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Reply to the reviewers

We appreciated the positive, detailed and helpful feedback from all three reviewers.

Reviewer 1.

Minor comments.

1. In the introduction, on page 2, the authors seem a little confused about the Plk1 Polo-box domain - text as written: "...kinase domain linked to tandem Polo-box domains (PBD)", and cite a review paper. Actually, there is only a single Polo-box domain in these kinases, which contains both Polo-boxes and a bit of the upstream linker region. The "PBD" terminology denotes his 2-Polo-box +linker structure. Perhaps it would be better here to cite the PBD structure (Elia et al., Cell, 2002) as a primary citation here.

Response: Thank you for finding this error, the text has been updated and the new citation included within the text on line 65.

1. Similarly, the line "...during the G2/M transition following successful DNA damage repair" cites the Seki et al paper, but those findings are shown in the Macurek et al paper, not the Seki et al paper.

_Response: _Thank you for finding this error, the new citation included within the text on line 69.

1. Using the model of the ternary complex as shown in Figure 1B, deletion constructs of Bora missing regions within the disordered loops, but still retaining the residues that bind the PBD, FW pocket and Aurora A, can be modeled and tested to see if such deletions can improve the ipTM scores and binding affinity.

Response: ____AlphaFold3 modelling was attempted with shorter regions of Bora to see the effect on the ipTM scores. Unfortunately, when Bora was reduced to shorter sequences, such as 18-88 or 18-45 modelled with 68-120, the models became inconsistent and of a low quality. Models were also created including the short region of Bora surrounding Ser252 that interacts with the polo box domain as well as Bora 18-120, but this had minimal effect on the calculated iPTM scores.

1. On page 5, "S112A" within the sentence "Unexpectedly, the F56A/W58A Bora was less efficiently phosphorylated on S112A (Supplementary Figure S11, F compared to H and Supplementary Table S4)." This should be "S112".

Response: ____Thank you for spotting this, the error has been corrected.

1. In the assays shown in Figure 2D, the presence of excess F56AW58A Bora that remained unphosphorylated on S112 may complicate the interpretation of the results. Can the authors show that the S112-phosphorylated F56AW68A Bora is predominantly bound to Aurora A in such a mixture, perhaps by NMR using labelled pS112 F56AW58A Bora and unlabeled S112 F56AW58A Bora?

_Response: _15N13C labelled of Bora 18-120 F56A W58A was produced and assigned. We then phosphorylated a sample using ERK2, tracking with NMR, and when the reaction had progressed to a 50:50 mixture of pSer112 and Ser112 (based on peak intensities) the kinase activity was quenched by addition of EDTA to sequester Mg2+. This produced a solution containing both pS112 and unphosphorylated S112 Bora species with marker peaks in HSQC spectra that could be used to directly compare Aurora-binding to the two species. Aurora-A was introduced to the sample and the peak intensities were monitored. Although both species are affected, there is much greater peak loss from the pS112 related peaks than those for unphosphorylated S112. This indicates that Aurora-A still preferentially binds pS112 Bora over S112 Bora when the F56A W58A mutation is present. This data has been included in Supplementary Figure S11.

1. Please expand Figure 3A to better show the FW pocket-forming residues on Plk1.

Response: ____Figure 3 has been amended to reduce the size of the sequence alignments so that 3A could be made slightly larger.

1. It would be helpful to label the peaks in the mass spectra in Fig. S11 with the phospho-species that they correspond to.

Response: ____This information has been added to the mass spectra in Fig. S11 (now supplementary Figure S14) to make them easier to view.

1. In the last paragraph on page 7, "see we" in the sentence "As well as a decrease in intensity around pSer112 in Bora, see we an overall effect with decreased intensity across most of the Bora sequence." Should be corrected to "we see".

Response: ____Thank you for spotting this, the error has been corrected.

1. While not required, it would be helpful if binding or Bora to Aurora A after Erk2 phosphorylation could be shown using fluorescence polarization or ITC to lend additional support to the NMR data for S112 and S59 phosphorylation and for CEP192 and TPX2 competition.

Response: ____This question has been partially answered in previous work by Tavernier et al. (2021), who showed improved binding of Aurora-A to Bora after Erk phosphorylation (by SPR), and they used labelled-TPX2 for a series of competition FP assays in that and the recent parallel study (Pillan et al. 2025).

We made initial efforts to perform additional FP assays using longer sections of Bora with different phosphorylation states but without success (perhaps due to the multisite-binding nature of the Bora–Aurora interaction, and difficulties with directly expressing phosphorylated Bora). The revised manuscript now includes some additional NMR data to show improved Bora–Aurora-A interaction after phosphorylation at Ser59 (Supplementary Figure S12).

1. The Aurora A phosphorylation motif has been further defined beyond that reported by the Pinna lab in 2005. Notably, the Ser-59 sequence on Bora (F-R-W-S-I), has, in addition to dominant selection for AR in the -2 position, both favorable -1 (W) and +1 (I) positions based on peptide library measurements (Alexander et al., Science Signaling 2011), further arguing that it may be an excellent Aurora A phosphorylation site.

Response: ____Thank you for highlighting this publication and how it further reinforces the likelihood of Ser59 being an effective substrate for Aurora-A, this should have been included in the original manuscript. This citation has now been included.

1. Have the authors tried to model the Drosophila melanogaster Aurora A-Bora-Polo complex to see if the Asn substitution of Bora Ser59, and the expected loss of the interactions between Bora pSer59 and Plk1 Arg59 and Aurora A Arg205 are compensated by other features?

Response: ____A ternary complex between the Drosophila melanogaster orthologues was modelled using AlphaFold3 (Uniprot code PLK1 (Q9VVR2 72-165), Aurora-A kinase (Q9VGF9) 151-411 and PLK1 (P52304 21-280)). This model was analysed using PDBe PISA to identify potential interactions between the three proteins, focusing on residues that are not conserved between the human and Drosophila sequences. From this model a potential salt bridge was identified between Drosophila Bora Lys120 and PLK1 Glu93 that would not occur in the human ternary complex given Lys120 is replaced with an asparagine. This could be an alternative (kinase-independent) method for improved Bora-PLK1 interaction. When comparing the Bora:Aurora-A side of the predicted interface and focusing on the short region of Bora in between Aurora-A and PLK1, there were no clear differences seen in the residues predicted to bind to Aurora-A. This modelling has been included in Supplementary Figure S10 C and D.

1. Given the relevance of the recent publication from Zhu et al. to this study, the authors may want to comment on, or test, the relative importance of PKA and Aurora A as a potential kinase for Bora S59. While those authors argue that PKA phosphorylates Bora on Ser-59, one could easily imagine a model in which either PKA or Aurora A could initially phosphorylate that site followed by a propagation step after initial Aurora A activation, in which Aurora A phosphorylation of Bora Ser-59 is the dominant process.

Response: ____A brief discussion of this recent publication has been added to the discussion, highlighting the similarities between the two publications and the importance of pSer59, as well as suggesting that in cellulo this modification could be achieved via more than one pathway. We also include some additional NMR data to show improved Bora–Aurora-A interaction after phosphorylation at Ser59 (Supplementary Figure S12).

Reviewer 2.

Minor comments.

Page 5: '... a K82R PLK1 mutant was used to increase the stability of the protein' - It is not clear how this mutation confers increased stability of the protein. The authors do not show any data to support this. Isn't the PLK1 K82R an ATP-binding-deficient, kinase-inactive mutant?

Response: ____Thank you for spotting this, the text has been updated to clarify that this version of PLK1 was used as it is acting as a substrate in the in vitro assay as we didn’t want to see any PLK1 activity within this assay.

All panels showing the Alphabridge diagram - it would be helpful if pictorial definitions of the colour codes were provided with corresponding score ranges (in addition to the description in the figure legend).

Response:____The AlphaBridge images have been updated to include details about the plDDT scores each of the different colours refer to.

Fig 2B - The Fluorescence anisotropy assay curves do not reach a plateau. Though the effect of mutation on binding affinity is pretty clear, if possible, I suggest including more data points at higher concentrations and estimating apparent Kd values.

__Response:____The direct binding assay was repeated with a higher concentration of PLK1 in order to try and see a top plateau. This was successful and has been included in Figure 2B (shown in black). The measured Kd was 24 ± 3 µM. __

The cartoon representation of the structures and molecular interfaces - better to avoid shadows, as they compromise the clarity of the figures, particularly the ones where side chains are shown in stick representation.

Response:____The structural images have been remade to remove the shadows and improve the clarity of the images.

It is important to discuss how the parallel studies by Verza et al. and Pillan et al. complement this study, highlighting similarities and differences.

Response:____References to these two publications and details on the similarities and differences seen are now included in the discussion.

Reviewer 3.

Major comments

It would be helpful to measure the level of pThr210 PLK1 in some experiments and graph the data. The current presentation is Fig. 2D-E is qualitative rather than quantitative.

Response:____Graphs displaying the levels of pThr210 produced in the assay are now shown in Supplementary Figure S4.

