses:

(1) The innovative task design and analyses, and the two independent samples of participants are clear strengths of the paper.

We thank the reviewer for this comment.

(2) The RSA analysis is not what I expected after I read the abstract and tile of the result section "The hippocampus represents abstract dimensions of affiliation and power". To me, the title suggests that the hippocampus has voxel patterns, which could be read out by a downstream area to infer the affiliation and power value, independent of the exact identity of the character in the current trial. The presented RSA analysis however presents something entirely different - namely that the affiliation trials and power trials elicit different activity patterns in the area indicated in Figure 3. What is the meaning of this analysis? It is not clear to me what is being "decoded" here and alternative explanations have not been considered. How do affiliation and power trials differ in terms of the length of sentences, complexity of the statements, and reaction time? Can the subsequent decision be decoded from these areas? I hope in the revision the authors can test these ideas - and also explain how the current RSA analysis relates to a representation of the "dimensions of affiliation and power".

We agree that this analysis needed to be better justified and explained. We have revised the text to clarify that by “represents the interaction decision trials along abstract social dimensions” we mean that hippocampal multivoxel patterns differentiate affiliation and power decisions in a way consistent with the conceptual framework of underlying latent dimensions. The analysis tests one simple prediction of this view – that on average these trial types are separable in the neural patterns. We have added details to the Methods, showing how the affiliation and power trials do not differ in word count or in sentiment, but do differ in their semantics, as assessed by a Large Language Model, as we expect from our task assumptions. Thanks to the reviewer’s comment, we also tested for and found a reaction time difference between affiliation and power trials, that we now control for.

(3) Overall, I found that the paper was missing some more fundamental and simpler RSA analyses that would provide a necessary backdrop for the more complicated analyses that followed. Can you decode character identity from the regions in question? If you trained a simple decoder for power and affiliation values (using the LLE, but without consideration of the sequential position as used in the spline analysis), could you predict left-out trials? Are affiliation and power represented in a way that is consistent across participants - i.e. could you train a model that predicts affiliation and power from N-1 subjects and then predict the Nth subject? Even if the answer to these questions is "no", I believe that they are important to report for the reader to get a full understanding of the nature of the neural representations in these areas. If the claim is that the hippocampus represents an "abstract" relationship space, then I think it is important to show that these representations hold across relationships. Otherwise, the claim needs to be adjusted to say that it is a representation of a relationship-specific trajectory, but not an abstract social space.

We appreciate this comment and agree on the value of clear, conceptually simple analyses. To address this concern, we have simplified our main analysis significantly by removing the spline-based analysis and substituting it with a multiple regression representational similarity analysis approach. We test whether within-character neural pattern distances scale with distance in social space (relationship-specific trajectories), and whether pattern distances across all characters scale with location distances when distances are globally standardized. We find evidence for both, consistent with a shared map-like coordinate system.

We agree that decoding character identity and an across-participant decoding approach could be informative. However, our current task is not well designed for such analyses and as such would complicate the paper. Although we agree that these questions are interesting, they would test questions that are outside the scope of this paper. 

(4) To determine that the location of a specific character can be decoded from the hippocampal activity patterns, the authors use a sequential analysis in a lowdimensional space (using local linear embedding). In essence, each trial is decoded by finding the pair of two temporally sequential trials that is closest to this pattern, and then interpolating the power/affiliation values linearly between these two points. The obvious problem with this analysis is that fMRI pattern will have temporal autocorrelation and the power and affiliation values have temporal autocorrelation. Successful decoding could just reflect this smoothness in both time series. The authors present a series of control analyses, but I found most of them to not be incisive or convincing and I believe that they (and their explanation of their rationale) need to be improved. For example, the circular shifting of the patterns preserves some of the autocorrelation of the time series - but not entirely. In the shifted patterns, the first and last items are considered to be neighboring and used in the evaluation, which alone could explain the poor performance. The simplest way that I can see is to also connect the first and last item in a circular fashion, even when evaluating the veridical ordering. The only really convincing control condition I found was the generation of new sequences for every character by shuffling the sequence of choices and re-creating new artificial trajectories with the same start and endpoint. This analysis performs much better than chance (circular shuffling), suggesting to me that a lot of the observed decoding accuracy is indeed simply caused by the temporal smoothness of both time series.

We thank the reviewer for emphasizing this important concern; we agree that we did not sufficiently address this in the initial submission. This concern is one main reason we removed the spline-based analysis and now use regression-based representational similarity analyses in its place. In the revision, we report autocorrelation-related analyses in the supplement, and via controls and additional analysis show that temporal distance (or its square) cannot explain the location-like effects. This substantially improves our ability to interpret the findings.

(5) Overall, I found the analysis of the brain-behavior correlation presented in Figure 5 unconvincing. First, the correlation is mostly driven by one individual with a large network size and a 6.5 cluster. I suspect that the exclusion of this individual would lead to the correlation losing significance. Secondly, the neural measure used for this analysis (determining the number of optimal clusters that maximize the overlap between neural clustering and behavioral clustering) is new, non-validated, and disconnected from all the analyses that had been reported previously. The authors need to forgive me for saying so, but at this point of the paper, would it not be much more obvious to use the decoding accuracy for power and affiliation from the main model used in the paper thus far? Does this correlate? Another obvious candidate would be the decoding accuracy for character identity or the size of the region that encodes affiliation and power. Given the plethora of candidate neural measures, I would appreciate if the authors reported the other neural measures that were tried (and that did not correlate). One way to address this would have been to select the method on the initial sample and then test it on the validation sample - unfortunately, the measure was not pre-registered before the validation sample was collected. It seems that the correlation was only found and reported on the validation sample?

We agree that this analysis was too complicated and under constrained, and thus not convincing. We think that removing this cluster-based analysis is the most conservative response to the reviewer’s concerns and have removed it from the revised paper.

Recommendations to the authors:

Reviewer #1 (Recommendations for the authors):

The manuscript's description of the shuffling analysis performed during decoding is currently ambiguous, particularly concerning the control variables. This ambiguity is present only in the Figure 4 legends and requires a more detailed explanation within the methods section. It is essential to clarify whether the permutation process was conducted within each character's data set or across multiple characters' data sets. If permutations were confined to within-character data, the conclusion would be that the hippocampus encodes context-specific information rather than providing a twodimensional common space.

We thank the reviewer for this comment. We have now removed the spline analysis due to these and other problems and have replaced it with representational similarity analyses that are both more rigorous and easier to interpret. We think these analyses allow us to make the claim that the characters are represented in a common space. 

In the methods, we explain the analyses (page 23-24, lines 475-500):

“We also expected the hippocampus to represent the different characters’ changing social locations, which are implicit in the participant’s choices. We used multiple regression searchlight RSA to test whether hippocampal pattern dissimilarity increases with social location distance, based on participant-specific trial-wise beta images where boxcar regressors spanned each trial’s reaction time.”

“We ran two complementary regression analyses to address two related questions. First, we asked whether the hippocampus represents how a specific relationship changes over time. For this analysis, for each participant and each searchlight, we computed character-specific (i.e., only for same character trial pairs) correlation distances between trial-wise beta patterns and Euclidean distances between the social location behavioral coordinates. Distances were zscored within character trial pairs to isolate character-specific changes. The second analysis asked whether the there is a common map-like representation, where all trials, regardless of relationship, are represented in a shared coordinate system. Here, we included all trial pairs and z-scored the distances globally. For both regression analyses, we included control distances to control for possible confounds. To account for generic time-related changes, we controlled for absolute scan-time difference, as this correlated with location distance across participants (see Temporal autocorrelation of hippocampal beta patterns in the supplement). Although the square of this temporal distance did not explain any additional variance in behavioral distances, we ran a robustness analysis including both temporal distance and its square and saw qualitatively the same clusters with similar effect sizes. As such, we report the main analysis only. We included binary dimension difference (0 = trial pairs of different dimension, 1 = trials pairs of the same dimension), to ensure effects could not be explained by dimension-related effects. In the group-level model, we controlled for sample and the average reaction time between affiliation and power decisions.”

In the results, we describe the results and our interpretation (pages 11-12, lines 185208):

“We have shown that the left hippocampus represents the affiliation and power trials differently, consistent with an abstract dimensional representation. Does it also represent the changing social coordinates of each character? To test this, we multiple-regression RSA searchlight to test whether left hippocampus patterns represent the characters’ changing social locations across interactions (see Figure 3). We restricted the distances to those from trial pairs from the same character and standardized the distances within character (see Figure 3BD). We controlled for temporal distance to ensure the effect was not explainable by the time between trials, and for whether the trials shared the same underlying dimension (affiliation or power; see Location similarity searchlight analyses for more details). At the group level, we controlled for sample and the average reaction time difference between affiliation and power trials. Using the same testing logic as the dimensionality similarity analysis, we first tested our hypothesis in the bilateral hippocampus and found widespread effects in both the left (peak voxel MNI x/y/z = -35/-22/-15, cluster extent = 1470 voxels) and right (peak voxel MNI x/y/z = 37/-19/-14, cluster extent = 1953 voxels) hemispheres. The whole-brain searchlight analysis revealed additional clusters in the left putamen (-27/-3/14, cluster extent = 131 voxels) and left posterior cingulate cortex (-10/-28/41, cluster extent = 304 voxels).”

“We then asked a second, complementary question: does the hippocampus represent all interactions, across characters, within a shared map? To test for this map-like structure, we repeated the analysis but now included all trial pairs, z-scoring distances globally rather than within character (Figure 3E-F). The remainder of the procedure followed the same logic as the preceding analysis. The hippocampus analysis revealed an extensive right hippocampal cluster (27/27/-14, cluster extent = 1667 voxels). The whole-brain analysis did not show any significant clusters.”

We also describe the results in the discussion (page 12, lines 220-226): 

“Then, we show that the hippocampus tracks the changing social locations (affiliation and power coordinates), above and beyond the effects of dimension or time; the hippocampus seemed to reflect both the changing within-character locations, tracking their locations over time, and locations across characters, as if in a shared map. Thus, these results suggest that the hippocampus does not just encode static character-related representations but rather tracks relationship changes in terms of underlying affiliation and power.”

The manuscript's description of the decoding analysis is unclear regarding the variability of the decoded positions. The authors appear to decode the position of a character along a spline, which raises the question of whether this position correlates with time, since characters are more likely to be located further from the center in later trials. There is a concern that the decoded position may not solely reflect the hippocampal encoding of spatial location, but could also be influenced by an inherent temporal association. Given that a character's position at time t is likely to be similar to its positions at t−1 and t+1, it is crucial that the authors clearly articulate their approach to separating spatial representation from temporal autocorrelation. While this issue may have been addressed in the construction of the test set, the manuscript does not seem to adequately explain how such biases were mitigated in the training set.

We agree that temporal confounding needs to be better accounted for, as our claims depend on space-like signals being separable from time-like ones. We address this in several ways in the revised manuscript.

First, we emphasize that this is a narrative-based task, where temporal structure is relevant. As such, our analyses aim to demonstrate that effects go beyond simple temporal confounds, like trial order or time elapsed.

Despite the temporal structure to the task, the decisions for the same character are spaced in time, and interleaved with other characters’ decisions, reducing the chance that a simple temporal confound could explain trajectory-related effects. We now describe the task better in the revised methods (page 16, lines 314-318):

“All six characters’ decision trials are interleaved with one another and with narrative slides. On average, after a decision trial for a given character, participants view ~11 narrative slides and complete ~3 decisions for other characters before returning to that same character, such that each character’s choices are separated by an average of ~20 seconds (range 12 seconds to 10 min).”

To address temporal autocorrelation in the fMRI time series, we used SPM’s FAST algorithm. Briefly, FAST models temporal autocorrelation as a weighted combination of candidate correlation functions, using the best estimate to remove autocorrelated signal.

We also now report the temporal autocorrelation profile of the hippocampal beta series in the supplement, including (pages 29-31, lines 593-656):

“The Social Navigation Task is a narrative-based task, where the relationships with characters evolve over time; trial pairs that are close in time may have more similar fMRI patterns for reasons unrelated to social mapping (e.g., slow drift). It is important to account for the role of time in our analyses, to ensure effects go beyond simple temporal confounds, like the time between decision trials. To aid in this, we quantified how fMRI signals change over time using a pattern autocorrelation function across decision trial lags. We defined the left and right hippocampus and the left and right intracalcarine cortex using the HarvardOxford atlas and thresholded them at 50% probability. We chose intracalcarine corex as an early visual control region that largely corresponds to primary visual cortex (V1), as it is likely to be driven by the visually presented narrative. We used the same trial-wise beta images as in the location similarity RSA (boxcar regressors spanning each decision trial’s reaction time). For each participant and region-of-interest (ROI), we extracted the decision trial-by-voxel beta matrix and quantified three kinds of temporal dependence: beta autocorrelation, multivoxel pattern correlation and multivoxel pattern correlation after regressing out temporal distance.”

“To estimate the temporal autocorrelation of the trial-wise beta values, we treated each voxel’s beta values as a time series across trials and measured how much a voxel’s response on one trial correlated (Pearson) with its response on previous trials. We averaged these voxel wise autocorrelations within each ROI. At one trial apart (lag 1), both the hippocampus and V1 showed small positive autocorrelations, indicating modest trial-to-trial carryover in response amplitude (see Supplemental figure 1) that by three trials apart was approximately 0.”

“Because our representational similarity analyses depend on trial-by-trial pattern similarity, we also estimated how multivoxel patterns were autocorrelated over time. For each lag, we computed the Pearson correlation between each trial’s voxelwise pattern and the pattern from the trial that many trials earlier, then averaged those correlations to obtain a single autocorrelation value for that lag. At one trial apart, both regions showed positive autocorrelation, with V1 having greater autocorrelation than the hippocampus; pattern correlations between trials 3 or 4 trials apart reduced across participants, settling into low but positive values. Then, for each participant and ROI, we regressed out the effect of absolute trial onset differences from all pairwise pattern correlations, to mirror the effects of controlling for these temporal distances in regressions. After removing this temporal distance component, the short lag pattern autocorrelation dropped substantially in both regions. The similarity in autocorrelation profiles between the two regions suggests that significant similarity effects in the hippocampus are unlikely to be driven by generic temporal autocorrelation.”

“Relationship between behavioral location distance and temporal distance “

“We also quantified how temporal distances between trials relates to their behavioral location distances, participant by participant. Our dimension similarity analysis controls for temporal distance between trials by design (see Social dimension similarity searchlight analysis), but our location similarity analysis does not. To decide on covariates to include in the analysis, we tested whether temporal distances can explain behavioral location distances. For each participant, we computed the correlations between trial pairs’ Euclidean distances in social locations and their linear temporal distances (“linear”) and the temporal distances squared (“quadratic”), to test for nonlinear effects. We then summarized the correlations using one-sample t-tests. The linear relationship was statistically significant (t₄₉ = 12.24, p < 0.001), whereas the quadratic relationship was not (t₄₉ = -0.55, p = 0.586). Similarly, in participant specific regressions with both linear and quadratic temporal distances, the linear effect was significant (t₄₉ = 5.69, p < 0.001) whereas the quadratic effect was not (t₄₉ = 0.20, p = 0.84). Based on this, we included linear temporal distances as a covariate in our location similarity analyses (see Location similarity searchlight analyses), and verified that adding a quadratic temporal distance covariate does not alter the results. Thus, the reported location-related pattern similarity effects go beyond what can be explained by temporal distance alone.”

How the free parameter of spectral clustering was determined, if there is any?

The interpretation of the number of hippocampal activity clusters is ambiguous. It is suggested that this number could fluctuate due to unique activity patterns or the fit to behaviorally defined trajectories. A lower number of clusters might indicate either a noisier or less distinct representation, raising the question of the necessity and interpretability of such a complex analysis. This concern is compounded by the potential sensitivity of the clustering to the variance in Euclidean distances of each trial's position relative to the center. If a character's position is consistently near the center, this could artificially reduce the perceived number of clusters. Furthermore, the manuscript should address whether there is any correlation between the number of clusters and behavioral performance. Specifically, what are the implications if participants are able to perform the task adequately with a smaller number of distinct hippocampal representation states?

The rationale for conducting both cluster analysis and position decoding as separate analyses remains unclear. While cluster analysis can corroborate the findings of position decoding, it is not apparent why the authors chose to include trials across characters for cluster analysis but not for decoding analysis. An explanation of the reasoning behind this methodological divergence would help in understanding the distinct contributions of each analysis to the study's findings.

The paper by Cohen et al. (1997), which provides the questionnaire for measuring the social network index, is not cited in the references. Upon reviewing the questionnaire that the author may have used, it appears that the term "social network size" does not refer to the actual size but to a score or index derived from the questionnaire responses. It may be more appropriate to replace the term "size" with a different term to more accurately reflect this distinction.

Thank you for seeking these clarifications. Given the complexity of this analysis, we have decided to drop it to focus instead on our dimension and location representational similarity analysis results.

Reviewer #2 (Recommendations for the authors):

How did the participants' decisions on previous trials influence the future trials that the subjects saw? If the different participants were faced with different decision trials, then how did you compare their decision? If two participants made the same decisions, would they have seen exactly the same sequence of trials (see point X on how the trial sequence was randomized).

All participants experience the same narrative, with the same decisions (i.e., the same available options); their choices (i.e., the options they select) are what implicitly shape each character’s affiliation and power locations, and thus each character’s trajectory. In other words, the narrative is fixed; what changes is the social coordinates assigned to each trial’s outcome depending on the participant’s choice of how to interact from the two narrative options. This means that we can meaningfully compare participants' neural patterns, given that every participant received the same text and images throughout.

We have now added details on the narrative structure, replacing more ambiguous statements with a clearer description (page 16, lines 309-318):

“The sequence of trials, including both narrative and decision trials, were fixed across participants; all that differs are the choices that the participants make. Narrative trials varied in duration, depending on the content (range 2-10 seconds), but were identical across participants. Decision trials always lasted 12 seconds, with two options presented until the participant made a choice, after which a blank screen was presented for the remainder of the duration. All six characters’ decision trials are interleaved with one another, and with the narrative slides. On average, after a decision trial for a given character, participants view ~11 narrative slides and complete ~3 decisions for other characters before returning to another decision with the same character, such that each character’s choices are separated by an average of ~20 seconds (ranging from 12 seconds to 10 min).”

Figure 2B: I assume that "count" is "count of participants"? It would be good to indicate this on the axis/caption.

Thank you for noting this. We have now removed this figure to improve the clarity of our figures. 

We have shown that the hippocampus represents the interaction decision trials along abstract social dimensions, but does it track each relationship's unique sequence of abstract social coordinates?". Please clarify what you mean by "represents the interaction decision trials”.

By “represents the interaction decision trials along abstract social dimensions”, we mean that when the participant makes a choice during the social interactions the hippocampal patterns represent the current social dimension of the choice (affiliation vs power). In other words, the hippocampal BOLD patterns differentiate affiliation and power decisions, consistent with our hypothesis of abstract social dimension representation in the hippocampus. We have clarified this (page 11, lines 185-187):

“We have shown that the left hippocampus represents the affiliation and power trials differently, consistent with an abstract dimensional representation.”

Page 8: "Hippocampal sequences are ordered like trajectories": It is not entirely clear to me what is meant by the split midpoint. Is this the midpoint of the piece-wise linear interpolation between two points, or simply the mean of all piecewise splines from one character? If the latter, is the null model the same as simply predicting the mean affiliation and power value for this character? If yes, please clarify and simplify this for the reader.

Page 8: "Hippocampal sequences track relationship-specific paths". First, I was misled by the "relationship-specific". I first understood this to mean that you wanted to test whether two relationships (i.e. the identity of the partner) had different representations in Hippocampus, even if the power/affiliation trajectories are the same. I suggest changing the title of this section.

The analysis in this section also breaks any temporal autocorrelation of measured patterns - so I am not sure if this is a strong analysis that should be interpreted at all. This analysis seems to not address the claim and conclusion that is drawn from it. I assume that the random trajectories have different choices and different affiliation/power values than the true trajectories. So the fact that the true trajectories can be better decoded simply shows that either choices or affiliation and power (or both) are represented in the neural code - but not necessarily anything beyond this.

Page 9: "Neural trajectories reflect social locations, not just choices". The motivation of this analysis is not clear to me. As I understand this analysis, both social location and choices are changed from the real trajectories. How can it then show that it reflects social locations, not just the choices?

Figure 4 caption: "on the -based approximation" Is there a missing "point"-[based] here?

We agree with the reviewer that this analysis is hard to interpret and does not adequately address concerns regarding temporal autocorrelation, and as such we have removed it from the manuscript. We describe the new results that include controlling for temporal distance between trials (pages 11-12, lines 185-208):

“We have shown that the left hippocampus represents the affiliation and power trials differently, consistent with an abstract dimensional representation. Does it also represent the changing social coordinates of each character? To test this, we multiple-regression RSA searchlight to test whether left hippocampus patterns represent the characters’ changing social locations across interactions (see Figure 3). We restricted the distances to those from trial pairs from the same character and standardized the distances within character (see Figure 3BD). We controlled for temporal distance to ensure the effect was not explainable by the time between trials, and for whether the trials shared the same underlying dimension (affiliation or power; see Location similarity searchlight analyses for more details). At the group level, we controlled for sample and the average reaction time difference between affiliation and power trials. Using the same testing logic as the dimensionality similarity analysis, we first tested our hypothesis in the bilateral hippocampus and found widespread effects in both the left (peak voxel MNI x/y/z = -35/-22/-15, cluster extent = 1470 voxels) and right (peak voxel MNI x/y/z = 37/-19/-14, cluster extent = 1953 voxels) hemispheres. The whole-brain searchlight analysis revealed additional clusters in the left putamen (-27/-3/14, cluster extent = 131 voxels) and left posterior cingulate cortex (-10/-28/41, cluster extent = 304 voxels).”

“We then asked a second, complementary question: does the hippocampus represent all interactions, across characters, within a shared map? To test for this map-like structure, we repeated the analysis but now included all trial pairs, z-scoring distances globally rather than within character (Figure 3E-F). The remainder of the procedure followed the same logic as the preceding analysis. The hippocampus analysis revealed an extensive right hippocampal cluster (27/27/-14, cluster extent = 1667 voxels). The whole-brain analysis did not show any significant clusters.”

We emphasize that the results are robust to the inclusion of temporal distance squared, in the methods (pages 23-24, lines 493-496):

“Although the square of this temporal distance did not explain any additional variance in behavioral distances, we ran a robustness analysis including both temporal distance and its square and saw qualitatively the same clusters with similar effect sizes.”

Page 8: last paragraph: The text sounds like you have already shown that you can decode character identity from the patterns - but I do not believe you have it this point. I would consider this would be an interesting addition to the paper, though.

This section has been removed, and we have been careful to not imply this in the current version of the manuscript. While we agree a character identity decoding would enrich our argument, we do not believe our task is well-suited to capture a character identity effect. Each character only has 12 decision trials, and these trials are partially clustered in time - this is one problem of temporal autocorrelation that we thank the reviewers for pushing us to consider in more detail. Dimension and location patterns, on the other hand, are more natural to analyze in our task, especially in representational similarity analyses that test whether the relevant differences scale with neural distances.

Page 14ff: Why is "Analysis section" not part of "Materials and Methods"? I believe adding the analysis after a careful description of the methods would improve the clarity of this section.

We agree with the reviewer and have now consolidated these two sections.

Two or three examples of Affiliation and Power decision trials should be provided, so the reader can form a more thorough understanding of how these dimensions were operationalized. For the RSA analysis, it is important to consider other differences between these two types of trials.

We agree that adding examples will clarify the operationalization of these dimensions. We now include example affiliation and power trials in a table (page 17-18).

We thank the reviewer for noting the need to rule out alternative hypotheses; we have added several such tests. Affiliation and power trials were not different in word count (page 17, lines 329-332):

“To ensure that any observed neural or behavioral differences were not confounded by trivial features of the text, we tested for differences between the affiliation and power trials (where the two options are concatenated). There were no differences in word count (affiliation average = 26.6, power average = 25.6; t-test p = 0.56).”

They were also not different in their sentiment, as assessed by a Large Language Model (LLM) analysis (page 17, lines 332-335): 

“The text’s sentiment also did not differ between these trial types (t-test p = 0.72), as quantified by comparing sentiment compound scores (from most negative, −1, to most positive, +1), using a Large Language Model (LLM) specialized for sentiment analysis [26]. “

The affiliation and power trials were different in terms of semantic content, consistent with our assumptions (page 17, lines 337-347):

“Our framework assumes that affiliation and power trials differ in their semantic content–that is, in the conceptual meaning of the text, beyond word count or sentiment. To test this assumption, we used an LLM-based semantic embedding analysis. Each decision trial was embedded into a semantic vector. We then measured the cosine similarity between pairs of trials and calculated the difference between average within-dimension similarity (affiliation-affiliation and power-power comparisons) and average between-dimension similarity (affiliationpower comparisons) and assessed its statistical significance with permutation testing (1,000 shuffles of trial labels). As expected, decision trials of the same dimension were more similar to each other than trials of different dimension, across multiple LLMs (OpenAI’s text-embedding-3-small [27]: similarity difference = 0.041, p < 0.001; all-MiniLM-L12-v2 [28]: similarity difference = 0.032, p < 0.001).”

The affiliation and power trials were different in average reaction time. To control for this difference in the dimension RSA analysis, we added each participant’s absolute value reaction time difference between the trial types as a covariate. The results were nearly identical to what they were before. We updated the text to reflect this new control (page 23, lines 471-474):

“However, there was a significant difference in the average reaction time between affiliation and power decisions across participants (t₄₉ = 6.92, p < 0.001; affiliation mean = 4.92 seconds (s), power mean = 4.51 s), so we controlled for this in the group-level analysis.”

The exact implementation and timing of the behavioral tasks should be described better. How many narrative trials were intermixed with the decision trials? Which characters were they assigned to? How was the sequence of trials determined? Was it fixed across participants, or randomized?

We agree that additional details are helpful. In the Methods, we now describe this with more detail (page 16, lines 301-318):

“There are two types of trials: “narrative” trials where background information is provided or characters talk or take actions (a total of 154 trials), and “decision” trials where the participant makes decisions in one-on-one interactions with a character that can change the relationship with that character (a total of 63 trials). On each decision, participants used a button response box to select between the two options. The options (1 or 2, assigned to the index and middle fingers) choice directions (+/-1 arbitrary unit on the current dimension) were counterbalanced.”

“The sequence of trials, including both narrative and decision trials, were fixed across participants; all that differs are the choices that the participants make. Narrative trials varied in duration, depending on the content (range 2-10 seconds), but were identical across participants. Decision trials always lasted 12 seconds, with two options presented until the participant made a choice, after which a blank screen was presented for the remainder of the duration. All six characters’ decision trials are interleaved with one another, and with the narrative slides. On average, after a decision trial for a given character, participants view ~11 narrative slides and complete ~3 decisions for other characters before returning to another decision with the same character, such that each character’s choices are separated by an average of ~20 seconds (ranging from 12 seconds to 10 min).”

What is the exact timing of trials during fMRI acquisition - i.e. how long were the trials, what was the ITI, were there long phases of rest to determine the resting baseline? These are all important factors that will determine the covariance between regressors and should be reported carefully. Ideally, I would like to see the trial-by-trial temporal auto-correlation structure across beta-weights to be reported.

We thank the reviewer for asking for this clarification. We have added the following text to clarify the trial timing (page 16, lines 314-318):

“All six characters’ decision trials are interleaved with one another and with narrative slides. On average, after a decision trial for a given character, participants view ~11 narrative slides and complete ~3 decisions for other characters before returning to that same character, such that each character’s choices are separated by an average of ~20 seconds (range 12 seconds to 10 min).”

We now describe the temporal autocorrelation patterns in the supplement, including how we decided on how to control for temporal distance in representational similarity analyses (pages 29-31, lines 593-656):

“The Social Navigation Task is a narrative-based task, where the relationships with characters evolve over time; trial pairs that are close in time may have more similar fMRI patterns for reasons unrelated to social mapping (e.g., slow drift). It is important to account for the role of time in our analyses, to ensure effects go beyond simple temporal confounds, like the time between decision trials. To aid in this, we quantified how fMRI signals change over time using a pattern autocorrelation function across decision trial lags. We defined the left and right hippocampus and the left and right intracalcarine cortex using the HarvardOxford atlas and thresholded them at 50% probability. We chose intracalcarine corex as an early visual control region that largely corresponds to primary visual cortex (V1), as it is likely to be driven by the visually presented narrative. We used the same trial-wise beta images as in the location similarity RSA (boxcar regressors spanning each decision trial’s reaction time). For each participant and region-of-interest (ROI), we extracted the decision trial-by-voxel beta matrix and quantified three kinds of temporal dependence: beta autocorrelation, multivoxel pattern correlation and multivoxel pattern correlation after regressing out temporal distance.”

“To estimate the temporal autocorrelation of the trial-wise beta values, we treated each voxel’s beta values as a time series across trials and measured how much a voxel’s response on one trial correlated (Pearson) with its response on previous trials. We averaged these voxel wise autocorrelations within each ROI. At one trial apart (lag 1), both the hippocampus and V1 showed small positive autocorrelations, indicating modest trial-to-trial carryover in response amplitude (see Supplemental figure 1) that by three trials apart was approximately 0.”

“Because our representational similarity analyses depend on trial-by-trial pattern similarity, we also estimated how multivoxel patterns were autocorrelated over time. For each lag, we computed the Pearson correlation between each trial’s voxelwise pattern and the pattern from the trial that many trials earlier, then averaged those correlations to obtain a single autocorrelation value for that lag. At one trial apart, both regions showed positive autocorrelation, with V1 having greater autocorrelation than the hippocampus; pattern correlations between trials 3 or 4 trials apart reduced across participants, settling into low but positive values. Then, for each participant and ROI, we regressed out the effect of absolute trial onset differences from all pairwise pattern correlations, to mirror the effects of controlling for these temporal distances in regressions. After removing this temporal distance component, the short lag pattern autocorrelation dropped substantially in both regions. The similarity in autocorrelation profiles between the two regions suggests that significant similarity effects in the hippocampus are unlikely to be driven by generic temporal autocorrelation.”

“Relationship between behavioral location distance and temporal distance “

“We also quantified how temporal distances between trials relates to their behavioral location distances, participant by participant. Our dimension similarity analysis controls for temporal distance between trials by design (see Social dimension similarity searchlight analysis), but our location similarity analysis does not. To decide on covariates to include in the analysis, we tested whether temporal distances can explain behavioral location distances. For each participant, we computed the correlations between trial pairs’ Euclidean distances in social locations and their linear temporal distances (“linear”) and the temporal distances squared (“quadratic”), to test for nonlinear effects. We then summarized the correlations using one-sample t-tests. The linear relationship was statistically significant (t₄₉ = 12.24, p < 0.001), whereas the quadratic relationship was not (t₄₉ = -0.55, p = 0.586). Similarly, in participant specific regressions with both linear and quadratic temporal distances, the linear effect was significant (t₄₉ = 5.69, p < 0.001) whereas the quadratic effect was not (t₄₉ = 0.20, p = 0.84). Based on this, we included linear temporal distances as a covariate in our location similarity analyses (see Location similarity searchlight analyses), and verified that adding a quadratic temporal distance covariate does not alter the results. Thus, the reported location-related pattern similarity effects go beyond what can be explained by temporal distance alone.”

Third and last argument is that they were scolded and told racist but that didn't sit with their views of themselves or their field. They aimed to protect it, and dismissed other cases as cherry picked anecdotes or as being a necessary part of the system, their system, their identity.

There's this non-homogeneous group of white privilege people that yearn to continue playing these types of games, and that may even see themselves as activists when buying them. These may be big mainstream titles, but much like in cinema and TV, their budgets are also big. They know, and they don't mind, they wish these games be as larger and ambitious as possible, ever bigger, and more complex, and continuously "improving", and "innovating" in this sense. They see defending this kind of consumption as defending their identity, defending who they are, defending dark comedy and freedom of speech... freedom of speech, at which point does it become hate speech? Why should their tone for people that have no skin in the game and who aim to get rid of their identity, of their way of living, without asking? You see how both sides have self-reinforcing narratives, and they may even acknowledge this, and although many left-wingers would love to parse out this radically big titles, instead of talking it out and recognising the current exclusionary and biased present (not perpetuating endless debates), some prominent white privilege people push a zero-sum incompatibility competition narrative where one must survive, and it will be them.

You can't expect a person who's played 5000 hours, to quit Fifa overnight.

Reviewer #1 (Public review):

Summary:

The manuscript by Mengxing et al., reports an assessment of three first-order thalamic nuclei (auditory, visual, somatosensory) in a 3 x 2 factorial design to test for specificity of responses in first-order thalamic nuclei to linguistic processing particularly in the left hemisphere. The conditions are reading, speech production, and speech comprehension and their respective control conditions. The authors report the following results:

(1) BOLD-response analyses: left MGB linguistic vs non-linguistic significant; left LGN linguistic vs non-linguistic significant. There is no hemisphere x stimulus interaction.

(2) MVPA: left MGB linguistic vs. non-linguistic significant; bilateral VLN linguistic vs. non-linguistic significant; significant lateralisation in MGB (left MGB responses better classified linguistic vs. non-linguistic in contrast to right).

(3) Functional connectivity: there is, in general, connectivity between the thalamic ROIs and the respective primary cortices independent of linguistics.

Strengths:

The study has a clear and comprehensive design and addresses a timely topic. First-order thalamic nuclei and their interaction with the respective cerebral cortex area are likely key to understanding how perception works in a world where one has to compute highly dynamic stimuli often in an instant. Speech is a prime example of an ecologically important, extremely dynamic, and complex stimulus. The field of the contribution of cerebral cortex-thalamic loops is wide open, and the study presents a solid approach to address their role in different speech modalities (i.e., reading, comprehension, production).

Weaknesses:

I see two major overall weaknesses in the manuscript in its current form:

(1) Statistics:

Unfortunately, I have doubts about the solidity of the statistics. In the analyses of the BOLD responses, the authors do not find significant hemisphere x stimulus interactions. In my view, such results would pre-empt doing a post-hoc t-test. Nevertheless, the authors motivate their post-hoc t-test by 'trends' in the interaction and prior hypotheses. I see two difficulties with that. First, the origin of the prior hypotheses is somewhat unclear (see also the comment below on hypotheses), and the post-hoc t-test is not corrected for multiple comparisons. I find that it is a pity that the authors did not derive more specific hypotheses grounded in the literature to guide the statistical testing, as I think these would have been available, and the response properties of the MGB and LGN also make sense in light of them. In addition, I was wondering whether the MVPA results would also need to be corrected for the three tests, i.e., the three ROIs.

Hypotheses:

In my view, it is relatively unclear where the hypotheses precisely come from. For example, the paragraph on the hypotheses in the introduction (p. 6-7) is devoid of references. I also have the impression that the hypotheses are partly not taking into account previous reports on first-order thalamic nuclei involvement in linguistic vs. non-linguistic processing. For example, the authors test for lateralisation of linguistic vs. non-linguistic responses in all nuclei. However, from previous literature, one could derive the hypothesis that the lateralisation in MGB for speech might be there - previous work shows, for example, that speech recognition abilities consistently correlate with left MGB only (von Kriegstein et al., 2008 Curr Biol; Mihai et al., 2019 eLife). In addition, the involvement of the MGB in speech in noise processing is present in the left MGB (Mihai et al., 2021, J Neuroscience). Developmental dyslexia, which is supposed to be based on imprecise phonological processing (Ramus et al., 2004 TiCS), has alterations in left MGB (Diaz et al., 2012 PNAS; Galaburda et al., 1994 PNAS) and left MGB connections to planum temporale (Tschentscher et al., 2019 J Neurosci) as well as altered lateralisation (Müller-Axt et al., 2025 Brain). Conversely, in the LGN, I'm not aware of any studies showing lateralisation for speech. See, for example, Diaz et al., 2018, Neuroimage, where there are correlations of LGN task-dependent modulation with visual speech recognition behaviour in both LGNs. Thus, based on this literature, one could have predicted the result pattern displayed, for example, in Figure 3A at least for MGB and LGN.

In summary, the motivation for the different hypotheses needs to be carved out more and couched into previous literature that is directly relevant to the topic. The above paragraph is, of course, my view on the topic, but currently, the paper lacks different literature as references to fully understand where the hypotheses are derived from.

Reviewer #2 (Public review):

Summary:

This study investigates the involvement of first-order thalamic nuclei in language-related tasks using task-based fMRI in a 3 × 2 design contrasting linguistic and non-linguistic versions of reading, speech comprehension, and speech production. By focusing on the LGN, MGN, and VLN and combining activation, connectivity, lateralization, and multivariate pattern analyses, the authors aim to characterize modality-specific and language-related thalamic contributions.

Strength:

A major strength of the work is its hypothesis-driven and multimodal analytical approach, and the modality-specific engagement of first-order thalamic nuclei is robust and consistent with known thalamocortical organization. This is a very sound study overall.

Weaknesses:

However, several conceptual issues complicate the interpretation of the results as evidence for linguistic modulation per se. A central concern relates to the operationalization of the linguistic versus non-linguistic contrast. In the present design, linguistic and non-linguistic stimuli differ along multiple dimensions beyond linguistic content. For example, written words and scrambled images differ in spatial frequency structure, edge composition, contrast regularities, and familiarity, while intelligible speech and acoustically scrambled sounds differ substantially in temporal and spectral statistics. This is particularly relevant given that first-order thalamic nuclei such as the LGN are known to be highly sensitive to low-level sensory properties. As a result, observed differences in thalamic responses may reflect sensitivity to stimulus properties rather than linguistic processing per se, and this limits the specificity of claims regarding linguistic modulation.

Relatedly, although the manuscript frequently refers to effects "depending on the linguistic nature of the stimuli," the statistical evidence for linguistic versus non-linguistic modulation is uneven across analyses. Whole-brain contrasts collapse across stimulus type and primarily test modality effects. Similarly, the primary ROI analyses of activation amplitude are collapsed across linguistic and non-linguistic conditions and convincingly demonstrate modality-specific engagement of thalamic nuclei, but do not in themselves provide evidence for linguistic modulation. Linguistic effects emerge only in later, more targeted analyses focusing on hemispheric lateralization and multivariate pattern classification, and these effects are nucleus-, modality-, and analysis-specific rather than general. Taken together, these results suggest that linguistic modulation constitutes a secondary and selective finding, whereas modality-specific task engagement represents the primary and most robust outcome of the study.

An additional interpretational issue concerns task engagement and attention. The tasks differ substantially in cognitive demands (e.g., passive reading and listening versus overt speech production), and linguistic and non-linguistic blocks may differ systematically in salience or engagement. This is particularly important given prior evidence, cited by the authors, that LGN and MGN activity can be modulated by task demands and attention. In the absence of behavioral measures indexing task engagement or compliance, it is difficult to determine whether differences between linguistic and non-linguistic conditions reflect linguistic processing per se or are mediated by attentional factors.

Finally, while the manuscript emphasizes the novelty of evaluating thalamic involvement in language, thalamic contributions to language have been documented previously in both lesion and functional imaging studies. The contribution of the present work, therefore, lies less in establishing thalamic involvement in language per se, and more in its focus on specific first-order nuclei, its multimodal design, and its combination of univariate, connectivity, and multivariate analyses. Moderating claims of novelty would help place the findings more clearly within the existing literature.

Reviewer #1 (Public review):

Summary:

Thach et al. report on the structure and function of trimethylamine N-oxide demethylase (TDM). They identify a novel complex assembly composed of multiple TDM monomers and obtain high-resolution structural information for the catalytic site, including an analysis of its metal composition, which leads them to propose a mechanism for the catalytic reaction.

In addition, the authors describe a novel substrate channel within the TDM complex that connects the N-terminal Zn²-dependent TMAO demethylation domain with the C-terminal tetrahydrofolate (THF)-binding domain. This continuous intramolecular tunnel appears highly optimized for shuttling formaldehyde (HCHO), based on its negative electrostatic properties and restricted width. The authors propose that this channel facilitates the safe transfer of HCHO, enabling its efficient conversion to methylenetetrahydrofolate (MTHF) at the C-terminal domain as a microbial detoxification strategy.

Strengths:

The authors provide convincing high-resolution cryo-EM structural evidence (up to 2 Å) revealing an intriguing complex composed of two full monomers and two half-domains. They further present evidence for the metal ion bound at the active site and articulate a plausible hypothesis for the catalytic cycle. Substantial effort is devoted to optimizing and characterizing enzyme activity, including detailed kinetic analyses across a range of pH values, temperatures, and substrate concentrations. Furthermore, the authors validate their structural insights through functional analysis of active-site point mutants.

In addition, the authors identify a continuous channel for formaldehyde (HCHO) passage within the structure and support this interpretation through molecular dynamics simulations. These analyses suggest an exciting mechanism of specific, dynamic, and gated channeling of HCHO. This finding is particularly appealing, as it implies the existence of a unique, completely enclosed conduit that may be of broad interest, including potential applications in bioengineering.

Weaknesses:

Although the idea of an enclosed channel for HCHO is compelling, the experimental evidence supporting enzymatic assistance in the reaction of HCHO with THF is less convincing. The linear regression analysis shown in Figure 1C demonstrates a THF concentration-dependent decrease in HCHO, but the concentrations used for THF greatly exceed its reported KD (enzyme concentration used in this assay is not reported). It has previously been shown that HCHO and THF can couple spontaneously in a non-enzymatic manner, raising the possibility that the observed effect does not require enzymatic channeling. An additional control that can rule out this possibility would help to strengthen the evidence. For example, mutating the THF binding site to prevent THF binding to the protein complex could clarify whether the observed decrease in HCHO depends on enzyme-mediated proximity effects. A mutation which would specifically disable channeling could be even more convincing (maybe at the narrowest bottleneck).

Another concern is that the observed decrease in HCHO could alternatively arise from a reduced production of HCHO due to a negative allosteric effect of THF binding on the active site. From this perspective, the interpretation would be more convincing if a clear coupled effect could be demonstrated, specifically, that removal of the product (HCHO) from the reaction equilibrium leads to an increase in the catalytic efficiency of the demethylation reaction.

While the enzyme kinetics appear to have been performed thoroughly, the description of the kinetic assays in the Methods section is very brief. Important details such as reaction buffer composition, cofactor identity and concentration (Zn²⁺), enzyme concentration, defined temperature, and precise pH are not clearly stated. Moreover, a detailed methodological description could not be found in the cited reference (6), if I am not mistaken.

The composition of the complex is intriguing but raises some questions. Based on SDS-PAGE analysis, the purified protein appears to be predominantly full-length TDM, and size-exclusion chromatography suggests an apparent molecular weight below 100 kDa. However, the cryo-EM structure reveals a substantially larger complex composed of two full-length monomers and two half-domains.

Given the lack of clear evidence for proteolytic fragments on the SDS-PAGE gel, it is unclear how the observed stoichiometry arises. This raises the possibility of higher-order assemblies or alternative oligomeric states. Did the authors attempt to pick or analyze larger particles during cryo-EM processing? Additional biophysical characterization of particle size distribution - for example, using interferometric scattering microscopy (iSCAT)-could help clarify the oligomeric state of the complex in solution.

The authors mention strict symmetry in the complex, yet C2 symmetry was enforced during refinement. While this is reasonable as an initial approach, it would strengthen the structural interpretation to relax the symmetry to C1 using the C2-refined map as a reference. This could reveal subtle asymmetries or domain-specific differences without sacrificing the overall quality of the reconstruction.

In this context, the proposed catalytic role of Zn²⁺ raises additional questions. Why is a 2:1 enzyme-to-metal stoichiometry observed, and how does this reconcile with previous reports? This point warrants discussion. Does this imply asymmetric catalysis within the complex? Would the stoichiometry change under Zn²⁺-saturating conditions, as no Zn²⁺ appears to be added to the buffers? It would be helpful to clarify whether Zn²⁺ occupancy is equivalent in both active sites when symmetry is not imposed, or whether partial occupancy is observed.

The divalent ion Zn2+ is suggested to activate water for the catalytic reaction. I am not sure if there is a need for a water molecule to explain this catalytic mechanism. Can you please elaborate on this more? As one aspect, it might be helpful to explain in more detail how Zn-OH and D220 are recovered in the last step before a new water molecule comes in.

Overall, the authors were successful in advancing our structural and functional understanding of the TDM complex. They suggest an interesting oligomeric complex composition which should be investigated with additional biophysical techniques.

Additionally, they provide an intriguing hypothesis for a new type of substrate channeling. Additional kinetic experiments focusing on HCHO and THF turnover by enzymatic proximity effects would strengthen this potentially fundamental finding. If this channeling mechanism can be supported by stronger experimental evidence, it would substantially advance our understanding and knowledge of biologic conduits and enable future efforts in the design of artificial cascade catalysis systems with high conversion rate and efficiency, as well as detoxification pathways.

Reviewer #2 (Public review):

Summary:

The manuscript reports a cryo-EM structure of TMAO demethylase from Paracoccus sp. This is an important enzyme in the metabolism of trimethylamine oxide (TMAO) and trimethylamine (TMA) in human gut microbiota, so new information about this enzyme would certainly be of interest.

Strengths:

The cryo-EM structure for this enzyme is new and provides new insights into the function of the different protein domains, and a channel for formaldehyde between the two domains.

Weaknesses:

(1) The proposed catalytic mechanism in this manuscript does not make sense. Previous mechanistic studies on the Methylocella silvestris TMAO demethylase (FEBS Journal 2016, 283, 3979-3993, reference 7) reported that, as well as a Zn2+ cofactor, there was a dependence upon non-heme Fe2+, and proposed a catalytic mechanism involving deoxygenation to form TMA and an iron(IV)-oxo species, followed by oxidative demethylation to form DMA and formaldehyde.

In this work, the authors do not mention the previously proposed mechanism, but instead say that elemental analysis "excluded iron". This is alarming, since the previous work has a key role for non-heme iron in the mechanism. The elemental analysis here gives a Zn content of about 0.5 mol/mol protein (and no Fe), whereas the Methylocella TMAO demethylase was reported to contain 0.97 mol Zn/mol protein, and 0.35-0.38 mol Fe/mol protein. It does, therefore, appear that their enzyme is depleted in Zn, and the absence of Fe impacts the mechanism, as explained below.

The proposed catalytic mechanism in this manuscript, I am sorry to say, does not make sense to me, for several reasons:

(i) Demethylation to form formaldehyde is not a hydrolytic process; it is an oxidative process (normally accomplished by either cytochrome P450 or non-heme iron-dependent oxygenase). The authors propose that a zinc (II) hydroxide attacks the methyl group, which is unprecedented, and even if it were possible, would generate methanol, not formaldehyde.

(ii) The amine oxide is then proposed to deoxygenate, with hydroxide appearing on the Zn - unfortunately, amine oxide deoxygenation is a reductive process, for which a reducing agent is needed, and Zn2+ is not a redox-active metal ion;

(iii) The authors say "forming a tetrahedral intermediate, as described for metalloproteinase", but zinc metalloproteases attack an amide carbonyl to form an oxyanion intermediate, whereas in this mechanism, there is no carbonyl to attack, so this statement is just wrong.

So on several counts, the proposed mechanism cannot be correct. Some redox cofactor is needed in order to carry out amine oxide deoxygenation, and Zn2+ cannot fulfil that role. Fe2+ could do, which is why the previously proposed mechanism involving an iron(IV)-oxo intermediate is feasible. But the authors claim that their enzyme has no Fe. If so, then there must be some other redox cofactor present. Therefore, the authors need to re-analyse their enzyme carefully and look either for Fe or for some other redox-active metal ion, and then provide convincing experimental evidence for a feasible catalytic mechanism. As it stands, the proposed catalytic mechanism is unacceptable.

(2) Given the metal content reported here, it is important to be able to compare the specific activity of the enzyme reported here with earlier preparations. The authors do quote a Vmax of 16.52 µM/min/mg; however, these are incorrect units for Vmax, they should be µmol/min/mg. There is a further inconsistency between the text saying µM/min/mg and the Figure saying µM/min/µg.