Have the authors measured the binding affinity of the F/W mutant Bora for PLK1 using the assay in Fig. 2B? Likewise, for Fig. 7 the S59 mutant could be tested to see if it affects PLK1 binding or activation.

Response:____The direct binding assay has been repeated with the use of a FAM-Bora peptide that incorporates the F56A W58A mutation which shows reduced binding (Figure 2B, shown in blue). A version of the Bora peptide phosphorylated on Ser59 was also tested in the direct binding assay and this shows a similar affinity for PLK1 to the wild-type sequence (Figure 2B, shown in red compared to the wild-type shown in black).

It would be helpful if measurements of pThr210 PLK1 for all conditions were shown in the graph Fig. 7F.

Response:____This graph has been updated to include the levels of phosphorylation seen for PLK1 in all of the conditions tested.

Minor comments

I found Figure S1B easier to understand than Fig S1A and Fig 1A-B. Some of the supplemental data Fig. S1C-E could be moved to a revised Figure 1, dropping the current Fig. 1A-B. Can the interaction plots (Fig. S1C-D) be rotated to have the same original at the top and order of proteins (i.e. Bora > Aurora A > {plus minus} PLK1 depending on the plot).

Response:____Figure 1 and S1 have been rearranged to hopefully make them easier to understand, with all AlphaFold3 models of the full-length sequences kept in the supplementary figure and the focus in 1B just on the truncated model. The AlphaBridge plots have been rotated as suggested.

Figure 3F. Typo "Strongyl" not "Strongly".

Response:____Thank you for spotting this, this has been corrected in the updated manuscript.

Figure 3 could be supplemental material.

Response:__Thank you for your suggestion, but we have decided to keep this as a main figure.

Fig. 7E. Run a positive control reaction +ERK2 on the second gel to allow direct comparison of pThr210 across all the conditions tested.

Response:____These samples have been rerun on the same membrane and the levels of phosphorylation have been quantified and included in Figure 7F.

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Referee #3

Evidence, reproducibility and clarity

Summary.

Miles and co-workers have carried out a careful and high-quality study of the activation mechanisms of the mitotic kinase PLK1. Multiple proteins have been implicated in PLK1 activation and localisation as cell enter and pass through mitosis. Initial activation of PLK1 is promoted by a complex of Bora with another kinase Aurora A. Later in mitosis, this activated PLK1 associates with mitotic spindle and centrosome proteins regulating different aspects of mitosis and cytokinesis. In this study, Miles et al. extend previous work on this question by proposing and testing detailed models for Bora/Aurora A-mediated activation of PLK1 to elucidate the mechanism of this reaction.

Using the latest Alphafold they generate a series of models of the PLK1/Bora/Aurora A complex to home in on the key regions mediating interactions of the three proteins. This approach suggests an arrangement where the first ~120 amino acids of Bora wrap Aurora A and create an interaction surface for the N-terminal kinase domain of PLK1. This orients Thr210 in PLK1 towards Aurora A creating a situation likely favourable for phosphorylation, although has the authors discuss there are some caveats to this. A further prediction of the modelling helps explain the requirement for Bora phosphorylation to promote the interaction with Aurora A. This data is presented in Fig. 1 and Fig. S1-S3.

In the subsequent figures the details of this model are tested using biochemical assays and structural biology methods to validate key predictions. First the PLK1 interaction with Bora was shown to require the conserved F/W motif of Bora and a conserved pocket close to R106 on PLK1 (Fig. 2 and 3). In reconstituted PLK1 activation assays the F/W motif mutant Bora showed greatly attenuated pThr210 phosphorylation. This reaction also required phosphorylation of Bora at S112, presumably due to the interaction with Aurora A. An R106A mutant PLK1 showed reduced binding to Bora and reduced kinase activation. This data is clear and provides compelling support for the model.

Using NMR the authors then investigate the interaction between Bora and Aurora A, and more specifically the requirement for Bora phosphorylation at Ser112. The NMR data in Fig. 4 and Fig. 6 provide good support for the Alphafold model. A helpful comparison with known Aurora A binding proteins is also shown to highlight the way CEP192, TPX2 and TACC3 contact a series of conserved pockets on the surface of Aurora A which are common to the Bora interaction. S59 phosphorylation by Aurora A is also shown to play an important role in contacting PLK1 and is required for pThr210 phosphorylation.

In summary, the authors have made valuable progress in working out details of the PLK1 activation mechanism, that extends previous work in the field.

Major comments.

It would be helpful to measure the level of pThr210 PLK1 in some experiments and graph the data. The current presentation is Fig. 2D-E is qualitative rather than quantitative.

Have the authors measured the binding affinity of the F/W mutant Bora for PLK1 using the assay in Fig. 2B? Likewise, for Fig. 7 the S59 mutant could be tested to see if it affects PLK1 binding or activation.

It would be helpful if measurements of pThr210 PLK1 for all conditions were shown in the graph Fig. 7F.

Minor comments.

I found Figure S1B easier to understand than Fig S1A and Fig 1A-B. Some of the supplemental data Fig. S1C-E could be moved to a revised Figure 1, dropping the current Fig. 1A-B. Can the interaction plots (Fig. S1C-D) be rotated to have the same original at the top and order of proteins (i.e. Bora > Aurora A > {plus minus} PLK1 depending on the plot). Figure 3F. Typo "Strongyl" not "Strongly". Figure 3 could be supplemental material. Fig. 7E. Run a positive control reaction +ERK2 on the second gel to allow direct comparison of pThr210 across all the conditions tested.

Significance

Timely and orchestrated activation of multiple mitotic protein kinases is crucial for the alignment and segregation of chromosomes, and for the process of cell division. In this study the authors explore how activation of the mitotic kinase PLK1 is triggered by another mitotic kinase Aurora A, and the role played by a scaffold protein Bora.

Strengths: Detailed analysis of mechanism using biochemical and structural approaches.

Limitations: The study is focussed on the biochemical and structural mechanisms rather than the cellular outcomes. Some data would benefit from additional quantitative measurement.

Relevance: Cancer and cell biology due to the role of Aurora A in many cancers.

Reviewer expertise: Biochemistry, molecular and cell biology.

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Referee #2

Evidence, reproducibility and clarity

Summary:

PLK1 is one of the master regulators of cell division. The activation of PLK1 requires the activation loop phosphorylation at T210, mediated by Aurora A kinase. However, Aurora A phosphorylation of PLK1 T210 requires Bora, one of the several activators of Aurora A kinase. While the molecular requirement of Aurora A kinase and Bora for PLK1 activation is well established, the mechanistic understanding of how Bora facilitates PLK1 activation by Aurora A has remained an important open question for a long time. Exploiting the latest development in AI-driven structure prediction, three independent studies provide a structural and mechanistic basis for PLK1 activation by Aurora A and Bora. Here, Miles et al. have generated AlphaFold models, further characterised some of the interfaces using NMR, and validated the contribution of intermolecular interactions at suggested interfaces in vitro using recombinant proteins in kinase assays. Overall, this is a well-executed work providing important new insights into our understanding of the activation of the critical regulator of cell division, PLK1. However, as the authors have highlighted in the discussion section, one limitation of this modelling study is that the models still do not entirely explain how these interactions facilitate the phosphorylation of Thr210ur, as this residue is oriented far away from Aurora A's active site for the reaction to take place. Despite this limitation, I believe this is an important work that advances our understanding significantly.

Comments:

Experimental data satisfactorily support claims. Hence, most of my comments are minor in nature.

Points to consider during revision:

Page 5: '... a K82R PLK1 mutant was used to increase the stability of the protein' - It is not clear how this mutation confers increased stability of the protein. The authors do not show any data to support this. Isn't the PLK1 K82R an ATP-binding-deficient, kinase-inactive mutant?

All panels showing the Alphabridge diagram - it would be helpful if pictorial definitions of the colour codes were provided with corresponding score ranges (in addition to the description in the figure legend).

Fig 2B - The Fluorescence anisotropy assay curves do not reach a plateau. Though the effect of mutation on binding affinity is pretty clear, if possible, I suggest including more data points at higher concentrations and estimating apparent Kd values.

The cartoon representation of the structures and molecular interfaces - better to avoid shadows, as they compromise the clarity of the figures, particularly the ones where side chains are shown in stick representation.

It is important to discuss how the parallel studies by Verza et al. and Pillan et al. complement this study, highlighting similarities and differences.

Significance

As highlighted in the summary, a mechanistic understanding of how PLK1 is activated by Aurora A kinase and its activator Bora has remained a long-standing open question. As PLk1 is one of the major regulators of cell division, which exerts its function (via phosphorylating numerous substrates) during different stages of mitosis, understanding its activation mechanism is of critical interest for those working on the cell cycle in general and cell division in particular. A key limitation of this study is the lack of any cellular functional evaluation of the interaction interfaces.