(3) The consumption of formaldehyde to form methylene-THF is potentially interesting, but the authors say "HCHO levels decreased in the presence of THF", which could potentially be due to enzyme inhibition by THF. Is there evidence that this is a time-dependent and protein-dependent reaction? Also in Figure 1C, HCHO reduction (%) is not very helpful, because we don't know what concentration of formaldehyde is formed under these conditions; it would be better to quote in units of concentration, rather than %.

(4) Has this particular TMAO demethylase been reported before? It's not clear which Paracoccus strain the enzyme is from; the Experimental Section just says "Paracoccus sp.", which is not very precise. There has been published work on the Paracoccus PS1 enzyme; is that the strain used? Details about the strain are needed, and the accession for the protein sequence.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Thach et al. report on the structure and function of trimethylamine N-oxide demethylase (TDM). They identify a novel complex assembly composed of multiple TDM monomers and obtain high-resolution structural information for the catalytic site, including an analysis of its metal composition, which leads them to propose a mechanism for the catalytic reaction.

In addition, the authors describe a novel substrate channel within the TDM complex that connects the N-terminal Zn²-dependent TMAO demethylation domain with the C-terminal tetrahydrofolate (THF)-binding domain. This continuous intramolecular tunnel appears highly optimized for shuttling formaldehyde (HCHO), based on its negative electrostatic properties and restricted width. The authors propose that this channel facilitates the safe transfer of HCHO, enabling its efficient conversion to methylenetetrahydrofolate (MTHF) at the C-terminal domain as a microbial detoxification strategy.

Strengths:

The authors provide convincing high-resolution cryo-EM structural evidence (up to 2 Å) revealing an intriguing complex composed of two full monomers and two half-domains. They further present evidence for the metal ion bound at the active site and articulate a plausible hypothesis for the catalytic cycle. Substantial effort is devoted to optimizing and characterizing enzyme activity, including detailed kinetic analyses across a range of pH values, temperatures, and substrate concentrations. Furthermore, the authors validate their structural insights through functional analysis of active-site point mutants.

In addition, the authors identify a continuous channel for formaldehyde (HCHO) passage within the structure and support this interpretation through molecular dynamics simulations. These analyses suggest an exciting mechanism of specific, dynamic, and gated channeling of HCHO. This finding is particularly appealing, as it implies the existence of a unique, completely enclosed conduit that may be of broad interest, including potential applications in bioengineering.

Weaknesses:

Although the idea of an enclosed channel for HCHO is compelling, the experimental evidence supporting enzymatic assistance in the reaction of HCHO with THF is less convincing. The linear regression analysis shown in Figure 1C demonstrates a THF concentration-dependent decrease in HCHO, but the concentrations used for THF greatly exceed its reported KD (enzyme concentration used in this assay is not reported). It has previously been shown that HCHO and THF can couple spontaneously in a non-enzymatic manner, raising the possibility that the observed effect does not require enzymatic channeling. An additional control that can rule out this possibility would help to strengthen the evidence. For example, mutating the THF binding site to prevent THF binding to the protein complex could clarify whether the observed decrease in HCHO depends on enzyme-mediated proximity effects. A mutation which would specifically disable channeling could be even more convincing (maybe at the narrowest bottleneck).

We agree with the reviewer that HCHO and THF can react spontaneously in a non-enzymatic manner, and our experiments were not intended to demonstrate enzymatic channeling. The linear regression analysis in Figure 1C was designed solely to confirm that HCHO reacts with THF under our assay conditions. Accordingly, THF was titrated over a broad concentration range starting from zero, and the observed THF concentration–dependent decrease in HCHO reflects this chemical reactivity.

We do not interpret these data as evidence that the enzyme catalyzes or is required for the HCHO–THF coupling reaction. Instead, the structural observation of an enclosed channel is presented as a separate finding. We have clarified this point in the revised text to avoid overinterpretation of the biochemical data (page 2, line 16).

Another concern is that the observed decrease in HCHO could alternatively arise from a reduced production of HCHO due to a negative allosteric effect of THF binding on the active site. From this perspective, the interpretation would be more convincing if a clear coupled effect could be demonstrated, specifically, that removal of the product (HCHO) from the reaction equilibrium leads to an increase in the catalytic efficiency of the demethylation reaction.

We agree that, in principle, a decrease in detectable HCHO could also arise from an indirect effect of THF binding on enzyme activity. However, in our study the experiment was not designed to assess catalytic coupling or allosteric regulation. The assay in question monitors HCHO levels under defined conditions and does not distinguish between changes in HCHO production and downstream consumption.

Additionally, we do not interpret the observed decrease in HCHO as evidence that THF binding enhances catalytic efficiency, or that removal of HCHO shifts the reaction equilibrium. Instead, the data are presented to establish that HCHO can react with THF under the assay conditions. Any potential allosteric effects of THF on the demethylation reaction, or kinetic coupling between HCHO removal and catalysis, are beyond the scope of the current study, and are not claimed.

While the enzyme kinetics appear to have been performed thoroughly, the description of the kinetic assays in the Methods section is very brief. Important details such as reaction buffer composition, cofactor identity and concentration (Zn²⁺), enzyme concentration, defined temperature, and precise pH are not clearly stated. Moreover, a detailed methodological description could not be found in the cited reference (6), if I am not mistaken.

Thank you for the suggestion. We have added reference [24] to the methodological description on page 8. The Methods section has been revised accordingly on page 8 under “TDM Activity Assay,” without altering the Zn²⁺ concentration.

The composition of the complex is intriguing but raises some questions. Based on SDS-PAGE analysis, the purified protein appears to be predominantly full-length TDM, and size-exclusion chromatography suggests an apparent molecular weight below 100 kDa. However, the cryo-EM structure reveals a substantially larger complex composed of two full-length monomers and two half-domains.

We appreciate the reviewer’s careful analysis of the apparent discrepancy between the biochemical characterization and the cryo-EM structure. This issue is addressed in Figure S1, which may have been overlooked.

As shown in Figure S1, the stability of TDM is highly dependent on protein and salt conditions. At 150 mM NaCl, SEC reveals a dominant peak eluting between 10.5 and 12 mL, corresponding to an estimated molecular weight of ~170–305 kDa (blue dot, Author response image 1). This fraction was explicitly selected for cryo-EM analysis and yields the larger complex observed in the reconstruction. At lower salt concentrations (50 mM) or higher (>150 mM NaCl), the protein either aggregates or elutes near the void volume (~8 mL).

SDS–PAGE analysis detects full-length TDM together with smaller fragments (~40–50 kDa and ~22–25 kDa). The apparent predominance of full-length protein on SDS–PAGE likely reflects its greater staining intensity per molecule and/or a higher population, rather than the absence of truncated species.

Author response image 1.

Given the lack of clear evidence for proteolytic fragments on the SDS-PAGE gel, it is unclear how the observed stoichiometry arises. This raises the possibility of higher-order assemblies or alternative oligomeric states. Did the authors attempt to pick or analyze larger particles during cryo-EM processing? Additional biophysical characterization of particle size distribution - for example, using interferometric scattering microscopy (iSCAT)-could help clarify the oligomeric state of the complex in solution.

Cryo-EM data were collected exclusively from the size-exclusion chromatography fraction eluting between 10.5 and 12 mL. This fraction was selected to isolate the dominant assembly in solution. Extensive 2D and 3D particle classification did not reveal distinct classes corresponding to smaller species or higher-order oligomeric assemblies. Instead, the vast majority of particles converged to a single, well-defined structure consistent with the 2 full-length + 2 half-domain stoichiometry.

A minor subpopulation (~2%) exhibited increased flexibility in the N-terminal region of the two full-length subunits, but these particles did not form a separate oligomeric class, indicating conformational heterogeneity rather than alternative assembly states (Author response image 2). Together, these data support the 2+2½ architecture as the predominant and stable complex under the conditions used for cryo-EM. Additional techniques, such as iSCAT, would provide complementary information, but are not required to support the conclusions drawn from the SEC and cryo-EM analyses presented here.

Author response image 2.

The authors mention strict symmetry in the complex, yet C2 symmetry was enforced during refinement. While this is reasonable as an initial approach, it would strengthen the structural interpretation to relax the symmetry to C1 using the C2-refined map as a reference. This could reveal subtle asymmetries or domain-specific differences without sacrificing the overall quality of the reconstruction.

We thank the reviewer for this thoughtful suggestion. In standard cryo-EM data processing, symmetry is typically not imposed initially to minimize potential model bias; accordingly, we first performed C1 refinement before applying C2 symmetry. The resulting C1 reconstructions revealed no detectable asymmetry or domain-specific differences relative to the C2 map. In addition, relaxing the symmetry consistently reduced overall resolution, indicating lower alignment accuracy and further supporting the presence of a predominantly symmetric assembly.

In this context, the proposed catalytic role of Zn²⁺ raises additional questions. Why is a 2:1 enzyme-to-metal stoichiometry observed, and how does this reconcile with previous reports? This point warrants discussion. Does this imply asymmetric catalysis within the complex? Would the stoichiometry change under Zn²⁺-saturating conditions, as no Zn²⁺ appears to be added to the buffers? It would be helpful to clarify whether Zn²⁺ occupancy is equivalent in both active sites when symmetry is not imposed, or whether partial occupancy is observed.

The observed ~2:1 enzyme-to-Zn²⁺ stoichiometry likely reflects the composition of the 2 full-length + 2 half-domain (2+2½) complex. In this assembly, only the core domains that are fully present in the complex contribute to metal binding. The truncated or half-domains lack the Zn²⁺ binding domain. As a result, only two metal-binding sites are occupied per assembled complex, consistent with the measured stoichiometry.

We note that Zn²⁺ was not deliberately added to the buffers, so occupancy may not reflect full saturation. Based on our cryo-EM and biochemical data, both metal-binding sites in the full-length subunits appear to be occupied to an equivalent extent, and no clear evidence of asymmetric catalysis is observed under these current experimental conditions. Full Zn²⁺ saturation could potentially increase occupancy, but was not explored in these experiments.

The divalent ion Zn²⁺ is suggested to activate water for the catalytic reaction. I am not sure if there is a need for a water molecule to explain this catalytic mechanism. Can you please elaborate on this more? As one aspect, it might be helpful to explain in more detail how Zn-OH and D220 are recovered in the last step before a new water molecule comes in.

Thank you for your suggestion. We revised our text in page 2 as bellow.

Based on our structural and biochemical data, we propose a structurally informed working model for TMAO turnover by TDM (Scheme 1). In this model, Zn²⁺ plays a non-redox role by polarizing the O–H bond of the bound hydroxyl, thereby lowering its pK_a. The D220 carboxylate functions as a general base, abstracting the proton to generate a hydroxide nucleophile. This hydroxide then attacks the electrophilic N-methyl carbon of TMAO, forming a tetrahedral carbinolamine (hemiaminal) intermediate. Subsequent heterolytic cleavage of the C–N bond leads to the release of HCHO. D220 then switches roles to act as a general acid, donating a proton to the departing nitrogen, which facilitates product release and regenerates the active site. This sequence allows a new water molecule to rebind Zn²⁺, enabling subsequent catalytic turnovers. This proposed pathway is consistent with prior mechanistic studies, in which water addition to the azomethine carbon of a cationic Schiff base generates a carbinolamine intermediate, followed by a rate-limiting breakdown to yield an amino alcohol and a carbonyl compound, in the published case, an aldehyde (Pihlaja et al., J. Chem. Soc. Perkin Trans. 2, 1983, 8, 1223–1226).

Overall, the authors were successful in advancing our structural and functional understanding of the TDM complex. They suggest an interesting oligomeric complex composition which should be investigated with additional biophysical techniques.

Additionally, they provide an intriguing hypothesis for a new type of substrate channeling. Additional kinetic experiments focusing on HCHO and THF turnover by enzymatic proximity effects would strengthen this potentially fundamental finding. If this channeling mechanism can be supported by stronger experimental evidence, it would substantially advance our understanding and knowledge of biologic conduits and enable future efforts in the design of artificial cascade catalysis systems with high conversion rate and efficiency, as well as detoxification pathways.

Reviewer #2 (Public review):

Summary:

The manuscript reports a cryo-EM structure of TMAO demethylase from Paracoccus sp. This is an important enzyme in the metabolism of trimethylamine oxide (TMAO) and trimethylamine (TMA) in human gut microbiota, so new information about this enzyme would certainly be of interest.

Strengths:

The cryo-EM structure for this enzyme is new and provides new insights into the function of the different protein domains, and a channel for formaldehyde between the two domains.

Weaknesses:

(1) The proposed catalytic mechanism in this manuscript does not make sense. Previous mechanistic studies on the Methylocella silvestris TMAO demethylase (FEBS Journal 2016, 283, 3979-3993, reference 7) reported that, as well as a Zn2+ cofactor, there was a dependence upon non-heme Fe²⁺, and proposed a catalytic mechanism involving deoxygenation to form TMA and an iron(IV)-oxo species, followed by oxidative demethylation to form DMA and formaldehyde.

In this work, the authors do not mention the previously proposed mechanism, but instead say that elemental analysis "excluded iron". This is alarming, since the previous work has a key role for non-heme iron in the mechanism. The elemental analysis here gives a Zn content of about 0.5 mol/mol protein (and no Fe), whereas the Methylocella TMAO demethylase was reported to contain 0.97 mol Zn/mol protein, and 0.35-0.38 mol Fe/mol protein. It does, therefore, appear that their enzyme is depleted in Zn, and the absence of Fe impacts the mechanism, as explained below.

The proposed catalytic mechanism in this manuscript, I am sorry to say, does not make sense to me, for several reasons:

(i) Demethylation to form formaldehyde is not a hydrolytic process; it is an oxidative process (normally accomplished by either cytochrome P450 or non-heme iron-dependent oxygenase). The authors propose that a zinc (II) hydroxide attacks the methyl group, which is unprecedented, and even if it were possible, would generate methanol, not formaldehyde.

(ii) The amine oxide is then proposed to deoxygenate, with hydroxide appearing on the Zn - unfortunately, amine oxide deoxygenation is a reductive process, for which a reducing agent is needed, and Zn2+ is not a redox-active metal ion;

(iii) The authors say "forming a tetrahedral intermediate, as described for metalloproteinase", but zinc metalloproteases attack an amide carbonyl to form an oxyanion intermediate, whereas in this mechanism, there is no carbonyl to attack, so this statement is just wrong.

So on several counts, the proposed mechanism cannot be correct. Some redox cofactor is needed in order to carry out amine oxide deoxygenation, and Zn²⁺cannot fulfil that role. Fe²⁺ could do, which is why the previously proposed mechanism involving an iron(IV)-oxo intermediate is feasible. But the authors claim that their enzyme has no Fe. If so, then there must be some other redox cofactor present. Therefore, the authors need to re-analyse their enzyme carefully and look either for Fe or for some other redox-active metal ion, and then provide convincing experimental evidence for a feasible catalytic mechanism. As it stands, the proposed catalytic mechanism is unacceptable.

We thank the reviewer for the detailed and thoughtful mechanistic critique. We fully agree that Zn²⁺ is not redox-active, and cannot directly mediate oxidative demethylation or amine oxide deoxygenation. We acknowledge that the oxidative step required for the conversion of TMAO to HCHO is not explicitly resolved in the present study. Accordingly, we have revised the manuscript to remove any implication of Zn²⁺-mediated redox chemistry, and have eliminated the previously imprecise analogy to zinc metalloproteases.

We recognize and now discuss prior biochemical work on TMAO demethylase from Methylocella silvestris (MsTDM), which proposed an iron-dependent oxidative mechanism (Zhu et al., FEBS 2016, 3979–3993). That study reported approximately one Zn²⁺ and one non-heme Fe²⁺ per active enzyme, implicated iron in catalysis through homology modeling and mutagenesis, and used crossover experiments suggesting a trimethylamine-like intermediate and oxygen transfer from TMAO, consistent with an Fe-dependent redox process. However, that system lacked experimental structural information, and did not define discrete metal-binding sites.

In contrast,

(1) Our high-resolution cryo-EM structures and metal analyses of TDM consistently reveal only a single, well-defined Zn²⁺-binding site, with no structural evidence for an additional iron-binding site as in the previous report (Zhu et al., FEBS 2016, 3979–3993).

(2) To investigate the potential involvement of iron, we expressed TDM in LB medium supplemented with Fe(NH₄)₂SO₄ and determined its cryo-EM structure. This structure is identical to the original one, and no EM density corresponding to a second iron ion was observed. Moreover, the previously proposed Fe²⁺-binding residues are spatially distant (Figure S6).

(3) ICP-MS analysis shows undetectable Iron, and only Zinc ion (Figure S5).

(4) Our enzyme kinetics analysis with the TDM without Iron is comparable to that of from MsTDM (Figure 1A). The differences in Km and Vmax we propose is due to the difference in the overall sequence of the enzymes. Please also see comment at the end on a new published paper on MsTDM.

While we cannot comment on the MsTDM results, our ‘experimental’ results do not support the presence of an iron-binding site. Our data indicate that this chemistry is unlikely to be mediated by a canonical non-heme iron center as proposed for MsTDM. We therefore revised our model as a structural framework that rationalizes substrate binding, metal coordination, and product stabilization, while clearly delineating the limits of mechanistic inference supported by the current data.

The scheme 1 and proposal mechanism section were revised in page 4. Figure S6 was added.

(2) Given the metal content reported here, it is important to be able to compare the specific activity of the enzyme reported here with earlier preparations. The authors do quote a Vmax of 16.52 µM/min/mg; however, these are incorrect units for Vmax, they should be µmol/min/mg. There is a further inconsistency between the text saying µM/min/mg and the Figure saying µM/min/µg.

Thank you for the correction. We converted the V_max unit to nmol/min/mg. and revised the text in page 2. We also compared with the value of the previous report in the TDM enzyme by revising the text on page 2. See also the note on a newly published manuscript and its comparison.

(3) The consumption of formaldehyde to form methylene-THF is potentially interesting, but the authors say "HCHO levels decreased in the presence of THF", which could potentially be due to enzyme inhibition by THF. Is there evidence that this is a time-dependent and protein-dependent reaction? Also in Figure 1C, HCHO reduction (%) is not very helpful, because we don't know what concentration of formaldehyde is formed under these conditions; it would be better to quote in units of concentration, rather than %.

We appreciate this important point. We have revised Figure 1C to present HCHO levels in absolute concentration units. While the current data demonstrate reduced detectable HCHO in the presence of THF, we agree that distinguishing between HCHO consumption and potential THF-mediated enzyme inhibition would require dedicated time-course and protein-dependence experiments. We have therefore revised the description to avoid overinterpretation and limit our conclusions to the observed changes in HCHO concentration in page 2, line 18-19.

(4) Has this particular TMAO demethylase been reported before? It's not clear which Paracoccus strain the enzyme is from; the Experimental Section just says "Paracoccus sp.", which is not very precise. There has been published work on the Paracoccus PS1 enzyme; is that the strain used? Details about the strain are needed, and the accession for the protein sequence.

Thank you for this comment. We now indicate that the enzyme is derived from Paracoccus sp. DMF and provide the accession number for the protein sequence (WP_263566861) in the Experimental Section (page 8, line 4).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The ITC experiment requires a ligand-into-buffer titration as an additional control. Also, maybe I misunderstood the molar ratio or the concentrations you used, but if you indeed added a total of 4.75 μL of 20 μM THF into 250 μL of 5 μM TDM, it is not clear to me how this leads to a final molar ratio of 3.

We thank the reviewer for this suggestion. A ligand-into-buffer control ITC experiment was performed and is now included in Figure S8C, which shows no realizable signal.

Regarding the molar ratio, it is our mistake. The experiment used 2.45 μL injections of 80 μM THF into 250 μL of 5 μM TDM. This corresponds to a final ligand concentration of ~12.8 μM, giving a ligand-to-protein molar ratio of ~2.6. We revised our text in page 9, ITC section.

(2) Characterization/quality check of all mutant enzymes should be performed by NanoDSF, CD spectroscopy or similar techniques to confirm that proteins are properly folded and fit for kinetic testing.

We appreciate the reviewer’s suggestion. All mutant proteins, including D220A, D367A, and F327A, were purified with yields similar to the wild-type enzyme. Additionally, cryo-EM maps of the mutants show well-defined density and overall structural integrity consistent with the wild-type. These findings indicate that the introduced mutations do not significantly affect protein folding, supporting their use for kinetic analysis. While NanoDSF might reveal differences in thermal stability due to mutations, it does not provide structural information. Our conclusions are not based on minor differences in thermostability. Our cryo-EM structures of the mutants offer much more reliable structural data than CD spectroscopy.

(3) Best practice would suggest overlapping pH ranges between different buffer systems in the pH-dependence experiments to rule out buffer-specific effects independent of pH.

We thank the reviewer for this helpful suggestion. We agree that overlapping pH ranges between different buffer systems can be valuable for excluding buffer-specific effects. In this study, the pH-dependence experiments were intended to provide a qualitative assessment of pH sensitivity rather than a detailed analysis of buffer-independent pKa values. While we cannot fully exclude minor buffer-specific contributions, the overall trends observed were reproducible and sufficient to support the conclusions drawn. We have added a clarifying statement to the revised manuscript to reflect this consideration, page 2, line 12.

(4) Structural comparison revealed high similarity to a THF-binding protein, with superposition onto a T protein.": It would be nice to show this as an additional figure, as resolution and occupancy for THF are low.

We thank the reviewer for this suggestion. To address this point, we have revised Figure S6 by adding an additional panel (C, now is Figure S7C) showing the structural superposition of TDM with the THF-binding T protein. This comparison is included to better illustrate the structural similarity, despite the limited resolution and partial occupancy of THF density in our map.

(5) Editing could have been done more thoroughly. Some spelling mistakes, e.g. "RESEULTS", "redius", "complec"; kinetic rate constants should be written in italic (not uniform between text and figures); Prism version is missing; Vmax of 16.52 µM/min/mg - doublecheck units; Figure S1B: The "arrow on the right" might have gone missing.

We corrected the spelling in page 2 ~ line 10, page 5 ~ line 34, page 6 ~ line40. Prism version was added. The arrow was added into figure S1B. The Vmax unit is corrected to nmol/min/mg.

Reviewer #2 (Recommendations for the authors):

(1) The authors must re-examine the metal content of their purified enzyme, looking in particular for Fe or another redox-active metal ion, which could be involved in a reasonable catalytic mechanism.

We thank the reviewer for this suggestion and have carefully re-examined the metal content of TDM. Elemental analyses by EDX and ICP-MS consistently detected Zn²⁺ in purified TDM (Zn:protein ≈ 1:2), whereas Fe was below the detection limit across multiple independent preparations (Fig. S5A,B). To assess whether iron could be incorporated or play a functional role, we expressed TDM in E. coli grown in LB medium supplemented with Fe(NH₄SO₄)₂ and performed activity assays in the presence of exogenous Fe²⁺. Neither condition resulted in enhanced enzymatic activity.

Consistent with these biochemical data, all cryo-EM structures reveal a single, well-defined metal-binding site coordinated by three conserved cysteine residues and occupied by Zn²⁺, with no evidence for an additional iron species or other redox-active metal site.

(2) The specific activity of the enzyme should be quoted in the same units as other literature papers, so that the enzyme activity can be compared. It could be, for example, that the content of Fe (or other redox-active metal) is low, and that could then give rise to a low specific activity.

Thank you for the suggestion, we quoted the enzyme units as similar with previous report. and revised the text in in page 2.

Since the submission of our paper a new report on MsTDM has been published (Cappa et al., Protein Science 33(11), e70364). It further supports our findings. First, the reported kinetic parameters using ITC (Vmax = 0.309 μmol/s, approximately 240 nmol/min/mg; Km = 0.866 mM) are comparable to our observed (156 nmol/min/mg and 1.33 mM, respectively) in the absence of exogenous iron. Second, the optimal pH for enzymatic activity similar to that observed in our paraTDM. Third, the reported two-state unfolding behavior is consistent with our cryo-EM structural observations, in which the more dynamic subunits appear to destabilize prior to unfolding of the core domains. Based on these findings, we now propose that Zn²⁺ appears to function primarily as an organizational cofactor at the core catalytic domain (revised Scheme 1).

La verdadera ruptura fue la transición de un proyecto entre amigos a una compañía auténtica, para Shah y su equipo, el desafío no consistía en el aspecto técnico del lenguaje, que ya era excelente; sino en cómo hacer que sobreviviera: convertir un código gratuito en una empresa sostenible. Fue una decisión, abandonar el experimento académico y transformarlo en un instrumento profesional que hoy emplean empresas de gran tamaño en cualquier parte del mundo.

A veces la frustración en cualquier campo es el combustible de las mejores innovaciones tecnológicas y el caso Julia y sus creadores no es exención.

Ellos lo saben, con tantos desarrollos en proceso y la tecnología corriendo a grandes pasos, su proyecto no tendrá final, siempre estará en constante evolución, e impactando en su proceso.

Bueno, siento que como todo lo novedoso siempre tiene un declive, entre los aparatos tecnológicos o lenguajes de programación o simplemente en este mundo de tendencias, todo tiene un cierto declive si no se innova, aunque también puede ser momento de una nueva revolución si se desea. Bueno si ven que se puede mejorar o dar un nuevo salto en su necesidad.

Reviewer #1 (Public review):

This study by Radziun and colleagues investigates the effects of using a hand-augmentation device on mental body representations. The authors use a proprioceptive localisation task to measure metric representations of finger length before and after participants wear the device, and then before and after they learn to use the device, which extends the lengths of the fingers by 10 cm. The authors find changes between different time points, which they interpret as evidence for three distinct forms of plasticity: one related to simply wearing the device, one related to learning to use it, and an aftereffect after taking the device off. A control experiment with a similar device, which does not lengthen the fingers, showed the first and third of these forms of plasticity, but not the second.

This study takes an interesting approach to a timely and theoretically significant issue. The study appears to be appropriately designed and conducted. There are, however, some points which require clarification.

(1) The nature of the localization task is unclear. On its face, the task appears to involve localization of each landmark within the 2-dimensional surface of the touchscreen. However, the regression analysis presupposes that localization is made in a 1-dimensional space. Figure S2 shows that three lines are presented on the screen above the index, middle, and ring fingers, which I imagine the participant is meant to use as a guide. But it is at least conceivable that the perceived location or orientation of the finger might not correspond exactly to these lines. While the method can deal gracefully with proximal-distal translations of the fingers (i.e., with the intercept parameter of the regression), it isn't clear how the participant is supposed to respond if their proprioceptive perception of finger location is translated left-right or rotated relative to the lines on the screen. I also worry that presenting a long, thin line to represent each finger on the screen may not be a neutral method and may prime participants to represent the finger as long and thin.

(2) The task used here fits within a wider family of tasks in the literature using localization judgments of multiple landmarks to map body representations. I feel that some discussion of this broader set of tasks and their use to measure body representation and plasticity is notably absent from the paper. It is also striking to me that some of the present authors have themselves recently criticized the use of landmark localization methods as a measure of represented body size and shape (Peviani et al, 2024, Current Biology). It is therefore surprising to see them use this task here as a measure of represented finger length without commenting on this issue.

(3) 18 participants strikes me as a relatively small sample size for this type of study. It weakens the manuscript that the authors do not provide any justification, or even comment on, the sample size. This is especially true as participants are excluded from the entire sample, and from specific analyses, on rather post-hoc grounds.

(4) I have some concerns about the interpretation of contraction in stage 2. The authors claim that wearing the finger extended produces "a contraction",i.e., an "under-representation" (page 12). But in both experiments, regression slopes in stage 2 were not significantly different from 1 (i.e., 0.98 [SE: 0.07] in Exp 1a and 1.04 [SE: 0.09] in Experiment 1b). So how can that be interpreted as "under-representation"?

(5) I also have concerns about the interpretation of the stretch that is claimed to occur following training. In Exp 1a, regression slopes in stage 3 are on average 1.15. That is LESS than in the pretest at stage 1 (mean: 1.16). The idea of stretch only comes about because of the lower slopes in stage 2, which the authors have interpreted as reflecting contraction. So what the authors call stretch and a 2nd form of plasticity could just be the contraction from stage 2 wearing off or dissipating, since perceived finger length in stage 3 just appears to return to the baseline level seen in stage 1. While the authors describe their results in terms of three distinct forms of plasticity, these are not in fact statistically independent. The dip in regression slopes in stage 2 is interpreted as evidence for two distinct plasticity effects, which I do not find convincing.

(6) The distinction between plasticity at stage 3 (which appears specific to augmentation) and plasticity at stage 4 (which does not appear specific, as it also occurs in Experiment 1b) feels strained. This feels like a very subtle distinction, and the theoretical significance of it is not convincingly developed.

(7) The reporting of statistics is not always consistent. For example, 95%CIs are presented for regression slopes in stages 1, 3, and 4, but not for stage 2. Statistics are performed on regression slopes, except for one t-test on page 7 comparing lengths in cm. Estimates of effect size would be nice additions to statistical tests.

(8) Minor point: On page 4, the authors write, "These included sorting colored blocks, stacking a Jenga tower, and sorting pegs into holes; the latter task required fine-grained manipulation and was used as our outcome measure of motor learning." This suggests that peg sorting was the outcome measure, but in Figure 1D, Jenga is presented as the outcome measure.

Reviewer #2 (Public review):

Summary:

This study aimed to explore dynamic changes in the somatosensory representation of both the body and artificial body parts. The study investigated how proprioceptive localisation along the finger changes when participants wear, actively use, and then remove a hand augmentation device - a rigid finger-extension. By mapping perceived target locations along the biological finger and the extension across multiple stages, the authors aim to characterise how the somatosensory system updates our spatial body representation during and after interaction with body augmentation technology.

Strengths:

The manuscript addresses an interesting question of how augmentation devices alter proprioceptive localisation abilities. Conceptually, the work moves beyond classic tool-use paradigms by focusing on a device that is used with the hand to extend the fingers' abilities (versus a tool that is simply used by the hand), and by attempting to map perceived spatial structure across both biological and artificial segments within the same framework.

A major strength is the multi-stage design, which samples localisation abilities at baseline, the beginning of device wear, post-training, and immediately post-removal. This provides a richer characterisation of short-term adaptation compared to a simple pre/post comparison. The dense sampling across stages and target locations generates a rich behavioural dataset that will be valuable to readers interested in somatosensory body representation. The within-subject, counterbalanced control session further strengthens interpretability, providing a useful comparison for interpreting stage-dependent effects, and to probe how functional training shapes changes in the perceptual representations. Finally, the augmentation device itself appears carefully engineered, with thoughtful design decisions regarding wearability, including comfort and customised fit. The manuscript is also communicated clearly, with transparent reporting of analyses and succinct figures that make the pattern changes across stages straightforward to evaluate.

Weaknesses:

There is conceptual ambiguity in how the regression outcomes are interpreted in relation to perceived length and spatial integration. The manuscript treats regression slope as a proxy for "length perception" and discards the intercept as "spatial bias," but in this localisation task translation (intercept) and scaling (slope) are coupled: changes in anchoring at the proximal baseline (intercept) or distal endpoint can generate slope differences without uniform rescaling across the mapped surface. Relatedly, the analyses do not establish whether the reported effects are global across targets or disproportionately driven by the most distal locations. This limits the strength of inferences about "partitioning" or "reallocation" of representational space across biological and artificial segments. Some interpretive statements also appear stronger than the evidence supports (e.g., describing the stage 2 bio-extension map as "geometrically accurate", despite Bayes factors that provide only anecdotal support for no difference from true length). Extensive repeated judgements to a fixed set of locations may additionally stabilise response strategies or anchoring even without feedback, complicating the separation of body-representation change from task-specific calibration.

The manuscript would also benefit from clearer conceptual framing of what the device is and what its training probes are. The device is described variably as an "artificial finger" versus a rigid "finger extension," with different implications for perception and function. In addition, the training tasks appear to emphasise manipulation and dexterity more than scenarios requiring an extended reachable workspace (indeed, participants appear to have performed at least as well, if not better, in the control training), which brings into question whether participants explored the device's intended functionality and possible proprioceptive consequences. The control experiment is thoughtfully designed to test whether functional training contributes to the stage 3 changes, but because localisation is not performed while wearing the short device, the design does not resolve whether the stage 2 change and the post-removal aftereffect are specific to the augmentative extension versus more general consequences of wearing a device on the finger (and the following possible distorted distal cues).

Finally, the immediate post-removal aftereffects are intriguing, but the mechanistic interpretation remains underspecified. As presented within the internal model framework, the magnitude and consistency of the aftereffect following brief exposure are difficult to reconcile with the stability expected from a lifetime biological finger model, and because the aftereffect is assessed only immediately after removal, its time course and functional significance remain unclear.

POetry at the eisteddfod, while with a medieval flavour, sought to use the past to legitimise themselves

The art style was also used to assert dominance - not exactly very nationalist is it

SLAYYY very important

Author response:

The following is the authors’ response to the previous reviews

Public Review:

Reviewer #1 (Public review):

Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI oncogene. Ewing sarcoma cells are addicted to this chimeric transcription factor, which represents a strong therapeutic vulnerability. Unfortunately, targeting EWS-FLI has proven to be very difficult and better understanding how this chimeric transcription factor works is critical to achieving this goal. Towards this perspective, the group had previously identified a DBD-𝛼4 helix (DBD) in FLI that appears to be necessary to mediate EWS-FLI transcriptomic activity. Here, the authors used multi-omic approaches, including CUT&tag, RNAseq, and MicroC to investigate the impact of this DBD domain. Importantly, these experiments were performed in the A673 Ewing sarcoma model where endogenous EWS-FLI was silenced, and EWS-FLI-DBD proficient or deficient isoforms were re-expressed (isogenic context). They found that the DBD domain is key to mediate EWS-FLI cis activity (at msat) and to generate the formation of specific TADs. Furthermore, cells expressing DBD deficient EWS-FLI display very poor colony forming capacity, highlighting that targeting this domain may lead to therapeutic perspectives.

This new version of the study comprises as requested new data from an additional cell line. The new data has strengthened the manuscript. Nevertheless, some of the arguments of the authors pertaining to the limitations of immunoblots to assess stability of the DBD constructs or the poor reproducibility of the Micro C data remain problematic. While the effort to repeat MicroC in a different cell line is appreciated, the data are as heterogeneous as those in A673 and no real conclusion can be drawn. The authors should tone down their conclusions. If DBD has a strong effect on chromatin organization, it should be reproducible and detectable. The transcriptomic and cut and tag data are more consistent and provide robust evidence for their findings at these levels. 

We agree that the Micro-C data have more apparent heterogeneity within and across cell lines as compared to other analyses such as our included CUT&Tag and RNA-seq. We addressed the possible limitations of the technique as well as inherent biology that might be driving these findings in our previous responses. Despite the poor clustering on the PCA plots, our analysis on differential interacting regions, TADs and loops remain consistent across both cell lines. We are confident that these findings reflect the context of transcriptional regulation by the constructs, therefore the role of the alpha-helix in modulating chromatin organization. To address the concerns raised by the editors and reviewers for the strength of the conclusions we drew from the Micro-C findings we have made changes to the language used to describe them throughout the manuscript. Find these changes outlined below.

• On lines 70-71, "is required to restructure" was changed to "is implicated in restructuring of"

• On line 91, "is required for" was changed to "participates in"

• On line 98, "is required for" changed to "is potentially required for"

• On line 360-361, "is required for restructuring" changed to "participates in restructuring"

Concerning the issue of stability of the DBD and DBD+ constructs, a simple protein half-life assay (e.g. cycloheximide chase assay) could rule out any bias here and satisfactorily address the issue.

While we generally agree that a cycloheximide assay is a relatively simple approach to look at protein half-life, as we discussed last me the assays included in this paper are performed at equilibrium and rely on the concentration of protein at the me of the assay. This is particularly true for assays involving crosslinking, like Micro-C. As discussed in our prior response, western blots are semi quantitative at best, even when normalized to a housekeeping protein. In analyzing the relative protein concentration of DBD vs. DBD+ with relative protein intensities first normalized to tubulin and using the wildtype EWSR1::FLI1 rescue as a reference point, we find that there is no statistical difference in the samples used for micro-C here (Author responseimage 1A) or across all of the samples that we have used for publication (Author response image 1B). This does show that DBD generally has more variable expression levels relative to wildtype EWSR1::FLI1, and this is consistent with our experience in the lab.

Nonetheless, we did attempt to perform the requested cycloheximide chase experiment to determine protein stability. Unfortunately, despite an extensive number of troubleshooting attempts, we have not been able to get good expression of DBD for these experiments. The first author who performed this work has left the lab and we have moved to a new lab space since the benchwork was performed. We continue to try to troubleshoot to get this experimental system for DBD and DBD+ to work again. When we tried to look at stability of DBD+ following cycloheximide treatment, there did appear to be some difference in protein stability (Author response image 2). However, these conditions are not the same conditions as those we published, they do not meet our quality control standards for publication, and we are concerned about being close to the limit of detection for DBD throughout the later timepoints. Additional studies will be needed with more comparable expression levels between DBD and DBD+ to satisfactorily address the reviewer concerns.

Author response image 1.

Expression Levels of DBD and DBD+ Across Experiments. Expression levels of DBD and DBD+ protein based on western blot band intensity normalized by tubulin band intensity. Expression levels are relative to wildtype EWSR1::FLI1 rescue levels and are calculated for (A) A673 samples used for micro-C and (B) all published studies of DBD and DBD+. P-values were calculated with an unpaired t-test.

Author response image 2.

CHX chase assay to determine the stability of DBD and DBD+. (A) Knock-down of endogenous EWSR1::FLI1 detected with FLI1 ab and rescue with DBD and DBD+ detected with FLAG ab. (B) CHX chase assay to determine the stability of DBD and DBD+ in A-673 cells with quantification of the protein levels (n=3). Error bars represent standard deviation. The half-lives (t1/2) of DBD and DBD+ were listed in the table.

Suggestions:

The Reviewing Editor and a referee have considered the revised version and the responses of the referees. While the additional data included in the new version has consolidated many conclusions of the study, the MicroC data in the new cell line are also heterogeneous and as the authors argue, this may be an inherent limitation of the technique. In this situation, the best would be for the authors to avoid drawing robust conclusions from this data and to acknowledge its current limitations.

As discussed above, we have changed the language regarding our conclusions from micro-C data to soften the conclusions we draw per the Editor’s suggestion.

The referee and Reviewing Editor also felt that the arguments of the authors concerning a lack of firm conclusions on the stability of EWS-FLI1 under +/-DBD conditions could be better addressed. We would urge the authors to perform a cycloheximide chase type assay to assess protein half-life. These types of experiments are relatively simple to perform and should address this issue in a satisfactory manner.

As discussed above, we do not feel that differences in protein stability would affect the results here because the assays performed required similar levels of protein at equilibrium. Our additional analyses in this response shows that there are not significant differences between DBD and DBD+ levels in samples that pass quality control and are used in published studies. However, we attempted to address the reviewer and editor comments with a cycloheximide chase assay and were unable to get samples that would have passed our internal quality control standards. These data may suggest differences in protein stability, but it is unclear that these conditions accurately reflect the conditions of the published experiments, or that this would matter with equal protein levels at equilibrium.

Note d'Information : Priorités de la Protection de l’Enfance et Justice des Mineurs

Synthèse de l'Exécutif

Ce document synthétise les orientations stratégiques et les réformes engagées par le ministère de la Justice pour renforcer la protection de l’enfance et moderniser la justice des mineurs.

Les points clés incluent :

• Urgence et Rapidité : Réduction des délais de jugement (passés de 18 mois à 8,7 mois en quatre ans) et création d'une ordonnance de protection provisoire permettant au procureur de statuer en 72 heures.

• Refonte du Placement : Fermeture des Centres Éducatifs Fermés (CEF) publics au profit des Unités de Placement de la Jeunesse et de l'Éducation (UJPE), mettant l'accent sur la continuité pédagogique (52 semaines/an).

• Moyens Humains Massifs : Création de 1 600 postes au ministère de la Justice, dont 50 nouveaux cabinets de juges des enfants en deux ans et 70 postes à la Protection Judiciaire de la Jeunesse (PJJ).

• Évolutions Législatives : Soutien à l'imprescriptibilité des crimes sexuels sur mineurs, à la présence obligatoire de l'avocat pour l'enfant, et volonté de réformer l'« excuse de minorité » pour les crimes les plus graves.

• Protection contre les Fléaux Modernes : Lutte contre la prostitution des mineurs (6 prostituées sur 10 sont mineures), interdiction des téléphones portables en centres de placement, et encadrement du protoxyde d'azote.

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1. Renforcement de la Protection des Enfants Victimes

Urgence Judiciaire et Mesures de Sûreté

L'accent est mis sur la nécessité d'une justice qui s'adapte au rythme de l'enfant.

• Ordonnance de protection provisoire : Un nouveau dispositif permet au procureur d'agir en 72 heures pour protéger immédiatement un mineur, avec des interdictions de contact et l'attribution provisoire du logement au parent protecteur.

Le juge dispose ensuite de 8 jours pour être saisi et de 15 jours pour statuer.

• Loi du 18 mars 2024 : Prévoit le retrait automatique de l'autorité parentale pour les parents condamnés pour crime ou violence sexuelle sur leur enfant, ainsi que l'élargissement de la suspension de l'exercice de cette autorité dès la mise en examen.

Accompagnement et Droits des Mineurs

• Avocat pour l'enfant : Soutien à la présence obligatoire d'un avocat en assistance éducative.

Une expérimentation avec les barreaux est envisagée avant une généralisation législative.

• Unités d'Accueil Pédiatrique (UAPED) : Déploiement en cours sur tout le territoire pour améliorer le recueil de la parole et le soin des victimes.

• Chiens d'assistance judiciaire : Passage de 10 à une trentaine de chiens actuellement, avec un objectif de 100 chiens (un par département) d'ici un à deux ans pour apaiser les enfants lors des procédures.

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2. Réforme de la Justice Pénale des Mineurs

Équilibre entre Sanction et Éducation

La doctrine ministérielle refuse l'opposition entre ces deux concepts.

• La sanction comme acte éducatif : « La sanction fait partie de l'éducation. La sanction toute seule n'est pas un but en soi [...] et une éducation sans aucun interdit mène au n'importe quoi. »

• Efficacité du Code de la Justice Pénale des Mineurs (CJPM) : Les délais entre les faits et la sanction ont été divisés par deux en quatre ans (8,7 mois en 2024 contre 18 mois en 2020).

Transformation des Structures de Placement

Le constat sur les Centres Éducatifs Fermés (CEF) est jugé sévère : coût élevé (30 à 50 % de plus), taux de fugue identique aux centres classiques, et déshérence éducative (seulement 5 à 10 heures de cours par semaine).

• Création des UJPE : Ces nouvelles unités fusionnent les anciens foyers et les CEF pour garantir un parcours de reconstruction pédagogique.

• Recrutement de professeurs techniques : Réouverture d'un concours pour 40 professeurs dépendant directement du ministère de la Justice afin d'assurer 26 heures de cours par semaine, 52 semaines sur 52, y compris durant les vacances scolaires.

• Santé et Addictions : Recrutement de 60 infirmiers pour pallier les carences de soins psychiatriques et de prise en charge des addictions dans les centres de placement.

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3. Moyens et Organisation de la Justice

Augmentation des Effectifs

Le budget de la Justice permet une hausse inédite des moyens humains :

• Magistrature : Création de 50 cabinets de juges des enfants supplémentaires en deux ans (notamment à Bobigny, Cambrai, Alès).

Actuellement, certains cabinets gèrent entre 400 et 500 dossiers.

• PJJ : Recréation de 70 postes, permettant de renforcer les effectifs là où ils baissaient depuis 20 ans (ex: Marseille, Île-de-France).

• Milieu Ouvert : Réaffectation de 150 éducateurs vers le milieu ouvert pour ramener la charge de travail à environ 23 dossiers par agent (contre 25 auparavant).

Unité de Commandement

Le système actuel est jugé trop fragmenté (plusieurs ministères concernés, compétences partagées avec les départements pour l'ASE).

Une volonté de meilleure coordination, voire d'unité de responsabilité, est exprimée.

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4. Enjeux de Société et Nouvelles Menaces

Violences Sexuelles et Imprescriptibilité

• Fin de la prescription : Avis favorable pour l'imprescriptibilité des crimes sexuels sur mineurs, ainsi que pour les crimes de sang (assassinats).

• Prostitution des mineurs : Un constat alarmant montre que 60 % des prostituées en France sont mineures.

Des unités dédiées au sein de la PJJ sont opérationnelles depuis trois mois pour lutter contre ce fléau et les réseaux de proxénétisme.

Sécurité Numérique et Addictions

• Interdiction des téléphones : La nouvelle circulaire de politique éducative et pénale impose l'interdiction des téléphones portables dans les chambres des centres de placement pour protéger les mineurs des prédations numériques (trafiquants, proxénètes).

• Protoxyde d'azote : Soutien à la pénalisation du transport et de l'achat en ligne (en dehors du cadre médical), alors que les intoxications ont triplé entre 2020 et 2023.

Débats sur la Responsabilité Pénale

• Excuse de minorité : Position favorable à la fin de l'automatisme de l'atténuation de peine pour les crimes les plus graves (assassinats, tortures) commis par des mineurs de 13 à 15 ans.

Cela nécessiterait une évolution constitutionnelle tout en préservant la spécialisation du jugement des mineurs.

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5. Données Clés et Statistiques

| Indicateur | Donnée Source | | --- | --- | | Délai moyen de jugement (2020) | 18 mois | | Délai moyen de jugement (2024) | 8,7 mois | | Dossiers par cabinet de juge des enfants | 400 à 500 (moyenne) | | Proportion de mineurs parmi les prostitués | 60 % | | Nombre de mineurs à l'ASE | 400 000 (dont 200 000 placés) | | Heures de cours en CEF | < 10h/semaine (contre 26h en milieu classique) | | Placements chez des tiers de confiance | < 9 % (19 000 jeunes) |

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Citations Marquantes

« L'enfant ne vit pas au rythme d'un dossier administratif ou d'un dossier judiciaire. [...] 4 mois pour un mineur c'est une vie. »

« Nous devrions pouvoir en grande partie avoir honte de la façon dont on traite une partie de ces enfants notamment à l'aide sociale à l'enfance. »

« Le placement doit protéger et pas rendre encore plus vulnérable. »

« La sanction fait partie de l'éducation. [...] Une éducation sans jamais aucun interdit mène au n'importe quoi. »

Everyone brings something valuable to the community. I love this sentences and I believe it's something to remember not only for language learning but about all aspects of community building in international education.

ABCA4 variant revealed but not target of research and doesn't seem to amount to anything Variantc.3113C>T p.(Ala1038Val)

Nature is not external to humanity and it is certainly not harmonious in the sense that everyone gets along, but ecologies function in a way where threads pulls every which way and affect every lifeform. We can prescribe that life is neutral and ecologies being "harmonious" is neutral, but we have to contend with our accountability ot each other and to the suffering of other humans and other species (read: individuals) that we can reasonably perceive as being harmed unnaturally.

I agree with this statement, I am proud of Jourdon for listening to his wife and being smart with his request. Not only is he aiming to get money, but he's doing it in a way that can be a double edge sword for the "master". this puts the master in a situation where he truly has to ponder. Dose he sacrifice his ego and put it to the side giving them their well-deserved money, or dose he reject their offer but lose the chance of them coming back.

relate the way the city is handled

We're all very sympathetic to Caliban, but he does attempt to use Miranda as a bargaining chip here, specifically selling her out for sexual purposes to Stephano. Its interesting that Miranda is made a sexual object by Caliban and Prospero, who hates Caliban for his attempted rape of her. Ferdinand isn't sexless, but is far less threatening as a white guy and prince. Prosper approves of him for those reasons, as well as the fact that he's in control, ]since he wants Ferdinand to fall in love with Miranda so he can exploit their courtship for power.

A majority of women also felt supported in their workplace, but at lower rates than those reporting feeling comfortable. Women between 18 and 24 felt most supported in their workplace, at 71 percent, which is interesting as they were the age group that felt least comfortable (73 percent).

Putting things online doesn’t automatically fix the problem. It might look like progress, but if there’s no real explanation or context, people still won’t understand what they’re looking at or why it matters. Just uploading images isn’t the same as making history truly accessible.