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Referee #1

Evidence, reproducibility and clarity

Miles et al. used a combination of AlphaFold modeling, biochemical assays of mutant constructs and NMR spectroscopy to model the ternary complex of Aurora A, Bora and Plk1, and elucidate how Bora can act as a molecular bridge that facilitates the phosphorylation of the activation loop Thr210 within Plk1 by Aurora A. Their studies identified an interaction between residues 52-73 within Bora and the 'FW' pocket on the N-terminal lobe of Plk1, which binds Phe56 and Trp58 of Bora. Additionally, Ser59 of Bora was identified as a good Aurora A substrate using a Bora peptide array, and pSer59 was predicted to form bridging interactions with Aurora Arg205 and Plk1 Arg59. This was supported by NMR and biochemical assays. In addition, the authors validate that phosphorylation of Ser-112 on Bora enhances stabilization of the Aurora A-Bora complex Overall, the model revealed novel details of the interactions within the Aurora A-Bora-Plk1 ternary complex that are supported by the biochemical and NMR data. The work will be of significant interest to basic scientists whose work involves protein kinase signaling, cell division/mitosis, signal transduction, and cancer biology. We recommend publication of this manuscript with the following minor changes and additions.

1. In the introduction, on page 2, the authors seem a little confused about the Plk1 Polo-box domain - text as written: "...kinase domain linked to tandem Polo-box domains (PBD)", and cite a review paper. Actually, there is only a single Polo-box domain in these kinases, which contains both Polo-boxes and a bit of the upstream linker region. The "PBD" terminology denotes his 2-Polo-box +linker structure. Perhaps it would be better here to cite the PBD structure (Elia et al., Cell, 2002) as a primary citation here.
2. Similarly, the line "...during the G2/M transition following successful DNA damage repair" cites the Seki et al paper, but those findings are shown in the Macurek et al paper, not the Seki et al paper.
3. Using the model of the ternary complex as shown in Figure 1B, deletion constructs of Bora missing regions within the disordered loops, but still retaining the residues that bind the PBD, FW pocket and Aurora A, can be modeled and tested to see if such deletions can improve the ipTM scores and binding affinity.
4. On page 5, "S112A" within the sentence "Unexpectedly, the F56A/W58A Bora was less efficiently phosphorylated on S112A (Supplementary Figure S11, F compared to H and Supplementary Table S4)." This should be "S112".
5. In the assays shown in Figure 2D, the presence of excess F56AW58A Bora that remained unphosphorylated on S112 may complicate the interpretation of the results. Can the authors show that the S112-phosphorylated F56AW68A Bora is predominantly bound to Aurora A in such a mixture, perhaps by NMR using labelled pS112 F56AW58A Bora and unlabeled S112 F56AW58A Bora?
6. Please expand Figure 3A to better show the FW pocket-forming residues on Plk1.
7. It would be helpful to label the peaks in the mass spectra in Fig. S11 with the phospho-species that they correspond to.
8. In the last paragraph on page 7, "see we" in the sentence "As well as a decrease in intensity around pSer112 in Bora, see we an overall effect with decreased intensity across most of the Bora sequence." Should be corrected to "we see".
9. While not required, it would be helpful if binding or Bora to Aurora A after Erk2 phosphorylation could be shown using fluorescence polarization or ITC to lend additional support to the NMR data for S112 and S59 phosphorylation and for CEP192 and TPX2 competition.
10. The Aurora A phosphorylation motif has been further defined beyond that reported by the Pinna lab in 2005. Notably, the Ser-59 sequence on Bora (F-R-W-S-I), has, in addition to dominant selection for AR in the -2 position, both favorable -1 (W) and +1 (I) positions based on peptide library measurements (Alexander et al., Science Signaling 2011), further arguing that it may be an excellent Aurora A phosphorylation site.
11. Have the authors tried to model the Drosophila melanogaster Aurora A-Bora-Polo complex to see if the Asn substitution of Bora Ser59, and the expected loss of the interactions between Bora pSer59 and Plk1 Arg59 and Aurora A Arg205 are compensated by other features?
12. Given the relevance of the recent publication from Zhu et al. in https://doi.org/10.1038/s41467-025-63352-y to this study, the authors may want to comment on, or test, the relative importance of PKA and Aurora A as a potential kinase for Bora S59. While those authors argue that PKA phosphorylates Bora on Ser-59, one could easily imagine a model in which either PKA or Aurora A could initially phosphorylate that site followed by a propagation step after initial Aurora A activation, in which Aurora A phosphorylation of Bora Ser-59 is the dominant process.

-Dan Lim and Michael Yaffe

Significance

The work is well done and clearly presented.

This article by researchers at UW discuss the mass wave of misinformation in regards to the coronavirus. In this article, one of the researchers reveals that often, misinformation during massive events such as the Covid 19 out break are due to people's growing fear and anxiety. Because of the uncertainty, people are often seeking solutions, which therefore help exhilarate information they may find shocking or comforting. This is why its important to always fact check data before fully believing what you see online.

This article talks about how even if profiles may not share much information about themselves, their sexual orientation can still be deduced by their contact list, or "friends" list online. This that simple things like your network of who you know can reveal about yourself. It was revealed in the article that researches were also able to build bots which can collect information just like this to keep tabs on people. This goes for show that although you may think you are protected, you're online data can reveal things you may not want to share.

I found Catherine Stinson’s “The Dark Past of Algorithms That Associate Appearance and Criminality” especially compelling — it highlights how seemingly neutral data-mining efforts (for example facial recognition or risk scoring) embed deep historical biases and reinforce harmful associations. It makes me reflect: when we apply mining methods in social-media contexts, it’s not just about data quality but also which associations we’re willing to carry forward.

Reading this article had me very surprised. But also kind of not. It was about how Facebook tracks and stores data on people that aren't even users on the Facebook platform. I had no idea about Facebook tracking people even if they didn't have a Facebook account. But I totally believe it and it makes sense to me. These social media companies cross so many boundaries just to try and get a little more money. It disturbs me hearing that Facebook can track people that aren't Facebook users and I can't believe this is normalized.

保留元素在矩阵中的位置信息

I like this part. The author says we should not just follow the minimum rules. We should try to create a city that feels peaceful for everyone. It's not just about avoiding fines; it's about making the community a better place to live.

This is a very useful and specific solution. It explains a technical way to reduce noise—using special windows. It even says how much noise it can block (around 40 dB).

This statistic is very powerful. It shows that in just five years, the number of complaints in New York City more than doubled. This proves that the problem is not getting better; it is getting much worse. It is a clear trend.

This is a direct link between bar noise and health problems. It's not just about being annoyed. The noise causes real mental health issues like anxiety and sleep problems for many people. This is very strong evidence for my research.

This part is important because it explains why we can't just close all the bars. They are important for the city's culture and for tourism. This shows that the problem is not simple; we have to find a balance between nightlife and quiet for residents.

This is a important statistic from the study. It gives specific numbers for how loud bars are. 80 dBA is very loud, like a garbage disposal. This is solid data I can use to show how big the problem is.

eLife Assessment

This manuscript investigates inter-hemispheric interactions in the olfactory system of Xenopus tadpoles. Using a combination of electrophysiology, pharmacology, imaging, and uncaging, the transection of the contralateral nerve is shown to lead to larger odor responses in the unmanipulated hemisphere, and implicates dopamine signaling in this process. The study uses a rich and sophisticated array of tools to investigate olfactory coding and uncovers valuable mechanisms of signaling. However, the data is incomplete, with a few of the conclusions not being well-supported by the data; the interpretation should be adjusted with some caveats, or additional experiments should be done to support these conclusions.

Reviewer #1 (Public review):

In this study, the authors investigate LFP responses to methionine in the olfactory system of the Xenopus tadpole. They show that this response is local to the glomerular layer, arises ipsilaterally, and is blocked by pharmacological blockade of AMPA and NMDA receptors, with little modulation during blockade of GABA-A receptors. They then show that this response is translently enlarged following transection of the contralateral olfactory nerve, but not the optic lobe nerve. Measurement of ROS- a marker of inflammation- was not affected by contralateral nerve transection, and LFP expansion was not affected by pharmacological blockade of ROS production. Imaging biased towards presynaptic terminals suggests that the enlargement of the LFP has a presynaptic component. A D2 antagonist increases the LFP size and variability in intact tadpoles, while a GABA-B antagonist does not. On this basis, the authors conclude that the increase driven by contralateral nerve transection is due to DA signaling.

Overall, I found the array of techniques and approaches applied in this study to be creatively and effectively employed. However, several of the conclusions made in the Discussion are too strong, given the evidence presented. For example, the authors state that "The observed potentiation was not related to inflammatory mediators associated to inury, because it was caused by a release of the inhibition made by D2 dopamine receptor present in OSN axon terminals." This statement is too strong - the authors have shown that D2 receptors are sufficient to cause an increase in LFP, but not that they are required for the potentiation evoked by nerve transection. The right experiment here would be to get rid of the D2 receptors prior to transection and show that the potentiation is now abolished. In addition, the authors have not shown any data localizing D2 receptors to OSN axon terminals.