This part highlights the disconnect between theory and reality. Scholars debate what an archive means, but real Black historical materials are sitting on shelves and not accessible to the communities they belong to. It shows how discussion does not always translate into action.

discussed in class - it is a key point in history - changed the path of things 1769 - start of Cali missions 1869 - the year the railroad connected Cali to the rest of the country - two events that expeditated the growth of Cali.

Tidbits: - oranges, grapes, horses, cows - all came to Cali. via the Spanish (Cows brought up from Baja.) - there is agriculture before the Spanish but the variety of crops that Cali is now famous for traces back to the Spanish. - student comments on how the water changes may have affected crops (the distribution of water and its movement) -> Gastil disagrees as Spanish did not make any aqueducts or anything. They did make some dams, some aquifers.

Father Serra - short and bald, very charismatic - 'moved people,' a gifted preacher; had followers and taught other priests - speaking to physicality: walked thousands of miles. a martyr. endurance. - lived here for 15 years. contributed to the building of 9 missions in those years.

• was still a colonizer, did contribute to behavior (abuse and death of NAs). but still wanted to love a life according to God. modeling himself after GOD
• "Most idealistic man" - Gastil There is a need to discuss Serra carefully. He contributed greatly but he was still part of the problem. You have to acknowledge both! Acknowledge the nuance.
• judge actions within the time.

add to other notes later: - Wintu and Shasta not within mission system - tribes outside: were wiped out during gold rush era - mission system helped tribes survive the times (in the long run) - does not excuse mission system but they seem to have helped in some respects

Enrique's stake is a sense of identity as a Mexican American individual and safety when confronted at a border checkpoint. He was cautious and aware of the reality of his scenario. At the end, he showed greater clarity about his own identity and confidence in his voice to speak up. He had shown great change, and even though this experience wasn't pleasant, the ability to continue and speak up about a hurtful issue he was able to persevere after reflecting.

The story centers on a moment where Kathi Kinnear is telling herself what she can’t do. She is limiting her capabilities in her own mind, which is a problem she was facing in the beginning of her hardship. The real stakes are whether she will accept those limitations or challenge them. If she fails to surpass her limitations, she reinforces doubt and dwells in that state of despair. If she succeeds in pushing past her own limits, she reshapes her identity. In the end, Kathi was able to see light through these dark times in her memory and persevere past what once restrained her.

screaming! yes

this is sooooo good!

Are these the two demonstrations of the Research as Inquiry Framework ?

First phishing. Fishing with an F. There's a way to catch fish using bait like worms. It's fun, except for the worms. Fishing with a Ph isn't fun. It's scary internet stuff. Phishing is a way for bad people to catch your private details, like your bank account number or passwords. The bait they use is lies. Here's how phishing works, say you get an email. It looks like it's from someone you trust, like your bank, but it's not. It tells you to confirm your bank details or your account may be closed. Scary. You click on the link and go to a website. It looks like a real Bank website. But it's not. You enter your details and someone uses them to steal your identity and buy things with your money. Which is not nice. Here's how to be safe from fishing. Number one, your bank will never ask you to confirm your details via an email, like ever. This is the most obvious way to spot a fishing attempt if you receive an email like this, suspecting. Don't click it. Number two, look for your name. Phishing messages say things like, um, dear valued customer. If it doesn't say your name, don't click it. Number three. Look at the URL in your browser browser window if the URL looks like a different name from the name of the company. Don't click it. Number four. Rest your mouse pointer on the link that will show the real web address. If it doesn't look like the proper company name, don't click it. Number five, look for spelling mistakes. If there are any spelling mistakes or the email doesn't look professional? Don't click it. Number six, get security software that includes anti-phishing and identity protection features like Norton 360 or Symantec. Best of all, just don't use links in emails to get to websites like ever. Always type the URL instead. Thank you for looking at our quick guide to scary internet stuff to allow to be safe.

Would it surprise you to learn that millions of computers in the U.S are infected with malware? That's a lot of computers. So, what's malware and why should you care? Malware short for malicious software includes viruses and spyware that get installed on your computer or mobile device without you knowing it. Criminals use malware to steal personal information and commit fraud. For example, they may use malware to steal the login information for your online accounts or to hijack your computer and use it to send spam, an infected computer can lead to serious problems like identity theft. The good news? There's a lot you can do to protect yourself and your your computer. One of the most important steps you can take install security software from a reliable company and set it to update automatically. The bad guys constantly develop new ways to attack your computer, so your software must be up to date to work, set your operating system, and your web browser to update automatically, too. If you're not sure how, use the help function and search for automatic updates. Don't buy security software and response to unexpected calls or messages, especially if they say they scanned your computer and found malware scammers. Send messages like these to trick you into buying worthless software, or worse, downloading malware. What else can you do? Use a pop-up blocker and don't click on links and pop- ups. Don't click on links or open attachments and emails, unless you know what they are. Even if the emails seem to be from friends or family. Download software only from websites you know and trust. Free stuff may sound appealing, but free downloads can hide malware. Make sure your web browser security setting is high enough to detect unauthorized downloads. For example, use at least the medium security setting. Even if you take precautions, malware can find its way onto your computer. So, be on the lookout for these signs. Your computer runs slowly. Drains its battery quickly. Displays, unexpected errors or crashes. It won't shut down or restart. It serves a lot of pop-ups, takes you to web pages you didn't, visit changes your home page, or creates new icons or toolbars without your permission. If you suspect malware, stop doing things that require passwords or personal info. Such as online shopping or banking. Use a different computer, maybe one at work, or at your local library to change your passwords. Update your security software and run a system scan. Delete files that Flags as malware. If you can't fix the problem on your own? Get help from a professional. Your computer manufacturer or internet service provider may offer free tech support. If not, contact a company or retail store that provides tech support. Keep in mind the most important thing you can do to prevent malware is to keep your computer software up to date. And remember, it's easy to find trusted information about computer security

That's why Innovative startups are popping up around the world, solving the security problem with Biometrics. We actually use the vein patterns in your eye. Eye verify discovered that the blood vessels in the whites of your eyes are as unique as the worlds on the tips of your fingers. So the iris, many people know the iris. They expect that to be a good biometric, and it is but actually takes infrared light and an infrared camera. Which phones don't have? We use just an ordinary phone, enter my name. Gosh, this is fast. Done. Eye Verified caught the attention of Samsung Sprint and Wells Fargo. They all invested now. The Kansas City-based company is using that backing to crack two markets where security is Paramount. Number one is Enterprises. The companies want to protect access to their networks. There's a breach like every day, the second one is, you know, broadly, mobile banking or mobile financial Services. According to one forecast by 2019, five, and a half billion people will use Biometric authentication on mobile and wearable devices. MasterCard has been experimenting with biometrics, too, and recently tested an app using voice and facial recognition for 14, 000 e-commerce transactions. MasterCard also invested in Bionam. This Toronto-based startup has developed a wristband, the 79 Nymi that harnesses what may be the most secure Biometric of all your heartbeat. You look at all of the little Peaks and valleys in the shape of that waveform that's unique to you. We've now tied this to you. It's in an active State because it's no, it's still on your rest, right? And now, we can have it do stuff. For example, we can have it unlock a phone. That's the beauty of persistent identity. Imagine how seamless life would be if logging onto your email, unlocking your car, or checking into a flight where as easy as strapping on a wristband and verifying your ECG. The device does the rest is binary to kill the password. I don't know if you use that word too much, but probably.

Passwords are everywhere. We use them to access our money or Communications, even our social lives. At first, we used one password for everything, but that wasn't good enough. So, we started making our passwords even more complicated and began using password managers to organize the dozens or hundreds of the unique passwords we used. But no matter how complex our password system wise, it was never enough to prevent account takeover because all it took was one fishing email or database exploit and your password was out in in the world.

AGAIN -> citizens / participants see it as NATIONAL GOVTS FAULT FOR NEGATIVE EFFECTS OF EUROZONE CRISIS -> on both sides of creditor/debtor isle - Blame for bailouts / austerity measures in EIR and ESP are on NATIONAL GOVTS FORT CREATING THE CONDITIONS FOR SUCH BAILOUTS -> likewise in Germany / Austria on nat govts for asceding to said bailouts. (giving money too wilingly, also blame national govter IN EIR and ESP -> i.e., blaming those member sttaes for their own fuck ups and the crisis writ large).

Notes that Germany / Asutria bailed them out, but felt crisis much less oainfully (didn't need austerity measures obv)

"When examining discussions of the Eurozone crisis in our groups, the first thing to note is that discussions of responsibility for the crisis were the one topic that revealed the most profound differences between the debtor states (Spain and Ireland) and the creditor states (Germany and Austria). Our Madrid and Dublin groups agreed that the austerity measures that were imposed in both countries as a consequence of bailout conditionality had had a very real negative effect on the citizens. Nevertheless, most participants insisted that these measures were to be seen as a consequence of mistakes by domestic actors – governments, banks, society as a whole."

In Germany and Austria, see it as NATIONAL issue _> are aware of crisis, but only participate in NATIONAL issue -> wary of DOMESTIC govt handing out too much money to EU, not about steering EU itself which, again, they largely see as too complicated a task to do so while making an informed decision.

Idea is that CITIIZENS HAVE LITTLE / NO POWER OVER EU, WHILE EU ITSELF HAS WAY TOO MUCH POWER / INFLUENCE OVER NATIONAL GOVTS -> IMPACT OF CRISIS SUMMARISED

idk basically the FRAME they see the EU through is summed up in some keywords: - economics - corruption - "good idea but poor execution"

"‘Good idea, but does not work in practice’ – an assessment of this kind was made in almost all of our focus groups, and usually went unopposed. What is also characteristic about the last speaker’s statement is a framing of European integration that emphasises national perspectives or differences. As in the exchange above, these perspectives were often defined culturally, especially in Germany and Austria, but equally important in this context were references to criteria of distributive justice between member states and/or one’s own state’s ability to defend and advance its interests."

See it also as some OTHER member states' faults -> works for THOSE countries (Italy) but not for OURS -> ethnonationalist / cultural distinctions here too invoked.

Notably, in focus groups 3/4 countries say dfisillusioned w/ politics IN GENRAL as oppsoed to only EU

In either case, MOST say they have no ideas how it works, SOME take conspiratorial attitude (not by today's standards, but still).

Interesting about voter turnout for EP elections (second category of analysis) - Basically, 2014 EP elections were lowest turnout on record despite Eurozone crisis. - BUT some countries had higher turnouts (Germany increase of 5%) -> QUESTION, why? Can you not conclude that Germany, as a creditor country, DID have a more involved electorate post-Eurozone? - In any case, says this IS NOT FULLY EVIDENCE OF EP ELEC†iON DECLINE -> because Western democraciues AT LARGE are on a downward slope of declining voter participation -> AND GAP IS NARROWING BETWEEN EP ELECTIONS AND MEMBER STATES, even if both on downard trajectory - But again, only in Germany is the closing of this gap attributable (maybe) to the crisis and to INCREASED turnout in EP elections.

I think hearing someone else read it aloud to me helps me hear it the way the reader would understand it. I haven't considered this technique before but will use it moving forward.

I haven't really considered the difference between revising and editing before. I assumed they were the same but they are not!

might be able to understand it, but you can only judge it if you are of the learned - who can understand what Spenser is writing, to understand the intent and invention of it

This is a great analogy as it says how taking the easier ways makes you more reliant while in contrast doing it yourself strengthens you and makes you cable of doing it on your own.

this is a good way to describe the situation as one side gives difficultly and more satisfaction when done vs the other where there is no difficulty at all.

Support systems for women in the music industry.

Women are experiencing harassment both in real life and online on social media--not for their talent but their attractiveness and physical looks, worth is judged based on appeal and sexual ability.

The french took a more hieracical route to promote assimilation. Th French divided colonies into cantons that did not correspind to pre colonial govenemnte units, with them sometimes intetionally breaking up old poltical units. - chiefes were not selected according to customaryprocedure, but were selected based on their education, lineage or loyalty to the french. - their function was basically to be a mouth piece to the locals, maintained no judicial authrity, and they eventualluy removed

Looking at this now, I can't help but think of how it could've been very inefficient to rely on goods from trade that would take this long to obtain. Getting to the Mediterranean Sea from the Indian Ocean at this time would have taken an extremely long time as it would require going all the way around the African continent, and it would take a very long time to get to the bottom of the continent from both sides.

Example of person working with Obsidian and Claude Code. Note that it does not use the Obsidian CLI access, but its API.

Same author some months back mentioned running Claude Code on a small Hetzner VPS (but just Claude Code, no models) so he could access it from anywhere (except offline obviously).

Laying out the overarching questions to be explored in the article as the research results are discussed. Main ideas of the whole article are expressed here; preceding text is priming the audience and providing background.

Comparison video of Claude Code using Anthropics cloud models vs local models on a M4 128GB. Still a heavy lift, fans spinning, memory usage almost at full capacity. But it works. Means that for my M1 16GB a smaller model is all that works, and you need to leave room for context loading too. For one-offs like code generation and for interactive in moving contexts there's different needs.

Warm-dry sagebrush steppe is a critically important habitat for Golden Eagles, serving as a nesting habitat, hunting ground (and primary habitat for prey). The health of this ecosystem is a key indicator of the Golden Eagle population's survival.

How to school districts ensure this occurs throughout? Is it only in some buildings but not other?

Enlargement was greater and longer-lasting in the smaller spines, and I’m wondering if this would change their response to stimulation at a later time (when their size is much larger than it was originally)? However, long-lasting enlargement was much higher in small spines than in large spines. This is super interesting. I would have thought that large spines would be easier to induce enlargement in than small ones. The fact that short-term enlargement occurred in all of the small spines and a large majority of the large spines indicates that the methods used were very effective, but is there a more impactful way that we could achieve higher levels of long-term enlargement, especially in larger spines? Would this improve the function of long-term memory?

This is crazy to think about, but it makes sense! Smaller spines can become larger spines, and these larger spines are involved in long-term memory which is much harder to be altered once it’s formed. It seems that short-term memory is much more prone to influence from outside factors, before it becomes a permanent, long-term memory or is cast out by the brain as unimportant information.

how...!?

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study by Howe and colleagues investigates the role of the posterolateral cortical amygdala (plCoA) in mediating innate responses to odors, specifically attraction and aversion. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. They show that specific glutamatergic neurons in the anterior and posterior regions of plCoA are responsible for driving attraction and avoidance, respectively, and that these neurons project to distinct downstream regions, including the medial amygdala and nucleus accumbens, to control these responses.

Strengths:

The major strength of the study is the thoroughness of the experimental approach, which combines advanced techniques in neural manipulation and mapping with high-resolution molecular profiling. The identification of a topographically organized circuit in plCoA and the connection between molecularly defined populations and distinct behaviors is a notable contribution to understanding the neural basis of innate motivational responses. Additionally, the use of functional manipulations adds depth to the findings, offering valuable insights into the functionality of specific neuronal populations.

Weaknesses:

There are some weaknesses in the study's methods and interpretation. The lack of clarity regarding the behavior of the mice during head-fixed imaging experiments raises the possibility that restricted behavior could explain the absence of valence encoding at the population level.

We agree with idea that head-fixation may alter the state of the animal and the neural encoding of odor. To address this, we have provided further analysis of walking behavior during the imaging sessions, which is provided in Figure S2. Overall, we could not identify any clear patterns in locomotor behavior that are odor-specific. Moreover, when neural activity was sorted depending on the behavioral state (walking, pausing or fleeing) we didn’t observe any apparent patterns in odor-evoked neural activity. This is now discussed in the Results and Limitations sections of the manuscript.

Furthermore, while the authors employ chemogenetic inhibition of specific pathways, the rationale for this choice over optogenetic inhibition is not fully addressed, and this could potentially affect the interpretation of the results.

The rationale was logistical. First, inhibition of over a timescale of minutes is problematic with heat generation during prolonged optical stimulation. Second, our behavioral apparatus has a narrow height between the ceiling and floor, making tethering difficult. This is now explained the results section. The trade-off of using chemogenetics is that we are silencing neurons and not specific projections. However, because we find that NAc- and MeA- projecting neurons have little shared collateralization, we believe the conclusion of divergent pathways still stands. This is now discussed in the Limitations section.

Additionally, the choice of the mplCoA for manipulation, rather than the more directly implicated anterior and posterior subregions, is not well-explained, which could undermine the conclusions drawn about the topographic organization of plCoA.

We targeted the middle region of plCoA because it contains a mixture of cell types found in both the anterior and posterior plCoA, allowing us to test the hypothesis that cell types, not intra plCoA location, elicit different responses. Had we targeted the anterior or posterior regions, we would expect to simply recapitulate the result from activation of random cells in each region. As a result, we think stimulation in the middle plCoA is a better test for the contribution of cell types. We have now clarified this in the text.

Despite these concerns, the work provides significant insights into the neural circuits underlying innate behaviors and opens new avenues for further research. The findings are particularly relevant for understanding the neural basis of motivational behaviors in response to sensory stimuli, and the methods used could be valuable for researchers studying similar circuits in other brain regions. If the authors address the methodological issues raised, this work could have a substantial impact on the field, contributing to both basic neuroscience and translational research on the neural control of behavior.

Reviewer #2 (Public review):

Summary:

The manuscript by the Root laboratory and colleagues describes how the posterolateral cortical amygdala (plCoA) generates valenced behaviors. Using a suite of methods, the authors demonstrate that valence encoding is mediated by several factors, including spatial localization of neurons within the plCoA, glutamatergic markers, and projection. The manuscript shows convincingly that multiple features (spatial, genetic, and projection) contribute to overall population encoding of valence. Overall, the authors conduct many challenging experiments, each of which contains the relevant controls, and the results are interpreted within the framework of their experiments.

Strengths:

- For a first submission the manuscript is well constructed, containing lots of data sets and clearly presented, in spite of the abundance of experimental results.

- The authors should be commended for their rigorous anatomical characterizations and posthoc analysis. In the field of circuit neuroscience, this is rarely done so carefully, and when it is, often new insights are gleaned as is the case in the current manuscript.

- The combination of molecular markers, behavioral readouts and projection mapping together substantially strengthen the results.

- The focus on this relatively understudied brain region in the context is valence is well appreciated, exciting and novel.

Weaknesses:

- Interpretation of calcium imaging data is very limited and requires additional analysis and behavioral responses specific to odors should be considered. If there are neural responses behavioral epochs and responses to those neuronal responses should be displayed and analyzed.

We have now considered this, see response above.

- The effect of odor habituation is not considered.

We considered this, but we did not find any apparent differences in valence encoding as measured by the proportion of neurons with significant valence scores across trials (see Figure 1J).

- Optogenetic data in the two subregions relies on very careful viral spread and fiber placement. The current anatomy results provided should be clear about the spread of virus in A-P, and D-V axis, providing coordinates for this, to ensure readers the specificity of each sub-zone is real.

We were careful to exclude animals for improper targeting. The spread of virus is detailed in Figures S3, S8 & S9.

- The choice of behavioral assays across the two regions doesn't seem balanced and would benefit from more congruency.

The choice of the 4-quadrant assay was used because this study builds off of our prior experiments that demonstrate a role for the plCoA in innate behavior. It is noteworthy that the responses to odor seen in this assay are generally in agreement with other olfactory behavioral assays, so one wouldn’t predict a different result. Moreover, the approach and avoidance responses measured in this assay are precisely the behaviors we wish to understand. We did examine other non-olfactory behavioral readouts (Figures S3, S8), and didn’t observe any effect of manipulation of these pathways.

- Rationale for some of the choices of photo-stimulation experiment parameters isn't well defined.

The parameters for photo-stimulation were based on those used in our past work (Root et al., 2014). We used a gradient of frequency from 1-10 Hz based on the idea that odor likely exists in a gradient and this was meant to mimic a potential gradient, though we don’t know if it exists. The range in stimulation frequencies appears to align with the actual rate of firing of plCoA neurons (Iurilli et al., 2017).

Reviewer #3 (Public review):

Summary:

Combining electrophysiological recording, circuit tracing, single cell RNAseq, and optogenetic and chemogenetic manipulation, Howe and colleagues have identified a graded division between anterior and posterior plCoA and determined the molecular characteristics that distinguish the neurons in this part of the amygdala. They demonstrate that the expression of slc17a6 is mostly restricted to the anterior plCoA whereas slc17a7 is more broadly expressed. Through both anterograde and retrograde tracing experiments, they demonstrate that the anterior plCoA neurons preferentially projected to the MEA whereas those in the posterior plCoA preferentially innervated the nucleus accumbens. Interestingly, optogenetic activation of the aplCoA drives avoidance in a spatial preference assay whereas activating the pplCoA leads to preference. The data support a model that spatially segregated and molecularly defined populations of neurons and their projection targets carry valence specific information for the odors. The discoveries represent a conceptual advance in understanding plCoA function and innate valence coding in the olfactory system.

Strengths:

The strongest evidence supporting the model comes from single cell RNASeq, genetically facilitated anterograde and retrograde circuit tracing, and optogenetic stimulation. The evidence clear demonstrates two molecularly defined cell populations with differential projection targets. Stimulating the two populations produced opposite behavioral responses.

Weaknesses:

There are a couple of inconsistencies that may be addressed by additional experiments and careful interpretation of the data.

Stimulating aplCoA or slc17a6 neurons results in spatial avoidance, and stimulating pplCoA or slc17a7 neurons drives approach behaviors. On the other hand, the authors and others in the field also show that there is no apparent spatial bias in odor-driven responses associated with odor valence. This discrepancy may be addressed better. A possibility is that odor-evoked responses are recorded from populations outside of those defined by slc17a6/a7. This may be addressed by marking activated cells and identifying their molecular markers. A second possibility is that optogenetic stimulation activates a broad set of neurons that and does not recapitulate the sparseness of odor responses. It is not known whether sparsely activation by optogenetic stimulation can still drive approach of avoidance behaviors.

We agree that marking specific genetic or projection defined neurons could help to clarify if there are some neurons have more selective valence responses. However, we are not able to perform these experiments at the moment. We have included new data demonstrating that sparser optogenetic activation evokes behaviors similar in magnitude as the broader activation (see Figure S4).

The authors show that inhibiting slc17a7 neurons blocks approaching behaviors toward 2-PE. Consistent with this result, inhibiting NAc projection neurons also inhibits approach responses. However, inhibiting aplCOA or slc17a6 neurons does not reduce aversive response to TMT, but blocking MEA projection neurons does. The latter two pieces of evidence are not consistent with each other. One possibility is that the MEA projecting neurons may not be expressing slc17a6. It is not clear that the retrogradely labeling experiments what percentage of MEA- and NACprojecting neurons express slc17a6 and slc17a7. It is possible that neurons expressing neither VGluT1 nor VGluT2 could drive aversive or appetitive responses. This possibility may also explain that silencing slc17a6 neurons does not block avoidance.

We have now performed RNAscope staining on retrograde tracing to better define this relationship. Although the VGluT1 and VGluT2 neurons have biased projections to the MeA and NAc, respectively, there is some nuance detailed in Figure S10. Generally, MeA projecting neurons are predominately VGluT2+, whereas NAc projecting have about 20% that express both. Some (less than 35%) retrogradely labeled neurons were not detected as VGluT1 or VGluT2 positive, suggesting that other populations could also contribute. We agree that the discrepancy between MeA-projection and VGluT2 silencing is likely due to incomplete targeting of the MeA-projecting population with the VGluT2-cre line. This is included in the Discussion section.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Main:

(1) For the head-fixed imaging experiments, what is the behavior of the mice during odor exposure? Could the weak reliability of individual neurons be due to a lack of approach or avoidance behavior? Could restricted behavior also explain the lack of valence encoding at the population level?

We agree that this is a limitation of head-fixed recordings. In the revised manuscript we did attempt to characterize their behavioral response, and look for correlations in odor representation. Although we did find different patterns of odor-evoked walking behavior, these patterns were not reliable or specific to particular odors (Figure S2). For example, one might expect aversive odors to pause walking or elicit a fast fleeing-like response, but we did not observe any apparent differences for locomotion between odors as all odors evoked a mixture of responses (Figure S2A-D, text lines 208-232). We then examined responses to odor depending on the behavioral state (walking, pausing or fleeing) and didn’t observe any apparent patterns in odor responses (Figure S2E,F). Lastly, we acknowledge in the text that the lack of valence encoding may be an artifact of head-fixation (see lines 849-857).

(2) For the optogenetic manipulations of Vglut1 and Vglut2 neurons, why was the injection and fiber targeted to the medial portion of the plCoA, if the hypothesis was that these glutamatergic neuron populations in different regions (anterior or posterior) are responsible for approach and avoidance? 

We targeted the middle region of plCoA because it contains a mixture of cell types found in both the anterior and posterior plCoA, allowing us to test the hypothesis that cell types, not intraplCoA location, elicit different responses. Had we targeted the anterior or posterior regions, we would expect to simply recapitulate the result from activation of random cells in each region. As a result, we think stimulation in the middle plCoA is a better test for the contribution of cell types. We have clarified this in the text (Lines 417-419).

Could this explain the lack of necessity with the DREADD experiments? 

For the loss of function experiments, a larger volume of virus was injected to cover a larger area and we did confirm targeting of the appropriate areas. Though, it is always possible that the lack of necessity is due to incomplete silencing.

Further, why was an optogenetic inhibition approach not utilized? 

Although optogenetic inhibition could have plausibly been used instead, we chose chemogenetic inhibition for two reasons: First, for minutes-long periods of inhibition, optical illumination poses the risk of introducing heat related effects (Owen et al., 2019). In fact, we first tried optical inhibition but controls were exhibited unusually large variance. Second, it is more feasible in our assay as it has a narrow height between the floor and lid that complicates tethering to an optic fiber. Past experiments overcame this with a motorized fiber retraction system (Root et al., 2014), but this is highly variable with user-dependent effects, so we found chemogenetics to be a more practical strategy. We have added a sentence to explain the rationale (see lines 561-563).

(3) The specific subregion of the nucleus accumbens that was targeted should be named, as distinct parts of the nucleus accumbens can have very different functions. 

We attempted to define specific subregions of the nucleus accumbens and found that plCoA projection is not specific to the shell or core, anterior or posterior, rather it broadly innervates the entire structure. We have added a note about this in manuscript (see lines 470-471). Given that we did not find notable subregion-specific outputs within the NAc, targeting was directed to the middle region of NAc, with coordinates stated in the methods. 

(4) Why was an intersectional DREADD approach used to inhibit the projection pathways, as opposed to optogenetic inhibition? The DREADD approach could potentially affect all projection targets, and the authors might want to address how this could influence the interpretation of the results.

This is partly addressed above in point 2. As for interpretation, we acknowledge that the intersectional approach silences the neurons projecting to a given target and not the specific projection and we have been careful with the wording. Although this may complicate the conclusion, we did map the collaterals for NAc and MeA projecting neurons and find that neurons do not appreciably project to both targets and have minimal projections to other targets. We have now taken care to state that we silence the neurons projecting to a structure, not silencing the projection, and we acknowledge this caveat. However, since the MeA- and NAcprojecting neurons appear to be distinct from each other (largely not collateralizing to each other), the conclusion that these divergent pathways are required still stands. We have added discussion of this in the Limitations section (see lines 859-863).

Minor:

(1) Line 402 needs a reference.

We have added the missing reference (now line 441).

(2) The Supplemental Figure labeling in the main text should be checked carefully.

Thank you for pointing this out. We have fixed the prior errors.

(3) Panel letter D is missing from Figure 2.

This has been fixed.

Reviewer #2 (Recommendations for the authors):

Major Concerns, additional experiments:

- In the calcium imaging experiments mice were presented with the same odor many times. Overall responses to odor presentations were quite variable and appear to habituate dramatically (Figure S1F). The general conclusion from these experiments are a lack of consistent valence-specific responses of individual neurons, but I wonder if this conclusion is slightly premature. A few potential explanatory factors that may need additional attention are: -First, despite recording video of the mouse's face during experiments, no behavioral response to any odor is described. Is it possible these odors when presented in head-fixed conditions do not have the same valence?

Yes, we agree that this is a possibility. We have added a discussion in the Limitations section (see lines 849-857). We have also added additional behavioral analysis discussed below.

On trials with neural responses are there behavioral responses that could be quantified? 

We have now added data in which we attempt to characterize their behavioral response, to look for correlations in odor representation (see lines 208-228). Although we did observe different patterns of odor-evoked walking behavior, these patterns were not reliable or specific to particular odors (Figure S2). One might expect aversive odors to pause walking or elicit a fast fleeing-like response, but we did not observe any apparent differences for locomotion between odors (Figure S2A-D). Next, we examined responses to odor depending on the behavioral state (walking, pausing or fleeing) and didn’t observe any meaningful differences in odor responses (Figure S2E,F). Lastly, we acknowledge that the odor representation may be different in freely moving animals that exhibit dynamic responses to odor (see lines 859-857).

- Habituation seems to play a prominent role in the neural signals, is there a larger contribution of valence if you look only at the first delivery (or some subset of the 20 presentations) of an odor type for a given trial? 

Indeed, we considered this, but we did not find any apparent differences in valence encoding as measured by the proportion of neurons with significant valence scores across trials (see Figure 1J).

- Is it reasonable to exclude valence encoding as a possibility when largely neurons were unresponsive to the positive valence odors (2PE and peanut) chosen when looking at the average cluster response (Figure 1F)? 

It is true that we see fewer neurons responding to the appetitive odors (Figure 1H) and smaller average responses within the cluster, but some neurons do respond robustly. If these were valence responses, we would predict that neural responses should be similarly selective, but we do not observe any such selectivity. The sparseness of responses to appetitive odors does cause the average cluster analysis (Figure 1F) to show muted responses to these odors, consistent with the decreased responsivity to appetitive odors. Moreover, single neuron response analysis reveals that a given neuron is not more likely to respond to appetitive or aversive odors with any selectivity greater than chance. For these reasons, we think it is reasonable to conclude an absence of valence responses, which is consistent with the conclusion from another report (Iurilli et al., 2017).

- While the preference and aversion assay with 4 corners is an interesting set-up and provides a lot of data for this particular manuscript. It would be helpful to test additional behaviors to determine whether these circuits are more conserved. As it stands the current manuscript relies on very broad claims using a single behavioral readout. Some attempts to use head-fixed approaches with more defined odor delivery timelines and/or additional valenced behavioral readouts is warranted.

We appreciate the suggestion, but are not able to perform these experiments at the moment. The choice of the 4-quadrant assay was used because it built off of our prior experiments that demonstrate a role for the plCoA in innate behavior. It is noteworthy that the responses to odor seen in this assay are generally in agreement with other olfactory behavioral assays, so one wouldn’t predict a different result. The approach and avoidance responses measured in this assay are precisely the behaviors we wish to understand. Moreover, we did examine other nonolfactory behavioral readouts (Figures S3, S8), and didn’t observe any effect of manipulation of these pathways. Lastly, we have tried to define parameters for head-fixed behavior that would permit correlation of neural responses with behavior, including longer stimulations and closed loop locomotion control of odor concentration, but were unsuccessful at establishing parameters that generated reliable behavioral responses. We acknowledge that one limitation of the study is the limited behavioral tests with two odors and whether the circuits are more broadly necessary for other odors. 

Minor comments:

• Please define PID in the Results when it is first introduced.

Done (see line 154)

• Line 412 Figure S5C-N should be Figure S6C-N.

Fixed. Now Figure S8C-N due to additional figures (see line 451).

• Throughout the Discussion it would be helpful if the authors referred to specific Figure panels that support their statements (e.g. lines 654-656 "[...] which is supported by other findings presented here showing that both VGluT2+ and VGluT1+ neurons project to MeA, while the projection to NAc is almost entirely composed of VGluT1+ neurons".

Thank you for the suggestion. We have added figure references in the discussion.

• Line 778 "producing" should be "produce".

Corrected (see line 840)

• The figures are very busy, especially all the manipulations. The authors are commended for including each data point, but they might consider a more subtle design (translucent lines only for each animal, and one mean dot for the SEM), just to reduce the overall clutter of an already overwhelming figure set. But this is ultimately left to the authors to resolve and style to their liking. 

Thank you for the suggestion. We have tried some different styles but like the original best.

Reviewer #3 (Recommendations for the authors):

If within reach, I suggest that the author determine the percentage of retrogradely labeled neurons to NAc or MEA that expresses GluT1 and GluT2. 

We have done this for the middle region plCoA that has the greatest mixture of cell types (See Figure S10, lines 504-517). We find that the MeA projecting neurons are mostly VGluT2+ with a minority that express both VGluT1 and VGlut2. NAc-projecting neurons are primarily VGluT1+ with about 20% expressing VGlut2 as well.

It would also be nice to sparse label of aplCoA and pplCoA using ChR2 to see if sparse activation drives approach or avoidance. 

We agree that it would be useful to vary the sparseness of the ChR2 expression, to see if produces similar results. We examined this using sparsely labeled odor ensembles, as previously done (Root et al., 2014). Briefly, we used the Arc-CreER mouse to label TMT responsive neurons with a cre-dependent ChR2 AAV vector targeted to the anterior or posterior regions, while previously we had broadly targeted the entirety of plCoA. We had established that this labeling method captures about half of the active cells detected by Arc expression, which is on the order of hundreds of neurons rather than thousands by broad cre-independent expression. Remarkably, we get effects similar in magnitude that are not significantly different from that with broader activation of the anterior or posterior domains (see new Figure S4, lines 267-288). It still remains possible that there is a threshold number of neurons that are necessary to elicit behavior, but that is beyond the scope of the current study. However, these data indicate that the effect of activating anterior and posterior domains is not an artifact of broad stimulation.

I think it's a bit of an exaggeration demonizing AI but I think we already know it's not alive and it says its happy to see us. It's like dora the explora saying how's your day going its just a cartoon and behind it is a voice actor that will never know you and your struggles in life.

I've heard that they got there data to train the AI illegally, but I don't think that's our problem I think that's between them and the person they stole from. As long as you inspect sources they use and credit them then that's not your issue. I think sometimes we dig deep into stuff thats none of our buisness. There are a lot of sketchy things going in the world and if we focus too much on its orgin we would die from boycotting everything. It's like if your boss does something illegally to get his money but your just working a normal job as long you don't use that money in a bad way then its none of your buisness what he does because your not directly assisting his act.

I guess there are details that you can find is profound that you won't find AI doing since it's mostly taking whats common but whose to say that It wont in the future make the small decisions we do. At the end of the day when we draw its from our in expereince in life and Ai just observing our observations

Reviewer #1 (Public review):

Summary:

This study set out to investigate potential pharmacological drug-drug interactions between the two most common antimalarial classes, the artemisinins and quinolines. There is strong rationale for this aim, because drugs from these classes are already widely-used in Artemisinin Combination Therapies (ACTs) in the clinic, and drug combinations are an important consideration in the development of new medicines. Furthermore, whilst there is ample literature proposing many diverse mechanisms of action and resistance for the artemisinins and quinolines, it is generally accepted that the mechanisms for both classes involve heme metabolism in the parasite, and that artemisinin activity is dependent on activation by reduced heme. The study was designed to measure drug-drug interactions associated with a short pulse exposure (4 h) that is reminiscent of the short duration of artemisinin exposure obtained after in vivo dosing. Clear antagonism was observed between dihydroartemisinin (DHA) and chloroquine, which became even more extensive in chloroquine-resistant parasites. Antagonism was also observed in this assay for the more clinically-relevant ACT partner drugs piperaquine and amodiaquine, but not for other ACT partners mefloquine and lumefantrine, which don't share the 4-aminoquinoline structure or mode of action. Interestingly, chloroquine induced an artemisinin resistance phenotype in the standard in vitro Ring-stage Survival Assay, whereas this effect was not as extensive for piperaquine.

The authors also utilised a heme-reactive probe to demonstrate that the 4-aminoquinolines can inhibit heme-mediated activation of the probe within parasites, which suggests that the mechanism of antagonism involves the inactivation of heme, rendering it unable to activate the artemisinins. Measurement of protein ubiquitination showed reduced DHA-induced protein damage in the presence of chloroquine, which is also consistent with decreased heme-mediated activation, and/or with decreased DHA activity more generally.

Overall, the study clearly demonstrates a mechanistic antagonism between DHA and 4-aminoquinoline antimalarials in vitro. It is interesting that this combination is successfully used to treat millions of malaria cases every year, which may raise questions about the clinical relevance of this finding. However, the conclusions in this paper are supported by multiple lines of evidence and the data is clearly and transparently presented, leaving no doubt that DHA activity is compromised by the presence of chloroquine in vitro. It is perhaps fortunate the that the clinical dosing regimens of 4-aminoquinoline-based ACTs have been sufficient to maintain clinical efficacy despite the non-optimal combination. Nevertheless, optimisation of antimalarial combinations and dosing regimens is becoming more important in the current era of increasing resistance to artemisinins and 4-aminoquinolines. Therefore, these findings should be considered when proposing new treatment regimens (including Triple-ACTs) and the assays described in this study should be performed on new drug combinations that are proposed for new or existing antimalarial medicines.

Strengths:

This manuscript is clearly written and the data presented is clear and complete. The key conclusions are supported by multiple lines of evidence, and most findings are replicated with multiple drugs within a class, and across multiple parasite strains, thus providing more confidence in the generalisability of these findings across the 4-aminoquinoline and peroxide drug classes.

A key strength of this study was the focus on short pulse exposures to DHA (4 h in trophs and 3 h in rings), which is relevant to the in vivo exposure of artemisinins. Artemisinin resistance has had a significant impact on treatment outcomes in South-East Asia, and is now emerging in Africa, but is not detected using a 'standard' 48 or 72 h in vitro growth inhibition assay. It is only in the RSA (a short pulse of 3-6 h treatment of early ring stage parasites) that the resistance phenotype can be detected in vitro. Therefore, assays based on this short pulse exposure provide the most relevant approach to determine whether drug-drug interactions are likely to have a clinically-relevant impact on DHA activity. These assays clearly showed antagonism between DHA and 4-aminoquinolines (chloroquine, piperaquine, amodiaquine and ferroquine) in trophozoite stages. Interestingly, whilst chloroquine clearly induced an artemisinin-resistant phenotype in the RSA, piperaquine only had a minor impact on the early ring stage activity of DHA, which may be fortunate considering that piperaquine is a currently recommended DHA partner drug in ACTs, whereas chloroquine is not.

The evaluation of additional drug combinations at the end of this paper is a valuable addition, which increases the potential impact of this work. The finding of antagonism between piperaquine and OZ439 in trophozoites is consistent with the general interactions observed between peroxides and 4-aminoquinolines, and it may be interesting to see whether piperaquine impacts the ring-stage activity of OZ439.

The evaluation of reactive heme in parasites using a fluorescent sensor, combined with the measurement of K48-linked ubiquitin, further support the findings of this study, providing independent read-outs for the chloroquine-induced antagonism.<br /> The in-depth discussion of the interpretation and implications of the results are an additional strength of this manuscript. Whilst the discussion section is rather lengthy, there are important caveats to the interpretation of some of these results, and clear relevance to the future management of malaria that require these detailed explanations.

Overall, this is a high quality manuscript describing an important study that has implications for the selection of antimalarial combinations for new and existing malaria medicines.

Weaknesses:

This study is an in vitro study of parasite cultures, and therefore caution should be taken when applying these findings to decisions about clinical combinations. The drug concentrations and exposure durations in these assays are intended to represent clinically relevant exposures, although it is recognised that the in vitro system is somewhat simplified and there may be additional factors that influence in vivo activity. This limitation is reasonably well acknowledged in the manuscript.

It is also important to recognise that the majority of the key findings regarding antagonism are based on trophozoite-stage parasites, and one must show caution when generalising these findings to other stages or scenarios. For example, piperaquine showed clear antagonism in trophozoite stages, but minimal impact in ring stages under these assay conditions.

A key limitation is the interpretation of the mechanistic studies that implicate heme-mediated artemisinin activation as the mechanism underpinning antagonism by chloroquine. This study did not directly measure the activation of artemisinins. The data obtained from the activation of the fluorescent probe are generally supportive of chloroquine suppressing the heme-mediated activation of artemisinins, and I think this is the most likely explanation, but there are significant caveats to consider. Primarily, the inconsistency between the fluorescence profile in the chemical reactions and the cell-based assay raise questions about the accuracy of this readout. In the chemical reaction, mefloquine and chloroquine showed identical inhibition of fluorescence, whereas piperaquine had minimal impact. On the contrary, in the cell, chloroquine and piperaquine had similar impacts on fluorescence, but mefloquine had minimal impact. This inconsistency indicates that the cellular fluorescence based on this sensor does not give a simple direct readout of the reactivity of ferrous heme, and therefore, these results should be interpreted with caution. Indeed, the correlation between fluorescence and antagonism for the tested drugs is a correlation, not causation. There could be several reasons for the disconnect between the chemical and biological results, either via additional mechanisms that quench fluorescence, or the presence of biomolecules that alter the oxidation state or coordination chemistry of heme or other potential catalysts of this sensor. It is possible that another factor that influences the H-FluNox fluorescence in cells also influences the DHA activity in cells, leading to the correlation with activity. It should be noted that H-FluNox is not a chemical analogue of artemisinins. It's activation relies on Fenton-like chemistry, but with a N-O rather that O-O bond, and it possesses very different steric and electronic substituents around the reactive centre, which are known to alter reactivity to different iron sources. Despite these limitations, the authors have provided reasonable justification for the use of this probe to directly visualise heme reactivity in cells, and the results are still informative.

Another interesting finding that was not elaborated by the authors is the impact of chloroquine in the DHA dose-response curves from the ring stage assays. Detection of artemisinin resistance in the RSA generally focuses on the % survival at high DHA concentrations (700 nM) as there is minimal shift in the IC50 (see Fig 2), however, chloroquine clearly induces a shift in the IC50 (~5-fold), where the whole curve is shifted to the right, whereas the increase in % survival is relatively small. This different profile suggests that the mechanism of chloroquine-induced antagonism may be different to the mechanism of artemisinin resistance. Current evidence regarding the mechanism of artemisinin resistance generally points towards decreased heme-mediated drug activation due to a decrease in hemoglobin uptake, which should be analogous to the decrease in heme-mediated drug activation caused by chloroquine. However, these different dose response curves suggest different mechanisms are primarily responsible. Additional mechanisms have been proposed for artemisinin resistance, involving redox or heat stress responses, proteostatic responses, mitochondrial function, dormancy and PI3K signalling among others. Whilst the H-FluNox probe generally supports the idea that chloroquine suppresses heme-mediated DHA activation, it remains plausible that chloroquine could induce these, or other, cellular responses that suppress DHA activity.

Impact:

This study has important implications for the selection of drugs to form combinations for the treatment of malaria. The overall findings of antagonism between peroxide antimalarials and 4-aminoquinolines in the trophozoite stage are robust, and the this carries across to the ring stage for chloroquine.

The manuscript also provides a plausible mechanism to explain the antagonism, although future work will be required to further explore the details of this mechanism and to rule out alternative factors that may contribute.

Overall, this is an important contribution to the field and provides a clear justification for the evaluation of potential drug combinations in relevant in vitro assays before clinical testing.

Reviewer #2 (Public review):

Summary:

This manuscript by Rosenthal and Goldberg investigates interactions between artemisinins and its quinoline partner drugs currently used for treating uncomplicated Plasmodium falciparum malaria. The authors show that chloroquine (CQ), piperaquine, and amodiaquine antagonize dihydroartemisinin (DHA) activity, and in CQ-resistant parasites, the interaction is described as "superantagonism," linked to the pfcrt genotype. Mechanistically, application of the heme-reactive probe H-FluNox indicates that quinolines render cytosolic heme chemically inert, thereby reducing peroxide activation. The work is further extended to triple ACTs and ozonide-quinoline combinations, with implications for artemisinin-based combination therapy (ACT) design, including triple ACTs.

Strengths:

The manuscript is clearly written, methodologically careful, and addresses a clinically relevant question. The pulsing assay format more accurately models in vivo artemisinin exposure than conventional 72-hour assays, and the use of H-FluNox and Ac-H-FluNox probes provides mechanistic depth by distinguishing chemically active versus inert heme. These elements represent important refinements beyond prior studies, adding nuance to our understanding of artemisinin-quinoline interactions.

Weaknesses:

Several points warrant consideration. The novelty of the work is somewhat incremental, as antagonism between artemisinins and quinolines is well established. Multiple prior studies using standard fixed-ratio isobologram assays have shown that DHA exhibits indifferent or antagonistic interactions with chloroquine, piperaquine, and amodiaquine (e.g., Davis et al., 2006; Fivelman et al., 2007; Muangnoicharoen et al., 2009), with recent work highlighting the role of parasite genetic background, including pfcrt and pfmdr1, in modulating these interactions (Eastman et al., 2016). High-throughput drug screens likewise identify quinoline-artemisinin combinations as mostly antagonistic. The present manuscript adds refinement by applying pulsed-exposure assays and heme probes rather than establishing antagonism de novo.

The dataset focuses on several parasite lines assayed in vitro, so claims about broad clinical implications should be tempered, and the discussion could more clearly address how in vitro antagonism may or may not translate to clinical outcomes. The conclusion that artemisinins are predominantly activated in the cytoplasm is intriguing but relies heavily on Ac-H-FluNox data, which may have limitations in accessing the digestive vacuole and should be acknowledged explicitly. The term "superantagonism" is striking but may appear rhetorical; clarifying its reproducibility across replicates and providing a mechanistic definition would strengthen the framing. Finally, some discussion points, such as questioning the clinical utility of DHA-PPQ, should be moderated to better align conclusions with the presented data while acknowledging the complexity of in vivo pharmacology and clinical outcomes.

Despite these mild reservations, the data are interesting and of high quality and provide important new information for the field.

Editor's Review of the Revision: The authors have provided a well-reasoned rebuttal to the comments of the three reviewers. Most of the changes were incorporated in their revised Discussion. Their data with the active heme probe H-FluNox are novel and the authors reveal interesting interactions between peroxide and 4-aminoquinoline-based antimalarials that open new avenues of research especially when considering antimalarial combinations that combine these chemical scaffolds. This study will be of broad interest to investigators studying and developing antimalarial drugs and combinations and the impact of Plasmodium falciparum resistance mechanisms. A minor recommendation would be that the authors state H-FluNox when referring to their small molecule probe in the abstract, so that it is captured in PubMed searches.

Reviewer #3 (Public review):

Summary:

The authors present an in vitro evaluation of drug-drug interactions between artemisinins and quinoline antimalarials, as an important aspect for screening the current artemisinin-based combination therapies for Plasmodium falciparum. Using a revised pulsing assay, they report antagonism between dihydroartemisinin (DHA) and several quinolines, including chloroquine, piperaquine (PPQ), and amodiaquine. This antagonism is increased in CQ-resistant strains in isobologram analyses. Moreover, CQ co-treatment was found to induce artemisinin resistance even in parasites lacking K13 mutations during the ring-stage survival assay. This implies that drug-drug interactions, not just genetic mutations, can influence resistance phenotypes. By using a chemical probe for reactive heme, the authors demonstrate that quinolines inhibit artemisinin activation by rendering cytosolic heme chemically inert, thereby impairing the cytotoxic effects of DHA. The study also observed negative interactions in triple-drug regimens (e.g., DHA-PPQ-Mefloquine) and in combinations involving OZ439, a next-generation peroxide antimalarial. Taken together, these findings raise significant concerns regarding the compatibility of artemisinin and quinoline combinations, which may promote resistance or reduce efficacy.

With the additive profile as the comparison and a lack of synergistic effect in any of the comparisons, it is hard to contextualize the observed antagonism. Including a known synergistic pair (e.g., artemisinin + lumefantrine) would have provided a useful benchmark to assess the relative impact of the drug interactions described.

Strengths:

This study demonstrates the following strengths:

• The use of a pulsed in vitro assay that is more physiologically relevant over the traditional 48h or 72h assays

• Small molecule probes, H-FluNox, and Ac-H-FluNox to detect reactive cytosolic heme, demonstrating that quinolines render heme inert and thereby block DHA activation.

• Evaluates not only traditional combinations but also triple-drug combinations and next-generation artemisinins like OZ439. This broad scope increases the study's relevance to current treatment strategies and future drug development.

• By using the K13 wild-type parasites, the study suggests that resistance phenotypes can emerge from drug-drug interactions alone, without requiring genetic resistance markers.