Similarly, the authors state, "the onset of LFP changes detected in glomeruli is determined by glutamate release from OSNs." Again, the authors have shown that blockade of AMPA/NMDA receptors decreases the LFP, and that uncaging of glutamate can evoke small negative deflections, but not that the intact signal arises from glutamate release from OSNs. The conclusions about the in vivo contribution of this contralateral pathway are also rather speculative. Acute silencing of one hemisphere would likely provide more insight into the moment-to-moment contributions of bilateral signals to those recorded in one hemisphere.

Author response:

Thank you for your time and for considering our manuscript as a Reviewed Preprint. We also would like to thank Reviewer 1 for their evaluation of our manuscript.

Here, we present a provisional response to reviewer comments and following their suggestions we will make an effort to: i) increase evidence for the role of dopamine in olfactory glomeruli and ii) delineate the circuit involved mediating the observed potentiation. Next, we briefly describe the set of experiments that are in progress or will be performed to improve our paper.

We will carry out immunostainings for tyrosine hydroxylase to certify that dopamine can be released on the genetically labelled glomerulus. There is a lack of good commercial antibodies for Xenopus (we already tried one and did not work, PA1-4679, Thermofisher scientific), but we will look for alternatives. In a previous set of experiments, we attempted to measure dopamine release in the glomerular layer by electroporating olfactory sensory neurons or olfactory bulb neurons with the dopamine sensors dLight1.1 (Addgene #111053) or dLight1.3 (Addgene # 111056). In our hands, fluorescence signals were extremely weak, barely undetectable. Similar results were obtained after electroporating the tectum or the rhombencephalon. We propose to repeat experiments using a more sensitive sensor such as GRAB_DA2m. Other approaches, such as performing single cell transcriptomics of olfactory sensory neurons might be considered to confirm the expression of D2 receptors.

We agree with the reviewer that we should obtain more lines of evidence in support for a presynaptic inhibition mediated by D2 receptors.To gain insight on the bilateral circuit mediating the observed potentiation of glomerular responses we are currently investigating the role of dorsolateral pallium neurons. In Xenopus tadpoles the lateral pallium plays an analogous role to the olfactory cortex in amniotes. Preliminary observations show that neurons located in this pallial region respond to ipsilateral stimulation of the olfactory epithelium and if damaged, a contralateral potentiation of glomerular output occurs. We aim to conclude this set of experiments and include it in the paper as we believe it clarifies the circuitry involved.

eLife Assessment

This valuable developmental study provides intriguing but incomplete evidence suggesting that, relative to adults, the enhancement of instrumental learning by Pavlovian bias is most pronounced in adolescence, while reward-induced memory enhancements are strongest in childhood. Although the authors tackle a key aspect of learning and motivation with rigorous experimental methods and sophisticated modeling techniques, there are substantial concerns about the absence of relevant analyses, the lack of accord between model-based and exploratory analyses, and the lack of an explanation for how the results cohere with inconsistent findings in the literature.

Reviewer #1 (Public review):

In this study, the authors aim to elucidate both how Pavlovian biases affect instrumental learning from childhood to adulthood, as well as how reward outcomes during learning influence incidental memory. While prior work has investigated both of these questions, findings have been mixed. The authors aim to contribute additional evidence to clarify the nature of developmental changes in these processes. Through a well-validated affective learning task and a large age-continuous sample of participants, the authors reveal that adolescents outperform children and adults when Pavlovian biases and instrumental learning are aligned, but that learning performance does not vary by age when they are misaligned. They also show that younger participants show greater memory sensitivity for images presented alongside rewards.

The manuscript has notable strengths. The task was carefully designed and modified with a clever, developmentally appropriate cover story, and the large sample size (N = 174) means their study was better powered than many comparable developmental learning studies. The addition of the memory measure adds a novel component to the design. The authors transparently report their somewhat confusing findings.

The manuscript also has weaknesses, which I describe in detail below.

It was not entirely clear to me what central question the researchers aimed to address. They note that prior studies using a very similar learning task design have reported inconsistent findings, but they do not propose a reason for why these inconsistent findings may emerge nor do they test a plausible cause of them (in contrast, for example, Raab et al. 2024 explicitly tested the idea that developmental changes in inferences about controllability may explain age-related change in Pavlovian influences on learning). While the authors test a sample of participants that is very large compared to many developmental studies of reinforcement learning, this sample is much smaller than two prior developmental studies that have used the same learning task (and which the authors cite - Betts et al., 2020; Moutoussis et al., 2018). Thus, the overall goal seems to be to add an additional ~170 subjects of data to the existing literature, which isn't problematic per se, but doesn't do much to advance our theoretical understanding of learning across development. They happen to find a pattern of results that differs from all three prior studies, and it is not clear how to interpret this.

Along those lines, the authors extend prior work by adding a memory manipulation to the task, in which trial-unique images were presented alongside reward outcomes. It was not clear to me whether the authors see the learning and memory questions as fundamentally connected or as two separate research questions that this paradigm allows them to address. The manuscript would potentially be more impactful if the authors integrated their discussion of these two ideas more. Did they have any a priori hypotheses about how Pavlovian biases may affect the encoding of incidentally presented images? Could heightened reward sensitivity explain both changes in learning and changes in memory? It was also not clear to me why the authors hypothesized that younger participants would demonstrate the greatest effects of reward on memory, when most of the introduction seems to suggest they might hypothesize an adolescent peak in both learning and memory.

As stated above, while the task methods seemed sound, some of the analytic decisions are potentially problematic and/or require greater justification for the results of the study to be interpretable.

Firstly, it is problematic not to include random participant slopes in the regression models. Not accounting for individual variation in the effects of interest may inflate Type I errors. I would suggest that the authors start with the maximal model, or follow the same model selection procedure they did to select the fixed effects to include for the random effects as well.

Secondly, the central learning finding - that adolescents demonstrate enhanced learning in Pavlovian-congruent conditions only - is interesting, but it is unclear why this is the case or how much should be made of this finding. The authors show that adolescents outperform others in the Pavlovian-congruent conditions but not the Pavlovian-incongruent conditions. However, this conclusion is made by analyzing the two conditions separately; they do not directly compare the strength of the adolescent peak across these conditions, which would be needed to draw this strong conclusion. Given that no prior study using the same learning design has found this, the authors should ensure that their evidence for it is strong before drawing firm conclusions.

It was also not clear to me whether any of the RL models that the authors fit could potentially explain this pattern. Presumably, they need an algorithmic mechanism in which the Pavlovian bias is enhanced when it is rewarded. This seems potentially feasible to implement and could help explain the condition-specific performance boosts.

I also have major concerns about the computational model-fitting results. While the authors seemingly follow a sound approach, the majority of the fitted lapse rates (Figure S10) are near 1. This suggests that for most participants, the best-fitting model is one in which choices are random. This may be why the authors do not observe age-related change in model parameters: for these subjects, the other parameter values are essentially meaningless since they contribute to the learned value estimate, which gets multiplied by a near-0 weight in the choice function. It is important that the authors clarify what is going on here. Is it the case that most of these subjects truly choose at random? It does seem from Figure 2A that there is extensive variability in performance. It might be helpful if the authors re-analyze their data, excluding participants who show no evidence of learning or of reward-seeking behavior. Alternatively, are there other biases that are not being accounted for (e.g., choice perseveration) that may contribute to the high lapse rates?

Parameter recovery also looks poor, particularly for gain & loss sensitivity, the lapse rate, and the Pavlovian bias - several parameters of interest. As noted above, this may be due to the fact that many of the simulations were conducted with lapse rates sampled from the empirical distribution. It would be helpful for the authors to a.) plot separately parameter recoverability for high and low lapse rates and b.) report the recoverability correlation for each parameter separately.

Finally, many of the analytic decisions made regarding the memory analyses were confusing and merit further justification.

(1) First, it seems as though the authors only analyze memory data from trials where participants "could gain a reward". Does this mean only half of the memory trials were included in the analyses? What about memory as a function of whether participants made a "correct" response? Or a correct x reward interaction effect?

(2) The RPE analysis overcomes this issue by including all trials, but the trial-wise RPEs are potentially not informative given the lapse rate issue described above.

(3) The authors exclude correct guesses but include incorrect guesses. Is this common practice in the memory literature? It seems like this could introduce some bias into the results, especially if there are age-related changes in meta-memory.

(4) Participants provided a continuum of confidence ratings, but the authors computed d' by discretizing memory into 'correct' or 'incorrect'. A more sensitive approach could compute memory ROC curves taking into account the full confidence data (e.g., Brady et al., 2020).

(5) The learning and memory tradeoff idea is interesting, but it was not clear to me what variables went into that regression model.

Reviewer #2 (Public review):

The authors of this study set out to investigate whether adolescents demonstrate enhanced instrumental learning compared to children and adults, particularly when their natural instincts align with the actions required in a learning task, using the Affective Go/No-Go Task. Their aim was to explore how motivational drives, such as sensitivity to rewards versus avoiding losses, and the congruence between automatic responses to cues and deliberate actions (termed Pavlovian-congruency) influence learning across development, while also examining incidental memory enhancements tied to positive outcomes. Additionally, they sought to uncover the cognitive mechanisms underlying these age-related differences through behavioral analyses and reinforcement learning models.