Weaknesses:

• The study would benefit from a future characterization of the molecular basis for the observed heme inactivation by quinolines to support this hypothesis - while the probe experiments are valuable, they do not fully elucidate how quinolines specifically alter heme chemistry at the molecular level.

• Suggestion of alternative combinations that show synergy could have improved the significance of the work. The invitro study did not include pharmacokinetic/pharmacodynamic modeling, hence it leaves questions about how the observed antagonism would manifest under real-world dosing conditions, necessitating furture work based on these findings.

Author response:

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

eLife Assessment

We appreciate the positive assessment. We recognize that since all of the work in this manuscript was done in vitro, there are reasonable concerns about the translatability of these data to clinical settings. These results should not directly inform malaria policy, but we hope that these data bring new considerations to the approach for choosing strategic antimalarial combinations. We have modified the manuscript to clarify this distinction.

Public Reviews

Reviewer #1 (Public Review):

We thank the reviewer for their thoughtful summary of this manuscript. It is important to note that DHA-PPQ did show antagonism in RSAs. In this modified RSA, 200 nM PPQ alone inhibited growth of PPQ-sensitive parasites approximately 20%. If DHA and PPQ were additive, then we would expect that addition of 200 nM PPQ would shift the DHA dose response curve to the left and result in a lower DHA IC50. Please refer to Figure 4a and b as examples of additive relationships in dose-response assays. We observed no significant shift in IC50 values between DHA alone and DHA + PPQ. This suggests antagonism, albeit not to the extent seen with CQ. We have modified the manuscript to emphasize this point. As the reviewer pointed out, it is fortunate that despite being antagonistic, clinically used artemisinin-4-aminoquinoline combinations are effective, provided that parasites are sensitive to the 4-aminoquinoline. It is possible that superantagonism is required to observe a noticeable effect on treatment efficacy (Sutherland et al. 2003 and Kofoed et al. 2003), but that classical antagonism may still have silent consequences. For example, if PPQ blocks some DHA activation, this might result in DHA-PPQ acting more like a pseudo-monotherapy. However, as the reviewer pointed out, while our data suggest that DHA-PPQ and AS-ADQ are “non-optimal” combinations, the clinical consequences of these interactions are unclear. We have modified the manuscript to emphasize the later point.

While the Ac-H-FluNox and ubiquitin data point to a likely mechanism for DHA-quinoline antagonism, we agree that there are other possible mechanisms to explain this interaction.  We have addressed this limitation in the discussion section. Though we tried to measure DHA activation in parasites directly, these attempts were unsuccessful. We acknowledge that the chemistry of DHA and Ac-H-FluNox activation is not identical and that caution should be taken when interpreting these data. Nevertheless, we believe that Ac-H-FluNox is the best currently available tool to measure “active heme” in live parasites and is the best available proxy to assess DHA activation in live parasites. These points are now addressed in the discussion section. Both in vitro and in parasite studies point to a roll for CQ in modulating heme, though an exact mechanism will require further examination. Similar to the reviewer, we were perplexed by the differences observed between in vitro and in parasite assays with PPQ and MFQ. We proposed possible hypotheses to explain these discrepancies in the discussion section. Interestingly, our data corelate well with hemozoin inhibition assays in which all three antimalarials inhibit hemozoin formation in solution, but only CQ and PPQ inhibit hemozoin formation in parasites. In both assays, in-parasite experiments are likely to be more informative for mechanistic assessment.

It remains unclear why K13 genotype influences RSA values, but not early ring DHA IC50 values. In K13^WT parasites, both RSA values and DHA IC50 values were increased 3-5 fold upon addition of CQ. This suggests that CQ-mediated resistance is more robust than that conferred by K13 genotype. However, this does not necessarily suggest a different resistance mechanism. We acknowledge that in addition to modulating heme, it is possible that CQ may enhance DHA survival by promoting parasite stress responses. Future studies will be needed to test this alternative hypothesis. This limitation has been acknowledged in the manuscript. We have also addressed the reviewer’s point that other factors, including poor pharmacokinetic exposure, contributed to OZ439-PPQ treatment failure.

Reviewer #2 (Public Review):

We appreciate the positive feedback. We agree that there have been previous studies, many of which we cited, assessing interactions of these antimalarials. We also acknowledge that previous work, including our own, has shown that parasite genetics can alter drug-drug interactions. We have included the author’s recommended citations to the list of references that we cited. Importantly, our work was unique not only for utilizing a pulsing format, but also for revealing a superantagonistic phenotype, assessing interactions in an RSA format, and investigating a mechanism to explain these interactions. We agree with the reviewer that implications from this in vitro work should be cautious, but hope that this work contributes another dimension to critical thinking about drug-drug interactions for future combination therapies. We have modified the manuscript to temper any unintended recommendations or implications.

The reviewer notes that we conclude “artemisinins are predominantly activated in the cytoplasm”. We recognize that the site of artemisinin activation is contentious. We were very clear to state that our data combined with others suggest that artemisinins can be activated in the parasite cytoplasm. We did not state that this is the primary site of activation. We were clear to point out that technical limitations may prevent Ac-H-FluNox signal in the digestive vacuole, but determined that low pH alone could not explain the absence of a digestive vacuole signal.

With regard to the “reproducibility” and “mechanistic definition” of superantagonism, we observed what we defined as a one-sided superantagonistic relationship for three different parasites (Dd2, Dd2 PfCRT^Dd2, and Dd2 K13^R539T) for a total of nine independent replicates. In the text, we define that these isoboles are unique in that they had mean ΣFIC50 values > 2.4 and peak ΣFIC50 values >4 with points extending upward instead of curving back to the axis. As further evidence of the reproducibility of this relationship, we show that CQ has a significant rescuing effect on parasite survival to DHA as assessed by RSAs and IC50 values in early rings.

Reviewer #3 (Public Review):

We thank the reviewer for their positive feedback. We acknowledge that no combinations tested in this manuscript were synergistic. However, two combinations, DHA-MFQ and DHA-LM, were additive, which provides context for contextualizing antagonistic relationships. We have previously reported synergistic and additive isobolograms for peroxide-proteasome inhibitor combinations using this same pulsing format (Rosenthal and Ng 2021). These published results are now cited in the manuscript.

We believe that these findings are specific to 4-aminoquinoline-peroxide combinations, and that these findings cannot be generalized to antimalarials with different mechanisms of action. Note that the aryl amino alcohols, MFQ and LM, were additive with DHA. Since the mechanism of action of MFQ and LM are poorly understood, it is difficult to speculate on a mechanism underlying these interactions.

We agree with the reviewer that while the heme probe may provide some mechanistic insight to explain DHA-quinoline interactions, there is much more to learn about CQ-heme chemistry, particularly within parasites.

The focus of this manuscript was to add a new dimension to considerations about pairings for combination therapies. It is outside the scope of this manuscript to suggest alternative combinations. However, we agree that synergistic combinations would likely be more strategic clinically.

An in vitro setup allows us to eliminate many confounding variables in order to directly assess the impact of partner drugs on DHA activity. However, we agree that in vivo conditions are incredibly more complex, and explicitly state this.

We agree that in the future, modeling studies could provide insight into how antagonism may contribute to real-world efficacy. This is outside the scope of our studies.

Recommendations for the Authors:

Reviewer #1 (Recommendations for the Authors):

The key weaknesses identified in this manuscript are described in the 'weaknesses' section of the public review. The major one is the inconsistency around the H-FluNox response in the chemical vs biological experiments. I can't think of a simple experiment to resolve this issue, but it is good that this data is openly provided in the manuscript. I believe there could be more discussion to clarify this limitation with the current study, and the conclusions, and particularly the title, should be softened regarding the mechanism of antagonism being based on heme reactivity.

We have softened the title and conclusions to take into account the limitations of our studies.

(1) Please double-check the definitions for isobologram interpretation. In most antimicrobial interaction studies, I see the threshold for antagonism at sumFIC50 of 1.5, or even 2. 1.25 is often interpreted as additive in many studies.

We acknowledge that different studies use various cutoff values. Our interpretations for additive versus antagonistic versus superantagonistic were based not only on mean ΣFIC50 values, but also isobologram shape. For example, the flat isoboles for MFQ-DHA were clearly distinct from the curved isoboles of PPQ-DHA. It is unclear what cutoff value(s) would be most clinically relevant.

(2) For the MFQ-PPQ interaction study, please make it clear that these drugs have very long half-lives (weeks), so the 4 h pulse assay isn't really relevant to their overall activity. It probably shows a slower onset of action, but there is plenty of drug remaining for many days in the clinical scenario, so perhaps the data from the traditional 48h assay is more relevant. The same consideration applies to OZ439, which may impact the interpretation of that data.

We have now included the half-lives of these compounds in the discussion section. Our intent was to use a pulsing format to make these isobolograms comparable with the other assays. It is important to note that pulses can reveal stronger phenotypes that might be missed with traditional methods. Thus, while 48 h assays may better mimic in vivo conditions, they could also mask important phenotypes.

Reviewer #3 (Recommendations for the Authors):

I have included most of my concerns in the public review. Below are some additional specific points for consideration:

(1) It is expected to include a synergistic combination as a control (e.g., artemisinin + lumefantrine) to contextualize the degree of antagonism observed. The experimental design should show some synergistic profiles in comparison. Adding a few experiments by including a synergistic control is needed.

Both MFQ-DHA and LM-DHA combinations were additive, which provides context for antagonistic combinations. This is now stated in the results section pertaining to Figure 1. We have also included a reference to our previous publication in which we demonstrated that proteasome inhibitor-peroxide combinations are synergistic to additive using this same pulsing format.

(2) Consider in vivo validation or pharmacokinetic/pharmacodynamic modeling to strengthen the translational relevance of the findings when it comes to doses and the IC50 correlations.

We agree that this would be useful to do in future, but it is outside the scope of the current study.

(3) It would be beneficial to include a discussion section on how the findings are generalizable to different Plasmodium falciparum genotypes (3D7, Dd2, MRA-1284) and their relevance.

Findings were consistent across three parasite backgrounds depending on PfCRT genotype. This point has been included in the discussion section. The background of these parasites is also provided in Table 1.

(4) Potential evaluation criteria to understand where certain combinations should be reconsidered can be included as a suggestion for the wider audience.

Our in vitro studies suggest that pulsing isobolograms would be a useful assay to include when evaluating combination therapies. While we believe that synergistic combinations would be more strategic than antagonistic combinations, we cannot provide evaluation criteria or make recommendations for reconsidering currently used combinations.

(5) Further elaborate on the mechanistic basis of heme inactivation by quinolines. If data are available, please include more data on the specificity of the process.

Despite our best efforts, we were unable to evaluate quinoline-heme interactions in parasites. Even in vitro, this interaction has remined elusive for decades. We agree that this would be an important future step towards supporting a specific mechanism for quinoline-DHA antagonism.

Reviewer #1 (Public review):

Summary:

Mitochondria encode a small set of proteins that are made inside the organelle by specialized ribosomes. When this mitochondrial translation system fails, oxidative phosphorylation is impaired, an outcome that is particularly harmful to energy-demanding tissues such as the heart. In this manuscript, the authors use a targeted CRISPR/Cas9 screen in cultured cells grown on galactose (a condition that forces reliance on oxidative phosphorylation) to identify genes required for mitochondrial activity. They highlight EOLA1, previously studied mainly in inflammatory contexts, as a top candidate.

Strengths:

The authors present data suggesting that EOLA1 is imported into mitochondria via an N-terminal targeting sequence and resides in the mitochondrial matrix. Loss of EOLA1 reduces oxygen consumption and is associated with altered mitochondrial ultrastructure. Mechanistically, affinity purification suggests interaction with mitochondrial elongation factors TUFM (mtEF-Tu), and RNA immunoprecipitation experiments enrich 12S mt-rRNA, consistent with a relationship to the small ribosomal subunit. Multiple assays, including sucrose-gradient profiling, reduced abundance of selected mtDNA-encoded proteins, and a click-chemistry labeling approach, support the conclusion that mitochondrial protein synthesis is decreased in EOLA1-deficient cells. Finally, whole-body Eola1 knockout mice show echocardiographic findings consistent with dilated cardiomyopathy and reduced levels of representative mitochondrially encoded proteins in cardiac tissue.

How to interpret the work:

The data support a role for EOLA1 in maintaining mitochondrial gene expression and oxidative phosphorylation capacity, and they plausibly implicate mitochondrial translation.

Weaknesses:

The main caveat is that the study does not yet establish how EOLA1 acts, whether it directly modulates translation elongation through TUFM, whether it is primarily required for mitoribosome biogenesis/rRNA stability, or whether it influences translation indirectly through mitochondrial stress pathways. The in vivo phenotype is intriguing, but without tissue-specific deletion/rescue and deeper cardiac pathology/mitochondrial functional measurements, it remains uncertain how directly the heart phenotype reflects a cardiomyocyte-autonomous defect in mitochondrial translation.

Reviewer #2 (Public review):

Summary:

In this study, the authors identify a previously uncharacterised regulator of mitochondrial function using a genetic screen and propose a role for this protein in supporting mitochondrial protein production. They provide evidence that the protein localises to mitochondria, interacts with components of the mitochondrial translation machinery, and is required for normal heart function in an animal model.

Strengths:

A major strength of the work is the use of multiple independent approaches to assess mitochondrial activity and protein production, which together provide support for the central conclusions. The in vivo data linking loss of this factor to impaired heart function are particularly compelling and elevate the relevance of the study beyond a purely cell-based context.

Weaknesses:

Given prior reports placing this protein outside mitochondria, its mitochondrial localisation would benefit from more rigorous and quantitative validation, and the proposed mechanism of the interaction with the mitochondrial translation machinery remains only partially explored. In addition, the physiological analysis is largely limited to the heart, leaving open questions about how broadly this pathway operates across tissues.

Major comments:

(1) Evidence for mitochondrial localization of EOLA1<br /> EOLA1 has previously been reported as a nuclear and cytosolic protein and is not annotated in MitoCarta 3.0, making rigorous validation of its mitochondrial localization particularly important. Although the authors provide several lines of evidence, interpretation is complicated by the use of different cell lines across localization, interaction, and functional experiments. Greater consistency in the cellular models used would strengthen the conclusions. The immunofluorescence analysis of tagged EOLA1 would also benefit from quantification across more cells and the inclusion of an additional mitochondrial marker (e.g., an outer membrane marker such as TOM20), as HSP60 staining can vary with mitochondrial state.

(2) Normalization of OCR measurements<br /> Clarification of how Seahorse oxygen consumption rate measurements were normalized (e.g., cell number or protein content) would aid interpretation, particularly given potential effects of Eola1 loss on cell growth.

(3) Linking interaction data to functional phenotypes<br /> Loss-of-function analyses are performed in mouse cell lines, whereas localization and interactome studies are conducted in human HEK293T cells. The absence of a human EOLA1 knockout model makes it difficult to directly connect the interaction data to the observed functional phenotypes. Additional validation or discussion of species conservation would improve clarity.

(4) Mechanistic interpretation of the EOLA1-TUFM-12S rRNA interaction<br /> The identification of TUFM and 12S mt-rRNA as EOLA1 interactors is an interesting finding; however, the basis for prioritizing TUFM among the many mitochondrial proteins identified in the interactome is not fully explained. Providing enrichment statistics and functional categorization of mitochondrial interactors would increase transparency. In addition, the proposed role of the ASCH domain in RNA binding would be strengthened by structure-informed or mutational analysis of the conserved RNA-binding motif.

(5) Interpretation of mitochondrial translation and protein abundance data<br /> Several assays supporting impaired mitochondrial translation would benefit from additional controls and quantification. The de novo mitochondrial translation assay (Fig. 3h) is not quantified, making it difficult to assess the magnitude and reproducibility of the effect. In addition, western blots showing reduced levels of mitochondrially encoded OXPHOS subunits (Figure 3g) lack a mitochondrial loading control (e.g., TOM20 or VDAC). Since loss of EOLA1 may affect mitochondrial mass, normalization to a mitochondrial marker is necessary. Relatedly, it would be informative to assess whether steady-state levels of mitoribosomal proteins (e.g., MRPS15, MRPL37) and nuclear-encoded OXPHOS subunits are altered upon Eola1 loss, both in knockout cell lines and in the knockout mouse.

(6) Physiological scope of the in vivo analysis<br /> The cardiac phenotype observed in the whole-body Eola1 knockout mouse is compelling, but the focus on a single tissue limits interpretation of EOLA1's broader physiological role. Examination of additional high-energy-demand tissues would help clarify whether the observed effects are heart-specific or more general. In addition, the presence of residual EOLA1 protein bands in western blots (Figure 4a) and remaining Eola1 transcripts in qRT-PCR analyses (Extended Figure 4e) from knockout tissues should be addressed. The authors should clarify whether these signals reflect incomplete knockout, alternative isoforms, antibody cross-reactivity, or technical background.

(7) Relationship to previously reported MT2A interaction<br /> Given prior reports of EOLA1 interaction with MT2A, a brief comment on whether MT2A was detected in the authors' co-immunoprecipitation experiments and how this relates to the proposed mitochondrial role would be useful.

Reviewer #3 (Public review):

The authors identified EOLA1 in a CRISPR/Cas9 screen for essential mitochondrial genes in a mouse B16-F10 cell line; however, no information on the library used for this screen or the list of all identified essential genes is provided. What was the p-value for EOLA1 in Figure 1b?

The authors show that EOLA1 is indeed a mitochondrial protein (using both mouse and human cell lines). It is valuable that the authors use different cell lines to investigate the function of this protein; however, this also presents a challenge, as four different cell lines (two mouse and two human) are used across individual experiments, with no consistency between them. Knock-out (KO) experiments were performed in mouse cell lines only, and human cell lines were used in overexpression experiments, in which EOLA1 was tagged with FLAG-HA. It would be beneficial if a knock-out were also generated in a human cell line to confirm the effect on the expression of mitochondria-encoded proteins, along with a rescue experiment in which the EOLA1 protein is reintroduced into KO cells.

Functional analysis of EOLA1: The authors performed affinity immunoprecipitation of FLAG-HA-tagged EOLA1 from stably overexpressing cells, and identified 202 co-immunoprecipitating proteins, of which 71 were known mitochondrial proteins; however, no list of these proteins is provided. Why did the authors choose TUFM? Were any mitochondrial ribosomal proteins co-immunoprecipitated, if EOLA1 is suggested to regulate translation? Were levels of TUFM affected in EOLA1-KO cells?

The authors continued to analyze mitochondrial ribosomes using sucrose gradient fractionation and in-vitro mitochondrial translation. However, there are several technical problems with the presented data: It has been established that mitochondrial ribosomes do not form polysomes in mammalian cells but rather perform translation as monosomes. The authors indirectly confirm this: almost no 12S or 16S rRNA (Fig. 3f) or MRP proteins (Extended data 3c) are present in "polysome" fractions. Although indeed 12S and 16S rRNAs are decreased in monosome fractions, the levels of mRNAs are not different between KO and WT cells, and neither is the migration of mitochondrial ribosomal proteins. As there is no loading control provided for the sucrose gradients blots (such as SDHA, VDAC), it is not possible to assess the overall levels of mitochondrial ribosomes. The gel presented for mitochondrial translation is of poor quality, as it is impossible to identify any of the expected 13 polypeptides. Although the intensity of the signal is weaker for KO, so is the intensity in the portion of Coomassie stained gel. A better-quality gel and quantification need to be provided to support the claims.

What is the difference between endogenous and exogenous RIP-qPCR? EOLA1 pulled down 12S rRNA without cross-linking (Figure 3d) or with UV-crosslinking (Figure 3e), however, both 12S and 16S rRNAs were enriched in UV-crosslinked cells (Figure 3c) and by UV-RIP-seq (Extended data 3b; although no control is provided here). Is no discussion offered for this observation? Is it possible that EOLA1 plays a role in the maturation of the mito-ribosome, rather than translation? Does EOLA1 co-migrate with the mito-ribosome on sucrose gradients?

Altogether, there is insufficient evidence to support the conclusion that EOLA1 plays a role in mitochondrial translation.

To investigate EOLA1 biological function, the authors created a whole-body EOLA1-/- mouse that exhibited no overall developmental abnormalities; however presented with an abnormal cardiac function. This is an ideal model to confirm prior observations in cellular models; however, apart from one western-blot for three mitochondrial encoded subunits, no other experiments were provided (such as measurements of the levels of 12S, or 16S rRNA, TUFM levels, ribosomes profile, mitochondrial translation, OXPHOS assembly, respirometry).

In Figure 2 g-i: TEM images are presented, but the method is not described, nor is any information on the cells used provided, nor is it clear how the circularity was determined. KO cells certainly look abnormal; however, are the authors sure that the indicated structures are mitochondria? They rather resemble autophagosomes/lysosomes with lamellar inclusions.

Microsoft has a commitment to entirely remove fossil fuel backup generation by 2030?

I know it's implied by the 100/100/0 statement, but I hadn't realised they had been explicit

Even US firms are using Chinese models because they are so much cheaper. They do not use them for everything, but where they can get away with lower cost options they will.

This was 2020, so around five years ago they started to do this.

price hikes of DRAM, due to pc laptop manufacturers having trouble in getting enough RAM. Shortages to keep going for 2026, after 2025. AI supply chain gobbling up the rest.

I'm trying this too

dysphoria is not just physical, but about emotions and desires (and their clash with environment) too.

I have experienced being on the viewer-side of users trauma dumping- whether it be in video formats, posted publicly for everyone to see and interact with, or under a comment section, where no one asked to read about their traumatic past. In many ways I can see how this may be therapeutic or even comforting, knowing that there are people out there to listen to and talk about your past with, however there are also cons to doing so. Most users when scrolling on social media somewhat expect there to be negative, sad, or frustrating stories that they will come across, however a lot of users may not feel comfortable being told personal stories and traumatic events that have happened to other people, as it could be triggering to their own past, or make them skeptical, fearing, or deeply uncomfortable. There have been various times where under a persons post where they're sharing their health journey from an eating disorder I will see users in the comments talking about all the ways in which they endulged in their unhealthy habits, and that could not only make users and the video poster uncomfortable, but even push them into reforming their bad habits. There's so many potential consequences sharing one's traumatic story online can bring, I find it would be much more efficient and beneficial to all users if these people would instead speak to a therapist or close friends (who are comfortable speaking about this with), rather than strangers on the internet who never asked to read or listen to your story. **

This is a really nice point since, yes, we do need evidence to support worthwhile arguments and to demonstrate how they relate to one another.

While yes I do agree this happens more than is should be, but it is only teachers who reveal too much about their personal lives and don't know how to keep professor and personal apart in their teaching jobs.

This is a very good point because yes, we do need evidence to back up points that are worth it, and we do need to show how they do connect.

I don't think there is really a reason for teachers to bring up their political beliefs but maybe in some cases.

This statement is very true but Roxbury Ma. was so successful because the residents knew better then to allow business people from outside the community come in and tell them what should be done. Che and other community members saved Roxbury by implementing macro social principles and including the community in the redevelopment of the town.

There is no generalized way to print cell in a cell compatible way but hopes to create a standard

I think it’s a bit sad and should change. If experienced writers and teachers helped new writers by giving them tips and corrections, we would have many more new novels. This would also help the economy and create new knowledge.

I hadn't thought about it that way, but I think It's right. Instead of just using the product, it would be much more interesting to create something myself

I don't see it exactly the same way, but I agree with part of it. I think writing is different from other talents. For example, being good at math comes from practice ,just repeating things over and over. But with writing, I believe people are born with that imagination; it’s a special gift you’re born with it

It makes sense that the cultural destruction of the native people in South America was not as successful as North America. The tactis used in North America seem much more brutal than the ones used in South America.

I wonder if there is something be gained but being so open about there being negative incentives to maximize engagement

What is gained for Elon stating this so openly. Is the hope that it pushes personal responsibility from the site and on individuals? I don’t believe this sentiment does an effective job but it’s interesting to reflect on the rhetoric.

We focus heavily on student success and ensuring students are supported academically, but teachers also need advisement. Resources that support educators while promoting student success are the most effective investments any school system can make, in my opinion.

These schools have excellent results, but the lessons and assessments that accompany their curriculum tend to take longer. This trade-off often results in less of the written curriculum being covered, but the content that is covered is usually absorbed at a higher cognitive level.