The study's major strengths lie in its rigorous methodological approach and comprehensive analysis. The use of mixed-effects logistic regression and beta-binomial regression models, with careful comparison of nested models to identify the best fit (e.g., a significant ΔBIC of 19), provides a robust framework for assessing age-related effects on learning accuracy. The task design, which separates action (pressing a key or holding back) from outcome type (earning money or avoiding a loss) across four door cues, effectively isolates these factors, allowing the authors to highlight adolescent-specific advantages in Pavlovian-congruent conditions (e.g., Go to Win and No-Go to Avoid Loss), supported by significant quadratic age interactions (p < .001). The inclusion of reaction time data and a behavioral metric of Pavlovian bias further strengthens the evidence, showing adolescents' faster responses and greater reliance on instinctual cues in congruent scenarios. The exploration of incidental memory, with a clear reward memory bias in younger participants (p < .001), adds a valuable dimension, suggesting a learning-memory trade-off that enriches the study's scope. However, weaknesses include minor inconsistencies, such as the reinforcement learning model's Pavlovian bias parameter not reflecting an adolescent enhancement despite behavioral evidence, and a weak correlation between learning and memory accuracy (r = -.17), which may indicate incomplete integration of these processes.

The authors largely achieved their aims, with the results providing convincing support for their conclusion that Pavlovian-congruency boosts instrumental learning in adolescence. The significant quadratic age effects on overall learning accuracy (p = .001) and in congruent conditions (e.g., p = .01 for Go to Win), alongside faster reaction times in these scenarios, convincingly demonstrate an adolescent peak in performance. While the reinforcement learning model's lack of an adolescent-specific Pavlovian bias parameter introduces a slight caveat, the behavioral and statistical evidence collectively align with the hypothesis, suggesting that adolescents leverage their natural instincts more effectively when these align with task demands. The incidental memory findings, showing younger participants' enhanced recall for reward-paired images, partially support the secondary aim, though the trade-off with learning accuracy warrants further exploration.

This work is likely to have an important impact on the field, offering valuable insights into developmental differences in learning and memory that could influence educational practices and psychological interventions tailored to adolescents. The methods, particularly the task's orthogonal design and probabilistic feedback, are useful to the community for studying motivation and cognition across ages, while the detailed regression analyses and reinforcement learning approach provide a solid foundation for future replication and extension. The data, including trial-by-trial accuracy and memory performance, are openly shareable, enhancing their utility for researchers exploring similar questions, though refining the model-parameter alignment could strengthen its broader applicability.

Author response:

We thank both reviewers for their thoughtful and constructive comments. To address this feedback, we plan to do the following:

Questions/Hypotheses: We will clarify the study’s motivation, central questions, and our hypotheses, with a particular focus on the integration across learning and memory.

Methods: To improve clarity and transparency, we will expand the Methods section and modify relevant figures to provide more explanation of the task, our decisions regarding data analysis approaches, and how they address our questions and hypotheses.

Learning Behavioral Analysis: As suggested by reviewers, we will fit and compare mixed-effects models with the maximal random effects structure for the within-subject variables and their interactions. We may simplify this structure as the data justify (i.e., if we encounter convergence problems or the random effects explain minimal variance). In the revision, we will also directly compare the adolescent peaks in performance across the conditions to support our conclusion that adolescents outperform people of other ages in the Pavlovian-congruent conditions.

Computational Modeling: We appreciate the reviewers’ close attention to the computational modeling methods, as it identified a small error in the reporting of the formulas we implemented. Specifically, the preprint’s softmax function had an error and should be printed as:

This correct parameterization can be seen in the Huys, 2018 public repository on line 48 here. As such, rather than indicating random choices, the lapse rates with estimated solutions close to one represent expected goal-directed behavior. That said, we acknowledge that parameter recovery indicated potential identifiability issues for some parameters, especially those with extreme values. We appreciate the reviewer’s suggestion to examine “learners” separately from “non-learners,” as has been done in prior work with adults (Cavanagh et al., 2013; Guitart-Masip et al., 2012). In this revision, we will investigate whether behavioral differences in learners vs. non-learners, among other potential explanations, accounts for the relatively poor parameter recovery. We will also explain more about why we selected these RL models, including how the Pavlovian policy works and why it adequately captures participants’ behavior.

Memory Behavioral Analysis: At the reviewers’ suggestion, we will expand our analysis of the learning-memory trade-off to fully explore this possible explanation. We will also explore the additional analyses that the reviewers suggested (e.g., ROC curves accounting for confidence ratings, analysis of correct vs. incorrect responses).

We are confident that these revisions will strengthen the work, and we are grateful to the reviewers for their thorough, insightful feedback. In the coming revision, we will provide a detailed point-by-point response to all comments and questions.

References

Cavanagh, J. F., Eisenberg, I., Guitart-Masip, M., Huys, Q., & Frank, M. J. (2013). Frontal Theta Overrides Pavlovian Learning Biases. The Journal of Neuroscience, 33(19), 8541–8548. https://doi.org/10.1523/JNEUROSCI.5754-12.2013

Guitart-Masip, M., Huys, Q. J. M., Fuentemilla, L., Dayan, P., Duzel, E., & Dolan, R. J. (2012). Go and no-go learning in reward and punishment: Interactions between affect and effect. NeuroImage, 62(1), 154–166. https://doi.org/10.1016/j.neuroimage.2012.04.024

Huys, Q. J. M. (2018). Bayesian Approaches to Learning and Decision-Making. In Computational Psychiatry (pp. 247–271). Elsevier. https://doi.org/10.1016/B978-0-12-809825-7.00010-9

man is too weak to even maintain the spirit of mutiny, needs to be whipped into submission

Christ thinks too highly of men whose nature condemns them to slavery

man will create other mysteries -find soothsayers, sorcery, heresy, atheism -- atheism here a miracle? miracle of rationality's triumph over mystery?

man doesn't want God but wants signs, wants miracles, wants proof

blessed are those who have not seen, and yet have believed

man are weak 'rebels'; can only be conquered by Miracle, Mystery and Authority

man needs more than bread but will to live

man only desires to renounce his freedom

men chiefly desire to submit to collective will, not follow their own desires

authority conquers the meek and gains their admiration

order of might makes right, despair of the meek forced to serve the mighty

first temptation: miracle -- turn stones into bread to seduce men to follow

Christ rejected the means that could make man happy (ie by limiting their freedom)

authority, submission

= The distribution of the sample proportion.

The distribution of X: is the set of the possible values that X can take, and how often they occur

No, the experiment did sampling withOUT replacement, because 50 balls were drawn simultaneously.

This type of tracking has always been something I've been curious about. I often notice that when I search something into google, or look for specific things on TikTok/Instagram, I soon will get ads, or posts about that thing I searched recommended to me. While I know that this is the apps taking my data and pumping out content related to it in order to get me more hooked to the app, I find this type of tracking invasive and weird. I also notice that I've been getting a lot of UW related content on my social media apps recently. So this makes me think that these apps track more then your searches, but things like your location, etc. as well.

Ai

I totally agree with this sentence because for some surveys even as a responder, I feel like the question is somehow swaying me in a certain direction to an answer. For example, when surveys are coming from school, but are not anonymous, I feel guided to say something good or only given certain choices of answer to choose from when I have other things I wish to answer as. But overall I think the idea is that it's really important to focus on creating a place where everyone feels safe and comfortable to truly express themselves.

I agree that double barreled questions shouldn't be asked. I think it can be confusing because the participant will wonder which concept to talk about if there are multiple asked in the question. I think when creating questions, this is something I have to closely pay attention to because I may not realise I am doing it. I wonder, if I am thinking a question I ask might be asking about two things, should I ask the question with one concept, and then just make the second concept a part of that question as a follow up, or make two separate questions for each concept?

I think these questions that are listed are all highly valuable questions. The reason why I think that is because I had been put in a lot of situations where I had to solve a problem in some kind of a way and many times I look over these factors. These are the most critical base of the solution process but I think are also very easy to miss and overlook them. Especially for "did anyone try to solve it in the past and fail?", I think this is really a strong question that needs to be pondered on and requires a lot of thinking/research based off other questions like "am I copying other person's solution/method?" "Can I learn out of their failures? Can I try it on my own?" and so on.

I think this makes an interesting point and also added another perspective. Usually when I do competitive analysis, I would usually come up with potential competition in my mind but never make an actual list of competition and comparison. Also the text mentioned that this will keep your research guided. This also added another perspective that you can use your comparison and competition to help you stay in your lane and I think that this also makes your solution go towards something that will solve a problem.

This idea really resonates with me because it highlights how important it is to research and analyze what already exists before creating something new. In UX design, it’s easy to jump straight into ideation, but taking the time to study competitors helps ensure that our design decisions are strategic rather than based on assumptions. By understanding what others have done, what works well and what doesn’t, we can build a stronger, more user-centered product that truly stands out.