This is often because we have used adult brain development models as our guide in education, rather than child development as our north star. Yes, we aim to reach the adult stages, but as our brains develop, educators and education professionals need to meet the basic needs of child development.

As I started my career in special education, I found that when you showed kids you cared, they typically responded. Now, the spike in per-pupil spending is nice, but it would be more important to know how these districts used the money. That is the important part of the equation. Money and resources are not the same thing. How you use the money to accumulate and use resources correctly is the key.

Closing the gap from below expectations to meet standards on a standardized test becomes the focus for kids who are behind. Instead of working backward and using creativity to spark a struggling child's imagination, educators often focus on skill- and drill-based methods. Sometimes it helps, but many times it just has children check out more.

Yes, areas of color are a very sad epidemic, but areas of poverty in all races are often held back. Poverty is one of the most cumbersome obstacles for all races. When your focus is on surviving or learning this math problem, survival instincts tend to kick in before the bigger picture view of education being the ticket to a better life.

Now I see the complexity of the argument. Because it is AI it does not display a real child. However I would argue that if you could create this you have the means to involve an actual child later.

An example a person threatens to kill another person. They don't actually kill the person, but they can still get in trouble with the law for the threat. They may not be charged with murder but there is still accountability. I think you could something similar in this scenario.

They technically still have possession of child porn which is illegal involving a real child or an AI generated. Also technically speaking all porn is created or produced for creative, imaginative purposes. That being said, an AI child is not far off.

I like this take.

If Benjamin Franklin had the opportunity to live his life again, he would do everything the same, save for fixing a few mistakes, but, even if he couldn't fix those mistakes, he'd still take the opportunity to live his life again.

This quote describes how hearing someone out, having a conversation with them can truly change and understand where someone may be coming from. These people may have been very adamant on who they were voting for, but just because Kathi and her friend decided to come inside talk and eat really helps convey the theme of hope. Hope is something that is needed when campaigning as sometimes it can be very bleak. That's why having hope, hearing people out, and showing sympathy along with determination are the perfect combination to have a good outlook for the future.

This is so interesting, as I have never heard about this before. It makes so much sense because when I am watching a show that is originally in a different language with an English dub, I can't stand how the audio doesn't match the way a person's mouth is moving. I never knew that the brain had to work harder, but now I know why it really bothers me.

Skip the 'key assumptions' part -- I don't think this gets it correctly anyways. E.g., comparisons of SD units shouldn't require linearity per se, that would be sufficient but not necessary. I'm not sure if interpersonal comparability in levels is necessary either -- if we had linearity and cardinality the measured changes wouldn't depend on the starting points

Claude rephrased these in simpler but less rigorous and imo less useful ways. This page should use the actual wording of the key questions on the page https://coda.io/d/Unjournal-Public-Pages_ddIEzDONWdb/Wellbeing-PQ_suPg8sEH#_luVrD0mE -- use quotes where possible, ellipse where necessary, and link or fold the details

I find 'useful and reliable' a bit too vague perhaps

perhaps useful for annotations too?

Podríamos considerar que se está hablando de comunicación científica, en específico, R&D collaboration (investigación y desarrollo). Ya que, se habla de varias instituciones trabajando juntas para lograr el avance de la ciencia.

Managers, but also can be applied to artists.

Maybe good information for the introduction but I need a global aspect of this.

Emily Eavis is co-organizer of the festival who has long advocated for equality in festivals:

-"We’re trying our best so the pipeline needs to be developed. This starts way back with the record companies, radio. I can shout as loud as I like but we need to get everyone on board."

Freelancing enhances abuse and discrimination.

-Causes power imbalances and strange working relationships.

It looks like there is progress for women, but there is still discrimination, misogyny, and sexual abuse in the music industry.

The industry is still considered a "boys club."

It is important to remember the minimun amount of sources needed, as some may get stuck relying on two, when more are often needed.

They work differently Hnf6 expression is not found in PDA and PanINs but Sox9 is absolutely required.

FUTURE DIRECTIONS

Kras activation may induce ADM and PanIN but it may take a long time. Pancreatitis speeds up this process.

Sox9 expression in acinar cells can destabilize it and promote ductal gene expression but will not fully transform.

SOX9 induces ductal genes in acinar cells.

MAIN TOPICS OF SOX9

PanINs similiar to acinar cells but originated in duct lesions were Alcian Blue+ and expressed Muc5AC and Claudin18 and lineage marker YFP.

Duct lesions more often associated in large ducts

Tests using CK19+ promoter activated oncogenic Kras in ductal cells. Previously suggested that PanINs rarely arise from ducts but that may be wrong.

There is a range of early, mucus-producing abnormal cell changes in the pancreatic ducts that are not cancer yet but can potentially develop into pancreatic cancer

They are trying to determine the origins of PDA using a PDA-initiating Kras mutation. Ductal and Stem-like centroacinar cells are refractory to oncogenic transformation, but acinar cells readily form PDA lesions

I want to include specific artists, but not sure if I want to include these people yet. This will be decided later on.

I didn't do much more than skim the appendix, but I wonder to what extent that community archives were surveyed for the purposes of this monograph? Along with what Millar states in the next reading, there are plenty of co-operatively developed written policies (between archivist / workers, institutions and representative communities, for example) that don't seem to slavishly adhere to the text and allow for wiggle room / interpretation.

Please support something that actual educators jointly request. Listen to your constituents and what they want, but please listen to the experts when they tell you what is needed.

This is beautifully said and completely true. In most aspects of professional life, leadership is taught as a matter of institutional control. In education, educators are often given a roll sheet and told, "Good luck." The leadership comes from trial and error and experience.

Until education is not viewed through a red or blue lens, arguments will always arise in the political realm. Along those lines are the obvious facts that we have unqualified politicians making educational laws and guidelines. Just because you were a student does not make you qualified to speak on educational policy any more than my attending a dental appointment makes me qualified to set dental guidelines.

Over time, an increasing number of responsibilities have been placed on them, leading to a decline in their effectiveness in their primary area of expertise. To foster a more productive environment, educators should be encouraged to exercise their creativity. They should not be burdened with additional roles such as social worker, police officer, counselor, or, in some cases, a substitute parental figure. Addressing this issue is important for improving the overall educational experience and supporting the best outcomes for students.

This, too, is leading to incredibly misleading information. It's no secret that this type of educational analysis is incredibly flawed, but with parents opting out of the information-gathering assessments, it's skewing the data. However flawed it is, when participation is limited, data is not accurately reflected.

no need for scare quotes. But note the calibration questions is one of the 2 aspects of this question.

Need to be changed to "better overall value but with better price"

We need to add a conclusion paragraph saying that, although ChMeetings and Churchtrac provide the same pricing model but ChMeetings is more value for money which can save you $85 per year (~25% saving per year)

Peter Naur, Programming as Theory Building 1985 https://doi.org/10.1016/0165-6074(85)90032-8

Programming as theory building 1985 in Zotero

While techdebt is about the accumulation of human decisions, and the resulting erosion of human understanding, the term itself suggests it is a property of the code itself, and that one could remove it from code.

To downplay inequalities, and barriers, and focus on objectivist fallacious measurements. It is most commonly thrown that money makes one happy, but that is a partly false statement. It's not money that motivates one to do work (necessarily), rather rationalisation. And the classical conundrum of how much you'd pay for a restaurant if everything is divided or on the house, doesn't consider the inherited circumstances that bias it.

INHERITANCE, SOCIALISATION, INVESTMENT. Check how much Catalan society invests in football athletes! Meritocracy or talentocracy disguise children exploitation (which is not too dissimilar to parents using their children for views on social media, and also perpetuates the family institution).

Meritocracy without changes from what we have now, is a marketing trick to switch the blame from companies to users. It's individuating, and hides corps, loans, rents, and stock markets. It hides Shirky principle and Matthew effect. It's a new shiny version of the classical pyramid of power.

And that's what's dark(ening), I'd say! It naturalises conservatism. By matching mainstream culture to individualistic desire, it establishes A Brave New World's premise: "I am giving you what you want". But desire is manufactured AND HISTORICAL (inherited, slightly molded through Overton window and Creeping normality), there was no choice, there is no choice, we have no free will, we are interdependent!

Remember, meritocracy was never meants as a means to pursue innovation and diversity, but as a replacement for traditions and aristocracy. So, what gets popular and is seen as deserving of merit, falls prey to popularity and survivorship biases. It would necessitate that marketing dissappeared, come on. It would need to equalise salary to avoid creating non-meritocratic gatekeeping and give true incentives for those innovating. But then... who would assess innovation? Exactly those who previously did, or the whole of population... which would maintain things as they were, unchallenged, keeping the status quo.

How do I say this... meritocarcy without breaking off with traditions, and without doing a hard reset, without destroying all inheritance and present structures, and without deeply changing culture, is grounds for ENSHITTIFIED MONOPOLIES. Violence besets violence, and privilege besets privilege. Meritocracy won't erase close ties, friendships, and networks. Meritocracy is a self-exploitation premise for enterpreneurship, that favours already consolidated and rich extroverted individuals. That is aggressive. That is monopolistic. That kills "healthy competition", or more than that, it kills cooperation. It kills care, and it kills interdependence.

It's winner-take-all. It's a coin with a face that says "I win", and a tails that says "You lose". Emperor Nero's conundrum, a Catch-22.

Because she was a woman.

It feels "fair", and it "works"... why shouldn't the people who work harder and have "the most" mechanotechnical capabilities be assigned to a job? In other words, as a friend of mine told, if we could have 3 Michelin star cooks only, why wouldn't we? It's an enticing idea, if we had these cooks with functional diversity, from different cultural backgrounds, skin tones, health, etc. it makes sense to begin with that we would assign resources to them.

But this ignores who we would be leaving. Further, we are skipping past what makes a 3-star chef, which is to say, it's NEVER a "chef", it's a WHOLE RESTAURANT. It's the location, ambience, the service, which often takes much much longer than a "typical" one, and requires many many more people (it's an spectacle in of itself, as they have minuscule dishes, and they often prepare them in front of people ONE BY ONE), plus, it essentialises consumption, as there is ONLY ONE 3-M Vegan restaurant in the world. It requires special utensils, learning, makes the process elitist and consumerist (telling you, you don't have to engage in it, leave that to experts), displacing hobbyism (the root of innovation), failure, spiral (not linear) learning processes, and many other externalities, like the type of exotic (highly limited) produce needed to make most recipes.

And that's accounting for the magical position that the process would be inclusive of everyone, and have enough chefs to feed the whole world. In what mind? Since we can't have this kind of home cook (or robot cook) for every person, we would have to rely on mass prepared dishes, probably inundating shelves with non-recyclable plastic containers to extend the food's life, these requiring a lot more carbon for transportation, and de-skilling people (less versatile, spitting at transference and imagination for other tasks, and reducing ability to make diverse stories and engage in interdisciplinary dialogue) who would pick food from a distant commodified service.

That's dark, I think. It speaks of how retribution is so engraved in our society, that we yearn for it even if false. But the dramatic ups and downs of this kind of life, as portrayed in shows like When Life gives you Tangerines, ridiculises oppression. It makes it invisible, as you don't lose a cared one in Minecraft...

Oh, the redundancy in onsite gen isn’t something I thought too much about before

Wow, this is waaay lower than I thought

Noted

Trick that should be noted.

Nicely put

This seems smart for trying to make the most money and resources out of it but will probably cause problems.

I like that this article gave a realistic view on how AI can help accelerate the efficiency of certain tasks but also has its limits. AI use can often be abused and is sometimes thought to be a cure all for any work, however it should be used as a supplement to learning instead of replacing the act of learning.

This quote pushes the agenda of AI resulting in real consequences and pushes something that is not per say fiction, but rather something that feels more like a 'force'. I chose this quote because it correlates with the narrative this subsection pushes about bias and prejudice, and how AI motivates ideas such as those.

I chose this sentence because it speaks a powerful message, more or so a point is trying to be proven. Which is that no matter how strong someone's judgement may be. A bias will be formed as a behavioural response, and to instead not accept that fact, but rather we should come to terms with.

This shows how slavery in the Caribbean was driven purely by profit even at the cost of human life.

When The English settlement disappeared the Indians took over. Although the English disappeared it's possible they went to live with the Indians so they could survive.

Instead of coming up with their own principles they used German ones and came up with a new name.

Context is important in explorative archeology. We might not know how a particular items were made hundreds of years ago but looking into past conditions and context is possible to develope a likely theory for how things were made in the past.

It is very important to understand that things are different in both time a culture. Realizing cultural roots can also help better attach ideas and goals to the things we view as different. For example we may not make an old recipe using the exact same methods but rather make it with modern appliances. So in some ways the recipe is different but the root is the same.

Description of how Activate keep their prompts, skills, databases, memory architecture under their own control and within their own environment.

Moving means wiring up another model or models, but the rest is kept as is.

• Detriots auto industry was a defining pull factor for black migrants
• to get elected detriot had some serious political barriers
• By 1970 the voting age percejt was almost 50, this made blacks BOP
• Detriots municipal election was at large and nonpartisan so people were not under the banners of parties and they had to win aross the city
• Per the agrument at the begging of the book, this is going to inhibit black politicans ability to get elected as well as the voting power of the population
• Black population is stepping mainly into this compettion between ford and the labor unions on the political level
• Oldest and largest chapters of the NAACP
• As in other cities, Black churches play a major role in all spheres of life, but politival and civil rights mobilization in general
• The ford motor company has a lot of say in who gets elected (maybe will chnage as their base of employees chnages) and before the major influx of the black population they are supporting discrimatory candidates
• 1920 election starts the immunization towards civil rights (or just anti racits) policy
• All or nothing kind of politics, because the black vote is in BOP, KKK will always mobolix=ze agaisntb and other candidate will always mobolize for
• Ford complicated the balncing of politica; preference and economic wellbeing
• In all of these examples the impratnt part is that the balk vote is non-trival, something to be feared or garnered, and this si before the true boom
• Explicit racism is orertty jarring for detriot
• Race becomes a wedge issue as the population and strength of BVAP increases
• "chnage his stance on racial issies once it became clear that Blak Detroiters we important"
• 1961 is the first victory for the Black community in determining the kayor, might go on to become the status quo
• In detriot, even in a system that was set up to limit their political voice (indeirectly), they could have been the BOP in 19/22 elections, with its least influential, still 50% of the time
• Politicians became more responsive to the opinions of Black politicians as the proportio of the oting age population increased
• Detriot had serious structrual barriers for balck people holding office
• It would take black politicians some time to get going but eventually they would have some very meanigful political representation
• black politicians were primarily concerned with anti-discrimination laws (jobs and housing)
• While not limited to civil reform, racial policy was importsnt at the begginbg as a means of reprsentation
• they were human, not monolithic and cared about a lot of things
• migrant legislators were particuarly concerned with the migrant concersn (hand up) which extended to non-black migrants as well
• locally, the political system made it hard for balck officials to be elected based on the support of blak homogoneus communiiues, which I remind you were fucking required by detriot whites
• outcome of the sytem was the exlusion of the black population from municipal eledted office
• detriot lagged in national representation (congress)
• while the population grew, conservative candidates won by convicing whites to vote reacially conservative
• the system in detriot rwally lagged the potential influence until there was a mass bloc

Of course, people listened to the priest but did not actually apply what he said.

• "The great migration unlocked Black Americans ability yo participate as full citizens in our democracy... in some instances for the first time"
• northern politiicians interactions with black voters chmaged as the population grew and there was an increase of Black elected officials
• partys responses were positive if they had no ther choice; black population would signifigantly affect the outcome
• highlight the agency of black people in the great migration
• its been studied through almost every angle except politics
• Black voters (when an important block) could seriously chnage the policiies that fficials took up, negative if they wwanted to alienatate the vite but positive to win it
• We are looking at top down change, how the motivations of the politicians chnaged
• secular realignment, parties are constanrtly changing all the time and can arrive at a completely differnt plcae over decades -politicians were mobolizing the black vote for the first time
• black colaitions were able to chnage the policies locally befire nationally (intuitive)
• we're gonna argue that the inlfux of migrants chnaged the place more than the place chnaged the migrants

Auteur : Manon Berriche, doctorante en sociologie au Medialab de Science Po.

Contexte : The Conversation, media en ligne qui fédère, sous la forme d'une association à but non lucratif, des établissements d'enseignement supérieur et de recherche. Des universitaire et des chercheurs en collaboration avec des journalistes proposent des articles d'analyse de l'actualité.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Manuscript number: RC-2025-03280

Corresponding author(s): Stephan Gruber

1. General Statements [optional]

First, we would like to thank the editor at Review Commons for the efficient handling of our manuscript. We also apologize for our delayed response.

We are grateful to all three reviewers for their careful evaluation of our work and for their constructive feedback, which will provide a valuable basis for improving the figures and the text, as described below. We expect to be able to complete the revision following the plan described below quickly.

We note that the reviewer reports (Rev. #1 and Rev. #3) made us realize that the manuscript text was misleading on the following point. Although we used the purified ATP hydrolysis–deficient Smc protein for sybody isolation, this does not restrict the selection to a specific conformation. As described in detail in Vazquez-Nunez et al. (Figure 5), this mutant displays the ATP-engaged conformation only in a smaller fraction of complexes (~25% in the presence of ATP and DNA), consistent with prior in vivo observations reported by Diebold-Durand et al. (Figure 5). Rather than limiting the selection to a particular configuration, our aim was to reduce the prevalence of the predominant rod state in order to broaden the range of conformations represented during sybody selection. Consistent with this interpretation, only a small number of isolated sybodies show strong conformation-specific binding in the presence or absence of ATP/DNA, as observed by ELISA (now included in the manuscript). We will revise the manuscript text accordingly to clarify this point.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

• *

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

Major comments: 1. Lack of direct in vitro binding measurements: The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And, do the sybodies affect the interaction of ScpAB with SMC? It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

We agree with the reviewer that adding such data would be reassuring and that obtaining solid data using purified components is not easy even for a smaller selection of sybodies. We have data that show direct binding of Smc to sybodies by various methods including ELISA, pull-downs and by biophysical methods (GCI). Initially, we omitted these data from the manuscript as we are convinced that the mapping data obtained with chimeric SMC proteins is more definitive and relevant. During the revision we will incorporate the ELISA data showing direct binding and also indicating a lack of preference for a specific state of Smc.

Many modes of sybody binding to Smc are plausible The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

We have attempted to map the binding by structure prediction, however, so far, even the latest versions of AlphaFold are not able to clearly delineate the binding interface. Indeed, many ways of binding are possible, including disruption of ScpAB interaction. However, since the main binding site is located on the SMC coiled coils, the later scenario would likely be an indirect consequence of altered coiled coil configuration, consistent with our current interpretation.

1. Sybody expression in vivo Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

We have tagged selected sybodies with gfp and performed live cell imaging. This showed that they are all roughly equally expressed and that they localize as foci in the cell presumably by binding to Smc complexes loaded onto the chromosome at ParB/parS sites. We will include this data in the revised version of the manuscript.

1. Sybodies should phenocopy ATP hydrolysis mutant of Smc The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

As eluded to above, we think that our selection gave rise to sybodies that bind various, possibly multiple Smc conformations. Consistent with this idea, the phenotypes are similar to null mutant rather than the ATP-hydrolysis defective EQ mutant, which display even more severe growth phenotypes. We will add the following notes to the text:

“These conditions favour ATP-engaged particles alongside the typically predominant ATP-disengaged rod-shaped state (add Vazquez Nunez et al., 2021).”

“ELISA data confirm that nearly all clones bind Smc-ScpAB; however, their binding shows little or no dependence on the presence of ATP or DNA.”

Minor comments: 1. It was surprising that no sybodies were found that could target both bacillus and spneu Smc. For example, sybodies targeting the head regions of Smc that might work in a more universal manner. Could the authors comment on the coverage of the sybodies across the protein structure?

It is rather common that sybodies (like antibodies and nanobodies) exhibit strong affinity differences between highly conserved proteins (> 90 % identity). The underlying reasons for such strong discrimination are i) location of less conserved residues primarily at the target protein surface and ii) the large interaction interface between sybody and target which offers multiple vulnerabilities for disturbance, in particular through bulky side chains resulting in steric clashes. Another frequently observed phenomenon is sybody binding to a dominant epitope, which also often applies to nanobodies and antibodies. A great example for this are the dominant epitopes on SARS-CoV-2 RBDs.

Growth curves (Fig. S3) show a large jump in recovery in growth under sybody induction conditions. Could the authors address this observation here and in the text?

We suppose that this recovery represents suppressor mutants and/or (more likely) improved growth in the absence of functional Smc during nutrient limitation (see Gruber et al., 2013 and Wang et al., 2013). We will add this statement to the text.

L41- Sentence correction: Loop can be removed. Ah, yes, sorry for this confusing error. Thank you. 4. L525 - bsuSmc 'E' :extra E can be removed. To do. Thank you. 5. References need to be properly formatted. To do. Thank you. 6. The authors should add in figure legend for Fig 1i) details on representation of the purple region, and explain the grey strokes for orientation of the loop. To do. 7. How many cells were analysed in the cell biological assays? Legends should include these information. To Be Included.

Reviewer #1 (Significance (Required)):

Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins have been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Review: "Single Domain Antibody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex" by Ophélie Gosselin et al, Review Commons RC-2025-03280 Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions. The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB. In summary, the authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes. Some specific comments: Line 75: "likely stabilizing otherwise rare intermediates of the conformational cycle." - sorry, why is that being concluded? Why not stabilizing longer-lived oncformations? We will clarify this statement!

Line 89: Sorry, possibly our lack of understanding: why first ribosome and then phage display?

Ribosome display offers to screen around 10^12 sybodies per selection round (technically unrestricted library size), while for phage display, the library size is restricted to around 10^9 sybodies due to the fact that production of a phage library requires transformation of the phagemid plasmid into E. coli, thereby introducing a diversity bottleneck. This is why the sybody platform starts off with ribosome display. It switches to phage display from round 2 onwards because the output of the initial round of ribosome display is around 10^6 sybodies, which can be easily transferred into the phage display format. Phage display is used to minimize selection biases. For more information, please consult the original sybody paper (PMID: 29792401).

Line 100: Why was only lethality selected? Less severe phenotypes not clear enough?

Yes, colony size is more difficult to score robustly, as the sizes of individual transformant colonies can vary quite widely. The number of isolated sybodies was at the limit of further analysis.

Line 106: Could it be tested somehow if convex and concave library sybodies fold in Bs?

We did not focus on the non-functional sybody candidates and only sybodies of the loop library turned out to cause functional consequences at the cellular level. Notably, we will include gfp-imaging showing that non-lethal sybodies are expressed to similar levels that toxic sybodies. Given the identical scaffold of concave and loop sybodies (they only differ in their CDR3 length), we expect that the concave sybodies fold in the cytoplasm of B. subtilis. For the convex sybodies exhibiting a different scaffold, this will be tested.

Line 125: Could Pxyl be repressed by glucose?

To our knowledge and experience, repression by glucose (catabolite repression) does not work well in this context in B. subtilis.

Line 131: The SMC replacement strain is a cool experiment and removes a lot of doubts!

Thank you! (we agree 😊)

Line 141: The mapping is good and looks reliable, but looks and feels like a tour de force? Of course, some cryo-EM would have been lovely (lines 228-229 understood, it has been tried!).

Yes, we have made several attempts at structural biology. Unfortunately, Smc-ScpAB is not well suited for cryo-EM in our hands and crystallography with Smc fragments and sybodies did not yield well-diffracting crystals.

Line 179: Mmmh. Do we not assume DNA binding on top of the dimerised heads to open the CC (clamp)?

We will clarify the text here.

Line 187: Having sybodies that presumably keep the CC together (closing) and some that do not allow them to come together correctly (opening) is really cool and probably important going forward.

Thank you!

Figure 1 Ai is not very colour-blind friendly.

We are sorry for this oversight. We will try to make the color scheme more inclusive. Thank you for the notification.

Optional: did the authors see any spontaneous mutations emerge that bypass the lethal phenotype of sybody expression?

No, we did not observe spontaneous mutations suppressing the phenotype, possibly due to the limited number of cell generations observed. We tried to avoid suppressors by limiting growth, but this may indeed be a good future approach for further fine map the binding sites and to obtain insights into the mechanism of inhibition.