Agreed. For the longest time I was searching for a "perfect framework" that would allow me to perform the most thorough competitive analysis. I then realized that the nature of the work was to show comparison, and that the methods should center around the stakeholders we're trying to communicate to.

Competitive analysis was something I wasn't aware of prior to this article, but after reading, I 100% agree with this statement. You can't just think about your customers when coming up with a solution design. Its also interesting that this is something that you will have to constantly think about. I wouldn't be surprised if it was the companies with good competitive analysis that are the one that stay afloat the longest.

I agree with this point. If you do a competitor analysis and find a competitor that is doing exactly what you want to do, what is the value in your new product? A good competitor analysis can ensure that you learn from others' mistakes, and address gaps that currently exist in the market. That's also why I think the question "why did they fail" is very important in understanding what previously went wrong when people tried to solve the problem.

I found the discussion of unintended versus intentional data poisoning especially striking — it reminded me of how a viral trend on a platform can distort a research survey in ways the authors likely never anticipated. One thing I’m wondering though: given that many social-media datasets are collected passively and opportunistically, how can researchers realistically detect when the data has already been poisoned by normal platform usage (rather than a malicious actor)?

eLife Assessment

This study provides a valuable contribution to understanding the functional and molecular organization of the medial nucleus accumbens shell in feeding. Using in vivo imaging, optogenetics, and genetic engineering, the authors present solid evidence for a rostro-caudal gradient in D1-SPN activity that refines earlier pharmacological models. The identification of Stard5 and Peg10 as molecular markers and the creation of a Stard5-Flp line represent meaningful advances for future circuit-specific studies. While stronger integration of molecular and functional results and additional analyses of other Stard5-expressing cell types (e.g., D2-SPNs, interneurons) would enhance completeness, the overall methodological rigor and convergence of findings make this a well-executed and informative study. This will be of interest to those interested in brain circuits, reward, emotion, and feeding behavior.

Reviewer #1 (Public review):

Summary:

This study examines how different parts of the brain's reward system regulate eating behavior. The authors focus on the medial shell of the nucleus accumbens, a region known to influence pleasure and motivation. They find that nerve cells in the front (rostral) portion of this region are inhibited during eating, and when artificially activated, they reduce food intake. In contrast, similar cells at the back (caudal) are excited during eating but do not suppress feeding. The team also identifies a molecular marker, Stard5, that selectively labels the rostral hotspot and enables new genetic tools to study it. These findings clarify how specific circuits in the brain control hedonic feeding, providing new entry points to understand and potentially treat conditions such as overeating and obesity.

Strengths:

(1) Conceptual advance: The work convincingly establishes a rostro-caudal gradient within the medNAcSh, clarifying earlier pharmacological studies with modern circuit-level and genetic approaches.

(2) Methodological rigor: The combination of fiber photometry, optogenetics, CRISPR-Cas9 genetic engineering, histology, FISH, scRNA-seq, and novel mouse genetics adds robustness, with complementary approaches converging on the central claim.

(3) Innovation: The generation of a Stard5-Flp line is a valuable resource that will enable precise interrogation of the rostral hotspot in future studies.

(4) Specificity of findings: The dissociation between appetitive and aversive conditions strengthens the interpretation that the observed gradient is restricted to feeding.

Weaknesses and points for clarification

(1) Role of D2-SPNs: Since D1 and D2 pathways often show opposing roles in feeding, testing, or discussing D2-SPN contributions would provide an important control and context. Since the claim is that Stard5 is expressed in both D1- and D2MSNs, it seems to contradict the exclusive role of D1R MSNs in authorizing food intake.

(2) Behavioral analyses:

a) In Figure 2, group differences in consumption appear uneven; additional analyses (e.g., lick counts across blocks and session totals) would strengthen interpretation.

b) The design and contribution of aversive assays to the main conclusions remain somewhat unclear and could be better justified.

c) The scope of behavior is mainly limited to consumption; testing related domains (motivation, reward valuation, and extinction) could broaden the significance.

(3) Molecular profiling:

a) Stard5 expression is present in both D1- and D2-SPNs; comparisons to bulk calcium signals and quantification of percentages across rostral and caudal cells would be helpful. The authors should establish whether these cells also express SerpinB2, an established marker of LH projecting neurons.

b) Verification of the Stard5-2A-Flp line (specificity, overlap with immunomarkers) should be documented more thoroughly.

c) The molecular analysis is restricted to a small set of genes; broader spatial transcriptomics could uncover additional candidate markers. See also above.

Reviewer #2 (Public review):

Summary:

Marinescu et al. combine in vivo imaging with circuit-specific optogenetic manipulation to characterize the anatomic heterogeneity of the medial nucleus accumbens shell in the control of food intake. They demonstrate that the inhibitory influence of dopamine D1 receptor-expressing neurons of the medial shell on food intake decreases along a rostro-caudal gradient, while both rostral and caudal subpopulations similarly control aversion. They then identify Stard5 and Peg10 as molecular markers of the rostral and caudal subregions, respectively. Through the development of a new mouse line expressing the flippase under the promoter of Stard5, they demonstrate that Stard5-positive neurons recapitulate the activity of D1-positive neurons of the rostral shell in response to food consumption and aversive stimuli.

Strengths:

This study brings important findings for the anatomical and functional characterization of the brain reward system and its implications in physiological and pathological feeding behavior. It is a well-designed study, technically sound, with clear and reliable effects. The generation of the new Stard5-Flp line will be a valuable tool for further investigations. The paper is very well written, the discussion is very interesting, addresses limitations of the findings, and proposes relevant future directions

Weaknesses:

At this stage, identification and characterization of the activity of Stard5-positive neurons is a bit disconnected from the rest of the paper, as this population encompasses both D1- and D2-positive neurons as well as interneurons. While they display a similar response pattern as D1-neurons, it remains to be determined whether their manipulation would result in comparable behavioral outcomes.

eLife Assessment

This study presents a valuable in-depth comparison of statistical methods for the analysis of ecological time series data, and shows that different analyses can generate different conclusions, emphasizing the importance of carefully choosing methods and of reporting methodological details. The evidence supporting the claims, based on simulated data for a two-species ecosystem, is solid, although testing on more complex datasets could be of further benefit. This paper should be of broad interest to researchers in ecology.

Reviewer #1 (Public review):

Summary:

The manuscript investigates methods for the analysis of time series data, in particular ecological time series. Such data can be analyzed using a myriad of approaches, with choices being made in both the statistical test performed and the generation of artificial datasets for comparison. The simulated data is for a two-species ecosystem. The main finding is that the rates of false positives and negatives strongly depend on the choices made during analysis, and that no one methodology is an optimal choice for all contexts. A few different scenarios were analyzed, including analysis with a time lag and communities with different species ratios.

Strengths:

The paper sets up a clear problem to motivate the study. The writing is easy to follow, given the dense subject matter. A broad range of approaches was compared for both statistical tests and surrogate data generation. The appendix will be helpful for readers, especially those readers hoping to implement these findings into their own work. The topic of the manuscript should be of interest to many readers, and the authors have put in extra effort to make the writing as clear as possible.

Weaknesses:<br /> The main conclusions are rather unsatisfying: "use more than one method of analysis", "be more transparent in how testing is done", and there is a "need for humility when drawing scientific conclusions". In fact, the findings are not instructions for how to analyze data, but instead highlight the extreme dependence of the interpretation of results on choices made during analysis. The conclusions reached in this study would be of interest to a specialized subset of researchers focused on the biostatistics of ecological data. Ending the article with a few specific recommendations for how to apply these conclusions to a broad range of datasets would increase the impact of the work.

Reviewer #2 (Public review):

Summary:

This manuscript tackles an important and often neglected aspect of time-series analysis in ecology - the multitude of "small" methodological choices that can alter outcomes. The findings are solid, though they may be limited in terms of generalizability, due to the simple use case tested.

Strengths:

(1) Comprehensive Methodological Benchmarking:

The study systematically evaluates 30 test variants (5 correlation statistics × 6 surrogate methods), which is commendable and provides a broad view of methodological behavior.

(2) Important Practical Recommendations:

The manuscript provides valuable real-world guidance, such as the superiority of tailored lags over fixed lags, the risks of using shuffling-based nulls, and the importance of selecting appropriate surrogate templates for directional tests.

(3) Novel Insights into System Dependence:

A key contribution is the demonstration that test results can vary dramatically with system state (e.g., initial conditions or abundance asymmetries), even when interaction parameters remain constant. This highlights a real-world issue for ecological inference.

(4) Clarification of Surrogate Template Effects:

The study uncovers a rarely discussed but critical issue: that the choice of which variable to surrogate in directional tests (e.g., convergent cross mapping) can drastically affect false-positive rates.

(5) Lag Selection Analysis:

The comparison of lag selection methods is a valuable addition, offering a clear takeaway that fixed-lag strategies can severely inflate false positives and that tailored-lag approaches are preferred.

(6) Transparency and Reproducibility Focus:

The authors advocate for full methodological transparency, encouraging researchers to report all analytical choices and test multiple methods.

Weaknesses / Areas for Improvement:

(1) Limited Model Generality:

The study relies solely on two-species systems and two types of competitive dynamics. This limits the ecological realism and generalizability of the findings. It's unclear how well the results would transfer to more complex ecosystems or interaction types (e.g., predator-prey, mutualism, or chaotic systems).

(2) Method Description Clarity:

Some method descriptions are too terse, and table references are mislabeled (e.g., Table 1 vs. Table 2 confusion). This reduces reproducibility and clarity for readers unfamiliar with the specific tests.

(3) Insufficient Discussion of Broader Applicability:

While the pairwise test setup justifies two-species models, the authors should more explicitly address whether the observed test sensitivities (e.g., effect of system state, template choice) are expected to hold in multi-species or networked settings.

(4) Lack of Practical Summary:

The paper offers great insights, but currently spreads recommendations throughout the text. A dedicated section or table summarizing "Best Practices" would increase accessibility and application by practitioners.

(5) No Real-World Validation:

The work is based entirely on simulation. Including or referencing an empirical case study would help illustrate how these methodological choices play out in actual ecological datasets.

Mijuel Johnson: Could the American Dream be different for each generation or ethnic/racial community?

Malinda Burke's experience: Fiscally conservative as in modern republican? Neo-liberalist view?

Polly Mann:Similar to Johnson: Is it different

"Homeownership and equity" What other factors limit someone from achieving that? -Student loans (how has that affected peoples financial gains?

David Hite: Allow for others to achieve it

Another mention of housing and the lack of accessibility for young people

Is it for everyone, is it for noone?

Have certain groups been excluded from the American Dream, is it attainable as a minority especially in the African American experience?

Middle class shrinking? What is a "comfortable life"

Sam Crane: Independent self-sufficiency? Could look at the ideas of neoliberalism and individual capital

Damian Conley's American Dream similar to Scott

Jack Ragheb's view: what paths have narrowed?

Roberto Lopez's American Dream

Scott Meyer's American Dream

Interesting statistic to search for

What resources do we no longer have that might have made the American Dream plausible back then, what do we have now?

The idealized belief brought from 1950s America and earlier beliefs

It is imperative to keep in mind these two entities coincide with each other. As our very country runs on democracy, this allows for politics to influence economy as people with power can be in positions that can have an impact on our economy. For example; U.S.-China trade war (2018-2020). The U.S. government, under President Donald Trump, imposed tariffs on hundreds of billions of dollars' worth of Chinese imports. The goal was to pressure China into changing their trade practices, protect American manufacturing, and reduce the trade deficit. The trades made Chinese goods more expensive in the U.S., which raised costs for American businesses and consumers. In response, China imposed retaliatory tariffs on U.S. exports like soybeans and cars, hurting American farmers and automakers. The uncertainty caused by the trade war slowed global investment and contributed to market volatility. It also pushed some companies to move production to other countries to avoid tariffs. Maybe taxing trades with other countries to keep manufacturing goods "American," wasn't the best idea for our economy.

Organizations like the BBC operated under a public service model, funded by license fees and guided by principles of impartiality, education, and cultural enrichment. This system reflected a time when broadcasting was seen as a national service, tightly regulated by the government to serve the public good. However, deregulation and the rise of neoliberal economic policies encouraged competition and privatization. By the 1990s and 2000s, with the expansion of satellite and digital broadcasting, streaming platforms like Netflix and Amazon Prime reshaped the media landscape. Production is often guided by audience demand, advertising revenue, and global market pressure rather than purely public service values.

I find it really concerning that Facebook decided not to notify users after such a massive data breach. It feels like they care more about protecting their reputation than protecting the people who use their platform. Even if the leaked information was already public, users still deserve to know when their data is being used or exposed in unsafe ways. I think this shows how weak data privacy laws are, especially when companies can make their own choices about whether to inform people.

This article explains how Facebook was storing a list of millions of Instagram passwords. They advertised it as tens of thousands, hoping to make the issue seem less intense. However, the number was truly in the millions. They stored the passwords in plain text, even though they were supposed to be encrypted. Records show that 20,000 Facebook employees had access to these passwords. Even though the situation seemed dire, Facebook didn't urge users to change their passwords and barely called attention to the incident. This shows how as users we must take privacy precautions into our own hands. The company will usually not be looking out for our privacy, so we must do it ourselves.

Pay close attention to the use of "artificial sense of scarcity," as it's message is unveiling a deeper meaning. It is essentially establishing the cooperate companies controlling the price of manufactured goods to prolong consumption. Companies commercialize products to mass produce creating a trait of value and importance of the products. Emphasizing buying the product as soon as possible before they go away. When companies see how a certain product is being sold they can analyze the sales performance and determine to raise prices according to consumer uptake. Therefore, establishing scarcity on products that can otherwise be viewed as trivial. They, essentially, have control over the market and the price of goods.

TEXT- This poster encourages people to focus less on the material aspect like tinsel and lights and instead welcoming others and showing compassion. This poster through the use of "holy" and "prayers" shows Christian values, reminding others that the true spirit of Christmas is love, connection not materials. The font is bond and overall makes someone focus on the text.

IMAGE- The background is diagonal gradient lines with yellow and sometimes orange, all coming together, symbolistic to people coming together. Lastly, there is a gradient of all the colors combined with the text "less tinsel more love" in cursive. This Overall the color usage gives off peace and more significance to the text.

(() => { // 以正文區為範圍，找不到就退而求其次 const scope = document.querySelector('#content3') || document.querySelector('#content') || document;

// 1) 去掉「打開字典」「顯示相似段落」等工具連結 scope.querySelectorAll('a.sprite-more, a.sprite-parallel').forEach(a => { // 這些連結常被一層 <div> 包著，一起移除比較乾淨 const wrap = a.closest('div'); if (wrap && wrap.contains(a)) wrap.remove(); else a.remove(); });

// 2) 去掉每行最左邊的行號超連結（class="popup" 的 1、2、...） scope.querySelectorAll('tr[id^="n"] td[align="right"] a.popup').forEach(a => a.remove());

// 3) 清掉行首那格若已空的殘留包裝 scope.querySelectorAll('tr[id^="n"] td[align="right"]').forEach(td => { if (!td.textContent.trim()) td.remove(); });

// 4) 去掉空的占位段落（例如

I've rethought what privacy actually means online. I've always assumed my personal information was private and secure, but this reflects the trust we've unwittingly placed in these platforms. Once information is uploaded online, is it truly private? Even if we delete something, a copy might still remain on the server. So, I'm now unsure if deletion is even necessary.

This example surprised me. Usually when I think of the idea of privacy, I think of it as something that needs to be respected at all costs across social media, especially by the company running the site. Each user's information and habits are their own and should not be easily viewable by the company. However, this example of death threats and harassment is something that most definitely has happened before and will happen again. In this instance, I would 100% want the company to be able to access my private messages. In this case, a disruption of privacy would be ethical, because it is in the intention of creating safety and protection. Instances like these should be the exceptions to the general rule of privacy.

skimming through the texts to obtain a sense of what you are reading

make sure the source is relevant and is reliable.

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Hi, I have a question about it.

Q1. pseudoinverse notebook uses JacobianWrtVariable.kQDot in the PseudoInverseController. However, JacobianWrtVariable.kV is used in the Pick notebook.

It means that qDot = v ?? So, is it okay to use either one ? - One confused thing : iiwa robot is 7 DOF robot. So, refer to the chapter 3 contents, position has 7 DOF (perhaps using quaternion), velocity has 6 DOF. How do we determine that qDot is not equal to v ? (Because I'm a beginner of the Drake, so I don't understand why qDot is equal to v in this example.)

Thanks.

eLife Assessment

This important work employed a recent, functional muscle network analysis for evaluating rehabilitation outcomes in post-stroke patients. While the research direction is relevant and suggests the need for further investigation, the strength of evidence supporting the claims is incomplete. Muscle interactions can serve as biomarkers, but improvements in function are not directly demonstrated, and the method's robustness is not benchmarked against existing approaches.

Reviewer #1 (Public review):

Summary:

This study addresses an important clinical challenge by proposing muscle network analysis as a tool to evaluate rehabilitation outcomes. The research direction is relevant, and the findings suggest further research. The strength of evidence supporting the claims is, however, limited: the improvements in function are not directly demonstrated, the robustness of the method is not benchmarked against already published approaches, and key terminology is not clearly defined, which reduces the clarity and impact of the work.

Comments:

There are several aspects of the current work that require clarification and improvement, both from a methodological and a conceptual standpoint.

First, the actual improvements associated with the rehabilitation protocol remain unclear. While the authors report certain quantitative metrics, the study lacks more direct evidence of functional gains. Typically, rehabilitation interventions are strengthened by complementary material (e.g., videos or case examples) that clearly demonstrate improvements in activities of daily living. Including such evidence would make the findings more compelling.

Second, the claim that the proposed muscle network analysis is robust is not sufficiently substantiated. The method is introduced without adequate reference to, or comparison with, the extensive literature that has proposed alternative metrics. It is also not evident whether a simpler analysis (e.g., EMG amplitude) might produce similar results. To highlight the added value of the proposed method, it would be important to benchmark it against established approaches. This would help clarify its specific advantages and potential applications. Moreover, several studies have shown very good outcomes when using AI and latent manifold analyses in patients with neural lesions. Interpreting the latent space appears even easier than interpreting muscle networks, as the manifolds provide a simple encoding-decoding representation of what the patient can still perform and what they can no longer do.

Third, the terminology used throughout the manuscript is sometimes ambiguous. A key example is the distinction made between "functional" and "redundant" synergies. The abstract states: "Notably, we identified a shift from redundancy to synergy in muscle coordination as a hallmark of effective rehabilitation-a transformation supported by a more precise quantification of treatment outcomes."

However, in motor control research, redundancy is not typically seen as maladaptive. Rather, it is a fundamental property of the CNS, allowing the same motor task to be achieved through different patterns of muscle activity (e.g., alternative motor unit recruitment strategies). This redundancy provides flexibility and robustness, particularly under fatiguing conditions, where new synergies often emerge. Several studies have emphasized this adaptive role of redundancy. Thus, if the authors intend to use "redundancy" differently, it is essential to define the term explicitly and justify its use to avoid misinterpretation.

Reviewer #2 (Public review):

Summary:

This study analyzes muscle interactions in post-stroke patients undergoing rehabilitation, using information-theoretic and network analysis tools applied to sEMG signals with task performance measurements. The authors identified patterns of muscle interaction that correlate well with therapeutic measures and could potentially be used to stratify patients and better evaluate the effectiveness of rehabilitation.

However, I found that the Methods and Materials section, as it stands, lacks sufficient detail and clarity for me to fully understand and evaluate the quality of the method. Below, I outline my main points of concern, which I hope the authors will address in a revision to improve the quality of the Methods section. I would also like to note that the methods appear to be largely based on a previous paper by the authors (O'Reilly & Delis, 2024), but I was unable to resolve my questions after consulting that work.

I understand the general procedure of the method to be: (1) defining a connectivity matrix, (2) refining that matrix using network analysis methods, and (3) applying a lower-dimensional decomposition to the refined matrix, which defines the sub-component of muscle interaction. However, there are a few steps not fully explained in the text.

(1) The muscle network is defined as the connectivity matrix A. Is each entry in A defined by the co-information? Is this quantity estimated for each time point of the sEMG signal and task variable? Given that there are only 10 repetitions of the measurement for each task, I do not fully understand how this is sufficient for estimating a quantity involving mutual information.

In the previous paper (O'Reilly & Delis, 2024), the authors initially defined the co-information (Equation 1.3) but then referred to mutual information (MI) in the subsequent text, which I found confusing. In addition, while the matrix A is symmetrical, it should not be orthogonal (the authors wrote AᵀA = I) unless some additional constraint was imposed?

(2) The authors should clarify what the following statement means: "Where a muscle interaction was determined to be net redundant/synergistic, their corresponding network edge in the other muscle network was set to zero."

(3) It should be clarified what the 'm' values are in Equation 1.1. Are these the co-information values after the sparsification and applying the Louvain algorithm to the matrix 'A'? Furthermore, since each task will yield a different co-information value, how is the information from different tasks (r) being combined here?

(4) In general, I recommend improving the clarity of the Methods section, particularly by being more precise in defining the quantities that are being calculated. For example, the adjacency matrix should be defined clearly using co-information at the beginning, and explain how it is changed/used throughout the rest of the section.

(5) In the previous paper (O'Reilly & Delis, 2024), the authors applied a tensor decomposition to the interaction matrix and extracted both the spatial and temporal factors. In the current work, the authors simply concatenated the temporal signals and only chose to extract the spatial mode instead. The authors should clarify this choice.

it is important to remember the location of where the information that you are citing and what paragraphs there in and the sentence to the period

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For anyone pursuing work in trade, energy, or mining, including my own job search in southern Africa, China’s rare earth policies will ripple across resource markets, affecting regional industries and investment flows

China’s licensing demands may backfire by pushing countries to create alternative supply networks

The move unsettles global markets, revealing how fragile international production systems have become

Beijing’s timing blindsided Washington, exposing U.S. dependence on Chinese materials

Trump’s threat of extreme tariffs escalates tensions and risks a new phase of the trade war

This quote captures the article’s theme. China is adopting the U.S.’s own economic tactics, perhaps more effectively

Analysts warn China’s leverage in critical minerals could outweigh U.S. power in technology

China is mirroring U.S. export controls by using its dominance over rare earths, a direct challenge to the US tech restrictions.

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add the Authers name and date it was published exact time and day if possible if t

essayant

alternatifs

qui nous

permet

Ouiiiiiii

voire

ainsi que

concernent

qui présente principalement deux problèmes ?

quelle suite ?

supprimer "ne jamais" : sans pourtant l'explorer est clair et correct

je n'ai pas bien compris qui est ce "on"

où ?

retracées

si elles existent, sont présentes

s'étend

il faut choisir : "Est-ce que le prologue est" OU "le prologue est-il"

comme des paratextes

Ouiiiiiiiiii

concernant (au sein d'un auteur n'est pas très correct, on dirait qu'on parle de l'intérieur de son corps)

références ?

destinataires ?

part

présente, dans la majorité des cas, un privilège ?

sortes

locale

"d'éditer précisément la petite pièce en deux scènes de La Conqueste de B**, précisément sur son privilège et son prologue" ? (pure suggestion, tu n'es bien sûr pas obligé de suivre)

nécessitait

fournit

je ne suis pas sûre de comprendre ce que ça veut dire

de ses permanences

Details: Website: Description Email: bconscenter.com.vn@gmail.com Zipcode: 75308 Phone: 0932221317 Address: Đường Thống Nhất, phường Đông Hoà, Thành phố Hồ Chí Minh

bconscentercity #bconscenter #bconsbinhandongtay #bconsthongnhat

be sure that you know about the different types of periodicals.

cnm has a data base that could help for research.

あああ

あああ

I feel like he's saying this too emotionally manipulate her

She was tired of him talking about the operation

This line shows the women trying to ease the tension between the two.

okay. my opinion, placing myself in the position of the child here (so if i was under the complete impression and belief that robovie was sentient), my answer would be YES it deserves civil liberties. (i should also preface this by saying that i think prisoners should have the right to vote and to receive compensation for their work) but robovie is not sentient so now what. what would robovie do with the money. money doesnt mean anything to it. and how would robovie know what to vote for. i feel like if a non-sentient robot COULD vote, it would be a huge conflict of interest for us humans because whos telling that robot what to do?

HOLY SHIT????

HOLY SHIT.

Holy shit.

oh my god......

okay so ACCORDING TO the child, who is taking all of this info at face value, robovie has thoughts and feelings and history and is able to care about things. CAN robovie care about things? No. Because Robovie is not sentient. Robovie is following a script that was meticulously composed by humans. But i digress. the child doesnt know this.

this page is all ok

Incredibly irresponsible.

也就是说，尽管字符串进行了byte转化，其内部存储时依旧是以单个字符为单位，可以通过列表下表索引和ord函数来获得相应的值。

字典的三种创建方式 1.dict1={1:'a','a':'b'}利用大括号里面封装键值对 2.dict1=dict([(1,'a'),('a','b')])利用dict()函数，里面存入序列对列表 3.dict1=dict(1='a','a'='b')利用dict()函数，里面传入键值对

This passage means that during the 1930s because of President Franklin D. Roosevelt’s New Deal programs many Black voters began switching their political support. Before this, most Black Americans had supported the Republican Party because Abraham Lincoln had ended slavery. But in the 1930s, many Black voters, especially in the North and West where they could vote freely, started supporting the Democratic Party, attracted by the New Deal’s promises of jobs and relief during the Great Depression. However, their hopes for racial equality and civil rights were disappointed. Despite campaigns by civil rights groups pushing for laws against

its very interesting that policies that attempted to stop "promiscuous or illicit sexual relatiopnships" would be accepted as a permissible legislative goal. That seemingly violates an individual liberty. After looking it up, I found out that extramarital sex was illegal in many states until the mid 20th century.

This paragraph lets in on an interesting interpretation of the first amendment. It opens up the possibility that rights that are not explicitly constitutionaly protected can be interpreted to be protected if deemed neccesary.

color- the swirling red and blue strokes resembles a twarn up flag, with the colors of patriotism. text- the lines describe a "borderless country" with broken things like a "worn table cloth" and a "broken loaf of bread." the lower case font makes it feel more personal and less nationalism. meaning- the text and image rejects nationalism and talks about unity.

What is easy to measure but hard to value? Wealth? Life? Time?

composition- the large text on the top feels like it shouting at the audence. color- the color is only limited to two colors. navy and white. This keeps the focuse on the message insead of distracting the audeance with the colors. meaning - justice from oppresstion requires strength and pain endurence, but that leads twards justice.

I was so suprised that the author uses ladder to represnts the fear of his alone.